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The effect of selected drugs on the vascular responses of the rat to localized cold 1954

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THE E F F E C T OF S E L E C T E D DRUGS ON THE V A S C U L A R RESPONSES OF THE RAT TO L O C A L I Z E D COLD G e o r g e E d w a r d S i n g e r A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o f t h e R e q u i r e m e n t s f o r t h e D e g r e e o f M a s t e r o f S c i e n c e i n The D e p a r t m e n t o f A n a t o m y 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 t h e s t a n d a r d r e q u i r e d f r o m c a n d i d a t e s f o r t h e d e g r e e o f M a s t e r o f S c i e n c e . M e m b e r s o f E x a m i n i n g C o m m i t t e e D e p a r t m e n t o f A n a t o m y THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A p r i l , 1 9 5 4 ABSTRACT Of Thesis f o r Master of Science Degree THE EFFECT OF SELECTED DRUGS ON THE VASCULAR RESPONSES OF THE RAT TO LOCALIZED COLD George Edward Singer Cold injury of both the dry and the moist types are of the utmost importance i n m i l i t a r y operations, and are l i k e l y to assume even greater importance i n the event of polar warfare. The modern l i t e r a t u r e on f r o s t b i t e began with Napoleon's retreat from Moscow i n 1812 when his surgeon, Baron Larrey recorded the disastrous event i n his memoirs. Since then i t has been the various wars of the western world that has provided the main stimulus f o r investigation into the cold problem. The experimental inves t i g a t i o n has effected l i t t l e p o s i t i v e benefit by way of management of an acute cold injury, but i t has served to break down many time honoured doctrines, e s p e c i a l l y the theory that slow thawing of a frozen limb provided the best treatment. The significance of the various events that occur i n the tissues during a freezing reaction are not agreed on by a l l workers, but the course of 'the reaction and e s p e c i a l l y the danger of the secondary effects during thawing are well known. The present investigation concerned an attempt to observe microscopically the vascular changes i n the r a t mesoappendix according to a technique of Zweifach. A cold point apparatus described by Hass and Taylor was u t i l i z e d A b s t r a c t , p.2 L o c a l i z e d Cold f o r exact f r e e z i n g of a c a p i l l a r y bed. The i n f l u e n c e of ten s e l e c t e d drugs on the r e a c t i o n s of the v a s c u l a r bed a f t e r f r e e z i n g was t e s t e d a l t e r n a t e l y w i t h c o n t r o l r a t s . Procaine, p r i s c o l i n e , b e n a d r y l , etamon, hydergine, apres- 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 h i s t - amine were t e s t e d . The c r i t e r i a f o r a drug e f f e c t i n c l u d e d delay i n onset of v a s c u l a r s t a s i s , l e s s e n i n g the r a t e and extent of the s t a s i s , and resumption of 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 s . No s i g n i f i c a n t d i f f e r e n c e was observed between the t r e a t e d and the c o n t r o l animals. In an e f f o r t to c o n f i r m t h i s i mpression g r o s s l y , the h i n d l e g s of r a t s were f r o z e n i n a carbon d i o x i d e and ether mixture at -20°C. f o r twenty seconds. The changes observed g r o s s l y f o l l o w i n g thawing were d e s c r i b e d num- e r i c a l l y and the a r b i t r a r y concept of an I n j u r y Index was u t i l i z e d which c o u l d be expressed g r a p h i c a l l y on a day by day b a s i s . Of the same ten drugs t e s t e d , p r o c a i n e , p r i s c o l i n e , b e n a d r y l , etarnon, hydergine, and a s c o r b i c a c i d were found to have no s i g n i f i c a n t e f f e c t . C h l o r - T r i p o l o n and h i stamine were found to have an adverse e f f e c t , and a p r e s o l i n e , and e s p e c i a l l y r u t i n , were found to have a prob- a b l y 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 . The v a l u e of r a p i d thawing i n water at 42°C. was c o n s i s t e n t l y confirmed throughout a l l gross experiments. TABLE OP CONTENTS T i t l e P a g e T a b l e o f C o n t e n t s A c k n o w l e d g e m e n t s I 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 D r u g s S e l e c t e d f o r T e s t i n g N e e d f o r C o n f i r m a t o r y G r o s s E x p e r i m e n t s I I M i c r o s c o p i c O b s e r v a t i o n 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 t h e R a t M e s o a p p e n d i x P h o t o g r a p h s - A p p a r a t u s The F r e e z i n g P o i n t F i g u r e - F r e e z i n g A p p a r a t u s E p i n e p h r i n e R e s p o n s e T e s t L o c a l i z e d C o l d I n j u r y E x p e r i m e n t s R e c o r d i n g o f O b s e r v a t i o n s C r i t e r i a F o r a D r u g E f f e c t C a r e o f A n i m a l s I I I P r e l i m i n a r y M i c r o s c o p i c E x p e r i m e n t s P r e l i m i n a r y C a p i l l a r y E x p e r i m e n t s I n i t i a l E p i n e p h r i n e R e s p o n s e T e s t s O b s e r v a t i o n s w i t h Compound F I V R e c o r d o f M i c r o s c o p i c O b s e r v a t i o n s P r o c a i n e P r o c a i n e r e p e a t e d P r o c a i n e P a r e n t e r a l P r i s c o l i n e P r o c a i n e P r i s c o l i n e E t a m o n H y d e r g i n e A p r e s o l i n e R u t i n A s c o r b i c a c i d B e n a d r y l C h l o r - T r i p o l o n H i s t a m i n e 3 Pr i s c o l i n e Repeated Benadryl Benadryl Repeated Benadryl Repeated Etamon Etamon Repeated Etamon Repeated Hydergine Apresoline Chlor-Tripolon Rutin Rutin Repeated Ascorbic Acid Histamine Histamine Repeated V Gross Frostbite Experiments 190 Reason f o r Gross Experiments Standard Freezing Injury Manner of Thawing Apparatus . Photograph - Apparatus Recording of Observations Injury Index Plate - Example of Injury Assessment Selection of Gross Experiments VI Record of Gross Observations 203 Duration of Injury and Rate of Thkw Procaine Procaine and P r i s c o l i n e Benadryl Colour Photographs Etamon and Hydergine Apresoline and Chlor-Tripolon Rutin Colour Photographs Ascorbic Acid Histamine Histamine Repeat VII Significance of the Data Obtained 308 Threshold Epinephrine Response Procaine Microscopically Time and Rate of Thawing Rutin Ascorbic Acid 4 VIII C o n c l u s i o n s R e a c h e d 317 Summary o f C o n c l u s i o n s A p p e n d i x 322 E q u i p m e n t f o r C o l d P o i n t C h e m i c a l s U s e d F o r m u l a f o r D r i p S o l u t i o n R e f e r e n c e s C i t e d 326 5 ACKNOWLEDGEMENTS Nearly every member of the Anatomy Department helped at some stage i n the work of t h i s project from Department Head to Animal Keeper. The work was guided throughout the year by Dr. S.M. Friedman, from whom I received constantly much help and i n s p i r a t i o n . Mr. G. Crosson and Mr. C. Blake deserve special 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 Faculty of Medicine provided much help i n two respects. I t was he who introduced me to the technique of observing micro- scopically the vasculature of the r a t mesoappendix and he guided me away from many p i t f a l l s during the early stages of the project. He also gave up his Christmas vacation to perform several of the experiments as acknowl- edged l a t e r . Appreciation i s here expressed f o r the supplies 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 injuries generally continue to present a challenge to our armed forces batt l ing 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 in Korea among the United States forces during the winter camp- aign of 1950-1951 when there were sixty thousand frost- bite casualties with six thousand amputations result ing. This definitely puts this problem into the category of a major battle hazard. The poss ib i l i ty , and even the probability of polar warfare in the near future forces greater attention to be paid to the problem of cold injury than has ibeelh^aid:.!:, The matter of acclimat- ization is being investigated as a preventive measure. Orr and Fainer outline several factors, more or less obvious, that w i l l help the soldier in the prevention of actual freezing injuries . In addition to the various physical agents, we would l ike to have some medication that could be administered to protect the combatant and c i v i l i a n alike against both the actual 7 freezing injury and also against the resultant immed- iate and chronic sequellae of frostbite 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 immediate- ly or later, often after the thawing has been complet- ed. Webster and Bigelow ( 2 ) writing for the recent special C i v i l Defence issue of the Canadian Medical Association Journal summarize the conflicting 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 for continued experimental work both in the laboratory and in controlled c l i n i c a l studies, and they seem to feel that the knowledge obtained so far i s barely laying the ground work for the great advances possible in this f i e l d . The Josiab. Macy Jr. Foundation has even start- ed a Conference on cold injury ( 3 ) . The best summary of the experimental work to date l i e s in the article prepared by Shumacker and Lempke ( 4 ) which gives a resume of the literature from as far back as 1917 right up to articles s t i l l in press in 1951. This is a rather extensive review and It forms the basis for the experimental work carried out for this thesis. 8 There are very few a r t i c l e s more recent t h a t are not c l i n i c a l , and since most of the experimental work now being performed i s financed through m i l i t a r y r e s e a r c h grants of one k i n d or another, r e p o r t s are very 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 of c o l d i n j u r y may be d i v i d - ed Into three broad c a t e g o r i e s . F i r s t l y might be mentioned general hypothermia, which i s i n c l u d e d only f o r completeness since i t uncommon, and s u r v i v a l from i t even more ra r e (5 ), although i t i s being i n v e s t i g - ated f o r anaesthetic uses i n c o n g e n i t a l heart surgery. Secondly i s the moist c o l d l e s i o n , the trench f o o t or immersion f o o t syndrome. Trench f o o t was p e c u l i a r l y a problem of the f i r s t World War, while immersion f o o t was the second World War v a r i e t y of the same c o n d i t i o n , being the r e s u l t of a prolonged v e r t i c a l posture w i t h an ambient temperature above f r e e z i n g . An apparently necessary requirement i s the need f o r moist c o l d , which conducts heat away from the exposed limb f a s t e r than does dry c o l d . The e f f e c t of the dependency of the limb i s of course to impede the blood c i r c u l a t i o n by d e l a y i n g venous r e t u r n . T h i r d l y i s the b i g problem of f c o s t b i t e . This 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 sufficient severity and duration to produce ice in the affected parts. This too had a counterpart in the second World War in the condition known as high altitude frostbite, charact- erized by a very rapid freezing at very low temperat- ures, such as the t a i l gunner of a fighter plane re- ceived when he took off his glove to adjust his gun, with his fingers immediately sticking to the metal and being deeply frozen. The frequent appearance in this brief class- i f i c a t i o n of such terms as trench foot, and high a l t - itude frostbite suggests that cold injury i s for a l l practical purposes a disease peculiarly associated with military operations. The modern literature on the 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 until the f i r s t World War that much effort or interest was put into trying to solve the frostbite problem. Sir 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 ice cryst- als intracellularly which caused the damage of freezing. 10 Experimental work during the second World War was centered mainly in trying to understand the physiol- ogy and the pathology of the frostbite lesion. Many are the papers discussing which was the more important of the various processes more or less going on simul- taneously. The whole process from i n i t i a l exposure to cold right through to the development of the late sequellae i s well discussed by Shumacker ( 4 ) and there seems to be l i t t l e value in repeating i t a l l here. The important aspect, not of course origin- ated by Shumacker, is the vascular component. In a freezing injury, naturally, cells 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 dir- ectly determine the extent of the damage resulting. It is the secondary changes following thawing that anethe gravest sequels to frostbite. After thawing (to quote from Shumacker) there develops a reactive hyperemia which may represent the reaction to slightly diffusible abnormal c e l l products resulting from direct cold injury or ischaemic injury. With the return of ' blood flow, oedema begins to form as protein-rich f l u i d escapes through the capillary walls rendered 11 h y p e r p e r m e a b l e b y t h e e f f e c t s o f t h e c o l d o r a n o x i a . The oedema p r o g r e s s i v e l y i n c r e a s e s a n d may r e a c h t h e m a x i m a l v o l u m e a l l o w e d b y t h e e x t e n s i l e l i m i t o f t h e e n c o m p a s s i n g s k i n . Cbtocidertsr l ly w i t h t h e f o r m a t i o n o f o e d e m a , s t a s i s d e v e l o p s i n t h e t r u e c a p i l l a r i e s . The f o r m e d e l e m e n t s o f t h e b l o o d a r e s e e n p r o g r e s s i v e l y t o f i l l t h e s m a l l v e s s e l s , b r i n g i n g t o ^ a h a l t t h e c i r c - u l a t i o n t h r o u g h t h e m . T h i s s t a s i s i s r e v e r s i b l e , a t t h e b e g i n n i n g a t l e a s t , s i n c e m e c h a n i c a l p r e s s u r e o r c h a n g e s i n b l o o d f l o w i n a d j a c e n t v e s s e l s w i l l b r e a k t h e r e d c e l l m a s s e s . T h e r e i s some c o n t r o v e r s y a s t o t h e c a u s e o f t h e s t a s i s . The v i e w m o s t w i d e l y h e l d i s t h a t t h e c a p i l l a r y w a l l s , r e n d e r e d h y p e r p e r m - e a b l e b y t h e i n j u r y m e r e l y f i l t e r t h e c e l l s o u t o f t h e b l o o d s t r e a m a s t h e s e r u m e n t e r s t h e i n t e r s t i t i a l s p a c e s . The s e c o n d s u g g e s t i o n i s t h a t s t a s i s m u s t b e t h e r e s u l t o f some c o l l o i d a l c h e m i c a l a l t e r a t i o n I n b l o o d u n r e l a t e d t o t h e c l o t t i n g m e c h a n i s m . The t h i r d p o s s i b i l i t y o f f e r e d c o n n e c t s s t a s i s w i t h s l u d g i n g w h e r e t h e r e i s p r e c i p i t a t e d o n t h e c e l l s a s t i c k y c o a t w h i c h c a u s e s t h e m t o a d h e r e t o o n e a n o t h e r a n d t o t h e w a l l s o f t h e v e s s e l s . S h u m a c k e r g i v e s t h e a r g u m e n t s f o r a n d a g a i n s t t h e s e v i e w s , w h i c h n e e d n o t b e m e n t i o n e d h e r e . T h i s summary f r o m S h u m a c k e r ' s d i s c u s s i o n p i c k s o u t t h e c o u r s e o f e v e n t s i n f r o s t b i t e t h a t o c c u r a t t h e 12 capillary level. Since studies were already i n progress at this University on the observation of the capillary responses in the rat mesoappendix (Warner, D.L., 7 ) and since Hass and Taylor had described an instrument ( 8 ) for applying a quant- itative hypothermal injury, It seemed very logical to study the behavior of the blood vessels in the rat mesoappendix after freezing as influenced by various medications applied both topically and par- enterally. Except for the technique of studying capillary responses to a local freezing injury as influenced by various drugs, which I have not found described in the literature, there i s nothing very original in the testing of the various drugs select- ed. Most of them have been used previously for gross freezing experiments. Ten were selected: procaine, Priscoline, Etamon, Hydergine, Apresoline, Rutin, Ascorbic acid, Benadryl, Chlor-Tripolon, and histamine. In view of the fact that some of these preparations are called by their trade names, a further discussion of the drugs i s in order. Drugs Tested in Cold Injury Experiments Procaine This is believed to paralyze peripheral con- 1 3 s t r i c t o r f i b e r s u p o n p e r i p h e r a l c o n t a c t w i t h t h e m . The d r u g was t e s t e d b o t h t o p i c a l l y a n d p a r e n t e r a l l y . T o p i c a l l y t h e r e i s no d i f f i c u l t y w i t h d o s a g e , a 0.b% t o 2.0% s o l u t i o n b e i n g g e n e r a l l y a d e q u a t e a s I s d e m o n s t r a t e d c l i n i c a l l y a l l t h e t i m e . T h e r e i s some t r o u b l e h o w e v e r , i n e s t i m a t i n g t h e e f f e c t i v e p a r e n t e r a l d o s e . The p r o c a i n e i s d e t o x i f i e d p r i n c i p a l l y i n t h e l i v e r , a n d t h i s i s r a p i d e n o u g h t o n e c e s s i t a t e f r e q u e n t o r b e t t e r , c o n t i n u o u s i n j e c t i o n s o f a s o l u t i o n o f a p p r o p - r i a t e c o n c e n t r a t i o n . G r i f f i t h ( 9 ) g i v e s 200 m g . i n t r a p e r i t o n e a l l y a s a s u i t a b l e d o s e t o a n a d u l t r a t f o r t h e r a p e u t i c o r t o p i c a l a c t i o n . 1 6 0 0 m g . i s t h e M . L . D . a n d 2 1 0 0 m g . i s t h e L . D ^ Q . 4 5 t o 5 5 m g . g i v e n i n t r a v e n o u s l y w i l l k i l l o f f t h e m a j o r i t y o f t h e a n i m a l s . On t h i s b a s i s t h e d o s a g e s u b s e q u e n t l y u s e d was c a l c u l a t e d . - N o t e t h a t p r o c a i n e i s h y d r o l y z e d a n d d e t o x i f i e d b y an enzyme i n t h e b l o o d t o g i v e p a r a - a m i n o b e n z o i c a c i d a n d d i e t h y l a m i n o e t h a n o w h i c h may h a v e i n t u r n a n i n d i r e c t a c t i o n o n t h e v e s s e l e n d o - t h e l i u m ( may a c t i n c o m p e t i t i o n w i t h h i s t a m i n e ) . P r i s c o l i n e T h i s i s b e n z a z o l i n e h y d r o c h l o r i d e , C i b a . I t a c t s as a v a s o d i l a t o r o f p e r i p h e r a l a r t e r i e s a n d a r t - e r i o l e s . I t i s a n a d r e n o l y t i c a g e n t , b l o c k i n g t h e 14 pressor without disturbing the depressor effect of epinephrine. The action is peripheral and there may also be some direct dilating effect on the vessel walls themselves (Beckman, 10). Dosage level based on approximate adult human dose. Etamon chloride Tetraethylammonium chloride, Parke-Davis, i s a depressant of both sympathetic and parasympathetic peripheral ganglia, with possibility of some locally exerted vasodilator action. Used at dosage level similar to reports tabulated by Shumacker ( 4 ). Hydergine Hydergine is not mentioned in 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 action on the peripheral circulation. Bluntschli and Goetz (12 ) showed that dihydroergocornine produced no constriction in the sympathectomized limb, and gave vasodilatation in the normally innervated limb, this being the f i r s t known ergot derivative acting purely as a sympathicolytic and also acting over higher o ; ; . r . . . p t 15 sympathetic centres rather than peripherally at the myoneural sympathetic junction, and they showed that the compound acted on the vascular centres in the medulla and or the hypothalamus. Rothlin of Basle a year earlier showed (13) a l l four of the dihyd- rogenated derivatives of ergot had this sympathico- adrenolytic activity, and Hurley used a preparation called CCK-179 containing equal parts of dihydro- ergocornine, dihydroergocristine, and dihydroerg©- kryptine, now put out by Sandoz as Hydergine. Hurley f e l t vasoconstriction (due to local response, local central nervous system reflex, and hypothalamic vaso- constriction due to cooled blood) could contribute greatly to tissue anoxia following frostbite, and that release of this could be of prime importance in the therapy of frostbite. He found, following exposure of sixty Sprague-Dawley and Wistar rats (distal seven centimetres of the t a i l s to -15°C for 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 significant improvement of each treated group over the controls. Hurley suggested that these dihydrogenated ergot alkaloids might prove to be of value in the treatment of high altitude frost- bite. The dosage of Hydergine used in the present experiments was nearly that used by Hurley. 16 Apresoline Hydralazine hydrochloride, Ciba, has an action claimed by the manufacturer to be largely central on the midbrain, serving to lower both the systolic and diastolic blood pressures, especially in hypertension, yet per- mitting increased blood flow through the kidneys. It has adrenergic blocking effects, adrenolytic and sympatho- lytic, against the pressor effects of epinephrine and nor-epinephrine. This drcug has not yet, to my knowledge^ been reported in frostbite experiments. L D 5 Q for white rats i s 3 4 mg. per kilogram body weight. The dosage level used was on the range ©f 2 mg. per animal. Rutin and Ascorbic acid It i s logical to try agents that reportedly reduce capillary permeability as well as f r a g i l i t y . Rutin is one of the vitamin P substances, and ascorbic acid i s of course vitamin C. The dosage selected for rutin was that reported in Shumacker's review. In the case of ascorbic acid, i t i s regarded as impossible to make rats deficient in this vitamin since they manu- facture their own, hence excessively large doses were used, which had the effect to be noted later in the gross experiments. Benadryl This antihistaminic agent has been used freq- uently before in frostbite ( 4 ) and the dosage level 1 7 supposedly effective in rats i s known. 0.43 mg. per kilogram body weight was used by Shumacker. Chlor-Tripolon This is an alternate antihistaminic, very powerful, and not previously reported in frostbite lesions experimentally. The dosage level selected was rather empirical at 5 mg. per animal. Histamine It was decided to attempt to assess the influence of histamine by virtue of the questionable findings with the antihistaminics. No stated effective dosage level could be found, and two values were sel- ected for t r i a l , 10 mg. and 50 mg. per kilogram body weight. Need for Confirmatory Gross Experiments As mentioned, i t was decided i t would be log- c i a l to carry on with microscopic observation of the blood vessels in the rat mesoappendix. The vessels at this location were selected primarily for conven- ience, since very beautiful preparations can be ob- tained with a minimum of effort, and with the equipment t© be described, such preparations can be maintained in a healthy condition for up to four hours. However, in cold injuries, the mesoappendix is seldom frozen, 18 and there i s no reason to expect that c a p i l l a r i e s that functioned one way i n the protected environment of the peritoneal cavity would function i n the same way i n the hind l e g of the same animal. Accordingly, the series of gross experiments were performed as described l a t e r i n an e f f o r t to correlate the findings In the two areas. 19 Microscopic Observation of the Rat Mesoappendix The Rat Mesoappendix There are numerous areas in various species of animals whe^e the fine details of the blood circulation can be observed clearly. Of these,the mesentery of the rat lying 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 sufficiently anaesthet- ized by an agent such as nembutal, and with a median abdominal incision 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 meso- appendix exposed for view with a low power microscope, and illuminated by transillumination. The Circulation of the Mesoappendix Without discussing the controversies concerning the anatomy of the capillary circulation that began in 1874 with Rouget, and have continued right up to the present, and w i l l be with us long into the future, the capillary bed structure as described by Chambers and Zweifach (22) was/shasis for identification and naming i of the various parts of the capillary beds observed in the mesoappendix. They b u i l t up a veritable anatomy of the capillary bed, and their scheme is here outlined. 20 T h e y f e l t t h e r a t m e s o a p p e n d i x was p r e d o m i n a n t l y a n u t r i t i v e f i e l ^ d , w i t h a c e n t r a l c h a n n e l a n d t r u e c a p - i l l a r i e s a s s i d e b r a n c h e s . The s m a l l a r b o r i z i n g a r t e r i e s f i n a l l y become w h a t C h a m b e r s a n d Z w e i f a c h t e r m t h e f i r s t d i v i s i o n o f t h e c a p i l l a r y b e d , t h e t e r m i n a l a r t e r i o l e s . T h e s e a r e c h a n n e l s t w e n t y t o t w e n t y - f i v e m i c r o n s i n d i a m - e t e r , w i t h a s i n g l e c o n t i n u o u s s m o o t h m u s c l e l a y e r . H e r e p u l s a t i o n s a r e r e l a t e d t o t h o s e i n t h e l a r g e r a r t e r i e s , b u t a r e l e s s r e g u l a r . S e c o n d l y i n l i n e o f b l o o d f l o w comes t h e c a p i l l a r y b e d p r o p e r . The m e t a r t e r i o l e i s f r o m e i g h t t o f i f t e e n m i c r o n s i n d i a m e t e r , i t h a s a t y p i c a l d i s c o n t i n u o u s m u s c l e c e l l c o a t , a n d i t shows v a s o m o t i o n w i t h s l o w c o n s t r i c t o r - d i l a t o r p h a s e s . The b l o o d t h e n f l o w s t h r o u g h t h e p r e c a p i l l a r y j u n c t i o n s o r s p h i n c t e r s . T h e s e a r e u s u a l l y t w i s t e d a n d a r e o u t f l o w i n g . T y p i c a l m u s c l e c e l l s a c t a s s p h i n c t e r s . V a s o m o t i o n h e r e i s i n d e p e n d e n t o f t h a t I n t h e a r t e r i o l e s . F i n a l l y t h e b l o o d p a s s e s i n t o t h e t r u e c a p i l l a r i e s . H e r e t h e r e i s p a s s i v e d i l a t a t i o n a n d t o n i c c o n s t r i c t i o n o f t h e e n d o - t h e l i a l c e l l s . T h e r e a r e m o r e r a p i d , b y - p a s s c h a n n e l s k n o w n as a r t e r i o - v e n o u s c h a n n e l s o r s h u n t s . The p r o x i m a l s e g m e n t o f a n a r t e r i o - v e n o u s c h a n n e l a p p a r e n t l y h a s a n a t y p i c a l t y p e o f s m o o t h m u s c l e c e l l t h a t s h o w s n o v a s o m o t i o n a n d r e s p o n d s o n l y t o a b n o r m a l s t i m u l i . T h e s e l e a d 21 Into p r e c a p i l l a r y junctions, or into true c a p i l l a r y junctions, and hence into the true c a p i l l a r i e s . The d i s t a l segment of the arterio-venous channel has a connective tissue coating that permits s l i g h t passive changes i n diameter. The post c a p i l l a r y junctions are inflowing, the same as the c a p i l l a r i e s are. Following either the true c a p i l l a r y channels or the arterio-venous channels, the blood enters the nonmuscular venule, which i s r e a l l y nothing more than a fusion of thorough- fare channels. This has a connective tissue covering, and s l i g h t passive changes i n diameter are permitted. Thirdly, and l a s t l y , i n the anatomy of the c a p i l l a r y bed are the muscular venules. These are twenty-five to t h i r t y microns i n diameter (the non-muscular venules were only f i f t e e n to twenty-five) and they are covered with both smooth muscle c e l l s and connective tissue. They are highly responsive and show varied contractions. Vasomotion i s a peculiar type of motor a c t i v i t y of the metarteriole and of i t s p r e c a p i l l a r i e s . These channels show i r r e g u l a r l y recurrent series of d i l a t a t i o n s and contractions at i n t e r v a l s varying from f i f t e e n sec- onds to three minutes. During active metarteriolar vaso- motion the sphincter-like closure 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 blood flow to the central 22 c h a n n e l s . T h e r e f o r e , t h e c e n t r a l a r t e r i o - v e n o u s c h a n n e l c a r r i e s o n t h e b a s a l w o r k o f t h e c a p i l l a r y b e d i n i s c h e m i a . The e n d o t h e l i a l w a l l o f t h e c a p i l l a r y p o s s e s s e s a c e r t a i n d e g r e e o f e l a s t i c i t y . The t r u e c a p i l l a r y s h o w s p a s s i v e c h a n g e s i n d i a m e t e r a s a c o n s e q u e n c e o f t h e v a r i a t i o n s o f t h e b l o o d f l o w t h r o u g h i t . When t h e p r e s s - u r e i n t h e c a p i l l a r i e s f a l l s t h e e n d o t h e l i a l c e l l t e n d s t o l o s e o i t s e x p a n d e d s t a t e , w h e r e - u p o n i t s n u c l e u s r o u n d s u p a n d c r e a t e s a b u l g e I n t o t h e l u m e n o f t h e c a p i l l a r y . N o t e t h a t c a p i l l a r i e s o f one " b e d " a l s o c o n n e c t w i t h o t h e r c a p i l l a r y " b e d s . " A c c o r d i n g t o C h a m b e r s ( 2 1 ) , t h e s m o o t h m u s c l e s p h i n c t e r c e l l s a r e a c t i v a t e d b y v a s o - c o n s t r i c t o r n e r v e s , w i t h t h e r e b e i n g no e v i d e n c e f o r v a s o d i l a t o r n e r v e s . A l s o a c c o r d i n g t o Z w e i f a c h a n d C h a m b e r s , v a s o - m o t i o n i s m a r k e d l y a f f e c t e d b y i r r i t a t i o n s u c h a s h a n d l i n g , s t r o k i n g , s t a b b i n g , b y n e r v e s u p p l y s u c h t h a t v a s o m o t i o n c e a s e s w h e n t h e n e r v e s u p p l y i s I n t e r r u p t e d , a n d b y d e e p a n a e s t h e s i a w i t h n e m b u t a l o r s i m i l a r a g e n t . I t c a n b e • i n c r e a s e d f u r t h e r b y a c u t e h a e m o r r h a g e , b y s y m p a t h e t i c n e r v e s t i m u l a t i o n , a n d b y i n t r a v e n o u s i n j e c t i o n s o f s u c h d r u g s as a d r e n a l i n , a n g i o t o n i n , a n d a d r e n a l c o r t i c a l e x t r a c t . I t c a n b e d e c r e a s e d ( w i t h some oedema f o r m a t i o n ) b y a t e m p e r a t u r e r i s e (37 .5 t o 4 1 G C ) , b y a t e m p e r a t u r e f a l l (10 t o 20 ° C ) , b y d i r e c t t r a u m a , a n d b y i n c r e a s e d 23 v i t a l a c t i v i t y a s m u s c u l a r e x e r c i s e o r g l a n d s e c r e t i o n . A g e n t s a s h i s t a m i n e , a d e n y l i c a c i d , a d e n o s i n e , k a l l i - k r e i n , a n d a c e t y l c h o l i n e a r e s u p p o s e d t o i n c r e a s e t h e c a p i l l a r y c i r c u l a t i o n w i t h o u t d e c r e a s i n g p r e c a p i l l a r y v a s o m o t i o n . I n t h e e p i n e p h r i n e r e s p o n s e , t h e m e t a r t - e r i o l e a n d p r e c a p i l l a r y s p h i n c t e r s c o n s t r i c t . I n c r e a s i n g t h e c o n c e n t r a t i o n g i v e s p r o g r e s s i v e l y f u r t h e r c o n s t r i c t i o n a l o n g t h e m e t a r t e r i o l e t o w a r d t h e d i s t a l c e n t r a l a r t e r i o l e . I n t h e h i s t a m i n e r e s p o n s e t h e r e i s d i l a t a t i o n o f t h e a r t - e r i o l e s , m e t a r t e r i o l e s , a n d p r e c a p i l l a r y s p h i n c t e r s . T h i s d i l a t a t i o n o c c u r s i n t h e c a p i l l a r i e s o n l y , i f t h e b l o o d was f l o w i n g t h r o u g h t h e m a t t h e t i m e . TWeapiKar^ Sphincter J)istaL Cha.nno.