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The effect of selected drugs on the vascular responses of the rat to localized cold Singer, George Edward 1954

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THE E F F E C T  OF S E L E C T E D  DRUGS  ON T H E V A S C U L A R  R E S P O N S E S OF T H E R A T TO L O C A L I Z E D COLD  George  A Thesis of  the  Edward  Submitted  in Partial  Requirements Master  Singer  of  for  the  Fulfilment Degree  of  Science  in The D e p a r t m e n t  of  Anatomy  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o the standard r e q u i r e d from c a n d i d a t e s f o r the degree o f Master of S c i e n c e .  Members o f E x a m i n i n g C o m m i t t e e Department o f Anatomy  THE U N I V E R S I T Y  OF B R I T I S H  April,  1954  COLUMBIA  ABSTRACT Of T h e s i s f o r Master of Science Degree THE EFFECT OF SELECTED DRUGS ON THE VASCULAR RESPONSES OF THE RAT  TO LOCALIZED COLD  George Edward S i n g e r Cold i n j u r y of both the dry and the moist types are of the utmost are l i k e l y  importance i n m i l i t a r y o p e r a t i o n s , and  to assume even g r e a t e r importance i n the event  of p o l a r warfare.  The modern l i t e r a t u r e on f r o s t b i t e  began w i t h Napoleon's  r e t r e a t from Moscow i n 1812 when h i s  surgeon, Baron L a r r e y r e c o r d e d the d i s a s t r o u s event i n h i s memoirs.  S i n c e then i t has been the v a r i o u s wars of  the western world that has p r o v i d e d the main s t i m u l u s f o r i n v e s t i g a t i o n i n t o the c o l d  problem.  The experimental i n v e s t i g a t i o n has e f f e c t e d p o s i t i v e b e n e f i t by way  little  o f management of an acute c o l d  i n j u r y , but i t has served to break down many time  honoured  d o c t r i n e s , e s p e c i a l l y the theory that slow thawing of a f r o z e n limb p r o v i d e d the b e s t treatment.  The  significance  of the v a r i o u s events that occur i n the t i s s u e s d u r i n g a f r e e z i n g r e a c t i o n are not agreed on by a l l workers,  but  the course o f 'the r e a c t i o n and e s p e c i a l l y the danger  of  the secondary e f f e c t s d u r i n g thawing are w e l l known. The p r e s e n t i n v e s t i g a t i o n concerned an attempt to observe m i c r o s c o p i c a l l y the v a s c u l a r changes i n the r a t mesoappendix a c c o r d i n g to a technique o f Zweifach. p o i n t apparatus d e s c r i b e d by Hass and T a y l o r was  A cold  utilized  Abstract,  p.2  Localized  for  exact  f r e e z i n g of  ten  s e l e c t e d d r u g s on  a f t e r f r e e z i n g was Procaine, oline,  a c a p i l l a r y bed. the  tested  chlor-tripolon, rutin,  included  delay  rate  extent  and  ulation  The  of  the  the h i n d ether  an  mixture  criteria  vessels.  effort  legs  to  and  basis.  priscoline,  confirm  the  ably  Of  the  be  and  bed  hist-  resumption of  and  this  the  circ-  control  impression  animals.  grossly,  i n a carbon d i o x i d e  an  The  Injury  drugs t e s t e d ,  etarnon, h y d e r g i n e ,  and  changes num-  Index a day  ascorbic  adverse e f f e c t ,  The  by  acid  and  to have a prob-  value  of  rapid  c o n s i s t e n t l y confirmed  experiments.  was  Chlor-Tripolon  were f o u n d  beneficial effect.  and  procaine,  effect.  t o have an  i n w a t e r a t 4 2 ° C . was  the  difference  e x p r e s s e d g r a p h i c a l l y on  significant  apres-  lessening  significant  especially rutin,  throughout a l l gross  and  t h a w i n g were d e s c r i b e d  same t e n  benadryl,  significant  thawing  stasis,  a r b i t r a r y concept of  h i s t a m i n e were f o u n d  apresoline,  acid,  at -20°C. f o r twenty seconds.  were f o u n d t o h a v e no and  No  o f r a t s were f r o z e n  u t i l i z e d which could day  vascular  of  f o r a drug e f f e c t  and  treated  observed g r o s s l y f o l l o w i n g erically  ascorbic  stasis,  o b s e r v e d between t h e  the  etamon, h y d e r g i n e ,  i n onset of v a s c u l a r  in static  In  of  influence  alternately with control rats.  p r i s c o l i n e , benadryl,  amine were t e s t e d .  was  reactions  The  Cold  TABLE OP CONTENTS  Title  Page  Table  of  Contents  Acknowledgements I  II  III  The C o l d P r o b l e m The C o l d P r o b l e m A s s e s s e d Drugs S e l e c t e d f o r T e s t i n g Rutin Procaine Ascorbic acid Priscoline Benadryl Etamon Chlor-Tripolon Hydergine Histamine Apresoline Need f o r C o n f i r m a t o r y Gross E x p e r i m e n t s Microscopic Observation The R a t M e s o a p p e n d i x The C i r c u l a t i o n o f t h e M e s o a p p e n d i x F i g u r e - The C a p i l l a r y B e d O b s e r v a t i o n o f the Rat Mesoappendix Photographs - Apparatus The F r e e z i n g P o i n t Figure - Freezing Apparatus E p i n e p h r i n e Response Test Localized Cold Injury Experiments Recording of Observations C r i t e r i a For a Drug E f f e c t Care of Animals Preliminary Microscopic Experiments Preliminary C a p i l l a r y Experiments I n i t i a l E p i n e p h r i n e Response Tests O b s e r v a t i o n s w i t h Compound F  IV Record of Microscopic Observations Procaine Procaine repeated Procaine Parenteral Priscoline  3  P r i s c o l i n e Repeated Benadryl Benadryl Repeated Benadryl Repeated Etamon Etamon Repeated Etamon Repeated Hydergine Apresoline Chlor-Tripolon Rutin R u t i n Repeated Ascorbic Acid Histamine Histamine Repeated V Gross F r o s t b i t e Experiments 190 Reason f o r Gross Experiments Standard F r e e z i n g I n j u r y Manner of Thawing Apparatus . Photograph - Apparatus Recording o f Observations I n j u r y Index P l a t e - Example of I n j u r y Assessment S e l e c t i o n o f Gross Experiments VI Record o f Gross O b s e r v a t i o n s D u r a t i o n of I n j u r y and Rate o f Thkw Procaine Procaine and P r i s c o l i n e Benadryl Colour Photographs Etamon and Hydergine A p r e s o l i n e and C h l o r - T r i p o l o n Rutin Colour Photographs Ascorbic Acid Histamine Histamine Repeat VII S i g n i f i c a n c e of the Data Obtained Threshold E p i n e p h r i n e Response Procaine M i c r o s c o p i c a l l y Time and Rate of Thawing Rutin Ascorbic Acid  203  308  4  VIII C o n c l u s i o n s Summary  Reached of Conclusions  Appendix Equipment f o r Cold P o i n t Chemicals Used Formula f o r Drip S o l u t i o n References  Cited  317 322  326  5  ACKNOWLEDGEMENTS Nearly every member of the Anatomy Department helped at some stage i n the work of t h i s p r o j e c t from Department Head to Animal Keeper.  The work was guided  throughout the year by Dr. S.M. Friedman, from whom I received c o n s t a n t l y much help and i n s p i r a t i o n .  Mr. G.  Crosson and Mr. C. Blake deserve s p e c i a l thanks f o r t h e i r i n t e r e s t and w i l l i n g assistance whenever c a l l e d upon to "lend a hand". Mr. D.L. Warner, a senior student i n the F a c u l t y of Medicine provided much help i n two r e s p e c t s .  I t was  he who introduced me to the technique o f observing micros c o p i c a l l y the v a s c u l a t u r e of the r a t mesoappendix and he guided me away from many p i t f a l l s during the e a r l y stages of the p r o j e c t .  He also gave up h i s Christmas  vacation to perform several of the experiments as acknowledged l a t e r . A p p r e c i a t i o n i s here expressed f o r the s u p p l i e s of Hydergine and Apresoline received from the Sandoz Corporation and the Ciba Company. G.E. Singer, A p r i l 1, 1954.  6  THE EFFECT OF SELECTED DRUGS ON THE VASCULAR RESPONSES OF THE RAT TO LOCALIZED COLD  THE COLD PROBLEM Frostbite and cold i n j u r i e s generally continue to present a challenge to our armed forces b a t t l i n g i n the colder regions of the earth.  Orr and Fainer ( 1 )  have recently reported on the t e r r i f i c t o l l i n Korea among the United States forces during the winter campaign of 1950-1951 when there were sixty thousand f r o s t bite casualties with six thousand amputations r e s u l t i n g . This d e f i n i t e l y puts this problem into the category of a major battle hazard.  The p o s s i b i l i t y ,  and even the  p r o b a b i l i t y of polar warfare i n the near future  forces  greater attention to be paid to the problem of cold injury than has ibeelh^aid:.!:,  The matter of acclimat-  i z a t i o n i s being investigated as a preventive measure. Orr and Fainer outline several factors, more or less obvious, that w i l l help the soldier i n the prevention of actual freezing i n j u r i e s .  In addition to the  various physical agents, we would l i k e to have some medication that could be administered to protect the combatant and c i v i l i a n a l i k e against both the actual  7  freezing injury and also against the resultant immediate and chronic sequellae of f r o s t b i t e once i t i s established.  At the moment no such medication i s  known, and a l l drugs suggested f a i l through being administered following the freezing, either immediatel y or l a t e r , often after the thawing has been completed.  Webster and Bigelow ( 2 ) writing f o r the recent  special C i v i l Defence issue of the Canadian Medical Association Journal summarize the c o n f l i c t i n g current theories regarding pathogenesis and treatment without actually stressing any special regimen.  They do  emphasize, as we a l l must, the value and necessity f o r continued experimental work both i n the laboratory and i n controlled c l i n i c a l studies, and they seem to f e e l that the knowledge obtained so f a r i s barely laying the ground work f o r the great advances possible i n t h i s field.  The Josiab. Macy J r . Foundation has even s t a r t -  ed a Conference on cold i n j u r y ( 3 ) .  The best  summary of the experimental work to date l i e s i n the a r t i c l e prepared by Shumacker and Lempke ( 4 ) which gives a resume of the l i t e r a t u r e from as f a r back as 1917 r i g h t up to a r t i c l e s s t i l l i n press i n 1951.  This  i s a rather extensive review and I t forms the basis for the experimental work carried out f o r this thesis.  8 There a r e v e r y few a r t i c l e s more r e c e n t t h a t a r e n o t clinical,  and s i n c e most o f the e x p e r i m e n t a l work now  b e i n g p e r f o r m e d i s f i n a n c e d through m i l i t a r y r e s e a r c h g r a n t s o f one k i n d o r a n o t h e r , r e p o r t s a r e v e r y  guard-  ed as h i g h l y s e c r e t .  THE COLD PROBLEM ASSESSED The acute v a r i e t y o f c o l d i n j u r y may be d i v i d ed I n t o t h r e e broad c a t e g o r i e s . mentioned g e n e r a l hypothermia,  F i r s t l y m i g h t be  which i s i n c l u d e d only  f o r completeness s i n c e i t uncommon, and s u r v i v a l f r o m i t even more r a r e (5 ), a l t h o u g h i t i s b e i n g  investig-  ated f o r a n a e s t h e t i c uses i n c o n g e n i t a l h e a r t surgery. Secondly i s the m o i s t c o l d l e s i o n , t h e t r e n c h f o o t o r immersion f o o t syndrome.  Trench f o o t was p e c u l i a r l y  a problem o f the f i r s t World War, w h i l e immersion f o o t was the second World War v a r i e t y o f t h e same c o n d i t i o n , b e i n g the r e s u l t o f a p r o l o n g e d v e r t i c a l p o s t u r e w i t h an ambient temperature necessary requirement  above f r e e z i n g .  An a p p a r e n t l y  i s t h e need f o r m o i s t  cold,  w h i c h conducts h e a t away from the exposed l i m b f a s t e r than does d r y c o l d .  The e f f e c t o f t h e dependency o f  the l i m b i s o f course t o impede the b l o o d by d e l a y i n g venous r e t u r n . problem o f f c o s t b i t e .  circulation  T h i r d l y i s the b i g  T h i s I s the c o n d i t i o n r e s u l t -  9  ing  from exposure to cold of s u f f i c i e n t severity and  duration to produce i c e i n the affected parts. too  This  had a counterpart i n the second World War i n the  condition known as high a l t i t u d e f r o s t b i t e , characterized by a very rapid freezing at very low temperatures, such as the t a i l gunner of a f i g h t e r plane r e ceived when he took o f f his glove to adjust h i s gun, with h i s fingers immediately s t i c k i n g to the metal and being deeply frozen. The frequent appearance i n t h i s b r i e f classi f i c a t i o n of such terms as trench foot, and high a l t itude f r o s t b i t e suggests that cold injury i s f o r a l l p r a c t i c a l purposes a disease p e c u l i a r l y associated with m i l i t a r y operations. the  The modern l i t e r a t u r e on  subject begins with Baron Larey's account ( 6 )  of Napoleon's casualties on the tragic retreat from Moscow i n 1812.  However, i t was not u n t i l the f i r s t  World War that much e f f o r t or i n t e r e s t was put into trying to solve the f r o s t b i t e problem.  S i r Thomas  Lewis was one of the main investigators of freezing injury at the time, and several papers preceded from his  laboratory on such ideas as supercooling of tissues  and the concept that i t was the formation of i c e crystals  i n t r a c e l l u l a r l y which caused the damage of freezing.  10  Experimental work during the second World War  was  centered mainly i n trying to understand the physiology and the pathology of the f r o s t b i t e l e s i o n .  Many  are the papers discussing which was the more important of the various processes more or l e s s going on simultaneously.  The whole process from i n i t i a l  exposure  to cold r i g h t through to the development of the l a t e sequellae i s well discussed by Shumacker ( 4 ) and there seems to be l i t t l e value i n repeating i t a l l here.  The important aspect, not of course o r i g i n -  ated by Shumacker, i s the vascular component.  In  a freezing injury, naturally, c e l l s and tissues w i l l be k i l l e d by the cold or by the anoxia accompanying the ischemia produced by the vasoconstriction.  But  the extent of the freezing influence does not d i r e c t l y determine the extent of the damage r e s u l t i n g . I t i s the secondary changes following thawing that anethe gravest sequels to f r o s t b i t e .  After thawing  (to quote from Shumacker) there develops a reactive hyperemia which may represent the reaction to s l i g h t l y d i f f u s i b l e abnormal c e l l products r e s u l t i n g from d i r e c t cold injury or ischaemic i n j u r y .  With the return of '  blood flow, oedema begins to form as p r o t e i n - r i c h f l u i d escapes through the c a p i l l a r y walls rendered  11  hyperpermeable  by the  effects  of  the  The oedema p r o g r e s s i v e l y i n c r e a s e s maximal  volume a l l o w e d by  encompassing oedema, formed to  stasis  the  ulation  beginning at  the to  held  red the  is  cell  that  s t r e a m as  spaces.  of  possibility is  stasis.  the  of  vessels.  This  to  of  some  the the  will  circat  one  events  break  widely  hyperpermout  of  cells  the  interstitial stasis  must  chemical alteration  stasis  or  controversy  cells  that  with  and to  the  The  be  In third  s l u d g i n g where  a sticky  another  which need not  summary f r o m  course  the  reversible,  rendered  is  Shumacker g i v e s  these views,  is  c l o t t i n g mechanism.  connects  adhere  The  progressively  The v i e w m o s t  walls,  p r e c i p i t a t e d on the  them to  the  to  is  serum e n t e r s  offered  causes  out  There  some c o l l o i d a l  blood unrelated  the  formation of  vessels  merely f i l t e r  the  of  since mechanical pressure  capillary  injury  the  seen  i n adjacent  the  limit  the  capillaries.  stasis  The s e c o n d s u g g e s t i o n  result  against  of  true  are  anoxia.  bringing to^ahalt  This  flow  the  blood  the  vessels,  masses.  cause  by the  there  blood  least,  i n blood  eable  the  the  through them.  changes  as  of  small  extensile  i n the  or  a n d may r e a c h  Cbtocidertsrlly w i t h  develops  elements  f i l l  the  skin.  the  cold  coat the  arguments  which walls  for  be m e n t i o n e d  and  here.  Shumacker's d i s c u s s i o n p i c k s in frostbite  that  occur  at  the  12  capillary level.  Since studies were already i n  progress at t h i s University on the observation of the c a p i l l a r y responses i n the r a t mesoappendix (Warner, D.L.,  7 ) and since Hass and Taylor had  described an instrument  ( 8 ) f o r applying a quant-  i t a t i v e hypothermal injury, I t seemed very l o g i c a l to study the behavior of the blood vessels i n the rat mesoappendix after freezing as influenced by various medications enterally.  applied both t o p i c a l l y and par-  Except f o r the technique of studying  c a p i l l a r y responses to a l o c a l freezing i n j u r y as influenced by various drugs, which I have not found described i n the l i t e r a t u r e , there i s nothing very o r i g i n a l i n the testing of the various drugs selected.  Most of them have been used previously f o r  gross freezing experiments.  Ten were selected:  procaine, P r i s c o l i n e , Etamon, Hydergine, Apresoline, Rutin, Ascorbic acid, Benadryl, Chlor-Tripolon, and histamine.  In view of the f a c t that some of these  preparations are c a l l e d by t h e i r trade names, a further discussion of the drugs i s i n order. Drugs Tested i n Cold Injury Experiments Procaine This i s believed to paralyze peripheral con-  13  strictor  fibers  The d r u g was Topically 2.0%  tested  there  is  however,  all  in  this  better, riate  the  is  is  t o p i c a l l y and  difficulty  rapid  the  and  as  or  2100  calculated. detoxified  b y an  in  turn  thelium  a  is  as  some  Is  the  in  the  action  55  of  to  give  on the  i n competition with  the  used  hydrolyzed  blood  the  mg.  subsequently  is  rat  is  majority  diethylaminoethano  an i n d i r e c t  ( may a c t  dosage  to  approp-  adult  45  procaine  enzyme  an  or  200 mg.  L . D ^ Q .  the  liver,  of  1600 mg.  off  dose.  the  action.  k i l l the  demonstrated  frequent  gives to  and  para-  w h i c h may  vessel  endo-  histamine).  Priscoline This acts  as  erioles.  is  benzazoline  a vasodilator It  is  an  hydrochloride,  of peripheral  adrenolytic  Ciba.  arteries  agent,  to  trouble  solution  dose  will  and  a  suitable  that  acid  of  a 0.b%  parenteral  necessitate  (9)  basis  - Note  amino b e n z o i c  is  Griffith  topical  mg.  On t h i s  to  them.  parenterally.  adequate  effective  injections  intravenously  animals.  with  w i t h dosage,  There  enough  continuous  therapeutic  given  time.  concentration.  M . L . D .  contact  detoxified principally in  intraperitoneally  have  no  estimating  The p r o c a i n e  for  both  solution being generally  clinically  and  upon p e r i p h e r a l  and  blocking  It artthe  was  14  pressor without disturbing the depressor e f f e c t of epinephrine.  The action i s peripheral and there may  also be some d i r e c t d i l a t i n g effect on the vessel walls themselves (Beckman, 10).  Dosage l e v e l based  on approximate adult human dose. Etamon chloride Tetraethylammonium chloride, Parke-Davis, i s a depressant of both sympathetic and parasympathetic peripheral ganglia, with p o s s i b i l i t y of some l o c a l l y exerted vasodilator action.  Used at dosage l e v e l  similar to reports tabulated by Shumacker ( 4 ). Hydergine Hydergine i s not mentioned i n Shumacker's review, probably because the f i r s t report by Hurley ( l l ) appeared just when the larger review was received for publication.  The use of the dihydrogenated ergot  alkaloids stemmed from basic pharmacological findings that these derivatives had a sympathicolytic on the peripheral c i r c u l a t i o n .  action  B l u n t s c h l i and Goetz  (12 ) showed that dihydroergocornine produced no c o n s t r i c t i o n i n the sympathectomized limb, and gave vasodilatation i n the normally innervated  limb, t h i s  being the f i r s t known ergot derivative acting purely as a sympathicolytic  and also acting over higher  o;;.r...pt  15  sympathetic centres rather than peripherally at the myoneural sympathetic junction, and they showed that the compound acted on the vascular centres i n the medulla and or the hypothalamus.  Rothlin of Basle  a year e a r l i e r showed ( 1 3 ) a l l four of the dihydrogenated derivatives of ergot had this  sympathico-  adrenolytic a c t i v i t y , and Hurley used a preparation c a l l e d CCK-179 containing equal parts of dihydroergocornine, dihydroergocristine, and dihydroerg©kryptine, now put out by Sandoz as Hydergine.  Hurley  f e l t vasoconstriction (due to l o c a l response, l o c a l central nervous system r e f l e x , and hypothalamic vasoconstriction  due to cooled blood) could contribute  greatly to tissue anoxia following f r o s t b i t e , and that release of this could be of prime importance i n the therapy of f r o s t b i t e .  He found, following exposure of  sixty Sprague-Dawley and Wistar rats ( d i s t a l seven centimetres of the t a i l s to -15°C f o r sixty  seconds)  that had received varying dosages of Hydergine, that there was s t a t i s t i c a l l y s i g n i f i c a n t improvement of each treated group over the controls.  Hurley suggested  that these dihydrogenated ergot alkaloids might prove to be of value i n the treatment of high altitude f r o s t bite.  The dosage of Hydergine used i n the present  experiments was nearly that used by Hurley.  16 Apresoline Hydralazine hydrochloride, Ciba, has an action claimed by the manufacturer to be l a r g e l y central on the midbrain,  serving to lower both the s y s t o l i c and d i a s t o l i c  blood pressures, especially i n hypertension, yet permitting increased blood flow through the kidneys.  I t has  adrenergic blocking effects, adrenolytic and sympatholytic, against the pressor effects of epinephrine and nor-epinephrine.  This drcug has not yet, to my knowledge^  been reported i n f r o s t b i t e experiments. rats i s 3 4 mg. per kilogram body weight.  L D  5  Q  f o r white  The dosage  l e v e l used was on the range ©f 2 mg. per animal. Rutin and Ascorbic acid I t i s l o g i c a l to t r y agents that reportedly reduce c a p i l l a r y permeability as well as f r a g i l i t y . Rutin i s one of the vitamin P substances, acid i s of course vitamin C.  and ascorbic  The dosage selected f o r  r u t i n was that reported i n Shumacker's review. the case of ascorbic acid, i t i s regarded  In  as impossible  to make rats d e f i c i e n t i n t h i s vitamin since they manufacture their own, hence excessively large doses were used, which had the e f f e c t to be noted l a t e r i n the gross experiments. Benadryl This antihistaminic agent has been used frequently before i n f r o s t b i t e ( 4 ) and the dosage l e v e l  17  supposedly e f f e c t i v e i n rats i s known.  0.43 mg.  per  kilogram body weight was used by Shumacker. Chlor-Tripolon This i s an alternate antihistaminic, very powerful, and not previously reported i n f r o s t b i t e lesions experimentally.  The dosage l e v e l selected  was rather empirical at 5 mg. per animal. Histamine I t was decided to attempt to assess the influence of histamine by virtue of the questionable findings with the antihistaminics.  No stated e f f e c t i v e  dosage l e v e l could be found, and two values were s e l ected f o r t r i a l , 10 mg.  and 50 mg. per kilogram body  weight. Need f o r Confirmatory Gross Experiments As mentioned, i t was decided i t would be l o g c i a l to carry on with microscopic observation of the blood vessels i n the r a t mesoappendix.  The vessels  at this l o c a t i o n were selected primarily f o r convenience, since very beautiful preparations can be obtained with a minimum of e f f o r t , and with the equipment t© be described, such preparations can be maintained i n a healthy condition f o r up to four hours.  However,  i n cold i n j u r i e s , the mesoappendix i s seldom frozen,  18 and  there i s no reason  to expect t h a t c a p i l l a r i e s  that  f u n c t i o n e d one way i n the p r o t e c t e d environment o f the p e r i t o n e a l c a v i t y would f u n c t i o n i n the same way i n the h i n d l e g o f the same animal.  Accordingly,  the s e r i e s  of gross experiments were performed as d e s c r i b e d i n an e f f o r t to c o r r e l a t e the f i n d i n g s I n the two areas.  later  19  Microscopic Observation of the Rat Mesoappendix The Rat Mesoappendix There are numerous areas i n various species of animals whe^e the f i n e d e t a i l s of the blood c i r c u l a t i o n can be observed c l e a r l y .  Of these,the mesentery of the  r a t l y i n g between the caecum and the terminal ileum i s one of the most accessible and most convenient to study. A l l that i s needed i s an animal s u f f i c i e n t l y anaesthetized by an agent such as nembutal, and with a median abdominal i n c i s i o n made with an instrument such as a pair of scissors, the caecum may be exteriorized and draped over a supporting loop with the so-called mesoappendix exposed f o r view with a low power microscope, and illuminated by transillumination. The Circulation of the Mesoappendix Without discussing the controversies concerning the anatomy of the c a p i l l a r y c i r c u l a t i o n that began i n 1874 with Rouget, and have continued r i g h t up to the present, and w i l l be with us long into the future, the c a p i l l a r y bed structure as described by Chambers and Zweifach (22) was/shasis f o r i d e n t i f i c a t i o n and naming i  of the various parts of the c a p i l l a r y beds observed i n the mesoappendix.  They b u i l t up a v e r i t a b l e anatomy  of the c a p i l l a r y bed, and their scheme i s here outlined.  20  They f e l t  the  a nutritive  fiel^d,  illaries  side  finally  as  become  division  of  These  are  eter,  with  but  are  less  central The  Chambers  twenty  to  to  regular.  eight  to  with  muscle  terminal  i n the  coat,  and  i t  slow c o n s t r i c t o r - d i l a t o r phases.  flows  through  These  are  muscle  the  usually  cells  independent  act of  blood passes  precapillary twisted as  that  into  and  the  the true  cells.  are  segment  of  atypical motion  type  of  are  only  vasomotion then  sphincters.  outflowing.  or  Typical  channel cell  abnormal  the  the  endochannels  has  shows no  stimuli.  is  The p r o x i m a l  apparently that  is  there  by-pass  shunts.  from  typical  Vasomotion here  channels  to  shows  constriction of  smooth muscle  and responds  a  The b l o o d  more r a p i d ,  an a r t e r i o - v e n o u s  flow is  Here  thelial  arterio-venous  Here  capillaries.  tonic  as  layer.  Finally  d i l a t a t i o n and  known  diam-  arterioles.  passive  There  in  arteries,  has  or  first  arterioles.  of blood  junctions  sphincters. In  the  larger  i t  cap-  arteries  The m e t a r t e r i o l e  i n diameter,  cell  term  smooth muscle  those  t h e c a p i l l a r y bed proper.  discontinuous  true  small arborizing  Secondly i n l i n e  microns  and  twenty-five microns  comes  fifteen  predominantly  channel  the  continuous  related  was  and Z w e i f a c h  c a p i l l a r y bed,  a single are  a  branches.  channels  pulsations  mesoappendix  with  what  the  rat  an  vaso  These  lead  21 Into p r e c a p i l l a r y j u n c t i o n s , or i n t o t r u e c a p i l l a r y j u n c t i o n s , and hence i n t o the true c a p i l l a r i e s .  The  distal  a  segment of the a r t e r i o - v e n o u s channel has  connective t i s s u e c o a t i n g t h a t permits changes i n diameter.  s l i g h t passive  The p o s t c a p i l l a r y j u n c t i o n s are  i n f l o w i n g , the same as the c a p i l l a r i e s are. e i t h e r the t r u e c a p i l l a r y channels channels,  Following  or the a r t e r i o - v e n o u s  the b l o o d enters the nonmuscular venule,  which i s r e a l l y n o t h i n g more than a f u s i o n of f a r e channels. and  T h i s has a connective t i s s u e c o v e r i n g ,  s l i g h t p a s s i v e changes i n diameter  T h i r d l y , and l a s t l y , bed  are p e r m i t t e d .  i n the anatomy of the  are the muscular venules.  to t h i r t y microns i n diameter  These are  capillary twenty-five  (the non-muscular  venules were only f i f t e e n to twenty-five)  and  are covered w i t h both smooth muscle c e l l s and tissue.  thorough-  they connective  They are h i g h l y r e s p o n s i v e and show v a r i e d  contractions. Vasomotion i s a p e c u l i a r type of motor of the m e t a r t e r i o l e and of i t s p r e c a p i l l a r i e s . channels  activity These  show i r r e g u l a r l y r e c u r r e n t s e r i e s of d i l a t a t i o n s  and c o n t r a c t i o n s at i n t e r v a l s v a r y i n g from f i f t e e n onds to three minutes.  sec-  During a c t i v e m e t a r t e r i o l a r vaso-  motion the s p h i n c t e r - l i k e c l o s u r e of the p r e c a p i l l a r i e s tends  to r e s t r i c t the c a p i l l a r y b l o o d flow to the c e n t r a l  22  channels. carries  Therefore,the  on the  ischemia. a  work o f  degree  changes  of  i n diameter  ure  i n the  capillaries falls  to  loseoits  up  and c r e a t e s  other the  the  blood  as  of  that  expanded  vasodilator Also  increased  stabbing,  cells  by acute  drugs  as  cell  the  press-  tends  nucleus  rounds  capillary.  connect  to  Chambers  with (21),  activated by evidence  vaso-  for  c a n be  a temperature 20  and Chambers,  supply such that  or  Interrupted,  similar  haemorrhage,  angiotonin, decreased  rise  ° C ) ,  (37.5  vaso-  to  by d i r e c t  by  ( w i t h some 41 C), G  trauma,  vasomotion  It  sympathetic of  such  cortical  oedema  by a  deep  c a n be •  injections  and a d r e n a l  handling,  and by  agent.  and by i n t r a v e n o u s  adrenalin,  to  the  b y i r r i t a t i o n s u c h as  supply i s  w i t h nembutal  stimulation,  (10  its  b e i n g no  shows  When t h e  also  are  Zweifach  by nerve  nerve  further  It  of  endothelial  "bed"  there  according to  nerve  extract.  capillary  i t .  lumen of  possesses  nerves.  c e a s e s when t h e  fall  with  the  in  capillary  The t r u e  According  sphincter  nerves,  the  channel  bed  a consequence  the  i s markedly affected  anaesthesia  of  through  one  "beds."  smooth muscle  stroking,  capillary  where-upon  Into  c a p i l l a r i e s of  constrictor  by  flow  state,  a bulge  capillary  motion  the  elasticity.  variations  Note  arterio-venous  The e n d o t h e l i a l w a l l  certain  passive  basal  central  formation)  temperature  and by  increased  23  vital  activity  Agents krein,  as and  capillary  histamine, acetyl  the  In  In  the  This  are  occurs  flowing  or  gland  to  decreasing  the  there  is  through  the  at  metart-  the  Increasing constriction  central  dilatation  capillaries  them  the  further  distal  the  precapillary  constrict.  and p r e c a p i l l a r y in  kalli-  increase  response,  progressively toward  secretion.  adenosine,  supposed  sphincters  response  metarterioles,  dilatation  b l o o d was  gives  metarteriole  histamine  erioles,  acid,  epinephrine  and p r e c a p i l l a r y  the  the  adenylic  choline  concentration  along  exercise  c i r c u l a t i o n without  vasomotion. eriole  as m u s c u l a r  of  arteriole. the  art-  sphincters. only,  i f  the  time.  TWeapiKar^  Sphincter  J)istaL Cha.nno.(  Capillary  Bed  24  is  true  Whether  a l l that  or not,  t h e y have mapped o u t  functioning of  action,  and  a beginner the  parts  units  to  the  they  find  his  bed,  and f r e q u e n t l y  describe the  and t h e i r  manner  to  readily  f u n c t i o n as  the  ( 7)  was  the  beginning of  observing capillary  hypertensive  with  compound F ,  thesis,  changes  and the  in  the  present  Much o f  description of  the  equipment  from  following his  unpublished  of l i v i n g  source  from  a four  hundred watt  in  lower r i g h t  One e n d o f of  technique  structures  a dismantled lantern  the  one  corner  projector  of  (18)  for  was u s e d .  is  trans-  A light  slide projector  b u l b was u s e d . hand  work.  thesis.  Generally Knisely's illumination  rats  same  for  the  over  this  taken  piece  they  Rat Mesoappendix  a p p a r a t u s he u s e d was  the  for  should.  Warner  taken  for  mesoappendix,  and Z w e i f a c h were  observed  various  was v e r y h e l p f u l  way a r o u n d i n t h e  As m e n t i o n e d n e a r  rendered  very well  such an u n d e r s t a n d i n g  The O b s e r v a t i o n o f  D.L.  and Z w e i f a c h  capillary  l a b e l l e d by Chambers  identified, said  of  Chambers  with  T h i s may be the  first  seen  photograph.  was  sealed  o f f by a  circular  inch board f i v e  inches  across.  A hole  made  in its  centre  for  the  (the  g l a s s - l i k e r o d may be  end o f  the  fused  c l e a r l y seen  in  quartz the  was rod  first  27 photograph going across from the projector towards, and actually between the hind feet of the r a t ) .  This rod  transmitted and "bent" the focused beam of l i g h t r i g h t to the desired l o c a t i o n .  This may be seen quite clearly  i n both photographs as a highlight on the mesoappendix approximately 1 cm. under the microscope nose. The quartz tube used was modelled after the improved tube described by Knisely (16).  I t was twelve  inches long, and the f i r s t ten inches were eleven mm. i n diameter. mm.  The l a s t two inches tapered down to about Shree  The l a s t h a l f - i n c h was curved upward at an angle of  ninety degrees.  The l a s t two inches were hollow, with a  one mm. channel down the shaft from a side arm. A L i e t z dissecting microscope was used, binocular as i l l u s trated and i t provided f o r magnifications of 12.5x, 5Gx, and 100 x. used.  The l a t t e r , 100 x, was the customary system  These lenses were mounted on a moveable nose-  piece, which was used primarily f o r observing various areas of the mesoappendix (and not f o r changing lens systems as designed) instead of moving the animal mounting. The i r r i g a t i o n f l u i d was maintained i n a four l i t r e brain j a r container, covered i n a water bath which was thermostatically controlled f o r about 43 C. G  The  solution was siphoned o f f and passed along two polyethylene tubes, one connecting to the side arm of the  28 quartz tube, the o t h e r ending i n a f i n e g l a s s tube was suspended  over the mesoappendix p r e p a r a t i o n i n a  moveable f a s h i o n . Illustrations. emerging  that  A l l t h i s i s c l e a r l y v i s i b l e i n the The p o l y e t h y l e n e tubes w i l l be seen  from a t h i c k rubber tube about one i n c h a c r o s s .  This was d e v i s e d to keep the d r i p s o l u t i o n warm u n t i l i t was d e l i v e r e d to the mesoappendix, and h o t water b a t h water was c i r c u l a t e d through t h i s rubber c a s i n g by means of  a simple r o t a r y puferp.  The d r i p s o l u t i o n was d e l i v e r e d  at  the mesoappendix a t a temperature  of approximately  37°C. The a n a e s t h e t i z e d animal would be g i v e n a median abdominal  i n c i s i o n and l a i d on i t s r i g h t s i d e on a l i t t l e  s h e l f of l u c i t e that was a t t a c h e d on the top o f a l u c i t e box one i n c h deep to c a t c h the expended d r i p  solution.  The caecum would be c a r e f u l l y e x t e r i o r i z e d , and the mesoappendix would then be draped over a l i t t l e  l u c i t e loop  two cm. across a t t a c h e d a t a s u i t a b l e h e i g h t above the s h e l f to a v o i d t e n s i o n on the mesoappendix. soaked c o t t o n packs then be packed  Warm s a l i n e  (not shown i n the i l l u s t r a t i o n ) would  around  the l o o p s o f bowel to keep them  warm and moist, and a l s o to support f u r t h e r the caecum. The warm d r i p s o l u t i o n was then s t a r t e d , which flowed over the mesoappendix from above.  S o l u t i o n a l s o flowed out  the channel i n the quartz tube, but i n as much as t h i s tube d i d n o t u s u a l l y touch the mesoappendix, t h i s  fluid  29  served from  l i t t l e more  gathering  beam o f  than  blood,  light.  keep  etc.,  the  adjusting  illustrate  the  that would  was n e c e s s a r y .  equipment  mentioned  c a n be p i c k e d o u t . loop  of  observer's  loop  (also  side  well  demarcated,  tube  showing at by  the  the  light  of  the  with its  end o f  the  quartz  s h i n i n g on the  prepared  At f i r s t  of  gelatin  i n the  as  cided  to  Locke  s o l u t i o n as  of  the  a plain  normal  discussed  add g e l a t i n e  thesis.  photographs and  a l l  The s e c o n d  visible,  alone,  translucent drip  on  but  not  too  quartz produced  mainly by  mesoappendix. solutions  through  by Zweifach  and g l u c o s e  This resulted  the  were  But a c c o r d i n g to  state  outlined in  but  picture  ex-  Ringer-Locke solution  (formula i n appendix)•  prolongation  The  The h i g h l i g h t i s tube  the  apparatus  caecum draped  Some d i f f i c u l t i e s w i t h perienced.  tube  the  h i g h l i g h t from the  centre).  the  the  obstruct  exceedingly well,  shows v e r y c l e a r l y t h e  not  end o f  Warner had p e r f e c t e d  and l i t t l e  points  to  the  the  (14),  both  i t  to  the  apLpendix a t  was the  addition was  de-  Ringerthe  end  i n much more p h y s i o l o g i c a l  preparations. The F r e e z i n g  Point  Running malleable m e t a l not  shown,  to  across tube  the  both photographs  is  a winding  l e a d i n g from a carbon d i o x i d e  right  of  the  pictures,  across  tank,  toward  30  the This  microscope  to  end i n  apparatus  is  the  carbon been (  dioxide  adapted  ) by  appendix  the at  operating  quick-section  according  to  inclusion of the  end  of  removed  from  the  control  needle  valve.  on  end  was  as  the  the  constructed  the  the  unit  Luer-Lok  unit  and  from a  sketched  in  has directions the  The f r e e z i n g  unit  Luer-Lok  Spencer  This  listed  thesis.  To t h i s  apparatus.  Taylor's  parts  microtome  of  valve  microtome.  Hass  the  was  Lok u n i t  a control  to  leave  was  plate  essentially  attached  syringes.  a  Luer-  Then  bel'ow w h i c h was  readily  fitting  co2 supply l i n e attaches here  19  needle 15  Hub o f 1 9 needle soldered to LuerLok f i t t i n g so tip of needle i s 1 o r 2 ram. p r o x i m a l to c l o s e d p o i n t o f 15 n e e d l e  Hass  and  Expanded COg d i s charges here to outside air  Taylor's  needle  C l o s e d by thin plate of solder  Cold  Point  Apparatus  31 attachable carbon  to  the  dioxide  rest  gas  expanding gas,  is  made.  of  the  needle imal  from  valve. the at  the  closed  the  point of  a l r e a d y been  to  the  the  closed  off point  to  onto  was  escape  the  closely  "jets",  spaced at  cold  could  tip  the  mesoappendix,  the  to  rate  closed then  just  be as  a rate  the  nineteen fifteen  into  just  side  o f gas  of  applied  highly  with  (the  the  seal  the  cold  •  expanded shaft  of  side-arm  to  the  level  shaft so  that  immediately proximal when t h e  fitting) fitting,  arm to flow,  to  just  larger  proximally  the  shaft  prox-  the  the  within  now the  with  fifte  air. short,  could  needle.  a suitable  the model i n the  with  outside  usually  fifteen  fifteen  and  a f r e e z i n g temperature tip  snow  ended  The g a s  fifteen  the  effect  by  needle  the  Luer-Lok  flow  cooling  controlled  an a p p r o p r i a t e  onto  through the  By c o n t r o l l i n g  obtained  of  locked  able  at  by a i r t i g h t  (at  the  c o n d u c t e d down t h e  fitting  n i n e t e e n was  needle  expanded gas and  nineteen  nineteen needle  fifteen  the  the  of  and  or probably  apparatus).  affixed  n e e d l e was l o c k e d the  the  The L u e r - L o k  point of the  of  end o f the  had  the  tip  of  valve  carbon dioxide  carbon dioxide,  The t i p  fifteen.  the  same w a y a s  c a r b o n d i o x i d e was  the  of  i n the  (needle  utilizing  number n i n e t e e n n e e d l e  to  point,  unit  The p r i n c i p l e  simple,  The l i q u i d  compressed  the  supply).  c o l d p o i n t was q u i t e of  of  site  photographs  be This  on is  the  32  demonstrating. localized  The p l a c i n g o f  very  accurately  just  the  just  about f i l l e d  the  desired  point at  the  field  was p l a c e d ,  the  point  the  the  way).  moist  film  This i n v o l v e d the the  area  the  field  tion.  of  field,  of  In  selected  the  resumed  way,  exactly at  and over  the at the  exactly point  needle  point  Epinephrine  the  and  on,  and  the  to  ice  then,  was  formation  100 p o w e r at  at  the  within  originally  centre  of  and  magnifica-  the  the  The f l o w  gas  of  the  could would  be  twenty-five warm d r i p  mesoappendix. but  area  Indicated  always  approximately  the  the  mesoappendix.  was  seconds  be  mesoappendix  later  bed  at  drip  frozen mesoappendix  thirty and  When  the  w h i c h was  expiry of  vessel.  on the  the  by c e n t e r i n g  of  could  affected at  microscope  warm s a l i n e  of  onto  point  due  be  capillary  c o n d i t i o n was  area  determined.  instantaneous,  (the  thickness  freezing  nearly  be  turned  remaining  microscope  boundaries  off  seconds,  still  the  mesoappendix  the  this  was  A freezing  whole  area for  a l w a y s be turned  the  The c o l d  freeze  whitening of  the  certain  could  microscope  The u s u a l  shown b y  any  gas  allowed to  of  by a suddeh  the  cold point the  c a p i l l a r y bed.  l o c a l i z e d over  swung o u t  in  through  100 p o w e r m a g n i f i c a t i o n .  precisely  the  only then  was  T h a w i n g was could  the  removed.  Response  Zweifach  (14) outlines  i n quite  careful  detail  his  33  epinephrine response response  to  threshold give  test.  vasotropic  response  a moderate  as  substances, just  through the  within to  fifteen  to  level  to  be  applied oftener  also  between  repeated  tissue  seconds.  to  the  the be  give  the  capillary  walls  of  stasis  the  or  trials. than  drug.  at  aration  beds  three minute  than  details  to  sticking  of  Blood  threshold epinephrine  states  seconds  not and  There  no  the  criteria  flowing  It through should in  the  capillary tests  after  the  w i t h marked  can prepside  test.  c r i t e r i a and response  the  should  or diapedesis  mesoappendix the  below  down c e r t a i n  materials  the  sensitizes  s h o u l d be no  sixty  Also,  within  intervals,  intermittent.  He f u r t h e r  fifty  response  i n a normal state.  There  i n v a l i d a t e the  These the  leukocytes  veiralte'Sr..  lays  of  solution should  epinephrine  response.  s h o u l d be  i s mounted.  reactions  of  epinephrine  flow  venules;?  The e p i n e p h r i n e  Zweifach  stagnation.  b e made m o r e  the  just  The warm d r i p  applications of  a minimum o f  slow  is  to  arterioles  arterioles  in six million  for ^considering a preparation must  to  the  substance  The t h r e s h o l d  in different  give a response.  restored  defines  terminal  sufficient  twenty  One p a r t  be  the  threshold  c a p i l l a r i e s and t r i b u t a r y  epinephrine varies  same t i s s u e .  the  a  and he  enough o f  narrowing of  and p r e c a p i l l a r i e s j u s t blood  He d i s c u s s e s  characteristics  were  of  great  34 value of  and u s e f u l n e s s  the  rat  as  the  actual  Cold  recorded  behavior effects the  of  of  vessels  for  total  area,  but  within  into  twenty  because from  the  the  few o f  "sludging" nature  of  occurred. observed 11 ) t h e  the  of  to  seconds  of  the  the and  the  compound  thesis,  relatively  as  easy.  thirty  animals  the  The .  Ice  at  process  The oedema  was n o t  of  the  As  area.  the  too  soon what  point in  was  "stasis".  i t  away  a termed  The  but  precise  constantly  could not  says  seen  a  thawing  cold  by Shumacker  thawing  Usually  observed  Shumacker  below  with  (called rapid  bloodwere  mentioned,,  the  overcome  melt  freezing  affected  after  determined,  as  the  w o u l d be the  injured  called  described  m i c r o s c o p i c a l l y , but elements  to  to  probably  seconds  and v e r y  also  thesis.  pattern.  w o u l d be the  a  with control  s l o w i n g w o u l d be  would appear,  described,  freezing  thawing  postcapillaries,  formed  in  mesoappendix  solid  warm d r i p was u s e d  this  of  a definite  within  mesoappendix)  the  was  perforated  present  hyperemia  thirty  test  of  vasoconstriction within  any v a s o c o n s t r i c t i o n  within  section  sections of  The o n s e t  rapid  seen  apparatus  would produce  degrees.  generalized  were  i n following  point  be  reactions  Experiments  tests  the  the  cold point  soon f i t t e d  cold  zero  Injury  the  g r e a t number  of  capillary  with epinephrine  following  performance  With  as  i n the  the  As w i l l  capillary tests  recorded  Localized  studying  mesoappendix.  preliminary P  in  (as  on  be page  progressively  35  to f i l l  the small v e s s e l s , b r i n g i n g to a h a l t the  circ-  u l a t i o n through them.  T h i s s t a s i s was  r e v e r s i b l e , at  the b e g i n n i n g at l e a s t ,  s i n c e mechanical p r e s s u r e or  changes i n the b l o o d flow i n adjacent v e s s e l s would break up the r e d c e l l masses. s t a s i s was  T h i s spread of the  a l s o c o n s t a n t l y observed,  and the  term  "spread" i s used i n f o l l o w i n g d e s c r i p t i o n s i n two ways, rather indiscrimaitely.  In one i n s t a n c e the spread  meant j u s t what i s i m p l i e d here, a p r o g r e s s i o n of the s t a s i s along a continuous channel. term was  More o f t e n the  employed to s i g n i f y the appearance of s t a s i s  i n s e p a r a t e n o t n e c e s s a r i l y connected ?  channels, somewhat  as an i n f e c t i o u s d i s e a s e spreads through a  neighbourhood,  although t h i s i s not s t r i c t l y a c o r r e c t usage a t a l l , even though convenient. experiments,  For the purposes  of the  the exact meaning of the term does not  really  matter. Recording of Observations In  a l l the r e c o r d s of the experimental o b s e r v a t i o n s  there w i l l be noted a b o r i n g and a p p a r e n t l y u s e l e s s repetition.  The d e c i s i o n was made e a r l y to i n d i c a t e v a r -  i a b l e s i n the technique w i t h i n the t e x t of the d e s c r i p t i o n s f o r each p r e p a r a t i o n , as opposed to d e s c r i b i n g the g e n e r a l method a t the beginning, and merely v a r i a t i o n s w i t h each animal.  the bare  I t seemed when the r e c o r d s  were w r i t t e n up that such v a r i a b l e s as how  much anaesth-  36  etic  was u s e d  exact of  Criteria  could  for At  The  the  spread  etc  not  a Drug  the  explaining,  what was meant medication  or  The  important,  section of  experiments  because  by an e f f e c t  due  to  for  the  onset If  or prevent  a valuable agent.  in  the  development  of  therapy  the  This  extent  The i d e a  to  that  stemmed p a r t l y  of  stasis, These  defining  influence of  just  some  vessels  T h i s was h e n c e  and cause regarded  In frostbite  field  as  a likely  therapy.  might but  i t  frozen  frostbite  the  the  in  the  in  stasis  facts  would  a restoration  extent  stasis,  that  mostly from  would  A drug  affected  s  a static  the  stasis.  fact  could  altogether  a drug' effective the  item  such a pronounced  vascular  which i t  from  was one  any m e d i c a t i o n t h a t  vascular  procaine  flushed over  is  applied toofor  but  effective  for  stasis  appearance  a reversible process,  small  the  the  stasis  its  the  of  necessarily delay or prevent  tissue.  onset  table.  treatment.  be  limit  dealing  resumed f l o w .  necessity  a freezing injury,  might  as  the  feature  not  data  a summary  of  time  stasis  appears  generalized stasis,  important.  delay  and  and s u c h a d i v e r s i t y  the  by such t i t l e s  considered of  administered,  i n d i c a t e d w i t h o u t much r e p e t i t i o n .  each  headed  stasis,  was  Effect  cold point  of  were  he  end o f  columns are  need  e x a c t l y how i t  time lapses,  details  with  and  that  dilate  the  attribute  was  the  circulation. in  drugs  37  Although these c r i t e r i a might appear to be f a i r l y o b j e c t i v e and s c i e n t i f i c , an i l l u s i o n .  such was i n f a c t  just  V a s c u l a r f i e l d s i n the r a t mesoappendix  were u n f o r t u n a t e l y found to have as much resemblence to each other as human f a c e s have to other human f a c e s . One mesoappendix might have only one o r two wide  areas  s p a r s e l y crossed by a few f i n e c a p i l l a r i e s t h a t c o u l d p o s s i b l y pass as c a p i l l a r y beds.  Other p r e p a r a t i o n s  would be j u s t crowded w i t h v e s s e l s going direction.  every p o s s i b l e  When t r y i n g to s p e c i f y as to what the extent  of the s t a s i s was, or how r a p i d the spread was, obvious t h a t there were d i f f i c u l t i e s .  i t is  On the whole, the  o b s e r v a t i o n of the mesoappendix was a very s u b j e c t i v e matter. A necessary  f e a t u r e of the m i c r o s c o p i c  experi-  ments l a y i n the d e c i s i o n to a l t e r n a t e when p o s s i b l e the t e s t and the c o n t r o l p r e p a r a t i o n s . f o u r hours to perform a m i c r o s c o p i c twelve animals.  Conditions  I t took g e n e r a l l y  experiment o f t e n o r  could and d i d change g r e a t l y  i n the room used f o r a l a b o r a t o r y over t h i s time.  Hence  the t e s t i n g o f a l l the c o n t r o l animals f o l l o w e d by t e s t i n g of the t e s t r a t s was d i s c a r d e d as i m p r a c t i c a l . Care of Animals Rats,  e i t h e r Wistar  or Hooded, i n the 150 to 25Q  gram weight range were obtained  i n batches o f f i f t y or so  38 when a v a i l a b l e u p o n r e q u i s i t i o n f r o m s e p a r a t e stock In  barns  a well  animal  elsewhere  cages  In  times each  campus.  They were  v e n t i l a t e d windowless i n s i d e h o l d i n g from  Fresh d r i n k i n g water all  on the  was  three  to  available  room,  healthy-and  i n a feeding well  i n these  trough.  in  s i x animals to  the  for  tiered each. at  kept  A l l animals  surroundings  housed  animals  a n d P u r i n a F o x Chow C h e c k e r s w e r e  cage  breeding  suspended stayed  Indefinite o  periods  of  time.  They were . s e p a r a t e d  a c c o r d i n g to  sex.-  39  Preliminary microscopic observations using Compound F (17 hydroxy-eortisone 21 acetate)  Preliminary c a p i l l a r y experiments In order t© gain some experience with the use of the microscope In connection with c a p i l l a r y observations, certain tests were performed using the c a p i l l a r y epinephrine response as described by Zweifach, B. W. (14),  Warner, D.L., the senior medical student ment-  ioned so frequently i n t h i s thesis as a part-time helper with the project, while performing experimental work i n connection with h i s graduating thesis f o r the Faculty of Medicine at this university, tested c a p i l l a r y  response  to various drugs including compound F., and he found certain effects due to the drug which greatly modified the c a p i l l a r y response to epinephrine.  He found ( 7  )  that whereas a 1:4 m i l l i o n (abbreviation f o r one part i n four m i l l i o n parts) d i l u t i o n was the threshold concentr a t i o n of epinephrine needed to e l i c i t  a vasomotor r e -  sponse as determined by metarteriole and p r e c a p i l l a r y constriction, that after i n j e c t i n g compound F. f o r a certain number of dayf the threshold response had decreased to 1:18 or 1:20 m i l l i o n d i l u t i o n of epinephrine. Because of the supposed delicacy of the technique of examining vascular responses under the micro-  40  scope, i t was f e l t that a r e p e t i t i o n of Warner's work, i n part, especially i f i t could be confirmed, would serve both as an i n i t i a t i o n  Into the technique and also  would serve i n a small measure to increase confidence i n the r e l i a b i l i t y of l a t e r observations with l o c a l i z e d cold  experiments.  I n i t i a l epinephrine response  tests  The purpose of the following tests was to t r y to determine the threshold epinephrine response of the metarterioles and p r e c a p i l l a r i e s i n the mesoappendix of the r a t .  Several adult male Wistar rats were selected  and set aside f o r use on several successive days.  The  apparatus was set up as described and a simple RingerLocke saline drip solution used.  Epinephrine chloride  solution 1:1000 was maintained i n a small f l a s k at the temperature of the water bath, and several test tubes of drip solution were also kept warm, with c a r e f u l l y measured quantities of drip solution to permit rapid preparation of the diluted epinephrine so as to minimize the tendency f o r the epinephrine to decompose. decomposition  This  of the epinephrine,especially when warm  and mixed with the drip solution,was a r e a l problem, and as w i l l be explained l a t e r possibly accounted f o r some of the c o n f l i c i n g r e s u l t s obtained. Animal 1  41  A normal adult male Wistar r a t weighing 135 grams was anaesthetized with 0.13 cc. of 0.6% ution administered intraperitoneally  nembutal s o l -  at zero minutes.  