( C a p i l l a r y B e d 24 W h e t h e r a l l t h a t C h a m b e r s a n d Z w e i f a c h d e s c r i b e i s t r u e o r n o t , t h e y h a v e m a p p e d o u t v e r y w e l l t h e v a r i o u s f u n c t i o n i n g u n i t s o f t h e c a p i l l a r y b e d , a n d t h e i r m a n n e r o f a c t i o n , a n d s u c h a n u n d e r s t a n d i n g was v e r y h e l p f u l f o r a b e g i n n e r t o f i n d h i s way a r o u n d i n t h e m e s o a p p e n d i x , f o r t h e p a r t s l a b e l l e d b y C h a m b e r s a n d Z w e i f a c h w e r e r e a d i l y i d e n t i f i e d , a n d f r e q u e n t l y o b s e r v e d t o f u n c t i o n as t h e y s a i d t h e y s h o u l d . The O b s e r v a t i o n o f t h e R a t M e s o a p p e n d i x A s m e n t i o n e d n e a r t h e b e g i n n i n g o f t h i s t h e s i s , D . L . W a r n e r ( 7 ) was o b s e r v i n g c a p i l l a r y c h a n g e s i n r a t s r e n d e r e d h y p e r t e n s i v e w i t h c o m p o u n d F , a n d t h e same a p p a r a t u s h e u s e d was t a k e n o v e r f o r t h e p r e s e n t w o r k . M u c h o f t h e f o l l o w i n g d e s c r i p t i o n o f t h e e q u i p m e n t i s t a k e n f r o m h i s u n p u b l i s h e d t h e s i s . G e n e r a l l y K n i s e l y ' s t e c h n i q u e ( 1 8 ) f o r t r a n s - i l l u m i n a t i o n o f l i v i n g s t r u c t u r e s was u s e d . A l i g h t s o u r c e f r o m a d i s m a n t l e d l a n t e r n s l i d e p r o j e c t o r w i t h a f o u r h u n d r e d w a t t b u l b was u s e d . T h i s may b e s e e n i n t h e l o w e r r i g h t h a n d c o r n e r o f t h e f i r s t p h o t o g r a p h . One e n d o f t h e p r o j e c t o r was s e a l e d o f f b y a c i r c u l a r p i e c e o f one i n c h b o a r d f i v e i n c h e s a c r o s s . A h o l e was made i n i t s c e n t r e f o r t h e e n d o f t h e f u s e d q u a r t z r o d ( t h e g l a s s - l i k e r o d may b e c l e a r l y s e e n i n t h e f i r s t   27 photograph going across from the projector towards, and actually between the hind feet of the rat). This rod transmitted and "bent" the focused beam of light right to the desired location. This may be seen quite clearly in 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). It was twelve inches long, and the f i r s t ten inches were eleven mm. in diameter. The last two inches tapered down to about Shree mm. The last half-inch was curved upward at an angle of ninety degrees. The last two inches were hollow, with a one mm. channel down the shaft from a side arm. A Lietz dissecting microscope was used, binocular as i l l u s - trated and i t provided for magnifications of 12.5x, 5Gx, and 100 x. The latter, 100 x, was the customary system used. These lenses were mounted on a moveable nose- piece, which was used primarily for observing various areas of the mesoappendix (and not for changing lens systems as designed) instead of moving the animal mounting. The irrigation f l u i d was maintained in a four l i t r e brain jar container, covered in a water bath which was thermostatically controlled for about 43GC. The solution was siphoned off and passed along two poly- ethylene tubes, one connecting to the side arm of the 28 quartz tube, the other ending i n a f i n e glass tube that was suspended over the mesoappendix preparation i n a moveable fashion. 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 I l l u s t r a t i o n s . The polyethylene tubes w i l l be seen emerging from a thick rubber tube about one inch across. This was devised to keep the drip solution warm u n t i l i t was delivered to the mesoappendix, and hot water bath water was ci r c u l a t e d through this rubber casing by means of a simple rotary puferp. The drip solution was delivered at the mesoappendix at a temperature of approximately 37°C. The anaesthetized animal would be given 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 side on a l i t t l e shelf of l u c i t e that was attached on the top of a l u c i t e box one inch deep to catch the expended drip solution. The caecum would be c a r e f u l l y exteriorized, and the meso- appendix would then be draped over a l i t t l e l u c i t e loop two cm. across attached at a suitable height above the shelf to avoid tension on the mesoappendix. Warm saline soaked cotton packs (not shown i n the i l l u s t r a t i o n ) would then be packed around the loops of bowel to keep them warm and moist, and also to support further the caecum. The warm drip solution was then started, which flowed over the mesoappendix from above. Solution also flowed out the channel i n the quartz tube, but i n as much as t h i s tube did not usually touch the mesoappendix, th i s f l u i d 29 s e r v e d l i t t l e m o r e t h a n t o k e e p t h e e n d o f t h e t u b e f r o m g a t h e r i n g b l o o d , e t c . , t h a t w o u l d o b s t r u c t t h e beam o f l i g h t . W a r n e r h a d p e r f e c t e d t h e a p p a r a t u s a n d l i t t l e a d j u s t i n g was n e c e s s a r y . The p h o t o g r a p h s i l l u s t r a t e t h e e q u i p m e n t e x c e e d i n g l y w e l l , a n d a l l p o i n t s m e n t i o n e d c a n b e p i c k e d o u t . The s e c o n d p i c t u r e shows v e r y c l e a r l y t h e l o o p o f c a e c u m d r a p e d o n t h e o b s e r v e r ' s s i d e o f t h e l o o p ( a l s o v i s i b l e , b u t n o t t o o w e l l d e m a r c a t e d , w i t h t h e h i g h l i g h t f r o m t h e q u a r t z t u b e s h o w i n g a t i t s c e n t r e ) . The h i g h l i g h t i s p r o d u c e d n o t b y t h e e n d o f t h e q u a r t z t u b e a l o n e , b u t m a i n l y b y t h e l i g h t s h i n i n g o n t h e t r a n s l u c e n t m e s o a p p e n d i x . Some d i f f i c u l t i e s w i t h d r i p s o l u t i o n s w e r e e x - p e r i e n c e d . A t f i r s t a p l a i n R i n g e r - L o c k e s o l u t i o n was p r e p a r e d ( f o r m u l a i n a p p e n d i x ) • B u t a c c o r d i n g t o t h e p r o l o n g a t i o n i n t h e n o r m a l s t a t e t h r o u g h t h e a d d i t i o n o f g e l a t i n a s d i s c u s s e d b y Z w e i f a c h ( 1 4 ) , i t was d e - c i d e d t o a d d g e l a t i n e a n d g l u c o s e b o t h t o t h e R i n g e r - L o c k e s o l u t i o n as o u t l i n e d i n t h e a p L p e n d i x a t t h e e n d o f t h e t h e s i s . T h i s r e s u l t e d i n m u c h m o r e p h y s i o l o g i c a l p r e p a r a t i o n s . The F r e e z i n g P o i n t R u n n i n g a c r o s s b o t h p h o t o g r a p h s i s a w i n d i n g m a l l e a b l e m e t a l t u b e l e a d i n g f r o m a c a r b o n d i o x i d e t a n k , n o t s h o w n , t o t h e r i g h t o f t h e p i c t u r e s , a c r o s s t o w a r d 30 t h e m i c r o s c o p e t o e n d i n a c o n t r o l v a l v e a p p a r a t u s . T h i s a p p a r a t u s i s t h e o p e r a t i n g u n i t f r o m a S p e n c e r c a r b o n d i o x i d e q u i c k - s e c t i o n m i c r o t o m e . T h i s h a s b e e n a d a p t e d a c c o r d i n g t o H a s s a n d T a y l o r ' s d i r e c t i o n s ( ) b y t h e i n c l u s i o n o f t h e p a r t s l i s t e d i n t h e a p p e n d i x a t t h e e n d o f t h e t h e s i s . The f r e e z i n g p l a t e was r e m o v e d f r o m t h e m i c r o t o m e u n i t t o l e a v e e s s e n t i a l l y t h e c o n t r o l n e e d l e v a l v e . To t h i s was a t t a c h e d a L u e r - L o k u n i t a s o n t h e e n d o f L u e r - L o k s y r i n g e s . T h e n was c o n s t r u c t e d t h e u n i t s k e t c h e d b e l ' o w w h i c h was r e a d i l y L u e r - L o k f i t t i n g c o 2 s u p p l y l i n e a t t a c h e s h e r e Hub o f 19 n e e d l e s o l d - e r e d t o L u e r - L o k f i t t i n g s o t i p o f n e e d l e i s 1 o r 2 ram. p r o x - i m a l t o c l o s e d p o i n t o f 1 5 n e e d l e 1 9 n e e d l e 1 5 n e e d l e E x p a n d e d COg d i s - c h a r g e s h e r e t o o u t s i d e a i r C l o s e d b y t h i n p l a t e o f s o l d e r H a s s a n d T a y l o r ' s C o l d P o i n t A p p a r a t u s 31 a t t a c h a b l e t o t h e r e s t o f t h e u n i t ( n e e d l e v a l v e a n d c a r b o n d i o x i d e g a s s u p p l y ) . The p r i n c i p l e o f t h e c o l d p o i n t was q u i t e s i m p l e , u t i l i z i n g t h e c o o l i n g e f f e c t o f e x p a n d i n g g a s , i n t h e same w a y a s c a r b o n d i o x i d e snow i s m a d e . The l i q u i d c a r b o n d i o x i d e , o r p r o b a b l y h i g h l y c o m p r e s s e d c a r b o n d i o x i d e was c o n d u c t e d down t h e s h a f t o f t h e n u m b e r n i n e t e e n n e e d l e a t a r a t e c o n t r o l l e d b y t h e n e e d l e v a l v e . The t i p o f t h e n i n e t e e n e n d e d j u s t p r o x - i m a l t o t h e c l o s e d t i p o f t h e f i f t e e n n e e d l e ( t h e c o l d • p o i n t , a t t h e e n d o f t h e a p p a r a t u s ) . The g a s e x p a n d e d f r o m t h e p o i n t o f t h e n i n e t e e n i n t o t h e l a r g e r s h a f t o f t h e f i f t e e n . The L u e r - L o k f i t t i n g w i t h t h e s i d e - a r m h a d a l r e a d y b e e n a f f i x e d b y a i r t i g h t s e a l t o t h e s h a f t o f t h e n i n e t e e n n e e d l e ( a t a n a p p r o p r i a t e l e v e l so t h a t t h e p o i n t o f t h e n i n e t e e n was j u s t i m m e d i a t e l y p r o x i m a l t o t h e c l o s e d o f f p o i n t o f t h e f i f t e e n w h e n t h e f i f t e e n n e e d l e was l o c k e d o n t o t h e L u e r - L o k f i t t i n g ) a n d w i t h t h e f i f t e e n n e e d l e l o c k e d o n t o t h e f i t t i n g , t h e now e x p a n d e d g a s was a b l e t o f l o w p r o x i m a l l y w i t h i n t h e f i f t e a n d t o e s c a p e t h r o u g h t h e s i d e a r m t o t h e o u t s i d e a i r . B y c o n t r o l l i n g t h e r a t e o f g a s f l o w , u s u a l l y w i t h s h o r t , c l o s e l y s p a c e d " j e t s " , a f r e e z i n g t e m p e r a t u r e c o u l d b e o b t a i n e d a t t h e c l o s e d t i p o f t h e f i f t e e n n e e d l e . T h i s c o l d t i p c o u l d t h e n b e a p p l i e d t o a s u i t a b l e s i t e o n t h e m e s o a p p e n d i x , j u s t a s t h e m o d e l i n t h e p h o t o g r a p h s i s 32 d e m o n s t r a t i n g . The p l a c i n g o f t h e c o l d p o i n t c o u l d b e l o c a l i z e d v e r y a c c u r a t e l y t h r o u g h t h e m i c r o s c o p e o n t o j u s t t h e d e s i r e d c a p i l l a r y b e d . The u s u a l c a p i l l a r y b e d j u s t a b o u t f i l l e d t h e f i e l d s h o w n b y t h e m i c r o s c o p e a t t h e 100 p o w e r m a g n i f i c a t i o n . The c o l d p o i n t c o u l d be p r e c i s e l y l o c a l i z e d o v e r a n y c e r t a i n v e s s e l . When t h e p o i n t was p l a c e d , t h e g a s was t u r n e d o n , a n d t h e a r e a a t t h e p o i n t a l l o w e d t o f r e e z e ( t h e wa rm s a l i n e d r i p was swung o u t o f t h e way). A f r e e z i n g c o n d i t i o n was I n d i c a t e d b y a s u d d e h w h i t e n i n g o f t h e a r e a due t o i c e f o r m a t i o n i n t h e m o i s t f i l m s t i l l r e m a i n i n g o n t h e m e s o a p p e n d i x . T h i s i n v o l v e d t h e w h o l e t h i c k n e s s o f t h e m e s o a p p e n d i x a n d t h e a r e a o f t h e m e s o a p p e n d i x a f f e c t e d was a l w a y s w i t h i n t h e f i e l d o f t h e m i c r o s c o p e a t t h e 100 p o w e r m a g n i f i c a - t i o n . I n t h i s w a y , b y c e n t e r i n g l a t e r a t t h e o r i g i n a l l y s e l e c t e d a r e a f o r f r e e z i n g w h i c h was a t t h e c e n t r e o f t h e f i e l d , t h e b o u n d a r i e s o f t h e f r o z e n m e s o a p p e n d i x c o u l d a l w a y s b e e x a c t l y d e t e r m i n e d . The f l o w o f g a s w o u l d b e t u r n e d o f f a t t h e e x p i r y o f a p p r o x i m a t e l y t w e n t y - f i v e s e c o n d s , a n d a t e x a c t l y t h i r t y s e c o n d s t h e wa rm d r i p was r e s u m e d o v e r t h e p o i n t a n d t h e m e s o a p p e n d i x . T h a w i n g was n e a r l y i n s t a n t a n e o u s , a n d t h e n , b u t o n l y t h e n c o u l d t h e n e e d l e p o i n t be r e m o v e d . E p i n e p h r i n e R e s p o n s e Z w e i f a c h ( 1 4 ) o u t l i n e s i n q u i t e c a r e f u l d e t a i l h i s 33 e p i n e p h r i n e r e s p o n s e t e s t . He d i s c u s s e s a t h r e s h o l d r e s p o n s e t o v a s o t r o p i c s u b s t a n c e s , a n d h e d e f i n e s t h e t h r e s h o l d r e s p o n s e a s j u s t e n o u g h o f t h e s u b s t a n c e t o g i v e a m o d e r a t e n a r r o w i n g o f t h e t e r m i n a l a r t e r i o l e s a n d p r e c a p i l l a r i e s j u s t s u f f i c i e n t t o s l o w t h e f l o w o f b l o o d t h r o u g h t h e c a p i l l a r i e s a n d t r i b u t a r y v e n u l e s ; ? w i t h i n f i f t e e n t o t w e n t y s e c o n d s . The t h r e s h o l d r e s p o n s e t o e p i n e p h r i n e v a r i e s i n d i f f e r e n t a r t e r i o l e s w i t h i n t h e same t i s s u e . One p a r t i n s i x m i l l i o n i s j u s t b e l o w t h e l e v e l t o g i v e a r e s p o n s e . The warm d r i p s o l u t i o n s h o u l d be r e s t o r e d b e t w e e n t r i a l s . The e p i n e p h r i n e s h o u l d n o t b e a p p l i e d o f t e n e r t h a n a t t h r e e m i n u t e i n t e r v a l s , a n d a l s o r e p e a t e d a p p l i c a t i o n s o f e p i n e p h r i n e s e n s i t i z e s t h e t i s s u e t o t h e d r u g . Z w e i f a c h l a y s down c e r t a i n c r i t e r i a f o r ^ c o n s i d e r i n g a p r e p a r a t i o n i n a n o r m a l s t a t e . I t m u s t g i v e t h e e p i n e p h r i n e r e s p o n s e . B l o o d f l o w i n g t h r o u g h t h e c a p i l l a r y b e d s s h o u l d b e i n t e r m i t t e n t . T h e r e s h o u l d b e a m i n i m u m o f l e u k o c y t e s s t i c k i n g o r d i a p e d e s i s i n t h e w a l l s o f t h e veira l te 'Sr . . T h e r e s h o u l d b e n o c a p i l l a r y s t a s i s o r s t a g n a t i o n . He f u r t h e r s t a t e s n o t e s t s c a n b e made m o r e t h a n f i f t y t o s i x t y s e c o n d s a f t e r t h e p r e p - a r a t i o n i s m o u n t e d . A l s o , m a t e r i a l s w i t h m a r k e d s i d e r e a c t i o n s i n v a l i d a t e t h e m e s o a p p e n d i x t e s t . T h e s e d e t a i l s o f t h e c r i t e r i a a n d c h a r a c t e r i s t i c s o f t h e t h r e s h o l d e p i n e p h r i n e r e s p o n s e w e r e o f g r e a t 34 v a l u e a n d u s e f u l n e s s i n s t u d y i n g t h e c a p i l l a r y r e a c t i o n s o f t h e r a t m e s o a p p e n d i x . A s w i l l b e s e e n i n t h e p r e l i m i n a r y c a p i l l a r y t e s t s w i t h e p i n e p h r i n e a n d c o m p o u n d P as r e c o r d e d i n t h e f o l l o w i n g s e c t i o n o f t h e t h e s i s , t h e a c t u a l p e r f o r m a n c e o f t h e t e s t was r e l a t i v e l y e a s y . L o c a l i z e d C o l d I n j u r y E x p e r i m e n t s W i t h t h e c o l d p o i n t a p p a r a t u s a s d e s c r i b e d , a g r e a t n u m b e r o f t e s t s w e r e p e r f o r a t e d w i t h c o n t r o l a n i m a l s a s r e c o r d e d i n f o l l o w i n g s e c t i o n s o f t h e t h e s i s . The . b e h a v i o r o f t h e v e s s e l s o f t h e m e s o a p p e n d i x t o t h e f r e e z i n g e f f e c t s s o o n f i t t e d i n t o a d e f i n i t e p a t t e r n . A s m e n t i o n e d , , t h e c o l d p o i n t w o u l d p r o d u c e s o l i d I c e a t p r o b a b l y b e l o w z e r o d e g r e e s . The o n s e t o f t h e f r e e z i n g w o u l d b e t o o r a p i d f o r t o t a l v a s o c o n s t r i c t i o n w i t h i n t h e a f f e c t e d a r e a , b u t w i t h i n t w e n t y t o t h i r t y s e c o n d s a f t e r t h e t h a w i n g a n y v a s o c o n s t r i c t i o n p r e s e n t w o u l d b e o v e r c o m e w i t h a g e n e r a l i z e d h y p e r e m i a w i t h i n t h e i n j u r e d a r e a . U s u a l l y w i t h i n t h i r t y s e c o n d s o f t h a w i n g ( c a l l e d r a p i d t h a w i n g b e c a u s e t h e warm d r i p was u s e d t o m e l t t h e c o l d p o i n t away f r o m t h e m e s o a p p e n d i x ) s l o w i n g w o u l d be o b s e r v e d i n a f e w o f t h e p o s t c a p i l l a r i e s , a n d v e r y s o o n w h a t was t e r m e d " s l u d g i n g " w o u l d a p p e a r , a l s o c a l l e d " s t a s i s " . The p r e c i s e n a t u r e o f t h i s p r o c e s s was n o t d e t e r m i n e d , b u t i t c o n s t a n t l y o c c u r r e d . The oedema d e s c r i b e d b y S h u m a c k e r c o u l d n o t b e o b s e r v e d m i c r o s c o p i c a l l y , b u t a s S h u m a c k e r s a y s ( a s o n p a g e 11 ) t h e f o r m e d e l e m e n t s o f t h e b l o o d w e r e s e e n p r o g r e s s i v e l y 35 to f i l l the small vessels, bringing to a h a l t the c i r c - u l a t i o n through them. This stasis was r e v e r s i b l e , at the beginning at l e a s t , since mechanical pressure or changes i n the blood flow i n adjacent vessels would break up the red c e l l masses. This spread of the stasis was also constantly observed, and the term "spread" i s used i n following descriptions i n two ways, rather indiscrimaitely. In one instance the spread meant just what i s implied here, a progression of the stasis along a continuous channel. More often the term was employed to s i g n i f y the appearance of s t a s i s i n separate ?not necessarily connected channels, somewhat as an infectious disease spreads through a neighbourhood, although this i s not s t r i c t l y a correct usage at a l l , even though convenient. For the purposes of the experiments, the exact meaning of the term does not r e a l l y matter. Recording of Observations In a l l the records of the experimental observations there w i l l be noted a boring and apparently useless r e p e t i t i o n . The decision was made early to indicate var- iables i n the technique within the text of the descript- ions f o r each preparation, as opposed to describing the general method at the beginning, and merely the bare variations with each animal. I t seemed when the records were written up that such variables as how much anaesth- 36 e t i c was u s e d a n d e x a c t l y how i t was a d m i n i s t e r e d , a n d e x a c t t i m e l a p s e s , e t c w e r e i m p o r t a n t , a n d s u c h a d i v e r s i t y o f d e t a i l s c o u l d n o t h e i n d i c a t e d w i t h o u t m u c h r e p e t i t i o n . C r i t e r i a f o r a D r u g E f f e c t A t t h e e n d o f e a c h s e c t i o n o f t h e d a t a d e a l i n g w i t h t h e c o l d p o i n t e x p e r i m e n t s a p p e a r s a summary t a b l e . The c o l u m n s a r e h e a d e d b y s u c h t i t l e s a s o n s e t o f s t a s i s , s p r e a d o f s t a s i s , g e n e r a l i z e d s t a s i s , r e s u m e d f l o w . T h e s e n e e d e x p l a i n i n g , b e c a u s e o f t h e n e c e s s i t y f o r d e f i n i n g j u s t w h a t was m e a n t b y a n e f f e c t due t o t h e i n f l u e n c e o f some m e d i c a t i o n o r t r e a t m e n t . The t i m e f o r t h e o n s e t o f t h e s t a s i s was one i t e m c o n s i d e r e d i m p o r t a n t . I f s t a s i s i s s u c h a p r o n o u n c e d f e a t u r e o f a f r e e z i n g i n j u r y , a n y m e d i c a t i o n t h a t c o u l d d e l a y s t a s i s o r p r e v e n t i t s a p p e a r a n c e a l t o g e t h e r w o u l d b e a v a l u a b l e a g e n t . T h i s a p p l i e d t o o f o r t h e e x t e n t i n t h e d e v e l o p m e n t o f t h e v a s c u l a r s t a s i s . A d r u g m i g h t n o t n e c e s s a r i l y d e l a y o r p r e v e n t v a s c u l a r s t a s i s , b u t i t m i g h t l i m i t t h e e x t e n t t o w h i c h i t a f f e c t e d t h e f r o z e n t i s s u e . The i d e a t h a t a d r u g ' s e f f e c t i v e i n f r o s t b i t e t h e r a p y s t emmed p a r t l y f r o m t h e f a c t t h a t t h e s t a s i s was a r e v e r s i b l e p r o c e s s , b u t m o s t l y f r o m t h e f a c t s t h a t p r o c a i n e f l u s h e d o v e r a s t a t i c f i e l d w o u l d d i l a t e t h e s m a l l v e s s e l s a n d c a u s e a r e s t o r a t i o n i n t h e c i r c u l a t i o n . T h i s was h e n c e r e g a r d e d a s a l i k e l y a t t r i b u t e i n d r u g s e f f e c t i v e I n f r o s t b i t e t h e r a p y . 37 Although these c r i t e r i a might appear to be f a i r l y objective and s c i e n t i f i c , such was i n f a c t just an i l l u s i o n . Vascular f i e l d s i n the rat mesoappendix were unfortunately found to have as much resemblence to each other as human faces have to other human faces. One mesoappendix might have only one or two wide areas sparsely crossed by a few f i n e c a p i l l a r i e s that could possibly pass as c a p i l l a r y beds. Other preparations would be just crowded with vessels going every possible d i r e c t i o n . When trying to specify as to what the extent of the sta s i s was, or how rapid the spread was, i t i s obvious that there were d i f f i c u l t i e s . On the whole, the observation of the mesoappendix was a very subjective matter. A necessary feature of the microscopic experi- ments lay i n the decision to alternate when possible the test and the control preparations. I t took generally four hours to perform a microscopic experiment of ten or twelve animals. Conditions could and did change greatly i n the room used f o r a laboratory over th i s time. Hence the testing of a l l the control animals followed by testing of the test rats was discarded as impractical. Care of Animals Rats, either Wistar or Hooded, i n the 150 to 25Q gram weight range were obtained i n batches of 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 b r e e d i n g s t o c k b a r n s e l s e w h e r e o n t h e c a m p u s . T h e y w e r e h o u s e d I n a w e l l v e n t i l a t e d w i n d o w l e s s i n s i d e r o o m , i n t i e r e d a n i m a l c a g e s h o l d i n g f r o m t h r e e t o s i x a n i m a l s e a c h . F r e s h d r i n k i n g w a t e r was a v a i l a b l e t o t h e a n i m a l s a t a l l t i m e s 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 k e p t s u s p e n d e d I n e a c h c a g e i n a f e e d i n g t r o u g h . A l l a n i m a l s s t a y e d h e a l t h y - a n d w e l l i n t h e s e s u r r o u n d i n g s f o r I n d e f i n i t e o p e r i o d s o f t i m e . T h e y w e r e . s e p a r a t e d a c c o r d i n g t o s e x . - 39 Preliminary microscopic observations using Compound F (17 hydroxy-eortisone 21 acetate) Preliminary capillary experiments In order t© gain some experience with the use of the microscope In connection with capillary observ- ations, certain tests were performed using the capillary epinephrine response as described by Zweifach, B. W. (14), Warner, D.L., the senior medical student ment- ioned so frequently in this thesis as a part-time helper with the project, while performing experimental work in connection with his graduating thesis for the Faculty of Medicine at this university, tested capillary response to various drugs including compound F., and he found certain effects due to the drug which greatly modified the capillary response to epinephrine. He found ( 7 ) that whereas a 1:4 million (abbreviation for one part in four million parts) dilution was the threshold concent- ration of epinephrine needed to e l i c i t a vasomotor re- sponse as determined by metarteriole and precapillary constriction, that after injecting compound F. for a certain number of dayf the threshold response had de- creased to 1:18 or 1:20 million dilution of epinephrine. Because of the supposed delicacy of the tech- nique of examining vascular responses under the micro- 40 scope, i t was f e l t that a repetition of Warner's work, in 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 in a small measure to increase confidence in the r e l i a b i l i t y of later observations with localized cold experiments. I n i t i a l epinephrine response tests The purpose of the following tests was to try to determine the threshold epinephrine response of the metarterioles and precapillaries in the mesoappendix of the rat. Several adult male Wistar rats were selected and set aside for use on several successive days. The apparatus was set up as described and a simple Ringer- Locke saline drip solution used. Epinephrine chloride solution 1:1000 was maintained in a small flask at the temperature of the water bath, and several test tubes of drip solution were also kept warm, with carefully measured quantities of drip solution to permit rapid preparation of the diluted epinephrine so as to minim- ize the tendency for the epinephrine to decompose. This decomposition of the epinephrine,especially when warm and mixed with the drip solution,was a real problem, and as w i l l be explained later possibly accounted for some of the conflicing results obtained. Animal 1 41 A normal adult male Wistar rat weighing 135 grams was anaesthetized with 0.13 cc. of 0.6% nembutal sol- ution administered intraperitoneally at zero minutes. At twenty-four minutes the preparation was mounted and ready on the microscope stage. The temperature of the drip solution was 33.5°C, and this was allowed to drip over the mesoappendix except when the test solution was flushed on with a medicine dropper. Approximately a. thirty seconds after the test solution was applied, the drip would be resumed un t i l the next tesfc. Long enough an interval was allowed for the preceding epinephrine to be washed away and for the vessel to recover from i t s effect before any more was used. An interval of about five minutes was considered suitable, and this was found to be adequate. At thirty-two minutes i;2 million epinephrine was applied and this gave a very strong response, classed as ////» At thirty-seven minutes 1:4 million gave a // response, at forty-two minutes 1:6 million gave no response. At forty-seven minutes a 1:4 million dilution gave a /// response. At fifty-two minutes a 1:5 million dilution gave a /// response. At fifty-seven minutes a 1;6 million dilution gave a very doubtful response, classed as plus or minus. On the basis of this, and with Warner>s agreement (he worked 42 with me for the f i r s t day) the threshold response was set at 1:5 million dilution. Animal 2 A normal adult male Wistar rat weighing 145 grams was anaesthetized with 0.13 cc. of 0,6% nembutal solution administered intraperltoneally at zero minutes. The animal was ready on the microscope by twenty-three minutes. At thirty minutes there was no response to 1:7 million epinephrine ( a l l dilutions were freshly prepared each time they were needed); at thirty-seven minutes no response to 1;6 million dilution; nor any response at forty minutes to 1:5 million. At forty-two minutes 1:4 million gave a / response two minutes later which lasted only a minute. At forty-seven minutes 1:5 million gave no response, but at fifty-two minutes 1:3 million gave a //. The preparation was discontinued at f i f t y - f i v e minutes. The temperature of the drip solution at the mesoappendix was 36°C. The threshold response was considered to be on the basis of this prep- aration 1:4 million dilution of epinephrine. Animal 3 On the following day, the same set-up was made ready as before, and a normal adult male Wistar rat 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 at twenty-three minutes with the drip going. At twenty-six minutes 1:6 million gave no response, but three minutes later 1:5 million gave a /// response. At thirty-two minutes 1:6 million gave a very doubtful effect, and the same with 1 :5 million four minutes later. At thirty-nine minutes 1:5 million gave no response, but at forty- two minutes i t gave a doubtful effect. At forty-five minutes a 1:4 million solution gave a /// effect. At forty-nine minutes 1:5 million gave no response, nor did 1:4 million two minutes later. Finally, at f i f t y - four minutes 1:5 million failed to give any effect. 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 for the epinephrine response was f e l t to be 1:5 million dilution. Animal 4 A normal adult male Wistar rat weighing 160 grams was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered intraperitoneally, at zero min- utes. At twenty-five minutes the preparation was mounted on the microscope stage and ready with the drip going at 36°G. 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 million dilution gave no response. At forty-three minutes a 1:5 million dilution gave a effect, which o was repeated at f i f t y minutes. At f i f t y - f i v e minues 1:5 million epinephrine had no effect, nor did 1;4 million at sixty minutes. One minute later a drop ©f plain ice water gave a vigourous //// response, and at sixty three minutes 1:4 million epinephrine gave a / response. The preparation was discontinued at / sixty-five minutes and the threshold response was considered to be 1*5 million dilution. Animal 5 A normal adult male Wistar rat weighing 175 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered intraperitoneally, at zero minutes. At twenty minutes the preparation was ready with the drip going. At twenty-four minutes a 1:6 million dilution gave no response. At thirty-two minutes 1:5 million epinephrine gave a / response. Five minutes later, on repeating the 1;5 million,,a doubtful effect was obtained. At forty-two minutes 1;4 million gave no response, but three minutes later the 1:4 million produced a // effect. It was noted (a) the saline 45 packs kept over the exposed bowel were too cool, and (b) there was some degree of general stasis in the mesoappendix f i r s t noticed about the forty-two minute period. There was also some blood loss from the abdominal incision which might have affected the general circulation. The preparation was discontinued at f i f t y minutes, and the threshold level for the epineph- rine response was f e l t to be again 1:5 million. 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 in the connecting tubing and the less heat i t lost on i t s way from the water bath) and the average temperature was about 37®C. Generally, in the following five animals much more spontaneous vasomotion was noted than at the previous temperature levels of 33 to 36°C. This may explain the slightly higher threshold levels obtained with this and the following preparations. A normal adult male Wistar rat, weight not recorded, was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered intraperitoneally. The temperature of the drip over the mesoappendix were 36.6°C. The preparation was ready at six minutes. At sixteen 46 minutes 1:7 million epinephrine gave a /// response, which was repeated five minutes later, and at twenty- five minutes 1;8 million gave a // response. A minute later plain drip solution gave no response, nor did 1:8 million dilution at twenty-eight minutes, nor did 1:7 million dilution at thirty-one minutes. 1:6 million failed to e l i c i t e any response at thirty-four minutes and 1:5 million did the smae at thirty-seven minutes. 1:4 million gave a / response at forty minutes, and at 43 minutes cold 1:100,000 dilution gave a //// response. For reasons not recorded, i t was - f e l t that 1;4 million was the threshold, and not 1;7 or 1:8 million v/as would appear to be. This opinion would have better support i f the interval between the applications of the epinephrine had been longer than three minutes. Animal 7 A normal adult male Wistar rat, weight not recorded was anaesthetized with 0.13 cc. of 0.6% nem- butal solution administered intraperitoneally, and fifteen minutes later 0.05 cc. had to be repeated to obtain a satisfactory depth of anaesthesia. The preparation was ready at twenty minutes, on the micro- scope, with the warm Ringer-Locke drip running. At 47 twenty-two minutes 1:10 million epinephrine gave no response, 1:8 million did not at twenty-six minutes, 1:7 million did not at twenty-nine minutes, and 1:6 million did not at thirty-two minutes. At thirty-five minutes 1:5 million dilution gave a very doubtful response, but 1;4 million gave a definite / response at forty minutes, but no response at forty-five min- utes. 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 million gave a // response. At f i f t y - s i x minutes 1:5 million f a i l e d to give any effect, but three minutes later 1»4 million gave a / response. Finally at sixty-two minutes, 1:100,000 cold epinephrine gave a very strong //// response. The preparation was discontinued at sixty-five minutes and the threshold was considered to be 1;4 million dilution of epinephrine. Animal 8 A nmrjnal adult male Wistar rat of unrecorded weight, was anaesthetized at zero minutes with 0.13 cc. of 0.6$ nembutal solution administered intraperi- 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 meso- appendix. At twenty-four minutes 1:6 million d i l - 48 ution of epinephrine gave a ///' response, and five minutes later 1;7 million also gave a /// response. The vascular stasis worsened gradually, and the preparation was discontinued with no results obtained. Animal 9 A normal adult male Wistar rat weighing 155 grams was anaesthetized at zero minutes with 0.13 cc. of 0.6$ nembutal solution administered intraperiton- eally, and the preparation was ready ten minutes later. At ten minutes 1:6 million dilution gave no response, but at fourteen minutes 1:5 million gave a / response. This was a very beautiful preparation to observe. At sixteen minutes 1:4 million gave no response, but five minutes later i t gave a doubtful response. At twenty- six minutes 1:3 million dilution gave a // response. The epinephrine was obviously getting brown, and was made up again from fresh 1;1000 stock epinephrine. At thirty-five minutes this fresh solution gave at 1:4 million dilution a // response. At forty minutes 1:5 million gave also a // response. At forty-four minutes 1:6 million gave no response, but a minute later 1*5 million gave a // response. The preparation was discontinued at f i f t y minutes, and i t was f e l t that 1:5 million was the threshold dilution for the epineph- 49 rine response. Animal 10 A normal adult male Wistar rat weighing 155 grams was anaesthetized with 0.13 cc. of 0.6$ nem- butal solution administered intraperitoneally, and i t was ready on the microscope stage with the warm drip going at five minutes. At seven minutes 0.05 cc of nembutal had to be repeated. At seventeen minutes 1:6 million dilution of epinephrine gave a j>ff response. 