At twenty-four minutes the preparation was mounted and ready on the microscope  stage.  The temperature  of the  drip solution was 3 3 . 5 ° C , and t h i s was allowed to drip over the mesoappendix except when the test solution flushed on with a medicine dropper.  was  Approximately a.  t h i r t y seconds after the test solution was applied, the drip would be resumed u n t i l the next tesfc.  Long enough  an i n t e r v a l was allowed f o r the preceding epinephrine to be washed away and f o r the vessel to recover from i t s effect before any more was used.  An i n t e r v a l of about  f i v e minutes was considered suitable, to be adequate.  and this was  At thirty-two minutes i;2  found  million  epinephrine was applied and this gave a very strong response, classed as ////»  At thirty-seven minutes  1:4 m i l l i o n gave a // response, at forty-two minutes 1:6 m i l l i o n gave no response.  At forty-seven minutes a  1:4 m i l l i o n d i l u t i o n gave a /// response.  At  fifty-two  minutes a 1:5 m i l l i o n d i l u t i o n gave a /// response. fifty-seven minutes a 1;6 m i l l i o n d i l u t i o n gave a very doubtful response, classed as plus or minus.  On the  basis of t h i s , and with Warner>s agreement (he worked  At  42  with me f o r the f i r s t day) the threshold response was set at 1:5 m i l l i o n d i l u t i o n . Animal 2 A normal adult male Wistar r a t weighing 145 grams was anaesthetized with 0.13 cc. of 0,6% solution administered i n t r a p e r l t o n e a l l y  nembutal  at zero minutes.  The animal was ready on the microscope by twenty-three minutes.  At t h i r t y minutes there was no response to  1:7 m i l l i o n epinephrine ( a l l d i l u t i o n s were freshly prepared each time they were needed); at thirty-seven minutes no response to 1;6 m i l l i o n d i l u t i o n ; nor any response at f o r t y minutes to 1:5 m i l l i o n .  At forty-two  minutes 1:4 m i l l i o n gave a / response two minutes l a t e r which lasted only a minute.  At forty-seven minutes  1:5 m i l l i o n gave no response, but at fifty-two 1:3 m i l l i o n gave a //. at f i f t y - f i v e minutes.  minutes  The preparation was discontinued The temperature of the drip  solution at the mesoappendix was 36°C.  The threshold  response was considered to be on the basis of t h i s preparation 1:4 m i l l i o n d i l u t i o n of epinephrine. Animal 3 On the following day, the same set-up was made ready as before, and a normal adult male Wistar r a t weighing 150 grams was anaesthetized with 0.13 cc of 0,6% nembutal  solution, at zero minutes.  The preparation  43 was ready on the microscope with the drip going.  at twenty-three minutes  At twenty-six minutes 1:6  m i l l i o n gave no response, but three minutes l a t e r m i l l i o n gave a /// response.  1:5  At thirty-two minutes  1:6 m i l l i o n gave a very doubtful effect, and the same with 1 5 m i l l i o n four minutes l a t e r .  At  :  thirty-nine  minutes 1:5 m i l l i o n gave no response, but at f o r t y two minutes i t gave a doubtful e f f e c t .  At f o r t y - f i v e  minutes a 1:4 m i l l i o n solution gave a /// e f f e c t .  At  forty-nine minutes 1:5 m i l l i o n gave no response, nor did 1:4 m i l l i o n two minutes l a t e r .  F i n a l l y , at f i f t y -  four minutes 1:5 m i l l i o n f a i l e d to give any e f f e c t . It was noted that flushing on the adrenaline too soon after the preceding application gaves an unsatisfactory response.  Possibly the preparation was kept too long.  On the basis of these observations, the threshold f o r the epinephrine response was f e l t to be 1:5  million  dilution. Animal 4 A normal adult male Wistar r a t weighing grams was anaesthetized with 0.13  cc. of 0.6$  solution administered intraperitoneally, utes.  160  nembutal  at zero min-  At twenty-five minutes the preparation was  mounted on the microscope going at 36°G.  stage and ready with the drip  At thirty-seven minutes 0.07  cc of  44  0.6% nembutal had to be repeated because of lightness of the anaesthesia.  At thirty-nine minutes a 1:6  m i l l i o n d i l u t i o n gave no response.  At forty-three  minutes a 1:5 m i l l i o n d i l u t i o n gave a  e f f e c t , which  o  was repeated at f i f t y minutes.  At f i f t y - f i v e minues  1:5 m i l l i o n epinephrine had no effect, nor d i d 1;4 m i l l i o n at sixty minutes.  One minute l a t e r a drop ©f  p l a i n i c e water gave a vigourous //// response, and at sixty three minutes 1:4 m i l l i o n epinephrine gave a / response. /  The preparation was discontinued at  s i x t y - f i v e minutes and the threshold response was considered to be 1*5 m i l l i o n d i l u t i o n . Animal 5 A normal adult male Wistar r a t weighing 175 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y ,  at zero minutes.  At twenty minutes the preparation was ready with the drip going.  At twenty-four minutes a 1:6 m i l l i o n  d i l u t i o n gave no response. At thirty-two minutes 1:5 m i l l i o n epinephrine gave a / response.  Five minutes  l a t e r , on repeating the 1;5 m i l l i o n , , a doubtful effect was obtained.  At forty-two minutes 1;4 m i l l i o n gave  no response, but three minutes l a t e r the 1:4 m i l l i o n produced a // e f f e c t .  I t was noted (a) the saline  45  packs kept over the exposed bowel were too cool, and (b) there was some degree of general s t a s i s i n the mesoappendix f i r s t noticed about the forty-two minute period.  There was also some blood loss from the  abdominal i n c i s i o n which might have affected the general circulation.  The preparation was discontinued at  f i f t y minutes, and the threshold l e v e l f o r the epinephrine response was f e l t to be again 1:5 m i l l i o n . Animal 6 The following day the same experiments were continued.  No attempt was made to control the temp-  erature of the drip solution f a l l i n g on the mesoappendix (the  faster the drip ran the shorter time i t was i n the  connecting tubing and the less heat i t l o s t on i t s way from the water bath) and the average temperature was about 37®C.  Generally, i n the following f i v e animals  much more spontaneous vasomotion was noted than at the previous temperature l e v e l s of 33 to 36°C.  This may  explain the s l i g h t l y higher threshold l e v e l s obtained with t h i s and the following preparations. A normal adult male Wistar r a t , weight not recorded, was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y .  The  temperature of the drip over the mesoappendix were 36.6°C. The preparation was ready at s i x minutes. At sixteen  46  minutes 1:7 m i l l i o n epinephrine gave a /// response, which was repeated f i v e minutes l a t e r , and at twentyf i v e minutes 1;8 m i l l i o n gave a // response.  A minute  l a t e r p l a i n drip solution gave no response, nor d i d 1:8 m i l l i o n d i l u t i o n at twenty-eight minutes, nor d i d 1:7 m i l l i o n d i l u t i o n at thirty-one minutes.  1:6  m i l l i o n f a i l e d to e l i c i t e any response at t h i r t y - f o u r minutes and 1:5 m i l l i o n d i d the smae at thirty-seven minutes.  1:4 m i l l i o n gave a / response at f o r t y  minutes, and at 43 minutes cold 1:100,000 d i l u t i o n gave a //// response.  For reasons not recorded, i t was  - f e l t that 1;4 m i l l i o n was the threshold, and not 1;7 or 1:8 m i l l i o n v/as would appear to be.  This opinion  would have better support i f the i n t e r v a l between the applications of the epinephrine had been longer than three minutes. Animal 7 A normal adult male Wistar r a t , weight not recorded was anaesthetized with 0.13 cc. of 0.6% nembutal solution administered intraperitoneally, and f i f t e e n minutes l a t e r 0.05 cc. had to be repeated to obtain a satisfactory depth of anaesthesia. The preparation was ready at twenty minutes, on the microscope, with the warm Ringer-Locke drip running. At  47 twenty-two minutes 1:10 m i l l i o n epinephrine gave no response, 1:8 m i l l i o n did not at twenty-six minutes, 1:7 m i l l i o n did not at twenty-nine minutes, and 1:6 m i l l i o n did not at thirty-two minutes.  At t h i r t y - f i v e  minutes 1:5 m i l l i o n d i l u t i o n gave a very doubtful response, but 1;4 m i l l i o n gave a d e f i n i t e / response at f o r t y minutes, but no response at f o r t y - f i v e minutes.  At forty-eight minutes cold 1:100,000 epineph-  rine gave a very strong //// response,  and at f i f t y -  three minutes 1:4 m i l l i o n gave a // response.  At  f i f t y - s i x minutes 1:5 m i l l i o n f a i l e d to give any e f f e c t , but three minutes l a t e r 1»4 m i l l i o n gave a / response.  F i n a l l y at sixty-two minutes, 1:100,000  cold epinephrine gave a very strong //// response. The preparation was discontinued at s i x t y - f i v e minutes and the threshold was considered to be 1;4 m i l l i o n d i l u t i o n of epinephrine. Animal 8 A nmrjnal adult male Wistar r a t of unrecorded weight, was anaesthetized at zero minutes with 0.13 cc. of 0.6$ nembutal solution administered i n t r a p e r i toneally, and the preparation was ready with warm drip going at fourteen minutes.  There was much vascular  stasis throughout the vascular f i e l d of the mesoappendix.  At twenty-four minutes 1:6 m i l l i o n d i l -  48  ution of epinephrine gave a ///' response, and f i v e minutes l a t e r 1;7 m i l l i o n also gave a /// response. The vascular stasis worsened gradually, and  the  preparation was discontinued with no r e s u l t s obtained. Animal 9 A normal adult male Wistar r a t weighing 155 grams was  anaesthetized at zero minutes with 0.13  cc.  of 0.6$ nembutal solution administered i n t r a p e r i t o n eally, and the preparation was ready ten minutes l a t e r . At ten minutes 1:6 m i l l i o n d i l u t i o n gave no response, but at fourteen minutes 1:5 m i l l i o n gave a / response. This was  a very b e a u t i f u l preparation to observe.  At  sixteen minutes 1:4 m i l l i o n gave no response, but f i v e minutes l a t e r i t gave a doubtful response.  At twenty-  six minutes 1:3 m i l l i o n d i l u t i o n gave a // response. The epinephrine was  obviously getting brown, and  was  made up again from f r e s h 1;1000 stock epinephrine. t h i r t y - f i v e minutes t h i s f r e s h solution gave at m i l l i o n d i l u t i o n a // response.  At  1:4  At f o r t y minutes  1:5 m i l l i o n gave also a // response.  At f o r t y - f o u r  minutes 1:6 m i l l i o n gave no response, but a minute l a t e r 1*5 m i l l i o n gave a // response. was  The preparation  discontinued at f i f t y minutes, and i t was  1:5 m i l l i o n was  f e l t that  the threshold d i l u t i o n f o r the epineph-  49 rine response. Animal 10 A normal adult male Wistar r a t weighing 155 grams was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered intraperitoneally, and i t was ready on the microscope going at f i v e minutes.  stage with the warm drip  At seven minutes 0.05 cc of  nembutal had to be repeated.  At seventeen minutes  1:6 m i l l i o n d i l u t i o n of epinephrine gave a  j>ff  response.  1:7 m i l l i o n gave a /// response at twenty minutes, and so did a 1:8 m i l l i o n solution at twenty-three  minutes.  At twenty-six minutes leukocytes were observed s t i c k i n g to the walls of the larger vessels, was discontinued as unphysiological. of the drip solution was 37°C.  and the preparation The temperature  No results were r e -  corded. Summary of Observations No.  Threshold epinephrine response i n millionths  1.  1:5  2.  14  3.  1:5  7.  1 4  4.  1:5  8.  No results  5.  1 5  9.  6.  1:4 ?  :  :  10  :  l 5 :  No results  50  Discussion of Observations These observations agree with Zweifach, (14 ) who  B.W.  stated a 1;6 m i l l i o n d i l u t i o n of epineph-  rine i s just below the l e v e l required f o r a vascular response according to h i s technique.  Here, with the  doubtful exception of Animal 6, no threshold was found lower that 1;5 m i l l i o n , and Incidentally, nohewwas found higher than 1:4 m i l l i o n , which i s rather an excellent result. It was learned that the requirements f o r a physiological preparation are f a i r l y s t r i c t , and some of the signs that a preparation was beginning to s p o i l were observed f i r s t hand.  These lessons were put to  good advantage l a t e r on the l o c a l cold experiments. Note that a p l a i n Ringer-Locke saline drip was employed. There were found l a t e r advantages to using a g e l a t i n glucose enriched and buffered drip solution. Observations with Compound F The purpose of the experiments here outlined was to test the e f f e c t compound P would have on the epinephrine threshold response i n the c i r c u l a t i o n of the  r a t mesoappendix. . 2.0 mg. of compound P i n saline  suspension i n a concentration of 25 mg. per cc. (that  51  i s 0.08 cc. per injection) administered i n t r a p e r i t o n e a l l y every day f o r approximately two weeks, was the contemplated dosage l e v e l , p r i o r to the microscopic observations. Eighteen normal adult male Wistar rats were s e l ected, and after marking the ears c h a r a c t e r i s t i c a l l y the i n i t i a l weights were taken as recorded on the Data Sheet.  The animals were divided into three groups of  six each so that the Injections could be started i n a staggered manner to f i n i s h the series on d i f f e r e n t days f o r convenience i n t e s t i n g .  The medication was given  to three test animals I n each group, and the remaining three were f o r controls to be examined alternately with the test animals.  At the end of the series of i n j e c t -  ions with compound P, the epinephrine threshold response test was performed on each r a t as i n the previous experiment.  Details f o r each animal are as follows:  Animal 1 (Control) The r a t was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y , having received no previous medication, and the preparation was ready on the microscope stage with the warm drip running at thirteen minutes.  At twenty-five minutes there was  a very doubtful response to 1 6 m i l l i o n d i l u t i o n of epi:  nephrine which could not be repeated at twenty-eight minutes.  At thirty-one minutes 1:5 m i l l i o n d i l u t i o n  52 Data Sheet  Animal3 1 to 6 Control 160 154 166  -  - 192 - T - 190 - T - 204 - T  Test 166 p p p p p p p p p p p p 164 P T 175 p p p p p p p p p p p p 180 P T 147 P P P P P P P P P P P P 150 P T I/p s/c Day 1 2 3 4 5 6 7 8 9 10 11 12 13 Animals 7 to 12 Control 144 155 146  -  - 206 - T - 214 - T - 211 - T  Test 162 p p p p p p p p p p p p 163 p T 172 P P P P P P P P P P P P 198 p T 153 P P P P P P P P P P P P 135 p T i/p s/c Day 1 2 3 4 5 6.7 8 9 10 11 12 13 Animals 15 to 18 Control 173 153 150  -  -  -  -  -  -  -  - 248 - T - 231 - ? - 227 - ?  Test 190 p p p p p p p p p p p p p p p p p 201 P ? 145 p p p p p p p p p p p p p p p p p 162 P ? 151 P P P P P P P P P P P P p p p p p 163 P ? i/p s/c Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 (-, means not administered, p, i n j e c t i o n , T, tested, ?, wasted, i / p , i n t r a p e r i t o n e a l l y , s/c subcutaneously)  53 gave a / response, and three minutes l a t e r the same d i l u t i o n gave a /// response.  The preparation was  discontinued and the threshold was recorded as 1:5 million. Animal 2 (Test) The r a t was anaesthetized with 0,13 cc of 0,6$ nembutal solution administered i n t r a p e r i t o n e a l l y after thirteen d a i l y intraperitoneal injections of compound F.  At f i f t e e n minutes 0.03 cc, of the nembutal had  to be repeated, and the preparation was ready at twent y - f i v e minutes with the warm drip running.  At t h i r t y -  million one minutes a l : 1 0 * d i l u t i o n gave a // response, and four minutes l a t e r 1:10 m i l l i o n d i l u t i o n gave a /// response.  At thirty-nine minutes 1:14 m i l l i o n gave  no response, and 1:10 m i l l i o n f a i l e d to give any response at f o r t y minutes but gave a /// three minutes later.  At f i f t y - o n e minutes 1;14 d i l u t i o n gave no million  response.  At f i f t y - f o u r minutes a l:10 gave a very A  doubtful response, and so d i d 1:8 m i l l i o n at f i f t y seven minutes. to be sure.  There was too much vascular s t a s i s  Probably solutions weaker than 1:10  m i l l i o n would have given a response i f they had been used f i r s t .  The results of this test had to be d i s -  counted i n view of the f a c t the preparation was not entirely physiological.  54 Animal 5 (Control) The r a t was anaesthetized with 0.15 ccof 0.6$ nembutal solution administered intraperitoneally at zero minutes, and at ten minutes 0.05 cc. of nembutal solution had to be repeated.  The preparation was  ready at twenty minutes with the warm drip running. At twenty-four minutes 1;6 m i l l i o n d i l u t i o n of epinephrine gave a very doubtful vascular response. At twenty-seven minutes 1;5 m i l l i o n gave a /// response which was repeated i n three more minutes.  At thirty-three  minutes 1:6 m i l l i o n d i d not give any response, and at thirty-seven minutes 1:5 m i l l i o n d i d not either.  At  f o r t y minutes 1-4 m i l l i o n gave a very doubtful e f f e c t . At forty-two minutes 1:100,000 d i l u t i o n gave a strong //// response.  At f o r t y - f i v e 1:4 m i l l i o n gave a ////  response, and at fifty-two minutes 1;5 m i l l i o n gave a /// response.  1*6 m i l l i o n gave a ///-response both at  fifty-two and f i f t y four minutes.  At f i f t y - s e v e n  minutes 1:7 m i l l i o n gave a //// response and at s i x t y one minutes 1:10 m i l l i o n gave also a //// response. F i n a l l y , at s i x t y - f i v e minutes p l a i n drip solution gave a ////, and the preparation was discontinued because vascular stasis was quite obvious.  Probably no results  after forty-two minutes were v a l i d , and 1«5 m i l l i o n was accepted as the threshold f o r this t e s t .  55  Animal 4 (Test) The r a t was anaesthetized with G.15 cc. of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y after thirteen d a i l y intraperitoneal injections of compound P.  At eighteen minutes the preparation was  ready with the warm drip running, and l:100million epinephrine solution gave a // response. million  At twenty-  three minutes a l : 1 4 " d i l u t i o n gave a // response, and at twenty-eight minutes a 1:16 m i l l i o n d i l u t i o n gave a /// response.  At thirty-three minutes p l a i n  drip was flushed over the mesoappendix with no apparent e f f e c t on the blood vessels.  At t h i r t y - f o u r  minutes 1;20 m i l l i o n d i l u t i o n was tested with no r e sponse.  At thirty-nine minutes 1:10 m i l l i o n gave  a doubtful response.  At forty-two minutes 1 16 :  m i l l i o n gave a /// response, and then at forty-seven minutes 1»18 was t r i e d again and i t gave a d e f i n i t e // response.  In two minutes 1;20 m i l l i o n gave no  e f f e c t but when 1:18 m i l l i o n was t r i e d at f i f t y - o n e minutes i t gave a /// response. preparation to work with.  This was a wonderful  The preparation was dis-  continued at f i f t y - f i v e minutes, and 1:18 m i l l i o n was accepted as the threshold l e v e l . Animal 5 (Control) The animal was anaesthetized with 0.13 cc. of  56  0.6$ nembutal solution administered  Intraperitoneally  at zero minutes, and the preparation was ready at ten minutes.  At twelve minutes 1:10 m i l l i o n d i l u t i o n of  epinephrine gave no response, nor d i d 1:8 m i l l i o n at f i f t e e n minutes, nor did 1:6 m i l l i o n at nineteen minutes.  However at twenty-three minutes 1:5 m i l l i o n  d i l u t i o n gave a /// response.  This was accepted as  a very d e f i n i t e threshold and the preparation was discarded at twenty-five minutes. Animal 6 (Test) The r a t was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y minutes after thirteen d a i l y intraperitoneal of compound P.  at zero  injections  At eleven minutes 0.05 cc. of nembutal  had to be repeated (subcutaneously) and the preparation was ready at nineteen minutes.  At twenty-five minutes  1:20 m i l l i o n d i l u t i o n of epinephrine gave n© vascular response.  At twenty-eight minutes 1*18 m i l l i o n gave  no response, but at thirty-one minutes 1:16 m i l l i o n gave a /// response.  Then at t h i r t y - f o u r minutes  1:18 m i l l i o n gave a // response, but at t h i r t y - e i g h t minutes 1:20 m i l l i o n gave no response.  On t h i s basis  1:18 m i l l i o n d i l u t i o n was accepted as the threshold  ,  and the preparation was discarded. Animal 7 (Control) The r a t was anaesthetized with 0.13 cc. of 0.6$  57  nembutal solution administered i n t r a p e r i t o n e a l l y at zero minutes, and at seven minutes the preparation was ready on the microscope stage with the warm drip running. At eight minutes 1:10 m i l l i o n epinephrine resulted i n no vascular response.  At eleven minutes 1«6 m i l l i o n  d i l u t i o n gave a /// response, and at f i f t e e n minutes 1;7 m i l l i o n gave a // response which was repeated i n three minutes.  At twenty-one minutes 1:10  gave no response.  million  At twenty-four minutes 1:8 m i l l i o n  gave a s l i g h t response.  In view of the f a c t the  preparation was possibly sensitized to epinephrine at this stage, although i t was otherwise an excellent preparation, 1:7 m i l l i o n was accepted as the threshold value.  The preparation was discontinued at twenty-  eight minutes. Animal 8 (Test) The r a t was anaesthetized with 0.15  cc. of  0.6%  nembutal solution administered i n t r a p e r i t o n e a l l y at zero minutes, and at twenty minutes 0.06  cc. was re-  peated, also by intraperitoneal i n j e c t i o n , and the preparation was ready at t h i r t y minutes.  At t h i r t y -  three minutes 1:20 m i l l i o n d i l u t i o n of epinephrine gave no response.  At thirty-seven minutes 1:18  gave a /, d e f i n i t e but s l i g h t , response.  million  At f o r t y min-  utes 1:16 m i l l i o n gave a // response, and at both three and s i x minutes l a t e r 1»16 m i l l i o n gave a / response.  58 1:18 m i l l i o n was accepted as the threshold value, and the preparation was discontinued at f i f t y minutes. Animal 9 (Control) The animal was anaesthetized with 0.15 cc. of 0.6$ nembutal solution administered  intraperitoneally  at zero minutes, and the preparation was ready at seven minutes with the warm drip running.  At eight  minutes 1:7 m i l l i o n epinephrine solution gave no vascular response, nor d i d 1;6 m i l l i o n at eleven minutes, nor d i d 1:5 m i l l i o n at fourteen minutes.  However, at  seventeen minutes, 1:4 m i l l i o n gave a d e f i n i t e / response. This was a good preparation with excellent active vasomotion.  At twenty-two minutes 1:4 m i l l i o n gave a //  response.  1;4 m i l l i o n was accepted as the threshold,  and the preparation was discontinued at twenty-five minutes. Animal 10 (Test) The animal was anaesthetized with 0.14 cc. of 0.6$ nembutal solution administered  intraperitoneally  at zero minutes, and at f i v e minutes 0.05 cc. were r e peated, with 0.03 cc. more at thirteen minutes. preparation was ready at eighteen minutes.  The  At nineteen  minutes 1:20 m i l l i o n epinephrine gave no apparent vascular reaction, but at twenty-two minutes 1;18 m i l l i o n gave a /// response.  1:18 m i l l i o n was accepted as the  threshold and the preparation was discontinued at twenty-five minutes.  59  Animal 11 (Control) The animal was anaesthetized with 0.14 cc. of 0.6$ nembutal solution administered  Intraperitoneally,  at zero minutes, with 0.05 cc. being repeated at both f i f t e e n minutes and twenty-one minutes.  At twenty-  eight minutes 1:7 m i l l i o n epinephrine gave a // response.  At thirty-one minutes 1:10 m i l l i o n gave a //  which was repeated three minutes l a t e r .  At t h i r t y -  seven minutes p l a i n warm drip gave no response, but immediately  after 1:10 m i l l i o n again gave a // response.  The physiological  nature of this preparation was not  recorded, and 1:10 m i l l i o n must be accepted as the threshold.  The preparation was discontinued at f o r t y  minutes. Animal 12 (Test) The animal was anaesthetized with 0.13 cc. of 0.6$ nembutal solution,  and the preparation was ready  thirteen minutes after the intraperitoneal  injection.  At fourteen minutes 1:20 m i l l i o n epinephrine d i l u t i o n gave a // response but some stasis developed i n the area being observed.  subsequently  At seventeen minutes 1:20  m i l l i o n gave a /// response, which was repeated at twenty minutes.  Vascular stasis continued to develop i n  the mesoappendix u n t i l the preparation was discontinued at twenty-five minutes.  1;20 m i l l i o n was accepted as  60  the  threshold  Animal  13  (Control)  The of  value.  a n i m a l was  0.6$ nembutal  eally cc.  at  -  more.  million  any v a s c u l a r  million old  At nine minutes, 1;6  gave  a / /  with  solution administered  zero minutes followed i n  aration, give  anaesthetized  after  epinephrine  response. and  this  cc.  intraperiton-  two m i n u t e s mounting the dilution  At nineteen was  0.14  accepted  by  prep-  failed  to  minutes as  0.05  the  1;5 thresh-  value.  Note With photograph  the  difficulties remaining  a n i m a l s 13 to  the  vascular  i n managing  the  camera,  be  wasted.  No.  animals  to  w e r e made  9  record of  five  Summary o f  18 attempts  responses, etc.,  Observations Kind  Threshold  11  Control  1:5  2.  Test  No  3.  Control  1:5  4.  Test  1:18  5.  (Control  1:5  6.  Test  1:18  7.  Control  1 7  8.  Test  1:18  :  dilution million  results'' million million million million million million  caused  to but the  61  Summary (Cont'd) No.  Kind  Threshold  dilution  Control  1:4 m i l l i o n  10.  Test  1 18 m i l l i o n  11.  Control  1:10 m i l l i o n  12.  Test  1 20 m i l l i o n  13.  Control  1:5 m i l l i o n  9.  :  :  D i s c u s s i o n o f Observations These o b s e r v a t i o n s  rather s a t i s f a c t o r i l y  confirmed Warner's f i n d i n g s as d e s c r i b e d ; t h a t  inject-  i n g compound P i n t o r a t s f o r a c e r t a i n number o f days decreased the epinephrine  t h r e s h o l d response from  a normal o f 1;4 m i l l i o n d i l u t i o n down to 1:18 m i l l i o n or 1 20 m i l l i o n :  dilution.  They a l s o form a base l i n e o f c a p i l l a r y p e r formance upon which l a t e r c a p i l l a r y behaviour was judged.  62 Record  of Microscopic  Here f o l l o w s microscopically outlined  a record  tested.  are  observations  Details of  given individually  The r e s u l t s  tested,  of  on the mesoappendix  previously.  injections  Observations  according to  are the  of  the  for  of  rat  time p e r i o d s each  grouped by the order  made  the  as and  animal  drugs  table  being  of  contents.  Procaine  Experimental  observations  (A summary o f in  table  Animal  1  form  after  the  Animal  cc o f  Intraperitoneally  Wistar  rat.  scope  stage  i n nine minutes.  any vasomotor  be  dilution  of  but the  two p e r c e n t  of blood  increased.  of  response  mesoappendix,  deterioration  rate  into  The p r e p a r a t i o n  million  later,  results  is  given  10.)  a 0.6% n e m b u t a l  jected  the  following  hydrochloride  (Test) 0.20  four  with procaine  flow  male  was r e a d y  micros  epinephrine  this  capillary  epinephrine.  One m i n u t e  on the  was  after  one to  in  give  circulation of attributed  Three  dropped  vessels  of  failed  f a i l u r e was  procaine  in-  a 280 gram a d u l t  A few drops  i n the  i n the  s o l u t i o n were  to  minutes  on,  and  the  g e n e r a l l y was n o t e d  this,  the  cold  point  to  63  was  a p p l i e d to a s e l e c t e d area of the mesoappendix  w i t h f r e e z i n g f o r f o r t y - f i v e seconds, which was owed a f t e r thawing by i n c r e a s e d b l o o d flow. cent p r o c a i n e was  foll-  Two  per-  dropped on the mesoappendix, but  v a s c u l a r s t a s i s was  observed  In a few c a p i l l a r y bedS  w i t h i n twenty-seconds of thawing, although the c e n t r a l venule was  functioning well.  By three and a h a l f  minutes a f t e r thawing, there was  s t a s i s i n the main  venule i n the i n j u r e d area, g i v i n g blockage g e n e r a l l y throughout freezing.  and  the whole area a f f e c t e d by  stasis the  Therefore i t would not seem as though the  p r o c a i n e delayed the onset of v a s o s t a s i s much f o l l o w i n g the f r e e z i n g i n j u r y .  Ten minutes l a t e r one  c a p i l l a r i e s were observed  or  two  to be f u n c t i o n i n g at the edge  of the i n j u r e d area, and three minutes a f t e r t h i s warm s a l i n e d r i p was was  stopped,  and two percent  i n s t i l l e d over the mesoappendix.  the  procaine  This r e s u l t e d i n  a more r a p i d flow i n the c a p i l l a r i e s around the p e r i phery of the i n j u r e d area, but there was  no  apparent  e f f e c t on any of the s t a s i s w i t h i n the f r o z e n area. minutes l a t e r more two p e r c e n t p r o c a i n e was  Pour  applied.  two minutes one l a r g e v e s s e l t h a t had been s t a t i c  In  was  f u n c t i o n i n g , and i n another minute a couple of small c a p i l l a r i e s at the edge of the i n j u r e d area were f l o w ing.  Two  minutes l a t e r more p r o c a i n e was  a p p l i e d , which  r e s u l t e d i n a s l i g h t t r i c k l e of b l o o d c o r p u s c l e s the main s t a t i c area.  through  Three minutes l a t e r more p r o c a i n e  64 resulted static  i n almost  area,  immediate  although only  o p e n i n g up  temporarily,  ulation  c l o s e d down a g a i n o n r e s u m i n g  drip.  Three minutes  up  the  same c h a n n e l s ,  in  the  vessels  The p r e p a r a t i o n after  the  Animal  was  initial  2  later with  tributary  w i t h 0.15  preparation  cc  of c a p i l l a r y  increased.  One m i n u t e  over  cold  freezing  the  for  field.  thirty  was l i m i t e d . was n o t e d but  to  within  o p e n e d up this  the  opened observed  channel. seconds  a p p l i e d to  a selected  the  venous  after  spread  four minutes and resumed  to of  Its  w h o l e a r e a was  in  area,  thawing, blood  an  but and  flow.  in stasis,  immediate followed  dropped  on  appeared the  the  adjacent  the  later  with  channels  thawing,  involve  again  area  to  was  still  P r o c a i n e was  P r o c a i n e was r e a p p l i e d , have  observed  resulted  affected  Two  mesoappendix,  more p r o c a i n e  This  small  later.  the  was  was  solution,  and one m i n u t e  dilatation.  minutes  flow  later,  seconds.  in  200 gram r a n g e  blood  throughout  Stasis  and a h a l f  was  a p p l i e d to  by marked v a s c u l a r  three  vasomotion  seven minutes  mesoappendix,  p o i n t was  vasoconstriction  the  again  of 0.6$ nembutal  was r e a d y  speed  the  saline  main venular  i n the  and  flushed  warm  circ-  freezing.  aqueous p r o c a i n e  be  the  the  discontinued thirty-five  percent the  this  main  (Test)  anaesthetized the  increased  the  since  more p r o c a i n e  to  A male W i s t a r r a t  and  of  first  process stasis channel,  channel  One m i n u t e w h i c h was  after not  65 greatly  r e l i e v e d by f u r t h e r  The p r e p a r a t i o n particularly een m i n u t e s Animal  5  was n o t  solution  after  the  Initial  administered was  be  of  solution  This  cc  the  stasis  stasis  adult  as  being  discarded  thirt-  injury.  in  the  of 0.6$  200  gram  nembutal  but  whole  the  prep-  mesoappendix  on mounting on  was n o t  the  influenced  by  procaine.  male W i s t a r r a t  anaesthetized administered  with  was r e a d y  applied  with freezing for  seconds that  of  in five  five  the  200  cc o f 0.6$  gram  nembutal  and  the  prep-  minutes.  The c o l d p o i n t  forty-five  seconds,  thawing stasis  had been  following  0.15  in  Intraperitoneally,  aration  the  procaine.  (Control^  r a n g e was  in  the  time  intraperitoneally,  two p e r c e n t  A normal  venules  was  Wistar rat  w i t h 0.15  i n vascular  stage.  applications  sixty  this  freezing  discontinued because  to  microscope  4  adult  anaesthetized  found  Animal  at  of  (Control)  r a n g e was  was  regarded  p h y s i o l o g i c a l , and i t  A normal male  aration  applications  was o b s e r v e d  frozen.  This  stasis  minutes,  and the  and in  was  within  the  did not  preparation  change was  discontinued. Animal  5  (Test) A normal  weight  r a n g e was  nembutal  was  male  Wistar rat,  anaesthetized  solution administered  preparation caine  adult  was r e a d y  in  applied locally  w i t h 0.15  in  the  cc o f  200 0.6$  intraperitoneally,  s i x minutes. on the  gram  and  Two p e r c e n t  mesoappendix,  and  the  prothe  66 rate of capillary erally  b l o o d f l o w was o b s e r v e d  increased.  Two m i n u t e s l a t e r  applied with freezing applied  immediately  forthirty  after  onds a f t e r  this.  blood flow  i n the i n j u r e d  vasodilatation  a h a l f minutes a f t e r  and  then  after  thirty  and  slowing  (and By  venule,  a l s o except  twelve  ulation  i n the remainThirty  increased blood flow  which,  static.  i n a l l except  was c o m p l e t e l y  seconds  i n the r e s t  over  resulting, Two m i n u t e s increased  one l a r g e  reopened by p r o c a i n e  i n t h e two above d e s c r i b e d s t a t i c  minutes a f t e r  q u i t e normal,  minutes  a r e a was i n v a s c u l a r  t h e warm s a l i n e d r i p was resumed, w i t h  and s t a s i s ,  connecting  third  a n d p r o c a i n e was f l u s h e d  o n l y one s m a l l a r e a r e m a i n e d  afterward  a  apparent,  d r i p was resumed, a n d n i n e t y  of the injured  t h e d r i p was s t o p p e d  with  b u t o n l y two  Three and a h a l f  der o f the v e s s e l s i n the f r o z e n a f e a .  the mesoappendix, w i t h  thawing, the  with  s t a s i s w i t h i n c r e a s e d vasomotion observed  later  sec-  t h a w i n g was any s t a s i s  seconds l a t e r .  one t h i r d  thirty  seconds a f t e r  o f the venules,  t h a w i n g t h e warm s a l i n e  seconds l a t e r  was  and a g a i n  o n l y i n two s m a l l c a p i l l a r i e s ,  involved  Procaine  a r e a was g r e a t l y s l o w e d ,  especially  and  t h e c o l d p o i n t was  seconds.  thawing,  Forty-five  t o be g e n -  the f r e e z i n g  areas).  injury the circ}  o f t h e m e s o a p p e n d i x was  apparently  and t h e p r e p a r a t i o n was d i s c o n t i n u e d .  Animal 6 (Control) A normal a _ d u l t male W i s t a r  r a t i n t h e 200 gram  w e i g h t r a n g e was a n a e s t h e t i z e d w i t h 0.15 c c o f 0.6$ nembutal  solution  administered  intraperitoneally,  and t h e  67 preparation point  was  followed after  was r e a d y  within  five  minutes.  applied with freezing for by immediate  rapid  thirty  thawing.  and a h a l f  thawing  still  stasis  t h e r e was  channel,  but  when t h e  spreading  a r a t i o n was 7  stasis  within  and  the  was  stasis  of  stasis,  was r e a d y  appear  warm s a l i n e  areas  of  the the  the  a half  prep-  200  gram  ninety  seconds  circulation,  but  i n these  was n o t e d  that  greatly  was  channels  opened up  increased  vascular  tone  the  general  affected,  area  of  spread open.  apparently  minutes  later  the  applied. and  after  stasis  was  i n comparison w i t h what  In  resumed  gave more  By ten minutes  One  with  remained  the  channels.  after  vessels.  mesoappendix had  d r i p was d i s c o n t i n u e d a n d p r o c a i n e  for  minutes  main channels  One a n d a h a l f  0.15  minutes.  d r i p was r e s u m e d ,  saline  many o f  with  mesoappendix,  i n small  circulation.  really  The  intraperiton-  in six  normal  it  there  applied with freezing  two and  to  although  The u n i n j u r e d  i n the  anaesthetized  flushed over  In  started  the  was  p o i n t was  seconds.  later  motion  venous  started  seconds.  Wistar rat  It  preparation  cold  minute the  after  i n one  solution administered  procaine  the  thirty-five thawing,  thirty  adult  was u s e d .  of 0.6$ nembutal  then  except  marked  (Test)  Two p e r c e n t and  seconds  discontinued.  weight range  eally,  no  minutes  warm R i n g e r d r i p was  A normal male  cc  seconds,  t h a w i n g t h e r e was v a s o c o n s t r i c t i o n and t h e n By t h r e e  Animal  cold  For twenty  vasodilatation.  was  The  vaso-  thawing not i t  had  68 been, the  and  i n comparison  the  surrounding  areas  of  200  gram  mesoappendix.  Animal  8  (Control) A  weight  normal  range  nembutal  solution stopped  was  was  and  ready  the  in five the  cold  started  injured  area.  resumed  which  within  administered  the  Two  was  and  completed  injured  cc  area.  The  drip  was  freezing of  thawing  in  later  the  the  drip  generalized vascular The  the  saline  rapidly  minutes  and  warm  seconds  spreading  the  0.6$  with  applied with  thirty  a half  of  intraperitoneally,  mesoappendix.  Within and  0.15  minutes,  p o i n t was  seconds.  had  r a t i n the  anaesthetized with  d r i p p i n g on  thirty  stasis  a d u l t male Wistar  solution  preparation  for  with  p r e p a r a t i o n was  was  stasis  discontin-  ued. Animal  9  (Test) A  weight  normal  range  nembutal  warm  percent  was  noted. stasis  ready  procaine  was  for thirty  iately  administered  drip  The  cold  was  two  i n seven running.  p o i n t was  and  and  observed,  The  greatly  a half and  0.15  cc  200  of  gram  0.6$  intraperitoneally, minutes,  locally next  mounted, stopped,  over  was  the  with two  mesofreez-  flow  thawing,  v a s o m o t i o n was  the  and  applied  increased blood after  and and  applied, with  procaine  minutes  good  r a t i n the  T h i s was  instilled  seconds.  afterward, By  Wistar  anaesthetized with  saline  appendix. ing  was  solution  preparation the  a d u l t male  immedwas no  apparent  69 throughout t h e i n j u r e d a r e a . applied,  P r o c a i n e was a g a i n r e -  and by f i v e and a h a l f m i n u t e s a f t e r thawing,  t h e r e was s t i l l  excellent blood flow with  but no s t a s i s had d e v e l o p e d . c a p i l l a r y s t a s i s generalized  vasomotion,  Two m i n u t e s l a t e r s l i g h t over the i n j u r e d a r e a was  r e a d i l y o b l i t e r a t e d with h a l f percent procaine.  One  minute l a t e r the warm s a l i n e d r i p was s t a r t e d w i t h immedi a t e g e n e r a l c a p i l l a r y s t a s i s r e s u l t i n g i n the i n j u r e d area.  The d r i p was stopped, w i t h s u b - t o t a l  of b l o o d f l o w i n the whole f i e l d . discontinued  resumption  The p r e p a r a t i o n  t e n m i n u t e s a f t e r the i n i t i a l  was  freezing  Injury. Animal 10  (Control)  A normal a d u l t male W i s t a r r a t i n the 200 gram w e i g h t range was used.  I t was a n a e s t h e t i z e d w i t h 0.15  cc o f 0.6$ nembutal s o l u t i o n a d m i n i s t e r e d i n t r a p e r i t o n eally.  Ten minutes l a t e r t h e c o l d p o i n t was  to a s e l e c t e d  applied  a r e a o f the mesoappendix w i t h f r e e z i n g f o r  t h i r t y seconds.  Quite extensive v a s o - d i l a t a t i o n  ed the thawing, and w i t h i n n i n e t y  seconds o f thawing,  s t a s i s s t a r t e d w h i c h r a p i d l y became e x t e n s i v e . and  follow-  By f o u r  a h a l f minutes f o l l o w i n g thawing t h e whole i n j u r e d  a r e a was i n s t a s i s except f o r one l a r g e c h a n n e l . preparation  was d i s c o n t i n u e d  a f t e r thawing.  The  f i v e and a h a l f minutes  70 Summary of Results witfe Procaine No.  Onset Stasis  Spread  Generalized  Flow Resum'd  1  Test  20"  Yes  3i«  ///  2  Test  3£*  No  Finally  ///  3  Control No r e s u l t s , preparation  4  Control 45"  Rapid  Yes  Hone  5  Test  Slight  Drip caused  ///  6  Control 3*'  Total stasis when drip started after thawing  7  Test  When saline drip resumed  ///  8  Control 30"  Rapid when drip resumed  None  9  Test  10  2*'  Control 90"  Rapid  discarded  General when drip resumed  ///  4-|'  None  Discussion of Results The u n s u i t a b i l i t y of p l a i n Ringer-Locke saline solution as a perfusion drip i s r e a d i l y apparent.  In  spite of this uncontrolled factor, i t i s f e l t that two percent procaine applied l o c a l l y to the mesoappendix both before and after a standard freezing injury with the cold point f o r t h i r t y seconds could be r e l i e d on to both delay the onset of stasis and reopen clogged and s t a t i c vessels oneethe s t a s i s had occurred. The average length of time required f o r the onset of stasis i n the test animals was nearly three and a h a l f minutes after thawing, whereas i n the control runs the corresponding  time was approximately ninety  71 seconds.  Thus i t would appear that two percent procaine  delays the onset of vascular s t a s i s i n a f r o z e n c a p i l l a r y bed a f t e r a p p l i c a t i o n of the c o l d p o i n t w i t h f r e e z i n g f o r t h i r t y seconds by an average of two minutes. Before f r e e z i n g of the c a p i l l a r y bed, two percent procaine a p p l i e d t o p i c a l l y to the mesoappendix gave g r e a t l y increased f l o w of blood i n a l l v e s s e l s observed, both i n v e l o c i t y and i n apparent volume of f l o w .  This was  thought to be due to a v a s o d i l a ^ t o r y e f f e c t , w i t h the s i t e of a c t i o n not determined.  The procaine could act d i r -  e c t l y on the small blood vessels or through i n h i b i t i o n of the v a s o c o n s t r i c t o r impulses.  I t was important to  observe that a d d i t i o n a l t o p i c a l a p p l i c a t i o n of two percent procaine f r e q u e n t l y aided or caused the opening up of channels i n s t a s i s , presumably through r e l e a s i n g v a s o c o n s t r i c t i o n , and thereby a l l o w i n g a g r e a t e r volume of blood at a more e f f e c t i v e h y d r o s t a t i c and p u l s i l e pressure to f l u s h the v e s s e l f r e e of the sludge of c e l l s . I t was noted on resuming the d r i p a f t e r f r e e z i n g that vascular s t a s i s i n many channels developed almost immediately, but often such s t a s i s could be overcome by l o c a l a p p l i c a t i o n of two percent procaine. I t was thought the p l a i n s a l i n e d r i p s o l u t i o n used aided the t r a n s f e r of the l i q u i d component of the blood through the damaged c a p i l l a r y w a l l s , and hence increased the sludging of c e l l s i n the v e s s e l s . Repeat Experiments w i t h Procaine Hydrochloride In t h i s instance, the above t r i a l s were repeated  72 using  a Ringer-Locke  gelatin sults  and g l u c o s e .  is  Animal  saline  A summary o f  given i n table  11  r a n g e was  nembutal  was r e a d y  two p e r c e n t  and  the  then  cold  within  re-  17.  p o i n t was  in  0.20  the  300  cc o f  subcutaneously  twenty-eight over  the  gram  0.6$ and  the  minutes. mesoappendix  applied with freezing  for  seconds.  was n o t  sufficiently  had  and r o l l e d  four  with general  times,  mesoappendix, 12  off  the  vascular  stasis  male W i s t a r r a t  anaesthetized  Twenty minutes and  the  Ringer-locke two p e r c e n t  seconds.  i t  The^ratcc.l&wed i t s  microscope  later  a n i m a l was  stage  three  throughout  or  the  300  cc o f  ready  gram  0.6$ suppos-  cc more n e m b u t a l  was  i n four minutes.  The  d r i p was d i s c o n t i n u e d  p r o c a i n e was f l u s h e d o v e r  p o i n t was  the  intramuscularly,  0.10  gelatine-glucose  in  with 0.20  solution administered  The c o l d  discarded because  (Test)  r a n g e was  edly.  be  making e v a l u a t i o n i m p o s s i b l e .  A normal adult  nembutal  to  anaesthetized.  intestines,  given,  with  was f l u s h e d  The p r e p a r a t i o n  weight  following  Animal  male W i s t a r r a t  anaesthetized  Procaine  Animal  after  solution administered  preparation  thirty  form  the  with  (Test)  A normal adult weight  solution modified  the  and  mesoappendix.  applied with freezing for  thirty  P r o c a i n e was f l u s h e d o n i m m e d i a t e l y  after,  tt at  which  of  cells.  time  the  Within  vessels sixty  were  closed tight  seconds  of  and  empty  thawing, a few  blood  73  c e l l s were flowing through.  Procaine was reapplied  and by three minutes of thawing ninety percent of the vessels i n the injured area were open and functioning. Restarting the gelatine-glucose drip did not effect any further s t a s i s . Further tests were performed with this preparation.  Thirty seconds after the drip was restarted  the injured area was about ninety percent normal with a few small c a p i l l a r i e s i n s t a s i s .  Sixty seconds l a t e r  two percent procaine was applied.  This put the whole  area into s t a s i s , which recovered e n t i r e l y with resumption of the warm d r i p , but shortly after, developed i n the whole injured area.  stasis re-  Further procaine  gave only a l i t t l e restoration of c i r c u l a t i o n which was aided by resuming flow of the d r i p .  This was a very  interesting preparation to watch, and i t showA that the Ringer-Locke drip containing gelatine much more p h y s i o l o g i c a l .  and glucose i s  Note that the procaine was  an e n t i r e l y aqueous solution,  and not a saline one.  Animal 13 (Control) A normal adult male Wistar rat i n the 300 gram weight range was anaesthetized with 0.20 cc of  0.6$  nembutal solution administered intramuscularly and the preparation was ready i n twelve minutes, with the warm drip solution running over the mesoappendix.  The cold  point was applied to a selected area with freezing for t h i r t y seconds.  Within t h i r t y seconds of thawing there  was much vascular s t a s i s , and within two and a half min-  74 utes  of  thawing the  whole i n j u r e d  and i r r e v e r s i b l e s t a s i s large  channels.  Animal  14  except  in  was  was  anaesthetized  ready  in  15  stasis  from  range  was  solution,  aesthetic  was n o t  was r e g a r d e d 16  0.6$ The  but  the  whole mesentery  was  kicking.  male W i s t a r r a t  and was n o t deep  i n the  with 0.30 ready  enough.  300  cc o f  because  gram  0.6$  the  an-  The i n t r a m u s c u l a r  Wistar rat  anaesthetized  solution administered  preparation  was  flushed  was  over  the  the  drip  and  with  freezing for  ready  t h e r e was  injured  area.  thirty  was  0.20  after  seconds.  complete  thawing,  cc o f  300  route  gram  0.6$  intraperitoneally,  a p p l i e d to  stoppage  P r o c a i n e was  after  i n the  seven minutes  later.  Procaine  stopping  the  the  Thirty of  and  mesoappendix seconds  saline  one h a l f  of  after  circulation in  a p p l i e d t w i c e w i t h no  warm g e l a t i n e - g l u c o s e  minutes  with  mesoappendix  cold point  thawing  two  minutes,  animal's  a d u l t male  was  the  When t h e  cc o f  gram  (Test)  range  nembutal  300  as i m p r a c t i c a l .  A normal weight  the  the  intramuscularly.  the  anaesthetized  nembutal  Animal  main  (Test)  A normal adult weight  three  in  w i t h 0.20  thirty-one  discarded because  vascular  Animal  was  male W i s t a r r a t  solution administered  preparation animal  two o r  complete  (Test)  range  nembutal  for  was i n  1  A normal adult weight  area  d r i p was the  the  effect.  restarted  static  chann-  75 els  opened,  and i n another minute s i x t y - f i v e  were f u n c t i o n i n g .  In one and a h a l f minutes p r o c a i n e  gave stoppage of f l o w . fifteen  percent  When the warm d r i p was r e s t a r t e d  seconds l a t e r the whole i n j u r e d area was f l o w i n g  one hundred p e r c e n t , were n o t e d .  although two small ecchymotic  areas  Two minutes l a t e r p r o c a i n e stopped b l o o d  flow i n the i n j u r e d a r e a , and resumption of the restarted i t  drip  completely.  Animal 17 ( C o n t r o l ) A normal a d u l t male" Wis t a r r a t i n the 300 gram weight range was a n a e s t h e t i z e d  w i t h 0.20  cc of 0.6$ nem-  b u t a l s o l u t i o n a d m i n i s t e r e d i n t r a p e r i t o n e a l l y , and i n e i g h t minutes the p r e p a r a t i o n was ready w i t h the flowing.  drip  The c o l d p o i n t was a p p l i e d w i t h f r e e z i n g  t h i r t y seconds,  and t h i r t y seconds a f t e r  for  thawing there  was general small c a p i l l a r y s t a s i s throughout the i n j u r e d area.  By f o u r minutes a f t e r thawing the s t a s i s was  spreading to l a r g e r channels,  and i n another minute  s t a s i s was g e n e r a l and n e a r l y complete.  the  Two and a h a l f  minutes l a t e r p r o c a i n e was a p p l i e d and a v e r y marked r e c o v e r y o c c u r r e d , although i t was i n c o m p l e t e . was i n c r e a s e d vasomotion,  There  r a t h e r than any vasospasm.  Three minutes l a t e r s t a s i s r e t u r n e d and the p r e p a r a t i o n wasdiscontinued.  76 Summary o f No.  Results Onset  with  Stasis  Procaine Spread  Generalized  11  Test  No r e s u l t s  because  12  Test  Minimal  Minimal  None  //'/'  13  Control  Rapid  2&»  None  14  Test  No r e s u l t s  because  of  general  stasis  15  Test  No r e s u l t s  because  of general  stasis  16  Test  Unusual  17  Control  30"  of  general  Flow  Resumed  stasis  / / /  30"  Immediate  30"  None  Conclusions These procaine  additional results  are  too  variable  significance. glucose  to In  The v a l u e  the the  case  warming ( or  drip  fluid  dosage  was  the  this  run,  is  ) of  be  of  the  any and  apparent.  some v a l u e frozen  Procaine  procaine  in  immed-  area  with  warm  rather  high  that  on a p a r t  was u s e d  the  in  following  the  H i s use to  administered were  per-  s t u d e n t who w a s h e l p i n g  with  time b a s i s .  could not  here used  (Parenteral)  k i l o g r a m body w e i g h t ,  medical  elsewhere.  stasis"  with  Note  by a s e n i o r  and o b s e r v a t i o n s used  to  adding gelatine  animal 16,  thawing  o f 2 0 0 mg p e r  project  of  few  applied  suggested.  parenterally. formed  of  Observations In  too  R i n g e r - L o c k e s o l u t i o n was  iate  Further  and  with locally  be of  imply  His  tests  methods  identical with the  term  stasis  those  "general  throughout  the  77 vessels in  the  of  the  whole mesoappendix,  injured  Animal  1  area  as  is  grams was i n j e c t e d procaine,  anaesthetized  with  subcutaneously  0.1  was  a p p l i e d to  appendix with immediately minute one  quarter  Animal  the  180 grams  0.6$ nembutal  spread  to  prepara-  and the the  cold  meso-  followed  began w i t h i n involve injured  female nonpregnant  was a n a e s t h e t i z e d  animal expired before 5  with  s o l u t i o n by subcutaneous  one  about area.  Hooded  rat  0.1  of  cc  injection.  any o b s e r v a t i o n s  could  be  (Test) nonpregnant  w e i g h i n g 170 grams was cc of  two p e r c e n t  injected  any o b s e r v a t i o n s  Animal  4  could  be  female  Hooded  subcutaneously  procaine.  before  The a n i m a l  rat  with  expired  made.  (Test) A normal adult  weighing 1.6  Stasis  was  solution The  seconds  c a p i l l a r i e s i n the  A normal adult  1.7  of  two  2 (Control)  weighing  Animal  area  thirty  and i t  A normal adult  The  cc o f  animal  microscope stage  freezing for  of  the  injection.  a selected  thawing,  1.6  cc of 0.6$ nembutal  by r a p i d thawing.  after  w e i g h i n g 160  with  and i n t e n m i n u t e s  was m o u n t e d o n t h e  point  merely  elsewhere.  male Hooded r a t  administered by subcutaneous tion  than  (Test) A normal adult  percent  implied  rather  cc o f  160 grams  was  a two p e r e e n t  nonpregnant injected procaine  female  Hooded  subcutaneously solution.  In  rat  with five  made.  78  minutes butal  i t  was  anaesthetized  solution administered  and when r e a d y  the  scope  The c o l d  area  stage. of  onds,  the  Within  of  area  preparation tip  seconds  to  of  was  rapid  involve  half  i n three minutes  develop  stasis,  venules  were w i d e l y  but  the  of of  (see  definition  weighing  -was m o u n t e d  for  was  solution,  on the  a p p l i e d to  freezing  it  adult  170 grams  0.6$ nembutal  Stasis  re-  vessels  injured  drip.  observed, i n the  inj-  Note  that  area  did  the  artrjot  collecting  stasis  T h e r e was no  i n the  was  general  mesoappendix.  (Control) A normal  thawing  sec-  Resumption of flow  i n one medium s i z e d v e n u l e . above)  selected  thirty  was  c a p i l l a r i e s and  seen  was  a  micro-  gelatine-glucose  thawing.  affected.  injection,  thawing through  the  i n the  nem-  on the  a p p l i e d to  thawing s t a s i s  and m e t a r t e r i o l e s  5  of 0.6$  was m o u n t e d  warm R i n g e r - L o c k e  erioles  Animal  cc  by subcutaneous  by immediate  the  thirty  which s p r e a d  0.07  mesoappendix w i t h f r e e z i n g f o r  followed  sumption  ured  with  nonpregnant  anaesthetized  a selected  seconds,  through resumption developed  spread  to  area  general  stasis  of  noted  or  cc  of  preparation  The c o l d  point  mesoappendix  with  followed  by immediate  rapid  the  in forty-five  by f o u r minutes  the  rat  the  of  involve nearly  injured  stage.  