1:7 million gave a /// response at twenty minutes, and so did a 1:8 million solution at twenty-three minutes. At twenty-six minutes leukocytes were observed sticking to the walls of the larger vessels, and the preparation was discontinued as unphysiological. The temperature of the drip solution was 37°C. No results were re- corded. Summary of Observations No. Threshold epinephrine response in millionths 1. 1:5 2. 1 :4 3. 1:5 7. 1 :4 4. 1:5 8. No results 5. 1 :5 9. l : 5 6. 1:4 ? 10 No results 50 Discussion of Observations These observations agree with Zweifach, B.W. (14 ) who stated a 1;6 million dilution of epineph- rine i s just below the level required for a vascular response according to his technique. Here, with the doubtful exception of Animal 6, no threshold was found lower that 1;5 million, and Incidentally, nohewwas found higher than 1:4 million, which i s rather an excellent result. It was learned that the requirements for 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 spoil were observed f i r s t hand. These lessons were put to good advantage later on the local cold experiments. Note that a plain Ringer-Locke saline drip was employed. There were found later advantages to using a gelatin- glucose enriched and buffered drip solution. Observations with Compound F The purpose of the experiments here outlined was to test the effect compound P would have on the epinephrine threshold response in the circulation of the rat mesoappendix. . 2.0 mg. of compound P in saline suspension in a concentration of 25 mg. per cc. (that 51 i s 0.08 cc. per injection) administered intraperitoneally every day for approximately two weeks, was the contemplated dosage level, prior to the microscopic observations. Eighteen normal adult male Wistar rats were sel- ected, and after marking the ears characteristically 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 different days for convenience in testing. The medication was given to three test animals In each group, and the remaining three were for controls to be examined alternately with the test animals. At the end of the series of inject- ions with compound P, the epinephrine threshold response test was performed on each rat as i n the previous exper- iment. Details for each animal are as follows: Animal 1 (Control) The rat was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered intraperitoneally, 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 million dilution of epi- nephrine which could not be repeated at twenty-eight minutes. At thirty-one minutes 1:5 million dilution 52 Data Sheet Animal3 1 to 6 Control 160 - - 192 - T 154 - - 190 - T 166 - - 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 - - 206 - T 155 - - 214 - T 146 - - 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 - - - - - - - 248 - T 153 - - - - - - - - 231 - ? 150 - - - - - - - 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, injection, T, tested, ?, wasted, i/p, intraperitoneally, s/c subcutaneously) 53 gave a / response, and three minutes later the same dilution gave a /// response. The preparation was discontinued and the threshold was recorded as 1:5 million. Animal 2 (Test) The rat was anaesthetized with 0,13 cc of 0,6$ nembutal solution administered intraperitoneally after thirteen daily intraperitoneal injections of compound F. At fifteen minutes 0.03 cc, of the nembutal had to be repeated, and the preparation was ready at twen- ty-five minutes with the warm drip running. At thirty- million one minutes a l:10*dilution gave a // response, and four minutes later 1:10 million dilution gave a /// response. At thirty-nine minutes 1:14 million gave no response, and 1:10 million f a i l e d to give any response at forty minutes but gave a /// three minutes later. At fifty-one minutes 1;14 dilution gave no million response. At fifty- f o u r minutes a l:10Agave a very doubtful response, and so did 1:8 million at f i f t y - seven minutes. There was too much vascular stasis to be sure. Probably solutions weaker than 1:10 million would have given a response i f they had been used f i r s t . The results of this test had to be dis- counted in view of the fact the preparation was not entirely physiological. 54 Animal 5 (Control) The rat 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 million dilution of epi- nephrine gave a very doubtful vascular response. At twenty-seven minutes 1;5 million gave a /// response which was repeated in three more minutes. At thirty-three minutes 1:6 million did not give any response, and at thirty-seven minutes 1:5 million did not either. At forty minutes 1-4 million gave a very doubtful effect. At forty-two minutes 1:100,000 dilution gave a strong //// response. At forty-five 1:4 million gave a //// response, and at fifty-two minutes 1;5 million gave a /// response. 1*6 million gave a ///-response both at fifty-two and f i f t y four minutes. At fifty-seven minutes 1:7 million gave a //// response and at sixty- one minutes 1:10 million gave also a //// response. Finally, at sixty-five minutes plain drip solution gave a ////, and the preparation was discontinued because vascular stasis was quite obvious. Probably no results after forty-two minutes were valid, and 1«5 million was accepted as the threshold for this test. 55 Animal 4 (Test) The rat was anaesthetized with G.15 cc. of 0.6$ nembutal solution administered intraperitoneally after thirteen daily 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. At twenty- million three minutes a l:14"dilution gave a // response, and at twenty-eight minutes a 1:16 million dilution gave a /// response. At thirty-three minutes plain drip was flushed over the mesoappendix with no appar- ent effect on the blood vessels. At thirty-four minutes 1;20 million dilution was tested with no re- sponse. At thirty-nine minutes 1:10 million gave a doubtful response. At forty-two minutes 1:16 million gave a /// response, and then at forty-seven minutes 1»18 was tried again and i t gave a definite // response. In two minutes 1;20 million gave no effect but when 1:18 million was tried at fifty-one minutes i t gave a /// response. This was a wonderful preparation to work with. The preparation was dis- continued at f i f t y - f i v e minutes, and 1:18 million was accepted as the threshold level. 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 million dilution of epinephrine gave no response, nor did 1:8 million at fifteen minutes, nor did 1:6 million at nineteen minutes. However at twenty-three minutes 1:5 million dilution gave a /// response. This was accepted as a very definite threshold and the preparation was discarded at twenty-five minutes. Animal 6 (Test) The rat was anaesthetized with 0.13 cc. of 0.6$ nembutal solution administered intraperitoneally at zero minutes after thirteen daily intraperitoneal injections of compound P. 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 million dilution of epinephrine gave n© vascular response. At twenty-eight minutes 1*18 million gave no response, but at thirty-one minutes 1:16 million gave a /// response. Then at thirty-four minutes 1:18 million gave a // response, but at thirty-eight minutes 1:20 million gave no response. On this basis 1:18 million dilution was accepted as the threshold , and the preparation was discarded. Animal 7 (Control) The rat was anaesthetized with 0.13 cc. of 0.6$ 57 nembutal solution administered intraperitoneally 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 million epinephrine resulted in no vascular response. At eleven minutes 1«6 million dilution gave a /// response, and at fifteen minutes 1;7 million gave a // response which was repeated i n three minutes. At twenty-one minutes 1:10 million gave no response. At twenty-four minutes 1:8 million gave a slight response. In view of the fact the preparation was possibly sensitized to epinephrine at this stage, although i t was otherwise an excellent prep- aration, 1:7 million was accepted as the threshold value. The preparation was discontinued at twenty- eight minutes. Animal 8 (Test) The rat was anaesthetized with 0.15 cc. of 0.6% nembutal solution administered intraperitoneally at zero minutes, and at twenty minutes 0.06 cc. was re- peated, also by intraperitoneal injection, and the preparation was ready at thirty minutes. At thirty- three minutes 1:20 million dilution of epinephrine gave no response. At thirty-seven minutes 1:18 million gave a /, definite but slight, response. At forty min- utes 1:16 million gave a // response, and at both three and six minutes later 1»16 million gave a / response. 58 1:18 million 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 million epinephrine solution gave no vasc- ular response, nor did 1;6 million at eleven minutes, nor did 1:5 million at fourteen minutes. However, at seventeen minutes, 1:4 million gave a definite / response. This was a good preparation with excellent active vaso- motion. At twenty-two minutes 1:4 million gave a // response. 1;4 million 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 five minutes 0.05 cc. were re- peated, with 0.03 cc. more at thirteen minutes. The preparation was ready at eighteen minutes. At nineteen minutes 1:20 million epinephrine gave no apparent vasc- ular reaction, but at twenty-two minutes 1;18 million gave a /// response. 1:18 million 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 fifteen minutes and twenty-one minutes. At twenty- eight minutes 1:7 million epinephrine gave a // resp- onse. At thirty-one minutes 1:10 million gave a // which was repeated three minutes later. At thirty- seven minutes plain warm drip gave no response, but immediately after 1:10 million again gave a // response. The physiological nature of this preparation was not recorded, and 1:10 million must be accepted as the threshold. The preparation was discontinued at forty 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 million epinephrine dilution gave a // response but some stasis developed subsequently in the area being observed. At seventeen minutes 1:20 million gave a /// response, which was repeated at twenty minutes. Vascular stasis continued to develop in the mesoappendix until the preparation was discontinued at twenty-five minutes. 1;20 million was accepted as 60 t h e t h r e s h o l d v a l u e . A n i m a l 1 3 ( C o n t r o l ) The a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 4 c c . o f 0 . 6 $ n e m 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 a t z e r o m i n u t e s - f o l l o w e d i n two m i n u t e s b y 0 . 0 5 c c . m o r e . A t n i n e m i n u t e s , a f t e r m o u n t i n g t h e p r e p - a r a t i o n , 1 ; 6 m i l l i o n e p i n e p h r i n e d i l u t i o n f a i l e d t o g i v e a n y v a s c u l a r r e s p o n s e . A t n i n e t e e n m i n u t e s 1 ; 5 m i l l i o n g a v e a / / a n d t h i s was a c c e p t e d a s t h e t h r e s h - o l d v a l u e . N o t e W i t h a n i m a l s 1 3 t o 1 8 9 a t t e m p t s w e r e made t o p h o t o g r a p h t h e r e c o r d o f t h e v a s c u l a r r e s p o n s e s , b u t d i f f i c u l t i e s i n m a n a g i n g t h e c a m e r a , e t c . , c a u s e d t h e r e m a i n i n g f i v e a n i m a l s t o b e w a s t e d . Summary o f O b s e r v a t i o n s N o . K i n d T h r e s h o l d d i l u t i o n 11 C o n t r o l 1 : 5 m i l l i o n 2 . T e s t No r e s u l t s ' ' 3 . C o n t r o l 1 : 5 m i l l i o n 4 . T e s t 1 : 1 8 m i l l i o n 5 . ( C o n t r o l 1 : 5 m i l l i o n 6 . T e s t 1 : 1 8 m i l l i o n 7 . C o n t r o l 1 : 7 m i l l i o n 8 . T e s t 1 : 1 8 m i l l i o n 61 Summary (Cont'd) No. Kind Threshold d i l u t i o n 9. 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 Discussion of Observations These observations rather s a t i s f a c t o r i l y confirmed Warner's findings as described;that i n j e c t - ing compound P into rats f o r a cer t a i n number of days decreased the epinephrine threshold response from a normal of 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 d i l u t i o n . They also form a base l i n e of c a p i l l a r y per- formance upon which l a t e r c a p i l l a r y behaviour was judged. 62 R e c o r d o f M i c r o s c o p i c O b s e r v a t i o n s H e r e 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 m i c r o s c o p i c a l l y o n t h e m e s o a p p e n d i x o f t h e r a t a s o u t l i n e d p r e v i o u s l y . D e t a i l s o f t i m e p e r i o d s a n d i n j e c t i o n s a r e g i v e n i n d i v i d u a l l y f o r e a c h a n i m a l t e s t e d . The r e s u l t s a r e g r o u p e d b y t h e d r u g s b e i n g t e s t e d , a c c o r d i n g t o t h e o r d e r o f t h e t a b l e o f c o n t e n t s . P r o c a i n e E x p e r i m e n t a l o b s e r v a t i o n s w i t h p r o c a i n e h y d r o c h l o r i d e (A summary o f t h e f o l l o w i n g r e s u l t s i s g i v e n i n t a b l e f o r m a f t e r A n i m a l 1 0 . ) A n i m a l 1 ( T e s t ) 0 . 2 0 c c o f a 0.6% n e m b u t a l s o l u t i o n w e r e 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 i n t o a 2 8 0 g r a m a d u l t m a l e W i s t a r r a t . The p r e p a r a t i o n was r e a d y o n t h e m i c r o s s c o p e s t a g e i n n i n e m i n u t e s . A f e w d r o p s o f o n e i n f o u r m i l l i o n d i l u t i o n o f e p i n e p h r i n e f a i l e d t o g i v e a n y v a s o m o t o r r e s p o n s e i n t h e c a p i l l a r y 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 , b u t t h i s f a i l u r e was a t t r i b u t e d t o d e t e r i o r a t i o n o f t h e e p i n e p h r i n e . T h r e e m i n u t e s l a t e r , two p e r c e n t p r o c a i n e was d r o p p e d o n , a n d t h e r a t e o f b l o o d f l o w i n t h e v e s s e l s g e n e r a l l y was n o t e d t o b e i n c r e a s e d . One m i n u t e a f t e r t h i s , t h e c o l d p o i n t 6 3 was applied to a selected area of the mesoappendix with freezing for f o r t y - f i v e seconds, which was f o l l - owed after thawing by increased blood flow. Two per- cent procaine was dropped on the mesoappendix, but vascular s t a s i s was observed In a few c a p i l l a r y bedS within twenty-seconds of thawing, although the central venule was functioning well. By three and a hal f minutes afte r thawing, there was stasis i n the main venule i n the injured area, giving blockage and s t a s i s generally throughout the whole area affected by the freezing. Therefore i t would not seem as though the procaine delayed the onset of vasostasis much following the freezing i n j u r y . Ten minutes l a t e r one or two c a p i l l a r i e s were observed to be functioning at the edge of the injured area, and three minutes after t h i s the warm saline drip was stopped, and two percent procaine was i n s t i l l e d over the mesoappendix. This resulted i n a more rapid flow i n the c a p i l l a r i e s around the p e r i - phery of the injured area, but there was no apparent e f f e c t on any of the s t a s i s within the frozen area. Pour minutes l a t e r more two percent procaine was applied. In two minutes one large vessel that had been s t a t i c was functioning, 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 injured area were flow- ing. Two minutes l a t e r more procaine was applied, which resulted i n a s l i g h t t r i c k l e of blood corpuscles through the main s t a t i c area. Three minutes l a t e r more procaine 64 r e s u l t e d i n a l m o s t i m m e d i a t e o p e n i n g u p o f t h e m a i n s t a t i c a r e a , a l t h o u g h o n l y t e m p o r a r i l y , s i n c e t h e c i r c - u l a t i o n c l o s e d down a g a i n o n r e s u m i n g t h e warm s a l i n e d r i p . T h r e e m i n u t e s l a t e r m o r e p r o c a i n e a g a i n o p e n e d up t h e same c h a n n e l s , w i t h i n c r e a s e d v a s o m o t i o n o b s e r v e d i n t h e v e s s e l s t r i b u t a r y t o t h i s m a i n v e n u l a r c h a n n e l . 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 t h i r t y - f i v e s e c o n d s a f t e r t h e i n i t i a l f r e e z i n g . A n i m a l 2 ( T e s t ) A m a l e W i s t a r r a t i n t h e 2 0 0 g r a m r a n g e was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n , a n d t h e p r e p a r a t i o n was r e a d y s e v e n m i n u t e s l a t e r . Two p e r c e n t a q u e o u s p r o c a i n e was a p p l i e d t o t h e m e s o a p p e n d i x , a n d t h e s p e e d o f c a p i l l a r y b l o o d f l o w was o b s e r v e d t o b e i n c r e a s e d . One m i n u t e l a t e r , m o r e p r o c a i n e was f l u s h e d o v e r t h e m e s o a p p e n d i x , a n d o n e m i n u t e s t i l l l a t e r t h e 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 a r e a w i t h f r e e z i n g f o r t h i r t y s e c o n d s . T h i s r e s u l t e d i n i m m e d i a t e v a s o c o n s t r i c t i o n t h r o u g h o u t t h e a f f e c t e d a r e a , f o l l o w e d b y m a r k e d v a s c u l a r d i l a t a t i o n . P r o c a i n e was d r o p p e d o n t h e f i e l d . S t a s i s i n s m a l l v e n o u s c h a n n e l s a p p e a r e d t h r e e a n d a h a l f m i n u t e s a f t e r t h a w i n g , b u t t h e p r o c e s s was l i m i t e d . P r o c a i n e was r e a p p l i e d , a n d t h e s t a s i s was n o t e d t o h a v e s p r e a d t o i n v o l v e a n a d j a c e n t c h a n n e l , b u t w i t h i n f o u r m i n u t e s o f t h a w i n g , t h e f i r s t c h a n n e l o p e n e d up a n d r e s u m e d I t s b l o o d f l o w . One m i n u t e a f t e r t h i s t h e w h o l e a r e a was a g a i n i n s t a s i s , w h i c h was n o t 6 5 g r e a t l y r e l i e v e d b y f u r t h e r a p p l i c a t i o n s o f t h e p r o c a i n e . The p r e p a r a t i o n was n o t r e g a r d e d a t t h i s t i m e a s b e i n g p a r t i c u l a r l y p h y s i o l o g i c a l , a n d i t was d i s c a r d e d t h i r t - e e n m i n u t e s a f t e r t h e I n i t i a l f r e e z i n g i n j u r y . A n i m a l 5 ( C o n t r o l ) A n o r m a l m a l e a d u l t W i s t a r r a t i n t h e 2 0 0 g r a m r a n g e was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 , b u t 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 b e c a u s e t h e w h o l e m e s o a p p e n d i x was f o u n d t o b e i n v a s c u l a r s t a s i s o n m o u n t i n g o n t h e m i c r o s c o p e s t a g e . T h i s s t a s i s was n o t i n f l u e n c e d b y a p p l i c a t i o n s o f two p e r c e n t p r o c a i n e . A n i m a l 4 ( C o n t r o l ^ A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 2 0 0 g r a m r a n g e was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 , a n d t h e p r e p - a r a t i o n was r e a d y i n f i v e m i n u t e s . 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 f o r t y - f i v e s e c o n d s , a n d w i t h i n s i x t y s e c o n d s o f t h a w i n g s t a s i s was o b s e r v e d i n t h e v e n u l e s t h a t h a d b e e n f r o z e n . T h i s s t a s i s d i d n o t c h a n g e i n t h e f o l l o w i n g f i v e m i n u t e s , a n d 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 . A n i m a l 5 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t , i n t h e 2 0 0 g r a m 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 . 1 5 c c o f 0 . 6 $ n e m 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 , a n d t h e p r e p a r a t i o n was r e a d y i n s i x m i n u t e s . Two p e r c e n t p r o - c a i n e was a p p l i e d l o c a l l y o n t h e m e s o a p p e n d i x , a n d t h e 66 r a t e of c a p i l l a r y b l o o d flow was observed to be gen- e r a l l y i n c r e a s e d . Two minutes l a t e r 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. P r o c a i n e was a p p l i e d immediately a f t e r thawing, and again t h i r t y sec- onds a f t e r t h i s . F o r t y - f i v e seconds a f t e r thawing, the b l o o d flow i n the i n j u r e d area was g r e a t l y slowed, w i t h v a s o d i l a t a t i o n e s p e c i a l l y o f the venules, but onl y two and a h a l f minutes a f t e r thawing was any s t a s i s apparent, and then only i n two small c a p i l l a r i e s , w i t h a t h i r d i n v o l v e d t h i r t y seconds l a t e r . Three and a h a l f minutes a f t e r thawing the warm s a l i n e d r i p was resumed, and n i n e t y seconds l a t e r one t h i r d of the i n j u r e d area was i n v a s c u l a r s t a s i s w i t h i n c r e a s e d vasomotion observed i n the remain- der o f the v e s s e l s i n the f r o z e n a f e a . T h i r t y seconds l a t e r the d r i p was stopped and pr o c a i n e was f l u s h e d over the mesoappendix, w i t h i n c r e a s e d b l o o d f l o w r e s u l t i n g , and only one small area remained s t a t i c . Two minutes afterward the warm s a l i n e d r i p was resumed, w i t h i n c r e a s e d slowing and s t a s i s , which, i n a l l except one l a r g e connecting venule, was completely reopened by p r o c a i n e (and a l s o except i n the two above d e s c r i b e d s t a t i c a r e a s ) . By twelve minutes a f t e r the f r e e z i n g i n j u r y } t h e c i r c - u l a t i o n i n the r e s t of the mesoappendix was a p p a r e n t l y q u i t e normal, 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 6 ( C o n t r o l ) A normal a _ d u l t male Wistar r a t i n the 200 gram weight range was a n a e s t h e t i z e d w i t h 0.15 cc of 0.6$ nem- b u t a l 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 67 p r e p a r a t i o n was r e a d y w i t h i n f i v e m i n u t e s . 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 s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . F o r t w e n t y s e c o n d s a f t e r 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 a n d t h e n m a r k e d v a s o d i l a t a t i o n . B y t h r e e a n d a h a l f m i n u t e s a f t e r t h a w i n g t h e r e was s t i l l no s t a s i s e x c e p t i n one v e n o u s c h a n n e l , b u t when t h e warm R i n g e r d r i p was s t a r t e d t h e r e was s p r e a d i n g s t a s i s w i t h i n t h i r t y s e c o n d 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 . A n i m a l 7 ( T e s t ) A n o r m a l m a l e a d u l t W i s t a r r a t i n t h e 2 0 0 g r a m w e i g h t r a n g e was u s e d . I t was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 , a n d t h e p r e p a r a t i o n was r e a d y i n s i x m i n u t e s . Two p e r c e n t p r o c a i n e was f l u s h e d o v e r t h e m e s o a p p e n d i x , a n d t h e n t h e 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 - f i v e s e c o n d s . I n two a n d a h a l f m i n u t e s a f t e r t h a w i n g , s t a s i s s t a r t e d t o a p p e a r i n s m a l l v e s s e l s . One m i n u t e l a t e r t h e warm s a l i n e d r i p was r e s u m e d , w i t h s p r e a d o f t h e s t a s i s , a l t h o u g h t h e m a i n c h a n n e l s r e m a i n e d o p e n . The u n i n j u r e d a r e a s o f t h e m e s o a p p e n d i x h a d a p p a r e n t l y n o r m a l c i r c u l a t i o n . One a n d a h a l f m i n u t e s l a t e r t h e s a l i n e 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 was a p p l i e d . I n n i n e t y s e c o n d s many o f t h e c h a n n e l s o p e n e d u p a n d r e s u m e d c i r c u l a t i o n , b u t i n c r e a s e d v a s c u l a r t o n e g a v e m o r e v a s o - m o t i o n i n t h e s e c h a n n e l s . B y t e n m i n u t e s a f t e r t h a w i n g i t was n o t e d t h a t t h e g e n e r a l a r e a o f s t a s i s was n o t r e a l l y g r e a t l y a f f e c t e d , i n c o m p a r i s o n w i t h w h a t i t h a d 68 b e e n , and i n c o m p a r i s o n w i t h t h e s u r r o u n d i n g a r e a s o f t h e m e s o a p p e n d i x . A n i m a l 8 ( C o n t r o l ) A n o r m a l a d u l t m a l e 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$ n e m 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 t h e p r e p a r a t i o n was r e a d y i n f i v e m i n u t e s , w i t h warm s a l i n e s o l u t i o n d r i p p i n g on t h e m e s o a p p e n d i x . The d r i p was s t o p p e d and t h e 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 s e c o n d s . W i t h i n t h i r t y s e c o n d s o f t h a w i n g s t a s i s h a d s t a r t e d and was s p r e a d i n g r a p i d l y i n t h e i n j u r e d a r e a . Two and a h a l f m i n u t e s l a t e r t h e d r i p was resumed w h i c h c o m p l e t e d t h 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 w i t h i n t h e i n j u r e d a r e a . 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 . A n i m a l 9 ( T e s t ) A n o r m a l a d u l t m a l e 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$ n e m 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 t h e p r e p a r a t i o n was r e a d y i n s e v e n m i n u t e s , mounted, and w i t h t h e warm s a l i n e d r i p r u n n i n g . T h i s was s t o p p e d , and two p e r c e n t p r o c a i n e was i n s t i l l e d l o c a l l y o v e r t h e meso- a p p e n d i x . The c o l d p o i n t was n e x 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 s e c o n d s . The p r o c a i n e was a p p l i e d immed- i a t e l y a f t e r w a r d , and g r e a t l y i n c r e a s e d b l o o d f l o w was n o t e d . By two and a h a l f m i n u t e s a f t e r t h a w i n g , no s t a s i s was o b s e r v e d , and good v a s o m o t i o n was a p p a r e n t 69 throughout the i n j u r e d area. Procaine was again r e - a p p l i e d , and by f i v e and a h a l f minutes a f t e r thawing, there was s t i l l e x c e l l e n t blood f l o w w i t h vasomotion, but no s t a s i s had developed. Two minutes l a t e r s l i g h t c a p i l l a r y s t a s i s g e n e r a l i z e d over the i n j u r e d area was r e a d i l y o b l i t e r a t e d w i t h 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 immed- i a t e general 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 resumption of blood flow i n the whole f i e l d . The p r e p a r a t i o n was discontinued ten minutes a f t e r the i n i t i a l f r e e z i n g I n j u r y . Animal 10 (Control) A normal a d u l t male Wistar r a t i n the 200 gram weight range was used. I t was anaesthetized w i t h 0.15 cc 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 . Ten minutes l a t e r 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. Quite extensive v a s o - d i l a t a t i o n f o l l o w - ed the thawing, and w i t h i n n i n e t y seconds of thawing, s t a s i s s t a r t e d which r a p i d l y became extensive. By f o u r and a h a l f minutes f o l l o w i n g thawing the whole i n j u r e d area was i n s t a s i s except f o r one l a r g e channel. The pre p a r a t i o n was dis c o n t i n u e d f i v e and a h a l f minutes a f t e r thawing. 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 results, preparation discarded 4 Control 45" Rapid Yes Hone 5 Test 2*' 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 General when drip resumed /// 10 Control 90" Rapid 4-|' None Discussion of Results The unsuitability of plain Ringer-Locke saline solution as a perfusion drip i s readily apparent. In spite of this uncontrolled factor, i t i s f e l t that two percent procaine applied locally to the mesoappendix both before and after a standard freezing injury with the cold point for thirty seconds could be relied on to both delay the onset of stasis and reopen clogged and static vessels oneethe stasis had occurred. The average length of time required for the onset of stasis in the test animals was nearly three and a half minutes after thawing, whereas in 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 frozen c a p i l l a r y bed after application of the cold point with freezing f or t h i r t y seconds by an average of two minutes. Before freezing of the c a p i l l a r y bed, two percent procaine applied t o p i c a l l y to the mesoappendix gave greatly increased flow of blood i n a l l vessels observed, both i n v e l o c i t y and i n apparent volume of flow. This was thought to be due to a vasodila^tory e f f e c t , with the s i t e of action 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 vasoconstrictor impulses. I t was important to observe that additional t o p i c a l application of two per- cent procaine frequently aided or caused the opening up of channels i n s t a s i s , presumably through releasing vasoconstriction, and thereby allowing a greater volume of blood at a more eff e c t i v e hydrostatic and p u l s i l e pressure to f l u s h the vessel free of the sludge of c e l l s . I t was noted on resuming the drip after freezing that vascular st a s i s i n many channels developed almost immediately, but often such sta s i s could be overcome by l o c a l application of two percent procaine. I t was thought the p l a i n saline drip solution used aided the transfer of the l i q u i d component of the blood through the damaged c a p i l l a r y walls, and hence increased the sludging of c e l l s i n the vessels. Repeat Experiments with Procaine Hydrochloride In t h i s instance, the above t r i a l s were repeated 7 2 u s i n g a R i n g e r - L o c k e s a l i n e s o l u t i o n m o d i f i e d w i t h g e l a t i n a n d g l u c o s e . A summary o f t h e f o l l o w i n g r e - s u l t s i s g i v e n i n t a b l e f o r m a f t e r A n i m a l 1 7 . A n i m a l 11 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 3 0 0 g r a m 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 . 2 0 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y a n d t h e p r e p a r a t i o n was r e a d y w i t h i n t w e n t y - e i g h t m i n u t e s . P r o c a i n e two p e r c e n t was f l u s h e d o v e r t h e m e s o a p p e n d i x a n d t h e n t h e 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 s e c o n d s . The p r e p a r a t i o n h a d t o b e d i s c a r d e d b e c a u s e i t was n o t 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 . T h e ^ r a t c c . l & w e d i t s i n t e s t i n e s , a n d r o l l e d o f f t h e m i c r o s c o p e s t a g e t h r e e o r f o u r t i m e s , w i t h g e n e r a l v a s c u l a r s t a s i s t h r o u g h o u t t h e m e s o a p p e n d i x , m a k i n g e v a l u a t i o n i m p o s s i b l e . A n i m a l 1 2 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 3 0 0 g r a m 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 . 2 0 c c o f 0 . 6 $ n e m 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 m u s c u l a r l y , s u p p o s - e d l y . T w e n t y m i n u t e s l a t e r 0 . 1 0 c c m o r e n e m b u t a l w a s g i v e n , a n d t h e a n i m a l was r e a d y i n f o u r m i n u t e s . The R i n g e r - l o c k e g e l a t i n e - g l u c o s e d r i p was d i s c o n t i n u e d a n d two p e r c e n t p r o c a i n e was f l u s h e d o v e r t h e m e s o a p p e n d i x . 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 s e c o n d s . 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 a f t e r , tt a t w h i c h t i m e t h e v e s s e l s w e r e c l o s e d t i g h t a n d e m p t y o f c e l l s . W i t h i n s i x t y s e c o n d s o f t h a w i n g , a f e w b l o o d 73 cel ls were flowing through. Procaine was reapplied and by three minutes of thawing ninety percent of the vessels in the injured area were open and functioning. Restarting the gelatine-glucose drip did not effect any further stasis . Further tests were performed with this prep- aration. Thirty seconds after the drip was restarted the injured area was about ninety percent normal with a few small capi l laries in stasis. Sixty seconds later two percent procaine was applied. This put the whole area into stasis, which recovered entirely with resump- tion of the warm drip, but shortly after, stasis re- developed in the whole injured area. Further procaine gave only a l i t t l e restoration of c irculat ion which was aided by resuming flow of the drip. This was a very interesting preparation to watch, and i t showA that the Ringer-Locke drip containing gelatine and glucose is much more physiological. Note that the procaine was an entirely aqueous solution, and not a saline one. Animal 13 (Control) A normal adult male Wistar rat in the 300 gram weight range was anaesthetized with 0.20 cc of 0.6$ nembutal solution administered intramuscularly and the preparation was ready in twelve minutes, with the warm drip solution running over the mesoappendix. The cold point was applied to a selected area with freezing for thirty seconds. Within thirty seconds of thawing there was much vascular stasis, and within two and a half min- 74 u t e s o f t h a w i n g t h e w h o l e i n j u r e d a r e a was i n c o m p l e t e a n d i r r e v e r s i b l e s t a s i s e x c e p t f o r two o r t h r e e m a i n l a r g e c h a n n e l s . 