area  Hooded  with 0.08  and when r e a d y  microscope  thirty  female  all of  warm d r i p  seconds the  after  vessels  thawing.  resumption of  solution.  flow.  thawing in  and  the  T h e r e was  no  79 Animal  6  (Test) A normal adult  weighing 1.6  160 grams  cc o f  later  nembutal  a n i m a l was  of  procaine,  the  cold  tip  mesoappendix  Hooded female  subcutaneously  w i t h 0.10  a d m i n i s t e r e d by  was  with freezing  developed w i t h i n  three minutes  spread  to  one  injured or  involve  area.  7  adult  150 grams  was  nonpregnant  preparation  tip  was  was mounted  a p p l i e d to  a  thirty  thirty  thawing,  seconds the  of  on the  selected  with freezing for  vessels  area  seconds. with  quarter  of  utes.  T h e r e was no g e n e r a l  of  area  seconds.  thawing, vessels flow  and in  the  noted  Hooded female  i n the  w i t h 0.09  cc  of  subcutaneously, microscope. of  the  Stasis  spread  injured stasis  rat  to area  and  The  cold  mesoappendix developed involve  in  one  in five  min-  and no r e s u m p t i o n  of  noted.  Animal  8  (Test) A normal adult  w e i g h i n g 160 grams of  thirty  the  anaesthetized  solution administered  the  cc  micro-  (Control)  0.6$ nembutal  flow  of  0.6$  stasis.  A normal weighing  tenth  of  on the  T h e r e was n o r e s u m p t i o n o f  any g e n e r a l  Animal  about  cc  a selected  for  Stasis  minutes  subcutaneous  was m o u n t e d  a p p l i e d to  rat  with  and t w e n t y - f i v e  anaesthetized  The p r e p a r a t i o n  and  the  injected  solution also  injection. scope  was  two p e r c e n t  the  nonpregnant  two p e r c e n t  was  nonpregnant injected  procaine,  Hooded female  subcutaneously  and i n s e v e n t y - f i v e  rat  with  1.6  minutes  80 it  was  ution  anaesthetized  was m o u n t e d  selected  area  thirty  seconds.  ing  involve  to  injured was  cc  of 0.6$ nembutal  administered by subcutaneous  preparation a  w i t h 0.1  of  the  and  the  cold  tip  was  applied  developed  seven-eighths  of  N o resumption  for  i n one m i n u t e ,  the  vessels  of flow  or  in  to  spread-  the  general  stasis  observed.  Animal  9  (Control) A normal adult  weighing  170 grams  0.6$ nembutal  nonpregnant  was  Hooded female  anaesthetized  solution administered  c o l d p o i n t was  a p p l i e d to  with  a selected  followed  thawing.  in forty-five  Stasis  thawing, in  the  of  flow  Animal  and  spread  injured was 10  1.6  involve  area  of  No g e n e r a l  thirds stasis  of or  adult  160 grams  nonpregnant  was  two p e r c e n t  the  meso-  after the  vessels  resumption  injected procaine,  a n i m a l was  Hooded female  subcutaneously and i n  anaesthetized  with 0.09  to  area  mesoappendix  selected thirty  of  seconds,  the  followed  commenced w i t h i n to  involve  forty  four  short  with  with  of  cc  of  applied freezing  by immediate r a p i d seconds  rat  twenty-five  The c o l d p o i n t was  spreading  The  observed.  solution.  Stasis  of  rapid  seconds  0.6$ nembutal a  the  by immediate  two  cc  (Test)  cc o f  minutes  to  area.  A normal weighing  began  0.10  rat  subcutaneously.  appendix w i t h f r e e z i n g ,  for  The  mesoappendix w i t h f r e e z i n g  Stasis  area.  injection.  sol-  thawing.  thawing,  segments o f  capillary  81 in  the  injured  Animal  11  area  (a very  slight  (Control)  A normal  adult nonpregnant  w e i g h i n g 150 grams 0.6$ nembutal  was  anaesthetized  a p p l i e d to  immediate r a p i d  ing, the  and  spread  Animal  area  12  started  to  cc  of  by  170 grams  the  nembutal  of  was  the  mesoappendix  followed  involve  area  15  the  after  vessels  thawin  subcutaneously  was  seconds of  the  with  w i t h 0.10  cc o f  0.6$  subcutaneous a  selected  thirty  rapid thawing. thawing,  vessels  1.7  minutes  a p p l i e d to  after  rat  in  Stasis  and the  spread injured  minutes.  (Control) adult, nonpregnant  160 grams  0.6$ nembutal The c o l d  followed  Hooded female  and f i f t y - f i v e  by immediate  almost h a l f  A normal weighing  the  thawing.  with freezing for  i n approximately five  Animal  the  administered by  The c o l d p o i n t  commenced i n f i f t y to  injected  anaesthetized  solution also  seconds,  of  seconds  all  of  nonpregnant  procaine,  a n i m a l was  injection. area  area  seconds,  in thirty  three minutes  of  (Test)  two p e r c e n t  later  cc  subcutaneously.  thirty  involve nearly  A normal adult weighing  rat  thawing through resumption of  Stasis  frozen  with 0.10  a selected  mesoappendix w i t h f r e e z i n g f o r  warm d r i p .  Hooded female  solution administered  The c o l d p o i n t was  by  effect).  was  anaesthetized  solution administered  p o i n t was  Hooded f e m a l e  a p p l i e d to  w i t h 0.10  cc  rat of  subcutaneously.  a selected  area  of  the  82 mesoappendix w i t h f r e e z i n g f o r by  immediate r a p i d  warm d r i p . thawing, vessels  Stasis  the  seconds  followed  thawing through resumption of  spreading in  thirty  commenced i n t h i r t y to  involve  injured  area  three  the  seconds  quarters  after  of  i n three minutes  the  after  thawing. Animal  14  (Control)  A normal  adult  w e i g h i n g 180 grams was 0.6$ nembutal was  nonpregnant  Hooded female  anaesthetized  solution,  w i t h 0.10  and when r e a d y  a p p l i e d to  a  selected  with  freezing  for  thirty  iate  r a p i d thawing through resumption  Stasis  commenced i n  spread  to  area  involve  i n four  about  of  the  seconds,  thirty  half  the  of  point  mesoappendix  followed  seconds  cold  cc  of  after  vessels  b y immed-  the  warm  thawing, in  the  drip. and  injured  minutes.  Summary o f R e s u l t s w i t h Onset  Mo.  area  the  rat  Parenteral  Stasis Spread P i n a l  1  Test  60"  2  Control  Died,  no r e s u l t s  3  Test  Died,  no r e s u l t s  4  Test  30"  5  Control  45"  6  Test  7  Procaine Extent  P l o w Resumed  25$  None  Slow  50$  Some  Slow  95$ i n 4»  None  180"  10$  None  Control  30" "  25$ i n  5'  None  8  Test  60"  7/8  of  vessels  None  9  Control  45"  2/3  of  vessels  None  Slow  83 Summary C o n t ' d No.  Onset  Stasis  Spread P i n a l  Extent Flow  Resumed  10  Test  40"  4 capillaries  None  11  Control  30"  Almost  None  12  Test  50"  5 0 $ i n 5»  None  13  Control  30"  7 5 $ i n 3»  None  14  Control  30"  50$ i n 4«  None  Impression  of  Results  "The p r o c a i n e better  protected  injury  as  they  are  treated  against  performed.  observations  a l l i n 3«  were  animals  stasis  However,  slight  and i t  significant."  See  seemed  produced  by  differences was  to  be  freezing from  difficult  to  d i s c u s s i o n on page  control say  i f  311.  Priscoline  Experimental This of  1.0  d r u g was  the  Animal  Animal  1  with Priscoline  tested parenterally  mg P r i s c o l i n e p e r  summary o f after  observations  in a  dosage  k i l o g r a m body w e i g h t .  following  results  is  A  given i n table  form  9.  (Test) A normal  weighing  adult  170 grams  0.17  cc  of  0.17  mg.,  was  nonpregnant injected  female  subcutaneously  a diluted Priscoline solution and  anaesthetized administered  twenty-four with  0.15  cc  Wistar  minutes  later  injection.  with  containing the  of 0.6$ nembutal  by subcutaneous  rat  animal  was  solution  The  preparation  84 was r e a d y to  In nine minutes  a selected  for  thirty  stasis  started  of  the  stopped  were  static  controls  p r e p a r a t i o n was further Animal  change 2  cold  Within  to  thirty  involve  thawing.  continue a far  so f a r  to  shut  greater  tested  with 0.15  the  good.  reopen-  within  three vessels  down a n d r e s t a r t extent  a  Stasis  than  during this  was i n j e c t e d  any  blood  other  project. later  The  with  no  later  injection,  eleven minutes  later.  twelve minutes  after  tip  the  a n i m a l was  the  solution administered  first  c c more b e i n g  of  ecchymotic areas.  dose  a selected thirty  r a p i d thawing with seconds  0.17  anaesthetized  the  of nembutal area  of  seconds,  the  in  and  followed solution.  thawing there had  developed  later  stasis  the  meso-  warm d r i p  A minute  by  injected  The p r e p a r a t i o n was r e a d y  was a p p l i e d t o  thirty  rat  subcutaneously with  with 0.05  appendix w i t h f r e e z i n g f o r immediate  female Wistar  s o l u t i o n c o n t a i n i n g 0 . 1 7 mg,  cc of 0.6$ nembutal  subcutaneous  three  later  (Test)  a diluted Priscoline  Within  thawing  noted.  sixteen minutes  cold  minute  d i s c o n t i n u e d seven minutes  w e i g h i n g 170 grams  and  of  A minute l a t e r  A normal adult nonpregnant  cc o f  seconds  new v e s s e l s  applied  freezing  A minute  and by another  of  t i p was  mesoappendix w i t h  v e n u l e s was r a t h e r  minutes to  the  several venules.  c e r t a i n l y to  normal  the  reopened,  observed  flow,  in  extending  and a h a l f  of  seconds.  few v e n u l e s ing  area  and  was  by  noted  85 in  two c a p i l l a r y  short  venules  stasis  were  was g e n e r a l  area.  In  static,  another  four minutes  minutes  later  i n the  animal  was  blood.flow  and w i t h i n  throughout  the  a few v e s s e l s  several  sixty  whole o f  seconds the  much o f  smaller  the  stasis  channels.  had been  and s t a s i s .  The p r e p a r a t i o n  was  By an-  with  Pour minutes  overcome  of  later  the  as  ex-  the  slowing the  of  the  animal  circulation.  d i s c a r d e d twenty minutes  i n w h a t was r e g a r d e d  Two  In four minutes  a g a i n becomming a p n o e i c ,  more,  injured  had reopened.  was h y p e r p n o e i c w i t h r e s t o r a t i o n  ing,  minute  The a n i m a l h a d g a s p i n g r e s p i r a t i o n s .  other  cept  beds.  after  thaw-  a Cheyne-Stokes type  of  respiration. Animal  5  (Control) A normal adult  w e i g h i n g 165 grams 0.6$ nembutal the cold  was  anaesthetized  was r e a d y  a p p l i e d to  appendix with  a selected  freezing for  ing,  stasis  involved. static, only  the  By s i x t y  and the  thirty the  area  of  any c i r c u l a t i o n  capillary  bed  spread. open,  after  by  solution. after  w.as n o t e d .  discontinued eight minutes  meso-  seconds  several  shunts were  the  and The  followed  warm d r i p  another  slowly  of  seconds  seconds more,  arterio-venous  sumption of was  seconds  process  cc  subcutaneously,  commenced f o r t y - f i v e  and i n f i f t e e n  w i t h 0.15  rat  i n twenty-six minutes.  immediate r a p i d thawing w i t h Vascular  female Wistar  solution administered  preparation tip  was  nonpregnant  areas  In three and no The  thawing.  thawwas  were minutes re-  preparation  86 Animal  4  (Test) A normal adult nonpregnant  w e i g h i n g 190 grams was 0.19  cc of  0.19 mg.,  administered was r e a d y  by  in fifteen  was a p p l i e d t o  later  the  injection.  and a h a l f  a selected  with  animal  cc o f 0.6$ nembutal  subcutaneous  The  minutes,  area  of  preparation  and the  the  in  of  t h a w i n g and  spread  rapidly.  after  thawing the  stasis  became  ninety  seconds  generalized  over  the  injured  blood  f l o w was n o t e d .  after  t h a w i n g no f u r t h e r  p r e p a r a t i o n was Animal  5  Stasis  area.  preparation  the  thirty  was  later  anaesthetized  0.05  cc were  on the  The c o l d  minutes and  tip  Wistar  with 0.15  the  seconds  cc  repeated,  and  By a n o t h e r  two m i n u t e s  within  injured  area.  the  in  was  selected  started, the  of  microscope stage a p p l i e d to  a  which  stasis  spread  h a d become  By seven minutes  four  thirty  by immediate r a p i d thawing.  stasis  rat  subcutaneously.  mesoappendix w i t h f r e e z i n g f o r  followed  the  female  solution administered  was r e a d y  more m i n u t e s .  seconds,  in  discontinued.  160 grams  Eleven minutes  of  By  (Control)  0.6$ nembutal  area  with-  fairly  and a h a l f  was o b s e r v e d ,  A normal adult nonpregnant weighing  started  No r e s u m p t i o n  Within five change  cold  mesoappendix  thirty  seconds  seconds.  was  solution  with freezing for thirty  rat  solution containing  and t w e n t y - t w o m i n u t e s w i t h 0.15  Wistar  subcutaneously  a diluted Priscoline  anaesthetized  tip  injected  female  Within  rapidly. generalized after  thaw-  87 i n g no f u r t h e r  c h a n g e was n o t e d ,  was d i s c o n t i n u e d . was  No r e s u m p t i o n o f  preparation  any  circulation  noted.  Animal  6  (Control)  weighing  A normal adult  nonpregnant  220 grams was  anaesthetized  0.6$ nembutal were  stage  applied  to  freezing  a selected thirty  thawingjstasis process  Thirty ured  spread  changes Animal  7  d u r i n g the several  i n vascular  was n o t e d ,  areas  of cc  on the cold  micro-  tip  was  with  seconds  beds,  sixty  throughout  stasis.  after  cc  0.05  thirty  following  rat  and seconds.  the  inj-  No r e s u m p t i o n  p r e p a r a t i o n was  t h a w i n g w i t h no  of  of  dis-  further  observed.  (Test)  weighing  170 grams was i n j e c t e d  cc o f  0.17 mg., aesthetized  a diluted Priscoline  w i t h 0.15  with freezing for  a  the  rat  with  a n i m a l was  area  seconds.  of  an-  solution.  i n nine minutes,  selected  thirty  Wistar  solution containing  cc of 0.6$ nembutal  was r e a d y  was a p p l i e d t o  female  subcutaneously  and i n n i n e t e e n minutes  The p r e p a r a t i o n point  and the  Within  A normal adult nonpregnant  0.17  0.15  the mesoappendix  and the  seven minutes  being  of  seconds.  later  area were  continued  area  later,  d e v e l o p e d i n two c a p i l l a r y  seconds  circulation  with  The p r e p a r a t i o n was r e a d y  twelve minutes  for  female W i s t a r  s o l u t i o n and i n seven minutes  repeated.  scope  the  and the  the  and  the  cold  mesoappendix  Capillary  stasis  88 developed w i t h i n capillary  beds.  resumption  static.  being  cc of  two m i n u t e s  nonpregnant  with 0.15  was r e a d y  twelve minutes,  and the  selected  the  area  of  seconds  Vascular  the thawing,  by another  subcutaneously  venous  venous  a few more  on the  later  the  animal solution  microscope stage  t i p was  sixty  seconds  channel were  seconds three  a p p l i e d to  venules  became  for  beds  and  A minute  By s i x more  a r a t i o n was d i s c o n t i n u e d t e n m i n u t e s  a  thawing,  capillary  static,  in  thawing.  after  affected.  channel flushed free. small  with  cc o f 0 . 6 $ nembutal  cold  rat  solution containing  by immediate r a p i d  started sixty  Wistar  mesoappendix w i t h f r e e z i n g  followed  stasis  one l a r g e  minutes  and the  after  later  prep-  thawing.  9 (Control) A normal adult nonpregnant  weighing  170 grams was  0.6$ nembutal and  after  female  and t w e n t y - e i g h t m i n u t e s  preparation  Animal  and  no  noted.  a diluted Priscoline  was a n a e s t h e t i z e d  the  several  had occurred,  160 grams was i n j e c t e d  0.16 mg.,  and  two  (Test)  weighing  thirty  seconds  By another  changes  A normal adult  The  thawing i n  was d i s c o n t i n u e d t e n m i n u t e s  w i t h no f u r t h e r  0.16  of  ninety  o f any c i r c u l a t i o n  preparation  8  seconds  By another  b e d s h a d become  Animal  thirty  solution,  i n ten minutes  0.05  female  anaesthetized administered  w i t h 0.15  on the  rat  cc  of  subcutaneously,  cc were r e p e a t e d .  t i o n was m o u n t e d a n d r e a d y  Wistar  The  prepara-  microscope stage  in  89 six minutes,  and  the  ected  the  mesoappendix  area of  seconds, ive  area within  but  stasis thirty  seconds  after  was  seconds  of  of  still  any o f  could not  be  are  Summary o f No.  It  not  rapid  thawing.  By  fairly the  Onset  a  sel-  for  thawing. the  injured another flow,  circulation.. four  that  and  The  a half  such a  minutes  preparation  and  summary  thirty Extens-  good b l o o d  physiological,  i n c l u d e d i n the  Results  to  throughout  w o u l d seem  considered  applied  with freezing  died while being observed thawing.  ations  developed  t h e r e was  no r e s t o r a t i o n  animal  tip  f o l l o w e d by immediate  vascular  sixty  cold  the  observ-  table.  with Priscoline  Stasis  Spread  Generalized  Flow  Resumed  None  ///  Rapidly  pit  t  45"  Slowly  3»  None  Test  30"  Rapidly  90"  None  5  Control  30"  Rapidly  6  Control  30"  Rapidly  2»  None  7  Test  30"  Slowly  2»  None  8  Test  60"  Slowly  None  9  Control  30"  Rapidly 30" (Physiological State  1  Test  30*'  2  Test  90"  3  Control  4  Slight  f o r 3&»  None  t None Unlikely)  Comments T$ie a v e r a g e to  occur  value  after  w i t h the  difference  time w i t h the  rapid  t h a w i n g was  controls  between  the  was  34".  test  runs  test  animals  for  48»», w h e r e a s t h e There and  the  was  stasis same  l i t t l e  controls  in  90 the  length  stasis  to  of  develop  circulation with  the  would In  test  seem  that  the  parenterally weight.  methods those  onset the  used  project  the  Fourteen  When r e a d y  selected  after  stage,  area  mg p e r  following  of  the  imply  as  in  is  the  the the  by resumption  of  It  in  value gener-  of  that  be  given  these  tests  the  term  implied  : i  the  than  merely  elsewhere.  employed,  a l l being  gram w e i g h t  with  with  "general  throughout rather  help-  His  identical  a  stated  was  amount on  applied  with freezing  by immediate  rapid  normal  range.  a n i m a l was m o u n t e d  cold point  the  is  s t u d e n t who w a s  stasis  200  tested  k i l o g r a m body  time b a s i s .  mesoappendix  followed  or  results  Note  14.  anaesthetizing  and  so.  P r i s c o l i n e was  could not  solution,  seconds,  effected  the  2.0  animals were  Wistar rats  0.6$ nembutal microscope  area  only  significant  stasis,  whole mesoappendix,  injured  occurred  of  Priscoline  H i s use to  Resumption  bed.  on a p a r t  elsewhere.  of  male  o f no  tests,  observations  vessels  thirty  with  vascular  consistently  by a s e n i o r m e d i c a l  here used  adult  not  of vascular  Animal  is  the  area.  developed  vascular  stasis"  within  but  i n a dosage of  the  generalized  P r i s c o l i n e was  form after  and  had  following  performed  ing with  for  injured  stasis  A summary o f  table  were  the  Observations In  took  animals,  protecting  Further  i t in  after  d e l a y i n g the  ally  in  time  of the  to  a  for  thawing  warm R i n g e r - L o c k e g e l a t i n -  91 glucose  drip  ulatory  channels  Animal  1  solution.  The r e a c t i o n s  were noted  cc o f  c o n t a i n i n g 0.4 m g .  o f nembutal were  expired before  Animal  2  It  containing  started  with 0.2  sixty  involve  seconds  five  minutes.  c o u l d be  the  done.  or  solution  and e i g h t minutes  later  was  cc o f nembutal.  Vascular  after  the  s i x other  thawing,  capillary  cc o f nembutal  started  in thirty  ing  involve  three  field  in five  or  Animal. 4  i t  stasis  which  fields  stasis  an-  spread  within  or  resumption  noted.  Stasis  injured  sub-  (Control)  w i t h 0.20  stasis  but  injected  An a n i m a l w e i g h i n g 200 grams was  to  0.2  cc o f a d i l u t e d P r i s c o l i n e  T h e r e was no g e n e r a l  circulation 3  0.2  0.4 m g . ,  aesthetized  Animal  later  subcutaneously,  any t e s t i n g  sol-  (Test)  cutaneously with  of  injected  a diluted Priscoline  An a n i m a l w e i g h i n g 200 grams was  four  was  Thirteen minutes  injected  animal  to  circ-  outlined below:  a n i m a l was 190 g r a m s .  subcutaneously with 0.2  cc  the  (Test) Weight of  ution  as  of  injected seconds  quarters  of  minutes.  resumption of flow  anaesthetized  subcutaneously. after the  thawing,  capillaries in  T h e r e was no  noted  spread-  i n any  the  general  vessel.  (Test) An a n i m a l w e i g h i n g 190 grams was i n j e c t e d  cutaneously with 0.20 c o n t a i n i n g 0.4 m g . ,  cc and  of  a diluted Priscoline  ten minutes  later  i t  was  subsolution anaesth-  92  etized  w i t h 0.20  seconds  after  capillaries stasis  or  Animal  5  cc o f nembutal.  thawing,  i n the  with  injured  resumption  of  Stasis  spread  to  area.  of  minutes. flow  Animal  the  6  at  Stasis  thawing,  was  developed  spreading  c a p i l l a r i e s i n the general  anaesthetized  to  frozen  stasis,  within  involve area  three  in  and no  four  resumption  (Test) a n i m a l w e i g h i n g 200 grams was  Priscoline  stasis  general  noted.  cutaneously  was  after  T h e r e was no  An  it  the  circulation.  cc o f nembutal.  seconds  quarters  of  half  (Control)  0.20  twenty  involve  ninety  T h e r e was no  An a n i m a l w e i g h i n g 190 grams with  began  with  0.20  containing 0.4  anaesthetized was  which  cc o f  time  T h e r e was no  spread  and no  resumption of  Animal  7 (Control)  mg.,  of  and twenty minutes cc  of nembutal.  two m i n u t e s  two s m a l l  stasis,  later No  following  c a p i l l a r i e s became the  sub-  a diluted solution of  w i t h 0.20  observed f o r  injected  thawing,  static.  no g e n e r a l i z e d  c i r c u l a t i o n i n the  affected  stasis, channels.  An a n i m a l w e i g h i n g a p p r o x i m a t e l y 200 grams anaesthetized  w i t h 0.20  before  i t  could  Animal  8  (Test)  cc  be mounted  of nembutal, on the  microscope  An a n i m a l w e i g h i n g 210 grams cutaneously  w i t h 0.20  cc  of  but  was  i t  was  expired  stage.  injected  a diluted Priscoline  subsolution  93 containing  0.4 m g . ,  animal  anaesthetized  was  administered in  ninety  and t w e l v e m i n u t e s with  0.20  subcutaneously.  seconds  quarter  of  the  general  stasis  after  to  c a p i l l a r i e s i n the  the  cc of nembutal  Vascular  thawing,  later  stasis  involve  injured  and no r e s u m p t i o n o f  also  developed  about  one  area.  No  circulation  was  noted. Animal  9  (Control) A n a n i m a l w e i g h i n g 210 grams was  w i t h 0.17  cc of nembutal  injection, peated. ing  and  spread  injured  10  started  to  area.  circulation Animal  administered by  and t w e n t y m i n u t e s Stasis  later  thirty  involve  half  No g e n e r a l  was  anaesthetized  0.07  seconds of  stasis  the  subcutaneous  after  vessels  developed  the  then  the  slowed,  resumed  Animal  11  in  the any  Some o f cells  cc o f nembutal. and  spread  to  the  vessels  stopped  for  later  Stasis  involve  injured area.  clumped together,  and then  sub-  and twenty m i n u t e s  w i t h 0.22 seconds,  injected  a diluted solution of  c a p i l l a r i e s i n the  stasis.  stasis,  ulation  cc of  c o n t a i n i n g 0.4 m g . ,  in thirty  no g e n e r a l into  0.20  anaesthetized  of  thaw-  (Test)  Priscoline  third  the  noted.  cutaneously with  was  re-  or resumption of  A n a n i m a l w e i g h i n g 200 grams was  it  cc were  began and  one  There to  go  the  circ-  a few seconds,  flow.  (Control)  A n a n i m a l w e i g h i n g 200 grams was  was  anaesthetized  and  94 w i t h 0.20 Stasis volve  cc o f nembutal  developed i n almost  Animal  12  administered  thirty  a l l the  seconds,  spreading  c a p i l l a r i e s i n the  cutaneously mg.  w i t h 0.20  of  the  of  15  and  i n the  seconds of  the  Stasis  spread  injured  cc  of nembutal.  after  thawing,  vessels  i n the  to  injured  or  to  area.  also.  any r e s u m p t i o n  in  or  within three  thirty quarters  anaesthetized in forty-five  involve  nine  tenths  No g e n e r a l  stasis  circulation.  (Control)  cc o f nembutal  injection.  the  was  Two p e t e c h i a l  An a n i m a l w e i g h i n g 190 grams  after  was  started  spreading  developed,  with 0.20  it  administered  started involve  containing  later  also  sub-  area.  Stasis  were produced  14  area.  (Control)  hemorrhages  Animal  in-  injected  minutes  An a n i m a l w e i g h i n g 190 grams w i t h 0.20  was  cc o f nembutal  injection.  thawing,  vessels  Animal  frozen  a diluted solution  and f i f t e e n  w i t h 0.20  subcutaneous  seconds  cc o f  of P r i s c o l i n e ,  anaesthetized by  to  (Test)  An a n i m a l w e i g h i n g 200 grams  0.4  subcutaneously.  Stasis  thawing,  vessels  any r e s u m p t i o n  started  Summary o f R e s u l t s  with  after  to  injured i n the  anaesthetized  administered by  spreading  i n the  was  forty-five  involve  area.  three  seconds  quarters  No g e n e r a l  c i r c u l a t i o n was  Priscoline  subcutaneous  stasis  noted.  of  95 Onset  No.  Stasis  Spread  observation -  during  plow  Generalized no  Resumed  results  1  Test  Died  2  Test  60"  3  Control  30"  4  Test  90"  5  Control  30"  6  Test  120"  7  Control  Died  8  Test  90"  9  Control  30"  10  Test  30"  11  Control  30"  12  Test  30"  75$ o f  vessels  None  None  13  Control  45"  90$ o f  vessels  None  None  14  Control  45"  75$ o f  vessels  None  None  Comments  on  the to  70",  stasis  just  than  observer  of  None  None  caps  None  None  4'  None  None  None  None  during  caps  50$  4«  only  observation -  25$ o f of  1/3  caps  vessels of  caps  Rapid  of on  stasis the  no  results  None  None  None  None  None  None  General  twice  as  it  was  these  in  Its  affected  vessels  regarded  as  being  in  None  the  to the  the  onset  noted.  definite  of  the  extent  as  of  However,  of  the  treated  tested and  in  stasis  To q u o t e  stasis,  or  control  whereas  delay  No r e s u m p t i o n  enough  the  Priscoline  controls.  "Priscoline  extent. was  in  in  35" l a t e r ,  Also  less the  thawing  appeared  long.  tests, in  after  average  Priscoline  some d e l a y i n g  lessening  2 small  consistently  animals  in  5«  75$ i n  75$ i n  onset  runs,  was  None  in  50$ o f  occurred  test  None  beds  Observations  The animals  5-6  the resulted  in  flow  results  consistent  some in  the  are  not  enough  to  96 be r e a l l y  significant." Benadryl  Experimental  observations  Benadryl mg.  per  (10 mg. p e r  was p r e p a r e d this  0.5  cc  cc)  by p l a c i n g  way,  from  Ringer-Locke  0.2  Animal  (Control)  1  A normal adult  cc.  microscope selected  later  stage  area  per  of  stasis  preparation  the  of  water. about  size  injection.  a  for  The p H o f  s o l u t i o n was  a value of  7.0.  female  Wistar  w i t h 0.1  rat  cc  was n o t  (Test)  0.05  cold  p o i n t was  forty  seconds  and spread  of  injected. the  a p p l i e d to  after  a  for  thawing  somewhat,  a v e r y good one,  but  and f u r t h e r  the obs-  recorded.  A normal adult nonpregnant 110 grams  cc were  mesoappendix w i t h f r e e z i n g  appeared  Animal  0.55  dilution  p r e p a r a t i o n was m o u n t e d o n  Within  were n o t  weighing  cc  of reasonable  a further  the  ervations 2  19.8  animal ranged  anaesthetized  and the  seconds.  vascular  0.50  solution administered intramuscularly,  i n eight minutes  Seven minutes  into  nonpregnant  w e i g h i n g 130 grams was 0.6$ nembutal  to  of  and a 1:100  gelatin-glucose drip starting  level  10 c c S t e r i - v i a l s o f  a subcutaneous  before  thirty  a dosage  were u s e d ,  volume o f dose  adjusted  and  at  amount w h i c h w o u l d be  absorption the  tested  Benadryl  k i l o g r a m body w e i g h t .  Benadryl  In  was  with  was  cc o f a Benadryl  injected  female  Wistar  subcutaneously  s o l u t i o n c o n t a i n i n g 0.055  rat  with mg.,  97 and t w e n t y - s e v e n m i n u t e s etized with  0.1  In  ready  tip  of  the  cold  mesoappendix  Within one  the  thirty  capillary noted. channel minute  bed.  only  circulation  minute  and  blood  excellent.  field  for  Animal  5  0.65  nine  125 grams  large In  i n the  rest  of  was  a good one.  and  a half  was  venous  another and  excellent.  the  rather  four  closed off,  After  unchanged,  mesoappendix  small,  and  was  lightly but  the  The p r e p a r a t i o n  minutes  after  was  thawing.  was  nonpregnant injected  female  Wistar  subcutaneously  a s o l u t i o n c o n t a i n i n g 0 . 0 6 5 mg. o f  Thirty-eight  minutes  later  the  cc of 0.6$ nembutal  cutaneously,  and i n  mounted  and r e a d y  tip  a p p l i e d to  was  one  in  (Test)  cc o f  w i t h 0.1  was  was n o t e d  ecchymosis  conditions remained  A normal adult weighing  area  seconds.  making observation d i f f i c u l t ,  testing  discontinued  Slight  was  area  thawing  circulation.  The a n i m a l was  anaesthetized,  thirty  after  beds were  administered  preparation  stasis  and a h a l f  injured  three minutes flow  thawing  involved.  another the  after  anaesth-  a selected  freezing for  capillary  i n the  the  a p p l i e d to  and resumed  four  solution  B y two m i n u t e s  another  opened  was  with  beds were In  a n i m a l was  seven minutes  seconds  capillary  the  cc of 0 . 6 $ nembutal  intramuscularly. and  later  a n i m a l was  microscope  a selected  with Benadryl.  anaesthetized  solution administered  sixteen minutes  on the  rat  area  the  preparation  stage. of  the  sub-  The  was  cold  mesoappendix  98  with freezing for seconds a  after  couple  seconds three  of  larger  flow  capillary  in  four  minutes  after  opening  and c l o s i n g  large the  While and  beds  admittedly  area  was  beds were one  reopened  t h e r e were pieces  of  venous  enormously better  the  injured vein  by Benadryl.  the  blood  A minute  channels than  Benadryl.  wall,  By f o u r t e e n  was p r e s e n t ,  Animal  4  / weighing  the  previous i n the later  other two o f  be  the  after  was  off,  the  blood to reduced  thawing  Normal  A normal  adult  120 grams  cc  was  nonpregnant  female  anaesthetized  solution administered  administered  the  vasomotor  discontinued.  Wistar  w i t h 0.1  cc  rat of  intramuscularly,  subcutaneously.  beds  expected  tendencies  preparation  two  capillary  being greatly  minutes  been  (Control)  0.6$ nembutal and 0 . 0 5  and  By f o u r  One h a d of  s i t u a t i o n was e s s e n t i a l l y u n c h a n g e d . tone  sixty  closed  could  adhesive  in  normally.  small  cohesive properties  and e s p e c i a l l y t h e i r  sixty  channel had the  in  stasis  in stasis.  a couple  the  to  by another  and flowed  impression  stick  observed  By another  flowing.  treatment with  cells,  but  large  more,  without p r i o r of  was  intermittently for  two m i n u t e s  a few s h o r t  this  venules,  thawing the  c h a n n e l was f r e e  capillary  stasis  thirty  t h e s e was r e s u m e d w i t h  capillary  In  Within  beds r e m a i n i n g .  seconds  minutes.  seconds.  rapid thawing,  the  the  thirty  In  nine  99 minutes  the  microscope selected thirty there in  stage.  area  of  the  the  slight  injured area.  stasis  venous  the  side  of  venules.  other was  as  runs.  By f i v e  stasis,  and a m i n u t e  that  in stasis  flowed  apparently  preparation  after  rapidly  to  to  later  quite  large  toward  the  large  preparation  i n comparison w i t h  minutes  after  thawing,  channels  one  large  with venous  continuously,  normally.  site  channels  I n v o l v e the on t h i s  one  three  no  steadily progressing  venous  thawing  seconds  involve  two more m i n u t e s  Inflicted  for  rapid  sixty  the a  ecchymosis at  started  quite minimal  observed i n l a r g e r  was  of  circulation  The i n j u r y  regarded  seconds  Stasis  After  had reopened,  on  t i p was a p p l i e d t o  amount  t h a w i n g and s p r e a d beds.  and r e a d y  mesoappendix w i t h f r e e z i n g  By t h i r t y  a very  capillary  was  was m o u n t e d  The c o l d  seconds. was  after  preparation  vasomotion  intermittent channel,  reopened  Three minutes  was d i s c o n t i n u e d w i t h no f u r t h e r  the  and  later  the  changes  noted. Animal  5  (Test) A normal adult  weighing cc  of  140 grams  was i n j e c t e d  female  0.15  applied  to  the  cc o f 0.6$ nembutal  cutaneously, was r e a d y  later  and  on the  a n i m a l was  microscope stage. area  of  rat  the  later  0.7  Benadryl. anaesthetized  solution administered  t h i r t e e n minutes  a selected  Wistar  subcutaneously with  a s o l u t i o n c o n t a i n i n g 0.07 mg. of  Twnety-one minutes with  nonpregnant  the  sub-  preparation  The c o l d mesoappendix  tip with  was  100 f r e e z i n g f o r t h i r t y seconds, warm d r i p s o l u t i o n . there was  and r a p i d l y thawed w i t h  T h i r t y seconds a f t e r thawing  much c a p i l l a r y s t a s i s present,  and venous  o b s t r u c t i o n i n a f a i r l y l a r g e c i r c u m s c r i b e d area w i t h i n the i n j u r e d f i e l d .  A f t e r another  utes the s i t u a t i o n was  unchanged.  f i v e and a h a l f minActually  these  o b s e r v a t i o n s should be discounted because the whole mesoappendix was  more or l e s s i n v a s c u l a r s t a s i s .  Animal 6 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 0.6$ and  150  grams was  a n a e s t h e t i z e d w i t h 0.15  nembutal s o l u t i o n a d m i n i s t e r e d the p r e p a r a t i o n was  The c o l d t i p was  ready  cc of  subcutaneously,  eleven minutes l a t e r .  a p p l i e d to a s e l e c t e d area of the  mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds w i t h immediate r a p i d thawing a f t e r w a r d . seconds of thawing,  Within  c a p i l l a r y s t a s i s was  which spread r a p i d l y and i n another  thirty  observed  t h i r t y seconds f i f t y  p e r c e n t of the v e s s e l s i n the i n j u r e d area were i n v o l v e d . By another minute no r e v a s c u l a r i z a t i o n was the s t a t i c channels. p r e p a r a t i o n was  F i v e minutes a f t e r t h i s  discontinued.  channels were f l o w i n g w e l l , area was  unchanged.  observed  A few  in  the  arterio-venous  but g e n e r a l l y the  injured  Ninety p e r c e n t of the v e s s e l s  i n the i n j u r e d area were i n s t a s i s when the p r e p a r a t i o n was  discontinued.  101 Animal 7  (Teat) A normal a d u l t nonpregnant female W i s t a r r a t  weighing 0.6  120 grams was  i n j e c t e d subcutaneously  cc of a s o l u t i o n c o n t a i n i n g 0.06  mg.  of Benadryl.  T h i r t y - t h r e e minutes l a t e r the animal was w i t h 0.15  cc o f 0.6$  subcutaneously, was  nembutal s o l u t i o n  with  anaesthetized  administered  and w i t h i n nine minutes the p r e p a r a t i o n  ready f o r t e s t i n g .  The c o l d p o i n t was a p p l i e d  to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds and thawed r a p i d l y afterward.  immediately  W i t h i n t h i r t y seconds of thawing three  c a p i l l a r y beds had c l o s e d o f f .  I n another  thirty  seconds one l a r g e venous channel became s t a t i c , opened up again f i v e minutes l a t e r .  but  Nine minutes  a f t e r thawing one of the s t a t i c c a p i l l a r y beds opened up.  There was  no f u r t h e r change i n two more minutes  o b s e r v a t i o n and the p r e p a r a t i o n was  discontinued.  Animal 8 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 120 grams was 0.6$  a n a e s t h e t i z e d w i t h 0.15  nembutal s o l u t i o n a d m i n i s t e r e d  and the p r e p a r a t i o n was minutes.  cc ©f  subcutaneously,  ready f o r t e s t i n g i n twenty  The c o l d p o i n t was  a p p l i e d to a s e l e c t e d  area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y W i t h i n t h i r t y seconds of r a d p i d thawing, venous s t a s i s was  starting,  capillary  seconds. and  and i t spread r a p i d l y t o  c l o s e o f f approximately one h a l f of the c i r c u l a t i o n i n  102 another t h i r t y seconds.  By another minute o n l y  two  a r t e r i o - v e n o u s shunts were f l o w i n g i n the i n j u r e d a r e a . In another two and a h a l f minutes no f u r t h e r change i n the c i r c u l a t i o n was noted,  and the b l o o d f l o w i n the  r e s t of the mesoappendix was normal.  The p r e p a r a t i o n  was  after  d i s c o n t i n u e d s i x and a h a l f minutes  thawing.  Animal 9 (Test) A normal a d u l t nonpregnant weighing 115 grams was  female Wistar r a t  I n j e c t e d subcutaneously w i t h  0.575 cc o f a s o l u t i o n c o n t a i n i n g 0.0575 mg. Benadryl,  © f  and forty-tw© minutes l a t e r the animal  a n a e s t h e t i z e d w i t h 0.15  cc o f 0.6$  administered subcutaneously. ready i n e i g h t minutes  nembutal  was  solution  The p r e p a r a t i o n was  and the c o l d p o i n t was  applied  t© a s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  Hyperemia was noted t h i r t y  a f t e r r a p i d thawing,  and i n another t h i r t y  seconds  seconds  v a s c u l a r s t a s i s i n the i n j u r e d area spread to i n v o l v e two or three c a p i l l a r y beds.  A f t e r another s i x  minutes no f u r t h e r change was motion was  noted.  A c t i v e vaso-  apparent i n a l l channels t h a t were not  N© channels appeared  to reopen,  static.  the s t a t i c ones b e i n g  of the order of c a p i l l a r i e s and p r e v e n u l e s ,  forming  approximately f o r t y p e r c e n t of the v e s s e l s w i t h i n the i n j u r e d area.  The c i r c u l a t i o n i n the r e s t of the  mesoappendix was normal. thawing,  By n i n e minutes  ©ne c a p i l l a r y bed reopened,  d i d l i k e w i s e a minute l a t e r .  following  and another bed  The p r e p a r a t i o n was  103 d i s c o n t i n u e d f o u r minutes l a t e r . Summary o f Observations w i t h Benadryl Onset S t a s i s  Ho.  Spread  (0.5 mg/Kg)  Generalized  Plow Resumed  1  Control  40"  Yes  2  Test  30"  Yes  None  //  3  Test  30  Yes  None  ///  4  Control  30-60"  Rapid  5  Test  No r e s u l t s - Whole mesoappendix i n  6  Control  30"  7  Test  30"  8  Control  30"  9  Test  60"  Incomplete  Almost  3 Cap beds[  3 Cap bedsi  ?  Incomplete  Rapid  Rapid  None  Not  stasis None  ///  None A-V shunts None  None  ///  D i s c u s s i o n o f Observations Benadryl d i d n o t appear to have any great  effect  on the time r e q u i r e d f o r v a s c u l a r s t a s i s t o develop as compared w i t h c o n t r o l animals, appeared to l i m i t the spread o f the s t a s i s .  but i t There  was a l s o evidence o f i n c r e a s e d resumption o f flow i n the s t a t i c v e s s e l s .  However, there are o n l y f o u r  and f o u r c o n t r o l animals.  test  These numbers are f a r  too few f o r r e a l l y s i g n i f i c a n t c o n c l u s i o n s  to be drawn.  F u r t h e r t e s t i n g w i t h Benadryl i s i n d i c a t e d , e s p e c i a l l y w i t h a h i g h e r dosage level« A c t i v e vasomotion was found to be a prominent f e a t u r e i n the re-opened v e s s e l s i n the i n j u r e d area, i n d i c a t i n g t h a t the f r e e z i n g i n j u r y was unduly m i l d o r  104 t h a t the a n t i h l a t a m i n i c a c t i o n on the e n d o t h e l i a l must have been e f f e c t u a l to a c e r t a i n  cells  degree.  C o n t r o l animal number f o u r r e q u i r e s some comment i n t h a t the reopening of channels, as marked "?", meaning d o u b t f u l , might be e x p l a i n e d by the f a c t t h a t without doubt the i n j u r y by f r e e z i n g to t h i s p r e p a r a t i o n was r e a l l y minimal both as regards to d u r a t i o n and In s p i t e of such e x p l a n a t i o n , good r e s u l t s ,  and such  extent. apparent  i t would not seem j u s t i f i a b l e to c r e d i t  the a n t i h i s t a m i n e used w i t h such g r e a t e f f e c t u n l e s s the t e s t s were repeated. F u r t h e r Experimental Observations w i t h Benadryl I n the f o l l o w i n g t e s t s w i t h Benadryl, mg.  per k i l o g r a m body weight was  1.0  the dosage l e v e l  T h i s d i d not appear to have any adverse  used.  e f f e c t on the  animals. Animal 1 (Test) A normal a d u l t nonpregnant female Wistar r a t weighing 150 0.75  grams was  i n j e c t e d subcutaneously  cc of a s o l u t i o n c o n t a i n i n g 115 mg.  o f Benadryl,  and t h i r t y - f i v e minutes l a t e r the animal was w i t h 0.i?5 cc of 0.6$ cutaneously. minutes s t i l l  with  anaesthetized  nembutal s o l u t i o n a d m i n i s t e r e d sub-  Seven minutes l a t e r 0.05  ec and  five  l a t e r 0.025 cc o f nembutal were i n j e c t e d  a l s o subeutaneously.  The p r e p a r a t i o n was  ready  tw©  and a h a l f minutes a f t e r the l a s t nembutal i n j e c t i o n , and the c o l d p o i n t was  a p p l i e d to a s e l e c t e d area of the  105 mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. f o r one  s t a t i c c a p i l l a r y bed not near the i n j u r e d area,  no v a s c u l a r s t a s i s developed minutes o f thawing. thawing,  w i t h i n e i g h t and a h a l f  By eleven and a h a l f minutes o f  t h i s c a p i l l a r y bed was  i s again to be noted was  Except  open and f l o w i n g .  It  that t h i s p a r t i c u l a r c a p i l l a r y  w e l l o u t s i d e the l i m i t s o f the f r e e z i n g I n j u r y .  By f o u r t e e n and a h a l f minutes o f thawing there was f u r t h e r change, The  bed  and the p r e p a r a t i o n was  e f f e c t of the Benadryl,  i f any,  t h i s i s o l a t e d v e s s e l cannot be  no  discontinued.  on the s t a s i s i n  explained.  Animal 2 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 150  grams was  a n a e s t h e t i z e d w i t h 0.15  0.6%  nembutal s o l u t i o n ,  0.05  cc were i n j e c t e d ,  aneously.  ©f  and i n twenty minutes a f u r t h e r both b e i n g administered  The p r e p a r a t i o n was  t e n minutes,  ce  ready w i t h i n  and the c o l d p o i n t was  subcutanother  a p p l i e d to a s e l -  ected area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  There was  s t a s i s w i t h i n t h i r t y seconds  of thawing which spread r a p i d l y . the s t a s i s was  still  spreading,  h a l f minutes of thawing,  By another  minute  and w i t h i n one  and  o n l y the main a r t e r i o - v e n o u s  shunts were f l o w i n g w i t h i n the I n j u r e d area,  although  the c i r c u l a t i o n i n the r e s t o f the mesoappendix a p p a r e n t l y normal.  a  The p r e p a r a t i o n was  was  discontinued  three minutes l a t e r w i t h no apparent change b e i n g  noted.  106 There was no resumption o f b l o o d f l o w noted i n any s t a t i c channel. Animal 5 ( T e s t ) A normal a d u l t nonpregnant weighing 150 grams was 0.75  i n j e c t e d subcutaneously w i t h  cc o f a s o l u t i o n c o n t a i n i n g 115 mg.  and w i t h i n t h i r t y - t h r e e minutes e t i z e d w i t h 0.125  cc o f 0.6$  ed w i t h i n e i g h t minutes. i n two more minutes,  of Benadryl,  the animal was  nembutal  anaesth-  s o l u t i o n admin-  a f u r t h e r 0.04  i s t e r e d subcutaneously,  a  female Wistar r a t  cc b e i n g I n j e c t -  The p r e p a r a t i o n was  and the c o l d p o i n t was  ready  a p p l i e d t©  s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r  t h i r t y seconds.  In one and a h a l f minutes  after  r a p i d thawing there was noted the s t a r t of a q u i c k l y spreading s t a s i s throughout the I n j u r e d a r e a .  By  three and a h a l f minutes  static  vessels flushed clear, minutes more, seven minutes  of thawing,  one ©f the  but shut down a g a i n .  In three  another channel f l u s h e d c l e a r , still  l a t e r another channel f l u s h e d c l e a r  i n a d i f f e r e n t p a r t o f the i n j u r e d a r e a . e x t e n s i o n o f any s t a s i s was and a h a l f minutes  and  observed.  No  further  By twenty-one  a f t e r thawing l i t t l e f u r t h e r change  was noted and the p r e p a r a t i o n was d i s c o n t i n u e d . Animal 4 ( C o n t r o l ) A normal a d u l t nonpregnant weighing 120 grams was 0.6$ nembutal  female W i s t a r r a t  a n a e s t h e t i z e d w i t h 0.15  s o l u t i o n and i n e i g h t minutes  cc o f  another  107 0.025 cc were I n j e c t e d a l s o subcutaneously. f o u r minutes the p r e p a r a t i o n was p o i n t was  ready,  seconds of thawing there was the i n j u r e d a r e a .  spread noted by another more,  and the c o l d  a p p l i e d to a s e l e c t e d area of the mesoappendix  w i t h f r e e z i n g f o r t h i r t y seconds.  throughout  Within  Within  thirty  s t a s i s which spread There was  two minutes.  still  rapidly further  In three minutes  one of the main s t a t i c Venous channels opened and  resumed c i r c u l a t i o n a p p a r e n t l y n o r m a l l y . three minutes t h e r e was the p r e p a r a t i o n was a f t e r thawing,  By  another  no f u r t h e r change noted,  and  d i s c o n t i n u e d ten and a h a l f minutes  a t which time i t was  noted another  small  venous channel had reopened. Animal 5 ( T e s t ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 150  grams was  i n j e c t e d subcutaneously  cc o f a d i l u t e d s o l u t i o n c o n t a i n i n g >15  mg.  and t h i r t y - o n e minutes l a t e r the animal was w i t h 0.15  cc o f 0.6$  cutaneously, minutes l a t e r .  with  0.75  of Benadryl, anaesthetized  nembutal s o l u t i o n a d m i n i s t e r e d sub-  w i t h 0.04  cc more being i n j e c t e d ten  The p r e p a r a t i o n was  ready two minutes  a f t e r the second nembutal i n j e c t i o n and the c o l d p o i n t was  a p p l i e d to a s e l e c t e d area of the mesoappendix w i t h  f r e e z i n g f o r t h i r t y seconds. r a p i d thawing,  W i t h i n t h i r t y seconds o f  s t a s i s i n one venous channel was  which backed up i n t o the i n j u r e d a r e a .  In  noted  another  108 minute a second venous channel acted s i m i l a r l y ,  and  the same w i t h a t h i r d w i t h i n the next minute. f o u r and a h a l f minutes  a f t e r thawing  a j ? e a was p r a c t i c a l l y i n s t a s i s . observed to reopen.  By  the whole i n j u r e d  No channels were  The p r e p a r a t i o n was d i s c o n t i n u e d .  Animal 6 ( C o n t r o l ) A normal  a d u l t nonpregnant  weighing 140 grams was 0.6$  female W i s t a r r a t  a n a e s t h e t i z e d w i t h 0.15  ce of  nembutal s o l u t i o n a d m i n i s t e r e d subcutaneously,  the p r e p a r a t i o n was c o l d p o i n t was  ready w i t h i n twelve minutes.  and The  a p p l i e d to a s e l e c t e d area of the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds, r a p i d l y thawed.  S t a s i s was  t h i r t y seconds of thawing, by s i x and a h a l f minutes  and  then  observed to s t a r t w i t h i n  and i t spread s l o w l y so t h a t a f t e r thawing o n l y three ven-  u l e s and c a p i l l a r i e s were observed to be i n s t a s i s .  In  a l l p r o b a b i l i t y the f r e e z i n g i n j u r y was  By  too s l i g h t .  another minute there was no f u r t h e r change noted and the p r e p a r a t i o n was d i s c o n t i n u e d . Animal 7 ( T e s t ) A normal  a d u l t nonpregnant  weighing 135 grams was 0.65  female W i s t a r r a t  i n j e c t e d subcutaneously w i t h  cc of a d i l u t e d s o l u t i o n c o n t a i n i n g 0.13  Benadryl,  and i n twenty-four minutes  a n a e s t h e t i z e d w i t h 0.10 i n j e c t e d subcutaneously, i n seven minutes. a r a t i o n was  mg.  the animal  cc of 0.6$ nembutal  of was  solution  w i t h 0.025 cc b e i n g r e p e a t e d  In another f i v e minutes  ready and the c o l d p o i n t was  th© prep-  applied with  109 f r e e z i n g f o r t h i r t y seconds to a s e l e c t e d area o f the mesoappendix. beginning  W i t h i n t h i r t y seconds o f thawing,  c a p i l l a r y s t a s i s was observed  which i n another  t h i r t y seconds had become r a t h e r g e n e r a l except f o r l a r g e a r t e r i o v e n o u s channels.  By another  many s m a l l e r channels  b u t n o t much change  took p l a c e i n the next  reopened,  three minutes.  f i v e minutes  Twenty-six  minutes a f t e r thawing one l a r g e venous channel  that  appeared to be i n I r r e v e r s i b l e s t a s i s opened up and resumed a p p a r e n t l y normal c i r c u l a t i o n . t i o n was d i s c o n t i n u e d i n another  The p r e p a r a -  e i g h t minutes w i t h  l i t t l e f u r t h e r change except f o r some g e n e r a l  extens-  i o n o f the s t a s i s w i t h i n the i n j u r e d area o f the mesoappendix. Animal 8 ( T e s t ) A normal a d u l t nonpregnant female Wistar r a t weighing 140 grams was i n j e c t e d subcutaneously  with  0.70 cc o f a d i l u t e d s o l u t i o n c o n t a i n i n g 0.14 mg. o f Benadryl  and i n f o r t y - s e v e n minutes the animal  a n a e s t h e t i z e d w i t h 0.125 cc o f 0.6$ nembutal administered ready  subcutaneously.  i n another  was  solution  The p r e p a r a t i o n was  twenty minutes and the c o l d p o i n t was  a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds, thawing.  S t a s i s f i r s t appeared three and a h a l f  minutes a f t e r thawing, aries.  f o l l o w e d by immediate r a p i d  and i n only three small  I n three minutes more,  capill-  one o f t h e . c a p i l l a r i e s  110 ©pened up w i t h resumption ulation.  o f a p p a r e n t l y normal  By another f o u r minutes the s t a s i s  to i n v o l v e two  c a p i l l a r i e s connecting adjacent  venous shunts.  The p r e p a r a t i o n was  circspread arterio-  d i s c o n t i n u e d one  minute l a t e r w i t h no f u r t h e r change being noted. Animal 9 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 0.6$  140  grams was  a n a e s t h e t i z e d w i t h 0.15  nembutal s o l u t i o n a d m i n i s t e r e d  cc o f  subcutaneously,  w i t h 0.025 c c . being repeated i n e l e v e n minutes.  The  p r e p a r a t i o n was  the  c o l d t i p was  ready i n another three minutes and  a p p l i e d to a s e l e c t e d area of the mesoapp-  endix w i t h f r e e z i n g f o r t h i r t y seconds.  Within  t h i r t y seconds of thawing s t a s i s had s t a r t e d , spread r a p i d l y .  By another  v e s s e l s were observed f u r t h e r change was  s i x minutes a few l a r g e  to be slowing,  noted.  and i t  but r e a l l y  The p r e p a r a t i o n was  continued s i x and a h a l f minutes a f t e r  little dis-  thawing.  Animal 10 ( C o n t r o l ) JS normal a d u l t nonpregnant female Wistar r a t weighing 160 grams was 0.6$  a n a e s t h e t i z e d w i t h 0.15  nembutal s o l u t i o n a d m i n i s t e r e d  and was  ready on the microscope  three minutes.  The f i e l d was  f a t i n the mesoappendix, was  v i s a b l e t© warrant  The c o l d t i p was  cc of  subcutaneously,  stage w i t h i n twentysomewhat obscured  by  but enough o f the c i r c u l a t i o n  c o n t i n u i n g w i t h the p r e p a r a t i o n .  a p p l i e d to a s e l e c t e d area of the  Ill mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds,  and  r a p i d l y developing  seconds  ©f thawing.  stasis started within thirty  N© r e s t o r a t i o n o f f l o w was observed i n  any o f the v e s s e l s ,  and the p r e p a r a t i o n was d i s -  continued f i v e and a h a l f minutes a f t e r thawing. Animal 11 (Test) A normal a d u l t nonpregnant female Wistar r a t weighing 135 grams was i n j e c t e d subcutaneously  with  0.65 e c . o f a d i l u t e d s o l u t i o n c o n t a i n i n g 0.13 mg. ©f Benadryl, and i n t h i r t y - f i v e minutes the animal a n a e s t h e t i z e d w i t h 0.125 cc o f 0.6% nembutal administered ready lied  subcutaneously.  was  solution  The p r e p a r a t i o n was  i n eleven minutes and the c o l d p o i n t was appto a s e l e c t e d area ©f the mesoappendix w i t h  f r e e z i n g f o r t h i r t y seconds,  followed  immediately  afterward w i t h r a p i d thawing.  In t h i r t y  seconds  a f t e r thawing there was observed  beginning  s t a s i s of  the c i r c u l a t i o n i n small v e s s e l s which spread  rapidly  to become more o r l e s s g e n e r a l i n the i n j u r e d area w i t h i n f o u r and a h a l f minutes o f thawing. v e s s e l s re-opened to r e s t o r e c i r c u l a t i o n ,  No and the  p r e p a r a t i o n was d i s c o n t i n u e d two minutes l a t e r . Summary o f Observations No.  Onset S t a s i s  1  Test  None  2  C o n t r o l 30"  w i t h Benadryl  (1.0 mg./Kg)  Spread  Generalized  Flow Resumed  None  None  Yes ?  Rapidly  l£»  None  112 Summary (Cont'd) Ne.  Onset S t a s i s l£»  Spread  Flow Resumed  3  Test  4  Control 30  5  Test  6  Control 3 0  7  Test  30"  Rapidly  60  8  Test  3&»  None  None  //  9  C o n t r o l 30"  Rapidly  None  None  10  C o n t r o l 30"  Rapidly  60"  None  11  Test  Rapidly  n  30" a  30"  Rapidly  Generalized None  ///  Rapidly  /  Rapidly  4&"  Non©  Slowly  None  None  u  4^»  None  D i s c u s s i o n o f Observations Benadryl i n j e c t e d t o the extent o f 1.0 mg. p e r k i l o g r a m body weight had no apparent adverse e f f e c t s on the r a t s t e s t e d . From the somewhat a r b i t r a r y v a l u e s i n the above summary t a b l e ,  the b e n e f i c i a l e f f e c t s o f Benadryl  i n the t e s t animals were I n c o n s i s t e n t ,  and they  c o n f l i c t e d w i t h e q u a l l y i n c o n s i s t e n t values  i n the  controls. The p a r t i c u l a r v a s c u l a r area chosen f o r i n j u r y by a p p l i c a t i o n of the f r e e z i n g p o i n t ,  as w e l l as the  exact d u r a t i o n o f the f r e e z i n g are r a t h e r u n c o n t r o l l a b l e v a r i a b l e s e n t e r i n g i n t o c o n s i d e r a t i o n o f the s i g n i f i c a n c e o f these r e s u l t s .  