1 A n i m a l 14 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 3 0 0 g r a m 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 . 2 0 c c o f 0 . 6 $ n e m 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 m u s c u l a r l y . The p r e p a r a t i o n was r e a d y i n t h i r t y - o n e m i n u t e s , b u t t h e a n i m a l was d i s c a r d e d b e c a u s e t h e w h o l e m e s e n t e r y was i n v a s c u l a r s t a s i s f r o m t h e a n i m a l ' s k i c k i n g . A n i m a l 1 5 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 3 0 0 g r a m 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 . 3 0 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n , a n d was n o t r e a d y b e c a u s e t h e a n - a e s t h e t i c was n o t d e e p e n o u g h . The i n t r a m u s c u l a r r o u t e was r e g a r d e d a s i m p r a c t i c a l . A n i m a l 1 6 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t i n t h e 3 0 0 g r a m 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 . 2 0 c c o f 0 . 6 $ n e m 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 , a n d t h e p r e p a r a t i o n was r e a d y s e v e n m i n u t e s l a t e r . P r o c a i n e was f l u s h e d o v e r t h e m e s o a p p e n d i x a f t e r s t o p p i n g t h e d r i p a n d t h e c o l d p o i n t was a p p l i e d t o t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . T h i r t y s e c o n d s a f t e r t h a w i n g t h e r e w a s c o m p l e t e s t o p p a g e o f c i r c u l a t i o n i n t h e i n j u r e d a r e a . P r o c a i n e was a p p l i e d t w i c e w i t h n o e f f e c t . When t h e warm g e l a t i n e - g l u c o s e s a l i n e d r i p was r e s t a r t e d two m i n u t e s a f t e r t h a w i n g , one h a l f o f t h e s t a t i c c h a n n - 75 els opened, and i n another minute s i x t y - f i v e percent were funct ion ing . In one and a h a l f minutes procaine gave stoppage of flow. When the warm drip was re s tar ted f i f t e e n seconds l a t e r the whole in jured area was flowing one hundred percent, although two small ecchymotic areas were noted. Two minutes l a t e r procaine stopped blood flow i n the in jured area, and resumption of the dr ip res tar ted i t completely. Animal 17 (Control) A normal adult male" Wis tar r a t i n the 300 gram weight range was anaesthetized with 0.20 cc of 0.6$ nem- buta l so lut ion administered i n t r a p e r i t o n e a l l y , and i n eight minutes the preparat ion was ready with the d r i p f lowing. The cold point was appl ied with freez ing for t h i r t y seconds, and t h i r t y seconds af ter thawing there was general small c a p i l l a r y s tas i s throughout the i n j u r e d area. By four minutes a f ter thawing the s ta s i s was spreading to larger channels, and i n another minute the s tas i s was general and near ly complete. Two and a h a l f minutes l a t e r procaine was appl ied and a very marked recovery occurred, although i t was incomplete. There was increased vasomotion, rather than any vasospasm. Three minutes l a t e r s tas i s returned and the preparat ion wasdiscontinued. 76 Summary o f R e s u l t s w i t h P r o c a i n e N o . O n s e t S t a s i s S p r e a d G e n e r a l i z e d F l o w R e s u m e d 11 T e s t No r e s u l t s b e c a u s e o f g e n e r a l s t a s i s 1 2 T e s t M i n i m a l M i n i m a l None / / ' / ' 1 3 C o n t r o l 3 0 " R a p i d 2&» None 14 T e s t No r e s u l t s b e c a u s e o f g e n e r a l s t a s i s 1 5 T e s t No r e s u l t s b e c a u s e o f g e n e r a l s t a s i s 1 6 T e s t U n u s u a l / / / 17 C o n t r o l 3 0 " I m m e d i a t e 3 0 " None C o n c l u s i o n s T h e s e a d d i t i o n a l r e s u l t s w i t h l o c a l l y a p p l i e d p r o c a i n e a r e t o o v a r i a b l e a n d t o o f e w t o b e o f a n y s i g n i f i c a n c e . The v a l u e o f a d d i n g g e l a t i n e a n d g l u c o s e t o t h e R i n g e r - L o c k e s o l u t i o n was a p p a r e n t . I n t h e c a s e o f a n i m a l 1 6 , some v a l u e i n i m m e d - i a t e w a r m i n g ( o r t h a w i n g ) o f t h e f r o z e n a r e a w i t h warm d r i p f l u i d was s u g g e s t e d . F u r t h e r O b s e r v a t i o n s w i t h P r o c a i n e ( P a r e n t e r a l ) I n t h i s r u n , p r o c a i n e was u s e d i n t h e r a t h e r h i g h d o s a g e o f 2 0 0 mg p e r k i l o g r a m b o d y w e i g h t , a d m i n i s t e r e d p a r e n t e r a l l y . N o t e t h a t t h e f o l l o w i n g t e s t s w e r e p e r - f o r m e d b y a s e n i o r m e d i c a l s t u d e n t who was h e l p i n g w i t h t h e p r o j e c t o n a p a r t t i m e b a s i s . H i s m e t h o d s a n d o b s e r v a t i o n s c o u l d n o t be i d e n t i c a l w i t h t h o s e u s e d e l s e w h e r e . H i s u s e o f t h e t e r m " g e n e r a l s t a s i s " i s h e r e u s e d t o i m p l y s t a s i s t h r o u g h o u t t h e 77 v e s s e l s o f t h e w h o l e m e s o a p p e n d i x , r a t h e r t h a n m e r e l y i n t h e i n j u r e d a r e a a s i s i m p l i e d e l s e w h e r e . A n i m a l 1 ( T e s t ) A n o r m a l a d u l t m a l e H o o d e d r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 6 c c o f t w o p e r c e n t p r o c a i n e , a n d i n t e n m i n u t e s t h e a n i m a l was 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 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n . The p r e p a r a - t i o n was m o u n t e d o n t h e m i c r o s c o p e s t a g e a n d t h e 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 a r e a o f t h e m e s o - a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s f o l l o w e d i m m e d i a t e l y b y r a p i d t h a w i n g . S t a s i s b e g a n w i t h i n o n e m i n u t e a f t e r t h a w i n g , a n d i t s p r e a d t o i n v o l v e a b o u t one q u a r t e r o f t h e c a p i l l a r i e s i n t h e i n j u r e d a r e a . A n i m a l 2 ( C o n t r o l ) A n o r m a l a d u l t f e m a l e n o n p r e g n a n t H o o d e d r a t w e i g h i n g 1 8 0 g r a m s was 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 $ n e m b u t a l s o l u t i o n b y s u b c u t a n e o u s i n j e c t i o n . The a n i m a l e x p i r e d b e f o r e a n y o b s e r v a t i o n s c o u l d b e m a d e . A n i m a l 5 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e H o o d e d r a t w e i g h i n g 1 7 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 7 c c o f two p e r c e n t p r o c a i n e . The a n i m a l e x p i r e d b e f o r e a n y o b s e r v a t i o n s c o u l d b e m a d e . A n i m a l 4 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e H o o d e d r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 6 c c o f a two p e r e e n t p r o c a i n e s o l u t i o n . I n f i v e 7 8 m i n u t e s i t was a n a e s t h e t i z e d w i t h 0 . 0 7 c c o f 0 . 6 $ n e m - 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 b y s u b c u t a n e o u s i n j e c t i o n , a n d when r e a d y t h e p r e p a r a t i o n was m o u n t e d o n t h e m i c r o - s c o p e s t a g e . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c - o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g t h r o u g h r e - s u m p t i o n o f t h e warm R i n g e r - L o c k e g e l a t i n e - g l u c o s e d r i p . W i t h i n t h i r t y s e c o n d s o f t h a w i n g s t a s i s was o b s e r v e d , w h i c h s p r e a d t o i n v o l v e h a l f o f t h e v e s s e l s i n t h e i n j - u r e d a r e a i n t h r e e m i n u t e s o f t h a w i n g . N o t e t h a t a r t - e r i o l e s a n d m e t a r t e r i o l e s i n t h e i n j u r e d a r e a d i d r jo t d e v e l o p s t a s i s , b u t t h e c a p i l l a r i e s a n d t h e c o l l e c t i n g v e n u l e s w e r e w i d e l y a f f e c t e d . R e s u m p t i o n o f f l o w was s e e n i n o n e m e d i u m s i z e d v e n u l e . T h e r e was no g e n e r a l ( s e e d e f i n i t i o n a b o v e ) s t a s i s i n t h e m e s o a p p e n d i x . A n i m a l 5 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e H o o d e d r a t w e i g h i n g 1 7 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 0 8 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n , a n d when r e a d y t h e p r e p a r a t i o n -was m o u n t e d o n t h e m i c r o s c o p e s t a g e . 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g t h r o u g h r e s u m p t i o n o f t h e w a r m d r i p s o l u t i o n . S t a s i s d e v e l o p e d i n f o r t y - f i v e s e c o n d s a f t e r t h a w i n g a n d i t s p r e a d t o i n v o l v e n e a r l y a l l t h e v e s s e l s i n t h e i n j u r e d a r e a b y f o u r m i n u t e s o f t h a w i n g . T h e r e was n o g e n e r a l s t a s i s n o t e d o r r e s u m p t i o n o f f l o w . 79 A n i m a l 6 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 6 c c o f two p e r c e n t p r o c a i n e , a n d t w e n t y - f i v e m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n a l s o a d m i n i s t e r e d b y s u b c u t a n e o u s i n j e c t i o n . The p r e p a r a t i o n was m o u n t e d o n t h e m i c r o - s c o p e a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . S t a s i s d e v e l o p e d w i t h i n t h r e e m i n u t e s o f t h a w i n g , a n d s p r e a d t o i n v o l v e a b o u t o n e t e n t h o f t h e v e s s e l s i n t h e i n j u r e d a r e a . T h e r e was n o r e s u m p t i o n o f f l o w n o t e d o r a n y g e n e r a l s t a s i s . A n i m a l 7 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 5 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 0 9 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y , a n d t h e p r e p a r a t i o n was m o u n t e d o n t h e m i c r o s c o p e . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . S t a s i s d e v e l o p e d i n t h i r t y s e c o n d s o f t h a w i n g , w i t h s p r e a d t o i n v o l v e one q u a r t e r o f t h e v e s s e l s i n t h e i n j u r e d a r e a i n f i v e m i n - u t e s . T h e r e was n o g e n e r a l s t a s i s a n d n o r e s u m p t i o n o f f l o w n o t e d . A n i m a l 8 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 6 c c o f two p e r c e n t p r o c a i n e , a n d i n s e v e n t y - f i v e m i n u t e s 80 i t was 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 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n . The p r e p a r a t i o n was m o u n t e d a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . S t a s i s d e v e l o p e d i n one m i n u t e , s p r e a d - i n g t o i n v o l v e s e v e n - e i g h t h s o f t h e v e s s e l s i n t h e i n j u r e d a r e a . N o r e s u m p t i o n o f f l o w o r g e n e r a l s t a s i s was o b s e r v e d . A n i m a l 9 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 7 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y . 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 a r e a o f t h e m e s o - a p p e n d i x w i t h f r e e z i n g , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . S t a s i s b e g a n i n f o r t y - f i v e s e c o n d s a f t e r t h a w i n g , a n d s p r e a d t o i n v o l v e two t h i r d s o f t h e v e s s e l s i n t h e i n j u r e d a r e a . No g e n e r a l s t a s i s o r r e s u m p t i o n o f f l o w was o b s e r v e d . A n i m a l 10 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 6 c c o f two p e r c e n t p r o c a i n e , a n d i n t w e n t y - f i v e m i n u t e s t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 0 9 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n . 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . S t a s i s commenced w i t h i n f o r t y s e c o n d s o f t h a w i n g , s p r e a d i n g t o i n v o l v e f o u r s h o r t s e g m e n t s o f c a p i l l a r y 8 1 i n t h e i n j u r e d a r e a ( a v e r y s l i g h t e f f e c t ) . A n i m a l 11 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 5 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y . 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g t h r o u g h r e s u m p t i o n o f t h e warm d r i p . S t a s i s s t a r t e d i n t h i r t y s e c o n d s a f t e r t h a w - i n g , a n d s p r e a d t o i n v o l v e n e a r l y a l l t h e v e s s e l s i n t h e f r o z e n a r e a b y t h r e e m i n u t e s o f t h a w i n g . A n i m a l 1 2 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 7 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 1 . 7 c c o f two p e r c e n t p r o c a i n e , a n d f i f t y - f i v e m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n a l s o a d m i n i s t e r e d b y s u b c u t a n e o u s i n j e c t i o n . 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . S t a s i s commenced i n f i f t y s e c o n d s a f t e r t h a w i n g , a n d s p r e a d t o i n v o l v e a l m o s t h a l f o f t h e v e s s e l s i n t h e i n j u r e d a r e a i n a p p r o x i m a t e l y f i v e m i n u t e s . A n i m a l 1 5 ( C o n t r o l ) A n o r m a l a d u l t , n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 6 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y . 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 a r e a o f t h e 82 m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g t h r o u g h r e s u m p t i o n o f t h e warm d r i p . S t a s i s commenced i n t h i r t y s e c o n d s a f t e r t h a w i n g , s p r e a d i n g t o i n v o l v e t h r e e q u a r t e r s o f t h e v e s s e l s i n t h e i n j u r e d a r e a i n t h r e e m i n u t e s a f t e r t h a w i n g . A n i m a l 1 4 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t H o o d e d f e m a l e r a t w e i g h i n g 1 8 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 0 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n , a n d w h e n r e a d y t h e 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d - i a t e r a p i d t h a w i n g t h r o u g h r e s u m p t i o n o f t h e wa rm d r i p . S t a s i s commenced i n t h i r t y s e c o n d s a f t e r t h a w i n g , a n d s p r e a d t o i n v o l v e a b o u t h a l f t h e v e s s e l s i n t h e i n j u r e d a r e a i n f o u r m i n u t e s . Summary o f R e s u l t s w i t h P a r e n t e r a l P r o c a i n e M o . O n s e t S t a s i s S p r e a d P i n a l E x t e n t P l o w R e s u m e d 1 T e s t 6 0 " S l o w 2 5 $ N o n e 2 C o n t r o l D i e d , no r e s u l t s 3 T e s t D i e d , n o r e s u l t s 4 T e s t 3 0 " S l o w 5 0 $ Some 5 C o n t r o l 4 5 " S l o w 9 5 $ i n 4 » None 6 T e s t 1 8 0 " 1 0 $ None 7 C o n t r o l 3 0 " " 2 5 $ i n 5 ' N o n e 8 T e s t 6 0 " 7 / 8 o f v e s s e l s N o n e 9 C o n t r o l 4 5 " 2 / 3 o f v e s s e l s None 8 3 Summary C o n t ' d N o . O n s e t S t a s i s S p r e a d P i n a l E x t e n t F l o w R e s u m e d 10 T e s t 4 0 " 4 c a p i l l a r i e s None 11 C o n t r o l 3 0 " A l m o s t a l l i n 3« None 1 2 T e s t 5 0 " 5 0 $ i n 5» None 1 3 C o n t r o l 3 0 " 7 5 $ i n 3» None 1 4 C o n t r o l 3 0 " 5 0 $ i n 4 « None I m p r e s s i o n o f R e s u l t s "The p r o c a i n e t r e a t e d a n i m a l s s e e m e d t o b e b e t t e r p r o t e c t e d a g a i n s t s t a s i s p r o d u c e d b y f r e e z i n g i n j u r y a s p e r f o r m e d . H o w e v e r , d i f f e r e n c e s f r o m c o n t r o l o b s e r v a t i o n s w e r e s l i g h t a n d i t was d i f f i c u l t t o s a y i f t h e y a r e s i g n i f i c a n t . " S e e d i s c u s s i o n o n p a g e 3 1 1 . P r i s c o l i n e E x p e r i m e n t a l o b s e r v a t i o n s w i t h P r i s c o l i n e T h i s d r u g was t e s t e d p a r e n t e r a l l y i n a d o s a g e o f 1 . 0 mg P r i s c o l i n e p e r k i l o g r a m b o d y w e i g h t . A summary o f t h e f o l l o w i n g r e s u l t s i s g i v e n i n t a b l e f o r m a f t e r A n i m a l 9 . A n i m a l 1 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 7 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 1 7 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0 . 1 7 m g . , a n d t w e n t y - f o u r m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n . The p r e p a r a t i o n 84 was r e a d y I n n i n e m i n u t e s a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . W i t h i n t h i r t y s e c o n d s o f t h a w i n g s t a s i s s t a r t e d i n s e v e r a l v e n u l e s . A m i n u t e l a t e r a f e w v e n u l e s r e o p e n e d , a n d b y a n o t h e r m i n u t e t h e r e o p e n - i n g o f t h e s t a t i c v e n u l e s was r a t h e r g o o d . S t a s i s s t o p p e d e x t e n d i n g t o i n v o l v e new v e s s e l s w i t h i n t h r e e a n d a h a l f m i n u t e s o f t h a w i n g . A m i n u t e l a t e r v e s s e l s w e r e o b s e r v e d t o c o n t i n u e t o s h u t down a n d r e s t a r t b l o o d f l o w , c e r t a i n l y t o a f a r g r e a t e r e x t e n t t h a n a n y o t h e r n o r m a l c o n t r o l s s o f a r t e s t e d d u r i n g t h i s p r o j e c 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 s e v e n m i n u t e s l a t e r w i t h n o f u r t h e r c h a n g e n o t e d . A n i m a l 2 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 7 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 1 7 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0 . 1 7 m g , a n d s i x t e e n m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n , w i t h 0 . 0 5 c c m o r e b e i n g i n j e c t e d e l e v e n m i n u t e s l a t e r . The p r e p a r a t i o n was r e a d y i n t w e l v e m i n u t e s a f t e r t h e f i r s t d o s e o f n e m b u t a l a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o - a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g w i t h t h e warm d r i p s o l u t i o n . W i t h i n t h i r t y s e c o n d s o f t h a w i n g t h e r e h a d d e v e l o p e d t h r e e e c c h y m o t i c a r e a s . A m i n u t e l a t e r s t a s i s w as n o t e d 85 i n two c a p i l l a r y b e d s . I n a n o t h e r m i n u t e s e v e r a l s h o r t v e n u l e s w e r e s t a t i c , a n d w i t h i n s i x t y s e c o n d s m o r e , s t a s i s was g e n e r a l t h r o u g h o u t t h e w h o l e o f t h e i n j u r e d a r e a . 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 . B y a n - o t h e r f o u r m i n u t e s a f e w v e s s e l s h a d r e o p e n e d . Two m i n u t e s l a t e r m u c h o f t h e s t a s i s h a d b e e n o v e r c o m e e x - c e p t i n t h e s m a l l e r c h a n n e l s . I n f o u r m i n u t e s t h e a n i m a l was a g a i n b e c o m m i n g a p n o e i c , w i t h s l o w i n g o f t h e b l o o d . f l o w a n d s t a s i s . P o u r m i n u t e s l a t e r t h e a n i m a l 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 o f t h e 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 t w e n t y m i n u t e s a f t e r t h a w - i n g , i n w h a t was r e g a r d e d a s a C h e y n e - S t o k e s t y p e o f r e s p i r a t i o n . A n i m a l 5 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 6 5 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y , a n d t h e p r e p a r a t i o n was r e a d y i n t w e n t y - s i x m i n u t e s . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o - a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g w i t h t h e wa rm d r i p s o l u t i o n . V a s c u l a r s t a s i s commenced f o r t y - f i v e s e c o n d s a f t e r t h a w - i n g , a n d i n f i f t e e n s e c o n d s a n o t h e r c a p i l l a r y b e d was i n v o l v e d . B y s i x t y s e c o n d s m o r e , s e v e r a l a r e a s w e r e s t a t i c , a n d t h e p r o c e s s s l o w l y s p r e a d . I n t h r e e m i n u t e s o n l y t h e a r t e r i o - v e n o u s s h u n t s w e r e o p e n , a n d no r e - s u m p t i o n o f a n y c i r c u l a t i o n w.as n o t e d . 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 e i g h t m i n u t e s a f t e r t h a w i n g . 86 A n i m a l 4 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 9 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 1 9 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0 . 1 9 m g . , a n d t w e n t y - t w o m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n . The p r e p a r a t i o n was r e a d y i n f i f t e e n a n d a h a l f m i n u t e s , a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . S t a s i s s t a r t e d w i t h - i n t h i r t y s e c o n d s o f t h a w i n g a n d s p r e a d r a p i d l y . B y n i n e t y s e c o n d s a f t e r t h a w i n g t h e s t a s i s became f a i r l y g e n e r a l i z e d o v e r t h e i n j u r e d a r e a . No r e s u m p t i o n i n b l o o d f l o w was n o t e d . W i t h i n f i v e a n d a h a l f m i n u t e s a f t e r t h a w i n g n o f u r t h e r c h a n g e was o b s e r v e d , a n d 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 . A n i m a l 5 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 6 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y . E l e v e n m i n u t e s l a t e r 0 . 0 5 c c w e r e r e p e a t e d , a n d t h e p r e p a r a t i o n was r e a d y o n t h e m i c r o s c o p e s t a g e i n f o u r m o r e m i n u t e s . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . W i t h i n t h i r t y s e c o n d s s t a s i s s t a r t e d , w h i c h s p r e a d r a p i d l y . B y a n o t h e r two m i n u t e s t h e s t a s i s h a d become g e n e r a l i z e d w i t h i n t h e i n j u r e d a r e a . B y s e v e n m i n u t e s a f t e r t h a w - 87 i n g n o f u r t h e r c h a n g e was n o t e d , a n d 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 . No r e s u m p t i o n o f a n y c i r c u l a t i o n was n o t e d . A n i m a l 6 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 2 2 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n a n d i n s e v e n m i n u t e s 0 . 0 5 c c w e r e r e p e a t e d . The p r e p a r a t i o n was r e a d y o n t h e m i c r o - s c o p e s t a g e t w e l v e m i n u t e s l a t e r , a n d t h e c o l d t i p w as a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . W i t h i n t h i r t y s e c o n d s o f t h a w i n g j s t a s i s d e v e l o p e d i n two c a p i l l a r y b e d s , a n d t h e p r o c e s s s p r e a d d u r i n g t h e f o l l o w i n g s i x t y s e c o n d s . T h i r t y s e c o n d s l a t e r s e v e r a l a r e a s t h r o u g h o u t t h e i n j - u r e d a r e a w e r e i n v a s c u l a r s t a s i s . No r e s u m p t i o n o f c i r c u l a t i o n was n o t e d , a n d 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 s e v e n m i n u t e s a f t e r t h a w i n g w i t h no f u r t h e r c h a n g e s b e i n g o b s e r v e d . A n i m a l 7 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 7 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 1 7 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0 . 1 7 m g . , a n d i n n i n e t e e n m i n u t e s t h e a n i m a l was a n - a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n . The p r e p a r a t i o n was r e a d y i n n i n e m i n u t e s , a n d t h e 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . C a p i l l a r y s t a s i s 88 d e v e l o p e d w i t h i n t h i r t y s e c o n d s o f t h a w i n g i n two c a p i l l a r y b e d s . B y a n o t h e r n i n e t y s e c o n d s s e v e r a l b e d s h a d become s t a t i c . B y a n o t h e r two m i n u t e s n o r e s u m p t i o n o f a n y c i r c u l a t i o n h a d o c c u r r e d , a n d 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 t e n m i n u t e s a f t e r t h a w i n g , w i t h n o f u r t h e r c h a n g e s b e i n g n o t e d . A n i m a l 8 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 6 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 1 6 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0 . 1 6 m g . , a n d t w e n t y - e i g h t m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n The p r e p a r a t i o n was r e a d y o n t h e m i c r o s c o p e s t a g e i n t w e l v e m i n u t e s , a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . V a s c u l a r s t a s i s s t a r t e d s i x t y s e c o n d s a f t e r t h a w i n g , a n d b y a n o t h e r s i x t y s e c o n d s t h r e e c a p i l l a r y b e d s a n d one l a r g e v e n o u s c h a n n e l w e r e a f f e c t e d . A m i n u t e l a t e r t h e v e n o u s c h a n n e l f l u s h e d f r e e . B y s i x m o r e m i n u t e s a f e w m o r e s m a l l v e n u l e s became s t a t i c , a n d 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 t e n m i n u t e s a f t e r t h a w i n g . A n i m a l 9 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 7 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y , a n d i n t e n m i n u t e s 0 . 0 5 c c w e r e r e p e a t e d . The p r e p a r a - t i o n was m o u n t e d a n d r e a d y o n t h e m i c r o s c o p e s t a g e i n 89 s i x m i n u t e s , a n d t h e c o l d t i p was a p p l i e d t o a s e l - e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . E x t e n s - i v e v a s c u l a r s t a s i s d e v e l o p e d t h r o u g h o u t t h e i n j u r e d a r e a w i t h i n t h i r t y s e c o n d s o f t h a w i n g . B y a n o t h e r s i x t y s e c o n d s t h e r e was s t i l l f a i r l y g o o d b l o o d f l o w , b u t n o r e s t o r a t i o n o f a n y o f t h e c i r c u l a t i o n . . The a n i m a l d i e d w h i l e b e i n g o b s e r v e d f o u r a n d a h a l f m i n u t e s a f t e r t h a w i n g . I t w o u l d seem t h a t s u c h a p r e p a r a t i o n c o u l d n o t b e c o n s i d e r e d p h y s i o l o g i c a l , a n d t h e o b s e r v - a t i o n s a r e n o t i n c l u d e d i n t h e summary t a b l e . Summary o f R e s u l t s w i t h P r i s c o l i n e N o . O n s e t S t a s i s S p r e a d G e n e r a l i z e d F l o w R e s u m e d 1 T e s t 30*' S l i g h t f o r 3&» None /// 2 T e s t 9 0 " R a p i d l y p i t t 3 C o n t r o l 45" S l o w l y 3» N o n e 4 T e s t 3 0 " R a p i d l y 9 0 " N o n e 5 C o n t r o l 3 0 " R a p i d l y N o n e 6 C o n t r o l 3 0 " R a p i d l y 2» None 7 T e s t 3 0 " S l o w l y 2» N o n e 8 T e s t 6 0 " S l o w l y None t 9 C o n t r o l 3 0 " R a p i d l y 3 0 " None ( P h y s i o l o g i c a l S t a t e U n l i k e l y ) Comments T$ie a v e r a g e t i m e w i t h t h e t e s t a n i m a l s f o r s t a s i s t o o c c u r a f t e r r a p i d t h a w i n g was 48»», w h e r e a s t h e same v a l u e w i t h t h e c o n t r o l s was 34". T h e r e was l i t t l e d i f f e r e n c e b e t w e e n t h e t e s t r u n s a n d t h e c o n t r o l s i n 90 t h e l e n g t h o f t i m e i t t o o k f o r 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 t o d e v e l o p i n t h e i n j u r e d a r e a . R e s u m p t i o n o f c i r c u l a t i o n a f t e r s t a s i s h a d d e v e l o p e d o c c u r r e d o n l y w i t h t h e t e s t a n i m a l s , b u t n o t c o n s i s t e n t l y s o . I t w o u l d seem t h a t P r i s c o l i n e was o f n o s i g n i f i c a n t v a l u e I n d e l a y i n g t h e o n s e t o f v a s c u l a r s t a s i s , o r i n g e n e r - a l l y p r o t e c t i n g t h e v a s c u l a r b e d . F u r t h e r O b s e r v a t i o n s w i t h P r i s c o l i n e I n t h e f o l l o w i n g t e s t s , P r i s c o l i n e was t e s t e d p a r e n t e r a l l y i n a d o s a g e o f 2 . 0 mg p e r k i l o g r a m b o d y w e i g h t . A summary o f t h e f o l l o w i n g r e s u l t s i s g i v e n i n t a b l e f o r m a f t e r A n i m a l 14. N o t e t h a t t h e s e t e s t s w e r e p e r f o r m e d b y a s e n i o r m e d i c a l s t u d e n t who was h e l p - i n g w i t h t h e p r o j e c t o n a p a r t t i m e b a s i s . H i s m e t h o d s a n d o b s e r v a t i o n s c o u l d n o t b e i d e n t i c a l w i t h t h o s e u s e d e l s e w h e r e . H i s u s e o f t h e t e r m " g e n e r a l : i s t a s i s " i s h e r e u s e d t o i m p l y s t a s i s t h r o u g h o u t t h e v e s s e l s o f t h e w h o l e m e s o a p p e n d i x , r a t h e r t h a n m e r e l y w i t h i n t h e i n j u r e d a r e a as i s i m p l i e d e l s e w h e r e . F o u r t e e n a n i m a l s w e r e e m p l o y e d , a l l b e i n g n o r m a l a d u l t m a l e W i s t a r r a t s i n t h e 2 0 0 g r a m w e i g h t r a n g e . When r e a d y a f t e r a n a e s t h e t i z i n g w i t h a s t a t e d a m o u n t o f 0 . 6 $ n e m b u t a l s o l u t i o n , t h e a n i m a l was m o u n t e d o n t h e m i c r o s c o p e s t a g e , a n d t h e 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g e f f e c t e d b y r e s u m p t i o n o f t h e warm R i n g e r - L o c k e g e l a t i n - 91 g l u c o s e d r i p s o l u t i o n . The r e a c t i o n s o f t h e c i r c - u l a t o r y c h a n n e l s w e r e n o t e d a s o u t l i n e d b e l o w : A n i m a l 1 ( T e s t ) W e i g h t o f a n i m a l was 1 9 0 g r a m s . I t w a s i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 2 c c o f a d i l u t e d P r i s c o l i n e s o l - u t i o n c o n t a i n i n g 0.4 m g . T h i r t e e n m i n u t e s l a t e r 0 . 2 c c o f n e m b u t a l w e r e i n j e c t e d s u b c u t a n e o u s l y , b u t t h e a n i m a l e x p i r e d b e f o r e a n y t e s t i n g c o u l d b e d o n e . A n i m a l 2 ( T e s t ) A n a n i m a l w e i g h i n g 2 0 0 g r a m s w as i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0.4 m g . , a n d e i g h t m i n u t e s l a t e r i t was a n - a e s t h e t i z e d w i t h 0 . 2 c c o f n e m b u t a l . V a s c u l a r s t a s i s s t a r t e d s i x t y s e c o n d s a f t e r t h e t h a w i n g , w h i c h s p r e a d t o i n v o l v e f i v e o r s i x o t h e r c a p i l l a r y f i e l d s w i t h i n f o u r m i n u t e s . T h e r e was no g e n e r a l s t a s i s o r r e s u m p t i o n o f c i r c u l a t i o n n o t e d . A n i m a l 3 ( C o n t r o l ) A n a n i m a l w e i g h i n g 2 0 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l i n j e c t e d s u b c u t a n e o u s l y . S t a s i s s t a r t e d i n t h i r t y s e c o n d s a f t e r t h a w i n g , s p r e a d - i n g t o i n v o l v e t h r e e q u a r t e r s o f t h e c a p i l l a r i e s i n t h e i n j u r e d f i e l d i n f i v e m i n u t e s . T h e r e w as n o g e n e r a l s t a s i s o r r e s u m p t i o n o f f l o w n o t e d i n a n y v e s s e l . A n i m a l . 4 ( T e s t ) A n a n i m a l w e i g h i n g 1 9 0 g r a m s w as i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 0 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n c o n t a i n i n g 0.4 m g . , a n d t e n m i n u t e s l a t e r i t was a n a e s t h - 9 2 e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l . S t a s i s b e g a n n i n e t y s e c o n d s a f t e r t h a w i n g , w i t h s p r e a d t o i n v o l v e h a l f t h e c a p i l l a r i e s i n t h e i n j u r e d a r e a . T h e r e was no g e n e r a l s t a s i s o r r e s u m p t i o n o f c i r c u l a t i o n . A n i m a l 5 ( C o n t r o l ) A n a n i m a l w e i g h i n g 1 9 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l . S t a s i s d e v e l o p e d w i t h i n t w e n t y s e c o n d s a f t e r t h a w i n g , s p r e a d i n g t o i n v o l v e t h r e e q u a r t e r s o f t h e c a p i l l a r i e s i n t h e f r o z e n a r e a i n f o u r m i n u t e s . T h e r e was no g e n e r a l s t a s i s , a n d n o r e s u m p t i o n o f f l o w n o t e d . A n i m a l 6 ( T e s t ) A n a n i m a l w e i g h i n g 2 0 0 g r a m s was i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 0 c c o f a d i l u t e d s o l u t i o n o f P r i s c o l i n e c o n t a i n i n g 0 . 4 m g . , a n d t w e n t y m i n u t e s l a t e r i t was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l . No s t a s i s was o b s e r v e d f o r two m i n u t e s f o l l o w i n g t h a w i n g , a t w h i c h t i m e t w o s m a l l c a p i l l a r i e s became s t a t i c . T h e r e was n o s p r e a d o f t h e s t a s i s , n o g e n e r a l i z e d s t a s i s , a n d n o r e s u m p t i o n o f c i r c u l a t i o n i n t h e a f f e c t e d c h a n n e l s . A n i m a l 7 ( C o n t r o l ) A n 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 2 0 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l , b u t i t e x p i r e d b e f o r e i t c o u l d b e m o u n t e d o n t h e m i c r o s c o p e s t a g e . A n i m a l 8 ( T e s t ) A n a n i m a l w e i g h i n g 2 1 0 g r a m s was i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 0 c c o f a d i l u t e d P r i s c o l i n e s o l u t i o n 93 c o n t a i n i n g 0.4 m g . , a n d t w e l v e m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l a l s o a d m i n i s t e r e d s u b c u t a n e o u s l y . V a s c u l a r s t a s i s d e v e l o p e d i n n i n e t y s e c o n d s a f t e r t h a w i n g , t o i n v o l v e a b o u t o n e q u a r t e r o f t h e c a p i l l a r i e s i n t h e i n j u r e d a r e a . No g e n e r a l s t a s i s a n d n o r e s u m p t i o n o f c i r c u l a t i o n was n o t e d . A n i m a l 9 ( C o n t r o l ) A n a n i m a l w e i g h i n g 2 1 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 1 7 c c o f n e m b u t a l a d m i n i s t e r e d b y s u b c u t a n e o u s i n j e c t i o n , a n d t w e n t y m i n u t e s l a t e r 0 . 0 7 c c w e r e r e - p e a t e d . S t a s i s s t a r t e d t h i r t y s e c o n d s a f t e r t h e t h a w - i n g a n d s p r e a d t o i n v o l v e h a l f o f t h e v e s s e l s i n t h e i n j u r e d a r e a . No g e n e r a l s t a s i s o r r e s u m p t i o n o f a n y c i r c u l a t i o n was n o t e d . A n i m a l 1 0 ( T e s t ) A n a n i m a l w e i g h i n g 200 g r a m s was i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 0 c c o f a d i l u t e d s o l u t i o n o f P r i s c o l i n e c o n t a i n i n g 0.4 m g . , a n d t w e n t y m i n u t e s l a t e r i t was a n a e s t h e t i z e d w i t h 0 . 2 2 c c o f n e m b u t a l . S t a s i s d e v e l o p e d i n t h i r t y s e c o n d s , a n d s p r e a d t o i n v o l v e one t h i r d o f t h e c a p i l l a r i e s i n t h e i n j u r e d a r e a . T h e r e w a s n o g e n e r a l s t a s i s . Some o f t h e v e s s e l s b e g a n t o go i n t o s t a s i s , t h e c e l l s c l u m p e d t o g e t h e r , a n d t h e c i r c - u l a t i o n s l o w e d , a n d t h e n s t o p p e d f o r a f e w s e c o n d s , a n d t h e n r e s u m e d f l o w . A n i m a l 11 ( C o n t r o l ) A n a n i m a l w e i g h i n g 200 g r a m s was a n a e s t h e t i z e d 94 w i t h 0 . 2 0 c c o f n e m b u t a l a d m i n i s t e r e d s u b c u t a n e o u s l y . S t a s i s d e v e l o p e d i n t h i r t y s e c o n d s , s p r e a d i n g t o i n - v o l v e a l m o s t a l l t h e c a p i l l a r i e s i n t h e f r o z e n a r e a . A n i m a l 12 ( T e s t ) A n a n i m a l w e i g h i n g 2 0 0 g r a m s was i n j e c t e d s u b - c u t a n e o u s l y w i t h 0 . 2 0 c 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 . 4 m g . o f P r i s c o l i n e , a n d f i f t e e n m i n u t e s l a t e r i t was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l a l s o a d m i n i s t e r e d b y s u b c u t a n e o u s i n j e c t i o n . S t a s i s s t a r t e d w i t h i n t h i r t y s e c o n d s o f t h a w i n g , a n d s p r e a d t o i n v o l v e t h r e e q u a r t e r s o f t h e v e s s e l s i n t h e i n j u r e d a r e a . A n i m a l 1 5 ( C o n t r o l ) A n a n i m a l w e i g h i n g 1 9 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l . S t a s i s s t a r t e d i n f o r t y - f i v e s e c o n d s a f t e r t h a w i n g , s p r e a d i n g t o i n v o l v e n i n e t e n t h s o f t h e v e s s e l s i n t h e i n j u r e d a r e a . Two p e t e c h i a l h e m o r r h a g e s w e r e p r o d u c e d a l s o . No g e n e r a l s t a s i s d e v e l o p e d , o r a n y r e s u m p t i o n i n c i r c u l a t i o n . A n i m a l 14 ( C o n t r o l ) A n a n i m a l w e i g h i n g 1 9 0 g r a m s was a n a e s t h e t i z e d w i t h 0 . 2 0 c c o f n e m b u t a l a d m i n i s t e r e d b y s u b c u t a n e o u s i n j e c t i o n . S t a s i s s t a r t e d a f t e r f o r t y - f i v e s e c o n d s a f t e r t h a w i n g , s p r e a d i n g t o i n v o l v e t h r e e q u a r t e r s o f t h e v e s s e l s i n t h e i n j u r e d a r e a . No g e n e r a l s t a s i s o r a n y r e s u m p t i o n i n t h e c i r c u l a t i o n was n o t e d . Summary o f R e s u l t s w i t h P r i s c o l i n e 9 5 N o . O n s e t S t a s i s S p r e a d G e n e r a l i z e d p l o w R e s u m e d 1 T e s t D i e d d u r i n g o b s e r v a t i o n - no r e s u l t s 2 T e s t 6 0 " 5 - 6 b e d s i n 4« None N o n e 3 C o n t r o l 3 0 " 7 5 $ i n 5« N o n e None 4 T e s t 9 0 " 5 0 $ o f c a p s None None 5 C o n t r o l 3 0 " 7 5 $ i n 4 ' None N o n e 6 T e s t 1 2 0 " 2 s m a l l c a p s o n l y None None 7 C o n t r o l D i e d d u r i n g o b s e r v a t i o n - n o r e s u l t s 8 T e s t 9 0 " 2 5 $ o f c a p s None None 9 C o n t r o l 3 0 " 5 0 $ o f v e s s e l s None None 10 T e s t 3 0 " 1 / 3 o f c a p s N o n e None 1 1 C o n t r o l 3 0 " R a p i d G e n e r a l N o n e 12 T e s t 3 0 " 7 5 $ o f v e s s e l s None None 1 3 C o n t r o l 4 5 " 9 0 $ o f v e s s e l s None None 14 C o n t r o l 4 5 " 7 5 $ o f v e s s e l s None None Comments o n O b s e r v a t i o n s The o n s e t o f s t a s i s a f t e r t h a w i n g i n t h e c o n t r o l a n i m a l s o c c u r r e d o n t h e a v e r a g e 3 5 " l a t e r , w h e r e a s i n t h e t e s t r u n s , P r i s c o l i n e a p p e a r e d t o d e l a y t h e s t a s i s t o 7 0 " , j u s t t w i c e a s l o n g . A l s o t h e e x t e n t o f t h e s t a s i s was c o n s i s t e n t l y l e s s i n t h e P r i s c o l i n e t r e a t e d a n i m a l s t h a n i t was i n t h e c o n t r o l s . To q u o t e t h e o b s e r v e r o f t h e s e t e s t s , " P r i s c o l i n e a s t e s t e d r e s u l t e d i n some d e l a y i n g i n t h e o n s e t o f s t a s i s , a n d i n some l e s s e n i n g i n I t s e x t e n t . No r e s u m p t i o n o f f l o w i n t h e a f f e c t e d v e s s e l s was n o t e d . H o w e v e r , r e s u l t s a r e n o t r e g a r d e d as b e i n g d e f i n i t e e n o u g h o r c o n s i s t e n t e n o u g h t o 96 b e r e a l l y s i g n i f i c a n t . " B e n a d r y l E x p e r i m e n t a l o b s e r v a t i o n s w i t h B e n a d r y l B e n a d r y l was t e s t e d a t a d o s a g e l e v e l o f 0 . 5 0 m g . p e r k i l o g r a m b o d y w e i g h t . 10 c c S t e r i - v i a l s o f B e n a d r y l ( 1 0 m g . p e r c c ) w e r e u s e d , a n d a 1 : 1 0 0 d i l u t i o n was p r e p a r e d b y p l a c i n g 0 . 2 c c . i n t o 1 9 . 8 c c o f w a t e r . I n t h i s w a y , v o l u m e o f d o s e p e r a n i m a l r a n g e d a b o u t a 0 . 5 c c amoun t w h i c h w o u l d b e o f r e a s o n a b l e s i z e f o r a b s o r p t i o n f r o m a s u b c u t a n e o u s i n j e c t i o n . The p H o f t h e R i n g e r - L o c k e g e l a t i n - g l u c o s e d r i p s o l u t i o n was a d j u s t e d b e f o r e s t a r t i n g t o a v a l u e o f 7 . 0 . A n i m a l 1 ( C o n t r o l ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 3 0 g r a m s was 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 $ n e m 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 m u s c u l a r l y , a n d i n e i g h t m i n u t e s a f u r t h e r 0 . 0 5 c c w e r e i n j e c t e d . S e v e n m i n u t e s l a t e r t h e p r e p a r a t i o n was m o u n t e d o n t h e m i c r o s c o p e s t a g e a n d t h e 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 a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . W i t h i n f o r t y s e c o n d s a f t e r t h a w i n g v a s c u l a r s t a s i s a p p e a r e d a n d s p r e a d s o m e w h a t , b u t t h e p r e p a r a t i o n was n o t a v e r y g o o d o n e , a n d f u r t h e r o b s - e r v a t i o n s w e r e n o t r e c o r d e d . A n i m a l 2 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 1 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 5 5 c c o f a B e n a d r y l s o l u t i o n c o n t a i n i n g 0 . 0 5 5 m g . , 97 a n d t w e n t y - s e v e n m i n u t e s l a t e r t h e a n i m a l was 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 $ n e m 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 m u s c u l a r l y . I n s e v e n m i n u t e s t h e p r e p a r a t i o n was r e a d y a n d t h e c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . W i t h i n t h i r t y s e c o n d s a f t e r t h a w i n g s t a s i s was n o t e d i n one c a p i l l a r y b e d . B y two m i n u t e s a f t e r t h a w i n g f o u r c a p i l l a r y b e d s w e r e i n v o l v e d . S l i g h t e c c h y m o s i s was n o t e d . I n a n o t h e r m i n u t e a n d a h a l f one l a r g e v e n o u s c h a n n e l o p e n e d a n d r e s u m e d c i r c u l a t i o n . I n a n o t h e r m i n u t e o n l y f o u r c a p i l l a r y b e d s w e r e c l o s e d o f f , a n d c i r c u l a t i o n i n t h e i n j u r e d a r e a was e x c e l l e n t . A f t e r a n o t h e r t h r e e m i n u t e s c o n d i t i o n s r e m a i n e d u n c h a n g e d , a n d t h e b l o o d f l o w i n t h e r e s t o f t h e m e s o a p p e n d i x was e x c e l l e n t . The a n i m a l w as r a t h e r s m a l l , a n d l i g h t l y a n a e s t h e t i z e d , m a k i n g o b s e r v a t i o n d i f f i c u l t , b u t t h e f i e l d f o r t e s t i n g was a g o o d o n e . 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 a n d a h a l f m i n u t e s a f t e r t h a w i n g . A n i m a l 5 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 2 5 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 6 5 c c o f a s o l u t i o n c o n t a i n i n g 0 . 0 6 5 m g . o f B e n a d r y l . T h i r t y - e i g h t m i n u t e s l a t e r t h e a n i m a l was 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 $ n e m 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 s u b - c u t a n e o u s l y , a n d i n s i x t e e n m i n u t e s t h e p r e p a r a t i o n was m o u n t e d a n d r e a d y o n t h e m i c r o s c o p e s t a g e . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x 9 8 w i t h f r e e z i n g f o r t h i r t y s e c o n d s . W i t h i n t h i r t y s e c o n d s a f t e r r a p i d t h a w i n g , s t a s i s was o b s e r v e d i n a c o u p l e o f t h e l a r g e r v e n u l e s , b u t b y a n o t h e r s i x t y s e c o n d s t h e f l o w i n t h e s e was r e s u m e d w i t h s t a s i s i n t h r e e c a p i l l a r y b e d s r e m a i n i n g . B y a n o t h e r s i x t y s e c o n d s f o u r c a p i l l a r y b e d s w e r e i n s t a s i s . B y f o u r m i n u t e s a f t e r t h a w i n g t h e o n e l a r g e c h a n n e l h a d b e e n o p e n i n g a n d c l o s i n g i n t e r m i t t e n t l y f o r t h e p r e v i o u s t w o m i n u t e s . I n two m i n u t e s m o r e , t h e b l o o d i n t h e o t h e r l a r g e c h a n n e l was f r e e f l o w i n g . A m i n u t e l a t e r two o f t h e c a p i l l a r y b e d s r e o p e n e d a n d f l o w e d n o r m a l l y . W h i l e a d m i t t e d l y t h e r e w e r e a c o u p l e s m a l l c a p i l l a r y b e d s a n d a f e w s h o r t p i e c e s o f v e n o u s c h a n n e l s c l o s e d o f f , t h i s a r e a was e n o r m o u s l y b e t t e r t h a n c o u l d b e e x p e c t e d w i t h o u t p r i o r t r e a t m e n t w i t h B e n a d r y l . One h a d t h e i m p r e s s i o n o f t h e c o h e s i v e p r o p e r t i e s o f t h e b l o o d c e l l s , a n d e s p e c i a l l y t h e i r a d h e s i v e t e n d e n c i e s t o s t i c k t o t h e i n j u r e d v e i n w a l l , b e i n g g r e a t l y r e d u c e d b y B e n a d r y l . B y f o u r t e e n m i n u t e s a f t e r t h a w i n g t h e 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 . N o r m a l v a s o m o t o r t o n e was p r e s e n t , a n d 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 . A n i m a l 4 ( C o n t r o l ) / A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 2 0 g r a m s was 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 $ n e m 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 m u s c u l a r l y , a n d 0 . 0 5 c c a d m i n i s t e r e d s u b c u t a n e o u s l y . I n n i n e 99 m i n u t e s t h e p r e p a r a t i o n was m o u n t e d a n d r e a d y o n t h e m i c r o s c o p e s t a g e . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s . B y t h i r t y s e c o n d s a f t e r r a p i d t h a w i n g t h e r e was a v e r y s l i g h t a m o u n t o f e c c h y m o s i s a t o n e s i t e i n t h e i n j u r e d a r e a . S t a s i s s t a r t e d s i x t y s e c o n d s a f t e r t h a w i n g a n d s p r e a d r a p i d l y t o i n v o l v e t h r e e l a r g e c a p i l l a r y b e d s . A f t e r two m o r e m i n u t e s no c h a n n e l s h a d r e o p e n e d , s t a s i s s t e a d i l y p r o g r e s s i n g t o w a r d t h e v e n o u s s i d e o f t h e c i r c u l a t i o n t o I n v o l v e t h e l a r g e v e n u l e s . The i n j u r y I n f l i c t e d o n t h i s p r e p a r a t i o n was r e g a r d e d as q u i t e m i n i m a l i n c o m p a r i s o n w i t h t h e o t h e r r u n s . B y f i v e m i n u t e s a f t e r t h a w i n g , v a s o m o t i o n was o b s e r v e d i n l a r g e r v e n o u s c h a n n e l s w i t h i n t e r m i t t e n t s t a s i s , a n d a m i n u t e l a t e r one l a r g e v e n o u s c h a n n e l , t h a t was i n s t a s i s q u i t e c o n t i n u o u s l y , r e o p e n e d a n d f l o w e d a p p a r e n t l y n o r m a l l y . T h r e e m i n u t e s l a t e r 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 w i t h n o f u r t h e r c h a n g e s n o t e d . A n i m a l 5 ( T e s t ) A n o r m a l a d u l t n o n p r e g n a n t f e m a l e W i s t a r r a t w e i g h i n g 1 4 0 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 7 c c o f a s o l u t i o n c o n t a i n i n g 0 . 0 7 m g . o f B e n a d r y l . T w n e t y - o n e m i n u t e s l a t e r t h e a n i m a l was a n a e s t h e t i z e d w i t h 0 . 1 5 c c o f 0 . 6 $ n e m 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 s u b - c u t a n e o u s l y , a n d t h i r t e e n m i n u t e s l a t e r t h e p r e p a r a t i o n was r e a d y o n t h e m i c r o s c o p e s t a g e . The c o l d t i p was a p p l i e d t o a s e l e c t e d a r e a o f t h e m e s o a p p e n d i x w i t h 100 freezing for t h i r t y seconds, and rapidly thawed with warm drip solution. Thirty seconds after thawing there was much c a p i l l a r y s t a s i s present, and venous obstruction i n a f a i r l y large circumscribed area within the injured f i e l d . After another f i v e and a h a l f min- utes the si t u a t i o n was unchanged. Actually these observations should be discounted because the whole mesoappendix was more or less i n vascular s t a s i s . Animal 6 (Control) A normal adult nonpregnant female Wistar r a t weighing 150 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered subcutaneously, and the preparation was ready eleven minutes l a t e r . The cold t i p was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds with immediate rapid thawing afterward. Within t h i r t y seconds of thawing, c a p i l l a r y stasis was observed which spread rapi d l y and i n another t h i r t y seconds f i f t y percent of the vessels i n the injured area were involved. By another minute no revascularization was observed i n the s t a t i c channels. Five minutes after this the preparation was discontinued. A few arterio-venous channels were flowing well, but generally the injured area was unchanged. Ninety percent of the vessels i n the injured area were i n st a s i s when the preparation was discontinued. 101 Animal 7 (Teat) A normal adult nonpregnant female Wistar r a t weighing 120 grams was injected subcutaneously with 0.6 cc of a solution containing 0.06 mg. of Benadryl. Thirty-three minutes l a t e r the animal was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered subcutaneously, and within nine minutes the preparation was ready f o r t e s t i n g . The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds and thawed r a p i d l y immediately afterward. Within t h i r t y seconds of thawing three c a p i l l a r y beds had closed o f f . In another t h i r t y seconds one large venous channel became s t a t i c , but opened up again f i v e minutes l a t e r . Nine minutes after 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 further change i n two more minutes observation and the preparation was discontinued. Animal 8 (Control) A normal adult nonpregnant female Wistar r a t weighing 120 grams was anaesthetized with 0.15 cc ©f 0.6$ nembutal solution administered subcutaneously, and the preparation was ready f o r testing i n twenty minutes. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of radpid thawing, c a p i l l a r y and venous st a s i s was s t a r t i n g , and i t spread r a p i d l y to close 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 only two arterio-venous shunts were flowing i n the injured area. In another two and a h a l f minutes no further change i n the c i r c u l a t i o n was noted, and the blood flow i n the r e s t of the mesoappendix was normal. The preparation was discontinued s i x and a h a l f minutes after thawing. Animal 9 (Test) A normal adult nonpregnant female Wistar r a t weighing 115 grams was Injected subcutaneously with 0.575 cc o f a solution containing 0.0575 mg. © f Benadryl, and forty-tw© minutes l a t e r the animal was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered subcutaneously. The preparation was ready i n eight minutes and the cold point was applied t© a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Hyperemia was noted t h i r t y seconds after rapid thawing, and i n another t h i r t y seconds vascular s t a s i s i n the injured area spread to involve two or three c a p i l l a r y beds. After another six minutes no further change was noted. Active vaso- motion was apparent i n a l l channels that were not s t a t i c . N© channels appeared to reopen, the s t a t i c ones being of the order of c a p i l l a r i e s and prevenules, forming approximately f o r t y percent of the vessels within the injured 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. By nine minutes following thawing, ©ne c a p i l l a r y bed reopened, and another bed did likewise a minute l a t e r . The preparation was 103 discontinued four minutes l a t e r . Summary of Observations with Benadryl (0.5 mg/Kg) Ho. Onset Stasis Spread Generalized Plow Resumed 1 Control 40" Yes Incomplete None 2 Test 30" Yes None // 3 Test 30 Yes None /// 4 Control 30-60" Rapid Incomplete ? 5 Test No r e s u l t s - Whole mesoappendix i n s t a s i s 6 Control 30" Rapid Almost None 7 Test 30" 3 Cap beds [ None /// 8 Control 30" Rapid Not A-V shunts None 9 Test 60" 3 Cap beds i None /// Discussion of Observations Benadryl d i d not appear to have any great e f f e c t on the time required f o r vascular s t a s i s to develop as compared with control animals, but i t appeared to l i m i t the spread of the s t a s i s . There was also evidence of increased resumption of flow i n the s t a t i c vessels. However, there are only four test and four control animals. 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 conclusions to be drawn. Further testing with Benadryl i s indicated, especially with a higher dosage level« Active vasomotion was found to be a prominent feature i n the re-opened vessels i n the injured area, i n d i c a t i n g that the freezing injury was unduly mild or 104 that the antihlataminic action on the endothelial c e l l s must have been e f f e c t u a l to a c e r t a i n degree. Control animal number four requires some comment i n that the reopening of channels, as marked "?", meaning doubtful, might be explained by the f a c t that without doubt the i n j u r y by freezing to t h i s preparation was r e a l l y minimal both as regards to duration and extent. In spite of such explanation, and such apparent good r e s u l t s , i t would not seem j u s t i f i a b l e to c r e d i t the antihistamine used with such great e f f e c t unless the tests were repeated. Further Experimental Observations with Benadryl In the following tests with Benadryl, 1.0 mg. per kilogram body weight was the dosage l e v e l used. This did not appear to have any adverse e f f e c t on the animals. Animal 1 (Test) A normal adult nonpregnant female Wistar r a t weighing 150 grams was injected subcutaneously with 0.75 cc of a solution containing 115 mg. of Benadryl, and t h i r t y - f i v e minutes l a t e r the animal was anaesthetized with 0.i?5 cc of 0.6$ nembutal solution administered sub- cutaneously. Seven minutes l a t e r 0.05 ec and f i v e minutes s t i l l l a t e r 0.025 cc of nembutal were injected also subeutaneously. The preparation was ready tw© and a h a l f minutes after the l a s t nembutal i n j e c t i o n , and the cold point was applied to a selected area of the 105 mesoappendix with freezing f o r t h i r t y seconds. Except f o r one s t a t i c c a p i l l a r y bed not near the injured area, no vascular s t a s i s developed within eight and a h a l f minutes of thawing. By eleven and a h a l f minutes of thawing, t h i s c a p i l l a r y bed was open and flowing. I t i s again to be noted that t h i s p a r t i c u l a r c a p i l l a r y bed was well outside the l i m i t s of the freezing Injury. By fourteen and a h a l f minutes of thawing there was no further change, and the preparation was discontinued. The e f f e c t of the Benadryl, i f any, on the s t a s i s i n th i s i s o l a t e d vessel cannot be explained. Animal 2 (Control) A normal adult nonpregnant female Wistar r a t weighing 150 grams was anaesthetized with 0.15 ce ©f 0.6% nembutal solution, and i n twenty minutes a further 0.05 cc were injected, both being administered subcut- aneously. The preparation was ready within another ten minutes, and the cold point was applied to a s e l - ected area of the mesoappendix with freezing f o r t h i r t y seconds. There was s t a s i s within t h i r t y seconds of thawing which spread rapi d l y . By another minute the s t a s i s was s t i l l spreading, and within one and a h a l f minutes of thawing, only the main arterio-venous shunts were flowing within the Injured area, although the c i r c u l a t i o n i n the rest of the mesoappendix was apparently normal. The preparation was discontinued three minutes l a t e r with no apparent change being noted. 106 There was no resumption of blood flow noted i n any s t a t i c channel. Animal 5 (Test) A normal adult nonpregnant female Wistar r a t weighing 150 grams was injected subcutaneously with 0.75 cc of a solution containing 115 mg. of Benadryl, and within thirty-three minutes the animal was anaesth- etized with 0.125 cc of 0.6$ nembutal solution admin- i s t e r e d subcutaneously, a further 0.04 cc being Inject- ed within eight minutes. The preparation was ready i n two more minutes, and the cold point was applied t© a selected area of the mesoappendix with freezing f o r t h i r t y seconds. In one and a half minutes a f t e r rapid thawing there was noted the s t a r t of a quickly spreading s t a s i s throughout the Injured area. By three and a h a l f minutes of thawing, one ©f the s t a t i c vessels flushed clear, but shut down again. In three minutes more, another channel flushed clear, and seven minutes s t i l l l a t e r another channel flushed c l e a r i n a d i f f e r e n t part of the injured area. No further extension of any st a s i s was observed. By twenty-one and a half minutes aft e r thawing l i t t l e further change was noted and the preparation was discontinued. Animal 4 (Control) A normal adult nonpregnant female Wistar r a t weighing 120 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution and i n eight minutes another 107 0.025 cc were Injected also subcutaneously. Within four minutes the preparation was ready, and the cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing there was s t a s i s which spread r a p i d l y throughout the injured area. There was s t i l l further spread noted by another two minutes. In three minutes more, 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 apparently normally. By another three minutes there was no further change noted, and the preparation was discontinued ten and a h a l f minutes after thawing, at which time i t was noted another small venous channel had reopened. Animal 5 (Test) A normal adult nonpregnant female Wistar r a t weighing 150 grams was injected subcutaneously with 0.75 cc of a d i l u t e d solution containing >15 mg. of Benadryl, and thirty-one minutes l a t e r the animal was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered sub- cutaneously, with 0.04 cc more being injected ten minutes l a t e r . The preparation was ready two minutes after the second nembutal i n j e c t i o n and the cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of rapid thawing, s t a s i s i n one venous channel was noted which backed up into the injured area. In another 108 minute a second venous channel acted s i m i l a r l y , and the same with a t h i r d within the next minute. By four and a h a l f minutes aft e r thawing the whole injured aj?ea was p r a c t i c a l l y i n s t a s i s . No channels were observed to reopen. The preparation was discontinued. Animal 6 (Control) A normal adult nonpregnant female Wistar r a t weighing 140 grams was anaesthetized with 0.15 ce of 0.6$ nembutal solution administered subcutaneously, and the preparation was ready within twelve minutes. The cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds, and then r a p i d l y thawed. Stasis was observed to s t a r t within t h i r t y seconds of thawing, and i t spread slowly so that by s i x and a half minutes aft e r thawing only three ven- ules 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 freezing i n j u r y was too s l i g h t . By another minute there was no further change noted and the preparation was discontinued. Animal 7 (Test) A normal adult nonpregnant female Wistar r a t weighing 135 grams was injected subcutaneously with 0.65 cc of a d i l u t e d solution containing 0.13 mg. of Benadryl, and i n twenty-four minutes the animal was anaesthetized with 0.10 cc of 0.6$ nembutal solution injected subcutaneously, with 0.025 cc being repeated i n seven minutes. In another f i v e minutes th© prep- aration was ready and the cold point was applied with 109 freezing f o r t h i r t y seconds to a selected area of the mesoappendix. Within t h i r t y seconds of thawing, beginning 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 rather general except f o r large arteriovenous channels. By another f i v e minutes many smaller channels reopened, but not much change took place i n the next three minutes. Twenty-six minutes after thawing one large 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 apparently normal c i r c u l a t i o n . The prepara- t i o n was discontinued i n another eight minutes with l i t t l e further change except f o r some general extens- ion of the st a s i s within the injured area of the meso- appendix. Animal 8 (Test) A normal adult nonpregnant female Wistar r a t weighing 140 grams was injected subcutaneously with 0.70 cc of a d i l u t e d solution containing 0.14 mg. of Benadryl and i n forty-seven minutes the animal was anaesthetized with 0.125 cc of 0.6$ nembutal solution administered subcutaneously. The preparation was ready i n another twenty minutes and the cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds, followed by immediate rapid thawing. Stasis f i r s t appeared three and a h a l f minutes a f t e r thawing, and i n only three small c a p i l l - a r i e s . In three minutes more, one of t h e . c a p i l l a r i e s 110 ©pened up with resumption of apparently normal c i r c - u l a t i o n . By another four minutes the s t a s i s spread to involve two c a p i l l a r i e s connecting adjacent a r t e r i o - venous shunts. The preparation was discontinued one minute l a t e r with no further change being noted. Animal 9 (Control) A normal adult nonpregnant female Wistar r a t weighing 140 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered subcutaneously, with 0.025 cc. being repeated i n eleven minutes. The preparation was ready i n another three minutes and the cold t i p was applied to a selected area of the mesoapp- endix with freezing 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 started, and i t spread rapi d l y . By another six minutes a few large vessels were observed to be slowing, but r e a l l y l i t t l e further change was noted. The preparation was d i s - continued six and a h a l f minutes a f t e r thawing. Animal 10 (Control) JS normal adult nonpregnant female Wistar r a t weighing 160 grams was anaesthetized with 0.15 cc of 0.6$ nembutal solution administered subcutaneously, and was ready on the microscope stage within twenty- three minutes. The f i e l d was somewhat obscured by f a t i n the mesoappendix, but enough of the c i r c u l a t i o n was visable t© warrant continuing with the preparation. The cold t i p was applied to a selected area of the I l l mesoappendix with freezing f o r t h i r t y seconds, and ra p i d l y developing s t a s i s started within t h i r t y seconds ©f thawing. N© restoration of flow was observed i n any of the vessels, and the preparation was d i s - continued f i v e and a ha l f minutes afte r thawing. Animal 11 (Test) A normal adult nonpregnant female Wistar r a t weighing 135 grams was in j e c t e d subcutaneously with 0.65 ec. of a di l u t e d solution containing 0.13 mg. ©f Benadryl, and i n t h i r t y - f i v e minutes the animal was anaesthetized with 0.125 cc of 0.6% nembutal solution administered subcutaneously. The preparation was ready i n eleven minutes and the cold point was app- l i e d to a selected area ©f the mesoappendix with freezing f o r t h i r t y seconds, followed immediately afterward with rapid thawing. In t h i r t y seconds after 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 vessels which spread r a p i d l y to become more or l e s s general i n the injured area within four and a h a l f minutes of thawing. No vessels re-opened to restore c i r c u l a t i o n , and the preparation was discontinued two minutes l a t e r . Summary of Observations with Benadryl (1.0 mg./Kg) No. Onset Stasis Spread Generalized Flow Resumed 1 Test None None None Yes ? 