P o s s i b l e r e p e t i t i v e t e s t s on twe  or more areas o f the same mesoappendix would be an improvement,  although the time f a c t o r and the v i a b i l i t y  113 of the p r e p a r a t i o n would be b i g o b s t a c l e s . I t would seem t h a t Benadryl i n the dosage used had no s i g n i f i c a n t i n f l u e n c e beyond the e r r o r s i n h e r e n t i n the experiment.  TMs  c o n c l u s i o n agrees w i t h that  o b t a i n e d above when the dosage o f 0.5 mg. was  per Kilogram  tested.  F u r t h e r Experimental Observations w i t h Benadryl The f o l l o w i n g t e s t s were performed the i n f l u e n c e o f Benadryl,  to determine  i n a dosage l e v e l ®f 1.5  per k i l o g r a m of body weight,  administered by  mg.  subcut-  aneous i n j e c t i o n one h a l f hour b e f o r e the o b s e r v a t i o n of a f r e e z i n g i n j u r y on the mesoappendix. are summarized i n t a b l e form a f t e r animal  The  results  10.  Animal 1 ( T e s t ) A normal  a d u l t nonpregnant  weighing 130 grams was  i n j e c t e d subcutaneously w i t h  cc o f a d i l u t e d s o l u t i o n c o n t a i n i n g 0.195  0.39  Benadryl, was  female W i s t a r r a t  of  and twenty-nine minutes l a t e r the animal  a n a e s t h e t i z e d w i t h 0.15  ution.  mg.  cc o f 0.6$  The p r e p a r a t i o n was  and the c o l d p o i n t was  nembutal  ready i n twelve  sol-  minutes,  a p p l i e d to a s e l e c t e d a r e a of  the mesoappendix w i t h f r e e z i n g i n j u r y f e r t h i r t y seconds.  W i t h i n s i x t y seconds of thawing,  v a s c u l a r s t a s i s was i n the b l o o d f l o w . ing.  One  starting,  a slight  r e a l l y j u s t a slowing  By another minute i t was  channel was  spread-  observed to ©pen and resume  114 circulation.  By another  f u r t h e r spread.  two minutes there was no  T h i s i s ah e x c e l l e n t area ©f the  mesoappendix w i t h many small c a p i l l a r y channels and the f a c t t h a t the s t a s i s was so l i m i t e d , together  with  a f a i r l y s t r o n g f r e e z i n g i n j u r y c e r t a i n l y makes the observed  facts significant.  A f t e r another two  minutes one l a r g e connecting v e i n was i n s t a s i s ,  and  by e i g h t minutes a f t e r thawing some s t a s i s was app^arent i n other p a r t s ©f the mesoappendix,  so t h a t  the p r e p a r a t i o n was d i s c o n t i n u e d . Animal 2 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 140 grams was a n a e s t h e t i z e d w i t h 0.15 cc ©f 0.6$ nembutal s o l u t i o n administered  subcutaneously,  and the p r e p a r a t i o n was ready on the microscope In seventeen minutes.  stage  The c o l d p o i n t was a p p l i e d  to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. seconds o f thawing,  Stasis started within thirty and spread r a p i d l y .  By three  minutes a f t e r thawing the s t a s i s was s l o w l y  extending  but was by no means becoming g e n e r a l i z e d .  Some  ecchymoses were p r e s e n t .  Two minutes l a t e r ,  cept f o r the a r t e r i o - v e n o u s channels, generalized, noted.  and no reopening  ex-  s t a s i s was  o f the channels  was  Pour minutes l a t e r the p r e p a r a t i o n was d i s -  continued. Animal 3 (Test) A normal a d u l t nonpregnant female Wistar r a t  115 weighing 130 0.39  i n j e c t e d subcutaneously  and f o r t y - f i v e minutes l a t e r the  a n a e s t h e t i z e d w i t h 0.15  c c . o f 0.6$  u t i o n administered subcutaneously. was  with  ce. o f a d i l u t e d s o l u t i o n c o n t a i n i n g 0.195  Benadryl, was  grams was  mg.  ©f  animal  nembutal  sol-  The p r e p a r a t i o n  ready f i f t e e n minutes l a t e r and the c o l d t i p was  a p p l i e d to a s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds, r a p i d thawing.  There was  seconds of thawing, s t a r t e d i n one  f o l l o w e d by  immediate  n© s t a s i s w i t h i n n i n e t y  but i n a minute more,  stasis  small venous channel o n l y .  Two  minutes l a t e r t h i s s t a t i c venous channel opened f o r h a l f i t s length, eonneeting  and then was  f l u s h e d f r e e by  arteriole-capillary tributary.  another two minutes there was  a  After  no f u r t h e r change.  The c i r c u l a t o r y system i n the whole I n j u r e d area functioning well. was  was  F i v e minutes l a t e r the p r e p a r a t i o n  d i s c o n t i n u e d w i t h no f u r t h e r changes b e i n g noted.  Animal 4 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 0.6$  135 grams was  nembutal s o l u t i o n ,  injection, microscope was  a n a e s t h e t i z e d w i t h 6.15  c c . of  a d m i n i s t e r e d by subcutaneous  and the animal was  mounted and ready ©n  stage i n eleven minutes.  the  The c o l d p o i n t  a p p l i e d to a s e l e c t e d area o f the mesoappendix  w i t h f r e e z i n g f o r t h i r t y seconds.  There was  stasis  116 s t a r t i n g w i t h i n t h i r t y seconds which spread  rapidly.  Tw©  still  and a h a l f minutes l a t e r the s t a s i s was  spreading,  but net y e t generalized.  I n two m©re  minutes the s t a s i s had become r a t h e r g e n e r a l i z e d through out the i n j u r e d area, any channels continued  and no r e v a s c u l a r i z a t i o n o f  was n o t i c e d .  The p r e p a r a t i o n was d i s -  three minutes l a t e r w i t h no f u r t h e r changes  observed. Animal S (Test) A normal a d u l t nonpregnant female Wistar r a t weighing 135 grams was i n j e c t e d subcutaneously  with  0.39 c c . ©f a d i l u t e d s o l u t i o n c o n t a i n i n g 0.195 mg. o f Benadryl,  and t h i r t y - e i g h t minutes l a t e r the  animal was a n a e s t h e t i z e d w i t h 0.15 c c . o f 0.6$ nemb u t a l s o l u t i o n administered  subcutaneously.  The  c o l d t i p was a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. o c c u r r e d i n s e v e r a l venous channels  i n the i n j u r e d  area w i t h i n s i x t y seconds a f t e r thawing. another  Stasis  In  t h i r t y seconds there was fio f u r t h e r spread  i n the s t a s i s ,  although a good s i x t y p e r c e n t o f the  v e s s e l s i n the i n j u r e d area were a f f e c t e d .  I n three  more minutes no resumption o f c i r c u l a t i o n i n any s t a t i c v e s s e l was observed, ued  and the p r e p a r a t i o n was d i s c o n t i n -  two minutes l a t e r .  Animal 6 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t  117 weighing  170 grams was a n a e s t h e t i z e d w i t h 0.20 c c . o f  0.6$ nembutal s o l u t i o n administered by subcutaneous injection,  and the p r e p a r a t i o n was ready i n t h i r t e e n  minutes.  Tbe c o l d p o i n t was a p p l i e d three minutes  l a t e r to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  Stasis  developed  w i t h i n t h i r t y seconds o f thawing and spread r a p i d l y . By another t h i r t y seconds, had developed.  s e v e r a l eechymotic  By n i n e t y seconds a f t e r  areas  thawing,  s t a s i s had become r a t h e r g e n e r a l i z e d except i n the l a r g e shunt channels. resumption  By two minutes l a t e r ,  o f c i r c u l a t i o n had been noted,  s t a s i s was s t i l l  extending.  no  and the  The p r e p a r a t i o n was  d i s c o n t i n u e d f o u r minutes l a t e r . Animal 7 (Test) A normal a d u l t nonpregnant female Wistar r a t weighing 140 grams was i n j e c t e d subcutaneously  with  0.42 c c . of a d i l u t e d s o l u t i o n c o n t a i n i n g 0.21 mg. o f Benadryl,  and twenty-eight minutes l a t e r the  animal was a n a e s t h e t i z e d w i t h 0.15 cc o f 0.6$ nembutal s o l u t i o n , a d m i n i s t e r e d by subcutaneous i n j e c t i o n . The p r e p a r a t i o n was ready i n nine minutes,  and the  c o l d p o i n t was a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. t h i r t y seconds o f r a p i d thawing, bed was In s t a s i s .  Within  one small c a p i l l a r y  By another two minutes the  s t a s i s had spread s l o w l y to i n v o l v e s e v e r a l o t h e r c a p i l l a r y beds.  In three more minutes there was n©  118 f u r t h e r involvement. was noted,  N© resumption o f c i r c u l a t i o n  and although  t h i s area was v e r y s i m i l a r t©  the subsequent c o n t r o l p r p a r a t i o n , stasis.  By three more minutes,  discontinued,  t h e r e was much l e s s the p r e p a r a t i o n was  w i t h no f u r t h e r change.  There was  no apparent d i f f e r e n c e from the f o l l o w i n g c o n t r o l r u n . Animal 8 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 120 grams was a n a e s t h e t i z e d w i t h 0.15 c c . ©f 0.6$ nembutal s o l u t i o n ,  administered  subcutaneously,  and  the p r e p a r a t i o n was ready i n t h i r t e e n minutes.  The  c o l d p o i n t was a p p l i e d to a s e l e c t e d area o f the  mesoappendix w i t h f r e e z i n g ,  f o l l o w e d by r a p i d thawing.  W i t h i n t h i r t y seconds o f thawing s t a s i s s t a r t e d which spread r a p i d l y , ecchymoses.  w i t h the development als© o f a few Two and a h a l f minutes l a t e r there were  no f u r t h e r changes i n the s m a l l e r s t a t i c v e s s e l s . was i n t e r e s t i n g to observe the events Some o f the l a r g e r channels e n t r a t e d c e l l u l a r sludge, free. occurred.  It  i n this preparation.  would f i l l up w i t h concand then r e p e a t e d l y f l u s h  By f i v e more minutes no f u r t h e r changes had The area o f s t a s i s was q u i t e complete  w i t h i n the i n j u r e d f i e l d ,  b e i n g a patchy  of the s m a l l e r v e n u l e s .  Two minutes l a t e r the  p r e p a r a t i o n was d i s c o n t i n u e d , s t a t i c v e s s e l s was  noted.  involvement  and no reopening  o f the  119 Animal 9 (Test) A normal a d u l t nonpregnant female W i s t a r r a t weighing 130 grams was i n j e c t e d subcutaneously w i t h 0.39 cc o f a d i l u t e d Benadryl s o l u t i o n c o n t a i n i n g 0.195 mg.,  and t h i r t y - o n e minutes l a t e r the animal was  a n a e s t h e t i z e d w i t h 0.15 c c . o f 0.6$ nembutal administered by subcutaneous i n j e c t i o n . a r a t i o n was ready i n s i x t e e n minutes,  solution  The prepand the c o l d  p o i n t was a p p l i e d t o a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  There was  no s t a s i s p r e s e n t by t h i r t y seconds a f t e r r a p i d and a f t e r another minute, only. static.  thawing,  s t a s i s formed i n one venule  A minute l a t e r a second c a p i l l a r y bed became Two minutes l a t e r there were o n l y the two  c a p i l l a r y beds s t a t i c ,  and the r e s t o f the c i r c u l a t i o n  w i t h i n the i n j u r e d area appeared normal.  Two and a  h a l f minutes l a t e r no f u r t h e r change developed, no resumption o f c i r c u l a t i o n b e i n g noted?  with  and the  p r e p a r a t i o n was d i s c o n t i n u e d . Animal 10 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 130 grams was a n a e s t h e t i z e d w i t h 0.15 c c . o f 0.6$ nembutal  s o l u t i o n , a d m i n i s t e r e d subcutaneously,  and the p r e p a r a t i o n was ready i n e i g h t minutes.  The  c o l d p o i n t was a p p l i e d to a s e l e c t e d a r e a o f the mesoappendix w i t h f r e e z i n g , r a p i d thawing.  f o l l o w e d immediately by  W i t h i n t h i r t y seconds o f thawing  120 there was r a p i d l y spreading  stasis,  which became  g e n e r a l i z e d throughout the i n j u r e d area w i t h i n another minute.  No r e c a n a l i z a t i o n was noted,  and  the p r e p a r a t i o n was d i s c o n t i n u e d three minutes l a t e r . Summary o f Observations Onset S t a s i s  No.  w i t h Benadryl  (1.5 mg./Kg)  Spread  Generalized  Plow Resumed Slight  1  Test  60"  Slowly  None  2  Control  30"  Slowly  5«  3  Test  None  None  None  4  Control  30"  Rapidly  3'  5  Test  60"  Slowly  6  Control  30"  Rapidly  7  Test  30"  Slowly  Incomplete  N©ne  8  Control  30"  Rapidly  Incomplete  None  9  Test  90"  Slowly  2 Cap Beds  None  Control  30"  Rapidly  10  Discussion of Not  60$ i n 90" 90"  90"  None  None None None  None  Observations  c o n s i d e r i n g the t e s t animal, number t h r e e ,  which d i d n o t develop s t a s i s ,  the average time taken  f e r the t e s t p r e p a r a t i o n s t o develop s t a s i s was s i x t y seconds, j u s t twice as l o n g as f o r the c o n t r o l s , took t h i r t y .  F u r t h e r , the s t a s i s spread more s l o w l y  and was much l e s s complete than I n the case of. the controls.  which  There was n© d i f f e r e n c e as regards any  resumption o f f l e w . These c o n s i d e r a t i o n s would appear t© s h i f t A  121 the d e c i s i o n more f a v o u r a b l y towards Benadryl  having  a p r o t e c t i v e e f f e c t on the mesoappendix c i r c u l a t i o n a g a i n s t a standard f r e e z i n g  injury.  Etamon Experimental  o b s e r v a t i o n s w i t h Etamon C h l o r i d e  The f o l l o w i n g t r i a l s were f o r the purpose ®f t e s t i n g the e f f e c t o f Etamon c h l o r i d e ( T e t r a - e t h y l ammonium e h l o r i d e ) i n a dosage l e v e l o f 25 mg. k i l o g r a m body weight,  per  on the c a p i l l a r y beds of the  r a t mesoappendix a f t e r a standard f r e e z i n g  injury.  Normal a d u l t male Wistar r a t s were used, f o u r t e s t animals,  averaging 310  averaging 280  grams.  u s i n g Ringer-Locke pH was  grams,  and f o u r c o n t r o l s ,  The procedure  was  as b e f o r e ,  gelatine-glucose solution.  The  a d j u s t e d to the f i r s t ehange to p i n k u s i n g  sodium b i c a r b o n a t e and methyl r e d i n d i c a t o r . Etamon s o l u t i o n was  a d m i n i s t e r e d to a l l t e s t  at one  time, u s i n g 0.10  10 mg.  per animal, which i s s l i g h t l y l a r g e r  25 mg.  per k i l o g r a m .  subcutaneously.  The animals  ec. (0.1 gm./ce), p r o v i d i n g  The drug was  than  administered  A summary o f the f o l l o w i n g observ-  a t i o n s i s g i v e n i n t a b l e form a f t e r the second Animal 8. Animal 1  (Oontrol) 0.20  c c . of 0.6$  nembutal s o l u t i o n was  i s t e r e d i n t r a p e r i t o n e a l l y f o r anaesthesia,  admin-  and i n  122 seven minutes the p r e p a r a t i o n was  ready,  mounted on  the microscope stage w i t h the d r i p f l o w i n g .  One  and a h a l f minutes l a t e r a s e l e c t e d area of the mesoappendix was seconds.  f r o z e n w i t h the c o l d p o i n t f o r t h i r t y T h i r t y seconds a f t e r thawing there  noted a very marked hyperemia, the d r i p s o l u t i o n was  and  resumed.  then the flow  Within  g e n e r a l v a s c u l a r s t a s i s had  oped,  somewhat incomplete.  seconds more, was  some c l e a r i n g , but only  main e e n t r a l a r t e r i o l e was and  temporary, One  observed.  A  flowing uninterruptedly,  the v a s c u l a r s t a s i s seemed to be e n t i r e l y on  venous s i d e of the c i r c u l a t i o n . was  devel-  In n i n e t y  noted i n a few o f the l a r g e r channels.  minute l a t e r no f u r t h e r change was  d i s c o n t i n u e d two  Animal 2  The  preparation  and a h a l f minutes l a t e r .  animal was  a n a e s t h e t i z e d w i t h 0.20  nembutal s o l u t i o n one hour and  cc.  fourteen  minutes a f t e r the Etamon had been administered. p r e p a r a t i o n was ing.  ready e i g h t minutes a f t e r  A s t a t e of hyperemia was  of the mesoappendix. t i p was  the  (Test) The  o f 0.6$  ©f  another  t h i r t y seconds, which was  was  The  anaesthetiz-  noted i n the v e s s e l s  Three minutes l a t e r the c o l d  a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds,  w i t h r a p i d thawing and resumption of flow of the warm drip solution. s t a s i s was  Within s i x t y seconds o f thawing  observed to be commencing.  By a minute  and a h a l f more,  123 the c o n d i t i o n of the  i n the i n j u r e d area was  found  to be much b e t t e r than  w i t h the p r e v i o u s c o n t r o l animal, the v a r y i n g f a c t o r s ,  circulation  but c o n s i d e r i n g  the d i f f e r e n c e was  enough to be t r u l y s i g n i f i c a n t u n l e s s  not  great  present  c o n s i s t e n t l y i n a l l the t e s t and c o n t r o l animals. By f o u r and a h a l f minutes a f t e r thawing, had  spread somewhat,  but there was  the  stasis  s t i l l v e r y good  c i r c u l a t i o n throughout the i n j u r e d a r e a .  Two  minutes l a t e r a main venous channel which had been s t a t i c was  noted  to be open.  a l s o noted  throughout the mesoappendix i n s p i t e of  Etamon (dose too small?!). s m a l l e r s t a t i c channels s t i l l later,  Good vasomotion  was  A minute l a t e r many  resumed flow,  and two  e i g h t y p e r c e n t o f the channels  minutes  once  s t a t i c v/ere ©pen and f u n c t i o n i n g w e l l .  I n three  minutes (twelve and a h a l f minutes a f t e r  thawing),  g e n e r a l i z e d v a s c u l a r s t a s i s was area. and  Two  the  p r e s e n t i n the i n j u r e d  minutes l a t e r no f u r t h e r change o c c u r r e d ,  the p r e p a r a t i o n was  discontinued.  Animal 3 ( C o n t r o l ) The 0.6$  animal was  a n a e s t h e t i z e d w i t h 0.20  nembutal s o l u t i o n administered  intraperitoneally,  and i n seventeen minutes the p r e p a r a t i o n was The  c o l d t i p was  seconds.  cc. of  ready.  applied with f r e e z i n g f o r t h i r t y  By t h i r t y seconds a f t e r r a p i d thawing net  much hyperemia o c c u r r e d ,  but a f t e r s i x t y seconds,  124 vascular stasis started. t h i s s t a s i s was there was  In s i x t y seconds more,  spreading r a p i d l y ,  and by tw© minutes  a slow i n s i d i o u s spread to a l l v e s s e l s w i t h  no more vasomotion observed. d i s c o n t i n u e d one minute  The p r e p a r a t i o n  was  later.  Animal 4 (Test) The animal was 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.20  s o l u t i o n two hours and seven minutes  a f t e r the Etamon had been a d m i n i s t e r e d . minutes the p r e p a r a t i o n was ready, hyperemia was noted.  Within s i x moderate applied with  and then immediate  rapid  achieved as b e f o r e w i t h resumption o f the  warm s a l i n e d r i p . was  and  The c o l d t i p was  f r e e z i n g f o r t h i r t y seconds, thawing was  cc o f  A v e r y b e a u t i f u l c a p i l l a r y bed  available f o r observation.  n i n e t y seconds a f t e r thawing.  Stasis f i r s t  By another minute  s t a s i s spread over a l a r g e venous a r e a . minutes f u r t h e r , circulation.  By  eight  no v e s s e l s reopened o r resumed any Two minutes l a t e r a main v a s c u l a r  channel reopened,  and w i t h i n three more minutes i t  showed a c t i v e vasomotion. f o r ten minutes. resume f l o w .  appeared  No f u r t h e r change was n o t e d  P a r t of the venous area d i d n o t The p r e p a r a t i o n was  discontinued.  Animal 5 ( C o n t r o l ) The animal was 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.20  cc. of  s o l u t i o n administered i n t r a p e r i t o n e a l l y ,  125 and the p r e p a r a t i o n was ready i n t e n minutes.  The  c o l d t i p was a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds, and s t a s i s was observed to commence w i t h i n seconds o f thawing.  thirty  By another s i x t y seconds the  s t a s i s was complete and i r r e v e r s i b l e ,  and the  p r e p a r a t i o n was d i s c a r d e d three minutes l a t e r w i t h no f u r t h e r change observed. Animal 6 (Test) The animal was a n a e s t h e t i z e d w i t h 0.20 c c . of 0.6$ nembutal  s o l u t i o n administered i n t r a p e r i t o n e a l l y  three hours and seven minutes a f t e r the Etamon was injected.  The p r e p a r a t i o n was ready i n f i v e minutes,  and e x c e l l e n t vasomotion was noted.  The c o l d p o i n t  was a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds.  By  t h i r t y seconds a f t e r f r e e z i n g good b l o o d f l o w through the i n j u r e d area was s t i l l p r e s e n t .  S l i g h t and temp-  o r a r y v a s c u l a r s t a s i s was noted one minute l a t e r ,  and  i n another minute permanent s t a s i s was noted i n a few small c a p i l l a r i e s .  By another f o u r minutes the s i t -  u a t i o n was much the same,  w i t h some slowing o f the  f l o w i n one venous channel.  Three minutes l a t e r 2%  p r o c a i n e a t room temperature was i n s t i l l e d over the mesoappendix w i t h stoppage o f the whole c i r c u l a t i o n . The p r e p a r a t i o n was d i s c a r d e d f i v e minutes  later  ( f o u r t e e n and a h a l f minutes a f t e r thawing). Animal 7 ( C o n t r o l ) The animal was a n a e s t h e t i z e d w i t h 0.20 c c . o f  126 0.6$  nembutal s o l u t i o n and the p r e p a r a t i o n was  i n n i n e minutes.  The c o l d p o i n t was  f r e e z i n g f o r t h i r t y seconds. of thawing great hyperemia was s t a s i s developing another was  minute,  in stasis,  t h r e e minutes. were slowing, thawing,  ready  applied with  W i t h i n t h i r t y seconds noted,  with vascular  and spreading r a p i d l y *  Within  t h i r t y percent of the i n j u r e d area which d i d not i n c r e a s e by  another  Three minutes l a t e r l a r g e r v e s s e l s and by f o u r t e e n and a h a l f minutes a f t e r  over h a l f the v e s s e l s i n the i n j u r e d area were  in stasis.  The p r e p a r a t i o n was  discontinued four  minutes l a t e r . Animal 8  (Test) The  o f 0.6$  animal was  a n a e s t h e t i z e d w i t h 0.20  nembutal s o l u t i o n administered  cc.  intraperitoneally  f o u r hours and f i v e minutes a f t e r the Etamon had been given. and  The p r e p a r a t i o n was  the c o l d p o i n t was  seconds.  ready i n n i n e minutes,  applied with f r e e z i n g f o r t h i r t y  S t a s i s s t a r t e d three minutes a f t e r thawing,  and f o u r minutes l a t e r was  found  to be complete i n  some p a r t s of the i n j u r e d area, but temporary i n o t h e r s . The p r e p a r a t i o n was  d i s c o n t i n u e d one minute l a t e r .  (Summary t a b l e w i l l be found  a f t e r the f o l l o w i n g  s e r i e s o f repeat t e s t s ) Repeat Experiments w i t h Etamon C h l o r i d e The f o l l o w i n g i s a r e p e t i t i o n o f the above run,  but u s i n g 25 mg.  127 o f Etamon ( t e t r a - e t h y l ammonium  c h l o r i d e ) per t e s t animal, a d m i n i s t e r e d subcutaneously.  The t e s t animals averaged 315 grams, the  c o n t r o l s 290 grams, Wistar r a t s .  and a l l were normal a d u l t male  The h e a v i e s t o f the t e s t animals,  weighing 355 grams,  d i e d f i f t e e n minutes a f t e r i t s  i n j e c t i o n of Etamon,  and another s i m i l a r male r a t  weighing 330 grams was 25 mg.  substituted,  being given  o f Etamon a l s o subcutaneously.  Animal 1 ( C o n t r o l ) The animal was 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.20  cc o f  s o l u t i o n administered i n t r a p e r i t o n e a l l y ,  and the p r e p a r a t i o n was c o l d p o i n t was  ready i n e i g h t minutes.  applied with freezing f o r t h i r t y  and w i t h i n t h i r t y seconds o f thawing v a s c u l a r developed i n venous channels,  The seconds,  stasis  r a p i d l y beeomming •  g e n e r a l i z e d over the i n j u r e d a r e a .  There was  no  f u r t h e r change by another t h r e e minutes and the p r e p a r a t i o n was d i s c o n t i n u e d . Animal 2 ( T e s t ) The animal was 0.6% nembutal  a n a e s t h e t i z e d w i t h 0.20  solution Intraperitoneally  minutes a f t e r the Etamon was t i o n was  injected.  ready i n f o u r minutes  cc. of  thirty-two The p r e p a r a -  and the c o l d t i p was  app-  l i e d to the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  W i t h i n t h i r t y seconds of thawing some s m a l l  128  c a p i l l a r y stasis was apparent and by another t h i r t y seconds many large venous channels were i n s t a s i s . In two minutes slow blood flow was observed through one large venous channel that had been i n s t a s i s . During the following ten minutes i t was noted that the infeeding c a p i l l a r i e s could not keep up the flow and the c i r c u l a t i o n i n the whole of the injured area ceased.  These were adjacent venous systems,  and a  large part of the s t a s i s was undoubtedly due to the f a c t that the a r t e r i o l e between was injured by the freezing and I t s flow stopped. preparation was discontinued.  Two minutes l a t e r the L i t t l e essential  difference was noted here from the previous control preparation. Animal 3 (Control) The animal was anaesthetized with 0.20 ce. ©f 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y and the preparation was ready i n s i x minutes.  The  c©ld point was applied t© a selected area of the mesoappendix with freezing f o r t h i r t y seconds, t h i r t y seconds of thawing,  and within  generalized stasis was  observed over the entire injured area.  There was n©  further change after three and a h a l f minutes and the preparation was discarded. Animal 4 (T&st) The animal was anaesthetized with 0.20 cc of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y  129 one hour and f i v e minutes a f t e r the Etamon i n j e c t i o n . The p r e p a r a t i o n was p o i n t was  ready  i n s i x minutes and  the  cold  a p p l i e d t© a s e l e c t e d area of the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds. t h i r t y seconds of thawing there was  Within  general  slowing  i n the venous channels w i t h many of the  connecting  channels  Vascular  pumping b l o o d i n t o the a r e a .  s t a r t e d two another  f i v e minutes,  through one was  and a h a l f minutes a f t e r thawing.  channel,  unchanged.  although there was  stasis By  scant f l o w  g e n e r a l l y the degree o f s t a s i s  The p r e p a r a t i o n was  d i s c a r d e d seven  and a h a l f minutes a f t e r thawing. Animal 5 ( C o n t r o l ) The animal was 0.6$  a n a e s t h e t i z e d w i t h 0.20  nembutal s o l u t i o n and the p r e p a r a t i o n was  i n twenty minutes.  The c o l d p o i n t was  cc. of ready  a p p l i e d to a  s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. there was ing.  W i t h i n t h i r t y seconds of thawing  g e n e r a l i z e d i r r e v e r s a b l e venous s t a s i s  By tw©  and a h a l f minutes l a t e r ,  unchanged and the p r e p a r a t i o n was  start-  the f i e l d  was  discarded.  Animal 6 (Test) The 0.6$  animal was  a n a e s t h e t i z e d w i t h 0.20  nembutal s o l u t i o n administered  cc. of  intraperitoneally  one hour and fourty-f©ur minutes a f t e r the Etamon injected.  The p r e p a r a t i o n was  and the c o l d p o i n t was  was  ready i n e i g h t minutes  a p p l i e d to a s e l e c t e d area o f  130 the mesoappendix w i t h f r e e z i n g f o r t h i r t y  sedonds.  By t h i r t y seconds a f t e r thawing g r e a t hyperemia was noted g e n e r a l l y , b u t no s t a s i s appeared by two minutes a f t e r thawing.  There was a good b r i s k b l o o d flow  throughout the f r o z e n area.  Two minutes l a t e r  was noted i n one small venous channel, vasomotion i n o t h e r s .  stasis  and i n c r e a s e d  In another minute the stream  was noted to slow t e m p o r a r i l y i n a few o f the l a r g e r venous channels.  I n t h i r t y seconds s t a s i s was noted  to be s p r e a d i n g to a couple venous areas,  although  t h e r e was g e n e r a l l y a good c i r c u l a t i o n .  The prep-  a r a t i o n was d i s c a r d e d seven minutes a f t e r thawing w i t h no f u r t h e r changes observed. Animal 7 ( C o n t r o l ) The animal was a n a e s t h e t i z e d w i t h 0.20 c c . ©f 0.6$ nembutal  solution intraperitoneally,  b u t when  the p r e p a r a t i o n was mounted s i x minutes l a t e r ,  general  s t a s i s was p r e s e n t throughout the mesoappendix,  and  the animal was d i s c a r d e d . Animal 8 ( T e s t ) The animal was a n a e s t h e t i z e d w i t h 0.20 c c . ©f 0.6$ nembutal  s o l u t i o n administered i n t r a p e r i t o n e a l l y  two hours and e i g h t minutes a f t e r the Etamon i n j e c t i o n . The p r e p a r a t i o n was ready i n f i v e minutes.  The c o l d  p o i n t was a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  Small  c a p i l l a r y s t a s i s was noted w i t h i n t h i r t y seconds thawing.  after  By another s i x t y seconds there was slowing ©f  131 the f l o w and s t a s i s i n l a r g e venous channels. and  Three  a h a l f minutes l a t e r one l a r g e s t a t i c venous  channel opened up, did likewise,  and I n another t h i r t y seconds another  and flowed normally.  I t would seem  t h a t the l a r g e r channels stay open p r o v i d e d  there i s  any b l o o d d r a i n i n g i n t o them from the c a p i l l a r i e s .  The  p r e p a r a t i o n was d i s c a r d e d e i g h t minutes a f t e r thawing w i t h no f u r t h e r change b e i n g noted. Summary o f Observations w i t h Etamon Ho.  Onset S t a s i a  1  Control  60"  2  Test  60"  3  Control  60"  Spread  Generalized  Rapidly  -  Fast,  •-  60"  -  then slow  Flow Resumed Temporary i : larger ch  12£«  ///  2»  None  -  4  Test  90"  5  Control  30"  6  Test  7  Control  30"  Rapidly  90"  None  8  Test  180"  Steady  7»  None  1  Control  30""  Rapidly  60"  None  2  Test  30"  Slow  14»  3  Control  30"  4  Test  150"  5  Control  30""—  6  Test  240"  150"  None  / / a t 10«  90"  None  Rapidly  >90"  Incomplete by 6^' None  —  .  >  None  > At 4'  // a t 3«  30"  None  None  None  30"  None  None  None  132 Summary  (Cont'd)  No.  Onset S t a s i s  Spread  Generalized  Flow Resumed  7  Control  Animal d i s c a r d e d because of general s t a s i s  8  Test  30"  Slight  a t 5» & 5&»  None  D i s c u s s i o n of Observations V a r i a b l e e f f e c t s from p a r e n t e r a l i n j e c t i o n s of Etamon would be expected i n t h i s experiment was  w i t h succeeding animals,  between the time of i n i t i a l and the performance  s i n c e there  an i n c r e a s e d i n t e r v a l a d m i n i s t r a t i o n of the drug  of the t e s t .  However, a s u p e r f i c i a l  examination of the above r e s u l t s would suggest Etamon had a d e f i n i t e e f f e c t which was  s u s t a i n e d f o r up to f o u r  hours f o l l o w i n g a d m i n i s t r a t i o n .  On the average, w i t h the  t e s t animals, v a s c u l a r s t a s i s d i d not s t a r t f o r 110 a f t e r thawing, i n g time was  whereas w i t h the c o n t r o l s the  40 seconds,  correspong-  almost three times as l o n g .  would seem that the spread and extent of the s t a s i s much l e s s w i t h the Etamon t r e a t e d  seconds  It was  animals.  However, because the d r i p s o l u t i o n seemed a t the f i n i s h of the t e s t s to be more deeply p i n k i s h than would be expected f o r n e u t r a l i t y , i t s pH was e l e c t r i c pH meter.  t e s t e d on  The d e t e r m i n a t i o n was  a day or so f o l l o w i n g the t e s t s ,  not done f o r  the f l u i d b e i n g s t o r e d  i n t i g h t l y stoppered f l a s k s i n an e l e c t r i c When determined,  an  regrigerator.  the pH measured 8.9 which i s a d m i t t e d l y  somewhat past n e u t r a l i t y !  The exact e f f e c t of t h i s more  133 a l k a l i n e d r i p s o l u t i o n was not studied, ance of the above r e s u l t s was  but the s i g n i f i c -  c o n s i d e r e d i n v a l i d , and the  t e s t s were repeated, as f o l l o w s ,  using a drip  solution  p r e t e s t e d on an e l e c t r i c pH meter, and c a r e f u l l y b u f f e r e d to a pH of  7.10.  Sepeat Experiments w i t h Etamon In the f i r s t few of the f o l l o w i n g  tests,  Etamon was used at a dosage l e v e l of 100 mg. k i l o g r a m body weight.  per  The o b s e r v a t i o n s are summar-  i z e d i n t a b l e form at the end of Animal 13. Animal 1 (Test) A normal a d u l t nonpregnant female Wistar r a t was used, weighing 225 grams. c h l o r i d e was  i n j e c t e d subcutaneously,  minutes l a t e r 0.15  cc of 0.6$ nembutal  injected intraperitoneally. 0.05  22.5 mg.  c c . more was  injected  of Etamon  and f i f t e e n solution  was  F i v e minutes l a t e r also  intraperitoneally.  Within ten more minutes the animal was s u f f i c i e n t l y a n a e s t h e t i z e d to permit c o n t i n u i n g .  The mesoappendix  was mounted on the microscope stage and the c o l d p o i n t was  applied  w i t h f r e e z i n g f o r f o r t y seconds.  W i t h i n t h i r t y seconds of thawing a d i s p e r s e d f i n e c a p i l l a r y vasostasis some v a r i a b l e  was  observed,  and a minute l a t e r  s t a s i s was noted In- the l a r g e r channels.  By two and a h a l f minutes a f t e r thawing the s t a s i s  was  beginning to spread and i n another two minutes the s t a s i s  was  134 q u i t e g e n e r a l i z e d i n the i n j u r e d area,  although  the a r t e r i o l e s were p u l s a t i n g t© some degree. p r e p a r a t i o n was d i s c o n t i n u e d w i t h no f u r t h e r  The changes  being noted. Animal 2 (Test) A  A normal a d u l t nonpregnant female Wistar r a t  weighing 280 grams was used.  28 mg.  o f Etamon was  i n j e c t e d subcutaneously and w i t h twenty-three minutes 0.15 c c . o f 0.6$ nembutal s o l u t i o n was i n j e c t e d peritoneally.  intra-  The p r e p a r a t i o n had to be d i s c a r d e d  a f t e r mounting the mesoappendix because the c i r c u l a t i o n was t o t a l l y i n v i s i b l e due to the amount o f f a t present. Animal 5 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 240 grams was used.  0.15 c c . o f 0.6$ nem-  b u t a l s o l u t i o n was i n j e c t e d i n t r a p e r i t o n e a l l y ,  but  a f t e r mountingj the mesoappendix f i v e minutes l a t e r showed the animal had to be d i s c a r d e d because o f generalized vascular  stasis.  Animal 4 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 205 grams was 0.6$ nembutal s o l u t i o n , oneal i n j e c t i o n ,  a n a e s t h e t i z e d w i t h 0.15 c c . ©f administered by i n t r a p e r i t -  and an a d d i t i o n a l 0.05 c c . was i n -  j e c t e d i n ten minutes and again i n seven more minutes. A f t e r mounting the mesoappendix,  the c o l d p o i n t  was  135 a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds. t h i r t y seconds of thawing, u l a r s t a s i s was The  noted  r a p i d l y spreading  vasc-  throughout the i n j u r e d a r e a .  s m a l l e r venules became i n v o l v e d In n i n e t y sec-  onds more,  but the a r t e r i o l e s were u n a f f e c t e d  the a r t e r i o - v e n o u s  observed  to be  Circulation  a p p a r e n t l y normal i n the r e a t o f  the mesoappendix.  By three minutes a f t e r thawing  the c i r c u l a t i o n was area,  and  shunts to the l a r g e r v e i n s app-  eared to be f u n c t i o n i n g n o r m a l l y . was  Within  q u i t e occluded i n the I n j u r e d  except f o r the l a r g e r v e s s e l s .  l a t e r the p r e p a r a t i o n was  Tw@  minutes  d i s c o n t i n u e d w i t h n©  f u r t h e r change noted. Animal 5 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing an unrecorded nembutal s o l u t i o n was  amount.  0.15  c c . of  0.6$  Injected intraperitoneally.  A f t e r mounting the mesoappendix,  the p r e p a r a t i o n  had  to be d i s c a r d e d because the amount of f a t p r e s e n t t o t a l l y obscured  the  circulation.  Animal 6 (Test) A normal a d u l t nonpregnant female Wistar r a t weighing 165  grams was  c h l o r i d e was  i n j e c t e d subcutaneously.  was  used.  16.5  mg.  of Etamon The  animal  found dead i n i t s cage ten minutes l a t e r .  Animal 7 (Test) A normal a d u l t nonpregnant female Wistar r a t  136 weighing 155 grams.  15.5 mg. o f Etamon c h l o r i d e was  i n j e c t e d subcutaneously i n t o the dorsum o f the animal, and f i f t e e n minutes l a t e r 0.10 c c . o f 0.6$ nembutal s o l u t i o n was i n j e c t e d i n t r a p e r i t o n e a l l y .  F i v e min-  u t e s l a t e r the p r e p a r a t i o n was mounted on the microscope stage w i t h e x c e l l e n t vasomotion observed, animal d i e d one minute  b u t the  later.  Animal 8 (Test) A normal  a d u l t nonpregnant  weighing 185 grams was used.  female Wistar r a t  18.5 mg. o f Etamon  c h l o r i d e was i n j e c t e d subcutaneously.and twelve minutes l a t e r 0.085 c c . o f 0.6$ nembutal injected intraperitoneally. p r e p a r a t i o n was ready, and s l u g g i s h ,  s o l u t i o n was  Four minutes l a t e r t h e  w i t h the v a s c u l a r bed d i l a t e d  but some vasomotion was noted,  no s t a s i s was observed.  Tbe c o l d p o i n t was a p p l i e d  w i t h f r e e z i n g f o r t h i r t y seconds, seconds o f thawing,  although  and w i t h i n  thirty  c a p i l l a r y and venous s t a s i s was  observed t o have o c c u r r e d .  I n another minute the  c i r c u l a t i o n c l o s e d down completely w i t h i n the i n j u r e d area.  By seven and a h a l f minutes  after  thawing,  g e n e r a l s t a s i s was p r e s e n t throughout the mesoappendix, and i t was f e l t  t h i s animal should n o t be admitted t o  the d a t a because o f obvious t o x i c e f f e c t s from the s t a r t due to the Etamon. Note; From the above and p r e v i o u s o b s e r v a t i o n s , i t  137 would appear t h a t Etamon c h l o r i d e at a dosage l e v e l of 100 mg.  per k i l o g r a m was  l e a s t 50$ of the animals.  too t o x i c , k i l l i n g  at  Further t r i a l s w i l l  be  l i m i t e d to a s m a l l e r dosage. Animal 9  (Test) A normal a d u l t n o n p r e g n a n t female W i s t a r r a t  weighing 160  grams was  c h l o r i d e was  i n j e c t e d subcutaneously  onds l a t e r 0.11  used.  c c . o f 0.6$  8.0  ready  o f Etamon  and t h i r t y  nembutal s o l u t i o n  injected intraperitoneally. the p r e p a r a t i o n was  mg.  W i t h i n twelve  and the c o l d p o i n t  a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds. t h i r t y seconds a f t e r thawing, observed  i n a l l except  a r t e r i o - v e n o u s shunts, which was  noted  q u i t e hyperemic,  minutes was Within  the l a r g e v e s s e l s and  was  the  w i t h i n the i n j u r e d area, Two  minutes  t h a t the adjacent areas were although no  spread of the v a s c u l a r  s t a s i s o c c u r r e d w i t h i n another t h i s was  was  spreading s t a s i s  a r e l a t i v e l y small f i e l d .  l a t e r i t was  sec-  two minutes.  Generally  an e x c e l l e n t p r e p a r a t i o n as f a r as o b t a i n i n g  a good v a s c u l a r bed f o r o b s e r v a t i o n was  concerned.  At the edges of the s t a t i c area p u l s a t i o n s could be seen e n t e r i n g the c l o s e d channels No reopening a r a t i o n was  o f any v e s s e l s was  from a r t e r i o l e s .  observed.  The  prep-  d i s c o n t i n u e d e i g h t and a h a l f minutes  a f t e r thawing.  138 Animal 10  (Test)  A normal a d u l t nonpregnant female W i s t a r r a t weighing 165 grams was 8.6 mg. i t was  i n j e c t e d subcutaneously w i t h  o f Etamon c h l o r i d e ,  and i n f o u r t e e n minutes  a n a e s t h e t i z e d w i t h 0.115  c c . ©f 0.6$  s o l u t i o n administered i n t r a p e r i t o n e a l l y , ce. more b e i n g g i v e n i n f i v e minutes.  nembutal  w i t h 0.05 The p r e p a r a -  t i o n was ready w i t h i n t h i r t e e n minutes a f t e r the second nembutal  injection,  but the animal had to be  d i s c a r d e d because o f to© much v a s c u l a r s t a s i s genera l l y i n the mesoappendix. Animal 11  (Control)  A normal a d u l t nonpregnant female W i s t a r r a t weighing 165 grams was o f 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.125  solution intraperiteneally,  p r e p a r a t i o n was ready i n s i x minutes. p o i n t was  The  cc.  and the cold  a p p l i e d to a s e l e c t e d area o f the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds and w i t h i n t h i r t y seconds o f thawing,  s t a s i s had s t a r t e d and  some ecchymosis was p r e s e n t .  In another minute i t  was observed t h a t the s t a s i s was l i m i t e d to o n l y one c a p i l l a r y bed.  By t h r e e and a h a l f minutes  after  thawing the l a r g e r channels i n the area were slowing, but open.  This f i e l d  were v e r y comparable Animal 12  and the degree o f c o l d  injury  to the s i t u a t i o n i n Animal 9.  (Test)  A normal a d u l t nonpregnant female W i s t a r r a t  139 weighing  170 grams was  c h l o r i d e subcutaneously was  i n j e c t e d w i t h 8.5  mg.  o f Etamon  and w i t h i n f o u r minutes 4.0  mg.  g i v e n a d d i t i o n a l l y , making the t o t a l dosage about  75 mg.  per k i l o g r a m body weight.  l a t e r 0.10  c c . o f 0.6$  Fourteen minutes  nembutal s o l u t i o n was  i n t r a p e r i t o n e a l l y and i n another p r e p a r a t i o n was  ready.  injected  twelve minutes the  The b l o o d v e s s e l s i n the  mesoappendix were observed  to be v e r y much d i l a t e d ,  and hyperemic.  the c o l d p o i n t was a p p l i e d  However,  to a s e l e c t e d area w i t h f r e e z i n g f o r t h i r t y  seconds.  Within t h i r t y seconds of thawing a l l c i r c u l a t i o n i n the mesoappendix stopped. moribund,  and was  The animal was  considered  d i s c a r d e d without f u r t h e r  observ-  ation. Animal 13  (Test)  A normal a d u l t nonpregnant female Wistar r a t weighing 8.0  mg.  160 grams was  i n j e e t e d subcutaneously  of Etamon c h l o r i d e ,  l a t e r i t was  and f o u r t e e n minutes  a n a e s t h e t i z e d w i t h 0.075 c c . o f  nembutal s o l u t i o n administered The p r e p a r a t i o n was p o i n t was  with  0.6$  Intraperitoneally.  ready i n s i x minutes and the c o l d  a p p l i e d to a s e l e c t e d area of the mesoappend-  i x w i t h f r e e z i n g f o r t h i r t y seconds. seconds o f thawing there was which spread r a p i d l y ,  Within  thirty  beginning venous s t a s i s  and by two  a f t e r thawing the whole area was areas were observed to be normal,  and a h a l f minutes static.  Adjoining  w i t h t r i c k l i n g of  140 b l o o d Into the I n j u r e d area from the a r t e r i o l e s the p e r i p h e r y . No  The  at  s t a s i s d i d not spread f u r t h e r .  channels were observed to ©pen up and resume f l o w .  The p r e p a r a t i o n was minutes a f t e r  d i s c o n t i n u e d f i v e and a h a l f  thawing.  Summary of Observations Onset S t a s i s  No.  w i t h Etamon Spread  Generalized  Rapidly  5^«  Plow Resumed N©ne  1  Test  30"  2  Test  N©  r e s u l t , f a t obscured c i r c u l a t i o n  3  Control  No  r e s u l t , general vascular  4  Control  30"  Rapidly  5  Control  N©  r e s u l t , f a t obscured  6  Test  No  r e s u l t , anlmalfound dead In cage  7  Test  No  r e s u l t , animal d i e d on microscope  8  Test  No  r e s u l t , p r e p a r a t i o n n©t p h y s i o l o g i c a l  9  Test  30"  10  Test  No  11  Control  30"  12  Test  No  13  Test  30"  D i s c u s s i o n ©f  Rapidly  5»  None  circulation  30",  limited  r e s u l t , general vascular None  stasis  stage  N©ne  stasis  None  N©ne  r e s u l t , animal moribund on microscope Rapidly  2-^»  None  Observations  Etam©n c h l o r i d e i n excess o f 50 mg. gram body weight i s too t o x i e for p r a c t i c a l  per  kilo-  use.  The number of animals w i t h v a l i d data i n t h i s s e r i e s i s too l i m i t e d f o r any c o n c l u s i o n s to be made. There were o n l y three t e s t animals and Observations  two c o n t r o l s .  w i t h the c o n t r o l p r e p a r a t i o n s  141 agree f a i r l y w e l l w i t h c o n t r o l s i n o t h e r  experiments.  In view of t h i s , i t would seem t h a t no v a l u e above the e r r o r i n t r i n s i c w i t h the method c o u l d be c l a i m e d f o r Etamon. of s t a s i s ,  There, was no apparent d e l a y i n the onset no apparent decrease i n the spread or e x t e n t  of the s t a s i s ,  and no evidence t h a t Etamon enabled  any  o f the s t a t i c channels to ©pen up and resume c i r c u l a t i o n . Hydergine Experimental Observations w i t h  Hydergine  A summary of the f o l l o w i n g data appears i n t a b l e form a f t e r Animal  15.  Animal 1 ( T e s t ) A female non pregnant i n g 185 grams was used. eously w i t h 0.33  a d u l t Wistar r a t weigh-  I t was  injected  c c . c o n t a i n i n g 0.1 mg.  subcutan-  of  Hydergine.  This seemed to have some e f f e c t i n making the animal more d o c i l e , of 0.6$  and twenty-six minutes l a t e r ,  nembutal s o l u t i o n was  e a l l y f o r anaesthesia. p r e p a r a t i o n was  ready,  0.10  cc.  Injected intraperiton-  W i t h i n seven minutes the but the animal had to be  dis-  carded because a s l o w l y s p r e a d i n g v a s c u l a r s t a s i s observed throughout  was  the mesoappendix.  Animal 2 (Test) An a d u l t nonpregnant female Wistar r a t weighi n g 185 grams was  i n j e c t e d subcutaneously w i t h 0.33  cc.  142  c o n t a i n i n g 0.1 mg. o f Hydergine, minutes l a t e r ,  and twenty-three  the animal was a n a e s t h e t i z e d w i t h  0.075 c c . o f 0.6$ nembutal intraperitoneally.  s o l u t i o n administered  I n seven minutes the p r e p a r a t i o n  was ready, and the c o l d p o i n t was a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds.  W i t h i n t h i r t y seconds o f thawing  a r a p i d l y s p r e a d i n g g e n e r a l small v e s s e l s t a s i s was observed,  b u t noteworthy was the f a c t t h a t two and a  h a l f minutes a f t e r thawing, a few small v e s s e l s t h a t were I n v a s c u l a r s t a s i s appeared to reopen,  and by  ten minutes a f t e r thawing q u i t e a marked resumption of the c i r c u l a t i o n was noted i n many v e s s e l s  that  otherwise would c e r t a i n l y n o t have been expected t© ©pen up. served,  I t i s p o s s i b l e t h a t vasomotion was obbut i t i s more l i k e l y t h a t the resumption o f  flow was merely a matter o f a p a s s i v e f l u s h i n g o f the sludged c e l l s by the more e f f e c t i v e ^ h y d r o s t a t i c and p u l s a t i n g p r e s s u r e p e r m i t t e d by the a c t i o n o f the Hydergine• Animal 5 ( C o n t r o l ) An a d u l t nonpregnant female Wistar r a t weighi n g 185 grams was a n a e s t h e t i z e d w i t h 0.10 c c . o f 0.6$ nembutal  s o l u t i o n administered i n t r a p e r i t o n e a l l y ,  and the p r e p a r a t i o n was ready on the mieroscope stage w i t h i n seven minutes.  The c o l d p o i n t was a p p l i e d  to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds,  and immediately f o l l o w i n g thawing,  143 s t a s i s i n c a p i l l a r y beds and was  noted.  In another  v e i n s were slowing,  channels  t h i r t y seconds the l a r g e  and by two minutes a f t e r thawing  the small venous s t a s i s was i n j u r e d area.  small venous  slowly spreading over  A few l a r g e r venous channels  the  opened  up f o l l o w i n g a temporary a r r e s t i n flow, not e x a c t l y the same as the venous s t a s i s otherwise A f t e r f i v e minutes f o l l o w i n g thawing,  described. the  arterioles  and a r t e r i o - v e n o u s shunts i n the i n j u r e d area were a l l regarded  as f r e e f l o w i n g ,  and the r e s t of the  u l a t i o n i n the mesoappendix was The p r e p a r a t i o n was  regarded  circ-  as normal.  discarded.  Animal 4 (Test) An a d u l t nonpregnant female Wistar r a t weighing 170  grams was  i n j e c t e d w i t h 0.33  c o n t a i n i n g 0.1  mg.  subcutaneously, 0.075 ce. of 0.6$ peritoneally. was  ready,  c c . of  of a c t i v e p r i n c i p l e ,  Hydergine, administered  and twenty-eight minutes l a t e r , nembutal s o l u t i o n was  injected  intra-  W i t h i n e i g h t minutes the p r e p a r a t i o n  and the c o l d t i p was  a p p l i e d to a s e l e c t e d  area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y onds.  V a s c u l a r s t a s i s i n f i v e c a p i l l a r y beds  sec-  was  s t a r t i n g w i t h i n f o r t y - f i v e seconds a f t e r thawing, although t h i s apparent ends was  d e l a y i n onset ©f f i f t e e n  not c o n s i d e r e d s i g n i f i c a n t .  I t was  sec-  noted  t h a t s e v e r a l normally f u n c t i o n i n g c a p i l l a r y beds were  144 i n t e r s p e r s e d through  the s t a t i c ones.  h a l f minutes f o l l o w i n g f r e e z i n g ,  By two  and  s e v e r a l of the  a  static  venous and c a p i l l a r y systems " f l u s h e d " open w i t h a c t i v e vasomotion observed  i n the p r e c a p i l l a r i e s .  It  was  noted f i f t e e n minutes a f t e r thawing t h a t one l a r g e venous system f a i l e d to resume complete flow,  although  a f a i n t t r i c k l e of b l o o d c o r p u s c l e s d i d resume, p o s s i b l y because of the t w i s t i n g and moving of the animal under the l i g h t a n a e s t h e t i c . the p r e p a r a t i o n was  One minute l a t e r  d i s c o n t i n u e d because of g e n e r a l  v a s c u l a r slowing g e n e r a l l y throughout  the mesoappendix.  Animal 5 ( C o n t r o l ) An a d u l t nonpregnant female Wistar r a t weighi n g 145 grams was 0.6$  a n a e s t h e t i z e d w i t h 0.075 c c . of  nembutal s o l u t i o n administered  The p r e p a r a t i o n was p o i n t was  intraperitoneally.  ready i n ten minutes,  and the c o l d  a p p l i e d to a s e l e c t e d area of the mesoappend-  i x w i t h f r e e z i n g f o r t h i r t y seconds. seconds a f t e r thawing there was  Within  forty-five  venous slowing  and  s t a s i s w i t h r a p i d l y spreading s t a s i s i n the venules i n the I n j u r e d area. i n g the s t a s i s was  Two  and a h a l f minutes a f t e r thaw-  spreading but many normal  beds were f u n c t i o n i n g normally i n between, i n the v e r y centre of the I n j u r e d area.  capillary and a l s o  By seven and  a h a l f minutes f o l l o w i n g the thawing there was f u r t h e r change to be noted.  no  The s t a s i s d i d not become  145 g e n e r a l i z e d nor complete i n the a f f e c t e d a r e a .  The  r e s t of the v a s c u l a t u r e i n the mesoappendix appeared r e l a t i v e l y normal when the p r e p a r a t i o n was d i s c o n t i n ued. Animal 6  (Test) An a d u l t nonpregnant female W i s t a r r a t weigh-  i n g 145  grams was  i n j e c t e d subcutaneously  of Hydergine contained i n 0.33 cutaneously, 0.6$  administered  mg.  sub-  and i n t w e n t y - f i v e minutes 0.075 c c . o f  nembutal s o l u t i o n was  The p r e p a r a t i o n was animal had  cc,  w i t h 0.1  ready  injected Intraperitoneally. ten minutes l a t e r ,  but  the  to be d i s c a r d e d because o f g e n e r a l i z e d  v a s c u l a r s t a s i s and ecchymosis i n the mesoappendix. Animal 7  (Test) An a d u l t nonpregnant female Wistar r a t weigh-  i n g 140 grams was  i n j e c t e d subcutaneously  ©f Hydergine c o n t a i n e d i n 0.33 f i v e minutes 0.05 administered  ce. ©f 0.6$  cc,  f o u r minutes l a t e r ,  and  mg.  and i n twenty-  nembutal s o l u t i o n  Intraperitoneally,  r e p e a t e d f o u r minutes l a t e r .  w i t h 0.1  was  w i t h 0.025 c c . b e i n g The p r e p a r a t i o n was  the c o l d p o i n t was  readfc  applied  to a s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. ing, was  W i t h i n t h i r t y seconds a f t e r thaw-  small c a p i l l a r y s t a s i s spreading to other beds noted,  area was  and i n another minute a f a i r l y  extensive  i n s t a s i s w i t h o n l y one l a r g e a r t e r y f l o w i n g .  146 A c t u a l l y the i n j u r e d area i n t h i s p r e p a r a t i o n  was  about f o u r times as l a r g e as i n animals 1 t© 6, i t was  and  f e l t t h a t i f Hydergine had any r e a l v a l u e i n  a i d i n g the f l u s h i n g of c e l l s through the vessels,  the area i n s t a s i s was  the c e l l s to f l u s h through  to©  static  extensive f e r  such a l o n g channel  by  means o f a r t e r i o l e p u l s a t i o n s .  By twelve and a  h a l f minutes f o l l o w i n g thawing,  no f u r t h e r change  was  noted.  The r e s t of the c i r c u l a t i o n i n the  mesoappendix was  regarded  as good.  phery o f the s t a t i c areas a few  At the p e r i -  a r t e r i o l e s were ob-  served to be p u l s a t i n g i n t o the s t a t i c v e s s e l s . p r e p a r a t i o n was  The  d i s c o n t i n u e d three minutes l a t e r .  Animal 8 ( C o n t r o l ) An a d u l t nonpregnant female Wistar r a t weighi n g 115 grams was  a n a e s t h e t i z e d w i t h 0.05  c c . of  0.6$  nembutal s o l u t i o n administered i n t r a p e r i t o n e a l l y the p r e p a r a t i o n was c o l d t i p was  ready w i t h i n s i x minutes.  f o r t y - f i v e seconds of thawing a r a p i d l y vascular s t a s i s occurred. ©nly the main channels  shunts were observed were observed minutes,  The  a p p l i e d to a s e l e c t e d area of the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds.  ing,  and  spreading  By two minutes a f t e r thawand the a r t e r i o - v e n o u s  to be f l o w i n g ,  to be slowing.  no change was  Within  noted,  and many of these  A f t e r a f u r t h e r three w i t h no f u r t h e r spread  i n the s t a s i s , channels.  147 and no reopening of any v a s c u l a r  The warm s a l i n e d r i p was  not resumed w i t h  t h i s p r e p a r a t i o n u n t i l e i g h t minutes a f t e r and i t was n o t observed additional stasis,  a r a t i o n was  Immediate  thus s u p p o r t i n g the c o n t e n t i o n  t h a t the Ringer-Locke glucose added was  to e f f e c t any  thawing,  s o l u t i o n with gelatine  f a i r l y physiological.  and  The  d i s c a r d e d one minute l a t e r ,  prep-  w i t h good  b l o o d f l o w i n the r e s t o f the mesoappendix. Note; The pH ©f the Ringer-Locke g e l a t i n e and glucose was  drip containing  adjusted before  starting,  u s i n g a Beckman pH meter and a d j u s t i n g to a pH 7.10  of  w i t h sodium b i c a r b o n a t e powder.  Animal 9 (Test) An a d u l t nonpregnant female Wistar r a t weighi n g 175 grams was o f Hydergine,  i n j e c t e d subcutaneously w i t h 0.1  c o n t a i n e d i n 0.33  cc,  e i g h t minutes l a t e r the animal was 0.075 c c . o f 0.6$  twenty-  anaesthetized with  nembutal s o l u t i o n a d m i n i s t e r e d  Intraperitoneally. a r a t i o n was  and  mg.  ready,  W i t h i n e i g h t minutes the prepbut the animal was  discarded  s i n c e g e n e r a l i z e d v a s c u l a r s t a s i s , and much eechymosis were p r e s e n t on mounting the mesoappendix. Animal 10  (Control)  0  An a d u l t nonpregnant female Wistar r a t weigh-  148 i n g 185 grams was 0,6$  a n a e s t h e t i z e d w i t h Q.10  nembutal s o l u t i o n a d m i n i s t e r e d  ee. o f  intraperitoneally  and seven minutes l a t e r the p r e p a r a t i o n was The c o l d p o i n t was  ready.  a p p l i e d to a s e l e c t e d area of the  mesoappendix w i t h f r e e z i n g f o r t h i r t y  seconds.  W i t h i n t h i r t y seconds of thawing g e n e r a l i z e d venous s t a s i s was idly.  noted to be s t a r t i n g which spread rap-  I n one minute more the whole i n j u r e d area  in vascular stasis.  The p r e p a r a t i o n was  ued w i t h no f u r t h e r change,  was  discontin-  the remainder  o f the  mesoappendix a p p a r e n t l y having normal c i r c u l a t i o n . Animal 11  (Test)  An a d u l t nonpregnant female Wistar r a t weighi n g 165 grams was mg.  o f Hydergine  i n j e c t e d subcutaneously w i t h ( i n 0.33  minutes l a t e r i t was 0.6$  cc.) and  0.1  twenty-three  a n a e s t h e t i z e d w i t h 0.05  cc. of  nembutal s o l u t i o n a d m i n i s t e r e d i n t r a p e r i t o n e a l l y ,  w i t h 0.025 c c . more being g i v e n I n f o u r  minutes.  E l e v e n minutes a f t e r the f i r s t nembutal  injection,  the p r e p a r a t i o n was  mounted on the microscope  ready f o r o b s e r v a t i o n .  The  c o l d p o i n t was  stage, applied  to a s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds. thawing,  W i t h i n t h i r t y seconds o f  v a s c u l a r s t a s i s was  In one minute more,  observed  to be  many of the channels  ed c l o s e d were a g a i n f u n c t i o n i n g ,  starting.  t h a t appear-  and t h i s r e s t o r -  149 a t i o n o f c i r c u l a t i o n was not c o n s i d e r e d ctosbeodue to p a s s i v e vasomotion.  In another minute,  there were  ©nly a few of the c a p i l l a r y beds i n s t a s i s ,  and f i v e  and a h a l f minutes a f t e r thawing there were o n l y c a p i l l a r y beds i n s t a s i s . the  One minute l a t e r , one o f  c a p i l l a r y beds reopened w i d e l y ,  be f r e e l y f l o w i n g .  two  and appeared t©  By n i n e and a h a l f minutes  thawing there was  o n l y one c a p i l l a r y bed  vascular stasis,  and the p r e p a r a t i o n was  after  showing discontin-  ued w i t h the c i r c u l a t i o n i n the remainder o f the mesoappendix a p p a r e n t l y normal. Animal 12  (Test)  An a d u l t nonpregnant female W i s t a r r a t weighi n g 170 grams was  i n j e c t e d w i t h 0.1 mg.  c o n t a i n e d i n 0.33  c c . subcutaneously,  o f Hydergine and  twenty-  f i v e minutes l a t e r 0.075 c c . ©f 0.6$ nembutal u t i o n was  Injected i n t r a p e r i t o n e a l l y f o r anaesthesia.  In another seven minutes the p r e p a r a t i o n was but  sol-  ready,  the animal was d i s c a r d e d beeause of e x t e n s i v e  v a s c u l a r s t a s i s and eechymosis throughout the mesoappendix  circulation.  Animal 15  (Test)  An a d u l t nonpregnant female W i s t a r r a t weighi n g 150 grams was  i n j e c t e d subcutaneously w i t h 0.1  of Hydergine contained In 0.33 minutes l a t e r the animal was  ce.  mg.  Thirty-three  a n a e s t h e t i z e d w i t h 0.15  150 c c . o f 0.6$ nembutal s o l u t i o n administered i n t r a muscularly.  I n n i n e minutes the p r e p a r a t i o n was  ready and no v a s c u l a r s t a s i s was noted i n the c i r c u l a t i o n o f the mesoappendix.  The c o l d p o i n t was  a p p l i e d w i t h f r e e z i n g f o r t h i r t y seconds. f i r s t appeared  Stasis  s i x t y seconds a f t e r thawing,  only three c a p i l l a r y beds, i n g i n the l a r g e r v e i n s .  and i n  withjhowever^ some slowI n another two minutes a  few o f the c l o s e d a r t e r i o - v e n o u s shunts reopened a channel through. thawing,  At n i n e and a h a l f minutes a f t e r  a v e r y s h o r t venous channel reopened.  By  s i x minutes l a t e r there had o c c u r r e d no f u r t h e r changes. still  The same f o u r s t a t i c c a p i l l a r y beds were  shut down w i t h e x c e l l e n t a r t e r i o l e flow  them i n the a r t e r i o - v e n o u s channels. of the c i r c u l a t i o n appeared  normal,  hyperemia p r e s e n t throughout.  through  The remainder w i t h moderate  The p r e p a r a t i o n was  d i s c o n t i n u e d n i n e t e e n and a h a l f minutes a f t e r thawing. Animal 14 ( C o n t r o l ) An a d u l t nonpregnant female Wistar r a t weighi n g 150 grams was a n a e s t h e t i z e d w i t h 0.10 c c . o f 0.6$ nembutal s o l u t i o n a d m i n i s t e r e d i n t r a m u s c u l a r l y , and seven minutes l a t e r the dosage was r e p e a t e d w i t h immediate death o f the animal, presumably intravenous  injection.  through  151 Animal 15 ( C o n t r o l ) An a d u l t nonpregnant female Wistar r a t weighi n g 170 grams was a n a e s t h e t i z e d w i t h 0.10 c c . o f 0.6$ nembutal s o l u t i o n ,  administered  three d i v i d e d s i t e s ,  intramuscularly i n  and eleven minutes l a t e r the  p r e p a r a t i o n was ready on the microscope stage.  The  c o l d p o i n t was a p p l i e d w i t h f r e e z i n g to a s e l e c t e d s i t e i n the mesoappendix f o r t h i r t y seconds. was observed  Stasis  w i t h i n t h i r t y seconds o f thawing, and  was s l o w l y spreading to i n v o l v e i n the next two minutes s e v e r a l c a p i l l a r y beds w i t h i n the i n j u r e d area. Pour minutes l a t e r the p r e p a r a t i o n was d i s c o n t i n u e d , w i t h no f u r t h e r change b e i n g  noted.  Note; The  i n j e c t i o n o f the nembutal other  than  i n t r a p e r i t o n e a l l y appears to l e s s e n the i n c i d e n c e e f the v a s c u l a r s t a s i s which has been so w a s t e f u l o f animals  even before any t e s t i n g c o u l d be done.  Summary e f Observations No.  Onset S t a s i s  w i t h Hydergine Spread  Generalized  Flow Resumed  1  Test  No r e s u l t s , mesoappendix i n s t a s i s  2  Test  30"  3  Control  Immediate  4  Test  45"  Rapidly  F a i r l y gen'l  2»  Almost t o t a l  Not even  None  2£«, max by 10» None 2^' i n many _ areas  152 Summary  (Cont'd)  No.  Onset S t a s i s  Spread  Generalized  Rapidly  F l o w Resumed  5  Control  45"  None  6  Test  No r e s u l t s , m e s o a p p e n d i x i n s t a s i s  7  Test  30"  8  Control  45"  R a p i d w i t h i n 1' A l m o s t Rapidly  None  total  None  E x c e p t A-V  None  shunts 9  Test  No r e s u l t s , m e s o a p p e n d i x i n s t a s i s  10  Control  30"  11  Test  30"  Rapidly Finally,  90"  only  None  None  / / / a t 90"  1 Cap. b e d 12  Test  No r e s u l t s , m e s o a p p e n d i x I n s t a s i s  13  Test  60"  14  Control  No r e s u l t s ,  15  Control  30"  To f o u r  peritoneal induces  Slowly  i n j e c t i o n s i n case stasis  ary f a i l u r e vessels The  injection None  Hydergine to r e f r a i n from  intra-  the m a t e r i a l i n j e c t e d  b e h a v e d much as i n o t h e r  T h e r e was i n t h e c o n t r o l a n i m a l s o f any r e s t o r a t i o n o f c i r c u l a t i o n  o n c e t h e y went i n t o s t a s i s f o l l o w i n g  custom-  i n blood thawing.  H y d e r g i n e p r e t r e a t e d r a t s d i d n o t show a d e f i n i t e  tendency ulation ury.  nembutal  i n the mesoappendix.  Control preparations experiments.  / / a t 3'  Yes  with  seem w a r r a n t e d  vascular  None  intravenous  Discussion of Observations I t would  Cap b e d s  t h a t was c o n s i s t e n t t o w a r d r e s u m p t i o n o f c i r c after  the v a s o s t a t i c e f f e c t  o f the f r e e z i n g  O t h e r d i f f e r e n c e s were n o t c o n s i d e r e d  inj-  significant  153 following  thawing.  Apresoline Experimental  Observations w i t h A p r e s o l i n e  A summary o f the f o l b w i n g o b s e r v a t i o n s w i l l be found below i n t a b l e form a f t e r Animal 11. Preliminary test f o r t o x i c i t y 10 mg. o f A p r e s o l i n e was administered  subcut-  aneously t o a 200 gram nonpregnant a d u l t female Hooded r a t . obvious time,  I n n i n e t y minutes there were v e r y  s i g n s ©f a s e r i o u s r e a c t i o n .  At the same  2 mg. o f A p r e s o l i n e was s i m i l a r l y a d m i n i s t e r e d  to another animal o f the same s i z e , and i n n i n e t y minutes i t was a p p a r e n t l y normal. Animals  used A d u l t male Hooded r a t s , f i v e i n a l l , i n  weight range o f 190 t o 220 grams were chosen f o r testing,  and to these a t one time was a d m i n i s t e r e d  2 mg. o f A p r e s o l i n e subcutaneously. minutes,  By f i f t e e n  a l l f i v e of the i n j e c t e d animals were ob-  v i o u s l y i n f l u e n c e d by the drug, versely,  though n o t ad-  and were a l l l y i n g q u i e t l y i n t h e i r  cages.  F i v e a d u l t male Hooded r a t s i n the weight range o f 150 t© 250 grams were s e l e c t e d t© serve as controls, oline.  and these were n o t i n j e c t e d w i t h  Apres-  154 Animal 1 (Control) The animal was anaesthetized w i t h 0.1 cc. of nembutal s o l u t i o n 0.6$ subcutaneously,  and i n  f i v e minutes 0.125 cc. more were administered. S t a s i s s t a r t e d w i t h i n n i n e t y seconds of thawing. There was minimal spread a f t e r three minutes of thawing,  and the greatest p a r t of the i n j u r e d  area was normal.  Only twenty percent of the i n -  jured vessels'were i n s t a s i s .  There was no  f u r t h e r extension by s i x minutes a f t e r thawing. No resumption of f l o w was observed. Note; The Ringer-Locke s a l i n e d r i p s o l u t i o n w i t h g e l a t i n e and glucose had been buffered without use of the Beckman pH meter,  and i t was p o s s i b l e the  s o l u t i o n when used was s l i g h t l y too a l k a l i n e ,  but  i t appeared that the m a t e r i a l was f a i r l y p h y s i o l o g i c a l w i t h no s i g n i f i c a n t adverse e f f e c t s on the v e s s e l s being noted. Animal 2 (Test) The animal was anaesthetized w i t h 0.075 cc.  of 0.6$ nembutal s o l u t i o n which was augmented  i n f i f t e e n minutes by 0.05 cc. more.  N© s t a s i s  was observed w i t h i n t h i r t y seconds ©f thawing, the  f i r s t occurring three minutes a f t e r thawing.  Only one c a p i l l a r y bed was involved by four and a  155 h a i r minutes o f thawing.  T h i s i s an e x c e l l e n t l y  p r e s e r v e d f i e l d to observe.  No r e v a s c u l a r i z a t i o n  was noted, n o r any f u r t h e r change by seven and a h a l f minutes f o l l o w i n g thawing. Animal 5 ( C o n t r o l ) The  animal was a n a e s t h e t i z e d w i t h 0.125 c c .  of 0.6$ nembutal s o l u t i o n w i t h no f u r t h e r nembutal being r e q u i r e d .  Freezing occurred  forty-five  minutes a f t e r the nembutal i n j e c t i o n . ing,  After freez-  a small a r t e r i o l a r haemorrhage was noted,  which was n o t severe enough to a f f e c t the animal g e n e r a l l y o r the mesoappendix v a s c u l a t u r e i n p a r t icular.  A t nine and a h a l f minutes a f t e r the thaw-  i n g there was g e n e r a l i z e d v a s c u l a r s t a s i s out the i n j u r e d a r e a . were made.  No i n t e r m e d i a t e  through-  observations  These o b s e r v a t i o n s were not i n c l u d e d  In the summary t a b l e . Animal 4 (Test) The  animal was a n a e s t h e t i z e d w i t h 0.125 c c .  o f 0.6$ nembutal s o l u t i o n .  F r e e z i n g o c c u r r e d one  hour and f o r t y minutes a f t e r the A p r e s o l i n e administration.  The f r e e z i n g took f o r t y seconds Before  thawing c o u l d commence,  and except f o r one s t r o n g l y  f l o w i n g a r t e r i o - v e n o u s anastamosis,  t h e r e was  g e n e r a l i z e d v a s c u l a r s t a s i s i n the i n j u r e d area t o the extent o f e i g h t y p e r c e n t w i t h i n t h i r t y seconds  156 o f thawing.  There was no f u r t h e r changes noted  by f o u r and a h a l f minutes a f t e r thawing,  and n©  r e s t o r a t i o n o f c i r c u l a t i o n apparent. Animal 5 ( C o n t r o l ) There was n© s t a s i s apparent by f i v e  and  a h a l f minutes f o l l o w i n g thawing. Animal 6 (Test) The animal was of 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.125  solution.  cc.  S t a s i s commenced w i t h i n  t h i r t y seconds o f thawing.  Many channels reopened  and the b l o o d flowed f r e e l y through them.  By tw©  and a h a l f minutes a f t e r thawing a l l channels were restored.  T h i s was not the u s u a l type o f v a s c u l a r  stasis.  The whole i n j u r e d area became f r e e l y  flowing,  and remained so by seven add a h a l f min-  utes a f t e r thawing when the p r e p a r a t i o n was  dis-  continued. Note; Into fco-theru:thr.e:e male a d u l t Hooded r a t s was  i n j e c t e d 2.0 mg.  o f A p r e s o l i n e subcutaneously.  Animal 7 (Test) The animal was o f 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.15  solution,  and f r e e z i n g  cc.  injury  was i n f l i c t e d s e v e n t y - f i v e minutes a f t e r the Apreso l i n e was  administered.  S t a s i s commenced w i t h i n  t h i r t y seconds a f t e r thawing.  By n i n e t y  seconds  157 there was r a p i d spread to the whole o f the i n j u r e d area.  No f u r t h e r change o c c u r r e d by f i v e minutes  a f t e r thawing,  and n© r e s t o r a t i o n of the c i r c u l -  a t i o n was noted. Animal 8 ( C o n t r o l ) The animal was of 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.15  solution.  S t a s i s commenced  w i t h i n t h i r t y seconds o f thawing. of the i n j u r e d area was u t e s a f t e r thawing. u l a t i o n was  cc.  Fifty  percent  i n s t a s i s w i t h i n three min-  No r e s t o r a t i o n ©f the c i r c -  ©bserved.  Animal 9 (Test) The animal was of 0.6$ nembutal  a n a e s t h e t i z e d w i t h 0.15  solution.  Freezing occurred  cc. approx-  i m a t e l y n i n e t y minutes a f t e r a d m i n i s t r a t i o n o f the Apresoline. bed,  S t a s i s o c c u r r e d In o n l y one  capillary  and i n t h a t by s i x t y seconds a f t e r thawing.  There was  a l s o one small s i t e o f eechymosis.  There  were no f u r t h e r changes by f i v e minutes a f t e r thawing,  and no r e s t o r a t i o n o f the c i r c u l a t i o n .  Animal 10  (Test)  The animal was ofnembutal 0.6$  a n a e s t h e t i z e d w i t h 0.15  solution.  Freezing occurred  cc. approx-  i m a t e l y two hours a f t e r a d m i n i s t r a t i o n o f the Apresoline.  S t a s i s o c c u r r e d w i t h ' H h i r t y seconds o f thaw-  158 W i t h i n s i x t y seconds ©f thawing,  Ing.  percent o f the i n j u r e d area developed stasis.  fifty  vascular  Only one a r t e r i o - v e n o u s b r i d g e was f u n c t -  i o n i n g by three minutes a f t e r thawing. a t i o n o f the s t a t i c c i r c u l a t i o n was  N© r e s t o r -  observed.  Animal 11 ( C o n t r o l ) Tbe of  animal was a n a e s t h e t i z e d w i t h 0.10 c c .  0.6$ nembutal s o l u t i o n .  f i f t e e n seconds o f thawing,,  S t a s i s developed  within  and r a p i d l y spread t©  i n v o l v e t h i r t y p e r c e n t ©f the I n j u r e d area w i t h i n s i x t y seconds a f t e r thawing.  Much ecchymosis was  p r e s e n t i n the s t a t i c area.  The s t a s i s  slowly  spread to i n v o l v e f i f t y p e r c e n t o f the I n j u r e d area w i t h i n three minutes a f t e r thawing. a t i o n o f the c i r c u l a t i o n was  No r e s t o r -  noted.  Addendum I n the case o f Animal 2,  f r e e z i n g occurred  ©ne hour a f t e r the A p r e s o l i n e injecti©n. Animal 6,  I t was two hours and t e n minutes.  Summary ©f Observations No.  With  Onset S t a s i s 90"  with Apresoline Spread  Generalized  1  Control  Minimal  2  Test  3  Control  Results not included  4  Test  30"  Slowly  glow Resumed  20$ I n 3« 1 Cap. bed i n  80$ i n 30" 80$ i n 4£»  None None  None  159 Summary (Cont'd) Onset S t a s i s  No.  Spread  Generalized  Plow Resumed  5  Control  None by 5-|'  6  Test  30",  7  Test  30"  Rapidly  8  Control  30"  Steady  9  Test  60",  i n o n l y one c a p i l l a r y bed  None  10  Test  30"  50$ i n 60" 90$ i n 3»  None  11  Control  15"  30$ i n 60" 50$ I n 3»  None  but complete r e s t o r a t i o n by 2^» Complete by 90" None 50$ i n 3»  None  D i s c u s s i o n e f Observations No d i f f e r e n c e can be claimed between the beh a v i o r e f the t e s t and the c o n t r o l group i n r e s p e c t t o time b e f o r e onset o f s t a s i s , extent o f the s t a s i s , o r resumption o f flow i n s t a t i c  channels,  Chler-Tripolon Experimental  Observations  with Chlor-Tripolon  Into each o f s i x a d u l t nonpregnant female Wistar r a t s In the 150-200 gram weight range was i n j e c t e d subcutaneously 5 mg, o f C h l o r - t r i p o l o n maleate d i s s o l v e d i n water to a s t r e n g t h o f 50 mg. p e r c c , (0,1 c c , f o r each animal). one hour before p r e p a r a i n g observation. controls.  The i n j e c t i o n was made the animals f o r m i c r o s c o p i c  S i x s i m i l a r animals were s e l e c t e d as Eaeh animal was a n a e s t h e t i z e d  before  p r e p a r a t i o n w i t h a subcutaneous i n j e c t i o n o f 0.15 c c .  160 ©f 0.6$ nembutal  solution.  A summary o f the f e l l -  owing o b s e r v a t i o n s i n t a b l e form w i l l be found a f t e r Animal 12. Animal 1  The u s u a l d r i p s o l u t i o n was (Control)  Stasis started within ing.  used.  t h i r t y seconds o f thaw-  Twenty p e r c e n t o f the i n j u r e d area was i n  stasis within  s i x t y seconds a f t e r thawing, and s i x t y  percent within and c l o s e d  three minutes.  intermittently.  changes observed w i t h i n  One venule opened There were no  further  f i v e minutes a f t e r thawing.'  No r e s t o r a t i o n of the c i r c u l a t i o n was noted. Animal 2 (Test) Stasis started within thawing.  f i f t e e n seconds a f t e r  W i t h i n s i x t y seconds a f t e r thawing,  s t a s i s was p r e s e n t i n the whole of the i n j u r e d except f o r one l a r g e  arterio-venous bridge.  small ecchymoses were p r e s e n t w i t h i n  area Three  the f r o z e d  area.  No f u r t h e r change was noted w i t h i n f i v e minutes a f t e r thawing,  and no resumption o f f l o w was observed.  Animal 3  (Control) Stasis started within  thawing. involved  t h i r t y seconds a f t e r  S i x t y p e r c e n t o f the i n j u r e d area within  was  f o r t y - f i v e seconds a f t e r thawing.  A f t e r two minutes o n l y one l a r g e venous channel was flowing  within  the i n j u r e d area.  There was  f u r t h e r change noted a f t e r f o u r minutes,  no  and n© r e -  s t o r a t i o n o f c i r c u l a t i o n i n any s t a t i c c h a n n e l .  161 Animal 4 (Test) S t a s i s commenced s i x t y seconds a f t e r i n one c a p i l l a r y bed ©nly. one venule was  thawing,  A l i t t l e d i s t a n c e away  observed to be opening and  closing,  but t h i s v e s s e l became s t a t i c two and a h a l f a f t e r thawing.  Stasis  minutes  spread to o n l y ©ne ©ther  c a p i l l a r y bed by t h r e e and a h a l f minutes a f t e r thawing.  T h i s amount ©f r e a c t i o n  was  a rather less  than would be expected from the amount ©f i n the i n j u r e d  area.  By f i v e minutes  twenty p e r c e n t o f the I n j u r e d area was restoration  effect  vascularity  after  thawing  in stasis.  o f the c i r c u l a t i o n was noted.  N©  The Ringer-  Locke g e l a t i n - g l u c o s e d r i p was not observed to have any a p p r e c i a b l e harmful e f f e c t on the behaviour o f the v e s s e l s i n the p r e p a r a t i o n . Animal 5 ( C o n t r o l ) Stasis There was  rapid  of the i n j u r e d  started  t h i r t y seconds a f t e r  spread t© i n v o l v e t w e n t y - f i v e p e r c e n t area i n s t a s i s w i t h i n s i x t y  S i x t y pereent o f the area was a f t e r thawing.  thawing.  seconds.  s t a t i c by t h r e e minutes  Affew venules were observed to s t a r t  and stop t h e i r f l o w o f b l o o d , w i t h no r e a l s t a s i s i n these channels. minutes,  There was no f u r t h e r  change by f i v e  and no resumption of f l o w observed.  Animal 6 (Test) S t a s i s was  noted throughout the mesoappendix  162 on mounting and the p r e p a r a t i o n was d i s c a r d e d . Animal 7 ( C o n t r o l ) S t a s i s o c c u r r e d w i t h i n f i f t e e n seconds a f t e r thawing.  I t became g e n e r a l i z e d i n the i n j u r e d a r e a  w i t h i n f o r t y - f i v e seconds a f t e r thawing.  One ven-  ous channel was observed to be f l u s h i n g open every l i t t l e while.  The whole i n j u r e d a r e a was s t a t i c by  three and a h a l f minutes a f t e r thawing, er change was noted by f i v e minutes,  and no f u r t h -  w i t h no resumpt-  i o n o f f l e w apparent. Animal 8 (Test) S t a s i s s t a r t e d w i t h i n f i f t e e n seconds o f thawing, and became g e n e r a l i z e d throughout the i n j u r ed area by s i x t y seconds.  No resumption o f f l o w  was noted except i n a few o f the l a r g e r venules t h a t were n o t t o t a l l y s t a t i c .  No f u r t h e r change was  noted by f i v e minutes a f t e r thawing. Animal 9 ( C o n t r o l ) Stasis started within forty-five a f t e r thawing.  seconds  Twenty-percent o f the i n j u r e d a r e a  was i n v o l v e d w i t h i n two minutes a f t e r thawing, s e v e n t y - f i v e p e r c e n t i n f o u r minutes.  and  No resumption  of f l o w was n o t e d . Animal 10 (Test) S t a s i s s t a r t e d w i t h i n f i f t e e n minutes thawing,  after  and was t e t a l i n the i n j u r e d a r e a w i t h i n  163 s i x t y seconds except f o r one c e n t r a l venous channel t h a t stopped and s t a r t e d f o r another s i x t y only.  seconds  There was no f u r t h e r change noted by three  minutes a f t e r thawing. Animal 11 ( C o n t r o l ) S t a s i s s t a r t e d f o r t y - f i v e seconds a f t e r thawing.  Only one c a p i l l a r y bed became i n v o l v e d by  s t a s i s by three minutes a f t e r thawing. changes were noted by f i v e minutes a f t e r  No f u r t h e r thawing.  Animal 12 (Test) S t a s i s s t a r t e d s i x t y seconds a f t e r thawing, spreading by two minutes a f t e r thawing.  By f o u r  minutes a f t e r thawing e i g h t y p e r c e n t o f the i n j u r e d area was i n s t a s i s ,  and by f i v e minutes a f t e r thaw-  i n g the area was one hundred p e r c e n t s t a t i c .  N©  resumption o f f l o w was n o t e d . Summary o f Observations w i t h C h l o r - T r i p o l o n No.  Onset S t a s i s  Spread  Generalized  20% i n 60"  60$ I n 3'  None  90$ i n 60"  None  90$ i n 2«  None  1  Control  30"  2  Test  15"  3  Contrel  30"  60$ i n 45"  4  Test  60"  Slight  5  Control  30"  25$ i n 60"  6  Test  Preparation discarded  7  Control  15"  Rapid  Plow Resumed  Two beds i n 3^» 60$ I n 3'  General i n 45"  None None  100$ i n 3£» None  164 Summary (Cont'd) N©«  Onset S t a s i s  Generalized  Spread Rapid  Flew Resumed None  General i n 60"  8  Test  15  9  Control  45"  20$ i n 2'  75$ i n 4«  None  10  Test  15"  90$ i n 60"  100$ i n 2»  None  11  Control  45"  12  Test  60"  tt  None  1 Cap bed i n 3»  80$ i n 4'  100$ i n 5«  None None  D i s c u s s i o n o f Observations The  animals p r e t r e a t e d w i t h  c o u l d be claimed  Chlor-Tripolon  to d i f f e r i n no r e s p e c t from those  c o n t r o l animals n o t p r e t r e a t e d w i t h  Chlor-Tripolon,  Rutin Experimental Observations w i t h All  the o b s e r v a t i o n s  Rutin  regarding r u t i n micro-  s c o p i c a l l y were performed by a s e n i o r m e d i c a l h e l p i n g w i t h the p r o j e c t p a r t time.  student  H i s method o f  t i m i n g i s s l i g h t l y v a r i e d from t h a t used elsewhere, and he uses the term "general  s t a s i s " to apply to the  whole v a s c u l a r bed o f the mesoappendix mount,  whereas  the term i s used elsewhere to apply o n l y to the v e s s e l s w i t h i n the f i e l d a c t u a l l y f r o z e n by the c o l d p o i n t , the s o - c a l l e d i n j u r e d area.  When s t a s i s develops  throughout thewhole p r e p a r a t i o n , regarded as u n p h y s i o l o g i c a l , carded,  such a mounting i s  and the animal i s d i s -  and the r e s u l t s are n o t i n c l u d e d .  165 The t e s t animals were prepared w i t h r u t i n p r i o r te the experimental o b s e r v a t i o n by s e l e c t i n g six  normal  a d u l t male Wistar r a t s , p l a c i n g them i n  one cage, and p u t t i n g I n t e t h e i r d r i n k i n g water the ground up r u t i n t a b l e t s .  600 mg. o f r u t i n was  p l a c e d i n about 250 c c . o f water,  and the animals  were observed t o be d r i n k i n g the water orily.  satisfact-  T h i s was kept b e f o r e them f o r f i v e  full  days, b e s i d e s t h e i r u s u a l f e e d , and then they were t e s t e d i n the u s u a l manner as o u t l i n e d below. E i g h t s i m i l a r animals were s e l e c t e d as c o n t r o l s . These were managed i n a l i k e manner, but d i d n o t r e c e i v e any r u t i n .  The f o l l o w i n g o b s e r v a t i o n s are  summarized i n t a b l e form a f t e r Animal 14. Animal 1 ( C o n t r o l ) A male Wistar r a t weighing 220 grams t h a t had r e c e i v e d no p r i o r m e d i c a t i o n w i t h r u t i n was anaestheti z e d w i t h 0.15 c c . of 0.6$ nembutal s o l u t i o n subcutaneously. minutes l a t e r , of  The p r e p a r a t i o n was ready but was d i s c a r d e d because  the microscope  injected twelve  i t fell off  stage.  Animal 2 ( C o n t r o l ) A male Wistar r a t weighing 180 grams t h a t had r e c e i v e d no p r i o r treatment w i t h r u t i n was anaestheti z e d w i t h 0.15 c c . o f 0.6$ nembutal s o l u t i o n , ed subcutaneously,  inject-  but the p r e p a r a t i o n had to be  166 discarded  (reason not r e c o r d e d ) .  Animal 5 ( C o n t r o l ) An u n t r e a t e d male Wistar r a t of 190 was  a n a e s t h e t i z e d w i t h 0.15  ution.  c c . o f 0.6$  grams  nembutal  A s e l e c t e d area of the meaioappendix  sol-  was  f r o z e n f o r t h i r t y seconds w i t h the c o l d p o i n t apparatus, f o l l o w e d by immediate r a p i d thawing w i t h the warm d r i p s o l u t i o n which had been d i s c o n t i n u e d d u r i n g the f r e e z i n g procedure.  V a s c u l a r s t a s i s began w i t h i n  t h i r t y seconds a f t e r thawing and spread to i n v o l v e about s i x c a p i l l a r y beds by f i v e minutes a f t e r thawing.  There was  no g e n e r a l i z e d s t a s i s or any  appar-  ent r e s t o r a t i o n of b l o o d f l o w i n the s t a t i c v e s s e l s . Numerous c a p i l l a r i e s and one medium s i z e d venule were Involved. Animal 4 ( C o n t r o l ) A normal a d u l t male Wistar r a t o f 190 t h a t had r e c e i v e d no p r i o r m e d i c a t i o n was i z e d w i t h 0.15  c c . o f 0.6$  to be administered,  anaesthet-  nembutal s o l u t i o n .  teen minutes l a t e r a f u r t h e r 0.05  Seven-  c c . o f nembutal had  and the animal was  a n a e s t h e t i z e d f i f t e e n minutes l a t e r . area of the mesoappendix was  grams  satisfactorily A  selected  f r o z e n f o r t h i r t y sec-  onds f o l l o w e d by immediate r a p i d thawing. began w i t h i n t h i r t y seconds o f thawing,  Stasis and  spread  167 to i n v o l v e f i v e or s i x c a p i l l a r y beds i n two There was  no g e n e r a l  minutes.  s t a s i s and no resumption of  i n those v e s s e l s i n which s t a s i s occurred. a r i e s and  (Control) A 170  gram normal a d u l t male Wistar  r e c e i v e d no p r i o r  0.15  Capill-  small c o l l e c t i n g venules were i n v o l v e d .  Animal 5  had  flow  c c . o f 0.6$  i o n a l 0.05  treatment was  r a t which  anaesthetized  with  nembutal s o l u t i o n r e q u i r i n g an a d d i t -  c c . twice w i t h i n the f o l l o w i n g f i f t e e n min-  utes.  Vascular  after  thawing and  s t a s i s began w i t h i n t h i r t y seconds i n v o l v e d about ten c a p i l l a r y beds  w i t h i n f o u r minutes.  There was  or resumtpion o f flow.  no g e n e r a l  Capillaries  and  stasis  small ven-  u l e s were a f f e c t e d . Animal 6  (Test) A 170  gram normal a d u l t Wistar male r a t  t r e a t e d w i t h r u t i n was o f 0.6$  anaesthetized  w i t h 0.15  cc.  nembutal s o l u t i o n f o l l o w e d In f i f t e e n min-  utes by another 0.05  cc.  F o l l o w i n g r a p i d thawing,  v a s c u l a r s t a s i s began w i t h i n t h i r t y seconds and  spread  to i n v o l v e three or f o u r c a p i l l a r y beds by f o u r minutes  a f t e r thawing.  There was  R e s t o r a t i o n of c i r c u l a t i o n was venule two minutes a f t e r  no  general  noted i n a  thawing.  spread. collecting  Capillaries  small c o l l e c t i n g venules were i n v o l v e d i n the Animal 7  and  stasis.  (Control) A normal a d u l t male Wistar r a t o f 170  grams  168 p r e v i o u s l y u n t r e a t e d w i t h r u t i n was w i t h 0.15  c c . of 0.6$  anaesthetized  nembutal s o l u t i o n , without  a d d i t i o n a l nembutal b e i n g r e q u i r e d ,  which r e s u l t e d  i n a l i g h t l y a n a e s t h e t i z e d animal. o c c u r r e d i n t h i r t y seconds. of  c i r c u l a t i o n was  No  stasis  R e v e r s a l of d i r e c t i o n  noted i n some of the medium s i z e d  venules w i t h i n one minute of thawing, but no was  observed  Animal 8  any  anywhere In the  stasis  field.  (Test) A normal a d u l t male Wistar r a t of 170  previously treated with r u t i n , w i t h 0.17  c c . o f 0.6$  was  anaesthetized  nembutal s o l u t i o n ,  i n f i f t e e n minutes w i t h 0.07  followed  c c . more, b o t h  i o n s being g i v e n subcutaneously.  grams,  inject-  S t a s i s began w i t h i n  f o r t y - f i v e seconds of r a p i d thawing, and  spread  s l i g h t l y to i n v o l v e about three c a p i l l a r y beds w i t h i n f i v e minutes a f t e r thawing. i n v o l v e d only about one i n j u r e d area.  S t a s i s was  and  and  t h i r d of the v e s s e l s i n the  No g e n e r a l s t a s i s or r e s t o r a t i o n o f  c i r c u l a t i o n i n the s t a t i c v e s s e l s was illaries  slight  noted.  Cap-  small venules were i n v o l v e d i n the  stasis.  Animal 9 (Te3t) A normal a d u l t male Wistar r a t of 160 was  a n a e s t h e t i z e d w i t h 0.15  c c . of 0.6$  s o l u t i o n a f t e r f u l l pretreatment  nembutal  with r u t i n .  commenced s e v e n t y - f i v e seconds a f t e r thawing, spread to i n v o l v e two  grams  Stasis and  or three c a p i l l a r y beds i n f o u r  169 minutes.  About one  t h i r d of the v e s s e l s i n the  i n j u r e d area were i n v o l v e d . resumption i e s and  o f flow was  No general s t a s i s or  observed  to o c c u r .  Capillar-  small c o l l e c t i n g venules were a f f e c t e d .  Animal 10  (Test)  A normal a d u l t male Wistar r a t of 210 p r e v i o u s l y t r e a t e d w i t h r u t i n was 0.21  c c . o f 0.6$  cutaneously.  anaesthetized with  nembutal s o l u t i o n administered subV a s c u l a r s t a s i s began w i t h one minute  a f t e r r a p i d thawing,  and spread to i n v o l v e by f o u r  minutes a f t e r thawing o n l y f o u r or f i v e beds.  grams  About two  capillary  t h i r d s of the v e s s e l s i n the i n -  j u r e d area were i n v o l v e d . sumption of flow was  No g e n e r a l s t a s i s or r e -  observed.  Capillaries  small c o l l e c t i n g venules were i n v o l v e d i n the  and stasis.  Animal 11 ( C o n t r o l ) A normal a d u l t male Wistar r a t o f 220 gram3 not p r e v i o u s l y t r e a t e d w i t h r u t i n was w i t h 0.18  c c . o f 0.6$  anaesthetized  nembutal s o l u t i o n f o l l o w e d i n  twenty minutes w i t h 0.08  c c . more.  p i r e d during observation,  The animal  ex-  and no r e s u l t s are r e c o r d -  ed. Animal 12  (Test)  A normal a d u l t male Wistar r a t of 180  grams,  having r e c e i v e d p r i o r treatment w i t h r u t i n ©rally, was  a n a e s t h e t i z e d w i t h 0.20  c c . of 0.6$  s o l u t i o n i n j e c t e d subcutaneously.  nembutal  S t a s i s commenced  170 w i t h i n t h i r t y seconds a f t e r r a p i d thawing,  and i t  spread to i n v o l v e about s i x c a p i l l a r y beds w i t h i n three minutes a f t e r thawing.  Two  t h i r d s of  v e s s e l s In the i n j u r e d area were i n s t a s i s , was  the but  there  no g e n e r a l i z e d s t a s i s throughout the p r e p a r a t i o n .  C i r c u l a t i o n was  observed  C a p i l l a r i e s and  small venules were i n v o l v e d i n the  static  to be resumed i n two v e s s e l s .  process.  Animal 13  (Control)  A normal a d u l t male Wistar r a t o f 190 not p r e v i o u s l y t r e a t e d w i t h r u t i n was w i t h 0.20  c c . of 0.6$  subcutaneously.  grams  anaesthetized  nembutal s o l u t i o n  administered  S t a s i s commenced w i t h i n s i x t y -  f i v e seconds a f t e r thawing,  spreading  so as to i n -  v o l v e three c a p i l l a r y beds w i t h i n three minutes. About one  q u a r t e r of the c a p i l l a r i e s i n the i n j u r e d  area were i n v o l v e d .  There was  no  s t a s i s ott  i o n of c i r c u l a t i o n i n the s t a t i c channels. i l l a r i e s o n l y were i n v o l v e d i n the Animal 14  resumptCap-  process.  (Test)  A normal a d u l t male Wistar r a t of 190 p r e v i o u s l y t r e a t e d w i t h r u t i n , was 0.20  c c . o f 0.6$  cutaneously.  anaesthetized  nembutal s o l u t i o n administered  There was  discarded.  with sub-  generalized vascular stasis  p r e s e n t ©n mounting the mesoappendix, a r a t i o n was  grams  and the  prep-  171 Summary of Observations w i t h Onset; S t a s i s  No,  Rutin  Spread  Generalized  Plow Resumed  1  Control  No r e s u l t s , f e l l o f f microscope stage  2  Control  No r e s u l t s ,  3  Control  30"  6 beds i n 5»  None  None  4  Control  30"  5-6 beds I n 2«  None  None  5  Control  30"  10 beds i n 4'  None  None  6  Test  30"  3-4 beds i n 4«  None  7  Control  None  8  Test  45"  3 beds i n 5»  None  None  9  Test  90"  2-3 beds i n 4«  None  None  10  Test  60"  4-5 beds i n 4>  None  None  11  Control  No r e s u l t s ,  12  Test  30"  6 beds i n 3'  None  13  Control  65"  3 beds i n 3'  None  14  Test  No r e s u l t s ,  discarded  expired during  /  observation  / None  general s t a s i s on mounting  D i s c u s s i o n o f Observations The  s i x t e s t animals, f o r the f i v e days b e f o r e '  the experiments took p l a c e , were kept i n one cage and allowed  to d r i n k from the same d r i n k i n g b o t t l e s .  This  together w i t h the f a c t the r u t i n tended t o s e t t l e somewhat to the bottom o f the c o n t a i n e r probably an uneven d i s t r i b u t i o n o f the m e d i c a t i o n The  caused  among the r a t s .  average time a f t e r thawing f o r s t a s i s t o  commence i n f i v e t e s t animals was 50".  Substantially  the same time, 60", h e l d f o r f o u r c o n t r o l  animals (not  172 c o n s i d e r i n g Animal 7 i n which n© s t a s i s o c c u r r e d ) . In animals  6 and 12,  s t a t i c channels the observer  d e f i n i t e resumption o f f l o w i n  occurred.  To quote the ©pinion o f  of these experiments, "Rutin shows some  d e l a y i n g i n the onset of s t a s i s , but s l i g h t i f any decrease  i n the area i n v o l v e d .  channels  resumed f l o w .  In two  animals  the  These r e s u l t s cannot be  c o n s i d e r e d d e f i n i t e or C o n s i s t e n t enough to warrant p o s i t i v e conclusions, i n g , and  but the drug appears promis-  the t e s t s should be repeated w i t h care to  ensure an accurate dosage per  animal."  Further observations with Rutin S i x r a t s as noted below were f e d R u t i n caref u l l y so t h a t each r e c e i v e d t w e n t y - f i v e mg. f o r f o u r days, f o l l o w e d by ©ne hundred mg. f o r f o u r more days. from the m e d i c a t i o n . ected as c o n t r o l s .  per  They showed no adverse Seven other animals As mentioned these  were made by a s e n i o r medical  student.  t a b l e form appears a f t e r Animal Animal 1  per  day day  effect  were s e l -  observations A summary i n  13.  (Test) A normal a d u l t nonpregnant female Wistar r a t  weighing 190 o r a l l y was  grams, p r e v i o u s l y t r e a t e d w i t h  a n a e s t h e t i z e d w i t h 0.1  s o l u t i o n administered  c c . of 0.6$  subcutaneously.  rutin nembutal  Vascular  stasis  173 was  observed to occur immediately a f t e r r a p i d  thawing,  which spread to i n v o l v e seven e i g h t h s of the i n j u r e d area w i t h i n two and a h a l f 'minutes a f t e r thawing. generalized s t a s i s occurred,  No  and no resumption o f  c i r c u l a t i o n i n the s t a t i c areas was observed. Animal 2 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 200 grams was 0.6$ nembutal  solution.  a n a e s t h e t i z e d w i t h 0.1  cc. of  Vascular s t a s i s occurred  immediately a f t e r thawing which r a p i d l y spread t© i n v o l v e a l l v e s s e l s i n the i n j u r e d  area.  Animal 5 (Test) A normal a d u l t nonpregnant female W i s t a r r a t of 195 grams was nembutal  a n a e s t h e t i z e d w i t h 0.1  solution.  cc. of  0.6$  Stasis started within forty  seconds a f t e r thawing, and spread to I n v o l v e h a l f the v e s s e l s i n the i n j u r e d a r e a w i t h i n three minutes  after  thawing. Animal 4 ( C o n t r o l ) A normal a d u l t nonpregnant female W i s t a r r a t weighing 200 grams was 0.6$ nembutal  solution.  a n a e s t h e t i z e d w i t h 0.1  cc. of  The whole mesoappendix went  i n t o v a s c u l a r s t a s i s from the f r e e z i n g , and the prepa r a t i o n was  discarded.  Animal 5 (Test) A normal a d u l t nonpregnant female Wistar r a t  174 weighing 160 grams was of 0.6$  a n a e s t h e t i z e d w i t h 0.1  nembutal s o l u t i o n administered  cc.  subcutaneously.  S t a s i s began w i t h i n f o r t y seconds a f t e r thawing,  and  i n v o l v e d o n l y f o u r s h o r t segments o f c a p i l l a r i e s . Animal 6 ( C o n t r o l ) A normal a d u l t nonpregnant female Wistar r a t weighing 180 0.6$  grams was  a n a e s t h e t i z e d w i t h 0.1  nembutal s o l u t i o n .  cc. of  S t a s i s began i n f i f t y - f i v e  seconds a f t e r thawing and  spread  to i n v o l v e one  third  of the v e s s e l s i n the f r o z e n area by f o u r minutes a f t e r thawing. Animal 7  (Test) A normal a d u l t nonpregnant female Wistar r a t  weighing 200 0.6$  grams was  a n a e s t h e t i z e d w i t h 0.1  nembutal s o l u t i o n administered  S t a s i s developed  within f i f t y  spreading to i n v o l v e one injured Animal 8  cc. of  subcutaneously.  seconds a f t e r thawing,  q u a r t e r of the v e s s e l s i n the  area. (Control) A normal a d u l t nonpregnant female Wistar r a t  weighing 180 0.6$  grams was  a n a e s t h e t i z e d w i t h 0.1  nembutal s o l u t i o n .  S t a s i s began i n s i x t y  seconds a f t e r thawing and i n v o l v e d o n l y two capillaries. e r a l small  There was  capillaries.  cc. of  or three  resumption of f l o w i n sev-  175 Animal 9  (Teat) A normal a d u l t nonpregnant female Wiatar r a t  weighing n.6$  180  grams was  a n a e s t h e t i z e d w i t h 0.1  nembutal s o l u t i o n .  cc. of  Stasis started forty-five  seconds a f t e r r a p i d thawing, i n v o l v i n g three or f o u r s h o r t segments of Animal 10  capillaries.  (Control)  A normal a d u l t male Wistar r a t weighing grams was  a n a e s t h e t i z e d w i t h 0.1  s o l u t i o n administered  c c . o f 0.6$  subcutaneously.  (  200  nembutal  Stasis start-  ed w i t h i n one minute a f t e r thawing to i n v o l v e h a l f the v e s s e l s i n the i n j u r e d Animal 11  area.  (Test)  A normal a d u l t nonpregnant female Wiatar r a t weighing 190 of 0.6$  grams was  a n a e s t h e t i z e d w i t h 0.13  nembutal s o l u t i o n .  Stasis started sixty  seconds a f t e r r a p i d thawing and one  cc.  spread to i n v o l v e  t h i r d o f the v e s s e l s In the i n j u r e d area i n three  minutes. Animal 12  (Control)  A normal a d u l t male W i s t a r r a t weighing grams was  a n a e s t h e t i z e d w i t h 0.13  u t a l s o l u t i o n administered  c c . of 0.6$  subcutaneously.  began f u t h i r t y v e seconds a f t e r thawing and i n v o l v e two  200  nembStasis  spread  t h i r d s of the v e s s e l s i n the i n j u r e d  to area.  176 Animal 15 ( C o n t r o l ) A normal a d u l t male Wistar r a t weighing 200 grams was a n a e s t h e t i z e d w i t h 0.13 c c . o f 0.6$ nemb u t a l s o l u t i o n administered  subcutaneously.  began f o r t y - f i v e seconds a f t e r thawing,  Stasis  and spread  to i n v o l v e two t h i r d s o f the v e s s e l s i n the i n j u r e d area. Summary o f Observations Onset S t a s i s  No.  with  Rutin  Spread  Generalized 7/8  Plow Re None  1  Test  Immediate  Slow  2  C o n t r o l Immediate  Rapid  3  Test  4  C o n t r o l No r e s u l t s , p r e p a r a t i o n  5  Test  40"  Slow  Minimal  None  6  Control  55"  Slow  1/3  None  7  Test  50"  Slow  1/4  None  8  Control  60"  Slow  Minimal  //  9  Test  60"  Slow  1/2  None  10  Control  60"  Slow  1/2  None  11  Test  60"  Slow  1/3  None  12  Control  30"  Slow  3/4  None  13  Control  45"  Slow  2/3  None  Discussion of  40"  1  Complete  Slow  1/2  None None  discarded  Observations  As mentioned above,  these o b s e r v a t i o n s  using  177 r u t i n were performed by another worker. r e a d i n g the data,  Prom  i t would appear t h a t the f r e e z -  i n g i n j u r y might be a l i t t l e more severe than customary w i t h the o t h e r t e s t s .  was  Even though t h i s  would p r e v e n t comparison w i t h the c o n t r o l s of o t h e r experimemts, the t e s t and c o n t r o l animals of t h i s run  should be  comparable.  The time r e q u i r e d  f o r the onset o f s t a s i s  a f t e r the r a p i d thawing was  Identical f o r both  s e r i e s (average of s i x animals each), namely f o r t y seconds. the  The speed and extent o f the spread o f  sifeasis was  control  the same f o r both the t e s t and the  series.  I t i s a l s o i n t e r e s t i n g to note  t h a t the o n l y i n s t a n c e o f resumption of f l o w i n the  s t a t i c vessels  o c c u r r e d i n a c o n t r o l animal.  To quote the impressions o f the o r i g i n a l observer, "The r e s u l t s of t h i s experiment  indicate  t h a t r u t i n does not a l t e r the r e a c t i o n o f c a p i l l a r i e s i n the r a t mesoappendix a f t e r f r e e z i n g and r a p i d thawing." Ascorbic  Acid  T h i s drug was not t e s t e d  microscopically  because the observed e f f e c t s on a gross standard f r e e z i n g i n j u r y d i d not warrant f u r t h e r  Investigation.  178 Histamine Experimental Observations w i t h Histamine Histamine i n the dosage l e v e l o f 100 mg. p e r k i l o g r a m body weight was t e s t e d by p a r e n t e r a l i n j e c t ion.  Summary t a b l e o f the f o l l o w i n g o b s e r v a t i o n s  appears  a f t e r Animal  9.  Animal 1 (Test) A normal a d u l t male W i s t a r r a t weighing 272 grams was i n j e c t e d subcutaneously w i t h 0.27 c c . o f a d i l u t e d histamine s o l u t i o n c o n t a i n i n g 100 mg. p e r cc,  and f i f t e e n minutes l a t e r the animal was an-  a e s t h e t i z e d w i t h 0.075 c c o f 0.6$ nembutal i o n a d m i n i s t e r e d subcutaneously.  solut-  0.05 c c . o f  nembutal were repeated i n t e n minutes and another 0.025 c c . three minutes a f t e r w a r d s . t i o n was ready on the microscope  The p r e p a r a -  stage  minutes a f t e r the f i n a l i n j e c t i o n ,  seventeen  and the c o l d  p o i n t was a p p l i e d to a s e l e c t e d area o f the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds, by immediate r a p i d thawing.  followed  Generalized vascular  s t a s i s developed i n the i n j u r e d area w i t h i n s i x t y seconds  o f thawing.  Animal 2 ( T e s t ) A normal a d u l t male W i s t a r r a t weighing 241 grams was I n j e c t e d subcutaneously w i t h 0.24 c c o f a  179 solution  c o n t a i n i n g 100 mg. o f h i s t a m i n e p e r  and s i x t y - t h r e e etized  minutes  with 0.20  cc.  subcutaneously.  ready,  the  of  the  tip  was  by immediate  oped i n t h i r t y rapidly  to  three minutes  Animal  3  seconds,  Stasis  devel-  progressing  the  injured  area  thawing.  adult  was a n a e s t h e t i z e d  Wistar rat  w i t h 0.1  cc.  minutes  after  t i o n was r e a d y t i p was  a further that  immediate  in  the  0.05  i n another  a p p l i e d to  appendix w i t h f r e e z i n g by  0.05  area  cc.  was  nembutal  injection.  In  injected  in  more.  The  eight minutes,  for  within  thirty  and  seconds,  General thirty  and  prepara-  a selected portion of  rapid thawing.  injured  cc.  w e i g h i n g 235  of 0.6$  administered by subcutaneous  twenty minutes  cold  thirty  portion  (Control)  solution  five  of  was  selected  thawing,  percent  after  A normal male grams  a  freezing for  sixty  within  preparation  a p p l i e d to  after  anaesth-  s o l u t i o n admin-  rapid thawing.  seconds  involve  a n i m a l was  When t h e  mesoappendix w i t h  followed  the  o f 0.6$ nembutal  istered  cold  later  c c ,  the the  followed  stasis  seconds  meso-  developed  after  thaw-  ing. Animal  4  (Test) A normal adult  grams  was  injected  male W i s t a r r a t  subcutaneously  with  weighing  244  0.24  of  cc.  a  180 s o l u t i o n c o n t a i n i n g histamine, i n the of 100 mg.  per c c .  The  animal was  i n twenty minutes w i t h 0.20  concentration anaesthetized  c c . of 0.6$  nembutal  s o l u t i o n administered by subcutaneous i n j e c t i o n , and when the p r e p a r a t i o n was stage,  the c o l d t i p was  ready on the microscope  a p p l i e d to a s e l e c t e d area  of the mesoappendix w i t h f r e e z i n g f o r t h i r t y S t a s i s d i d not develop  seconds.  i n any v e s s e l w i t h i n seven  minutes a f t e r thawing, at which time the p r e p a r a t i o n was  discontinued.  Good vasomotion was  maintained  throughout the o b s e r v a t i o n p e r i o d . Animal 5  (Test) A normal a d u l t male Wistar r a t weighing  grams was  i n j e c t e d subcutaneously  a s o l u t i o n c o n t a i n i n g 100 mg. and i n twenty-eight i z e d w i t h 0.20  cc  w i t h 0.24  of 0.6$  cc. of  o f histamine per  minutes the animal was  247  cc,  anaesthet-  nembutal s o l u t i o n a l s o  administered by subcutaneous i n j e c t i o n .  S t a s i s dev-  eloped w i t h i n t h i r t y seconds of thawing,  and  spread  to i n v o l v e t h i r t y p e r c e n t of the i n j u r e d area w i t h i n f o u r minutes ©f thawing. Animal 6 ( C o n t r o l ) A normal a d u l t male Wistar r a t weighing grams was  a n a e s t h e t i z e d w i t h 0.15  c c . o f 0.6$  208  nem-  b u t a l s o l u t i o n administered by subcutaneous I n j e c t i o n .  181 The p r e p a r a t i o n was mounted on the microscope stage and the c o l d p o i n t was  a p p l i e d to a s e l e c t e d a r e a o f  the mesoappendix w i t h f r e e z i n g wor f o l l o w e d by immediate  thirty  r a p i d thawing.  seconds,  Stasis devel-  oped w i t h i n t h i r t y seconds a f t e r thawing,  rapidly  spreading t o become g e n e r a l i z e d w i t h i n the i n j u r e d area w i t h i n n i n e t y seconds a f t e r  thawing.  Animal 7 ( T e s t ) A normal a d u l t male W i s t a r r a t weighing 270 grams was  i n j e c t e d subcutaneously w i t h 0.27  cc. of a  histamine s o l u t i o n c o n t a i n i n g 100 mg.  per c c ,  i n t h i r t y - f i v e minutes the animal was  anaesthetized  w i t h 0.20  c c . o f 0.6$ nembutal  and  s o l u t i o n a l s o admin-  i s t e r e d by subcutaneous i n j e c t i o n .  The p r e p a r a -  t i o n was mounted on the microscope stage and the c o l d p o i n t was  a p p l i e d to a s e l e c t e d area o f the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds, by immediate r a p i d thawing.  followed  S t a s i s developed w i t h i n  f o r t y - f i v e seconds a f t e r thawing,  becomming q u i t e  g e n e r a l i z e d w i t h i n the i n j u r e d area. Animal 8 ( C o n t r o l ) A normal a d u l t male W i s t a r r a t weighing 286 grams was  a n a e s t h e t i z e d w i t h 0.25  butal solution,  c c . of a 0.6$ nem-  and when ready the p r e p a r a t i o n  mounted on the microscope stage.  was  The c o l d p o i n t  was.  182 a p p l i e d to a s e l e c t e d area of the mesoappendix w i t h f r e e z i n g f o r t h i r t y seconds, r a p i d thawing.  f o l l o w e d by immediate  S t a s i s developed w i t h i n s i x t y sec-  onds a f t e r thawing,  r a p i d l y becomming g e n e r a l i z e d  w i t h i n the i n j u r e d area by two  arid a h a l f minutes  a f t e r thawing. Animal 9 ( T e s t ) A normal a d u l t male Wistar r a t weighing grams was  i n j e c t e d w i t h 0.24  u t i o n c o n t a i n i n g 100 mg. was  per c c ,  a n a e s t h e t i z e d w i t h 0.20  solution. p o i n t was  c c . of a histamine and the  c c . of 0.6$  The p r e p a r a t i o n was  244 sol-  animal  nembutal  mounted and the c o l d  a p p l i e d to a s e l e c t e d area of the meso-  appendix w i t h f r e e z i n g f o r t h i r t y seconds.  Stasis  s t a r t e d w i t h i n t h i r t y seconds a f t e r thawing,  but at  no time d u r i n g the p e r i o d of o b s e r v a t i o n d i d i t spread to i n v o l v e more than t w e n t y - f i v e p e r c e n t o f the i n j u r e d a r e a . Summary o f Observations w i t h Histamine No.  P a r e n t e r a l ~'  Bpsetastasis  Spread  Generalized by 60"  Flow Resumed  1  Test  30"?  Rapid  None  2  Test  30"  Rapid  60$ i n 3'  None  3  Control  30"  Rapid  by 30"  None  183 Summary  (Cont'd)  No.  Onset S t a s i 3  4  Test *  5  Test  6  Spread  Generalized  Flow Resumed  None '30"  Slow  30$ i n 4»  None  Control  30"  Rapid  by 90"  None  7  Test  45"  Rapid  8  Control  60"  Rapid  9  Test  30"  Slowly  None  Yes by Sfrt  None  o n l y 25$  None  D i s c u s s i o n o f Observations These t e s t s w i t h p a r e n t e r a l h i s t a m i n e were somewhat o f a t r i a l of h i s t a m i n e ,  and the r e c o r d i n g was incomplete i n a  few r e s p e c t s . used,  to determine an e f f e c t i v e dosage  There were i n s u f f i c i e n t c o n t r o l  f o r one t h i n g .  animals  Some o f the d e t a i l s r e g a r d i n g  time i n t e r v a l s are l a c k i n g .  None the l e s s , i t would seem  t h a t h i s t a m i n e , as here used, had some d e f i n i t e  effects  on the b e h a v i o r o f b l o o d v e s s e l s a f t e r a l o c a l i z e d freezing injury.  