2 Control 30" Rapidly l£» None 112 Summary (Cont'd) Ne. Onset Stasis Spread Generalized Flow Resumed 3 Test l£» Rapidly None /// 4 Control 30 n Rapidly / 5 Test 30" Rapidly 4&" Non© 6 Control 30 a Slowly None None 7 Test 30" Rapidly 60 u 8 Test 3&» None None // 9 Control 30" Rapidly None None 10 Control 30" Rapidly 60" None 11 Test 30" Rapidly 4^» None Discussion of Observations Benadryl injected to the extent of 1.0 mg. per kilogram body weight had no apparent adverse e f f e c t s on the rats tested. From the somewhat a r b i t r a r y values i n the above summary table, the b e n e f i c i a l e f f e c ts of Benadryl i n the test animals were Inconsistent, and they c o n f l i c t e d with equally inconsistent values i n the controls. The p a r t i c u l a r vascular area chosen f o r i n j u r y by application of the freezing point, as well as the exact duration of the freezing are rather uncontrollable variables entering into consideration of the significance of these r e s u l t s . Possible r e p e t i t i v e tests on twe or more areas of the same mesoappendix would be an im- provement, although the time fa c t o r and the v i a b i l i t y 113 of the preparation would be b i g obstacles. I t would seem that Benadryl i n the dosage used had no s i g n i f i c a n t influence beyond the errors inherent i n the experiment. TMs conclusion agrees with that obtained above when the dosage of 0.5 mg. per Kilogram was tested. Further Experimental Observations with Benadryl The following tests were performed to determine the influence of Benadryl, i n a dosage l e v e l ®f 1.5 mg. per kilogram of body weight, administered by subcut- aneous i n j e c t i o n one h a l f hour before the observation of a freezing i n j u r y on the mesoappendix. The r e s u l t s are summarized i n table form aft e r animal 10. Animal 1 (Test) A normal adult nonpregnant female Wistar r a t weighing 130 grams was injected subcutaneously with 0.39 cc of a d i l u t e d solution containing 0.195 mg. of Benadryl, and twenty-nine minutes l a t e r the animal was anaesthetized with 0.15 cc of 0.6$ nembutal s o l - ution. The preparation was ready i n twelve minutes, and the cold point was applied to a selected area of the mesoappendix with freezing i n j u r y f e r t h i r t y sec- onds. Within sixt y seconds of thawing, a s l i g h t vascular s t a s i s was s t a r t i n g , r e a l l y just a slowing i n the blood flow. By another minute i t was spread- ing. One channel was observed to ©pen and resume 114 c i r c u l a t i o n . By another two minutes there was no further spread. This i s ah excellent area ©f the mesoappendix with many small c a p i l l a r y channels and the f a c t that the st a s i s was so limit e d , together with a f a i r l y strong freezing i n j u r y c e r t a i n l y makes the observed f a c t s s i g n i f i c a n t . After another two minutes one large connecting vein was i n s t a s i s , and by eight minutes afte r thawing some sta s i s was app^arent i n other parts ©f the mesoappendix, so that the preparation was discontinued. Animal 2 (Control) A normal adult nonpregnant female Wistar r a t weighing 140 grams was anaesthetized with 0.15 cc ©f 0.6$ nembutal solution administered subcutaneously, and the preparation was ready on the microscope stage In seventeen minutes. The cold point was applied to a selected area of the mesoappendix with freezing fo r t h i r t y seconds. Stasis started within t h i r t y seconds of thawing, and spread r a p i d l y . By three minutes afte r thawing the s t a s i s was slowly extending but was by no means becoming generalized. Some ecchymoses were present. Two minutes l a t e r , ex- cept f o r the arterio-venous channels, s t a s i s was generalized, and no reopening of the channels was noted. Pour minutes l a t e r the preparation was d i s - continued. Animal 3 (Test) A normal adult nonpregnant female Wistar r a t 115 weighing 130 grams was injected subcutaneously with 0.39 ce. of a d i l u t e d solution containing 0.195 mg. ©f Benadryl, and f o r t y - f i v e minutes l a t e r the animal was anaesthetized with 0.15 cc. of 0.6$ nembutal s o l - u t ion administered subcutaneously. The preparation was ready f i f t e e n minutes l a t e r and the cold t i p was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds, followed by immediate rapid thawing. There was n© s t a s i s within ninety seconds of thawing, but i n a minute more, st a s i s started i n one small venous channel only. 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, and then was flushed free by a eonneeting a r t e r i o l e - c a p i l l a r y t r ibutary. After another two minutes there was no further change. The c i r c u l a t o r y system i n the whole Injured area was functioning well. Five minutes l a t e r the preparation was discontinued with no further changes being noted. Animal 4 (Control) A normal adult nonpregnant female Wistar r a t weighing 135 grams was anaesthetized with 6.15 cc. of 0.6$ nembutal solution, administered by subcutaneous i n j e c t i o n , and the animal was mounted and ready ©n the microscope stage i n eleven minutes. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. There was st a s i s 116 st a r t i n g within t h i r t y seconds which spread r a p i d l y . Tw© and a h a l f minutes l a t e r the stasis was s t i l l spreading, but net yet generalized. In two m©re minutes the sta s i s had become rather generalized through out the injured area, and no revascularization of any channels was noticed. The preparation was d i s - continued three minutes l a t e r with no further changes observed. Animal S (Test) A normal adult nonpregnant female Wistar r a t weighing 135 grams was injected subcutaneously with 0.39 cc. ©f a d i l u t e d solution containing 0.195 mg. of Benadryl, and t h i r t y - e i g h t minutes l a t e r the animal was anaesthetized with 0.15 cc. of 0.6$ nem- butal solution administered subcutaneously. The cold t i p was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds. Stasis occurred i n several venous channels i n the injured area within s i x t y seconds after thawing. In another t h i r t y seconds there was fio further spread i n the s t a s i s , although a good s i x t y percent of the vessels i n the injured area were affected. In three more minutes no resumption of c i r c u l a t i o n i n any s t a t i c vessel was observed, and the preparation was discontin- ued two minutes l a t e r . Animal 6 (Control) A normal adult nonpregnant female Wistar r a t 117 weighing 170 grams was anaesthetized with 0.20 cc. of 0.6$ nembutal solution administered by subcutaneous i n j e c t i o n , and the preparation was ready i n th i r t e e n minutes. Tbe cold point was applied three minutes l a t e r to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Stasis developed within t h i r t y seconds of thawing and spread r a p i d l y . By another t h i r t y seconds, several eechymotic areas had developed. By ninety seconds aft e r thawing, s t a s i s had become rather generalized except i n the large shunt channels. By two minutes l a t e r , no resumption of c i r c u l a t i o n had been noted, and the st a s i s was s t i l l extending. The preparation was discontinued four minutes l a t e r . Animal 7 (Test) A normal adult nonpregnant female Wistar r a t weighing 140 grams was injected subcutaneously with 0.42 cc. of a d i l u t e d solution containing 0.21 mg. of Benadryl, and twenty-eight minutes l a t e r the animal was anaesthetized with 0.15 cc of 0.6$ nembutal solution, administered by subcutaneous i n j e c t i o n . The preparation was ready i n nine minutes, and the cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of rapid thawing, one small c a p i l l a r y bed was In s t a s i s . By another two minutes the sta s i s had spread slowly to involve several other c a p i l l a r y beds. In three more minutes there was n© 118 further involvement. N© resumption of c i r c u l a t i o n was noted, and although this area was very s i m i l a r t© the subsequent control prparation, there was much l e s s s t a s i s . By three more minutes, the preparation was discontinued, with no further change. There was no apparent difference from the following control run. Animal 8 (Control) A normal adult nonpregnant female Wistar r a t weighing 120 grams was anaesthetized with 0.15 cc. ©f 0.6$ nembutal solution, administered subcutaneously, and the preparation was ready i n thirteen minutes. The cold point was applied to a selected area of the mesoappendix with freezing, followed by rapid thawing. Within t h i r t y seconds of thawing stasis started which spread rapidly, with the development als© of a few ecchymoses. Two and a h a l f minutes l a t e r there were no further changes i n the smaller s t a t i c vessels. I t was i n t e r e s t i n g to observe the events i n th i s preparation. Some of the larger channels would f i l l up with conc- entrated c e l l u l a r sludge, and then repeatedly f l u s h f ree. By f i v e more minutes no further changes had occurred. The area of s t a s i s was quite complete within the injured f i e l d , being a patchy involvement of the smaller venules. Two minutes l a t e r the preparation was discontinued, and no reopening of the s t a t i c vessels was noted. 119 Animal 9 (Test) A normal adult nonpregnant female Wistar r a t weighing 130 grams was injected subcutaneously with 0.39 cc of a dil u t e d Benadryl solution containing 0.195 mg., and thirty-one minutes l a t e r the animal was anaesthetized with 0.15 cc. of 0.6$ nembutal solution administered by subcutaneous i n j e c t i o n . The prep- aration was ready i n sixteen minutes, and the cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds. There was no sta s i s present by t h i r t y seconds aft e r rapid thawing, and after another minute, s t a s i s formed i n one venule only. A minute l a t e r a second c a p i l l a r y bed became s t a t i c . Two minutes l a t e r there were only the two c a p i l l a r y beds s t a t i c , and the res t of the c i r c u l a t i o n within the injured area appeared normal. Two and a half minutes l a t e r no further change developed, with no resumption of c i r c u l a t i o n being noted? and the preparation was discontinued. Animal 10 (Control) A normal adult nonpregnant female Wistar r a t weighing 130 grams was anaesthetized with 0.15 cc. of 0.6$ nembutal solution, administered subcutaneously, and the preparation was ready i n eight minutes. The cold point was applied to a selected area of the meso- appendix with freezing, followed immediately by rapid thawing. Within t h i r t y seconds of thawing 120 there was ra p i d l y spreading s t a s i s , which became generalized throughout the injured area within another minute. No re c a n a l i z a t i o n was noted, and the preparation was discontinued three minutes l a t e r . Summary of Observations with Benadryl (1.5 mg./Kg) No. Onset Stasis Spread Generalized Plow Resumed 1 Test 60" Slowly None Sl i g h t 2 Control 30" Slowly 5« None 3 Test None None None 4 Control 30" Rapidly 3' None 5 Test 60" Slowly 60$ i n 90" None 6 Control 30" Rapidly 90" None 7 Test 30" Slowly Incomplete N©ne 8 Control 30" Rapidly Incomplete None 9 Test 90" Slowly 2 Cap Beds None 10 Control 30" Rapidly 90" None Discussion of Observations Not considering the test animal, number three, which did not develop s t a s i s , the average time taken f e r the test preparations to develop s t a s i s was s i x t y seconds, just twice as long as f o r the controls, which took t h i r t y . Further, the sta s i s spread more slowly and was much l e s s complete than In the case of. the controls. There was n© difference as regards any resumption of flew. These considerations would Aappear t© s h i f t 121 the decision more favourably towards Benadryl having a protective e f f e c t on the mesoappendix c i r c u l a t i o n against a standard freezing i n j u r y . Etamon Experimental observations with Etamon Chloride The following t r i a l s were f o r the purpose ®f testing the e f f e c t of Etamon chloride (Tetra-ethyl ammonium ehloride) i n a dosage l e v e l of 25 mg. per kilogram body weight, on the c a p i l l a r y beds of the r a t mesoappendix afte r a standard freezing i n j u r y . Normal adult male Wistar rats were used, four test animals, averaging 310 grams, and four controls, averaging 280 grams. The procedure was as before, using Ringer-Locke gelatine-glucose sol u t i o n . The pH was adjusted to the f i r s t ehange to pink using sodium bicarbonate and methyl red i n d i c a t o r . The Etamon solution was administered to a l l test animals at one time, using 0.10 ec. (0.1 gm./ce), providing 10 mg. per animal, which i s s l i g h t l y l a r g e r than 25 mg. per kilogram. The drug was administered subcutaneously. A summary of the following observ- ations i s given i n table form after the second Animal 8. Animal 1 (Oontrol) 0.20 cc. of 0.6$ nembutal solution was admin- 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, and i n 122 seven minutes the preparation was ready, mounted on the microscope stage with the drip flowing. One and a h a l f minutes l a t e r a selected area of the meso- appendix was frozen with the cold point f o r t h i r t y seconds. Thirty seconds after thawing there was noted a very marked hyperemia, and then the flow ©f the drip solution was resumed. Within another t h i r t y seconds, general vascular s t a s i s had devel- oped, which was somewhat incomplete. In ninety seconds more, some clearing, but only temporary, was noted i n a few of the l a r g e r channels. One minute l a t e r no further change was observed. A main eentral a r t e r i o l e was flowing uninterruptedly, and the vascular s t a s i s seemed to be e n t i r e l y on the venous side of the c i r c u l a t i o n . The preparation was discontinued two and a h a l f minutes l a t e r . Animal 2 (Test) The animal was anaesthetized with 0.20 cc. of 0.6$ nembutal solution one hour and fourteen minutes afte r the Etamon had been administered. The preparation was ready eight minutes afte r anaesthetiz- ing. A state of hyperemia was noted i n the vessels of the mesoappendix. Three minutes l a t e r the cold t i p was applied with freezing f o r t h i r t y seconds, with rapid thawing and resumption of flow of the warm drip solution. Within s i x t y seconds of thawing stasis was observed to be commencing. By a minute 123 and a h a l f more, the condition of the c i r c u l a t i o n i n the injured area was found to be much better than with the previous control animal, but considering the varying f a c t o r s , the difference was not great enough to be t r u l y s i g n i f i c a n t unless present consistently i n a l l the test and control animals. By four and a half minutes a f t e r thawing, the s t a s i s had spread somewhat, but there was s t i l l very good c i r c u l a t i o n throughout the injured area. 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. Good vasomotion was also noted throughout the mesoappendix i n spite of the Etamon (dose too small?!). A minute l a t e r many smaller s t a t i c channels resumed flow, and two minutes s t i l l l a t e r , eighty percent of the channels once s t a t i c v/ere ©pen and functioning w e l l . In three minutes (twelve and a h a l f minutes after thawing), generalized vascular s t a s i s was present i n the injured area. Two minutes l a t e r no further change occurred, and the preparation was discontinued. Animal 3 (Control) 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 , and i n seventeen minutes the preparation was ready. The cold t i p was applied with freezing f o r t h i r t y seconds. By t h i r t y seconds a f t e r rapid thawing net much hyperemia occurred, but after s i x t y seconds, 124 vascular s t a s i s started. In sixty seconds more, t h i s s t a s i s was spreading rapidly, and by tw© minutes there was a slow insidious spread to a l l vessels with no more vasomotion observed. The preparation was discontinued one minute l a t e r . Animal 4 (Test) The animal was anaesthetized with 0.20 cc of 0.6$ nembutal solution two hours and seven minutes after the Etamon had been administered. Within s i x minutes the preparation was ready, and moderate hyperemia was noted. The cold t i p was applied with freezing f o r t h i r t y seconds, and then immediate ra p i d thawing was achieved as before with resumption of the warm saline d r i p . A very be a u t i f u l c a p i l l a r y bed was available f o r observation. Stasis f i r s t appeared ninety seconds a f t e r thawing. By another minute sta s i s spread over a large venous area. By eight minutes further, no vessels reopened or resumed any c i r c u l a t i o n . Two minutes l a t e r a main vascular channel reopened, and within three more minutes i t showed active vasomotion. No further change was noted f o r ten minutes. Part of the venous area did not resume flow. The preparation was discontinued. Animal 5 (Control) 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 , 125 and the preparation was ready i n ten minutes. The cold t i p was applied with freezing f o r t h i r t y seconds, and s t a s i s was observed to commence within t h i r t y seconds of thawing. By another sixt y seconds the stas i s was complete and i r r e v e r s i b l e , and the preparation was discarded three minutes l a t e r with no further change observed. Animal 6 (Test) 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 three hours and seven minutes aft e r the Etamon was injected. The preparation was ready i n f i v e minutes, and excellent vasomotion was noted. The cold point was applied with freezing f o r t h i r t y seconds. By t h i r t y seconds after freezing good blood flow through the injured area was s t i l l present. S l i g h t and temp- orary vascular 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 four minutes the s i t - uation was much the same, with some slowing of the flow i n one venous channel. Three minutes l a t e r 2% procaine at room temperature was i n s t i l l e d over the mesoappendix with stoppage of the whole c i r c u l a t i o n . The preparation was discarded f i v e minutes l a t e r (fourteen and a h a l f minutes after thawing). Animal 7 (Control) The animal was anaesthetized with 0.20 cc. of 126 0.6$ nembutal solution and the preparation was ready i n nine minutes. The cold point was applied with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing great hyperemia was noted, with vascular s t a s i s developing and spreading rapidly* Within another minute, t h i r t y percent of the injured area was i n s t a s i s , which did not increase by another three minutes. Three minutes l a t e r larger vessels were slowing, and by fourteen and a h a l f minutes afte r thawing, over h a l f the vessels i n the injured area were i n s t a s i s . The preparation was discontinued four minutes l a t e r . Animal 8 (Test) 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 four hours and f i v e minutes after the Etamon had been given. The preparation was ready i n nine minutes, and the cold point was applied with f r e e z i n g f o r t h i r t y seconds. Stasis started three minutes a f t e r thawing, and four minutes l a t e r was found to be complete i n some parts of the injured area, but temporary i n others. The preparation was discontinued one minute l a t e r . (Summary table w i l l be found after the following series of repeat tests) Repeat Experiments with Etamon Chloride The following i s a r e p e t i t i o n of the above run, 127 but using 25 mg. of Etamon (tetra-ethyl ammonium chloride) per test animal, administered subcutan- eously. The test animals averaged 315 grams, the controls 290 grams, and a l l were normal adult male Wistar r a t s . The heaviest of the te s t animals, weighing 355 grams, died f i f t e e n minutes after 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 substituted, being given 25 mg. of Etamon also subcutaneously. Animal 1 (Control) 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 , and the preparation was ready i n eight minutes. The cold point was applied with freezing f o r t h i r t y seconds, and within t h i r t y seconds of thawing vascular s t a s i s developed i n venous channels, r a p i d l y beeomming • generalized over the injured area. There was no further change by another three minutes and the prep- aration was discontinued. Animal 2 (Test) The animal was anaesthetized with 0.20 cc. of 0.6% nembutal solution Intraperitoneally thirty-two minutes after the Etamon was injected. The prepara- ti o n was ready i n four minutes and the cold t i p was app- l i e d to the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing some small 128 capillary stasis was apparent and by another thirty seconds many large venous channels were in stasis. In two minutes slow blood flow was observed through one large venous channel that had been in stasis. During the following ten minutes i t was noted that the infeeding capillaries could not keep up the flow and the circulation in the whole of the injured area ceased. These were adjacent venous systems, and a large part of the stasis was undoubtedly due to the fact that the arteriole between was injured by the freezing and Its flow stopped. Two minutes later the preparation was discontinued. 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 intraperitoneally and the preparation was ready in six minutes. The c©ld point was applied t© a selected area of the meso- appendix with freezing for thirty seconds, and within thirty seconds of thawing, generalized stasis was observed over the entire injured area. There was n© further change after three and a half minutes and the preparation was discarded. Animal 4 (T&st) The animal was anaesthetized with 0.20 cc of 0.6$ nembutal solution administered intraperitoneally 129 one hour and f i v e minutes after the Etamon i n j e c t i o n . The preparation was ready i n six minutes and the cold point was applied t© a selected area of the meso- appendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing there was general slowing i n the venous channels with many of the connecting channels pumping blood into the area. Vascular s t a s i s started two and a hal f minutes afte r thawing. By another f i v e minutes, although there was scant flow through one channel, generally the degree of s t a s i s was unchanged. The preparation was discarded seven and a hal f minutes afte r thawing. Animal 5 (Control) The animal was anaesthetized with 0.20 cc. of 0.6$ nembutal solution and the preparation was ready i n twenty minutes. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing there was generalized irreversable venous s t a s i s s t a r t - ing. By tw© and a h a l f minutes l a t e r , the f i e l d was unchanged and the preparation was discarded. Animal 6 (Test) 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 one hour and fourty-f©ur minutes after the Etamon was injected. The preparation was ready i n eight minutes and the cold point was applied to a selected area of 130 the mesoappendix with freezing f o r t h i r t y sedonds. By t h i r t y seconds after thawing great hyperemia was noted generally, but no s t a s i s appeared by two minutes afte r thawing. There was a good brisk blood flow throughout the frozen area. Two minutes l a t e r s t a s i s was noted i n one small venous channel, and increased vasomotion i n others. In another minute the stream was noted to slow temporarily i n a few of the larg e r venous channels. In t h i r t y seconds s t a s i s was noted to be spreading to a couple venous areas, although there was generally a good c i r c u l a t i o n . The prep- aration was discarded seven minutes a f t e r thawing with no further changes observed. Animal 7 (Control) The animal was anaesthetized with 0.20 cc. ©f 0.6$ nembutal solution i n t r a p e r i t o n e a l l y , but when the preparation was mounted s i x minutes l a t e r , general st a s i s was present throughout the mesoappendix, and the animal was discarded. Animal 8 (Test) The animal was anaesthetized with 0.20 cc. ©f 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y two hours and eight minutes after the Etamon i n j e c t i o n . The preparation was ready i n f i v e minutes. The cold point was applied to a selected area of the meso- appendix with freezing 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 within t h i r t y seconds aft e r thawing. By another s i x t y seconds there was slowing ©f 131 the flow and s t a s i s i n large venous channels. Three and a h a l f minutes l a t e r one large s t a t i c venous channel opened up, and In another t h i r t y seconds another did likewise, and flowed normally. I t would seem that the larger channels stay open provided there i s any blood draining into them from the c a p i l l a r i e s . The preparation was discarded eight minutes a f t e r thawing with no further change being noted. Summary of Observations with Etamon Ho. Onset Stasia Spread Generalized Flow Resumed 1 Control 60" Rapidly 60" Temporary i : - - larger ch 2 Test 60" 12£« /// 3 Control 60" Fast, then 2» None •- slow - 4 Test 90" 150" None //a t 10« 5 Control 30" Rapidly 90" None 6 Test >90" Incomplete by 6^' None 7 Control 30" Rapidly 90" None 8 Test 180" Steady 7» None 1 Control 30"" Rapidly 60" None 2 Test 30" Slow 14» // at 3« 3 Control 30" — . > 30" None 4 Test 150" None None None 5 Control 30""— > 30" None 6 Test 240" At 4' None None 132 Summary (Cont'd) No. Onset Stasis Spread Generalized Flow Resumed 7 Control Animal discarded because of general st a s i s 8 Test 30" S l i g h t None at 5» & 5&» Discussion of Observations Variable effects from parenteral injections of Etamon would be expected i n this experiment since there was with succeeding animals, an increased i n t e r v a l between the time of i n i t i a l administration of the drug and the performance of the test. However, a s u p e r f i c i a l examination of the above res 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 sustained for up to four hours following administration. On the average, with the test animals, vascular stasis did not st a r t f o r 110 seconds after thawing, whereas with the controls the correspong- ing time was 40 seconds, almost three times as long. I t would seem that the spread and extent of the stasis was much les s with the Etamon treated animals. However, because the drip solution seemed at the f i n i s h of the tests to be more deeply pinkish than would be expected f o r n e u t r a l i t y , i t s pH was tested on an e l e c t r i c pH meter. The determination was not done f o r a day or so following the tests, the f l u i d being stored i n t i g h t l y stoppered flasks i n an e l e c t r i c regrigerator. When determined, the pH measured 8.9 which i s admittedly somewhat past n e u t r a l i t y ! The exact ef f e c t of this more 133 alkaline drip solution was not studied, but the s i g n i f i c - ance of the above r e s u l t s was considered i n v a l i d , and the tests were repeated, as follows, using a drip solution pretested on an e l e c t r i c pH meter, and c a r e f u l l y buffered to a pH of 7.10. Sepeat Experiments with Etamon In the f i r s t few of the following tests, Etamon was used at a dosage l e v e l of 100 mg. per kilogram body weight. The observations are summar- ized i n table form at the end of Animal 13. Animal 1 (Test) A normal adult nonpregnant female Wistar rat was used, weighing 225 grams. 22.5 mg. of Etamon chloride was injected subcutaneously, and f i f t e e n minutes l a t e r 0.15 cc of 0.6$ nembutal solution was injected i n t r a p e r i t o n e a l l y . Five minutes l a t e r 0.05 cc. more was injected also i n t r a p e r i t o n e a l l y . Within ten more minutes the animal was s u f f i c i e n t l y anaesthetized to permit continuing. The mesoappendix was mounted on the microscope stage and the cold point was applied with freezing f o r f o r t y seconds. Within t h i r t y seconds of thawing a dispersed f i n e c a p i l l a r y vasostasis was observed, and a minute l a t e r some variable stasis was noted In- the larger channels. By two and a half minutes after thawing the st a s i s was beginning to spread and i n another two minutes the stasis 134 was quite generalized i n the injured area, although the a r t e r i o l e s were pulsating t© some degree. The preparation was discontinued with no further changes being noted. Animal 2 (Test) A A normal adult nonpregnant female Wistar r a t weighing 280 grams was used. 28 mg. of Etamon was injected subcutaneously and with twenty-three minutes 0.15 cc. of 0.6$ nembutal solution was injected i n t r a - p e r i t oneally. The preparation had to be discarded after mounting the mesoappendix because the c i r c u l a t - ion was t o t a l l y i n v i s i b l e due to the amount of f a t present. Animal 5 (Control) A normal adult nonpregnant female Wistar r a t weighing 240 grams was used. 0.15 cc. of 0.6$ nem- butal solution was injected i n t r a p e r i t o n e a l l y , but after mountingj the mesoappendix f i v e minutes l a t e r showed the animal had to be discarded because of gen- era l i z e d vascular s t a s i s . Animal 4 (Control) A normal adult nonpregnant female Wistar r a t weighing 205 grams was anaesthetized with 0.15 cc. ©f 0.6$ nembutal solution, administered by i n t r a p e r i t - oneal i n j e c t i o n , and an additional 0.05 cc. was i n - jected i n ten minutes and again i n seven more minutes. After mounting the mesoappendix, the cold point was 135 applied with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing, r a p i d l y spreading vasc- ular s t a s i s was noted throughout the injured area. The smaller venules became involved In ninety sec- onds more, but the a r t e r i o l e s were unaffected and the arterio-venous shunts to the larger veins app- eared to be functioning normally. C i r c u l a t i o n was observed to be apparently normal i n the reat of the mesoappendix. By three minutes after thawing the c i r c u l a t i o n was quite occluded i n the Injured area, except f o r the larger vessels. Tw@ minutes l a t e r the preparation was discontinued with n© further change noted. Animal 5 (Control) A normal adult nonpregnant female Wistar r a t weighing an unrecorded amount. 0.15 cc. of 0.6$ nembutal solution was Injected i n t r a p e r i t o n e a l l y . After mounting the mesoappendix, the preparation had to be discarded because the amount of f a t present t o t a l l y obscured the c i r c u l a t i o n . Animal 6 (Test) A normal adult nonpregnant female Wistar r a t weighing 165 grams was used. 16.5 mg. of Etamon chloride was injected subcutaneously. The animal was found dead i n i t s cage ten minutes l a t e r . Animal 7 (Test) A normal adult nonpregnant female Wistar r a t 136 weighing 155 grams. 15.5 mg. of Etamon chloride was injected subcutaneously into the dorsum of the animal, and f i f t e e n minutes l a t e r 0.10 cc. of 0.6$ nembutal solution was injected i n t r a p e r i t o n e a l l y . Five min- utes l a t e r the preparation was mounted on the microscope stage with excellent vasomotion observed, but the animal died one minute l a t e r . Animal 8 (Test) A normal adult nonpregnant female Wistar r a t weighing 185 grams was used. 18.5 mg. of Etamon chloride was injected subcutaneously.and twelve min- utes l a t e r 0.085 cc. of 0.6$ nembutal solution was injected i n t r a p e r i t o n e a l l y . Four minutes l a t e r the preparation was ready, with the vascular bed d i l a t e d and sluggish, but some vasomotion was noted, although no s t a s i s was observed. Tbe cold point was applied with freezing f o r t h i r t y seconds, and within t h i r t y seconds of thawing, c a p i l l a r y and venous s t a s i s was observed to have occurred. In another minute the c i r c u l a t i o n closed down completely within the injured area. By seven and a h a l f minutes aft e r thawing, general stasis was present throughout the mesoappendix, and i t was f e l t this animal should not be admitted to the data because of obvious toxic e f f e c t s from the st a r t due to the Etamon. Note; From the above and previous observations, i t 137 would appear that Etamon chloride at a dosage l e v e l of 100 mg. per kilogram was too toxic, k i l l i n g at l e a s t 50$ of the animals. Further t r i a l s w i l l be l i m i t e d to a smaller dosage. Animal 9 (Test) A normal adult nonpregnant female Wistar r a t weighing 160 grams was used. 8.0 mg. of Etamon chloride was injected subcutaneously and t h i r t y sec- onds l a t e r 0.11 cc. of 0.6$ nembutal solution was injected i n t r a p e r i t o n e a l l y . Within twelve minutes the preparation was ready and the cold point was applied with freezing f o r t h i r t y seconds. Within t h i r t y seconds a f t e r thawing, spreading s t a s i s was observed i n a l l except the large vessels and the arterio-venous shunts, within the injured area, which was a r e l a t i v e l y small f i e l d . Two minutes l a t e r i t was noted that the adjacent areas were quite hyperemic, although no spread of the vascular s t a s i s occurred within another two minutes. Generally th i s was an excellent preparation as f a r as obtaining a good vascular bed f o r observation was concerned. At the edges of the s t a t i c area pulsations could be seen entering the closed channels from a r t e r i o l e s . No reopening of any vessels was observed. The prep- aration was discontinued eight and a h a l f minutes after thawing. 