Although no d i f f e r e n c e was apparent  i n the time a f t e r thawing f o r s t a s i s t o s t a r t ,  the  extent and r a t e ©f spread o f the s t a s i s seemed to be much l e s s i n the histamine t r e a t e d animals.  It is  d o u b t f u l i f these f i n d i n g s are s i g n i f i c a n t i n view o f the o b j e c t i o n s here r a i s e d . The s e v e r i t y o f the f r e e z i n g i n j u r y would seem to be somewhat g r e a t e r here than i n other experiments*  184 F u r t h e r Observations  w i t h Histamine  The f o l l o w i n g t e s t s were performed t© study the e f f e c t o f histamine  a p p l i e d l o c a l l y ©ver the r a t  mesoappendix i n 1:1000 d i l u t i o n b e f o r e f r e e z i n g w i t h the c o l d p o i n t (the warm Ringer-Locke g e l a t i n - g l u c o s e d r i p being t e m p o r a r i l y d i s c o n t i n u e d f o r t h i s ,  but  of course b e i n g resumed f o l l o w i n g f r e e z i n g s© as t© e f f e c t an immediate r a p i d thawing,  and t© m a i n t a i n  a p h y s i o l o g i c a l p r e p a r a t i o n d u r i n g the o b s e r v a t i o n period). 150  The u s u a l technique was employed, and  gram normal a d u l t nonpregnant female Wistar r a t s  were employed, b e i n g a n a e s t h e t i z e d f i f t e e n minutes b e f o r e experimentation solution.  w i t h 0.15 c c . o f 0.6$ nembutal  A summary o f the f o l l o w i n g o b s e r v a t i o n s  appears i n t a b l e form a f t e r animal 10. Animal 1 ( C o n t r o l ) S t a s i s o c c u r r e d w i t h i n twenty seconds a f t e r thawing,  which r a p i d l y spread to i n v o l v e h a l f ©f the  v e s s e l s w i t h i n the i n j u r e d area by s i x t y seconds a f t e r i  thawing.  No resumption o f flow was observed  static vessels.  I t was noted  t h a t the l a r g e a r t e r i o -  venous b r i d g e s were f l o w i n g normally Injured  i n the  even through the  area.  Animal 2 ( T e s t ) Histamine I n 1:1000 c o n c n e t r a i o n was f l u s h e d  185 over the mesoappendix p r i o r to f r e e z i n g . observed  This  to g i v e g e n e r a l i z e d i n c r e a s e i n b l o o d f l o w .  A f t e r r a p i d thawing,  s t a s i s was  observed  i n a larger  venule w i t h i n s i x t y seconds a f t e r thawing. n i n e t y seconds a f t e r thawing, was in  was  found p r e s e n t ,  small c a p i l l a r y  stasis  but i n ©nly one c a p i l l a r y bed w i t h -  the i n j u r e d area.  noted up to two  By  There was  no f u r t h e r change  and a h a l f minutes a f t e r thawing when  the p r e p a r a t i o n was  discontinued.  Animal 5 ( C o n t r o l ) S t a s i s was thawing.  noted w i t h i n s i x t y seconds a f t e r  T h i s spread s l o w l y to I n v o l v e other  i l l a r y beds.  One  l a r g e venous channel c l e a r l y showed  hemoconcentration.  H a l f the i n j u r e d area was  to be i n s t a s i s by two minutes a f t e r thawing. t h i s time,  h i s t a m i n e , 1:1000, was  observation f i e l d ,  cap-  and t h i s was  instilled  found At  over  the  noted to cause some  d i l a t a t i o n ©f a venule w i t h resumed b l o o d f l o w In the l a r g e channel that had There was thawing.  shown the  hemoconcentration.  no f u r t h e r change by f i v e minutes a f t e r The h i s t a m i n e had no e f f e c t on the small  channels t h a t were d e f i n i t e l y s t a t i c .  N i n e t y sec-  onds a f t e r the histamine was  applied,  small venules resumed flow,  and the whole c a p i l l a r y  bed i t d r a i n e d resumed c i r c u l a t i o n .  one o f the  This process  186 was n o t d u p l i c a t e d elsewhere w i t h i n the i n j u r e d  area.  Animal 4 (Test) Histamine 1:1000 was f l u s h e d over the mesoappendix p r i e r to f r e e z i n g .  Generalized vascular  s t a s i s o c c u r r e d w i t h i n f i f t e e n seconds a f t e r  thawing.  One or two channels opened up and resumed b l o o d f l o w w i t h i n s i x t y seconds a f t e r  thawing,  b u t t h i s phen-  omenon i n v o l v e d only a s m a l l p o r t i o n o f the whole i n jured c i r c u l a t o r y  bed.  No f u r t h e r changes were ob-  served by f i v e minutes a f t e r  thawing.  Animal 5 ( C o n t r o l ) Small c a p i l l a r y bed s t a s i s was p r e s e n t t h i r t y seconds a f t e r  thawing.  A l a r g e r venous channel  showed a temporary slowing and s l u d g i n g o f b l o o d . f u r t h e r development was observed except t h a t l a r g e channel l a t e r became s t a t i c . was f l u s h e d over the area,  Ne  this  Histamine 1:1000  and w h i l e i t had no v i s i b l e  e f f e c t on t h i s l a r g e s t a t i c venule,  i t d i d cause the  c i r c u l a t i o n to r e f l o w i n the c a p i l l a r y bed t h a t  first  showed s t a s i s . Animal 6 (Test) r- •  There was g e n e r a l i z e d v a s c u l a r s t a s i s f i f t e e n seconds a f t e r r a p i d thawing.  within  No e f f e c t  u t a b l e to the histamine c o u l d be observed.  attrib-  There was  no resumption o f c i r c u l a t i o n i n any v e s s e l w i t h i n the i n j u r e d area.  187 Animal 7 ( C o n t r o l ) S t a s i s s t a r t e d w i t h i n f i f t e e n seconds a f t e r thawing, ured  and became g e n e r a l i z e d throughout the i n j -  area w i t h i n f o r t y - f i v e seconds.  channel showed i n t e r m i t t e n c y o f flow.  One venous Histamine  1:1000 when f l u s h e d over the area had no apparent effect. Animal 8 (Te3t) Vascular  stasis f i r s t  a f t e r r a p i d thawing. in  appeared n i n e t y seconds  Some i n t e r m i t t e n c y o f s t a s i s  a couple o f the l a r g e r channels was a t t r i b u t e d  to the histamine  i n s t i l l a t i o n p r i o r to f r e e z i n g ,  otherwise the s t a s i s r a p i d l y became g e n e r a l ^  but  through-  out the i n j u r e d a r e a . Animal 9 ( C o n t r o l ) S t a s i s o c c u r r e d w i t h i n t h i r t y seconds o f thawing and slowly became g e n e r a l i z e d w i t h i n n i n e t y seconds to i n v o l v e approximately s i x t y p e r c e n t I n j u r e d area. any  o f the  No r e s t o r a t i o n o f the c i r c u l a t i o n i n  s t a t i c v e s s e l was noted.  l a t e r w i t h histamine  1:1000,  On f l u s h i n g the f i e l d c i r c u l a t i o n resumed i n  a l a r g e venous channel but only t e m p o r a r i l y  and I n t e r -  mittently. Animal 10  (Test)  Tbe mesoappendix was f l u s h e d w i t h the warm  188 histamine 1:1000 solution, usual manner.  and then frozen In the  Stasis started within t h i r t y seconds  after rapid thawing,  but i n one c a p i l l a r y bed only,  with no spread of the stasis within ninety seconds after thawing.  Some sludging i n larger channels  was corrected completely by i n s t i l l i n g more histamine 1:1000 over the area,  and this e f f e c t was repeated  after the vessel sludged when the saline drip washed the histamine away.  No further change was noted by  f i v e minutes after thawing,  and the preparation was  discontinued. Summary of Observations No.  Onset Stasis  with Histamine (Topical) Spread  Generalized  Flow Resumed  1  Control  20"  Rapid  2  Test  60"  None  3  Control  60"  4  Test  5  Control  6  Test  7  Control  15"  8  Test  90"  9  Control  30"  Slow 60$ i n 90" None Histamine gave doubtful effect  Test  30"  None Histamine corrected sludging  10  50$ i n 60" None  None  Slow 50$ i n 2» None Histamine gave s l i g h t resumption of flow i n 15"  30"  None  /  Slow larger venules None Histamine gave s l i g h t resumption In flow i n 15"  None  Rapid i n 15" None Histamine gave no apparent effect Rapid  Yes  189 D i s c u s s i o n of Observations w i t h Histamine On the average,  the t e s t animals took f o r t y  seconds to develop s t a s i s ,  while the c o n t r o l s r e -  q u i r e d o n l y t h i r t y f o l l o w i n g r a p i d thawing. was however not c o n s i s t e n t enough to be  This  significant.  Histamine a p p l i e d t o p i c a l l y over the c o n t r o l p r e p a r a t i o n s a f t e r s t a s i s had o c c u r r e d f r e q u e n t l y o  p e r m i t t e d the r e s t o r a t i o n of c i r c u l a t i o n i n s t a t i c channels.  In comparing t h i s a c t i o n w i t h a s i m i l a r  one o b t a i n e d by f l u s h i n g p r o c a i n e over the mesoappendix, i t might be suggested that the e f f e c t could be o b t a i n e d through mere p h y s i c a l a c t i o n of the weight of the f l u i d b e i n g a p p l i e d from a h e i g h t . Histamine c o u l d not be d e f i n i t e l y c r e d i t e d w i t h any s i g n i f i c a n t b e n e f i c i a l e f f e c t on the b l o o d channels f o l l o w i n g a standard f r e e z i n g  injury.  190  Gross F r o s t b i t e  Experiments  Reason f o r Gross Experiments As mentioned p r e v i o u s l y , on page 18, there was no reason t o b e l i e v e that the c a p i l l a r i e s o f the r a t mesoappendix f u n c t i o n e d i n the same way as the c a p i l l a r i e s o f the r a t ' s h i n d l e g , and s i n c e i t was n o t the p e r i t o n e a l contents t h a t would be a f f e c t e d by f r o s t b i t e , i t was t h e r e f o r e n e c e s s a r y to t e s t the same t e n m e d i c a t i o n s i n order to f i n d out whether they would I n f l u e n c e the course of the l e s i o n s produced i n the h i n d l e g of the r a t by a standard c o l d i n j u r y .  Shumacker ( 4 ) v e r y complete-  l y summarizes the standard i n j u r i e s o f a g r e a t many gross f r e e z i n g experiments performed by s e v e r a l different investigators.  I n almost a l l o f these  the tendency was to compare the r e s u l t i n g  conditions  of a s e t of t e s t e x t r e m i t i e s a t a c e r t a i n p e r i o d a f t e r a s p e c i f i e d c o l d i n j u r y to the r e s u l t i n g conditions of a similar set of control  extremities  the same l e n g t h o f time a f t e r the same s p e c i f i e d cold injury.  As an example o f what i s meant^an  experiment o f Shumacker, Radigan, Ziperman, and Hughs ( 4 ) i s summarized.  The t a i l s o f s i x t y -  f o u r mice were f r o z e n f o r f i v e seconds a t - 1 5 ° C , the animals h a v i n g r e c e i v e d no treatment.  Four  191 animals (6.3$) showed what the authors termed excellent result  (grade 1 r e a c t i o n ) , n i n e (14.1$)  showed a good r e s u l t , and f i f t y - f i v e a poor r e s u l t  an  (grade 3 r e a c t i o n ) .  (85.9$) showed  Then t h i r t y - t w o  mice were g i v e n a drug (Benadryl, but the d e t a i l s are  not important) and put to the same f r e e z i n g  injury.  Two  animals  r e s u l t ^ f o u r animals  (6.3$) showed an e x c e l l e n t (12.5$) showed a good r e s u l t ,  and twenty-eight animals (87.5) showed a poor result.  There need be no d i s c u s s i o n of these  ures except to mention s i g n i f i c a n t because  fig-  t h a t they are o b v i o u s l y  of the numbers of animals  used.  However, i n a s s e s s i n g the f r o s t b i t e i n j u r y , and i n comparing  d i f f e r e n t animals, i t seemed as  something more was  needed.  though  Frostbite, like  any  other p a t h o l o g i c a l p r o c e s s , i s a dynamic p r o c e s s . We  cannot say to-day whether one i n d i v i d u a l i s  i n a b e t t e r c o n d i t i o n than another, as f a r as the d i s e a s e i n q u e s t i o n i s concerned*,unless" we know  how  both were y e s t e r d a y and they w i l l be tomorrow. rt  A c c o r d i n g l y , In the f o l l o w i n g pages there w i l l some attempt  to r e c o r d the gross f r o s t b i t e  appear  results  i n a dynamic manner, and the concept, an a r b i t r a r y concept, granted, w i l l be e x p l a i n e d .  192 Standard F r e e z i n g I n j u r y Although i t i s somewhat t r i t e to mention it,  f r e e z i n g i s f r e e z i n g , and the temperature a t  which the f r e e z i n g i n j u r y i s i n f l i c t e d matters l i t t l e p r o v i d e d the degree o f c o l d i s s u f f i c i e n t to f r e e z e the e x t r e m i t y w i t h i n a reasonable p e r i o d of  time.  In the data summarized by Shumacker  ( )  ambient temperatures v a r y i n g from -10°C. to -55°G. were used, p r o v i d e d u s u a l l y by some mixture w i t h carbon d i o x i d e snow and u s u a l l y ether i n a beaker i n t o which the e x t r e m i t y was l e n g t h of time.  dipped f o r a s t a t e d  By p a s s i n g carbon d i o x i d e gas  i n t o a small beaker of ether i t was found t h a t a temperature of from -15 G G  to -25°C. was  o b t a i n a b l e , and hence t h i s was  readily  the temperature  range used f o r the bulk of the experiments reviewed by Shumacker. was  T h e r e f o r e -20°C. p l u s or minus 5C.°  accepted as the temperature to be used f o r the  standard i n j u r y .  I t was not always p o s s i b l e to  o b t a i n on any one day a temperature e x a c t l y -20°C. but  i t was u s u a l l y p o s s i b l e to m a i n t a i n throughout  each day's work the same temperature, whether -15°C. or  -25°C.  I n a l l experiments the value o f -20°G.  w i l l be r e c o r d e d , and where n e c e s s a r y t h i s w i l l  be  q u a l i f i e d by some such statement as^'the animals s u f f e r e d a s l i g h t l y more severe f r e e z i n g I n j u r y than  193 was u s u a l l y the case?  T h i s might mean a f r e e z i n g  temperature o f -23°C. was used.  The d i f f i c u l t y  i n o b t a i n i n g the exact temperature each day l a y i n several factors.  The d i s c h a r g e n o z z l e f o r the  carbon d i o x i d e would f r e e z e up to a v a r y i n g extent from day to day.  The amount o f ether i n the beaker  would v a r y g r e a t l y , the warmth o f the room would be somewhat of a f a c t o r , e t c .  F o r t h i s reason,  the o b s e r v a t i o n s o f d i f f e r e n t experiments are hardly  comparable. The d u r a t i o n o f the standard i n j u r y was also-)  a problem, but t h i s was more e a s i l y s e t t l e d .  The  f i r s t gross experiment was a time experiment i n which the l e n g t h o f the f r e e z i n g v a r i e d from f i v e through twenty seconds to s%xty seconds. be seen l a t e r ,  seconds As w i l l  the f i v e second i n j u r y produced too  s l i g h t a r e a c t i o n f o r standard use, and the s i x t y second I n j u r y produced f a r too severe a r e a c t i o n . A c t u a l l y the twenty second i n j u r y ( p o s s i b l y due t o minor v a r i a t i o n s i n degree o f c o l d n e s s , or v a r i a t i o n s i n the amount o f the e x t r e m i t y immersed) was n o t always i d e a l , but there seemed l i t t l e v i r t u e i n r e d u c i n g the time to f i f t e e n  seconds.  So, w i t h these c o n s i d e r a t i o n s , a twenty second f r e e z i n g i n j u r y a t -20°C. was accepted as  194 the standard f r e e z i n g i n j u r y f o r the purposes of the experiments here performed. Manner of Thawing There always has been a g r e a t c o n t r o v e r s y over whether a f r o z e n e x t r e m i t y should be thawed slowly or r a p i d l y .  The time honoured method f o r  f r o z e n ears or noses  (which more o f t e n than n o t  were not " f r o z e n " i n the true sense of the word, but j u s t c o o l e d to the extent t h a t the subcutaneous f a t s o l i d i f i e d thus b l a n c h i n g the s k i n ) has been to rub the p a r t a c t i v e l y w i t h a h a n d f u l of snow.  T h i s was  one o f the " c e r t a i n w i d e l y accepted  concepts based almost e n t i r e l y upon armchair p h i l osophy of the p a s t " t h a t Shumacker c r i t i c i s e s  so  a c t i v e l y , and he perhaps more than any o t h e r p e r s o n has gather* enough evidence together to show t h a t t h i s i s erroneous and can be d i s c a r d e d , at l e a s t an experimental l e v e l .  on  Most of the experiments  reviewed by Shumacker g i v e Immersion o f the f r o z e n e x t r e m i t y i n warm water a t 42°C. f o r a s p e c i f i e d p e r i o d o t time, u s u a l l y two minutes, as the accepted method f o r r a p i d thawing. employed  These standards were  In the p r e s e n t experiments, and where  p o s s i b l e ( p r o v i d i n g enough animals were a v a i l a b l e ) both the r a p i d thawing a t 42°C. and o r d i n a r y at room temperature was  used.  This p r o v i d e d  thawing  195 a d d i t i o n a l c o n t r o l s e r i e s f o r almost every experiment. Apparatus The accompanying photograph  illustrates  an a c t u a l f r e e z i n g i n j u r y and r a p i d thawing I n progress.  The carbon d i o x i d e gas was r u n i n from  an o r d i n a r y gas c y l i n d e r through maleable metal tubine t o the c o n t r o l needle v a l v e ( t h i s was the adapted Spencer Carbon D i o x i d e Microtome apparatus as adapted f o r the m i c r o s c o p i c experiments, but w i t h the expansion c o l d p o i n t needles d i s c o n n e c t e d and a simple number n i n e t e e n s p i n a l a n a e s t h e t i c needle attached) and then through a l o n g needle i n t o a small 125 c c . beaker h a l f f i l l e d w i t h ether.  The gas b u b b l i n g i n overflowed the e t h e r ,  and a l a r g e r moat was p r o v i d e d to c a t c h the s p i l l a g e . A thermometer r e g i s t e r i n g to -30°C. was kept suspended i n the f r e e z i n g mixture and r e a d f r e q u e n t l y throughout the performance.  The animal was  a n a e s t h e t i z e d , and the h i n d l e g was extended and dipped i n t o the mixture almost to the f u r l i n e . G e n e r a l l y about t e n to twenty seconds were r e q u i r e d f o r the limb to c o o l down, and f r e e z i n g was i n d i c ated by a sudden b l a n c h i n g o f the s k i n and squirming of the animal.  Such a limb when f r o z e n was v e r y  197 brittle,  and c o u l d  piece  glass  of  similar  because  of  its  of  watch, be  started  used  for  moment from the  the time  hand  side  thawing.  this  (twenty  photograph  of plain on the  tap  in  and  used  of  instead  water  the  at  of  its  the  is  the  expiry  the  rapid  animalsjust  shown d i p p e d  a minute  treatment  preserving  kettle  the  beaker.  or  was  in  as  i n the  In actual  because  of  right  exactly 42°C.  for  the  removed  Gn t h e  the  and warm.  beaker  maintained  at  could  from  l i m b was  suspended  water  a larger the  of  stop  generally  depicted  treatment  thawing part  by a h e l p e r ,  is  thermometer  thoroughly limp  water  the  hind leg  freezing  The l e g was k e p t  of  and  a  that  counted  seconds).  a beaker  rapid  a b u t t o n ^ was  interval the  A  second hand  exactly  the  the  large  freezing mixture  of  much as  thickness.  blanching,  finished  until  across  The t i m i n g was  The f r o z e n  indicated  snapped  with pressing  timing.  of  be  two  practice performed was  larger  volume  t e m p e r a t u r e much more  constantly. Recording  the  Observations  The c o u r s e recorded the at  until  freezing  the  of  the  lapse  injury.  injured of  l i m b s was  twenty-four  The a n i m a l s  that  hours were  room temperature were m e r e l y p l a c e d back  cages  i n quietness,  except  for  not  any i n j e c t i o n  after  thawed in  their  they  198  would be r e c e i v i n g .  The f e e t o f s u c h a n i m a l s  r  would b e g i n t o thaw i m m e d i a t e l y , and any chance c o n t a c t w i t h t h e m e t a l o f t h e cage would a i d t h e warming so t h a t t h e l i m b would be l i m p w i t h i n f i v e minutes u s u a l l y . ten  (thawed)  I t would t a k e a n o t h e r  o r f i f t e e n m i n u t e s f o r t h e f o o t t o warm up,  and w i t h i n t h r e e o r f o u r hours t h e l e g would b e g i n to  show oedema.  U s u a l l y the s k i n would be s t i l l  b l u i s h , b u t i t would soon become engorged, p i n k , and warm. in  The f e e t o f t h e a n i m a l s thawed  rapidly  t h e warm water were p i n k and warm as soon as t h e y  were r e t u r n e d t o t h e i r cages, and w i t h i n t h e f o l l o w i n g c o u p l e o f hours t h e y would be s w o l l e n , b u t s t i l l p i n k and warm. On t h e f o l l o w i n g day, u s u a l l y a t t h e same time as t h e f r e e z i n g had been done t h e a n i m a l s were weighed, and t h e f e e t examined.  E x a m i n a t i o n was  made under f o u r c a t e g o r i e s : oedema, c o l o u r change, m o i s t u r e exudate o r s l o u g h , and gangrene o f t o e s or  other p a r t s o f the f o o t .  A c o n s t a n t comparison  e v a l u a t i o n was a t t e m p t e d , and o f c o u r s e t h e same p e r s o n d i d t h e examining, b u t when p o s s i b l e some o t h e r p e r s o n p r e s e n t would be asked t o c o r r o b o r a t e the  impressions.  Then under each c a t e g o r y , an  attempt was made t o a s s i g n a number t o each o b s e r -  199 vation. is  The number  outlined  as  scale  used  f o r each  category  follows: None  Oedema 1  Minimal  2  Moderate  3 4  Gangrene  Incomplete As f u l l y  rounded  as  possible  Normal  Colour  Exudate  apparent  1  Pinker  than  2  Red  3  Purple  4  Blue,  5  Black  0  Dry  1  Barely  2  Quite  3  Infected  normal  either cyanosis or beginning eschar Scab  not  graded.  moist moist  o r wet  ulcer  Blood  clot  not  0  None  apparent  1  One  5  Five  6  Front  7  W h o l e paw  8  Whole  or  graded  toe gangrenous toes half  slough.  or missing  gangrenous o f paw  or missing  gangrenous  gangrenous  or missing  i n j u r e d area gangrenous or missing  2G0 This at  the v e r y  start  preliminary just  c l a s s i f i c a t i o n was of  the  not  designed  experiments.  t e s t s were p e r f o r m e d i n o r d e r  exactly what changes t o e x p e c t ,  table  t h a t would b e s t  a d o p t e d as ible,  and  describe  o u t l i n e d was i n a few  instance,  One  the  or  to  i t i s not  two  determine  to c o n s t r u c t  lesions.  c e r t a i n l y not  respects  degree of  the  and  completely  The  the  only  the  best.  s w e l l i n g c o u l d have perhaps been  But  the  d i d not  only,  assessing  and  Such a g r a d i n g  s y s t e m as  the  limits On  injury print the  does n o t  cription.  give  the  d i d , but  o f what i s meant by The  word  within  example o f how The  the  the  colour  accurately  I t gives  "much" u n d e r t h e  the word  page  experiment.  c o l o u r c h a n g e s as  transparency  synonymous w i t h  a third  as  as  rough.'.;  accompanying  des-  oedema c o l u m n  "incomplete"  used before  for  d e g r e e oedema c h a n g e . Index As w i l l  the  the  accurate  c h a n g e s were g r a d e d i n p r a c t i c e .  original  Injury  e r r o r of  performed  the p r e c e d i n g  r a p i d , and  t h e f o l l o w i n g p a g e i s an  illustration  is  the  reasonably  experiment i n p h y s i c s .  o u t l i n e d on  at a l l times convenient,  method.  t o be  e x p e r i m e n t s as  e x a c t n e s s o f an  was  of  c h a n g e s had  the b i o l o g i c a l  r e q u i r e the  possFor  a water displacement  accurate  one  one  more a c c u r a t e l y m e a s u r e d by t h e method o f  a  be  n o t e d on  t h e f o l l o w i n g Example  term I n j u r y Index i s i n t r o d u c e d .  T h i s word,  sheet, Injury  201 Example of I n j u r y C a l i b r a t i o n 2 T h i s I s a photograph o f the f e e t o f group 5, which were f r o z e n f o r twenty seconds, then thawed slowly a t room temperature. The p i c t u r e was taken on day three, f o r t y e i g h t hours a f t e r the i n j u r y occurred.  Animal Oedema Colour Exudate Gangrene Much Blue Wet None 1 2 Much Red None Wet 3 Much Blue Wet None 4 Much Purple Wet None 5 Much Purple Wet None 6 Much Purple Wet None Converting these o b s e r v a t i o n s to the a r b i t r a r y number code, we have: Animal  0  C  E  1 2 3 4 5 6  3 3 3 3 3 3  4 2 4 3 3 3  2 2 2 2 2 2  18  19  12  Totals  G  The sum o f these f o u r t o t a l s , here 49, i s p l o t t e d on a l l subsequent graphs as the "INJURY INDEX". On the sample graph drawn on t h i s page, the components o f the o r d i n a t e are blocked i n f o r i l l u s t r a t i o n , b u t the other graphs would be too crowded w i t h these a d d i t i o n a l d e t a i l s which can be r e a d from the data t a b l e s when d e s i r e d .  50  Exu  late  40 I N J U 30 R Y  Col our  I • 20 D E X Oed  10  2 DAY  ema  3  202  Index i s merely an a r b i t r a r y number i n v e n t e d i n order t h a t the f r o s t b i t e changes, on a time b a s i s , c o u l d be r e p r e s e n t e d g r a p h i c a l l y without too much crowding and hence c o n f u s i o n on the graphs.  A sample graph i s  i n d i c a t e d w i t h the standard s c a l e used throughout a l l graphs, and the meaning o f the o r d i n a t e p o i n t s on the I n j u r y Index l i n e graph e x p l a i n e d . S e l e c t i o n of Gross Experiments Gross experiments were performed as o u t l i n e d i n f o l l o w i n g pages.  The d e t a i l s and purpose of each  experiment i s e x p l a i n e d under the write-up f o r the experiment.  Nine d i f f e r e n t runs were performed as  o u t l i n e d i n the Table o f Contents; (1) D u r a t i o n o f I n j u r y and r a t e o f thaw, (2) p r o c a i n e , (3) r e p e a t p r o caine, together w i t h P r i s c o l i n e ,  (4) B e n a d r y l , (5) E t -  amon and Hydergine, (6) A p r e s o l i n e and C h l o r - T r i p o l o n , (7) r u t i n ,  (8) a s c o r b i c a c i d , and (9) h i s t a m i n e .  The  reason f o r the p a r t i c u l a r grouping o f the two drugs Into one experiment was made only f o r convenience. reasons f o r the dosages  The  s e l e c t e d has a l r e a d y been e x p l a i n e d .  203  Record o f Gross O b s e r v a t i o n s  Here f o l l o w s a r e c o r d o f o b s e r v a t i o n s made g r o s s l y on the e f f e c t s o f a standard f r e e z i n g  injury  on the h i n d l e g o f the r a t as o u t l i n e d p r e v i o u s l y . The r e s u l t s are grouped by the drugs b e i n g  tested,  a c c o r d i n g t o the order o f the t a b l e o f c o n t e n t s .  D u r a t i o n o f I n j u r y and Rate o f Thaw Experimental O b s e r v a t i o n s w i t h Time and Rate o f Thaw The purpose of the experiment d e s c r i b e d here was to t e s t the e f f e c t s o f a standard c o l d i n j u r y as i n f l u e n c e d by the d u r a t i o n o f the f r o z e n s t a t e , and a l s o as i n f l u e n c e d by the r a t e o f thawing.  Thirty-  s i x normal a d u l t nonpregnant female W i s t a r r a t s were s e l e c t e d , and they were d i v i d e d i n t o s i x groups o f s i x animals each, w i t h each group designed as f o l l o w s : Group: 1.  A group t o r e c e i v e a f r e e z i n g  injury  o f f i v e seconds d u r a t i o n , w i t h r a p i d thawing I n water a t 4 2 C . G  2.  A group to r e c e i v e a f r e e z i n g  Injury  ©f twenty seconds d u r a t i o n , w i t h r a p i d thawing i n water a t 42®G. 3.  A gr©up t© r e c e i v e a f r e e z i n g  injury  204 of  3ixty  seconds d u r a t i o n , w i t h  r a p i d thawing i n water a t 42®C. 4.  A group to r e c e i v e a f r e e z i n g  injury  of f i v e seeonds d u r a t i o n , w i t h slow thawing a t room temperature. 5.  A group to r e c e i v e a f r e e z i n g of  injury  twenty seconds d u r a t i o n , w i t h  slow thawing a t room temperature. 6.  A group to r e c e i v e a f r e e z i n g of  injury  s i x t y seconds d u r a t i o n , w i t h  slow thawing a t room temperature. Although a l l groups are numbered the same throughout a l l o t h e r experiments, the  numbering  freezing,  i n this particular  i s independent.  On Day 1,  instance, the day o f  each animal i n a group was marked c h a r a c t -  e r i s t i c a l l y on the ear, and i n i t i a l weights were r e corded as on the Data Sheet.  A l l animals were g i v e n  by subcutaneous I n j e c t i o n from 0.1 c c . to 0.125 c c . of 0.6$ nembutal  solution.  Then, when a n a e s t h e t i z e d ,  one h i n d l e g was dipped i n a beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature down t© -20 G. G  to the  The f o o t was immersed up  the hock ( j u s t below the f u r l i n e ) ,  and as so©n as  s k i n blanched, i n d i c a t i n g the f o o t was completely  f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y squirmed, i n  205 s p i t e o f adequate  anaesthetic),  the time was counted  so t h a t the l e g was immersed and f r o z e n f o r e x a c t l y the r e q u i r e d l e n g t h o f time, depending on the group to which the animal belonged.  The animals o f Groups  4, 5, and 6 were p l a c e d i n t h e i r cages and the f e e t were allowed to thaw a t room temperature, approximately f i f t e e n o r twenty minutes limp and warm.  taking  to become  The animals o f groups 1, 2, and 3  were t r e a t e d by r a p i d thawing,  whereby the f r o z e n  f e e t were immersed immediately a t the e x p i r y o f the a p p r o p r i a t e time i n t e r v a l i n the e t h e r , i n t o maintained at e x a c t l y 42°C.  The f e e t were l e f t i n  the warm water f o r approximately one minute, thoroughly thawed and warm.  water  until  Then these animals were  a l s o r e t u r n e d to t h e i r cages and a l l t h i r t y s i x were l e f t q u i e t l y alone u n t i l of the nembutal. i n the t a b l e s ,  they wakened from the e f f e c t s  Observations were made as r e c o r d e d by the method d e s c r i b e d above f o r  e s t i m a t i n g the degree o f f r o s t b i t e i n j u r y under the c a t e g o r i e s o f oedema, c o l o u r change, exudate and slough, and  gangrene.  Data Sheets These t a b l e s are f a i r l y  self-explanatory,  b e i n g simple t a l l i e s by animals I n groups, days,  Day 1 being the day o f the f r e e z i n g .  n e c e s s a r i l y as compact as p o s s i b l e .  and by They are  Symbols a r e :  206 Grp  Gm  Gm  0 C £ G  Gm  0 C E G  Gm  1  250 174 168 232 204 224  260 169 171 236 201 220  1 2 2 2 1 1  1 1 A z 1 1 — 1  260 167 172 234 202 222  *1  1 1 1 •|-  - --  267 170 1 1 — A 172 2 233 1 1 198 1 1 _ 219 £ 1  204 234 175 231 180 210  228 237 175 225 182 207  3 3 3 3 3 3  2 2 1 1 1 1  1 1 1 1 1  225 229 176 227 180 201  3 3 3 3 3 3  2 1 1 2 2 2  1 1 1 1 1 1  234 228 177 226 182 202  3 2 3 3 2 3  2 1 — 1 2 2 2  194 248 230 183 238 175  200 251 241 186  4 3 3 3 2o2 3 173 3  2 2 2 3 2 2  1 1 1 1 1 — 1 — 1  -  191 242 233 180 229 170  3 3 3 3 4 3  2 3 2 4 2 3  2 1 2 1 2 2 — 2  192 238 230 176 224 170  3 3 3 3 3 3  3 4 3 4 3 3  2 2 2 1 2 — 2 — 2  220 240 164 195 205 215  215 235 168 196 203 210  3 3 3 3 3 3  2 2 1 2 3 2  1 1 1 1 2 1  -  216 232 162 191 199 210  2 3 2 2 3 3  2 3 1 2 2 2  2 2  - — 2 -  210 226 165 186 192 206  1 3 2 1 2 3  1 3 3 2 2 3  1 2 —  234 174 173 210 216 178  226 170 168 210 215 178  3 3 3 3 3 3  3 3 3 2 2  2 2 2 1 1 1  -  225 164 163 210 212 175  3 3 3 3 o 3  4 4 3 2 3 3  2 2 2 mm 2 — 2 — 2  214 155 157 205 204 176  4 3 3 3 3 3  4 3 3 3 3 3  2 2 _ 2 — 2 _ 2 1  235 215 240 199 238 197  232 214 242 199 228 197  3 3 3 3 3 1  4 4 4 4 4 4  2 mm 2 2 1 — 1 —  226 207 234 189 224 188  3 3 3 3 3 3  4 4 3 4 4 5  2 2 2 2 2 2  222 205 224 184 214 187  3 3 3 2 1 3  4 5 4 5 5 5  2 2 2 2 2 2  1  2  2  3  4  5  6  Day  -  - - _  —  -  -  --  3  1 — 1 — 1 — 1 2" 1  -  -  -  2 2  mm  -  -  1 _  —  -  —  8  0 C E G  ** * -- --  - -  4  - -  -- -  _  -  1 — 1 1  _  -  -  1 1 1 8  207  I  Grp 1  2  3  Gm  O C E G  Gm  O C E G  260 163 175 225 190 214  i£ - 1 1 - 4 1 - i - 1 1 - i 1 - -  4 4§ § - -  i  267 160 181 226 194 216  & & - 1 1 £ - -  225 224 172 211 179 194  2 1 1 2 1 2  215 226 178 214 175 198  1 1 11 1 1 1 1 1 1 1 2  2 1 1 1 1 2  1 -  -  - 1  -  Gm  224 230 179 223 176 203  O C E G  1 £ 1 1 1 1  1 1 1 1 1 2  -  -  186 3 236 3 226 3 Dead 221 3 171 3  3 2 3 2 3 2 -  178 3 3 3 224 3 3 1 231 3 3 3 -  184 3 3 3 226 3 2 1 232 2 3 3 -  3 2 3 2 -  226 3 3 3 172 3 3 3 -  228 3 3 1 170 3 3 1 -  204 218 168 187 188 204  2 2 1 i } 2  2 3 2 1 1 1  1 1 -  -  205 220 174 186 191 203  1 2 13 1 1 1 1 1 1 22  - -  210 230 180 188 195 203  3 3 - 4 13 1 1 1 - 1 1 - 2 1  5  210 157 160 202 200 157  3 3 3 3 3 3  4 3 3 2 3 3  3 2 1 1 3  3 4 4  211 158 163 200 200 186  3 3 3 2 3 3  3 3 3 3 3 4  -4 3 6 3 5 1 1 11  210 159 168 204 204 189  3 3 - 4 3 3 2 6 3 3 15 1 2 1 2 3-1 3 5-4  6  214 195 220 185 215 190  3 2 1 1 2 1  3 5 5 4 4 2  2 2 2 2 2 -  8 6 7 8 8 8  210 19v7 216 184 210 190  3 3 3 -  4 4 4 3 3 3  3 3 3 -  212 2 4 1 8 203 2 4 2 8 225 2 4 - 8 182 8 208 8 185 8  Day  5  4  6  - - 1 -  8 8 8 8 8 8  7  208 Grp» group,  Gm, grams,  0, ©edema,  E, exudate,  G, gangrene,  C, c o l o u r  change,  -, means n o t p r e s e n t . A l l  animals were d i s p o s e d o f f w i t h chl©rof©rm fumes on Day 7. Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which g i v e s the average weight f o r each group each day,  and under I , or Index,  g i v e s the t o t a l f o r the  i n j u r y changes o c c u r r i n g f o r t h a t group that day. described before,  As  t h i s concept o f an I n j u r y Index I s  j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes t o g e t h e r f o r each group each day. Note t h a t the v a l u e s f o r Group 3 on days 5, 6, and 7 are u n d e r l i n e d to i n d i c a t e an e x t r a p o l a t i o n to make up f o r the animal t h a t d i e d . Graphs The v a l u e s on the Summary Sheet a r e p l o t t e d on two simple l i n e graphs,  the f i r s t f o r the I n j u r y  Index changes from day to day, the weight changes. easier reading,  and the second f o r  These are done In c o l o u r s f o r  but each l i n e i s otherwise c l e a r l y  l a b e l l e d by the nature o f the i n j u r y ,  r a t h e r than by  group numbers. C o n d i t i o n o f the Animals There i s l i t t l e need t© i n c l u d e d e t a i l s o f the r a t s themselves. freezing,  W i t h i n a couple o f hours o f the  a l l f e e t were g r o s s l y ©edematous,  bright  209  Summary sheet Gm  Grp  Gm  Gm  I  Gm  1  209  209  14*  209  2  206  209  31  206  34  210  26  3  211  216  39  207  47  205  51  4  206  204  37  202  37  197  32  5  197  194  43  191  49  185  49  6  221  219  48  211  63  206  66  Day  1  2  Grp  Gm  I  I  209  4  3  Gm  I  Gm  I  1  204  9  207  6  208  2  201  18  201  14  206  12£  3  202  48  200  50  202  46  4  195  20  196  19  201  15  5  181  57  186  61  189  58  6  203  88  201  87  202  69  Day  5  6  7  I 9-  210  Rapid Thaw Yersu3 Slow Thaw - E f f e c t o f F r e e z i n g Time  \ *  60" Freeze Slow Thaw  J 20" Freeze Slow Thaw  60" Freeze F a s t Thaw  5" Freeze Slow Thaw 20" Freeze F a s t Thaw 5" F r e e z e F a s t Thaw  1  2  3  4 DAY  5  6  7  211  1  2  3  4 DAY  5  6  7  212 p i n k , warm, and e s s e n t i a l ^ n o n t e n d e r ,  although  a p p a r e n t l y numb f o r the animals hobbled around cages a p p a r e n t l y unaware o f the p o s i t i o n s injured feet.  their  of their  The day to day changes i n the appear-  ance of the o f the f e e t may be r e a d i l y imagined from the v a l u e s on the Data Sheets.  G e n e r a l l y the r a t s  d i d w e l l , and the graph showing weight changes i n Icates  a f a i r l y s i m i l a r response through a l l groups.  Individual  r a t weight changes mean l i t t l e .  D i s c u s s i o n of Observations The I n j u r y Index graph g i v e s a v e r y c l e a r s e p a r a t i o n o f the v a r i o u s groups. of 80 i n d i c a t e d  An Index readyjng  a v e r y severe r e a c t i o n ,  and I n t h i s  case the f e e t o f Group £'s animals a l l sloughed o f f to the l i m i t s o f the f r e e z i n g , other extreme,  whereas,  a t the  by Day 7 i t was almost i m p o s s i b l e  to d e t e c t any change I n the f e e t o f the animals o f Group 1,  except f o r a s l i g h t t h i c k e n i n g o f the s k i n  over the dorsum o f the paw. There I s f i r s t o f a l l , as would be expected, a worse i n j u r y produced the l o n g e r the limb i s f r o z e n . The e f f e c t o f the s i x t y second i n j u r y I s worse than f o r the twenty, five.  and the twenty worse than f o r the  T h i s response was c o n s i s t e n t throughout the  213 graph. Then there i s secondly, w e l l demonstrated, the g r e a t b e n e f i t to be d e r i v e d from r a p i d  thawing  of the f r o z e n limb i n warm water at 42°C. e f f e c t was  This,  a l s o c o n s i s t e n t throughout the graph.  Photographs On Day 5, nembutal  a f t e r a r e p e a t dosage o f the  anaesthetic,  c o l o u r by groups.  a l l f e e t were photographed i n  The p r i n t s o b t a i n e d were o f to©  poor a q u a l i t y to m e r i t i n c l u s i o n h e r e . the p i c t u r e o f Group 5, l a b e l l e d used e a r l i e r to i l l u s t r a t e how calculated.  However,  "Time 5" has been an I n j u r y Index i s  I t would be worthwhile to examine t h i s  page again In r e f e r e n c e to t h i s experiment. Procaine Experimental Observations w i t h Pr©caine H y d r o c h l o r i d e The purpose o f the experiment here d e s c r i b e d was  to t e s t the e f f e c t s o f a standard c o l d i n j u r y as  i n f l u e n c e d by p a r e n t e r a l i n j e c t i o n s a dosage l e v e l ©f 200 mg.  o f p r o c a i n e at  per k i l o g r a m body weight  administered h o u r l y p r i o r to the f r e e z i n g i n j u r y and f o r f o r t y - e i g h t hours a f t e r w a r d s .  T h i r t y normal  a d u l t male Wistar r a t s were s e l e c t e d , and they were d i v i d e d i n t o f i v e groups ©f s i x animals each, w i t h  214 each group designed as f o l l o w s : Group: 1.  A group to r e c e i v e a f r e e z i n g  injury  of twenty seconds d u r a t i o n w i t h slow thawing a t room temperature. 2.  A group to r e c e i v e o n l y the p r o c a i n e i n j e c t i o n s as o u t l i n e d .  3.  A group to r e c e i v e b o t h the p r o c a i n e i n j e c t i o n s and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h slow thawing a t room temperature.  4.  A group to r e c e i v e a f r e e z i n g  Injury  of twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42°G. 5.  A group to r e c e i v e b o t h the p r o c a i n e i n j e c t i o n s and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42°C.  These groups are numbered i d e n t i c a l l y throughout a l l subsequent gross experiments.  Note t h a t Group 1  here corresponds to Group 5 o f the l a s t experiment, and Group 4 here w i t h Group 2 t h e r e .  On day 1 o f  the experiment, the day of f r e e z i n g ,  each animal was  marked c h a r a c t e r i s t i c a l l y on the ear,  and  Initial  weights were r e c o r d e d as on the D*ta Sheet.  The  animals o f Groups 2, 3, and 5 r e c e i v e d t h e i r  initial  215 subcutaneous i n j e c t i o n o f p r o c a i n e s o l u t i o n p r e pared a t 20 mg. p e r c c . f o r ease i n computing doses.  Then, approximately twenty minutes l a t e r ,  the animals o f Groups 1, 3, 4, and 5 r e c e i v e d by subcutaneous i n j e c t i o n an a n a e s t h e t i z i n g dose o f 0.6$ nembutal 0.125  cc.  s o l u t i o n r a n g i n g from 0.1 c e . to  Then, when a n a e s t h e t i z e d , one h i n d l e g  of each animal i n these groups was dipped i n a beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature down t o -20°C. The f o o t was immersed up to the hock ( j u s t below the fur l i n e ) ,  and as soon as the s k i n blanched, I n d i c -  a t i n g the f o o t was completely f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y squirmed, i n s p i t e o f adequate a n a e s t h e t i c ) ,  the time was counted so t h a t  the l e g was immersed and f r o z e n f o r e x a c t l y twenty seconds by stop watch. 3  The animals o f Groups 1 and  were p l a c e d i n t h e i r cages and the f e e t were  owed to thaw a t room temperature,  all-  t a k i n g approxim-  a t e l y f i f t e e n o r twenty minutes to become limp and warm.  The animals o f groups 4 and 5 were t r e a t e d  by r a p i d thawing,  whereby the f r o z e n f e e t were imm-  ersed immediately a t the e x p i r y o f the twenty seconds i n t o water m a i n t a i n e d a t e x a c t l y 42®C.  The f e e t  were l e f t i n the warm water f o r approximately one  216 minute, u n t i l  thoroughly thawed and warm.  Then these  animals were a l s o r e t u r n e d to t h e i r cages and a l l thirty  were l e f t q u i e t l y alone (except f o r the h o u r l y  i n j e c t i o n s of p r o c a i n e ) u n t i l e f f e c t s of the nembutal.  they wakened from the  Observations were made as  recorded i n the t a b l e s , by the method d e s c r i b e d above for  e s t i m a t i n g the degree of f r o s t b i t e i n j u r y under  the c a t e g o r i e s of oedema, c o l o u r change, exudate and slough, and gangrene. Data sheets These t a b l e s are f a i r l y s e l f - e x p l a n a t o r y , being simple t a l l i e s by animals  i n groups, and  days, Day 1 being the day of f r e e z i n g . n e c e s s a r i l y as compact as p o s s i b l e .  They are Days 1 and  i n c l u d e a r e c o r d of the h o u r l y i n j e c t i o n s . symbols used are: 0, oedema,  Grp,  administered,  grams,  2  The qlh, hourly,  E, exudate and  slough,  P, f r e e z i n g , -, means not p r e s e n t or n o t N, f u l l  the dose of p r o c a i n e ,  value).  Gm,  C, c o l o u r change,  G, gangrene,  procaine  group,  by  dose of p r o c a i n e , one  £, one  half  q u a r t e r the dose of  (note t h a t £ i n the O C E G  columns means h a l f  A l l animals not a l r e a d y dead were disposed  r  of w i t h c h l o r o f o r m fumes on Day  4.  Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet  217 Data Sheet Grp Gm 1  2  3  4  5  Day  qlh  264 310 180 287 254 318  -p -P -p -p -p -p  410 295 348 241 267 268  N-NNNNN (Died) N-NimNN-----iii|ii£---i--|N-NNNNN  279 317 202 306 320 318  N-NNNNN t-t NPNNNNN i-(Died) NPNNNN*(Died). NPNNNNN-l l l l l l NFNNNN& £|£i§3*£H Died)  261 317 292 255 238 244  NFNNNNi -P -P -F -F -P -P  283 242 210 282 341 285 1  '  —  •  HHiili  N-NN]Om---^Hfnii N-NNNNN  ?-?  ?"?  llllillll llllSISi  NFNNNN}  NPNNNNN  NPNNNNN-N NPNNNNN  niiil—i"?  UHHil  • l l l l l l l  i-llilll  NFNNNNN-^--li|||H| NPNNNNN-N NPNNNNN  1-1  IHIHI  (Died)-  £-£  §-£ §-§  *-#  £-§  Gm  O C E G  260 304 166 290 246 320  3 3 3 3 3 3  3 2 3 2 2 2  qlh  11122 • 2-  285  3 3 5  2 4 8  f - f 254 230 192 2^2 1 3 0 53 3 2 3 0 8 2  *  3 1  262 312 286 250 232 240  3 3 3 3 3 3  1 1 1 1 1 1  1 1 1 1 1 1  272 2 i l 1  232 2§1 204 2 1 -  262 3 1 1  335 2^1 2  £|—(Died)  ml':  >  218  Data Sheet Gm  0C EG  Gm  0 C E G-  270 310 165 295 252 323  33 32 33 33 33 33  260 303 164 292 249 311  3 3 3 3 3 3  Grp q l h 1  2  (Dead) i^JJJLii.i  ts  (Dead) (Dead) (Dead) (Dead) --lii-i-iji-:  1|— 4  --•  5  - i  \ \  n u i i i m i i i i i . i 444444444444444 I  ; (Dead) Day 2 (Cont)  4 3 4 3 4 4  2 3 1 2 2 2 — 3  -  259 303 221 245 212 (Dying)  272  i i i t i l l - - - 3  2 2 2 1 1 2  296 2 3 2 297 2 4 -  281 (Dead wt) 274 2 3 1 -  266 306 289 249 238 240  3 1 2 1 31 31 1 3 11 31 1  267 283 289 239 229 244  2 1 1 1 3 2 2 2 1-  270 1 1 230 2 1 199 2 - -  262 218 196 237  2 1 - -(Dead) 21 -(Dead) 2 1 --(Dead) 31 -  ocp 6  0  0  (Dead) 3  * -i _ <-> ± -  219  Summary Sheet Grp  Gm  Gm  1  269  264 41  269 46  263 55  2  284  270  259  246  3  319  306 40  296 38  277 36  4  268  264 30  265 26  258 22  5  254  242 24  239 16£  226  Day  1  which day,  I  Gm  3  2  I  Gm  I  4  g i v e s the average weight f o r each group  each  and under I , o r Index, g i v e s the t o t a l f o r the  i n j u r y changes o c c u r r i n g f o r that group t h a t day.  As  d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t . a n a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes apparent together f o r each group each day.  Except f o r Groups 1 and 3 where no animals  e x p i r e d , a l l I v a l u e s had t o be e x t r a p o l a t e d up t o correspond to s i x animals p e r group. e x p l a i n e d below,  As w i l l be  t h i s s e r i o u s l y reduces the s i g n i f  i c a n c e o f the d a t a .  220  Graphs The v a l u e s on the Summary Sheet are p l o t t e d on two simple l i n e graphs,  the f i r s t f o r the I n j u r y  Index changes from day t o day, the  weight changes.  and the second f o r  These are done In c o l o u r s f o r  e a s i e r r e a d i n g (except f o r the Time graphs o f the l a s t experiment these and a l l succeeding gross graphs have i d e n t i c a l c o l o u r s ) ,  but each l i n e i s otherwise  c l e a r l y l a b e l l e d by the n a t u r e o f the i n j u r y and treatment r a t h e r than by group numbers. C o n d i t i o n o f the Animals I t w i l l be obvious from the Data Sheets t h a t the of  p r o c a i n e i n the dosage g i v e n took a t e r r i b l e the animals.  toll  Many, a f t e r the s i x t h dose,were  comatose and i n c o n v u l s i o n s .  T h i s e x p l a i n s why such  d r a s t i c r e d u c t i o n s i n the m e d i c a t i o n were n e c e s s a r y . Many d i e d ,  and o f those s u r v i v i n g , many s a t l i m p l y  huddled i n t h e i r cages,weakly p r o t e s t i n g every d i s turbance. of  The day to day changes i n the appearance  the f e e t may be imagined from the v a l u e s on the  Data Sheets.  These d© n o t r e f l e c t the p i t i f u l  cond-  i t i o n o f the animals. D i s c u s s i o n o f Observations P r o c a i n e i n the dosage used I s too t o x i c t o be p r a c t i c a l .  221  2  3  D A Y  4  1  2  3 D A Y  4  222  The I n j u r y Index graph confirms the i m p r e s s i o n t h a t r a p i d thawing a t 42 G reduces the s e v e r i t y o f the 0  injury reaction.  This i s c o n s i s t e n t b o t h w i t h the  p r o c a i n e t r e a t e d groups and w i t h the c o n t r o l groups. Procaine treatment a d m i n i s t e r e d as o u t l i n e d a p p a r e n t l y r e s u l t e d i n l e s s i n j u r y than was shown by the c o n t r o l s .  The f a c t t h a t the graphs f o r the p r o -  c a i n e t r e a t e d animals l i e under the c o r r e s p o n d i n g c o n t r o l graphs supports t h i s c l a i m .  However, two  animals never make a group, and the l a c k o f any s i g n i f i c a n c e i n these graphs i s r e a l i z e d .  They are i n -  cluded merely f o r i n t e r e s t . The weight graphs a t t e s t to the t o x i c of the p r o c a i n e .  effects  The p r o c a i n e t r e a t e d animals show  a steady and r a p i d weight l e s s , w h i l e the c o n t r o l s tend to m a i n t a i n t h e i r weight more s t e a d i l y .  P r o c a i n e and P r i s c o l i n e Experiments w i t h P r o c a i n e (Repeat) and P r i s c o l i n e The purpose o f the experiment here d e s c r i b e d was to t e s t the e f f e c t s o f a standard c o l d i n j u r y on the h i n d l e g s o f r a t s as i n f l u e n c e d by two d i f f e r e n t drugs.  Because o f the t o x i c i t y and m o r t a l i t y o f the  223 p r o c a i n e a t the dosage l e v e l used i n the l a s t experiment (200 mg. p e r k i l o g r a m body weight) i t was  de-  c i d e d n e c e s s a r y to repeat the experiment u s i n g the p r o c a i n e a t h a l f the dosage, v i z . 100 mg. per k i l o gram, but administered by subcutaneous i n j e c t i o n three h o u r l y .  The e f f e c t s of p r i s c o l i n e were a l s o  t e s t e d at a dosage l e v e l o f 50 mg.  per k i l o g r a m body  weight a l s o administered subcutaneously at three hourly i n t e r v a l s .  T h i r t y normal a d u l t nonpregnant  female W i s t a r r a t s were s e l e c t e d , and they were c l e a r l y d i v i d e d i n t o two groupings;  one o f e i g h t e e n  animals f o r t e s t i n g w i t h p r o c a i n e , and the o t h e r o f twelve animals for. the P r i s c o l i n e t e s t s .  Each d i v -  i s i o n was d i v i d e d i n t o groups of s i x animals each, w i t h each group designed as f o l l o w s ; Group;  2.  A group to r e c e i v e o n l y the p r o c a i n e i n j e c t i o n s as o u t l i n e d .  4.  A group to r e c e i v e a f r e e z i n g  injury  of twenty seconds d u r a t i o n w i t h o r a p i d thawing i n water a t 42 C. 5.  A group to r e c e i v e b o t h the p r o c a i n e i n j e c t i o n s and a f r e e z i n g I n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42®C.  Group;  4.  A group to r e c e i v e a f r e e z i n g  injury  224 of  twenty seconds d u r a t i o n w i t h  o r a p i d thawing i n water a t 42 C. (Group) 5.  A group to r e c e i v e both P r i s c o l i n e i n j e c t i o n s as o u t l i n e d and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing In water at 42°C.  These group numbers are i d e n t i c a l throughout a l l gross experiments ( except the f i r s t ) . f r e e z i n g was  done,  On the day the  Day 1, the animals o f each group  were marked c h a r a c t e r i s t i c a l l y on the ear,  and  i n i t i a l weights were r e c o r d e d as on the Data Sheet. The animals o f Group 2 and both Groups 5 r e c e i v e d initial  i n j e c t i o n s of t h e i r a p p r o p r i a t e drug.  p r o c a i n e s o l u t i o n was cc.  two  The  i n a c o n c e n t r a t i o n of 10 mg.  and the P r i s c o l i n e was  2 mg. p e r c c .  per  Then one  hour approximately a f t e r the second dose o f the drugs the  animals of both Groups 4 and b o t h Groups 5 were  a n a e s t h e t i z e d w i t h 0.1  to 0J25 c c . o f 0.6$  s o l u t i o n a d m i n i s t e r e d subcutaneously. etized, was  nembutal  When anaesth-  one h i n d l e g o f each animal i n these groups  dipped i n a beaker o f ether through which carbon  d i o x i d e gas was b u b b l i n g to b r i n g the temperature down o to  -20 C.  The f o o t was  below the f u r l i n e ) ,  immersed up to the hock  (just  and as soon as the s k i n blanched,  225 i n d i c a t i n g the f o o t was completely f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y squirmed, i n s p i t e o f adequate a n a e s t h e t i c ) , the  the time was counted so t h a t  l e g was immersed and f r o z e n f o r e x a c t l y  seconds by stop watch. r a p i d thawing,  twenty  A l l animals were t r e a t e d by  whereby the f r o z e n f e e t were Immersed  immediately a t the e x p i r y o f the twenty seconds i n t o water maintained a t e x a c t l y 42°C. left  The f e e t were  i n the warm water f o r approximately one minute,  u n t i l thoroughly thawed and warm.  Then these animals  were r e t u r n e d to t h e i r cages and l e f t  q u i e t l y alone  (except f o r the three h o u r l y i n j e c t i o n s ) u n t i l they wakenSd from the e f f e c t s o f the nembutal.  Observ-  a t i o n s were made as r e c o r d e d i n the t a b l e s , b y t the method d e s c r i b e d above f o r e s t i m a t i n g the degree o f f r o s t b i t e i n j u r y under the c a t e g o r i e s o f oedema, c o l o u r change, exudate and slough, and gangrene. Data sheets These t a b l e s are f a i r l y being simple t a l l i e s days,  self-explanatory,  by animals i n groups, and by  Day 1 being the day o f f r e e z i n g .  n e c e s s a r i l y as compact as p o s s i b l e .  They are  Days 1, 2, and  3 i n c l u d e a r e c o r d o f the three h o u r l y i n j e c t i o n s . The symbols used a r e :  G-rp, group,  Gm, grams,  226 Data  Sheet  Procaine Grp 2  4  5  Day  Gm  q3h  197 184 194 178 178 181  NN-NNNNNNNNN  188 195 170 186 173 191  __  201 189 170 207 201 194  NNFNNNNNNNNN NNFNNNNNNNNN NNF (Died) NNFNNNNNNNNN NNFNNNNNNNNN NNFNNNNNNNNN  0 C E G  NN-mOTONNNNN  NN-NNNNNNNNN NN-10JNNNNNNN NN-NNNNNNNNN NN-NNNNNNNNN p  ~ F (Died) p--— -.-p—--  Gm  NNNNNN  182 175 175 168 165 168  NNNN  177 3 1 1 187 3 4 2 _  <*• tm  NNNNNN NNNNNN NNNNNN NNNNNN NNNNNN 3 2 1 p o  o  —  Ct  3 2 1 — 3 3 mm 2 3  NNNN  NNNN NNNN NNNN NNNN  175 3 2 1 161 3 3 1 181 2 1 - -  ca  mm  ————  3 1 _ 3 1 1 -  NNNNNN NNNNNN  187 3 1 _ _ 174 2 1 - -  NNNN  3 2 - 3 2 1 3 1 1 mm  NNNNNN  190 3 3 - 187 3 2 - 182 3 1 - -  NNNN NNNN NNNN  NNNNNN  NNNNNN  2  1  0 C E G  q3h  q3h  NNNN  3  Priscoline Grp  Gm  4  192 146 182 195 198 195  5  185 204 197 184 205 206  Day  1  q3h •P •P •P -P -P -P PPPPPPPPPPPP PPPPPPPPPPPP PPPPPPPPPPPP PPPPPPPPPPPP PPPPPPPPPPPP PPPPPPPPPPPP  O C E G 3 3 3 3 3 3  3 1 3 1 3 2 2 2 12 2 2  3 3 3 3 3 3  3 1 2 3 2 2  1 1 2 1  -  q3h  PPPPPP PPPPPP PPPPPP PPPPPP PPPPPP PPPPPP  Gm  O C E G  187 150 175 192 194 180  3 3 3 3 3 3  2 1 3 1 3 1 2 1 1 2-  181 198 190 179 198 200  3 3 3 3 3 3  3 2 1 3 2 1  q3h  PPPP PPPP PPPP PPPP PPPP PPPP  227 Data Sheet Procaine Gm  0 C E G  175 168 165 163 155 159 168 2 2 178 3 3 (Dead) 168 2.2 155 2 3 176 1 1  Gm  0 C E G  Day  1 2 —  174 2 2 2 «. 176 2 4 3 6  173 2 2 2 _ 184 2 3 — 6  175 1 2 3 186 1 2 - 6  - -  176 1 2 1 — 164 1 3 180 1 1  162 1 1 168 1 2 — 186 1 1  _  190 2 1 174 2 2  198 1 1 178 1 2 —  -  190 2 1 190 3 3 2 190 2 1  1 1  - _  —  - ---  6  0 C E G  0 C E G  Gm  0 C E G  205 132 176 185 204 181  1 1 3 1 1 1  2 2 3 1 2 1  185 197 188 170 196 194  2 1 1 1 2 1  3 1 3 1 — 2 2 2 1 —  1 3 1 _ 2 3 - 3 4 2 — mm 1 _ 2 — — 2 2  175 188 184 172 194 197  3 2 1 3 3 3  3 1 1 2 2 2  - -  178 190 181 172 195 187  3 1 2 2 2 2  1  1 1 1 1 —  -  228 2 3 - 198 2 2 1 208 2 3 212 2 2 1 • 214 2 2 3 10  Gm 196 131 172 192 202 180  -  - _ -  194 2 3 192 1 1 — 197 1 1  21-  —-———-—^——  2 a. 3 4 2 2 - 2 2 -  -  mm  _  187 1 2 184 1 4 202 1 1  Priscoline  2 3 3 1 2 2  - -  - - -  8  184 146 171 188 184 178  4  0 C E G  217 216 212 217 208 211  i  5  Gm  176 170 170 177 174 175  4  4  0 C E G  188 182 178 178 174 174  179 2 1 167 2 1 (Dead) 182 3 3 1 181 2 1 1 176 2 2 1  Grp Gm  Gm  6  -  -  - -  3 1 3 2 2 2  1  _  —  -  2  mm  -  —  2 1 -  8  -  1 1 -  -  -  1 —  •--  - -  Gm  0 G E G  228 140 193 214 235 200  1 1 2 1 1 1  2 1 3 1 2 2  202 207 210 194 210 205  2 1 1 1 2 1  2 2 2 2 3 3  10  — 1 3 — — — — —  -  mm  -  mm  3 2 3 2  — a*  1  —  228 Data Sheet Procaine Grp Gm  0 C E G  Gm  0 C E G  207 205 201 199 201 194  Gm  0 C E G  2  201 203 198 207 194 193  4  172 1 2 3 181 1 1 6 (Dead) 169 1 3 167 1 3 3 189 1  -  180 1 2 3 mm 186 1 1 6  - -  173 1 1 2 175 2 3 197  190 183 200  _ 210 1 — 180 1 3 (Dead) 190 2. 3 202 2 2 1 199 2 2 2  219 1 1 184 1 2 1 —  219 1 197 1 1 2  -  -  5  -  -  -  Day 11  220 210 213 205 200 200  -  -- - -  -  -  192 mm 189 1 1  - 6 1 - — - — 1 - - - mm  mm  -  192 1 3 1 204 2 2 1 — 205 3 2 3  207 2 2 3 5 220 1 2 1 206 2 2 1 5  12  13  -  Priscoline Grp Gm 4  0 C E G  Gm  0 C E G  - - --  214 134 182 193 230 191  1 2 1 2 2 3 2 — 1 — 1 1 1  210 149 186 210 214 210  mm 1 1 1 2 1 1 2 2 3 2  1  180 194 197 189 202 198  1 2 2  200 206 214 180 210 200  1 — 1 1 — 1 — 1 1 1 — 1 2 3 2 — — 1  212 1 2 _ 133 1 2 1 178 1 3 1 193 1 221 2 184 2  -  5  Gm  0 C E G  180 194 197 184 195 194  Day 11  1 2 1 1 2 2 2 2 1 2  -  _  - 2 3 2 mm  12  - -  -  - 21  -  - —  1 1 2 1 2 2 3 1 2 1  mm  _  -  -  -  13  - _ — — - - - - mm  mm  -  -  229  q3h, every three hours, change,  0, oedema,  E, exudate and slough,  N, i n j e c t i o n of p r o c a i n e ,  G, gangrene,  P, f r e e z i n g ,  not p r e s e n t o r n o t a d m i n i s t e r e d , Priscoline.  C, c o l o u r  -, means  P, i n j e c t i o n o f  A l l s u r v i v i n g animals were d i s p o s e d  of on Day 13 w i t h c h l o r o f o r m fumes. Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which g i v e s the average weight f o r each group day,  each  and under I , or Index, g i v e s the t o t a l f o r the  I n j u r y changes o c c u r r i n g f o r that group that day.  As  d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes apparent t o g e t h e r f o r each group each day.  Where an animal d i e d ,  the I n j u r y Index  was e x t r a p o l a t e d to r a i s e the value to what c o u l d be expected f o r a complete  group.  Graphs The v a l u e s on the Summary Sheet are p l o t t e d on two simple l i n e graphs,  the f i r s t f o r p r o c a i n e  and the second f o r P r i s c o l i n e .  The weight curves and  the I n j u r y curves are p u t onto the same c o r r e s p o n d i n g graph,  and p l o t t e d a g a i n s t time.  The X marks on the  graphs I n d i c a t e missed days to p e r m i t the graph to  230 Summary Sheet Procaine Grp  Gm  2  185  4  187  5  Gm  I  I  Gm  I  Gm  I  Gm  I  172  164  179  174  37  175 36  168 31  174 37  175 29  198  30  184 26  177 27  187 25  192 17  Day  1  2  3  4  6  8  Grp  Gm  2  177  199  201  208  4  187 34  176 31  182 28  191 12  5  212 28  198 25  201 29  210 37  10  11  12  13  Day  Gm  I  Gm  I  I  Gm  I  Priscoline Grp  Gm  I  Gm  4  185  42  180 35  177 30  179 26  181 22  5  197  36  191 34  185 31  184 31  188 23  Day  1  2  3  4  6  8  Grp  Gm  I  Gm  I  I  Gm  Gm  I  I  Gm  Gm  I  4  210 22  185 17  191 17  196 16  5  205 33  191 24  193 24  202 15  10  11  12  13  Day  I  Gm  I  231  10  1  2  3  4  X 6  X 8 X 10  D A Y  11  12  13  232  210  1  2  3  4  X 6  D A Y  X 8 X  10  11  12  13  233  be contained  w i t h i n one page.  These graphs are  done i n c o l o u r s f o r e a s i e r r e a d i n g  ( a l l graphs have  i d e n t i c a l c o l o u r s except f o r the Time graphs of first  experiment),  but  each l i n e i s otherwise  l a b e l l e d by the nature of the i n j u r y and  the clearly  treatment  r a t h e r than by group numbers. C o n d i t i o n of the Animals The  animals r e c e i v i n g p r o c a i n e  were i n much  b e t t e r c o n d i t i o n on the reduced dosage r e c e i v e d l e s s often.  However, they were somewhat a f f e c t e d ,  appearing r a t h e r out or s o r t s , and l e s s a c t i v e than h e a l t h y r a t s should be. observable i l l  The  e f f e c t on  P i r s c o l i n e had  no  the group of animals r e c e i v -  ing i t . D i s c u s s i o n o f Observations A c a s u a l i n s p e c t i o n of the I n j u r y Index graphs f o r procaine that procaine  and P r i s c o l i n e would g i v e the protected  P r i s c o l i n e permitted question  the r a t l e g s and  a worse i n j u r y .  that  The  key  the to  the  l i e s i n the f a c t t h a t I n j u r y Index l i n e s f o r  the c o n t r o l groups, Groups 4, and  impression  on b o t h the  procaine  the P r i s c o l i n e graphs should be more s i m i l a r than  they are,  considering  the f a c t they are f o r I d e n t i c a l  groups r e c e i v i n g the standard  freezing injury, follow-  234 ed by r a p i d thawing, without any m e d i c a t i o n . u a l l y there was l i t t l e  Act-  s i g n i f i c a n t d i f f e r e n c e i n the  appearance of the v a r i o u s groups o f animals. Weight changes were compa^rable between the v a r i o u s groups,  i n d i c a t i n g t h a t none were a d v e r s e l y  a f f e c t e d by the drugs.  Benadryl Experimental O b s e r v a t i o n s w i t h Benadryl The purpose o f the experiment here d e s c r i b e d was to t e s t the e f f e c t s o f a standard c o l d i n j u r y as I n f l u e n c e d by p a r e n t e r a l i n j e c t i o n s o f Benadryl a t a dosage l e v e l o f 2 mg. p e r k i l o g r a m body weight admini s t e r e d subcutaneously a t three hour i n t e r v a l s  prior  to the f r e e z i n g i n j u r y and f o r f o r t y - e i g h t hours a f t erwards.  T h i r t y - s i x normal a d u l t nonpregnant female  Wistar r a t s were s e l e c t e d ,  and they were d i v i d e d  i n t o f i v e groups w i t h each group designed as f o l l o w s : Group: 1.  A group of e i g h t animals to serve as a .control group and to r e c e i v e a f r e e z i n g i n j u r y of twenty seconds d u r a t i o n w i t h slow thawing a t room temperature.  2.  A group o f f i v e animals to serve as a  235 c o n t r o l a l s o , and to r e c e i v e o n l y the Benadryl i n j e c t i o n s as o u t l i n e d . 3.  A group of e i g h t animals to r e c e i v e b o t h the Benadryl i n j e c t i o n s and a f r e e z i n g i n j u r y o f twenty  seconds  w i t h slow thawing at room temperature. 4.  A group of e i g h t animals to serve as a c o n t r o l , to r e c e i v e a f r e e z i n g  Inj-  u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42°G. 5.  A group o f seven animals (one of the e i g h t o r i g i n a l l y s e t a s i d e d i e d when b e i n g a n a e s t h e t i z e d withnembutal, was  and  e l i m i n a t e d completely from the  r e c o r d s ) to r e c e i v e b o t h the Benadryl i n j e c t i o n s and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n with<> r a p i d thawing i n water at 42°C. The l a r g e r number o f animals b e i n g used was d e c i d e d on because s e v e r a l other animals i n the same room had chest c o n d i t i o n s and i t was f e a r e d a few of the animals i n t h i s experiment might l a t e r develop i n f e c t i o n have to be d i s c a r d e d ,  and  hence two e x t r a r a t s were added  to each group as spares.  Group 2 i s small because  were not enough animals to f i l l  the d e s i r e d quota.  there These  236 groups are numbered i d e n t i c a l l y throughout a l l subsequent gross experiments. iment, the day o f f r e e z i n g ,  On Day 1 of the expereach animal was marked  c h a r a c t e r i s t i c a l l y on the ear, and i n i t i a l were r e c o r d e d as on the Data Sheet.  weights  The animals o f  Groups 2, 3, and 5 r e c e i v e d t h e i r i n i t i a l  subcutan-  eous i n j e c t i o n o f Benadryl s o l u t i o n prepared by ©f dilutlon the A  Parke-Davis product c o n t a i n i n g 10 mg.  per c c . to a more convenient s t r e n g t h o f 2 mg. p e r cc.  and each animal was g i v e n 0.1 c c . f o r each 100  grams of body weight.  By u s i n g a t u b e r c u l i n  graduated i n hundredths  o f a c c . i t was a simple matter  to i n j e c t the c o r r e c t  syringe  amount j u s t by g i v i n g the animal's  weight i n 1/100's of a c c .  The i n j e c t i o n s were g i v e n  every three hours f o r a t o t a l o f e i g h t e e n i n j e c t i o n s , l a s t i n g from h a l f p a s t t e n o ' c l o c k of Day 1 to h a l f p a s t one o ' c l o c k on Day 3, as noted on the Data Sheet. Groups 2 and 3 missed t h e i r i n j e c t i o n once, a t h a l f past seven o ' c l o c k on the evening o f Day 2 because o f a shortage o f the drug. cages,  The animals were kept i n f i v e  a l l the animals of one group i n the same cage. At l e a s t one hour a f t e r the i n i t i a l  Benadryl  i n j e c t i o n the animals i n Groups 1, 3, 4, and 5 were g i v e n subcutaneously from 0.07 c c . to 0.1 c c . o f nembutal leg  solution.  0,6%  Then, when a n a e s t h e t i z e d , one h i n d  o f each animal i n these groups was dipped i n a  237  beaker ©f ether through which carbon d i o x i d e gas was ©  b u b b l i n g to b r i n g the temperature down to -20 C. f o o t was Immersed up to the hock line),  The  ( j u s t below the f u r  and as soon as the s k i n blanched, i n d i c a t i n g  the  f o o t was completely f r o z e n ( a t t h i s moment the  rat  i n v a r i a b l y squirmed, i n s p i t e o f adequate anaesth-  e s i a ) , the time was counted so t h a t the l e g was immersed and f r o z e n f o r e x a c t l y twenty seconds by stop watch.  The animals  Groups 1 and 3 were  p l a c e d i n t h e i r cages and the f e e t were allowed t© thaw a t room temperature, t a k i n g approximately f i f t e e n or  twenty minutes to become limp and warm.  The animals  of  groups 4 and 5 were t r e a t e d by r a p i d thawing,  where-  by the f r o z e n f e e t were immersed Immediately a t the e x p i r y o f the twenty seconds i n t o water m a i n t a i n e d a t e x a c t l y 42°C. for  The f e e t were l e f t i n the warm water  approximately one minute, u n t i l thoroughly thawed  and warm.  Then these animals were a l s o r e t u r n e d to  t h e i r cages and a l l t h i r t y - s i x were l e f t q u i e t l y alone (except f o r the three h o u r l y i n j e c t i o n s o f Benadryl) u n t i l they wakened from the e f f e c t s o f the nembutal. Observations were made as r e c o r d e d on the Data Sheets, by the method d e s c r i b e d above f o r e s t i m a t i n g the degree of  f r o s t b i t e i n j u r y under the c a t e g o r i e s o f oedema,  238 c o l o u r change, exudate and slough, and gangrene. Data Sheets These t a b l e s are f a i r l y  self-explanatory,  b e i n g simple t a l l i e s by animals i n groups, and by days, Day 1 b e i n g the day o f f r e e z i n g . n e c e s s a r i l y as compact as p o s s i b l e .  They a r e Days 1 and 2  i n c l u d e a r e c o r d o f the three h o u r l y i n j e c t i o n s . symbols used a r e : Grp, group, three hours,  0, oedema,  Gm, grams,  and slough,  G, gangrene,  not  p r e s e n t o r not a d m i n i s t e r e d ,  B, Benadryl i n j e c t i o n .  q3h, every  G, c o l o u r change,  ate  The  E, exud-  P, f r e e z i n g ,  -, means  N, a n a e s t h e t i c ,  A l l s u r v i v i n g animals were  d i s p o s e d o f on Day 10 w i t h c h l o r o f o r m fumes. Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which i n t h i s experiment does two t h i n g s . of  Because  the u n u s u a l l y l a r g e number o f animals per group,  the  Summary Sheet f i r s t  by groups, for day.  summates the v a l u e s each day  and under I , or Index, g i v e s the t o t a l  the i n j u r y changes o c c u r r i n g f o r t h a t group  that  As d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y  Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding all  the p a t h o l o g i c a l changes apparent t o g e t h e r f o r  each group each day.  I n the lower h a l f of the  Summary Sheet the v a l u e s are i n t e r p o l a t e d down f o r a  239 Data Sheet Grp  Gm  q3h  0 C E G  1  233 288 284 298 250 317 276 276  -NP -NP -NP -NP -NP -NP -NP -NP  3 3 3 3 3 3 3 3  280 325 272 272 253  B — BBBBBBBB B—BBBBBBBB B—BBBBBBBB B—BBBBBBBB B--BBBBBBBB  218 326 305 308 272 190 280 188  BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB  3 3 3 3 3 3 3 3  1 1 3 3 3 2 3 2  302 260 308 250 315 274 280 280  -NP -NP -NP -NP -NP -NP -NP  3 3 3 3 3 3 3 3  1 1 1 1 1 1 1 1  -  -  232 275 254 214 268 246 242 196  BNP Died BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB BNPBBBBBBBB  3 3 3 3 3 3 3  1 1 1 2 -  -  1 1  -  Day  1  3 2 4 1 2 1 3 1 1 3  q3h  0 C E G  -  2 1 2 1 2  3 3 3 3 3 3 3 3  -  2 1  BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB  2  -  -  2  -  BBBBBBBB BBBBBBBB BBBBBBBB BBBBBBBB BBBBBBBB BBBBBBBB BBBBBBBB BBBBBBBB  - — — —  -  mm  - -  1 - —  - -  -—-  BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB BB-BBBBB  3 3 3 3 3 3 3 3  3 4 4 4 4 4 5 5  2 2 2 2 2 2 3 2  3 3 3 3 3 3 3 3  1 1 3 1 1 1 1 1  1 1 1 1 1 1  3 3 3 3 3 3 3  2 1 2 3 2 1 2  1 1 1 1 2  3  _  1 1 1 1 1 5 2 — —  1 _  — —  1  -  — 2 -  mm  240 Data Sheet rrp Gm  q3h 0 C E G  216 280 280 280 240 •305 265 270  B B B B B B B B  2  290 330 260 270 250  B B B B, B  3  210 305 290 300 260 175 260 165  B B B B B B B B  1  4  2 1 2 2 1 2 2 — 1  -  0 C E G  215 275 280 260 230 290 260 270  3 3 2 1 3 3 3 3  3 3 5 5 3 4 3 3  2 1 1 1 3 2 mm 1 2 1 mm  -  -  -  280 320 265 270 250 5 5 5 5 5 3 5 5  2 3 2 2 3 3 3 2  300 250 — 310 240 — 295 — 275 275 _ 270  2 1 3 2 2 3 2 2  1 1 3 3 1 3 1 2  (Dead) 265 B„ 240 B 220 B 260 B 240 B 230 B 180 B  3 2 1 3 2 1 3 3 1 2 3 2 1 1 3 1 Died  -  Day  4 3 5 3 3 3 4 3  3 3 3 3 3 2 3 2  -  5  3 3 3 2 3 3 3 3  Gm  3(cont) 4  Gm  0 C E G  210 250 270 270 230 285 250 260  3 2 3 2 3 3 2 3  5 5 5 5 5 5 5 3  —  3  — 4 — 5 — 5  5  — 5 — 4  1  mm  Gm  0 C E G  205 260 270 280 225 280 245 255  3 2 3 2 2 3 2 3  3 2 5 1 4 1 2 5 5 1 1 5 2 4 1 0 5 5 3  1 2 2 3 2 2 2 2  2 2 2 2 2 2 2 1  3 3 3 5 3 3 4 5  4 1 1 2 1 4 2 — 1 5 1 4  200 280 280 285 250 180 260 170  2 2 2 3 3 1 2 2  3 5 3 5 4 2 5 2  1 5 4 2 2 5 3 4 2 — 5 1  200 285 285 265 245 170 260 180  3 2 2 3 3 1 3 3  -2 — -1 1 — — --  295 245 300 230 280 265 275 255  1 1 2 1 1 2 1 1  1 1 3 1 — 3 2 mm 1 2 2 — 1 1 1 mm  280 250 265 235 290 260 260 250  _  1 2 2 3 1 2 1 1  1 1 2 1  280 250 280 225 305 250 260 270  1 -  -- -  260 230 220 250 230 235  2 2 2 1 1 2  2 3 3 4 1 1  235 225 220 240 230 230  1 1 1 1 1  2 -  —  5 2 M  -  -  -  - -  -_  1 1 - — — -  240 235 220 250 235 230  -  -  -  275 310 265 270 250  280 320 260 270 245  205 325 280 290 250 175 260 160  4 1  -  1 1 1 1 1 1 1  2 1 2 4 — 2 1  1 1 Qj 1  -  -  -  -  -1 1 — -— -  1 mm 1 .-1 2 a 1 1  -  mm  10  1 1 1 1 1 1 — 1 1 1 1  -  _  4  — 4  1 3 2 5 2 5  -  — — 5  1 5  - - 1 — -1 — —  — — — —  2 1 3 2 2 1 —  - -- mm  241 T o t a l s Sheet I  Gm  64  2080 59  2025 91  1385  1375  95  1945 70  1905 85  2215 42  36  2145 29  2090 27  1455 42  32  1425 26  1410 22  Gm  I  Gm  1  2222  45  2136 66  2  1402  3  2087  44  1965 86  4  2269  32  5  1526  29  Grp  Day  1  Grp  Gm  1400  3  2  4  I  Gm  I  7  5  I  1  2020 75  2  1370  3  1890 73  4  2110 13  5  1380 16  Day  I  10 Summary Sheet Gm  I  Gm  1  277  33  267 49  2  280  3  261  33  246 64  4  284  24  5  254  25  Day  1  Grp  I  Gm  48  260 45  253 69  252 57  277  275  274  72  243 52  238 63  236 54  277 30  27  268 21  261 21  264  242 37  32  237 26  235 22  230 16  I  280  2  3  4  5  I  Gm  7  I  Gm  10  I  9  242 t h e o r e t i c a l s i x animal group,  and average  weights  were obtained by d i v i d i n g the t o t a l weight f o r the group by the number o f animals i n the group. Photographs On Day 3, at approximately the afternoon,  three o'clock i n  a l l animals were again a n a e s t h e t i z e d  w i t h the same dosage o f 0.6$ nembutal s o l u t i o n , were photographed i n c o l o u r .  and  Colour p r i n t s o f  Groups 1, 2, 3, and 4 are attached below, but unfortu n a t e l y the p i c t u r e o f Group 5 was improperly exposed and d i d n o t develop.  The f e e t can be com-  pared w i t h the corresponding e n t r i e s on the Data Sheets on Day 3.  245  244  Graphs The f i n a l i n t e r p o l a t e d v a l u e s on the Summary Sheet are p l o t t e d on two simple l i n e graphs, the f i r s t f o r the I n j u r y Index changes from day to day, and the second- f o r the c o r r e s p o n d i n g weight  changes.  These are done i n c o l o u r s f o r e a s i e r r e a d i n g (except f o r the Time graphs o f the f i r s t experiment, these and a l l o t h e r gross graphs have I d e n t i c a l  colours),  but each l i n e i s otherwise c l e a r l y l a b e l l e d by the nature of the i n j u r y and treatment r a t h e r than by group numbers. C o n d i t i o n of the Animals The animals remained i n g e n e r a l good h e a l t h throughout the d u r a t i o n o f the experiment.  As  will  be apparent from the Weight graph, the Benadryl t r e a t e d animals d i d not l o s e weight any more than the c o n t r o l animals.  Th^e behaxior and appearance o f  the animals d i d not s u f f e r much. D i s c u s s i o n of the Observations I t was  f e l t t h a t Benadryl as a d m i n i s t e r e d ,  had a c o n s i s t e n t l y harmful e f f e c t on the c l i n i c a l course o f the f r o ^ s t b l t e l e s i o n s as produced In t h i s experiment.  T h i s was  r e a d i l y apparent by c a s u a l  o b s e r v a t i o n of the animals i n the v a r i o u s groups, as w e l l as apparent from a study of the d a t a o b t a i n e d and the graph drawn from i t .  Although the c o l o u r  245  1  2  3  4  7  5 D  A  T  10  246  J  5  5 D A Y  7  10  247 photographs are incomplete f o r group 5, groups and  3 may  be  compared, and  I n j u r y graph f o r day The  t h e i r p o s i t i o n s on  3 placed  the  lines).  p r e v i o u s l y noted b e n e f i c i a l e f f e c t s o f  the r a p i d thawing at 42°G. was and  (dotted  1  w i t h Groups 4 and  v e r s e l y a f f e c t e d by i n a l l respects  5,  again confirmed,  although group 5 was  ad-  the Benadryl, both groups were  l e s s a f f e c t e d by  the f r o s t b i t e a f t e r  r a p i d thawing than wereethe animals i n Groups 1  and  3 which were thawed at room temperature. All  the groups showed a s i g n i f i c a n t average  weight l o s s , except the c o n t r o l group r e c e i v i n g Benadryl o n l y .  T h i s l o s s was  the  a t t r i b u t e d s o l e l y to  the e f f e c t s of the f r e e z i n g i n j u r y , b o t h through probably pain, The a f f e c t e d the  and  toxic effects l o c a l l y .  f a c t t h a t an a n t i h i s t a m i n i c  drug  adversely  course of the l e s i o n s produced by a stand-  ard f r o s t b i t e i n j u r y suggested b o t h the need f o r r e peating  the experiment w i t h another  (Chlor-Tripolon)  and  a l s o the p o s s i b i l i t y t h a t , i f  Benadryl a n t a g o n i z i n g h i s t a m i n e or a substance l i b e r a t e d by  it  histamine-like  the trauma of f r e e z i n g , a drug  l i k e histamine should be i n the  antihistaminic  investigated for i t s role  t i s s u e r e a c t i o n , and  f o r any p o s s i b l e  influence  c o u l d have on the course of the f r o s t b i t e l e s i o n s .  248  Etamon and Hydergine Experimental O b s e r v a t i o n s w i t h Etamon and Hydergine The purpose of the experiment here was  described  to t e s t the e f f e c t s o f a standard c o l d i n j u r y as  influenced  by p a r e n t e r a l  Injections  o f two d i f f e r e n t  drugs.  Etamon c h l o r i d e  ( t e t r a - e t h y l ammonium c h l o r -  ide) was  to be t e s t e d at a dosage l e v e l o f 50  mg.  per k i l o g r a m body weight a d m i n i s t e r e d subcutaneously at three hour i n t e r v a l s , p r i o r to the f r e e z i n g and f o r f o r t y - e i g h t hours a f t e r w a r d s . was  injury  Hydergine  to be t e s t e d i n d i f f e r e n t animals at a dosage  level  of 1 c c . per animal a d m i n i s t e r e d a l s o subcutaneously at three hour I n t e r v a l s , p r i o r to'the f r e e z i n g and f o r f o r t y - e i g h t hours a f t e r w a r d s .  injury  Twenty-four  normal a d u l t male W i s t a r r a t s were s e l e c t e d ,  and they  were d i v i d e d i n t o f o u r groups w i t h each group designed as  follows: Group: 4.  A group o f s i x animals to serve as a control, to receive  a freezing  ury o f twenty seconds d u r a t i o n  inj-  with  r a p i d thawing i n water a t 42©C. 5.  A group o f s i x animals to r e c e i v e b o t h the Etamon I n j e c t i o n s  and a  f r e e z i n g i n j u r y of twenty seconds dura t i o n w i t h r a p i d thawing i n water  249  at 4.  42°G.  Another group of s i x animals to serve as a c o n t r o l , to r e c e i v e a f r e e z i n g i n j u r y o f twenty  seconds  d u r a t i o n w i t h r a p i d thawing a t 42°C. 5.  A group o f s i x animals to r e c e i v e b o t h the Hydergine i n j e c t i o n s and a f r e e z i n g i n j u r y of twenty  seconds  d u r a t i o n w i t h r a p i d thawing i n water at  42°C.  The group numbers are kept u n i f o r m throughout a l l the gross experiments, hence e x p l a i n i n g the d u p l i c a t i o n . There w i l l be no ambiguity because the group b e i n g d i s c u s s e d w i l l be e x a c t l y r e f e r r e d t o .  On the t h i r d  day p r i o r to the f r e e z i n g (Day -3 on the Data S h e e t s ) , each animal was marked c h a r a c t e r i s t i c a l l y on the ear, and i n i t i a l weights were r e c o r d e d .  On Day 1, the  day of the f r e e z i n g , weights were a g a i n r e c o r d e d and then the animals o f b o t h Groups 5 r e c e i v e d initial  their  subcutaneous i n j e c t i o n s o f e i t h e r Etamon of  Hydergine. - T h i s was a t a q u a r t e r o f t e n o ' c l o c k . The r e s p e c t i v e i n j e c t i o n s were c o n t i n u e d every t h r e e hours u n t i l a q u a r t e r a f t e r t e n o ' c l o c k on Day 3, w i t h no doses midsed.  The r e c o r d of these i n j e c t i o n s  was n o t put onto the Data Sheets.  The animals were  250 kept i n f o u r cages,  a l l animals o f one group i n the  same cage. At the  l e a s t one hour a f t e r the i n i t i a l  injections  animals were g i v e n ( a l l animals, t h a t i s ) subcut-  aneously from 0.07 to 0.12 c c . o f 0.6$ nembutal solution.  Then when a n a e s t h e t i z e d , one h i n d l e g o f  each animal was dipped i n a beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature down t o -20°C. to  The f o o t was immersed up  the hock ( j u s t below the f u r line),- and as soon  as the s k i n blanched, i n d i c a t i n g the f o o t was complete l y f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y i n s p i t e o f adequate  squirmed,  a n a e s t h e s i a ) , the time was  counted so that the l e g was immersed and f r o z e n f o r e x a c t l y twenty seconds by stop watch. were then a l l t r e a t e d by r a p i d thawing,  The animals whereby the  f r o z e n f e e t were immersed immediately a t the e x p i r y of  the twenty seconds i n t o water maintained a t  e x a c t l y 42°C. for  The f e e t were l e f t i n the warm water  approximately one minute, u n t i l thoroughly thawed  and warm.  Then the animals were r e t u r n e d to t h e i r  cages and were l e f t q u i e t l y alone (except f o r the three h o u r l y i n j e c t i o n s ) u n t i l e f f e c t s o f the nembutal.  they wakened from the  Observations were made as  r e c o r d e d on the Data Sheets,  by the method d e s c r i b e d  above f o r e s t i m a t i n g the degree o f f r o s t b i t e intiury  251 under the c a t e g o r i e s of oedema, c o l o u r exudate and slough, and  change,  gangrene.  Data Sheets These t a b l e s are f a i r l y  self-explanatory,  b e i n g simple t a l l i e s by animals i n groups, and by days, Day 1 b e i n g the day o f f r e e z i n g . n e c e s s a r i l y as compact as p o s s i b l e . for  Etamon i s p l a c e d on the f i r s t ,  Hydergine i s p l a c e d on the second. are:  G-rp, group,  change,  Gm,  grams,  A l l the d a t a and the d a t a f o r The symbols used  0, oedema,  E, exudate and slough,  -, means not p r e s e n t .  They are  G,  C, c o l o u r  gangrene,  A l l s u r v i v i n g animals were  d i s p o s e d o f on Day 13 w i t h c h l o r o f o r m fumes. Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which gives the average weight f o r each group  each  day, and under I , or Index, g i v e s the t o t a l f o r the i n j u r y changes o c c u r r i n g f o r t h a t group that day. As d e s c r i b e d b e f o r e , t h i s concept of an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the  p a t h o l o g i c a l changes apparent t o g e t h e r f o r each  group each day.  I n view of the f a c t none o f the  animals d i e d , no e x t r a p o l a t i o n was n e c e s s a r y .  As  noted on the Summary Sheet, the top h a l f a p p l i e s to Etamon, and the remainder to Hydergine.  252 D a t a Sheet - Etamon  Gm  Gm  Gm  0 C E G  Gm  0 C E G  168 184 171 175 173 156  170 193 176 183 191 157  163 180 166 172 178 151  3 3 3 3 2 3  1 2 — 1 _ — 2 1 _ 2 1 2  165 186 167 176 182 156  3 2 3 2 1 3  2 1 1 2 1 2 1 2 1  168 198 180 167 176 174  180 197 184 169 172 177  172 199 178 166 170 174  3 3 3 3 3 3  2 2 2 1 2 3  173 204 181 168 171 176  3 3 3 3 3 3  2 1 _ 2 2 1 1 1 2 1 —  Day  -3  1  2  Grp  Gm  0 C E G  Gm  0 C E G  Gm  161 184 163 180 179 150  2 2 1 2 2 1 2 1 1 2 1  2 2 -  -  161 190 166 182 188 159  2 2 2 1 — 2 2 1 1 2 1 1 2  -  ---  178 1 2 3 206 — — — 175 2 2 3 205 — _ _ 200 — — _ mm 182  168 198 180 163 167 173  2 2 2 2 2 2  1 2 1 2  172 203 182 169 167 176  2 1 1 1 2 2  2 2 — — 2 _ — 2 2 1 1 2  190 _ — 218 194 1 — — 184 1 _ 189 1 2 — 195 — 2 2  Grp 4  |  f 5  4  5  Day  -  2 1 2 2 2 3  —  -  -  -  1 1 1 1 1  mm  mm  -  —  ---  3  —  -  --  0 C E  --  -  -  13  253  Data Sheet - Hydergine  Gm  0 C E G  - —-  315 352 329 284 260 273  3 3 3 2 3 3  2 2 2 2 2 2  1 1  1 1 1 1  281 291 273 202 254 281  3 3 3 3 3 3  2 1 1 3 3 2  1 1 1  Gm  Gm  Gm  0 C E G  4  330 362 336 268 264 272  308 342 320 273 257 270  316 350 329 281 259 275  3 3 3 3 3 3  2 _ 2 1 1 2 2 1 2 1  5  289 295 279 208 267 297  282 296 285 203 266 296  283 295 279 204 263 290  3 3 3 3 3 3  2 2 2 3 3 1  Day  -3  1  2  Grp  Gm  0  C E G  Gm  0 C E G  311 346 325 286 258 275  3 3 2 2 2 2  3 3 3 2 1 3  1 2 3 1 1  311 344 326 282 258 275  2 3 2 2 2 2  3 3 2 2 2 3  1 2  273 287 268 193 261 275  3 3 3 3 3  3 2 2 3 2 8 1  1 2 1 1 ±  279 290 269 201 257 279  2 3 3 3 3 3  3 1 3 3 3 2  Grp  4  5  Day  4  _  —  _ -  _ — -  — -— - mm  - 1 -1 -1 1 -  —  3  5  Gm  0 C E G  - 3 — - 1 -  _  322 343 343 329 275 285  2 2 2 1 2  2  _  293 298 272 221 285 303  1 2 2  -2 1 2 2  -  13  —- ——1 1 2 - - —1 1 - 2, _  2 1 1 1  mm  2 3 _ 2 1 1 2 2 1 2  - -  254 Summary Sheet Etamon Gm  Gm  Gm  1  Gm  1  Gm  I  4  171  178  168  29  172  27  170  21  5  177  180  177  35  179  31  175  30  Day  -3  1  2  Grp  Gm  1  Gm  1  4  174  19  191  13  5  178  23  196  9  Day  5  Grp  3  4  13  Hydergine Gm  Gm  Gm  I  Gm  1  Gm  I  4  305  295  301  32  302  33  300  37  5  273  271  271  35  264  35  265  36  Day  -3  1  2  Grp  Gm  1  Gm  1  4  299  35  316  15  5  263  41  279  26  Day  5  Grp  13  3  4  255 Graphs The v a l u e s on the Summary Sheet are p l o t t e d d i r e c t l y on the two simple l i n e graphs, the f i r s t for  Etamon and the second f o r Hydergine,  Weight  and I n j u r y Index changes are p l o t t e d a g a i n s t the days.  The graphs had to be f o r e s h o r t e n e d between  Days 5 and 13 i n order to c o n t a i n them w i t h i n page at the s c a l e chosen.  one  The graphs are done i n  c o l o u r s f o r e a s i e r r e a d i n g (except f o r the Time graphs of the f i r s t  experiment, these and a l l o t h e r  gross graphs have i d e n t i c a l c o l o u r s ) , but each l i n e i s otherwise c l e a r l y l a b e l l e d by the nature o f the i n j u r y and treatment r a t h e r than by group numbers. C o n d i t i o n of the Animals The animals remained w e l l throughout the d u r a t i o n of the experiment.  Initially  the Etamon  had a subduing e f f e c t on the behaviour of the r a t s , but  l a t e r they became n o r m a l l y a c t i v e .  The Etamon  i n j e c t i o n s were somewhat p a i n f u l , c a u s i n g s q u e a l i n g each time the m a t e r i a l was  injected.  The Hydergine  had no apparent e f f e c t on the animals r e c e i v i n g i t . D i s c u s s i o n o f the Observations The i m p r e s s i o n c l i n i c a l l y was  t h a t Etamon  had no i n f l u e n c e on the course o f the f r o s t b i t e ury,  inj-  and that Hydergine worsened the course of the  injury.  256  -1  1  2  3  4  5  13  257  -1  1  2  3  4  5  258 On  the Etamon graph the I n j u r y Index l i n e f o r the  Etamon t r e a t e d group remained s l i g h t l y above the control l i n e , later crossing i t .  However w i t h  the Hydergine t r e a t e d Index l i n e there i s a l a t e r divergence. Noteworthy i s the f a c t t h a t both c o n t r o l i n j u r y Index l i n e s are s i m i l a r , which they  should  be i n that s i m i l a r groups, Groups 4, were b o t h treated i d e n t i c a l l y .  I f the mean of these  two  r e d l i n e s were drawn i n , the r e s u l t i n g l i n e would o n l y serve  to accentuate the d i f f e r e n c e s  described  above. The weight graphs run e s s e n t i a l l y p a r a l l e l f o r a l l groups.  Apresoline Experiments w i t h A p r e s o l i n e  and  Chlor-Tripolon  and  Chlor-Tripolon  The purpose of the experiment here was  to t e s t the e f f e c t s o f a standard  described  c o l d i n j u r y as  i n f l u e n c e d by p a r e n t e r a l I n j e c t i o n s of two d i f f e r e n t drugs.  A p r e s o l i n e was  to be  t e s t e d a t a dosage l e v e l  o f 2 mg.  per animal administered  subcutaneously at  three hour i n t e r v a l s , p r i o r to the f r e e z i n g i n j u r y and f o r was  to be  f o r t y - e i g h t hours afterwards.  Chlor-Tirpolon  t e s t e d i n d i f f e r e n t animals at a dosage l e v e l  259 of 5 mg. per animal administered a l s o subcutaneously at three hour i n t e r v a l s , p r i o r to the f r e e z i n g i n j u r y and f o r f o r t y - e i g h t hours a f t e r w a r d s .  E i g h t e e n normal  a d u l t nonpregnant female W i s t a r r a t s were  selected,  and they were d i v i d e d i n t o three groups o f s i x animals each, w i t h each group designed as f o l l o w s : Group:  4.  A group o f s i x animals to serve as a c o n t r o l , to r e c e i v e  a freezing  i n j u r y o f twenty seconds  duration  w i t h r a p i d thawing i n water a t 42°C. 5.  A group o f s i x animals to r e c e i v e b o t h the A p r e s o l i n e i n j e c t i o n s and a f r e e z i n g i n j u r y o f twenty seconds duration  w i t h r a p i d thawing i n water  at 42oc. 5.  A group o f s i x animals to r e c e i v e b o t h the C h l o r - T r i p o l o n  injections  and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water  at 42°C. The group numbers are kept u n i f o r m throughout a l l the gross experiments, hence e x p l a i n i n g  the d u p l i c a t i o n .  There w i l l be no ambiguity because the group b e i n g d i s c u s s e d w i l l be e x a c t l y r e f e r r e d t o .  On Day 1 o f the  experiment, the day o f f r e e z i n g , each animal was marked  260 c h a r a c t e r i s t i c a l l y on the ear, and i n i t i a l were recorded as on the Data Sheet.  weights  The animals o f  both Groups 5 r e c e i v e d t h e i r a p p r o p r i a t e  initial  i n j e c t i o n s at t e n o ' c l o c k and again a t one o ' c l o c k . At eal  three o ' c l o c k a l l animals r e c e i v e d by i n t r a p e r i t o n i n j e c t i o n an a n a e s t h e t i z i n g dose o f 0 . 6 $ nembutal  solution of 0.075 cc.  Then when a n a e s t h e t i z e d , one  h i n d l e g o f each animal was dipped i n a beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature down to - 2 0 C.  The f o o t was  immersed up to the hock ( j u s t below the f u r l i n e ) , and as soon as the s k i n blanched, i n d i c a t i n g the f o o t was completely f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y squirmed, i n s p i t e o f adequate a n a e s t h e s i a ) , the time was  counted so that the l e g was immersed and f r o z e n  for  e x a c t l y twenty seconds by stop watch.  The animals  were then a l l t r e a t e d by r a p i d thawing, whereby the f r o z e n f e e t were immersed Immediately a t the e x p i r y of  the twenty seconds i n t o water m a i n t a i n e d a t e x a c t l y  42©c.  The f e e t were l e f t i n the warm water f o r approx-  i m a t e l y one minute,-  u n t i l thoroughly thawed and warm.  Then the animals were r e t u r n e d to t h e i r cages and were l e f t q u i e t l y alone (except f o r the three h o u r l y ions) u n t i l nembutal.  inject-  they wakened from the e f f e c t s o f the Observations were made as r e c o r d e d on the  261 Data Sheets, by the method d e s c r i b e d above f o r e s t i m a t i n g the degree of f r o s t b i t e i n j u r y under the c a t e g o r i e s of oedema, c o l o u r change, exudate and slough, and gangrene.  The animals were kept i n three cages,  all  animals of one group i n the same cage.  On Day  all  animals were d i s p o s e d of w i t h c h l o r o f o r m fumes.  1^_4  Data Sheets These t a b l e s are f a i r l y  self-explanatory,  b e i n g simple t a l l i e s by animals i n groups, and by days, Day 1 b e i n g the day of f r e e z i n g . n ~ •?• c * . = ?a n e c e s s a r i l y as compact as p o s s i b l e .  They are  Days 1, 2, and 3  i n c l u d e a r e c o r d of the three h o u r l y i n j e c t i o n s . symbols used a r e :  Grp, group,  three hours,  0, oedema,  and slough,  G, gangrene,  administered, ection,  Gm,  q3h, every  C, c o l o u r change,  E, exudate  -, means not p r e s e n t o r n o t  P, f r e e z i n g i n j u r y ,  G, C h l o r - T r i p o l o n  grams,  The  A, A p r e s o l i n e  inj-  injection.  Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which g i v e s the average weight f o r each group  each  day, and under I, or Index, g i v e s the t o t a l f o r the i n j u r y changes o c c u r r i n g f o r that group t h a t day.  As  d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes apparent t o g e t h e r f o r each group  262 Data Sheet Apresoline Grp  Gin q3h  Gm  O C E G  4  155 158 153 133 131 132  — — — — — —  141 143 134 145 140 145  3 3 3 3 3 3  2 1 2 4 4 2  1 -  5  163 159 162 167 163 161  AAPAAAAAAAAAA AAPAAAAAAAAAA AAPAAAAAAAAAA AAPAAAAAAAAAA AAPAAAAAAAAAA AAPAAAAAAAAAA  153 146 148 157 151 144  3 3 3 3 3 3  1 1 1 1 1 1  - -  Day  P p p P p p  1  q3h  -  - - - -  AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA  2  Gm  O C E G  145 141 131 146 143 150  3 2 3 3 3 3  1 1 2 4 4 2  -  — — — — — —  152 146 151 156 147 149  3 3 3 2 3 3  1 1 1 - 1 - 1 - 1  AA AA AA AA AA AA  © 1 1  q3h  3  Chlor-Tripolon Grp 5  Day  Gm  q3h  Gm  O C E G  140 132 175 189 151 136  CCPCC—CCCCC CCPCC—CCCCC CCPCC—CCCCC CCPCC—CCCCC CCPCC CCCCC CCPCC—CCCCC  132 121 161 171 142 126  2 3 3 3 3 3  1  2  3 3 3 2 2 2  - - 1 - -  q3h C C C C C G  Gm  O C E G  q3h  125 115 155 162 137 120  1 3 3 3 3 3  CCh CC CC CC CC CC  3  5 3 4 2 3 3  2 1  1 -  263 Data Sheet Apresoline Grp Gm 0 C E G 4  5  Day  155 153 148 146 147 150  2 1 2 3 3 3  1 _ 1 2 4 3 1 1  155 145 156 161 159 149  2 2 2 1 1 1  1 _ 1 1 1 1 1 2  - -— — - _ -  0 C E G  Gm  0 C E G  158 157 153 152 153 157  1 1 1 1 2 3 4 3 3 1 2 1  -  160 164 159 158 155 162  1 2 2 1  162 150 158 163 153 155  1 1 1 2 2 2 2 1 1 1 1 2  —  174 169 173 180 165 168  1 2 2 1 2 1 1 2 1 1 1 1 2 2  GM  -  -  -  —-  -  - - —  5  4  -  2 1 2 2 2 6 2 2 2 2  - mm  mm  -—-  9  Gm  0 C E G  163 167 159 158 159 162  _  - - -1 1 - 1 1 1 6 1 2 - 2 2 2 4 -  1  _  181 159 174 180 163 168  1 2 1 1 2 2 1 1 1  *•  - - --1 - 1, 2 2 -  11  Chi or•-Tripolon Grp Gm 5  Day  121 107 145 152 128 112 4  0 C E G _  2 3 2 2 1  _ 5 1 6 2 1 2 1 5 3 2 1 5 2 3  —  Gm  0 C E G  Gm  0 C E G  123 108 148 156 134 112  _  125 122 152 175 150 113  1 2 1 1 1 1  5  2 2 2 3 1  5 1 1 1 4 5  mm  2  —  2 1 1  5 7 4 5 1 6  9  7 1 1 7 1 2 6 1 6 1 6 7 1 mm  — -  Gm  0 C E G  130 123 154 1 _ 182 153 1 1 122  7  - — 7 - 6 - ——6 —6 - - -7 mm  11  264 Data Sheet Apresoline Grp 4  Gm  166 _ 1 - 1 164 - - - 164 — 1 — — 143 1 1 - 6 142 2 2 - 2 135 1 2 — 198 166 187 186 169 178  Day  0 C E G  1 1 1 2 1 2  - - -  1 2  14 Chlor-Tripolon  Grp  Gm  0 C E G  5  134 135 175 194 162 125  1 2 2 1 2 2  Day  14  7 1 — 7 1 1 2 6 1 — 6 1 1 6 2 — 7  265 Summary Sheet Apresoline Grp  Gm  Gm  I  Gm  I  Gm  I  Gm  I  Gm  I  4  144  141  34  143  33  147  27  155  23  160  29  5  163  150  24  155  23  153  17  157  17  172  20  Day  1  2  3  4  5  9  Chlor-Tripolon Grp  Gm  Gm  I  Gm  I  Gm  I  Gm  I  Gm  I  5  154  142  35  136  40  128  49  130  61  139  54  Day  1  2  3  4  Apresoline Grp  Gm  I  Gm  I  4  161  25  152  20  5  171  18  181  13  Day  11  Grp  Gm  I  Gm  I  144  42  154  59  5 Day  11  14  14  Chior-Tripolon  5  9  266 each day.  I n view of the f a c t that none of the  animals d i e d , no e x t r a p o l a t i o n was n e c e s s a r y . Graphs The v a l u e s on the Summary Sheet are d i r e c t l y p l o t t e d onto the two simple l i n e graphs,  the f i r s t  being f o r I n j u r y Index changes by days, and the second f o r weight changes a l s o by days.  The  graphs  had to be f o r e s h o r t e n e d between Days 5 and 14 i n order to c o n t a i n them w i t h i n one page a t the s c a l e chosen.  The graphs are done i n c o l o u r s f o r e a s i e r  r e a d i n g (except f o r the Time graphs o f the f i r s t experiment, these and a l l o t h e r g r o s s graphs have i d e n t i c a l c o l o u r s ) , but each l i n e i s otherwise c l e a r l y l a b e l l e d by the nature o f the i n j u r y and treatment r a t h e r than by group numbers. C o n d i t i o n of the Animals The animals r e c e i v i n g A p r e s o l i n e were a p p a r e n t l y q u i t e u n a f f e c t e d by the drug.  However, the C h l o r -  T r i p o l o n q u i t e a d v e r s e l y a f f e c t e d the r a t s r e c e i v i n g i t . As w i l l be noted from the Data Sheets, of  q u i t e a few  the i n j e c t i o n s w i t h C h l o r - T r i p o l o n had to be missed  because of s e r i o u s I l l n e s s i n the animals. none e x p i r e d ,  However  and a l l r e c o v e r e d to an a p p a r e n t l y normal  s t a t e a f t e r the C h l o r - T r i p o l o n was  discontinued.  267  10  1  2  3  4  5  D A T  9  -  11  -  -  14  268  1  2  3  4  5  D A Y  9  -  11  -  -  14  269  Discussion of the Observations There was only one untreated  c o n t r o l group  used i n t h i s experiment, but i t behaved i n a t y p i c a l manner, and the I n j u r y Index l i n e graph f o r t h i s group I s comparable w i t h the s i m i l a r graph i n other experiments.  Generally speaking Apresoline  favour-  ably i n f l u e n c e d the behaviour of the i n j u r e d f e e t of those animals r e c e i v i n g i t . impression c l i n i c a l l y ,  This was the general  and the Apresoline I n j u r y  Index graph bears t h i s out.  However, the Chlor-  T r i p o l o n t r e a t e d animals were made very much worse a f t e r the f r e e z i n g i n j u r y .  This r e a c t i o n i s much  greater than I t was f o r Benadryl.  I t i s possible  the general e f f e c t s the Chlor-Tripolon had on the animals could have i n f l u e n c e d the h e a l i n g of the injured feet. The weight graph confirms the f a c t that the Chlor-Tripolon had an adverse e f f e c t g e n e r a l l y on the animals r e c e i v i n g i t . Rutin Experimental Observations w i t h Rutftn The purpose of the experiment here described was to t e s t the e f f e c t s of a standard c o l d i n j u r y as  270 i n f l u e n c e d by r u t i n administered o r a l l y .  Thirty  normal a d u l t male W i s t a r r a t s were s e l e c t e d f o r the t e s t and they were d i v i d e d i n t o f i v e groups of s i x animals each, w i t h each group designed as f o l l o w s : Group: 1.  A c o n t r o l group to r e c e i v e a f r e e z i n g i n j u r y of twenty seconds d u r a t i o n w i t h slow thawing a t room temperature.  2.  Another c o n t r o l group to r e c e i v e o n l y the o r a l r u t i n m e d i c a t i o n .  3.  A group to r e c e i v e b o t h the r u t i n and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h slow thawing at room temperature.  4.  A c o n t r o l group to r e c e i v e o n l y a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water at 42°C.  5.  A group to r e c e i v e b o t h the r u t i n and a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42°C.  As noted p r e v i o u s l y , these groupings are the same throughout a l l gross experiments.  On Days -9, -7, and  271  -5, b e i n g the n i n t h , seventh, and f i f t h days p r i o r to f r e e z i n g , a l l animals i n groups 2, 3, and 5 r e c e i v e d 100 c c . o f water c o n t a i n i n g 100 mg o f r u t i n (two ground up 50 mg. compressed r u t i n  tablets).  Each animal was i n a separate cage, eaeh r e c e i v i n g i t s own i n d i v i d u a l water supply c o n t a i n i n g the r u t i n . On Day -4, b e i n g the f o u r t h day b e f o r e the f r e e z i n g , the c o n c e n t r a t i o n o f the r u t i n s o l u t i o n was i n c r e a s e d so t h a t each animal o f Groups 2, 3, and 5 r e c e i v e d each day 100 mg. o f r u t i n contained i n 35 c c . T h i s was administered on eaeh o f Days -4, -3, -2, and -1, and o f course the same s o l u t i o n was g i v e n d a i l y afterwards u n t i l the animals were disposed o f on day 6.  No r e c o r d was kept o f the p r e c i s e amount o f  r u t i n consumed by the animals r e c e i v i n g i t .  There  was a l i t t l e wastage, and s e v e r a l o f the animals some o f t h e i r s o l u t i o n each day. felt  left  However, i t was  that i t c o u l d be s a f e l y claimed each animal  took  at l e a s t 50 mg. o f r u t i n p e r day, and many took as much as 90 or 95 mg. of  Although no r e c o r d was kept  the f a c t , i t was noted t h a t the animals were some-  what c o n s i s t e n t i n whether o r n o t they drank up a l l t h e i r r u t i n s o l u t i o n each day.  A l l animals i n  Groups 1 and 4 were kept s i x to a cage, t h a t i s , one cage f o r each o f these two groups. were weighed on Days 1, 3, and 5.  A l l animals  272 On Day 1, the day o f the f r e e z i n g , the animals of  Groups 1, 3, 4, and 5 were g i v e n subcutaneously  from 0.1 to 0.125 c c . o f 0.6$ nembutal  solution.  Then when the animals were a n a e s t h e t i z e d , one h i n d leg  o f each animal i n these groups was dipped i n a  beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature  down to -20°C.  The f o o t was immersed up to the hock ( j u s t below the fur  l i n e ) , and as soon as the s k i n blanched,  ing  the f o o t was completely f r o z e n ( a t t h i s moment  the r a t i n v a r i a b l y squirmed,  indicat-  i n s p i t e o f adequate  a n a e s t h e t i c ) , the time was counted so that the l e g was  immersed and f r o z e n f o r e x a c t l y twenty  seconds by  o  stop watch.  The animals o f Groups 1 and 3 were  p l a c e d i n t h e i r cages and the f e e t were allowed t e thaw a t room temperature, or  twenty minutes  t a k i n g approximately f i f t e e n  to become limp and warm.  The  animals o f groups 4 and 5 were t r e a t e d by r a p i d thawing,  whereby the f r o z e n f e e t were immersed  at  the e x p i r y of the twenty  at  e x a c t l y 42°C.  for  immediately  seconds i n t o water maintained  The f e e t were l e f t i n the warm water  approximately one minute, u n t i l thoroughly thawed  and warm.  Then these animals were a l s o r e t u r n e d t©  t h e i r cages and a l l t h i r t y were l e f t q u i e t l y alone u n t i l they wakened from the e f f e c t s o f the nembutal.  273  Observations were made as recorded i n the Data Sheet, by the method d e s c r i b e d b e f o r e f o r e s t i m a t i n g the degree o f f r o s t b i t e i n j u r y under the c a t e g o r i e s o f oedema, c o l o u r change,  exudate, slough, and gangrene.  Data Sheet This table i s f a i r l y  self-explanatory, being  a simple t a l l y by animals i n groups, and by days, Dayl being the day o f f r e e z i n g . compact as p o s s i b l e . group,  Gm, grams,  exudate and slough,  I t i s n e c e s s a r i l y as  The symbols used a r e ; 0, oedema,  Grp,  C, c o l o u r change,  G, gangrene,  P, f r e e z i n g ,  E, -,  means n o t p r e s e n t . Summary Sheet F o l l o w i n g the Data Sheet i s a Summary Sheet which g i v e s the average weight f o r each group  each  day, and under I , or Index, g i v e s the t o t a l f o r the i n j u r y changes o c c u r r i n g f o r t h a t group t h a t day. As d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes apparent together f o r each group each day.  Except f o r Group 2, where o f course no  f r eezing was performed, and no Index number--wilife appear. Because no animals e x p i r e d no e x t r a p o l a t i o n was necessary.  274 Data Sheet  Grp Gm 1  230 270 280 255 245 260  0 C E G 3 3 2 3 3 3  2 1 3 2 2 2  2 1  2 2 2 2  2 — _  2  240 235 250 240 255 200  3  215 240 235 195 210 235  2 3 2 2 2  2 2 1 1 1  2 1 1 1 2  190 215 240 265 230 245  3 2 3 3 2 3  2 2 1 1 2 1  2 2 2 1 —  250 235 230 245 205 245  2 1 2 2 2 1  1 1 — 1 1 1 1 1  4  5  Day 1  Gm  0 G E G  0 C E G  210 255 250 230 225 240  2 3 3 3 2 3  3 3 3 3 3 2  2 2 1 2 2 1 2 1 2 1 2 1 1  -  -  —  3 3 3 3 3 2  3 1 3 3 1 3 3 2  — -  235 200 205 230 245 200 222 2 —  2  -  2 1  — — — mm  —  -  -— --  Gm  0 C E G  220 245 250 210 220 240  3 2 3 2 3 2  3 3 3 1 3 2  2 1 3 3 1 1 2 2 2  2 3 2 1 1 1  -  —  0 C E G 3 2 3 2 3 1  2 _ 1 2 3 3 1 2 1 3 3 2 mm  --  —-  240 225 235 235 255 215  -  -  -  -  210 230 200 190 215 205  2 2 2 2 2 2  2 2 1 2 1 2 1 2  -  3 3 3 2 1 2  2 3 2 2 2 2  3 2 1 1 1 2 2 2  210 235 210 180 215 225  2 2 3 1 1 1  2 3 2 2 1 1 1 1  -  2 2 1 1 1 1  2 3 1 1 1 1  185 205 230 260 215 240  111 1 1 1 — 2 2 1 2 1 1 — 1 2 2 — 1 1  2 1 2 1 1 1  2 1 1 2 1 1  2  190 210 220 270 220 245  2 2 2 1 1 — 2 2 2 1 1 — — 1 2 1 2 2  2 1 1 1 1 1  2 _ 2 1 2 2 1 — 2 — 2 1  225 210 210 215 180 230  1  1 1 — 1 2 2 2  _  _  ;3  1 2 2 1 1 2  --  --  —  2 2 2  2 1 1 1  1 — —  ---  -  2 1 1 1  -  -  2 2 —  1  mm  2  — -  2 --  -  -  _ 230 1 205 — 1 210 1 2 2 215 175 2 2 2 — 250  --  5  -  1 2 3 1 — 1 1 1  mm  -  ---  1 2 2 1 1 2 6  -  1  -  275  Summary Sheet I  Gm  I  Gm  1  257  42  235 36  2  237  3  222  31  208 34  31  212 33  31  4  231  35  223 21  25  216 24  22  5  235  17  228 13  16  214 13  10  1  2  3  4  5  6  Day  I  I  Gm  53  231 47  I  Grp  37  234  237  Graphs The v a l u e s appearing on the Summary Sheet above were p l o t t e d d i r e c t l y onto two simple l i n e graphs, the f i r s t f o r the I n j u r y Index changes from day to day, and the second f o r the weight  changes.  These are done i n c o l o u r s f o r e a s i e r r e a d i n g (except for  the Time graphs o f the f i r s t  experiment, these and  all  succeeding gross graphs have i d e n t i c a l  colours,  and o f course, a l l are on the same s c a l e ) , b u t each l i n e i s otherwise c l e a r l y l a b e l l e d by the n a t u r e o f the  i n j u r y and treatment r a t h e r than by group numbers.  C o n d i t i o n o f the Animals All  animals remained i n e x c e l l e n t c o n d i t i o n f o r  276  1  2  3  4  D A Y  5  6  277  260  1  2  5 D A Y  4  5  278 the  duration of  rutin  the  experiment.  They drank  their  solution readily.  Photographs On D a y 4, animals with solution, as  a l l the  to  feet  with  Sheet,  4.  to  the  and a l s o w i t h  anaesthetizing  cc o f 0 . 6 $  the  recorded for the  below.  appearance  Injury  the  nembutal  were photographed  colour prints  compare values  again  0.125  feet  i n d i c a t e d by the  interesting  Day  0.1  after  of  in It  colour is  these  Day 4 on the  Index graph  for  Data  279  280 D i s c u s s i o n of Observations I t was  f e l t both c l i n i c a l l y  and  through  o b s e r v a t i o n of the Data Sheet and graphs t h a t r u t i n as a d m i n i s t e r e d had a c o n s i s t e n t l y b e n e f i c i a l on the c l i n i c a l  course o f f r o s t b i t e l e s i o n s as p r o -  duced i n t h i s experiment. development  effect  T h i s c o n s i s t e d i n decreased  o f oedema, l e s s c o l o u r change, l e s s  slough, and l e s s gangrene.  The I n j u r y Index  skin graph  l i n e s are w e l l and c o n s i s t e n t l y separated. The p r e v i o u s l y noted b e n e f i c i a l e f f e c t o f r a p i d thawing i n water a t 42 G. was Q  again confirmed,*  and w i t h r u t i n , the animals r e c e i v i n g the drug were i n a l l r e s p e c t s lefis a f f e c t e d by the I n j u r y than those not  r e c e i v i n g the drug, although b o t h groups were  r a p i d l y thawed. R u t i n as a d m i n i s t e r e d appeared  to have no  ad-  v e r s e e f f e c t s on the animals r e c e i v i n g i t . A l l o f the animals on the average i n each group showed a s i g n i f i c a n t weight l o s s , except f o r the c o n t r o l group r e c e i v i n g r u t i n o n l y .  The r o l e t h a t l i m i t e d  fluid  i n t a k e p l a y e d i n t h i s weight l o s s was not assessed. Note; The s e n i o r m e d i c a l student who the  was h e l p i n g w i t h  p r o j e c t p a r t time performed the a c t u a l f r e e z i n g ©f  281  the f e e t , and w h i l e he f r o z e them more or l e s s u n i f o r m l y throughtout the r u t i n s e r i e s , h i s degree of i n j u r y was only about e i g h t y p e r c e n t as severe as the i n j u r y used elsewhere In the experiments. Although i t i s easy to w r i t e about a standard c o l d i n j u r y , the a c t u a l performance o f the same i s a f f l i c t e d w i t h many minor v a r i a t i o n s . Ascorbic Acid Experimental O b s e r v a t i o n s w i t h A s c o r b i c  Acid  The purpose o f the experiment here d e s c r i b e d was to t e s t the e f f e c t s o f a standard c o l d  injury  as i n f l u e n c e d by p a r e n t e r a l i n j e c t i o n s o f a s c o r b i c a c i d a t a dosage l e v e l o f 50 mg. administered subcutaneously i n 1 c c . amounts twice a day f o r f i v e days p r e c e d i n g the f r e e z i n g i n j u r y and f o r f i v e afterwards.  days  Forty-two normal a d u l t nonpregnant  female W i s t a r r a t s were s e l e c t e d , and they were d i v i d ed i n t o seven groups o f s i x animals each, w i t h each group designed as f o l l o w s : Group: 1.  A c o n t r o l group to r e c e i v e a f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h slow thawing a t room temperature.  2.  A group to r e c e i v e only the a s c o r b i c  282 a c i d i n j e c t i o n s as o u t l i n e d . 3.  A group to r e c e i v e both the a s c o r b i c a c i d i n j e c t i o n s and a f r e e z i n g ury  inj-  o f twenty seconds d u r a t i o n w i t h  slow thawing at room temperature. 4.  A group to r e c e i v e a f r e e z i n g of  injury  twenty seconds d u r a t i o n w i t h  r a p i d thawing i n water at 42°G. 5.  A group to r e c e i v e both the a s c o r b i c a c i d i n j e c t i o n s and a f r e e z i n g ury  inj-  o f twenty seconds d u r a t i o n w i t h  r a p i d thawing i n water a t 42°C. 6.  A c o n t r o l group f o r m i c r o s c o p i c obse r v a t i o n to r e c e i v e no a s c o r b i c  7.  acid.  A group f o r m i c r o s c o p i c  observation  to  Injections  r e c e i v e the a s c o r b i c  as o u t l i n e d . These groups, w i t h the e x c e p t i o n o f Groups 6 and 7 are numbered i d e n t i c a l l y throughout a l l gross experiments. Note the m i c r o s c o p i c work was not performed as planned on these l a s t two groups, but they were c a r r i e d along u n t i l Day 3, and t h e i r weights were i n c l u d e d i n the Data Sheets as c o n t r o l s .  On the f i f t h day b e f o r e the  f r e e z i n g , Day -5, each animal was marked c h a r a c t e r i s t -  283 i c a l l y on the ear, and i n i t i a l weights were r e c o r d e d as on the Data Sheets.  The animals o f Groups 2, 3,  5, and 7 a l l r e c e i v e d t h e i r i n i t i a l  subcutaneous  i n j e c t i o n s o f a s c o r b i c a c i d at n i n e o ' c l o c k .  The  i n j e c t i o n s were continued a t nine o ' c l o c k b o t h morning and evening u n t i l n i n e o'clock i n the morning o f Day 3, w i t h the i n j e c t i o n s being missed once, the evening dose on Day -1. Data Sheets.  D a i l y weights were recorded as on the  On Day 1, the day o f the f r e e z i n g , the  animals of Groups 1, 3, 4, and 5, a t a q u a r t e r p a s t ten  o ' c l o c k , were a n a e s t h e t i z e d w i t h an i n t r a p e r i t o n -  eal  i n j e c t i o n o f 0.075 to 0.1 cc o f 0.6$ nembutal  solution. of  Then, when a n a e s t h e t i z e d , one h i n d l e g  each animal i n these groups was dipped i n a  beaker o f ether through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature  down to -20®G.  The f o o t was immersed up to the hock ( j u s t below the fur  line),  and as soon as the s k i n blanched, I n d i c -  a t i n g the f o o t was completely f r o z e n ( a t t h i s moment the r a t i n v a r i a b l y squirmed, i n s p i t e o f adequate a n a e s t h e t i c ) , the time was counted  so t h a t  the l e g was immersed and f r o z e n f o r e x a c t l y twenty seconds by stop watch.  