138 Animal 10 (Test) A normal adult nonpregnant female Wistar r a t weighing 165 grams was injected subcutaneously with 8.6 mg. of Etamon chloride, and i n fourteen minutes i t was anaesthetized with 0.115 cc. ©f 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y , with 0.05 ce. more being given i n f i v e minutes. The prepara- t i o n was ready within thirteen minutes after the second nembutal i n j e c t i o n , but the animal had to be discarded because of to© much vascular s t a s i s gener- a l l y i n the mesoappendix. Animal 11 (Control) A normal adult nonpregnant female Wistar r a t weighing 165 grams was anaesthetized with 0.125 cc. of 0.6$ nembutal solution i n t r a p e r i t e n e a l l y , and the preparation was ready i n six minutes. The cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds and within t h i r t y seconds of thawing, s t a s i s had started and some ecchymosis was present. In another minute i t was observed that the s t a s i s was l i m i t e d to only one c a p i l l a r y bed. By three and a h a l f minutes after thawing the larger channels i n the area were slowing, but open. This f i e l d and the degree of cold i n j u r y were very comparable to the s i t u a t i o n i n Animal 9. Animal 12 (Test) A normal adult nonpregnant female Wistar r a t 139 weighing 170 grams was injected with 8.5 mg. of Etamon chloride subcutaneously and within four minutes 4.0 mg. was given a d d i t i o n a l l y , making the t o t a l dosage about 75 mg. per kilogram body weight. Fourteen minutes l a t e r 0.10 cc. of 0.6$ nembutal solution 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 and i n another twelve minutes the preparation was ready. The blood vessels i n the mesoappendix were observed to be very much d i l a t e d , and hyperemic. However, the cold point was applied to a selected area with fr e e z i n g for 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. The animal was considered moribund, and was discarded without further observ- ation. Animal 13 (Test) A normal adult nonpregnant female Wistar r a t weighing 160 grams was injeeted subcutaneously with 8.0 mg. of Etamon chloride, and fourteen minutes l a t e r i t was anaesthetized with 0.075 cc. of 0.6$ nembutal solution administered Intraperitoneally. The preparation was ready i n s i x minutes and the cold point was applied to a selected area of the mesoappend- i x with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing there was beginning venous s t a s i s which spread r a p i d l y , and by two and a h a l f minutes after thawing the whole area was s t a t i c . Adjoining areas were observed to be normal, with t r i c k l i n g of 140 blood Into the Injured area from the a r t e r i o l e s at the periphery. The s t a s i s d i d not spread further. No channels were observed to ©pen up and resume flow. The preparation was discontinued f i v e and a h a l f minutes afte r thawing. Summary of Observations with Etamon No. Onset Stasis Spread Generalized Plow Resumed 1 Test 30" Rapidly 5^« N©ne 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 s t a s i s 4 Control 30" Rapidly 5» None 5 Control N© r e s u l t , f a t obscured c i r c u l a t i o n 6 Test No r e s u l t , anlmalfound dead In cage 7 Test No r e s u l t , animal died on microscope stage 8 Test No r e s u l t , preparation n©t p h y s i o l o g i c a l 9 Test 30" Rapidly 30", l i m i t e d N©ne 10 Test No r e s u l t , general vascular s t a s i s 11 Control 30" None None N©ne 12 Test No r e s u l t , animal moribund on microscope 13 Test 30" Rapidly 2-̂ » None Discussion ©f Observations Etam©n chloride i n excess of 50 mg. per k i l o - gram body weight i s too toxie for p r a c t i c a l use. The number of animals with v a l i d data i n t h i s series i s too l i m i t e d f o r any conclusions to be made. There were only three test animals and two controls. Observations with the control preparations 141 agree f a i r l y well with controls i n other experiments. In view of t h i s , i t would seem that no value above the error i n t r i n s i c with the method could be claimed for Etamon. There, was no apparent delay i n the onset of s t a s i s , no apparent decrease i n the spread or extent of the s t a s i s , and no evidence that Etamon enabled any of 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 with Hydergine A summary of the following data appears i n table form a f t e r Animal 15. Animal 1 (Test) A female non pregnant adult Wistar r a t weigh- ing 185 grams was used. I t was injected subcutan- eously with 0.33 cc. containing 0.1 mg. of Hydergine. This seemed to have some e f f e c t i n making the animal more docile , and twenty-six minutes l a t e r , 0.10 cc. of 0.6$ nembutal solution was Injected i n t r a p e r i t o n - e a l l y f o r anaesthesia. Within seven minutes the preparation was ready, but the animal had to be d i s - carded because a slowly spreading vascular s t a s i s was observed throughout the mesoappendix. Animal 2 (Test) An adult nonpregnant female Wistar r a t weigh- ing 185 grams was injected subcutaneously with 0.33 cc. 1 4 2 containing 0.1 mg. of Hydergine, and twenty-three minutes l a t e r , the animal was anaesthetized with 0.075 cc. of 0.6$ nembutal solution administered in t r a p e r i t o n e a l l y . In seven minutes the preparation was ready, and the cold point was applied to a s e l - ected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing a r a p i d l y spreading general small vessel s t a s i s was observed, but noteworthy was the f a c t that two and a ha l f minutes after thawing, a few small vessels that were In vascular s t a s i s appeared to reopen, and by ten minutes a f t e r thawing quite a marked resumption of the c i r c u l a t i o n was noted i n many vessels that otherwise would c e r t a i n l y not have been expected t© ©pen up. I t i s possible that vasomotion was ob- served, but i t i s more l i k e l y that the resumption of flow was merely a matter of a passive fl u s h i n g of the sludged c e l l s by the more effective^hydrostatic and pulsating pressure permitted by the action of the Hydergine• Animal 5 (Control) An adult nonpregnant female Wistar r a t weigh- ing 185 grams was anaesthetized with 0.10 cc. of 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 on the mieroscope stage within seven minutes. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds, and immediately following thawing, 143 st a s i s i n c a p i l l a r y beds and small venous channels was noted. In another t h i r t y seconds the large veins were slowing, and by two minutes after thawing the small venous s t a s i s was slowly spreading over the injured area. A few larger venous channels opened up following a temporary arrest i n flow, not exactly the same as the venous s t a s i s otherwise described. After f i v e minutes following thawing, the a r t e r i o l e s and arterio-venous shunts i n the injured area were a l l regarded as free flowing, and the rest of the c i r c - u l a t i o n i n the mesoappendix was regarded as normal. The preparation was discarded. Animal 4 (Test) An adult nonpregnant female Wistar r a t weighing 170 grams was injected with 0.33 cc. of Hydergine, containing 0.1 mg. of active p r i n c i p l e , administered subcutaneously, and twenty-eight minutes l a t e r , 0.075 ce. of 0.6$ nembutal solution was injected i n t r a - p e r i t o n e a l l y . Within eight minutes the preparation was ready, and the cold t i p was applied to a selected area of the mesoappendix with freezing f o r t h i r t y sec- onds. Vascular s t a s i s i n f i v e c a p i l l a r y beds was s t a r t i n g within f o r t y - f i v e seconds aft e r thawing, although this apparent delay i n onset ©f f i f t e e n sec- ends was not considered s i g n i f i c a n t . I t was noted that several normally functioning c a p i l l a r y beds were 144 interspersed through the s t a t i c ones. By two and a h a l f minutes following freezing, several of the s t a t i c venous and c a p i l l a r y systems "flushed" open with active vasomotion observed i n the p r e c a p i l l a r i e s . I t was noted f i f t e e n minutes a f t e r thawing that one large 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 blood corpuscles did resume, possibly because of the twisting and moving of the animal under the l i g h t anaesthetic. One minute l a t e r the preparation was discontinued because of general vascular slowing generally throughout the mesoappendix. Animal 5 (Control) An adult nonpregnant female Wistar r a t weigh- ing 145 grams was anaesthetized with 0.075 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 preparation was ready i n ten minutes, and the cold point was applied to a selected area of the mesoappend- i x with f r e e z i n g f o r t h i r t y seconds. Within f o r t y - f i v e seconds after thawing there was venous slowing and s t a s i s with r a p i d l y spreading s t a s i s i n the venules i n the Injured area. Two and a h a l f minutes after thaw- ing the stasis was spreading but many normal c a p i l l a r y beds were functioning normally i n between, and also i n the very centre of the Injured area. By seven and a h a l f minutes following the thawing there was no further change to be noted. The s t a s i s did not become 145 generalized nor complete i n the affected area. The re s t of the vasculature i n the mesoappendix appeared r e l a t i v e l y normal when the preparation was discontin- ued. Animal 6 (Test) An adult nonpregnant female Wistar rat weigh- ing 145 grams was injected subcutaneously with 0.1 mg. of Hydergine contained i n 0.33 c c , administered sub- cutaneously, and i n twenty-five minutes 0.075 cc. of 0.6$ nembutal solution was injected Intraperitoneally. The preparation was ready ten minutes l a t e r , but the animal had to be discarded because of generalized vascular s t a s i s and ecchymosis i n the mesoappendix. Animal 7 (Test) An adult nonpregnant female Wistar r a t weigh- ing 140 grams was injected subcutaneously with 0.1 mg. ©f Hydergine contained i n 0.33 c c , and i n twenty- f i v e minutes 0.05 ce. ©f 0.6$ nembutal solution was administered Intraperitoneally, with 0.025 cc. being repeated four minutes l a t e r . The preparation was readfc four minutes l a t e r , and the cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds after thaw- ing, small c a p i l l a r y s t a s i s spreading to other beds was noted, and i n another minute a f a i r l y extensive area was i n stasis with only one large artery flowing. 146 Actually the injured area i n t h i s preparation was about four times as large as i n animals 1 t© 6, and i t was f e l t that i f Hydergine had any r e a l value i n aiding the flushing of c e l l s through the s t a t i c vessels, the area i n stasis was to© extensive f e r the c e l l s to f l u s h through such a long channel by means of a r t e r i o l e pulsations. By twelve and a h a l f minutes following thawing, no further 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. At the p e r i - phery of the s t a t i c areas a few a r t e r i o l e s were ob- served to be pulsating into the s t a t i c vessels. The preparation was discontinued three minutes l a t e r . Animal 8 (Control) An adult nonpregnant female Wistar r a t weigh- ing 115 grams was anaesthetized with 0.05 cc. of 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 within six minutes. The cold t i p was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds. Within f o r t y - f i v e seconds of thawing a r a p i d l y spreading vascular s t a s i s occurred. By two minutes a f t e r thaw- ing, ©nly the main channels and the arterio-venous shunts were observed to be flowing, and many of these were observed to be slowing. After a further three minutes, no change was noted, with no further spread 147 i n the s t a s i s , and no reopening of any vascular channels. The warm saline drip was not resumed with th i s preparation u n t i l eight minutes after thawing, and i t was not observed to e f f e c t any Immediate additional s t a s i s , thus supporting the contention that the Ringer-Locke solution with gelatine and glucose added was f a i r l y p h y s i o l o g i c a l . The prep- aration was discarded one minute l a t e r , with good blood flow i n the r e s t of the mesoappendix. Note; The pH ©f the Ringer-Locke drip containing gelatine and glucose was adjusted before s t a r t i n g , using a Beckman pH meter and adjusting to a pH of 7.10 with sodium bicarbonate powder. Animal 9 (Test) An adult nonpregnant female Wistar r a t weigh- ing 175 grams was injected subcutaneously with 0.1 mg. of Hydergine, contained i n 0.33 c c , and twenty- eight minutes l a t e r the animal was anaesthetized with 0.075 cc. of 0.6$ nembutal solution administered Intraperitoneally. Within eight minutes the prep- aration was ready, but the animal was discarded since generalized vascular s t a s i s , and much eechymosis were present on mounting the mesoappendix. Animal 10 (Control) 0 An adult nonpregnant female Wistar r a t weigh- 148 ing 185 grams was anaesthetized with Q.10 ee. of 0,6$ nembutal solution administered i n t r a p e r i t o n e a l l y and seven minutes l a t e r the preparation was ready. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing generalized venous st a s i s was noted to be s t a r t i n g which spread rap- i d l y . In one minute more the whole injured area was i n vascular s t a s i s . The preparation was discontin- ued with no further change, the remainder of the mesoappendix apparently having normal c i r c u l a t i o n . Animal 11 (Test) An adult nonpregnant female Wistar r a t weigh- ing 165 grams was injected subcutaneously with 0.1 mg. of Hydergine ( i n 0.33 cc.) and twenty-three minutes l a t e r i t was anaesthetized with 0.05 cc. of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y , with 0.025 cc. more being given In four minutes. Eleven minutes after the f i r s t nembutal i n j e c t i o n , the preparation was mounted on the microscope stage, ready f o r observation. The cold point was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Within t h i r t y seconds of thawing, vascular s t a s i s was observed to be s t a r t i n g . In one minute more, many of the channels that appear- ed closed were again functioning, and t h i s r e s t o r - 149 ation of c i r c u l a t i o n was not considered ctosbeodue to passive 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 after thawing there were only two c a p i l l a r y beds i n s t a s i s . One minute l a t e r , one of the c a p i l l a r y beds reopened widely, and appeared t© be f r e e l y flowing. By nine and a h a l f minutes aft e r thawing there was only one c a p i l l a r y bed showing vascular s t a s i s , and the preparation was discontin- ued with the c i r c u l a t i o n i n the remainder of the mesoappendix apparently normal. Animal 12 (Test) An adult nonpregnant female Wistar r a t weigh- ing 170 grams was injected with 0.1 mg. of Hydergine contained i n 0.33 cc. subcutaneously, and twenty- f i v e minutes l a t e r 0.075 cc. ©f 0.6$ nembutal s o l - ution 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 preparation was ready, but the animal was discarded beeause of extensive vascular s t a s i s and eechymosis throughout the meso- appendix c i r c u l a t i o n . Animal 15 (Test) An adult nonpregnant female Wistar r a t weigh- ing 150 grams was injected subcutaneously with 0.1 mg. of Hydergine contained In 0.33 ce. Thirty-three minutes l a t e r the animal was anaesthetized with 0.15 150 cc. of 0.6$ nembutal solution administered i n t r a - muscularly. In nine minutes the preparation was ready and no vascular st a s i s was noted i n the c i r c - u l a t i o n of the mesoappendix. The cold point was applied with freezing f o r t h i r t y seconds. Stasis f i r s t appeared s i x t y seconds a f t e r thawing, and i n only three c a p i l l a r y beds, withjhowever^ some slow- ing i n the larger veins. In another two minutes a few of the closed arterio-venous shunts reopened a channel through. At nine and a ha l f minutes after thawing, a very short venous channel reopened. By six minutes l a t e r there had occurred no further changes. The same four s t a t i c c a p i l l a r y beds were s t i l l shut down with excellent a r t e r i o l e flow through them i n the arterio-venous channels. The remainder of the c i r c u l a t i o n appeared normal, with moderate hyperemia present throughout. The preparation was discontinued nineteen and a h a l f minutes a f t e r thaw- ing. Animal 14 (Control) An adult nonpregnant female Wistar r a t weigh- ing 150 grams was anaesthetized with 0.10 cc. of 0.6$ nembutal solution administered intramuscularly, and seven minutes l a t e r the dosage was repeated with immediate death of the animal, presumably through intravenous i n j e c t i o n . 151 Animal 15 (Control) An adult nonpregnant female Wistar r a t weigh- ing 170 grams was anaesthetized with 0.10 cc. of 0.6$ nembutal solution, administered intramuscularly i n three divided s i t e s , and eleven minutes l a t e r the preparation was ready on the microscope stage. The cold point was applied with freezing to a selected s i t e i n the mesoappendix f o r t h i r t y seconds. Stasis was observed within t h i r t y seconds of thawing, and was slowly spreading to involve i n the next two min- utes several c a p i l l a r y beds within the injured area. Pour minutes l a t e r the preparation was discontinued, with no further change being noted. Note; The i n j e c t i o n of the nembutal other than i n t r a p e r i t o n e a l l y appears to lessen the incidence ef the vascular stasis which has been so wasteful of animals even before any testing could be done. Summary ef Observations with Hydergine No. Onset Stasis 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" Rapidly F a i r l y gen'l 2£«, max by 10» 3 Control Immediate 2» Almost t o t a l None 4 Test 45" Not even None 2^' i n many _ areas 152 Summary (Cont'd) No. Onset S t a s i s Spread G e n e r a l i z e d Flow Resumed 5 C o n t r o l 45" R a p i d l y None None 6 Test No r e s u l t s , mesoappendix i n s t a s i s 7 Test 30" Rapid w i t h i n 1' Almost t o t a l None 8 C o n t r o l 45" R a p i d l y Except A-V None shunts 9 Test No r e s u l t s , mesoappendix i n s t a s i s 10 C o n t r o l 30" R a p i d l y 90" None 11 Tes t 30" F i n a l l y , o n l y None /// at 90" 1 Cap. bed 12 Test No r e s u l t s , mesoappendix In s t a s i s 13 Test 60" To f o u r Cap beds None // a t 3' 14 C o n t r o l No r e s u l t s , i ntravenous nembutal i n j e c t i o n 15 C o n t r o l 30" Slowly Yes None D i s c u s s i o n of Observations w i t h Hydergine I t would seem warranted to r e f r a i n from i n t r a - p e r i t o n e a l i n j e c t i o n s i n case the m a t e r i a l i n j e c t e d induces v a s c u l a r s t a s i s i n the mesoappendix. C o n t r o l p r e p a r a t i o n s behaved much as i n other experiments. There was i n the c o n t r o l animals custom- ary f a i l u r e of any 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 b l o o d v e s s e l s once they went i n t o s t a s i s f o l l o w i n g thawing. The Hydergine p r e t r e a t e d r a t s d i d not show a d e f i n i t e tendency that was c o n s i s t e n t toward resumption of c i r c - u l a t i o n a f t e r the v a s o s t a t i c e f f e c t of the f r e e z i n g i n j - u r y . Other d i f f e r e n c e s were not c o n s i d e r e d s i g n i f i c a n t 153 following thawing. Apresoline Experimental Observations with Apresoline A summary of the folbwing observations w i l l be found below i n table form after Animal 11. Preliminary test f o r t o x i c i t y 10 mg. of Apresoline was administered subcut- aneously to a 200 gram nonpregnant adult female Hooded r a t . In ninety minutes there were very obvious signs ©f a serious reaction. At the same time, 2 mg. of Apresoline was s i m i l a r l y administered to another animal of the same size , and i n ninety minutes i t was apparently normal. Animals used Adult male Hooded ra t s , f i v e i n a l l , i n weight range of 190 to 220 grams were chosen f o r testing, and to these at one time was administered 2 mg. of Apresoline subcutaneously. By f i f t e e n minutes, a l l f i v e of the injected animals were ob- viously influenced by the drug, though not ad- versely, 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. Five adult male Hooded rats i n the weight range of 150 t© 250 grams were selected t© serve as controls, and these were not injected with Apres- o l i n e . 154 Animal 1 (Control) The animal was anaesthetized with 0.1 cc. of nembutal solution 0.6$ subcutaneously, and i n f i v e minutes 0.125 cc. more were administered. Stasis started within ninety seconds of thawing. There was minimal spread af t e r three minutes of thawing, and the greatest part of the injured area was normal. Only twenty percent of the i n - jured vessels'were i n s t a s i s . There was no further extension by s i x minutes after thawing. No resumption of flow was observed. Note; The Ringer-Locke saline drip solution with gelatine and glucose had been buffered without use of the Beckman pH meter, and i t was possible the solution 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 material was f a i r l y physiolog- i c a l with no s i g n i f i c a n t adverse effects on the vessels being noted. Animal 2 (Test) The animal was anaesthetized with 0.075 cc. of 0.6$ nembutal solution 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 within t h i r t y seconds ©f thawing, the f i r s t occurring three minutes after 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 of thawing. This i s an e x c e l l e n t l y preserved f i e l d to observe. No revascularization was noted, nor any further change by seven and a half minutes following thawing. Animal 5 (Control) The animal was anaesthetized with 0.125 cc. of 0.6$ nembutal solution with no further nembutal being required. Freezing occurred f o r t y - f i v e minutes after the nembutal i n j e c t i o n . After f r e e z - ing, a small a r t e r i o l a r haemorrhage was noted, which was not severe enough to af f e c t the animal generally or the mesoappendix vasculature i n part- i c u l a r . At nine and a h a l f minutes afte r the thaw- ing there was generalized vascular s t a s i s through- out the injured area. No intermediate observations were made. These observations were not included In the summary table. Animal 4 (Test) The animal was anaesthetized with 0.125 cc. of 0.6$ nembutal solution. Freezing occurred one hour and f o r t y minutes a f t e r the Apresoline admin- i s t r a t i o n . The freezing took f o r t y seconds Before thawing could commence, and except f o r one strongly flowing arterio-venous anastamosis, there was generalized vascular s t a s i s i n the injured area to the extent of eighty percent within t h i r t y seconds 156 of thawing. There was no further changes noted by four and a h a l f minutes after thawing, and n© restoration of c i r c u l a t i o n apparent. Animal 5 (Control) There was n© s t a s i s apparent by f i v e and a h a l f minutes following thawing. Animal 6 (Test) The animal was anaesthetized with 0.125 cc. of 0.6$ nembutal solution. Stasis commenced within t h i r t y seconds of thawing. Many channels reopened and the blood flowed f r e e l y through them. By tw© and a h a l f minutes after thawing a l l channels were restored. This was not the usual type of vascular s t a s i s . The whole injured area became f r e e l y flowing, and remained so by seven add a half min- utes aft e r thawing when the preparation was d i s - continued. Note; Into fco-theru:thr.e:e male adult Hooded rats was injected 2.0 mg. of Apresoline subcutaneously. Animal 7 (Test) The animal was anaesthetized with 0.15 cc. of 0.6$ nembutal solution, and freezing i n j u r y was i n f l i c t e d seventy-five minutes a f t e r the Apres- oline was administered. Stasis commenced within t h i r t y seconds a f t e r thawing. By ninety seconds 157 there was rapid spread to the whole of the injured area. No further change occurred by f i v e minutes after thawing, and n© re s t o r a t i o n of the c i r c u l - ation was noted. Animal 8 (Control) The animal was anaesthetized with 0.15 cc. of 0.6$ nembutal solution. Stasis commenced within t h i r t y seconds of thawing. F i f t y percent of the injured area was i n s t a s i s within three min- utes aft e r thawing. No restoration ©f the c i r c - u l a t i o n was ©bserved. Animal 9 (Test) The animal was anaesthetized with 0.15 cc. of 0.6$ nembutal solution. Freezing occurred approx- imately ninety minutes aft e r administration of the Apresoline. Stasis occurred In only one c a p i l l a r y bed, and i n that by s i x t y seconds after thawing. There was also one small s i t e of eechymosis. There were no further changes by f i v e minutes a f t e r thaw- ing, and no restoration of the c i r c u l a t i o n . Animal 10 (Test) The animal was anaesthetized with 0.15 cc. ofnembutal 0.6$ solution. Freezing occurred approx- imately two hours aft e r administration of the Apres- o l i n e . Stasis occurred with'Hhirty seconds of thaw- 158 Ing. Within s i x t y seconds ©f thawing, f i f t y percent of the injured area developed vascular s t a s i s . Only one arterio-venous bridge was funct- ioning by three minutes after thawing. N© restor- ation of the s t a t i c c i r c u l a t i o n was observed. Animal 11 (Control) Tbe animal was anaesthetized with 0.10 cc. of 0.6$ nembutal solution. Stasis developed within f i f t e e n seconds of thawing,, and ra p i d l y spread t© involve t h i r t y percent ©f the Injured area within s i x t y seconds aft e r thawing. Much ecchymosis was present i n the s t a t i c area. The sta s i s slowly spread to involve f i f t y percent of the Injured area within three minutes a f t e r thawing. No re s t o r - ation of the c i r c u l a t i o n was noted. Addendum In the case of Animal 2, freezing occurred ©ne hour after the Apresoline injecti©n. With Animal 6, I t was two hours and ten minutes. Summary ©f Observations with Apresoline No. Onset Stasis Spread Generalized glow Resumed 1 Control 90" Minimal 20$ In 3« None 2 Test Slowly 1 Cap. bed i n None 3 Control Results not included 4 Test 30" 80$ i n 30" 80$ i n 4£» None 159 Summary (Cont'd) No. Onset Stasis Spread Generalized Plow Resumed 5 Control None by 5-|' 6 Test 30", but complete re s t o r a t i o n by 2^» 7 Test 30" Rapidly Complete by 90" None 8 Control 30" Steady 50$ i n 3» None 9 Test 60", i n only 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$ In 3» None Discussion ef Observations No difference can be claimed between the be- havior ef the test and the control group i n respect to time before onset of s t a s i s , extent of the s t a s i s , or resumption of flow i n s t a t i c channels, Chler-Tripolon Experimental Observations with Chlor-Tripolon Into each of six adult nonpregnant female Wistar rats In the 150-200 gram weight range was i n - jected subcutaneously 5 mg, of Chlor-tripolon maleate dissolved i n water to a strength of 50 mg. per cc, (0,1 cc, f o r each animal). The i n j e c t i o n was made one hour before preparaing the animals f o r microscopic observation. Six s i m i l a r animals were selected as controls. Eaeh animal was anaesthetized before preparation with a subcutaneous i n j e c t i o n of 0.15 cc. 160 ©f 0.6$ nembutal solution. A summary of the f e l l - owing observations i n table form w i l l be found aft e r Animal 12. The usual drip solution was used. Animal 1 (Control) Stasis started within t h i r t y seconds of thaw- ing. Twenty percent of the injured area was i n st a s i s within s i x t y seconds after thawing, and s i x t y percent within three minutes. One venule opened and closed i n t e r m i t t e n t l y . There were no further changes observed within f i v e minutes after thawing.' No res 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 f i f t e e n seconds aft e r thawing. Within sixty seconds aft e r thawing, stasis was present i n the whole of the injured area except f o r one large arterio-venous bridge. Three small ecchymoses were present within the frozed area. No further change was noted within f i v e minutes after thawing, and no resumption of flow was observed. Animal 3 (Control) Stasis started within t h i r t y seconds a f t e r thawing. Sixty percent of the injured area was involved within f o r t y - f i v e seconds after thawing. After two minutes only one large venous channel was flowing within the injured area. There was no further change noted after four minutes, and n© re- storation of c i r c u l a t i o n i n any s t a t i c channel. 161 Animal 4 (Test) Stasis commenced si x t y seconds after thawing, i n one c a p i l l a r y bed ©nly. A l i t t l e distance away one venule was observed to be opening and closing, but t h i s vessel became s t a t i c two and a h a l f minutes af t e r thawing. Stasis spread to only ©ne ©ther c a p i l l a r y bed by three and a h a l f minutes aft e r thaw- ing. This amount ©f reaction was a rather l e s s e f f e c t than would be expected from the amount ©f v a s c u l a r i t y i n the injured area. By f i v e minutes a f t e r thawing twenty percent of the Injured area was i n s t a s i s . N© restoration of the c i r c u l a t i o n was noted. The Ringer- Locke gelatin-glucose drip was not observed to have any appreciable harmful e f f e c t on the behaviour of the vessels i n the preparation. Animal 5 (Control) Stasis started t h i r t y seconds a f t e r thawing. There was rapid spread t© involve twenty-five percent of the injured area i n s t a s i s within s i x t y seconds. Sixty pereent of the area was s t a t i c by three minutes after thawing. Affew venules were observed to s t a r t and stop t h e i r flow of blood, with no r e a l s t a s i s i n these channels. There was no further change by f i v e minutes, and no resumption of flow observed. Animal 6 (Test) Stasis was noted throughout the mesoappendix 162 on mounting and the preparation was discarded. Animal 7 (Control) Stasis occurred within f i f t e e n seconds a f t e r thawing. I t became generalized i n the injured area within f o r t y - f i v e seconds aft e r thawing. One ven- ous channel was observed to be fl u s h i n g open every l i t t l e while. The whole injured area was s t a t i c by three and a h a l f minutes after thawing, and no f u r t h - er change was noted by f i v e minutes, with no resumpt- ion of flew apparent. Animal 8 (Test) Stasis started within f i f t e e n seconds of thawing, and became generalized throughout the i n j u r - ed area by si x t y seconds. No resumption of flow was noted except i n a few of the larger venules that were not t o t a l l y s t a t i c . No further change was noted by f i v e minutes aft e r thawing. Animal 9 (Control) Stasis started within f o r t y - f i v e seconds after thawing. Twenty-percent of the injured area was involved within two minutes aft e r thawing, and seventy-five percent i n four minutes. No resumption of flow was noted. Animal 10 (Test) Stasis started within f i f t e e n minutes aft e r thawing, and was t e t a l i n the injured area within 163 s i x t y seconds except f o r one central venous channel that stopped and started f o r another s i x t y seconds only. There was no further change noted by three minutes a f t e r thawing. Animal 11 (Control) Stasis started f o r t y - f i v e seconds after thaw- ing. Only one c a p i l l a r y bed became involved by stas i s by three minutes aft e r thawing. No further changes were noted by f i v e minutes after thawing. Animal 12 (Test) Stasis started s i x t y seconds after thawing, spreading by two minutes aft e r thawing. By four minutes after thawing eighty percent of the injured area was i n s t a s i s , and by f i v e minutes a f t e r thaw- ing the area was one hundred percent s t a t i c . N© resumption of flow was noted. Summary of Observations with Chlor-Tripolon No. Onset Stasis Spread Generalized Plow Resumed 1 Control 30" 20% i n 60" 60$ In 3' None 2 Test 15" Rapid 90$ i n 60" None 3 Contrel 30" 60$ i n 45" 90$ i n 2« None 4 Test 60" Sl i g h t Two beds i n 3^» None 5 Control 30" 25$ i n 60" 60$ In 3' None 6 Test Preparation discarded 7 Control 15" General i n 45" 100$ i n 3£» None 164 Summary (Cont'd) N©« Onset Stasis Spread 8 Test 15tt 9 Control 45" 10 Test 15" 11 Control 45" 12 Test 60" Generalized Flew Resumed None None None None None Rapid General i n 60" 20$ i n 2' 75$ i n 4« 90$ i n 60" 100$ i n 2» None 1 Cap bed i n 3» 80$ i n 4' 100$ i n 5« Discussion of Observations The animals pretreated with Chlor-Tripolon could be claimed to d i f f e r i n no respect from those control animals not pretreated with Chlor-Tripolon, Rutin Experimental Observations with Rutin A l l the observations regarding r u t i n micro- sc o p i c a l l y were performed by a senior medical student helping with the project part time. His method of timing i s s l i g h t l y varied from that used elsewhere, and he uses the term "general s t a s i s " to apply to the whole vascular bed of the mesoappendix mount, whereas the term i s used elsewhere to apply only to the vessels within the f i e l d a c t u a l l y frozen by the cold point, the so-called injured area. When stasis develops throughout thewhole preparation, such a mounting i s regarded as unphysiological, and the animal i s d i s - carded, and the re s u l t s are not included. 