The animals o f Groups 1 and  3 were p l a c e d i n t h e i r cages and the f e e t were allowed to  thaw a t room temperature,  t a k i n g approximately  284  f i f t e e n o r twenty minutes t o become limp and warm. The animals of groups 4 and 5 were t r e a t e d by r a p i d thawing, whereby the f r o z e n f e e t were  immersed  immediately a t the e x p i r y of the twenty seconds i n t o water maintained a t e x a c t l y 4 2 C . G  The f e e t were  l e f t i n the warm water f o r approximately one minute, u n t i l thoroughly thawed and warm.  Then these  animals were als© r e t u r n e d to t h e i r cages and a l l animals were l e f t q u i e t l y alone u n t i l they wakened from the e f f e c t s o f the nembutal.  Observations were  made as recorded i n the t a b l e s , by the method d e s c r i b ed above f o r e s t i m a t i n g the degree o f f r o s t b i t e from the c a t e g o r i e s o f oedema, c o l o u r change, exudate and slough, and gangrene.  The animals o f Groups 6 and 7  were destroyed ©n Day 3 and the remainder on Day 8, a l l by c h l o r o f o r m fumes. Data Sheets These t a b l e s are f a i r l y  self-explanatory,  being simple t a l l i e s by animals i n groups, and by days, Day 1 being the day o f f r e e z i n g . e s s a r i l y as compact  as p o s s i b l e .  There i s i n c l u d e d a  record of a l l ascorbic a c i d i n j e c t i o n s . used are:  Grp, group,  They are nec-  The symbols  Gm, grams, b i d , twice a day,  285 Data Sheet Gm  bid  -P-  —  196 198 180 230 201 212  -F-F-F-F-  205 201 218 216 203 219  Cccccc-  201 196 217 205 194 206  C-C c-c c-c c-c c-c c-c  cc cc cc cc cc cc  233 225 210 229 229 224  cccccc-  227 226 212 229 233 225  CPC CFC CFC CPC CFC CFC  --  213 224 201 193 212 227  --  218 226 203 193 215 232  -F-F-F-F-F-F-  Gm  bid  Gm  bid  Gm  bid  Gm  190 192 172 219 195 206  191 195 176 217 199 208  _ _  _  ~-  193 198 180 221 204 210  _ mm  —  193 198 177 221 204 212  —  193 198 181 227 205 212  2  200 199 208 203 190 208  197 194 209 205 198 214  CC CC cc cc cc cc  200 189 215 212 198 219  CC CC cc cc cc cc  201 195 220 212 201 217  CC cc cc cc cc cc  3  215 220 206 212 220 203  225 220 209 217 222 209  cc cc cc cc cc cc  225 224 212 221 229 212  cc cc cc cc cc cc  232 231 210 225 233 220  4  204 218 200 185 208 219  204 215 201 187 211 219  201 209 204 198 200 190  Grp Gm 1  5  6  7  — — — —  mm  -. —  mm mm  bid — — —  --  —P<—  —  208 220 201 190 209 221  --  210 222 202 195 210 220  201 214 204 194 206 195  cc cc cc cc cc cc  206 221 206 199 210 192  CC cc cc cc cc cc  205 229 200 202 211 194  GG  206 233 203 200 214 192  cccccc-  206 230 205 199 210 196  CFC CFC CPC CPC CPC CPC  219 200 216 185 174 182  220 200 214 186 174 186  __  __  --  230 203 218 189 186 188  __ _  —  226 199 217 189 186 186  __  —  226 200 211 191 179 191  __  —  216 200 211 182 178 187  210 206 200 195 193 192  214 210 205 198 195 196  cc cc cc cc cc cc  217 207 206 201 196 199  cc cc cc cc cc cc  214 219 203 212 196 207  cc cc cc cc cc cc  213 218 196 207 196 203  cccccc-  212 217 199 210 196 196  G-C C-C C-C C-C C-C C-C  Day -5  -4  — — —  — —  -3  -mmm  — —_  — ——  —  -2  cc cc cc cc cc  -1  — mm mm  — mm mm  — — — —  1  286 Data Sheet G r p Gm  1  2  3  4  5  6  7  Day  199 199 180 228 200 217  0 C E G  bid  Gm  0 C E G  bid  Gm  0 C E G  4 4 4 4 4 4  __  196 199 181 223 201 218  4 4 4 4 4 4  __  194 202 179 221 196 211  3 3 3 3 3 3  3 3 3 3 3 3  1 1 2 2 2 2  _  -  227 230 217 228 235 227  4 4 4 4 4 4  3 3 3 3 3 3  2 2 2 2 2 2  217 225 203 187 214 232  4 4 4 3 4 4  1 1 1 3 1 3  2 2 2  -  —  -  1 1  •*  204 236 209 204 215 202  4 4 4 4 4 3  2 2 3 1 1 2  214 223 202 216 202 202 2  — —  -  203 199 223 199 186 198  235 204 208 195 192 194  —  -  -  —  2 1 1 2 1  cc cc cc cc  204 200 214 187 177 189  cc cc cc cc cc cc  220 221 213 219 223 217  4 3 3 3 4 4  4 4 4 4 4 3  211 219 195 183 206 226  4 4 3 3 4 4  1 1 1 2 3 — 1 1 3 1  202 226 202 203 210 198  3 3 3 3 3 3  3 2 2 2 1 2  -  -  -  - mm mm  CC  -  -  2 2 1 2 2 2  CC CC  mm mm  -  3 3 4 3 3 3  cc cc cc cc cc  --  231 204 221 188 191 196  cc cc cc cc cc cc  211 223 200 222 196 202  — — —  3  2 2 2 2 2 2  —  -  ——  — -  -  _ —  -  —  3 1 —  -  —  —  1 2  — -  -  cc cc cc cc cc cc cc cc cc cc  217 222 213 221 226 211  3 1 1 2 4 3  3 4 5 5 3 3  ,211 220 193 176 195 204  3 3 3 3 2 3  2 2 1 3 1 3  1 2 2  -  —  mm  —  —  201 223 203 203 214 199  3 3 3 3 2 2  3 3 3 2 2 2  2 2  —  —  —  1 1 2  -  — _— —  -  _  206 202 215 183 171 181  mm mm  -  2 2 1 1 1 2  CC CC  —  — —  3 4 4 2 3 3  cc cc cc cc cc cc  Gm  185 195 175 220 190 206 208 202 210 176 164 174  2  _  1 1 2 1 1 1 2 —  -  -  1  -  214 221 216 218 224 205 212 215 193 184 199 217 198 220 198 208 211 205  — mm mmmm mm mm  -- —  CC CC CC CC CC  cc  4  5  287 Data Sheet Grp 1  0 C E G  Gm  0 C E G  Gm  0 C E G  3 3 3 3 3 3  195 199 178 219 192 199  3 3 2 3 3 3  196 198 174 219 194 201  3 2 2 2 3 3  4 4 4 4 4 4  2 2 — 2 2 1 1 2  -  -  -  2  3  4  5  Day  3 4 4 4 4 4  1 2 1 2 2 2  4 4 2 5 1 5  214 205 219 167 156 164 2  3 1 1 2 3 3  4 4 4 4 4 4  1 -  3 3 3 3 2 3  3 3 1 4 1 3  2 3 2 1 1 3  -  3 3 3 2 2 2  2 2 2 2 2 2  3 3  -  2 2  1 1 2  7 6 3  -  -  3  -  -  5(Cont)  3 2 4 3 5 4  3 3 3 5 3 3  3 6 3 5 5 5  220 205 220 161 154 157  Gm  0 C E G  194 196 171 212 193 204  3 2 2 2 2 3  3 3 3 3 3 3  1 2 3 2 3 3  4 6 6 6 6 5  8 9 9 9 5 7  215 200 210 148 154 151  210 211 218 214 221 203  2 5 1 4 9 1 5 1 7 2 3 2 7 3 3 2 4 3 3 3 6  207 210 214 208 217 203  2 1 3 3 3 3  5 3 3 3 3 3  2 2 1 2 3 3  5 9 8 8 5 6  210 212 214 214 220 200  2 2 2 3 3 3  2 2 2 3 3 3  3 1 0 2 3 2  215 221 202 193 204 219  2 3 2 2 1 3  2 1 3 2 — 2 — 4 2 2 1 2 3 3  -  215 220 199 189 202 213  2 3 2 3 2 3  3 3 1 3 2 3  3 «, 2 2 2 2 2 3  -  213 211 197 188 202 211  1 2 2 2 2 3  3 3 2 3 2 3  2 mm 3 2 «M 2 3 1 3  202 217 208 209 218 211  3 3 3 2 2 1  2 3 3 3 — 2 — 3 2 — 2 — 2 2  205 216 208 207 219 203  3 3 3 2 1 1  2 3 3 2 2 3  3 mm 3 2 3 1 an 1 2  196 214 210 212 214 208  3 3 2 1 2 1  3 3 2 2 2 3  3 3 1 3 1 — 2 — 2 —  6  - - -  M  -  -  -  7  -  -  8  -  -  288 0,  oedema,  G, c o l o u r change,  G, gangrene, injury,  E, exudate and slough,  C, a s c o r b i c a c i d i n j e c t i o n ,  P,  freezing  -, means not p r e s e n t or not a d m i n i s t e r e d .  Summary Sheet F o l l o w i n g the Data Sheets Is a Summary Sheet which gives the average weight f o r each group  each  day, and under I, or I n j u r y Index, g i v e s the t o t a l f o r the i n j u r y changes o c c u r r i n g f o r t h a t group t h a t  day.  As d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes apparent together f o r each group each day.  S i n c e no animals e x p i r e d there was  no  extrapolating required. Graphs The v a l u e s on the Summary Sheet are p l o t t e d d i r e c t l y onto two simple l i n e graphs, the f i r s t f o r the day to day I n j u r y Index changes, f o r the c o r r e s p o n d i n g weight changes.  and the second These are  done i n c o l o u r s f o r e a s i e r r e a d i n g (the same c o l o u r s are used throughout a l l gross g r a p h s ) , but each l i n e i s otherwise c l e a r l y l a b e l l e d by the n a t u r e of the I n j u r y and treatment r a t h e r than by group numbers. In the case o f the weight graph, two s h o r t e r l i n e s are i n c l u d e d f o r Groups 6 and 7 .  I t haSjbecause o f  crowding,been n e c e s s a r y to I n d i c a t e Group numbers on a few of the c r o s s i n g  lines.  289 Summary Sheet Grp Gm  Gm  Gm  Gm  Gm  Gm  Gm  I  Gm  I  1  196  198  201  201  203  203  204 52  203 54  2  201  203  206  208  210  203  201  195  3  213  217  221  225  225  225 -  227 54  217 56  4  206  206  208  210  212  215  213 41  207 36  5  200  202  206  207  208  208  212 41  207 37  6  196  197  196  200  201  202  205  205  7  199  203  204  209  206  205  210  209  -4  -3  -2  -1  1  2  3  I  Gm  I  Gm  Day -5  Grp Gm  I  Gm  I  Gm  I  1  201 47  195 54  197 71  197 83  195 79  2  193  189  188  186  180  3  218 48  216 60  213 76  210 89  210 86  4  200 35  203 44  209 40  206 46  204 44  5  207 39  207 40  211 38  210 43  209 42  Day  4  5  6  7  8  290  90  ' A  8C  / /  v  Ascorbic j / Slow Tnaw / 70  I  I I U  6C  /  \ /  H D £ X  W  50  /  ' / / /  Y  I  v  I  / / Control / / Slow Thaw  J I  \ v  /  / \  Control Past Thaw  / /  40 Ascorbic Past Thaw 30  20  10 •4  -2  1  2 D  A  3 Y  4  5  6  7  8  291  -4  -2  1  2  3  4  5  6  7  8  292  C o n d i t i o n o f the Animals The a s c o r b i c a c i d i n j e c t i o n s as performed w i t h the dosage used caused a s k i n slough at the s i t e o f injection.  The s k i n over the d e p o s i t o f the drug  would t u r n b l a c k w i t h i n twenty-four to t h i r t y - s i x hours, and by the second day a c i r c u l a r piece o f s k i n and f u r would drop out, l e a v i n g a c l e a n c i r c u l a r T h i s must be a t t r i b u t a b l e t o a s t r i c t l y l o c a l w i t h no g e n e r a l i z e d r e a c t i o n s .  defect.  effect,  Except f o r the p a i n  of the i n j e c t i o n m a t e r i a l , the animals seemed o t h e r wise u n a f f e c t e d by the drug.  There i s no c l e a r - c u t  tendency toward weight changes apparent from the graphs t h a t would I n d i c a t e a g e n e r a l adverse  effect.  The c o n t r o l group, Group 2, r e c e i v i n g o n l y a s c o r b i c a c i d i n j e c t i o n s does show a marked weight l o s s  that  c o u l d be a t t r i b u t e d ©nly to the I n j e c t i o n s . B i s c u s s i o n o f Observations A s c o r b i c a c i d as a d m i n i s t e r e d caused  little  i n f l u e n c e on the course o f the l e s i o n s a f t e r the standard c o l d i n j u r y as performed.  I n the case o f  slow thawing groups, the a s c o r b i c a c i d l i n e i s a t all  times s l i g h t l y h i g h e r than the c o n t r o l , i\is als©  quite p a r a l l e l diversion. significant.  to i t except on Day 8 when there i s  I t i s doubtful i f t h i s difference i s I n the case o f the r a p i d thaw groups  293 the l i n e s c r o s s , but g e n e r a l l y remain c l o s e t o g e t h e r . So l i t t l e  e f f e c t d i d the a s c o r b i c a c i d have  t h a t i t was  clinically  decided u s e l e s s to perform the m i c r o s c o p i c  experiments, and as mentioned  the animals o f Groups  6 and 7 were destroyed on Day  3.  The I n j u r y graph g i v e s a dramatic  demonstrat0  i o n of the v a l u e of r a p i d thawing i n water a t 42 C. as c o n t r a s t e d w i t h thawing at room temperature.  The  c o n t r o l graphs are n o t q u i t e t y p i c a l of what u s u a l l y happend.  This would i n d i c a t e a s l i g h t l y more severe  f r e e z i n g i n j u r y than i s u s u a l l y  performed.  Histamine gxperimental Observations w i t h Histamine The purpose o f the experiment here d e s c r i b e d was  to t e s t the e f f e c t s o f a standard c o l d i n j u r y as  i n f l u e n c e d by p a r e n t e r a l i n j e c t i o n s o f h i s t a m i n e a t a dosage l e v e l o f 10 mg.  p e r k i l o g r a m body weight  administered subcutaneously a t three hour  Intervals  beginning immediately p r i o r to the f r e e z i n g  injury  and c o n t i n u i n g f o r f o r t y - e i g h t hours a f t e r w a r d s .  In  view of the f a c t an e f f e c t i v e dosage of h i s t a m i n e was unknown,  t h i s experiment was  run, and a c c o r d i n g l y , because  i n the n a t u r e of a t r i a l animals were s c a r c e ,  294 a v a r i e d c o l l e c t i o n of r a t s were used, w i t h almost no selection possible.  There were three groups o f s i x  animals, each group c o n t a i n i n g  an a d u l t normal male  Wistar, two normal a d u l t male Hoodeds, and three normal a d u l t nonpregnant female Hoodeds. group was  Although each  a mixture of breeds, each group was  s i m i l a r to each o t h e r .  fairly  The groups were designed as  follows: Group: 2.  A c o n t r o l group to r e c e i v e o n l y the h i s t a m i n e i n j e c t i o n s as o u t l i n e d .  4.  A c o n t r o l group a l s o to r e c e i v e  a  f r e e z i n g i n j u r y o f twenty seconds duration  w i t h r a p i d thawing i n water  at 42°C. 5.  A t e s t group to r e c e i v e b o t h the histamine i n j e c t i o n s as o u t l i n e d , and a f r e e z i n g i n j u r y of twenty seconds d u r a t i o n  w i t h r a p i d thawing  i n water a t 42°G. These groups have the same numbering  as s i m i l a r groups  throughout a l l the gross experiments.  D a i l y weights  were taken a f t e r each animal was c h a r a c t e r i s t i c a l l y marked on the ear, and r e c o r d e d on the Data Sheets. On Day 1, the day of the f r e e z i n g , the animals o f  295 Groups 2 and 5 r e c e i v e d t h e i r i n i t i a l  subcutaneous  i n j e c t i o n o f h i s t a m i n e , and an hour and a h a l f  later,  at h a l f p a s t t e n o ' c l o c k , thesanimals o f Groups 4 and 5 were a n a e s t h e t i z e d w i t h 0.1 c c . o f 0.6$ nembutal s o l u t i o n administered subcutaneously.  When anaesth-  e t i z e d , one h i n d l e g o f each animal i n these was  groups  dipped i n a beaker of ether through which carbon  d i o x i d e gas was b u b b l i n g to b r i n g the temperature down to -20°C.  The f o o t was immersed up to the hock  ( j u s t below the f u r l i n e ) ,  and as soon as the s k i n b l a n -  ched, i n d i c a t i n g the f o o t was completely f r o z e n , the time was counted so t h a t the l e g was immersed and f r o z e n f o r e x a c t l y twenty seconds by stop watch.  The  animals were then t r e a t e d by r a p i d thawing, whereby the f r o z e n f e e t were immersed immediately a t the exp i r y o f the twenty e x a c t l y 42°G. for  seconds  i n t o water maintained a t  The f e e t were l e f t  approximately one minute,  and warm.  until  I n the warm water thoroughly thawed  Then the animals were r e t u r n e d to t h e i r  cages, and were l e f t q u i e t l y alone (except f o r the three h o u r l y i n j e c t i o n s o f histamine) u n t i l from the e f f e c t s o f the nembutal.  they wakened  Observations were  made as r e c o r d e d on the Data Sheets, by the method d e s c r i b e d above f o r e s t i m a t i n g the degree o f f r o s t b i t e  296 i n j u r y under the c a t e g o r i e s of oedema, c o l o u r exudate and slough, and  change,  gangrene.  Data Sheets These t a b l e s are f a i r l y  self-explanatory,  being simple t a l l i e s by animals i n groups, and by days, Day 1 b e i n g the day of f r e e z i n g . n e c e s s a r i l y as compact as p o s s i b l e .  They are Days 1 and 2  i n c l u d e a r e c o r d o f the three h o u r l y i n j e c t i o n s . symbols used are;  Grp, group,  three hours,  0, oedema,  and slough,  G, gangrene,  m, male,  f , female,  Gm,  grams,  q3h, every  C, c o l o u r change, W, W i s t a r ,  The  E, exudate  H, Hooded,  H, h i s t a m i n e i n j e c t i o n , P, f r e e z i n g  -, means  not  p r e s e n t or not a d m i n i s t e r e d ,  injury.  All  s u r v i v i n g animals were d i s p o s e d o f on Day 8 w i t h  c h l o r o f o r m fumes. Summary Sheet F o l l o w i n g the Data Sheets i s a Summary Sheet which g i v e s the average weight f o r each group each day, and under I, o r I n j u r y Index, g i v e s the t o t a l f o r the  i n j u r y changes o c c u r r i n g f o r that group t h a t day.  As d e s c r i b e d b e f o r e , t h i s concept o f an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes  t o g e t h e r f o r each group each day.  Note that s e v e r a l v a l u e s had to be o b t a i n e d by e x t r a p -  9  —  — —  s s z2  2  s  e  --  *2T 391 3 391 2 8£T  mm mm  e s 2 r8T  —  -  I e  —  -  2 s 2 v9T S z 2 9ST 2 z 2 £91 I I I 203 s S 933 3 I 2 603  --  mm  — —  S £e  1 3 2 99T 1 3 2 29T 3 2 2 091 T f T f T 303 I 3 2 133 3 1 2 ITS  —  *6T 623 623 f-23  a oo  0 0  82T - 3 I LfZ - 3 3 3 09 T £9T - 3 3 -  T  -  T 3 3 T 3 — 3  T T 2 T 3 T  381 V  *  89T 29T 29T £81 6T3 033 99T 9frT 261 933 023 933  0  a  0  0 ras  T 3 2 9£T  HHHHHHHH  2  HHHHHHHH HHHHHHHH HHHHHHHH HHHHHHHH HHHHHHHH HHHHHHHH  3  T 3 3 T 3 3  3 f 09T T V 99T 2 2 £91 3 2 061 3 p 033 T * 8T3  98T v33 £33 323 0  «D  HHHHHHHffiMH HHHHHHHHHdH HHHHHHHHHiffl HHHHHHHHHiSH I.'.;'(..R'TCI.0.n«3H HHHHHHHHHiffl i 8  d  -  HHHHHHHHH-H HHHHHHHHH-H HHHHHHHHH-H HHHHHHHHH-H HHHHHHHHH-H HHHHHHHHH-H t*2D  (pBecr)  If!  a  T  ,  - T 3  HHHHHHHH HHHHHHHH HHHHHHHH HHHHHHHH  691  3 —  (q-troo) 3 ted  T i 2 *2T 3 T 2 9vT 3 2 3 091 T 2 2 991  69T £*T 26T 223 023 223  LftT  a  Til  3 3 2 28T  r£T  0  -  3 3 2 £21 3 I 2 i H 2 2 3 191  2  0 0  TOO  T-  2-  v-  Asa  9vT r9T 99T T£T £31 *8T  TvT v9T 99T 691 63T 6£T  £21 091 £9T 891 931 ££T  32T 02T 09T JH 69T 221 m ££T  9  691 £91 £91 86 T 933 233  £21 OvT 921 26 T 933 6T3  09T 29T 09T 08T 9T3 6T3  £91 69T 89T JH 081 233 mn frT3 mm  *  0£T 9*1 261 223 923 633  991 6f-T 06 T 223 823 633  T9T 62T ££T 023 923 6T3  39T 39T 261 JH 323 323 m 0T3  3  ra©  £63  TOD  mo  to©  298 Data Sheet Grp Gm 2  0 C E G  239 233 239 197 151 172  4  205 222 208 167 157 160 5  0 C E. G  Gm  242 237 244 195 150 175 2 2 1 2 2 3  2 3 2 2 2 3  1 2 2 3 3 3  186 2 3 (Dead) 181 2 2 153 2 3 157 3 2 144 2 2  Day  Gm  -  —  -  2 2 6 2 2 —  6  203 221 207 163 159 165  245 240 244 200 150 180 2 2 1 2 2 3  2 2 2 3 2 3  3 3 3 2  -  3  187 2 3 1 188 157 147 141  0 C E G  2 2 3 2  3 3 3 3  2 2 1 2 3 3  266 222 215 167 161 167  -  —  2 2 2 3 2 3  2 3 1 2 3 3  187 2 3 2  1 _ 3 6 3 3  _  -  -  193 2 3 2 152 2 3 2 7 134 2 3 3 (Died)  -  7  8 Summary Sheet  Grp Gm  Gm  Gm  Gm  Gm  I  Gm  I  Gm  I  2  197  192  201  201  198  197  201  4  182  183  175  187  182 43  184 43  187 36  5  156  156  160  156  157 40  154 37  158 43  -3  -1  1  I  Gm  Day -4 Grp Gm  I  Gm  2  205  205  4  186 36  187  5  160 46  164  Day  5  6  2 I  3 Gm  I  207  210  40  186 40  200 41  44  164 52  161 54  7  8  4  299 olation  i n order to make up f o r the animals t h a t  expired.  There i s , of course, no I n j u r y Index f o r Group 2.  j&raphsC • The v a l u e s on the Summary Sheet are p l o t t e d d i r e c t l y on the simple l i n e graph.  The upper p a r t  of the graph d e a l s w i t h the weight changes from day to day, and there i s f o r t h i s p a r t three l i n e s . lower p a r t c o n t a i n s the I n j u r y Index changes. are  The These  done i n c o l o u r s f o r e a s i e r r e a d i n g , but each l i n e  i s otherwise c l e a r l y l a b e l l e d by the n a t u r e of the injury  and treatment r a t h e r than by group numbers.  The same c o l o u r s and s c a l e h o l d f o r a l l g r o s s graphs. C o n d i t i o n of the Animals Two  o f the female Hooded r a t s o f Group 5 were  puny and d i d not s u r v i v e the f u l l course of the experiment. but  S e v e r a l of the o t h e r animals appeared  t h i s was  sickly,  c o n s i d e r e d q u i t e t r u t h f u l l y to be the  f a u l t of the animals themselves, r a t h e r than caused by the  demands of the experiment.  D i s c u s s i o n of Observations As j u s t mentioned a scrubby l o t .  They d i d so p o o r l y g e n e r a l l y t h a t i t  would n o t be s i g n i f i c a n t i o n s from them.  the animals used were r a t h e r  to d e r i v e any p o s i t i v e  conclus-  However, i t w i l l be noted that the  300  301  I n j u r y Index l i n e f o r histamine h i g h e r than f o r the c o n t r o l .  ( s o l i d green) i s  I t may  not be p o s s i b l e  to s t a t e the histamine i n f l u e n c e d the course of the f r o s t b i t e l e s i o n s a d v e r s e l y , but i t i s q u i t e true t h a t there was  no b e n e f i c i a l  effect.  As mentioned at the s t a r t , t h i s was  i n the nature of a t r i a l run.  experiment  I t o b v i o u s l y needed  r e p e a t i n g w i t h a more s u i t a b l e group of  animals.  Histamine Experimental Observations w i t h Histamine Tbe purpose of the experiment was  (repeat)  here  outlined  to r e p e a t the e f f e c t s of a standard c o l d i n j u r y as  i n f l u e n c e d by p a r e n t e r a l i n j e c t i o n s of h i s t a m i n e at a h i g h e r dosage l e v e l o f 50 mg.  per k i l o g r a m body  weight administered subcutaneously  at three hour  i n t e r v a l s beginning immediately p r i o r to the f r e e z i n g I n j u r y and c o n t i n u i n g f o r f o r t y - e i g h t hours a f t e r w a r d s . E i g h t s e n normal a d u l t maleWistar  r a t s were s e l e c t e d  and they were d i v i d e d i n t o three groups of s i x animals designed as f o l l o w s : Group: 2.  A c o n t r o l group to r e c e i v e o n l y the h i s t a m i n e i n j e c t i o n s as o u t l i n e d .  4.  A c o n t r o l group a l s o to r e c e i v e a  302  f r e e z i n g i n j u r y o f twenty seconds d u r a t i o n w i t h r a p i d thawing i n water at 5.  42°C.  A t e s t group to r e c e i v e b o t h the h i s t a m i n e i n j e c t i o n s as o u t l i n e d , and a f r e e z i n g I n j u r y of twenty seconds d u r a t i o n w i t h r a p i d thawing i n water a t 42°C.  These groups have the same numbering as s i m i l a r groups throughout a l l the gross experiments.  Each animal was  marked c h a r a c t e r i s t i c a l l y on the ear, and  initial  weights were recorded as on the Data Sheet. of  Groups 2 and 5 r e c e i v e d t h e i r i n i t i a l  The animals  subcutaneous  i n j e c t i o n of h i s t a m i n e , and an hour and a h a l f  later,  at  h a l f p a s t nine o ' c l o c k i n the evening, the animals  of  Groups 4 and 5 were a n a e s t h e t i z e d w i t h 0.1 cc o f  0.6$ nembutal s o l u t i o n administered  intraperitoneally.  When a n a e s t h e t i z e d , one h i n d l e g o f each animal i n these groups was dipped i n a beaker of e t h e r through which carbon d i o x i d e gas was b u b b l i n g to b r i n g the temperature down t© -20 G. G  the  The f o o t was immersed up to  hock ( j u s t below the f u r l i n e ) ,  and as soon as the  s k i n blanched, i n d i c a t i n g the f o o t was completely f r o z e n  303 the time was counted so t h a t the l e g was immersed and f r o z e n f o r e x a c t l y twenty  seconds by stop watch.  The  animals were then t r e a t e d by r a p i d thawing, whereby the f r o z e n f e e t were immersed immediately a t the exp i r y o f the twenty e x a c t l y 42°C. for  seconds i n t o water maintained a t  The f e e t were l e f t i n the warm water  approximately one minute, u n t i l thoroughly thawed  and warm.  Then the animals were r e t u r n e d to t h e i r  cages, and were l e f t q u i e t l y alone (except f o r the three h o u r l y i n j e c t i o n s o f histamine) u n t i l they wakened from the e f f e c t s o f the nembutal.  Observations were  made as recorded on the Data Sheet, by the method d e s c r i b e d above f o r e s t i m a t i n g the degree o f f r o s t b i t e I n j u r y under the c a t e g o r i e s o f oedema, c o l o u r change, exudate Data  and slough, and gangrene.  Sheets These t a b l e s are f a i r l y s e l f explanatory,  b e i n g simple t a l l i e s by animals i n groups, and by days, Day 1 being the day o f f r e e a i n g . e s s a r i l y as compact as p o s s i b l e .  They a r e nec-  Days 1, 2, and 3  i n c l u d e a r e c o r d o f the three h o u r l y i n j e c t i o n s . symbols used are:  Grp, group,  three hours,  0, oedema,  and slough,  G, gangrene,  Gm, grams,  C, c o l o u r change,  The  q3h, every E, exudate  H, histamine i n j e c t i o n ,  s  9 —  -  —  I 3 3 2 2 3 2 3 2 2 2 3 3 2 2 2 2  861 Z,*3 023 *23 9T3 681  -  3 3 2 66T 3 2 3 9*3 3 2 3 123 3 3 2 361 3 3 2 vI3 2 3 2 361  -  2 I 3 3 I  *33 163 961 **3 693 323  -  3 2 - I 3 3 13  2 833 I 002 3 103 3 923 - - I I i9S - I I I 833  - 3 2 2 933 3 3 063 - 1 2 2 203 - 1 3 2 1*3 - - I T TA3 - - I I £23  *33 **3 233 023 £63 T*3  *33 2*3 733 LZZ f6Z 1*3  mm  - 3 - - I - -  2 I 3 T T I I  mm  —  a 0 0  XBQ  0  3 0 0  2  HH HH HH  HH HH HH --  — — — -  mm mm  —  —  -  •» mm mm mm  mm  T I I I I I  T 3 3 3 3 3  I 3 - 3 I 3 T 3 3 — 3  -  2 361 2 3*3 2 233 * 023 * 603 2 *8I 2 2 2 2 2 3  HH HH HH HH HH HH  t*2b  £)  a 0 0  HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH  —  Z.03 Z.33 903 613 163 323  HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH HHHHHHHHH  mm mm  -  —  -  I T 3 I *• I 3 I I I I — T 3  -  A23  —  I I I I I  I I 3 3 I I  2 161  0 a 0 0  *02  A*3  * Z.I3 * * * * * *  3  O 3 0 0 rao decs  * 003 * 253 * *23  *  *  213 623 813 233 963  3  023 102 261 223 293 *33  it©  T 3 2 *6T 3 3 3 5*3 1 3 2 333 3 3 2 123 3 3 2 2T3 3 3 2 88T S  LZZ  563 803 Z,*3  1  £B<I  HHHHiffl HHHHi3H HHHHiSH HHHH3H HHHHiSH HHHHdH  603 193 633 623 6T3 88T  5  d-  *23 502 503 6*3  *23  aaa--—aa-  2*3  *  113 523 *T3 633 963 1*3  HHHH-H HHHH-H HHHH-H HHHH-H HHHH-H HHHH-H  903 **3 213 323 212 8*3  3  6LZ  rao  qgb  9LZ  TOOd«i£)  305 means not p r e s e n t or not a d m i n i s t e r e d , injury.  P,  freezing  A l l animals were d i s p o s e d of on Day 6 w i t h  c h l o r o f o r m fumes. Summary Sheet F o l l o w i n g the Data Sheet i s a Summary Sheet which g i v e s the average weight f o r each group  each  day, and under I, or I n j u r y Index, g i v e s the t o t a l f o r the  I n j u r y changes  o c c u r r i n g f o r t h a t gropp t h a t  day.  As d e s c r i b e d b e f o r e , t h i s concept of an I n j u r y Index i s j u s t an a r b i t r a r y number o b t a i n e d by adding a l l the p a t h o l o g i c a l changes  together f o r each group each day.  In as much as no animals d i e d , no e x t r a p o l a t i o n  was  r e q u i r e d to complete the groups f o r s i x animals. Graph.3 The v a l u e s on the Summary Sheet are p l o t t e d d i r e c t l y on the simple l i n e graph.  The upper p a r t  of the graph d e a l s w i t h the weight changes from day to day, and t h e r e i s f o r t h i s p a r t t h r e e l i n e s . lower p a r t c o n t a i n s the I n j u r y Index changes. are  The These  drawn i n c o l o u r s f o r e a s i e r r e a d i n g , but each l i n e  i s otherwise c l e a r l y l a b e l l e d by the n a t u r e of the injury  and treatment r a t h e r than by group numbers.  The same c o l o u r s and s c a l e h o l d f o r a l l g r o s s graphs.  306 Summary Sheet  Grp Gm  Gm  I  Gm  I  Gm  I  Gm  I  Gm  I  2  243  238  230  238  242  243  4  252  248 37  241 32  244 29  243 26  243 22  5  224  223 38  214 37  217 39  212 43  219 43  Day  1  2  3  4  5  6  C o n d i t i o n o f the Animals The p r e s e n t s e l e c t i o n o f r a t s , feeing a more mature group than the animals used i n the l a s t e x p e r i ment s u r v i v e d e x c e l l e n t l y , and were i n no way a d v e r s e l y a f f e c t e d by the histamine  injections.  D i s c u s s i o n o f Observations In view o f the f a c t a f u r t h e r experiment  with  histamine w i t h a b e t t e r group o f animals continued t o g i v e d i v e r g i n g I n j u r y Index graph l i n e s , w i t h the l i n e f o r the histamine on top, a l l adds s i g n i f i c a n c e to the c o n t e n t i o n t h a t h i s t a m i n e , as a d m i n i s t e r e d ^ a d v e r s e l y i n f l u e n c e s the course o f f r o s t b i t e l e s i o n s as produced in this  experiment.  307  D  E  X  10  I 1  2  3  4  D A Y  5  6  308  VTI S i g n i f i c a n c e o f Data  There i s no i n t e n t i o n o f a n a l y z i n g  statistic-  a l l y i n t h i s s e c t i o n o f the t h e s i s a l l of the data obt a i n e d so f a r .  Such would be a tremendous task, q u i t e  beyond the c a p a b i l i t i e s o f the w r i t e r , and c e r t a i n l y not j u s t i f i e d because o f the v e r y s u b j e c t i v e nature o f the o b s e r v a t i o n s , e s p e c i a l l y o f the m i c r o s c o p i c p a r t . However, c o n c l u s i o n s have been made from the data which do not have obvious  justification,  and i t i s proposed t o  study a few s e l e c t i o n s o f the data t o determine whether or n o t thiy&re s i g n i f i c a n t .  I n t h i s way the remainder o f  the data may be r e l a t e d to the analyzed m a t e r i a l . Threshold Epinephrine  Response  The main purpose behind  the p r e l i m i n a r y micro-  s c o p i c experiments i n P a r t I I I was to p r o v i d e a standard f o r m i c r o s c o p i c o b s e r v a t i o n o f the mesoappendix v a s c u l ature t h a t could be r e l a t e d to a l l subsequent o b s e r v a t i o n s . A c c o r d i n g l y , the data obtained must be c o n s i d e r e d and the Standard  E r r o r (S.E.) c a l c u l a t e d . On p a g e . 4 9  recorded,  we have a summary o f data which i s here  to the n e a r e s t m i l l i o n t h s d i l u t i o n .  to each i n d i v i d u a l number, —  x-x  X refers  X i s the mean, WZ, the sum, —  2  the d e v i a t i o n o f each number from the mean, (x-x) the  square of t h i s d e v i a t i o n .  309  Data  x  x  x-x  5 4 5 5 5 4 4 5  .4 .6 .4 .4 .4 .6 .6 .4  4.6  (x-x) .16 .36 .16 .16 .16 .36 .36 .16 £1.88  S . E . --JWE? V  !lM  n(n-l)  This being less than 2.0 ship between the values,  suggests a very close r e l a t i o n and the f a c t that twice the  Standard Error i s only .366,  also f a r less than 2.0  in-  dicates such a relationship even more strongly. Further along, In the same section, at pages sixty and sixty-one we have a comparison of the threshold epinephrine response between normal controls and made hypertensive with compound F.  animals  Here we have two  sets of figures recorded to the nearest m i l l i o n t h s of dilution; Control  5 5 5 7 4 10 5  Test  18 18 18 18 20  The difference between these sets i s highly s i g n i f i c a n t because there Is absolutely no overlap of any of the numbers, and i f none of the data overlap, the values  310 f o r twice the s i g n i f i c a n t e r r o r ©n e i t h e r s i d e o f the mean f o r b o t h groups c o u l d not p o s s i b l y o v e r l a p  either.  Procaine M l c r o a c Q p i e a l l ^ I n the $ile-roeeoplc ©xperiiwats u s i n g  topical  procaine,, t h e f i r s t d a t a o b t a i n e d p r o v i d e s two s e t s o f f i g u r e s , as c o p i e d below from peg© 70,  the numbers  i n d i c a t i n g the time i n seconds f o r the onset o f v a s c u l a r stasis; X Control  4§ 210 30 90  i  375  is;  94  Tost  20 210 90  ®Q  •:14S0 <  These Standard  860 172  x-x  (x-x)  49 116 64 4  8401 13466 4096 10 £  152 38 82 82 278  19969 S.33. 5  40,8  S.E.  75.9  23104 1444 8724 6724 77284 £  116280 "  E r r o r s ar© e x c e e d i n g l y l a r g e , and the spread  on e i t h e r s i d e o f the mean f o r the c o n t r o l group would be from 53.2 t o 130.3, and f o r the t e s t group f r o a 96.1 t o 247.9  There i s too much o v e r l a p f o r t h e r e t o b© any  r e a l s i g n i f i c a n t d i f f e r e n c e between th© two groups o f d a t a , and any significanee i s o n l y s l i g h t l y p o s s i b l e . However* we f i n d more f a v o u r a b l e data f o r p r o c a i n e  311 parenterally.  The  summary t a b l e on pages 82 and 83  gives  the f o l l o w i n g f i g u r e s , a l s o f o r time f o r onset o f s t a s i s i n seconds: Control  10 5 10 5 5 5  45 30 45 30 30 30 210  100 25 100 25 25 25  r  35 60 30 180 60 40 50  Test  (x-x)  x-x  X  10 40 110 10 30 20  3.16  S.E. =  21.6  100 400 12100 100 900 400  70  x  S.E. -  14000  420  The  300  spread of the Standard E r r o r about the mean f o r the  c o n t r o l group  i s 31.84  i t i s from 48.4 no o v e r l a p p i n g  to 38.16, and f o r the t e s t  to 91.6.  group  I t w i l l "be noted t h a t there i s  In these two ranges, which suggests a  probable s i g n i f i c a n t d i f f e r e n c e between the two However, the same spread u s i n g  twice the Standard E r r o r i s  f o r the c o n t r o l groups from 28.68 to 41.32 group I t i s from 26.8  to 113.2.  groups.  and f o r the t e s t  Here we n o t i c e  the range  f o r the t e s t v a l u e s embraces the spread f o r the c o n t r o l s . This g r e a t l y reduces the chances of the d i f f e r e n c e being s i g n i f i c a n t , although a d e f i n i t e d i f f e r e n c e i s s t i l l possible ;  312 The data thus f a r examined i n d i c a t e s the t r e n d in  the experiments  i n v o l v i n g the m i c r o s c o p i c o b s e r v a t i o n .  Time and Rate o f Thawing Concerning  the gross experiments,  the time and r a t e o f thawing  data to  we have  consider.  first The  graphs and the summary sheet are b u t two e x p r e s s i o n s o f the same summarized data (see pages 209 and 210). has  to be determined  i s whether any two graphs are s i g -  n i f i c a n t l y d i f f e r e n t from each o t h e r . we must f i r s t  What  To s e t t l e  this  e s t a b l i s h whether any two p o i n t s on the  graph l i n e s i n q u e s t i o n on the same day are s i g n i f i c a n t l y different. On Day 2, the I n j u r y Index v a l u e f o r Group 3 was 39 and f o r Group 6 i t was 48 (page 209).  These are two  p o i n t s r e l a t i n g the i n f l u e n c e o f the r a t e o f thaw i n a f r e e z i n g i n j u r y o f s i x t y seconds (broken blue  lines).  The data sheet f o r Day 2 (page 206) r e l a t i v e to the animals of  these groups g i v e s us, by summing the i n j u r y v a l u e s f o r  each animaln i n t u r n the f o l l o w i n g data; x Group 3  7 6 7 7 6 6 39 6.5  x-x  .2 (x-x)  .5 .5  .25 .25 .25 .25 .25 .25  .5  .5 .5 .5 1  »  5  0  S.E^r  .7  313  Group  6  z  x  x-x  9 9 9 8 8 5  1 1 1 0 0 3  1 1 1 0 0 9  48  £12  8 The  spread f o r  gives  a range  twice the for  Group 6 o f from no o v e r l a p o f strongly between to  the  data  lines  lines.  of of  line  of  comparisons.  Standard Error  there  Is  same  colour or line  of  colour would  only  is  one  difference  to  graph w i t h  fast  210)  would  o f f r e e z i n g and r a t e  thaw  dotted  the  a  the  five  twenty  ( b l u e b r o k e n and  involve of  or  meaningless  a comparison of  second freeze  apply  compare  colour with  lead  Is  very  solid  s l o w thaw a g a i n s t  duration  for  there  a significant  graph with  on page  mean  e x p e r i m e n t p r o v i d e d we  For instance,  lines  the and  that  second freeze  solid  .63  S uch a conclusion would  this  another  7.2  the  Comparing a s o l i d  dotted  to  The f a c t  that  the  5.8  about  8.63.  two g r o u p s .  a l l the  only  to  even t w i c e  suggestive the  Standard Error  Group 3 o f from  7.37  =  S.E.  red  two v a r i a b l e s ,  thaw.  Rutin As o  graphs page  for  274,  a further rutin will i t  will  be  example of be seen  s i g n i f i c a n t data,  considered. t h a t no  On t h e  the  Data  animals died.  Sheet,  The  data  314 for  Groups 1 and 3 on Day 6 g i v e s two sets of i n j u r y  v a l u e s by animals as below:  Group 1  I X  X  x-x  (x-x)  6 7 9 6 6 3  0 1 3 0 0 3  0 1 9 0 0 9  Z  37 6  3  5 10 3 3 3 3  I  27  X  19 S.E. — .8 .25 30.25 2.25 2.25 2.25 2.25  .5 5.5 1.5 1.5 1.5 1.5 £  39.50  4.5  S.E. -  1.2  The spread about the mean f o r Group 1 g i v e n by the Standard  e r r o r g i v e s a range o f from 5.2 to 6.8 and f o r Group  3 the c o r r e s p o n d i n g range would be from 3.3 t o 5.7.  Here  there i s some o v e r l a p which does reduce the s i g n i f i c a n c e of the  the d i f f e r e n c e c o n s i d e r a b l y , b u t s t i l l  a difference i n  behaviour o f the two groups on the p a r t i c u l a r day i n  question i s l i k e l y . has been examined,  Only one aspect of the complete graph and t h i s aspect was the most u n l i k e l y  appearing comparison, taken on Day 6 when the graph l i n e s i n q u e s t i o n were f a i r l y c l o s e t o g e t h e r . same two graphs are f u r t h e s t a p a r t .  On Day 4 the  Taking the data  from page 274 f o r these two groups, Groups 1 and 3 on Day 4 we have again i n j u r y t o t a l s by animals as f o l l o w s :  315 X  Group 1  10 9 10 9 9 6  r  53  X  9  Group 33  (x-x)  x-x  8 9 7 6 5 6  I  41  X  7  1 0 1 0 0 3  1 0 1 0 0 3  1 2 0 1 2 1  S.E.  =  .6  S.E.  -  .6  1 4 0 1 4 1  Here the spread on e i t h e r s i d e of the mean g i v e n by the Standard E r r o r g i v e s f o r Group 1 a range of 8.4 and f o r Group 3 a range of from 6.4  to 7.6.  to  9.6  Here we  see no o v e r l a p which i n d i c a t e s a s i g n i f i c a n t  difference  although examination o f the same spread p r o v i d e d  through  the more e x a c t i n g standard of u s i n g twice the Standard E r r o r g i v e s a range f o r Group 1 of Group 3 of from 5.8 of  to 8.2.  7.8  to 10.2  T h i s reduces  and f o r  the p r o b a b i l i t y  the d i f f e r e n c e b e i n g s i g n i f i c a n t , but does not necess-  a r i l y c a n c e l out the s i g n i f i c a n c e . both these analyses i n t o the graphs  Hence, i n c o r p o r a t i n g f o r slow thaw f r o s t b i t e  l e s i o n s permits us to say i t i s l i k e l y but not  entirely  probable that r u t i n r e s u l t s i n a s i g n i f i c a n t l y l e s s course over the time i n t e r v a l s t u d i e d .  severe  316  Ascorbic Acid Then, a t the other extreme, as i n the case w i t h a s c o r b i c a c i e , we have to c o n s i d e r whether the c o r r e s p onding graphs are s i m i l a r or not.  The d o t t e d l i n e s  on page 290, as are the s o l i d l i n e s , are running q u i t e parallel  to each o t h e r .  I t i s almost apparent,  without  c a l c u l a t i n g standard e r r o r t h a t the m e d i c a t i o n has n o t made the s l i g h t e s t d i f f e r e n c e on the course o f the f r o s t b i t e lesions.  However, the o t h e r comparison i s made even  more s i g n i f i c a n t , t h a t r a p i d thawing r e s u l t s i n an o b v i o u s l y decreased  s e v e r i t y i n the course o f f r o s t b i t e  lesions.  Conclusion This b r i e f examination done w i l l  o f the data as has been  serve to strengthen the v a l i d i t y of the c o n c l u s -  i o n s a r r i v e d at i n the f i n a l s e c t i o n o f t h i s t h e s i s . Al^.1 the data c e r t a i n l y c o u l d n o t have been analyzed, nor c o u l d the more e l a b o r a t e s t a t i s t i c a l methods d e a l i n g w i t h the degree o f p r o b a b i l i t y be a p p l i e d . speak w i t h rough g e n e r a l i t i e s .  At b e s t we can o n l y  317  V I I I Conclusions  This i n v e s t i g a t i o n was  Reached  an i n d i r e c t attempt to  determine whether a s p e c i f i c s e l e c t i o n o f ten medica t i o n s would or would not i n f l u e n c e f a v o u r a b l y  the  v a s c u l a r responses i n the f r o z e n e x t r e m i t i e s of the r a t . An i n d i r e c t approach was i t was  not  considered  n e c e s s a r y because  c o n s i d e r e d p r a c t i c a l to observe the v e s s e l s  i n the e x t r e m i t i e s . beginning,  A„s has been o u t l i n e d at  the v a s c u l a r bed  of the mesoappendix  s e l e c t e d as the s i t e f o r the m i c r o s c o p i c observations  at  to be compared w i t h  the s i t e of o r i g i n a l i n t e r e s t , the T h i s whole i n v e s t i g a t i o n was  i n t e r e s t i n g one gained  to be working on.  i n the m i c r o s c o p i c  ions.  Any the  the gross e f f e c t s  extremities. at a l l times a most  Much experience  was  examination of v a s c u l a r r e a c t -  Each of the p r e p a r a t i o n s  l e a s t h a l f an hour.  was  studies.  made at the mesoappendix p o r t i o n of  v a s c u l a r system had  the  studied required at  Such experience  certainly  provided  a d i f f e r e n t concept of the v a s c u l a r system than had  been  e n t e r t a i n e d p r e v i o u s l y by the w r i t e r . The f a c h was which was  t h r e s h o l d epinephrine  response t e s t of Zwei-  d u p l i c a t e d w i t h some h i g h degree o f  reliability,  very g r a t i f y i n g i n view of the f a c t such work  318 was of  the w r i t e r ' s f i r s t  experience w i t h microtechnique  t h i s n a t u r e , and a l s o the v a s o t r o p i c e f f e c t o f  compound F was c o r r o b o r a t e d w i t h Warner's f i n d i n g s , which was  a source o f keen s a t i s f a c t i o n to us both. As f a r as the i n f l u e n c e t h a t the v a r i o u s medic-  a t i o n s had on the v a s c u l a r system o f the mesoappendix a f t e r l o c a l i z e d f r e e z i n g was concerned,  i t is firstly  important to remember t h a t a l l the drugs t e s t e d were exp e c t e d to have some i n f l u e n c e .  Such i n f l u e n c e as was  noted was observed a c c o r d i n g to very h i g h l y impressions.  These impressions c o u l d n o t be proven to  be s t a t i s t i c a l l y experiments  subjective  significant.  A l l the m i c r o s c o p i c  r e s u l t e d i n the same c o n c l u s i o n ; any differ:ences  noted i n b e h a v i o r o f the c i r c u l a t i o n due t o treatment by one o f the t e n drug agents were o f d o u b t f u l s i g n i f i c a n c e . Because o f t h i s i t was d e c i d e d that the technique as empl o y e d f o r o b s e r v i n g the e f f e c t o f l o c a l i z e d c o l d  injury  on the v a s c u l a r bed o f the mesoappendix was a v e r y poor tool f o r investigating cold  injury.  In r e g a r d to the gross experiments  more d e f i n i t e  f i n d i n g s were found; 1.  As expectedjthe r a t e o f thawing  a frozen  extremity was a s i g n i f i c a n t f a c t o r i n determining the degree o f i n j u r y t h a t f r e e z i n g would produce. thawing  Warm  i n water a t 42°C. was the method o f r a p i d  selected.  thawing  319 2.  The d u r a t i o n of the f r o z e n s t a t e a l s o  a direct relationship lesions,  to the s e v e r i t y of the  bore  resulting  up to a maximum, o f course, of t o t a l gangrene  and subsequent amputation 3.  by the  Procaine was  animal.  found to be v e r y t o x i c  i n the  dosage l e v e l administered, and on a r e p e a t experiment i t was  found to p r o v i d e no s i g n i f i c a n t 4.  Priscoline  5.  Benadryl p r o v i d e d no  benefit.  p r o v i d e d no s i g n i f i c a n t  benefit  grossly.  I t was  suggested but not proved  significant  t h a t Benadryl might  have a d e t r i m e n t a l e f f e c t on the course of lesions.  benefit. actually  frostbite  ••• !  6.  Etamon and Hydergine  p r o v i d e d no  7.  A p r e s o l i n e p r o v i d e d some p o s s i b l y  significant  beflefit. significant  b e n e f i t , but C h l o r - T r i p o l o n as administered made the of  the f r o s t b i t e 8.  313,  injury  s i g n i f i c a n t l y more severe.  R u t i n has been c o n s i d e r e d a l r e a d y on page  and i t was  felt  t h a t r u t i n had a p r o b a b l y  b e n e f i t on the course o f the f r o s t b i t e 9.  course  Ascorbic acid  significant  lesions.  as administered had a b s o l u t e l y  no i n f l u e n c e whatever on the course of the f r o s t b i t e i n j u r y . 10. i c a n t harmful  Histamine was effect.  found to have a p r o b a b l y  signif-  320 The attempt  to f o l l o w the course o f a f r o s t b i t e  I n j u r y on a time b a s i s was made throughout a l l the gross experiments,  and i t was found q u i t e p r a c t i c a l t o p r e s e n t  the day by day changes on a simple l i n e graph.  However  to accomplish t h i s one a numbrical b a s i s , the a r b i t r a r y concept o f an I n j u r y Index was i n t r o d u c e d .  The a c t u a l  d e t e r m i n a t i o n o f an a l l i n c l u s i v e Index e v a l u a t i o n p r e sented some d i f f i c u l t i e s , b u t a workable  s o l u t i o n was  found and t h i s was employed throughout a l l the gross experiments. By way o f apparatus, the equipment f o r the m i c r o s c o p i c examination of the r a t mesoappendix was c o n s t r u c t e d a c c o r d i n g to Zweifach's d i r e c t i o n s , and the c o l d p o i n t f o r c o n t r o l l e d hypothermia  o f Hass and T a y l o r was made.  This equipment was found to work q u i t e  satisfactorily.  Summary of C o n c l u s i o n s 1.  U s e f u l n e s s o f the t h r e s h o l d e p i n e p h r i n e  response t e s t f o r v a s o t r o p i c substances was confirmed. 2.  The v a s c u l a t u r e of the r a t mesoappendix  was observed i n r e s p e c t to i t s r e a c t i o n s to a standard localized freezing injury.  P r o c a i n e , P r i s c o l i n e , Benadryl,  Etamon, Hydergine, A p r e s o l i n e , C h l o r - T r i p o l o n , R u t i n , A s c o r b i c a c i d , and Histamine f a i l e d to i n f l u e n c e these reactions  significantly. 3.  A dynamic g r a p h i c a l method was d e s c r i b e d  321 f o r r e c o r d i n g gross f r o s t b i t e l e s i o n s arbitrary  concept of an I n j u r y Index was 4.  Hydergine, and  5.  the  was  course of f r o s t b i t e l e s i o n s  C h l o r - T r i p o l o n and  The  consistantly  Histamine p r o b a b l y  siggrossly. had  effect.  A p r e s o l i n e , and  a beneficial 7.  the  introduced.  A s c o r b i c a c i d were found to have no  a s i g n i f i c a n t adverse 6.  and  P r o c a i n e , P r i s c o l i n e , Benadryl, Etamon,  n i f i c a n t e f f e c t on  had  grossly,  especially  Rutin probably  effect. value of r a p i d confirmed.  thawing i n water at 42°C.  322  APPENDIX  Equipment  f o r Hass and T a y l o r ' s Cold P o i n t Apparatus  1.  C y l i n d e r o f carbon d i o x i d e gas a t 1,000  pounds p r e s s u r e . 2.  F r e e z i n g attachment f o r Spencer carbon d i -  oxide microtome, Scientific  complete w i t h copper tube c o n n e c t i o n .  S u p p l i e s Co. L t d . , C a t . No. 50, p.630, Item  No. 38756. 3.  Hypodermic  needles as i n Becton, D i c k i n s o n and  Company (B.-D.) Cataloque f o r May, 1951.  4.  Item  LNR  19  Item  LNRS 15  G  3"  page 26.  G 1*"  page 26.  Connectors; Luer-Lok hose end connector, H/468L, Page 33 Adams Luer-Lok connector w i t h s i d e arm adapter, 429A, page 36.  5. Labour r e q u i r e d ; (1) A t t a c h H/468L to f r e e z i n g  attachment  (2) S o l d e r 429A a t proper s i t e on s h a f t of No.19  needle.  Chemicals Used Water, o r d i n a r y l a b o r a t o r y s i n g l e d i s t i l l e d c i t y supply ( c h l o r i n a t e d ) ,  from  sodium c h l o r i d e , potassium  c h l o r i d e , c a l c i u m c h l o r i d e , sodium b i c a r b o n a t e ,  sodium  323 dihydrogen  phosphate, glucose, a s h - f r e e g e l a t i n , a l l as  ordinary C P .  l a b o r a t o r y reagents  as a v a i l a b l e without  special ordering. Procaine h y d r o c h l o r i d e , Winthrop-Steams, as Novocain powder i n f i v e gram b o t t l e s . P r i s c o l i n e , Ciba Company L i m i t e d brand o f 2benzyl-imidazoline hydrochloride (benzazoline) a v a i l a b l e i n rubber 25  capped v i a l s of 10 c c ,  each c c . c o n t a i n i n g  mg. Benadryl h y d r o c h l o r i d e , Parke, Davis and Co.  Ltd.  brand of diphenhydramine h y d r o c h l o r i d e a v a i l a b l e i n 10 S t e r i - v i a l s each c c  c o n t a i n i n g 10  Etamon C h l o r i d e ,  cc.  mg.  Parke, Davis and  Co. L t d . brand  of tetraethylammonium c h l o r i d e a v a i l a b l e i n 20 c c . S t e r i v i a l s w i t h 100 mg. Hydergine,  per c c . Sandoz (Canada) L t d . brand of  p r o p o r t i o n s each of d i h y d r o e r g o c o r n i n e , t i n e , and d i h y d r o e r g o k r y p t i n e , c o n t a i n i n g 0.3  mg.  of the  mixture.  of 1-hydrazinophthalazine  C i b a Company L t d . brand  hydrochloride, available i n 1 cc.  ampoules c o n t a i n i n g 20 mg.  i n boxes of 5 ampoules.  Schering Corporation Limited  brand of chlorprohpenpyridamine pure powder.  dihydroergocris-  a v a i l a b l e i n 1 c c . ampoules  Apresoline Hydrochloride?  Chlor-Tripolon,  equal  maleate, obtained as  the  324 Rutin, 50 mg.  Parke, Davis and Co. L t d . , obtained as  compressed t a b l e t s i n b o t t l e of  500.  A s c o r b i c a c i d and Histamine phosphate  obtained  i n 1 gram v i a l s from B r i t i s h Drug Houses. Hydrocortone,  Merck and Co. L t d . brand of  h y d r o c o r t i s o n e acetate (compound F) a v a i l a b l e as the s a l i n e suspension i n s t e r i l e rubber-capped a i n i n g 25 mg.  v i a l s cont-  per c c .  E p i n e p h r i n e,  Connaught Medical Research Lab-  o r a t o r i e s 1:1000 s o l u t i o n of epinephrine h y d r o c h l o r i d e , a v a i l a b l e i n 30 c c . s t e r i l e rubber-capped Nembutal, No. 8612.  100  Abbott L a b o r a t o r i e s L i m i t e d ,  per c c .  Solution Sodium c h l o r i d e Potassium c h l o r i d e Calcium c h l o r i d e Sodium b i c a r b o n a t e Water Glucose Gelatin  All  36 gms. 1.68 .96 .80 4000 c c . 8 gms. 40  s o l i d i n g r e d i e n t s except g e l a t i n were weighed  out c a r e f u l l y and d i s s o l v e d i n the water. was  Item  cc. rubber diaphragm capped v i a l f o r  v e t e r i n a r y use c o n t a i n i n g 60 mg. Ringer-Locke  vials.  d i s s o l v e d i n a 1000  The  cc only, heated u n t i l  gelatin  completely  d i s s o l v e d , then f i l t e r e d under s u c t i o n and added to the r e s t of the s o l u t i o n . was notweighed  U s u a l l y the sodium b i c a r b o n a t e  out, but when the s o l u t i o n was  mixed, a  325  dropper f u l l  o f methyl r e d i n d i c a t o r was added to the  completed s o l u t i o n , added u n t i l  and then sodium b i c a r b o n a t e was  the f i r s t  change to r e d .  slowly  326 References C i t e d  (1)  Orr, K.D. and F a i n e r , D . C , C o l d . I n j u r y i n Korea During Winter o f 1950—51 Medicine 31 177-220,(may)1952 :  (2)  Webster, D.R. and Bigelow, W.G., I n j u r i e s Dueto Cold, F r o s t b i t e , Immersion Foot, and Hypothermia, Canad.M.A.ffi., C i v i l Defence Issue, 67: 534-538,(Dec)1952.  (3)  F e r r e r , M.Irene, ed., Trans. F i r s t Conference, Cold I n j u r y , June 4-5, 1951, J o s i a h Macy J r . Foundation, New York, 1952.  (4)  Shumacker,  (4a) E d i t o r i a l ,  H.B.Jr., and Lempke, R.E., Recent Advances i n F r o s t b i t e , Surgery 30:873-904,(Nov)1951 Frostbite, J.A.M.A. 148:940-941,(Mar.15)1952.  (5) Laufman, H., . _ Profound A c c i d e n t a l Hypothermia, J.A.M.A. 147-.1201-1212, (Nov.24)1951. (6)  L a r r e y , D.J., . Memoirs o f M i l i t a r y Surgery and Campaigns of the French Armies, Trans, by H a l l , R.W. B a l t i m o r e , J.Cushing, 1914, v o l . 1 1 , 156-164.  (7) Warner, D.L., , .. C a p i l l a r y Changes i n Rats Rendered HyperTensive by the A d m i n i s t r a t i o n o f Compound F. Unpublished Graduating T h e s i s , F a c u l t y o f . Medicine, U n i v e r s i t y o f B r i t i s h . C o l u m b i a , A p r i l , 1954. (8)  Hass, G.M. and T a y l o r , C.V., A Q u a n t i t a t i v e Hypothermal Method f o r the Production of Local Injury of Tissue, Arch. Path. 145:563-580, (May)1948.  327  (9)  Griffith,  J.Q. and F a r r i s , E . J . , eds., The Rat i n L a b o r a t o r y I n v e s t i g a t i o n , J.B. L i p p i n c o t t , P h i l a d e l p h i a , 1942.  (10)  Beckman,  H., Pharmacology In C l i n i c a l P r a c t i c e , W.B. Saunders, P h i l a d e l p h i a , 1953, p.208.  (11)  Hurley, L. A., ^-Roberts, J.E., Buchanan, A.R., and T i l l q u i s t , G., P r e l i m i n a r y I n v e s t i g a t i o n s of the Value of the Dihydrogenated A l k a l o i d s of E r g o t i n the Treatment of Experimental F r o s t b i t e , Surgery, Gynecology, and O b s t e t r i c s , 92: 303-308,(Mar)1951.  (12)  B l u n t s c h l i , H.J. and Goetz, R.H., The E f f e c t of Ergot D e r i v a t i v e s on the C i r c u l a t i o n i n Man With S p e c i a l Reference to Two New Hydrogenated Compounds (Dihydroergotamine and D i h y d r o e r g o c o r n i n e ) , Am. Heart J . , 35:873-894,1948.  (13)  Rothlin,  (14)  Zweifach,  E., Pharmacology of the N a t u r a l and Dihydrogenated A l k a l o i d s of E r g o t . B u l l . Acad. S u i s s . Sc.M., 2:249-273,1946-47. B.W., I n d i r e c t Methods f o r Regional Blood Plow: I, M i c r o s c o p i c Observations of C i r c u l a t i o n i n Rat Mesoappendix and Dog Omentum; Use i n Study of V a s o t r o p i c Substances, i n P o t t e r , V.R., ed., Methods i n M e d i c a l Research, 1:131-139,1948, Year Book Pub. Chicago,  (15) K n i s e l y ,  M.H., Method of I l l u m i n a t i n g I n t e r n a l Organs, Anat. Record, 64;499,1936.  (16) K n i s e l y ,  M.H., Improved Fused Quartz I l l u m i n a t i o n , Anat. Record, 58;73,1934, and 71 503,1938. :  (17) K n i s e l y ,  M.H., M i c r o s c o p i c Observations on T r a n s i l l u m i n e d Spleens, Proc.Soc.Exp.Biol.&.Med., 32 212,1934. :  328  (18)  K n i s e l y , M.H., i n Handbook of M i c r o s c o p i c a l Technique, McClung, C.E., ed., p.632-642.  (19)  Chambers,  R., Vasomotion i n Hemodynamics of Blood Capillary Circulation. Ann. N.Y. Acad. Sc., 49 549-552,(May)1948. :  (20)  Chambers,, R.,/.and Zweifach, B.W., Topography and F u n c t i o n of Mesenteric Capillary Circulation., Am. J . Anat. 75 173,(Sep)1944. :  (21)  Chambers,  R., Blood C a p i l l a r y C i r c u l a t i o n Under Normal C o n d i t i o n s and i n Traumatic Shock, Nature, London, 162:835-837,(Nov)1948.  (22)  Chambers, R. and Zweifach, B.W., F u n c t i o n a l A c t i v i t y of the Blood C a p i l l a r y Bed, With S p e c i a l Reference to V i s c e r a l Tissue. Ann. N.Y. Acad. Sc., 46 683-695,(Sep)1946. :  (23)  Luyet, i n An O u t l i n e o f General Physiology, H e i l b r u n n , L.V., Saunders, P h i l a d e l p h i a , 3rd ed., 1952, p.491.  

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