165 The test animals were prepared with r u t i n p r i o r te the experimental observation by sel e c t i n g si x normal adult male Wistar r a t s , placing them i n one cage, and putting Inte t h e i r drinking water the ground up r u t i n t a b l e t s . 600 mg. of r u t i n was placed i n about 250 cc. of water, and the animals were observed to be drinking the water s a t i s f a c t - o r i l y . This was kept before them f o r f i v e f u l l days, besides t h e i r usual feed, and then they were tested i n the usual manner as outlined below. Eight s i m i l a r animals were selected as controls. These were managed i n a l i k e manner, but did not receive any r u t i n . The following observations are summarized i n table form after Animal 14. Animal 1 (Control) A male Wistar r a t weighing 220 grams that had received no p r i o r medication with r u t i n was anaesthet- ized with 0.15 cc. of 0.6$ nembutal solution injected subcutaneously. The preparation was ready twelve minutes l a t e r , but was discarded because i t f e l l o f f of the microscope stage. Animal 2 (Control) A male Wistar r at weighing 180 grams that had received no p r i o r treatment with r u t i n was anaesthet- ized with 0.15 cc. of 0.6$ nembutal solution, i n j e c t - ed subcutaneously, but the preparation had to be 166 discarded (reason not recorded). Animal 5 (Control) An untreated male Wistar r a t of 190 grams was anaesthetized with 0.15 cc. of 0.6$ nembutal s o l - ution. A selected area of the meaioappendix was frozen f o r t h i r t y seconds with the cold point apparat- us, followed by immediate rapid thawing with the warm drip solution which had been discontinued during the freezing procedure. Vascular s t a s i s began within t h i r t y seconds after thawing and spread to involve about six c a p i l l a r y beds by f i v e minutes afte r thaw- ing. There was no generalized s t a s i s or any appar- ent restoration of blood flow i n the s t a t i c vessels. Numerous c a p i l l a r i e s and one medium sized venule were Involved. Animal 4 (Control) A normal adult male Wistar rat of 190 grams that had received no p r i o r medication was anaesthet- ized with 0.15 cc. of 0.6$ nembutal solution. Seven- teen minutes l a t e r a further 0.05 cc. of nembutal had to be administered, and the animal was s a t i s f a c t o r i l y anaesthetized f i f t e e n minutes l a t e r . A selected area of the mesoappendix was frozen f o r t h i r t y sec- onds followed by immediate rapid thawing. Stasis began within t h i r t y seconds of thawing, and spread 167 to involve f i v e or six c a p i l l a r y beds i n two minutes. There was no general s t a s i s and no resumption of flow i n those vessels i n which st a s i s occurred. C a p i l l - aries and small c o l l e c t i n g venules were involved. Animal 5 (Control) A 170 gram normal adult male Wistar r a t which had received no p r i o r treatment was anaesthetized with 0.15 cc. of 0.6$ nembutal solution requiring an addit- ional 0.05 cc. twice within the following f i f t e e n min- utes. Vascular s t a s i s began within t h i r t y seconds after thawing and involved about ten c a p i l l a r y beds within four minutes. There was no general s t a s i s or resumtpion of flow. C a p i l l a r i e s and small ven- ules were affected. Animal 6 (Test) A 170 gram normal adult Wistar male r a t treated with r u t i n was anaesthetized with 0.15 cc. of 0.6$ nembutal solution followed In f i f t e e n min- utes by another 0.05 cc. Following rapid thawing, vascular s t a s i s began within t h i r t y seconds and spread to involve three or four c a p i l l a r y beds by four min- utes after thawing. There was no general spread. Restoration of c i r c u l a t i o n was noted i n a c o l l e c t i n g venule two minutes afte r thawing. C a p i l l a r i e s and small c o l l e c t i n g venules were involved i n the s t a s i s . Animal 7 (Control) A normal adult male Wistar r a t of 170 grams 168 previously untreated with r u t i n was anaesthetized with 0.15 cc. of 0.6$ nembutal solution, without any additional nembutal being required, which resulted i n a l i g h t l y anaesthetized animal. No s t a s i s occurred i n t h i r t y seconds. Reversal of d i r e c t i o n of c i r c u l a t i o n was noted i n some of the medium sized venules within one minute of thawing, but no s t a s i s was observed anywhere In the f i e l d . Animal 8 (Test) A normal adult male Wistar r a t of 170 grams, previously treated with r u t i n , was anaesthetized with 0.17 cc. of 0.6$ nembutal solution, followed i n f i f t e e n minutes with 0.07 cc. more, both i n j e c t - ions being given subcutaneously. Stasis began within f o r t y - f i v e seconds of rapid thawing, and spread s l i g h t l y to involve about three c a p i l l a r y beds within f i v e minutes after thawing. Stasis was s l i g h t and involved only about one t h i r d of the vessels i n the injured area. No general s t a s i s or 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 the s t a t i c vessels was noted. Cap- i l l a r i e s and small venules were involved i n the s t a s i s . Animal 9 (Te3t) A normal adult male Wistar r a t of 160 grams was anaesthetized with 0.15 cc. of 0.6$ nembutal solution after f u l l pretreatment with r u t i n . Stasis commenced seventy-five seconds aft e r thawing, and spread to involve two or three c a p i l l a r y beds i n four 169 minutes. About one t h i r d of the vessels i n the injured area were involved. No general s t a s i s or resumption of flow was observed to occur. C a p i l l a r - ies and small c o l l e c t i n g venules were affected. Animal 10 (Test) A normal adult male Wistar r a t of 210 grams previously treated with r u t i n was anaesthetized with 0.21 cc. of 0.6$ nembutal solution administered sub- cutaneously. Vascular s t a s i s began with one minute after rapid thawing, and spread to involve by four minutes after thawing only four or f i v e c a p i l l a r y beds. About two thirds of the vessels i n the i n - jured area were involved. No general s t a s i s or re- sumption of flow was observed. C a p i l l a r i e s and small c o l l e c t i n g venules were involved i n the s t a s i s . Animal 11 (Control) A normal adult male Wistar r a t of 220 gram3 not previously treated with r u t i n was anaesthetized with 0.18 cc. of 0.6$ nembutal solution followed i n twenty minutes with 0.08 cc. more. The animal ex- pired during observation, and no r e s u l t s are record- ed. Animal 12 (Test) A normal adult male Wistar r a t of 180 grams, having received p r i o r treatment with r u t i n ©rally, was anaesthetized with 0.20 cc. of 0.6$ nembutal solution injected subcutaneously. Stasis commenced 170 within t h i r t y seconds a f t e r rapid thawing, and i t spread to involve about six c a p i l l a r y beds within three minutes a f t e r thawing. Two thirds of the vessels In the injured area were i n s t a s i s , but there was no generalized s t a s i s throughout the preparation. C i r c u l a t i o n was observed to be resumed i n two vessels. C a p i l l a r i e s and small venules were involved i n the s t a t i c process. Animal 13 (Control) A normal adult male Wistar rat of 190 grams not previously treated with r u t i n was anaesthetized with 0.20 cc. of 0.6$ nembutal solution administered subcutaneously. Stasis commenced within s i x t y - f i v e seconds after thawing, spreading so as to i n - volve three c a p i l l a r y beds within three minutes. About one quarter of the c a p i l l a r i e s i n the injured area were involved. There was no s t a s i s ott resumpt- ion of c i r c u l a t i o n i n the s t a t i c channels. Cap- i l l a r i e s only were involved i n the process. Animal 14 (Test) A normal adult male Wistar r a t of 190 grams previously treated with r u t i n , was anaesthetized with 0.20 cc. of 0.6$ nembutal solution administered sub- cutaneously. There was generalized vascular s t a s i s present ©n mounting the mesoappendix, and the prep- aration was discarded. 171 Summary of Observations with Rutin No, Onset ; Stasis 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 , discarded 3 Control 30" 6 beds i n 5» None None 4 Control 30" 5-6 beds In 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 , expired during observation 12 Test 30" 6 beds i n 3' None / 13 Control 65" 3 beds i n 3' None None 14 Test No r e s u l t s , general st a s i s on mounting Discussion of Observations The s i x test animals, f o r the f i v e days before ' the experiments took place, were kept i n one cage and allowed to drink from the same drinking b o t t l e s . This together with the f a c t the r u t i n tended to s e t t l e some- what to the bottom of the container probably caused an uneven d i s t r i b u t i o n of the medication among the r a t s . The average time aft e r thawing f o r s t a s i s to commence i n f i v e test animals was 50". Substantially the same time, 60", held f o r four control animals (not 172 considering Animal 7 i n which n© s t a s i s occurred). In animals 6 and 12, d e f i n i t e resumption of flow i n s t a t i c channels occurred. To quote the ©pinion of the observer of these experiments, "Rutin shows some delaying 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 involved. In two animals the channels resumed flow. These re s u l t s cannot be considered d e f i n i t e or Consistent enough to warrant p o s i t i v e conclusions, but the drug appears promis- ing, and the tests should be repeated with care to ensure an accurate dosage per animal." Further observations with Rutin Six ra t s as noted below were fed Rutin care- f u l l y so that each received twenty-five mg. per day f o r four days, followed by ©ne hundred mg. per day f o r four more days. They showed no adverse e f f e c t from the medication. Seven other animals were s e l - ected as controls. As mentioned these observations were made by a senior medical student. A summary i n table form appears after Animal 13. Animal 1 (Test) A normal adult nonpregnant female Wistar r a t weighing 190 grams, previously treated with r u t i n o r a l l y was anaesthetized with 0.1 cc. of 0.6$ nembutal solution administered subcutaneously. Vascular s t a s i s 173 was observed to occur immediately a f t e r rapid thawing, which spread to involve seven eighths of the injured area within two and a h a l f 'minutes after thawing. No generalized s t a s i s occurred, and no resumption of 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 (Control) A normal adult nonpregnant female Wistar r a t weighing 200 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution. 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© involve a l l vessels i n the injured area. Animal 5 (Test) A normal adult nonpregnant female Wistar r a t of 195 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution. Stasis started within f o r t y seconds after thawing, and spread to Involve h a l f the vessels i n the injured area within three minutes a f t e r thawing. Animal 4 (Control) A normal adult nonpregnant female Wistar r a t weighing 200 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution. The whole mesoappendix went into vascular s t a s i s from the freezing, and the prep- aration was discarded. Animal 5 (Test) A normal adult nonpregnant female Wistar r a t 174 weighing 160 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution administered subcutaneously. Stasis began within f o r t y seconds after thawing, and involved only four short segments of c a p i l l a r i e s . Animal 6 (Control) A normal adult nonpregnant female Wistar r a t weighing 180 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution. Stasis began i n f i f t y - f i v e seconds after thawing and spread to involve one t h i r d of the vessels i n the frozen area by four minutes after thawing. Animal 7 (Test) A normal adult nonpregnant female Wistar r a t weighing 200 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution administered subcutaneously. Stasis developed within f i f t y seconds after thawing, spreading to involve one quarter of the vessels i n the injured area. Animal 8 (Control) A normal adult nonpregnant female Wistar r a t weighing 180 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution. Stasis began i n s i x t y seconds aft e r thawing and involved only two or three c a p i l l a r i e s . There was resumption of flow i n sev- era l small c a p i l l a r i e s . 175 Animal 9 (Teat) A normal adult nonpregnant female Wiatar r a t weighing 180 grams was anaesthetized with 0.1 cc. of n.6$ nembutal solution. Stasis started f o r t y - f i v e seconds after rapid thawing, involving three or four short segments of c a p i l l a r i e s . Animal 10 (Control) A normal adult male Wistar r a t weighing 200 grams was anaesthetized with 0.1 cc. of 0.6$ nembutal solution administered subcutaneously. ( Stasis s t a r t - ed within one minute afte r thawing to involve h a l f the vessels i n the injured area. Animal 11 (Test) A normal adult nonpregnant female Wiatar r a t weighing 190 grams was anaesthetized with 0.13 cc. of 0.6$ nembutal solution. Stasis started s i x t y seconds afte r rapid thawing and spread to involve one t h i r d of the vessels In the injured area i n three minutes. Animal 12 (Control) A normal adult male Wistar rat weighing 200 grams was anaesthetized with 0.13 cc. of 0.6$ nemb- u t a l solution administered subcutaneously. Stasis began f u t h i r t y v e seconds afte r thawing and spread to involve two thirds of the vessels i n the injured area. 176 Animal 15 (Control) A normal adult male Wistar r a t weighing 200 grams was anaesthetized with 0.13 cc. of 0.6$ nem- butal solution administered subcutaneously. Stasis began f o r t y - f i v e seconds after thawing, and spread to involve two thirds of the vessels i n the injured area. Summary of Observations with Rutin No. Onset Stasis Spread Generalized Plow Re 1 Test Immediate Slow1 7/8 None 2 Control Immediate Rapid Complete None 3 Test 40" Slow 1/2 None 4 Control No res u l t s , preparation discarded 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 Observations As mentioned above, these observations using 177 r u t i n were performed by another worker. Prom reading the data, i t would appear that the fre e z - ing injury might be a l i t t l e more severe than was customary with the other t e s t s . Even though t h i s would prevent comparison with the controls of other experimemts, the test and control animals of t h i s run should be comparable. The time required f o r the onset of stasis a f t e r the rapid thawing was Identical f o r both series (average of six animals each), namely f o r t y seconds. The speed and extent of the spread of the sifeasis was the same f o r both the test and the control s e r i e s . I t i s also i n t e r e s t i n g to note that the only instance of resumption of flow i n the s t a t i c vessels occurred i n a control animal. To quote the impressions of 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 that r u t i n does not a l t e r the reaction of c a p i l l a r i e s i n the r a t mesoappendix afte r freezing and rapid thawing." Ascorbic Acid This drug was not tested microscopically because the observed effects on a gross standard freezing i n j u r y did not warrant further Investigation. 178 Histamine Experimental Observations with Histamine Histamine i n the dosage l e v e l of 100 mg. per kilogram body weight was tested by parenteral i n j e c t - ion. Summary table of the following observations appears after Animal 9. Animal 1 (Test) A normal adult male Wistar rat weighing 272 grams was injected subcutaneously with 0.27 cc. of a d i l u t e d histamine solution containing 100 mg. per c c , and f i f t e e n minutes l a t e r the animal was an- aesthetized with 0.075 c c of 0.6$ nembutal solut- ion administered subcutaneously. 0.05 cc. of nembutal were repeated i n ten minutes and another 0.025 cc. three minutes afterwards. The prepara- ti o n was ready on the microscope stage seventeen minutes afte r the f i n a l i n j e c t i o n , and the cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds, followed by immediate rapid thawing. Generalized vascular sta s i s developed i n the injured area within s i x t y seconds of thawing. Animal 2 (Test) A normal adult male Wistar r a t weighing 241 grams was Injected subcutaneously with 0.24 c c of a 1 7 9 s o l u t i o n c o n t a i n i n g 1 0 0 m g . o f h i s t a m i n e p e r c c , a n d s i x t y - t h r e e m i n u t e s l a t e r t h e a n i m a l was a n a e s t h - e t i z e d w i t h 0 . 2 0 c c . o f 0 . 6 $ n e m 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 s u b c u t a n e o u s l y . When t h e p r e p a r a t i o n was r e a d y , t h e c o l d t i p was a p p l i e d t o a s e l e c t e d p o r t i o n o f t h e m e s o a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . S t a s i s d e v e l - o p e d i n t h i r t y s e c o n d s a f t e r t h a w i n g , p r o g r e s s i n g r a p i d l y t o i n v o l v e s i x t y p e r c e n t o f t h e i n j u r e d a r e a w i t h i n t h r e e m i n u t e s a f t e r t h a w i n g . A n i m a l 3 ( C o n t r o l ) A n o r m a l m a l e a d u l t W i s t a r r a t w e i g h i n g 2 3 5 g r a m s was 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 $ n e m 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 b y s u b c u t a n e o u s i n j e c t i o n . I n t w e n t y m i n u t e s a f u r t h e r 0 . 0 5 c c . was i n j e c t e d a n d i n f i v e m i n u t e s a f t e r t h a t 0 . 0 5 c c . m o r e . The p r e p a r a - t i o n w a s r e a d y i n a n o t h e r e i g h t m i n u t e s , a n d t h e c o l d t i p w a s a p p l i e d t o a s e l e c t e d p o r t i o n o f t h e m e s o - a p p e n d i x w i t h f r e e z i n g f o r t h i r t y s e c o n d s , f o l l o w e d b y i m m e d i a t e r a p i d t h a w i n g . G e n e r a l s t a s i s d e v e l o p e d i n t h e i n j u r e d a r e a w i t h i n t h i r t y s e c o n d s a f t e r t h a w - i n g . A n i m a l 4 ( T e s t ) A n o r m a l a d u l t m a l e W i s t a r r a t w e i g h i n g 2 4 4 g r a m s was i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 2 4 c c . o f a 180 solution containing histamine, i n the concentration of 100 mg. per cc. The animal was anaesthetized i n twenty minutes with 0.20 cc. of 0.6$ nembutal solution administered by subcutaneous i n j e c t i o n , and when the preparation was ready on the microscope stage, the cold t i p was applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds. Stasis did not develop i n any vessel within seven minutes afte r thawing, at which time the preparation was discontinued. Good vasomotion was maintained throughout the observation period. Animal 5 (Test) A normal adult male Wistar rat weighing 247 grams was injected subcutaneously with 0.24 cc. of a solution containing 100 mg. of histamine per c c , and i n twenty-eight minutes the animal was anaesthet- ized with 0.20 c c of 0.6$ nembutal solution also administered by subcutaneous i n j e c t i o n . Stasis dev- eloped within t h i r t y seconds of thawing, and spread to involve t h i r t y percent of the injured area within four minutes ©f thawing. Animal 6 (Control) A normal adult male Wistar r a t weighing 208 grams was anaesthetized with 0.15 cc. of 0.6$ nem- butal solution administered by subcutaneous Injection. 181 The preparation was mounted on the microscope stage and the cold point was applied to a selected area of the mesoappendix with freezing wor t h i r t y seconds, followed by immediate rapid thawing. Stasis devel- oped within t h i r t y seconds a f t e r thawing, r a p i d l y spreading to become generalized within the injured area within ninety seconds after thawing. Animal 7 (Test) A normal adult male Wistar r a t weighing 270 grams was injected subcutaneously with 0.27 cc. of a histamine solution containing 100 mg. per c c , and i n t h i r t y - f i v e minutes the animal was anaesthetized with 0.20 cc. of 0.6$ nembutal solution also admin- i s t e r e d by subcutaneous i n j e c t i o n . The prepara- t i o n was mounted on the microscope stage and the cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds, followed by immediate rapid thawing. Stasis developed within f o r t y - f i v e seconds after thawing, becomming quite generalized within the injured area. Animal 8 (Control) A normal adult male Wistar r a t weighing 286 grams was anaesthetized with 0.25 cc. of a 0.6$ nem- butal solution, and when ready the preparation was mounted on the microscope stage. The cold point was. 182 applied to a selected area of the mesoappendix with freezing f o r t h i r t y seconds, followed by immediate rapid thawing. Stasis developed within s i x t y sec- onds after thawing, r a p i d l y becomming generalized within the injured area by two arid a h a l f minutes after thawing. Animal 9 (Test) A normal adult male Wistar r a t weighing 244 grams was injected with 0.24 cc. of a histamine s o l - ution containing 100 mg. per c c , and the animal was anaesthetized with 0.20 cc. of 0.6$ nembutal solution. The preparation was mounted and the cold point was applied to a selected area of the meso- appendix with freezing f o r t h i r t y seconds. Stasis started within t h i r t y seconds afte r thawing, but at no time during the period of observation did i t spread to involve more than twenty-five percent of the injured area. Summary of Observations with Histamine Parenteral ~' No. Bpsetastasis Spread Generalized Flow Resumed 1 Test 30"? Rapid by 60" None 2 Test 30" Rapid 60$ i n 3' None 3 Control 30" Rapid by 30" None 183 Summary (Cont'd) No. Onset Stasi3 Spread Generalized Flow Resumed 4 Test * None 5 Test '30" Slow 30$ i n 4» None 6 Control 30" Rapid by 90" None 7 Test 45" Rapid Yes None 8 Control 60" Rapid by Sfrt None 9 Test 30" Slowly only 25$ None Discussion of Observations These tests with parenteral histamine were somewhat of a t r i a l to determine an e f f e c t i v e dosage of histamine, and the recording was incomplete i n a few respects. There were i n s u f f i c i e n t control animals used, f o r one thing. Some of the d e t a i l s regarding time i n t e r v a l s are lacking. None the l e s s , i t would seem that histamine, as here used, had some d e f i n i t e e f f ects on the behavior of blood vessels after a l o c a l i z e d freezing i n j u r y . Although no difference was apparent i n the time aft e r thawing f o r st a s i s to s t a r t , the extent and rate ©f spread of the st a s i s seemed to be much l e s s i n the histamine treated animals. I t i s doubtful i f these findings are s i g n i f i c a n t i n view of the objections here raised. The severity of the freezing i n j u r y would seem to be somewhat greater here than i n other experiments* 184 Further Observations with Histamine The following tests were performed t© study the e f f e c t of histamine applied 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 before freezing with the cold point (the warm Ringer-Locke gelatin-glucose drip being temporarily discontinued f o r t h i s , but of course being resumed following freezing s© as t© eff e c t an immediate rapid thawing, and t© maintain a physiological preparation during the observation period). The usual technique was employed, and 150 gram normal adult nonpregnant female Wistar rats were employed, being anaesthetized f i f t e e n minutes before experimentation with 0.15 cc. of 0.6$ nembutal solution. A summary of the following observations appears i n table form aft e r animal 10. Animal 1 (Control) Stasis occurred within twenty seconds after thawing, which r a p i d l y spread to involve h a l f ©f the vessels within the injured area by si x t y seconds after i thawing. No resumption of flow was observed i n the s t a t i c vessels. I t was noted that the large a r t e r i o - venous bridges were flowing normally even through the Injured area. Animal 2 (Test) Histamine In 1:1000 concnetraion was flushed 185 over the mesoappendix p r i o r to free z i n g . This was observed to give generalized increase i n blood flow. After rapid thawing, s t a s i s was observed i n a larger venule within s i x t y seconds aft e r thawing. By ninety seconds after thawing, small c a p i l l a r y s t a s i s was found present, but i n ©nly one c a p i l l a r y bed with- i n the injured area. There was no further change noted up to two and a h a l f minutes afte r thawing when the preparation was discontinued. Animal 5 (Control) Stasis was noted within sixty seconds a f t e r thawing. This spread slowly to Involve other cap- i l l a r y beds. One large venous channel c l e a r l y showed hemoconcentration. Half the injured area was found to be i n s t a s i s by two minutes after thawing. At th i s time, histamine, 1:1000, was i n s t i l l e d over the observation f i e l d , and t h i s was noted to cause some d i l a t a t i o n ©f a venule with resumed blood flow In the large channel that had shown the hemoconcentration. There was no further change by f i v e minutes after thawing. The histamine had no e f f e c t on the small channels that were d e f i n i t e l y s t a t i c . Ninety sec- onds after the histamine was applied, one of the small venules resumed flow, and the whole c a p i l l a r y bed i t drained resumed c i r c u l a t i o n . This process 186 was not duplicated elsewhere within the injured area. Animal 4 (Test) Histamine 1:1000 was flushed over the meso- appendix p r i e r to freezing. Generalized vascular stasis occurred within f i f t e e n seconds afte r thawing. One or two channels opened up and resumed blood flow within sixty seconds a f t e r thawing, but t h i s phen- omenon involved only a small portion of the whole i n - jured c i r c u l a t o r y bed. No further changes were ob- served by f i v e minutes after thawing. Animal 5 (Control) Small c a p i l l a r y bed st a s i s was present t h i r t y seconds afte r thawing. A lar g e r venous channel showed a temporary slowing and sludging of blood. Ne further development was observed except that t h i s large channel l a t e r became s t a t i c . Histamine 1:1000 was flushed over the area, and while i t had no v i s i b l e e f f e c t on thi s large s t a t i c venule, i t did cause the c i r c u l a t i o n to reflow i n the c a p i l l a r y bed that f i r s t showed s t a s i s . Animal 6 (Test) r- • There was generalized vascular s t a s i s within f i f t e e n seconds afte r rapid thawing. No e f f e c t a t t r i b - utable to the histamine could be observed. There was no resumption of c i r c u l a t i o n i n any vessel within the injured area. 187 Animal 7 (Control) Stasis started within f i f t e e n seconds aft e r thawing, and became generalized throughout the i n j - ured area within f o r t y - f i v e seconds. One venous channel showed intermittency of flow. Histamine 1:1000 when flushed over the area had no apparent e f f e c t . Animal 8 (Te3t) Vascular s t a s i s f i r s t appeared ninety seconds a f t e r rapid thawing. Some intermittency of s t a s i s i n a couple of the larger channels was attributed to the histamine i n s t i l l a t i o n p r i o r to freezing, but otherwise the stasis r a p i d l y became general^ through- out the injured area. Animal 9 (Control) Stasis occurred within t h i r t y seconds of thawing and slowly became generalized within ninety seconds to involve approximately sixt y percent of the Injured area. No restoration of the c i r c u l a t i o n i n any s t a t i c vessel was noted. On fl u s h i n g the f i e l d l a t e r with histamine 1:1000, c i r c u l a t i o n resumed i n a large venous channel but only temporarily and Inter- mittently. Animal 10 (Test) Tbe mesoappendix was flushed with the warm 188 histamine 1:1000 solution, and then frozen In the usual manner. Stasis started within thirty seconds after rapid thawing, but in one capillary bed only, with no spread of the stasis within ninety seconds after thawing. Some sludging in 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 effect was repeated after the vessel sludged when the saline drip washed the histamine away. No further change was noted by five minutes after thawing, and the preparation was discontinued. Summary of Observations with Histamine (Topical) No. Onset Stasis Spread Generalized Flow Resumed 1 2 3 Control 20" Test 8 Test 60" Control 60" 4 Test 5 Control 30" 6 Test 7 Control 15" 90" 9 Control 30" 10 Test 30" Rapid 50$ i n 60" None None None None Slow 50$ i n 2» None Histamine gave slight resumption of flow in 15" / Slow larger venules None Histamine gave slight resumption In flow in 15" None Rapid i n 15" None Histamine gave no apparent effect Rapid Yes Slow 60$ in 90" Histamine gave doubtful effect None Histamine corrected sludging None 189 Discussion of Observations with Histamine On the average, the test animals took f o r t y seconds to develop s t a s i s , while the controls re- quired only t h i r t y following rapid thawing. This was however not consistent enough to be s i g n i f i c a n t . Histamine applied t o p i c a l l y over the control preparations after s t a s i s had occurred frequently o permitted the restoration of c i r c u l a t i o n i n s t a t i c channels. In comparing this action with a similar one obtained by f l u s h i n g procaine over the mesoappendix, i t might be suggested that the ef f e c t could be obtained through mere physical action of the weight of the f l u i d being applied from a height. Histamine could not be d e f i n i t e l y credited with 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 blood channels following a standard freezing i n j u r y . 190 Gross Frostbite Experiments Reason f o r Gross Experiments As mentioned previously, on page 18, there was no reason to believe that the c a p i l l a r i e s of the r a t mesoappendix functioned i n the same way as the c a p i l l a r i e s of the rat's hind leg, and since i t was not the peritoneal contents that would be affected by f r o s t b i t e , i t was therefore necessary to test the same ten medications i n order to f i n d out whether they would Influence the course of the lesions produced i n the hind l e g of the r a t by a standard cold injury. Shumacker (4 ) very complete- l y summarizes the standard i n j u r i e s of a great many gross freezing experiments performed by several d i f f e r e n t investigators. In almost a l l of these the tendency was to compare the r e s u l t i n g conditions of a set of test extremities at a certain period after a spec i f i e d cold i n j u r y to the r e s u l t i n g conditions of a si m i l a r set of control extremities the same length of time after the same sp e c i f i e d cold i n j u r y . As an example of what i s meant^an experiment of Shumacker, Radigan, Ziperman, and Hughs ( 4 ) i s summarized. The t a i l s of s i x t y - four mice were frozen f o r f i v e seconds at -15°C, the animals having received no treatment. Four 191 animals (6.3$) showed what the authors termed an excellent r e s u l t (grade 1 reaction), nine (14.1$) showed a good r e s u l t , and f i f t y - f i v e (85.9$) showed a poor r e s u l t (grade 3 re a c t i o n ) . Then thirty-two mice were given a drug (Benadryl, but the d e t a i l s are not important) and put to the same freezing injury. Two animals (6.3$) showed an excellent result^ four animals (12.5$) showed a good r e s u l t , and twenty-eight animals (87.5) showed a poor r e s u l t . There need be no discussion of these f i g - ures except to mention that they are obviously s i g n i f i c a n t because of the numbers of animals used. However, i n assessing the f r o s t b i t e injury, and i n comparing d i f f e r e n t animals, i t seemed as though something more was needed. Frost b i t e , l i k e any other pathological process, i s a dynamic process. We cannot say to-day whether one i n d i v i d u a l i s i n a better condition than another, as f a r as the disease i n question i s concerned*,unless" we know how both were yesterday and r tthey w i l l be tomorrow. Accordingly, In the following pages there w i l l appear some attempt to record the gross f r o s t b i t e r e s u l t s 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 explained. 192 Standard Freezing Injury Although i t i s somewhat t r i t e to mention i t , freezing i s freezing, and the temperature at which the freezing injury i s i n f l i c t e d matters l i t t l e provided the degree of cold i s s u f f i c i e n t to freeze the extremity within a reasonable period of time. In the data summarized by Shumacker ( ) ambient temperatures varying from -10°C. to -55°G. were used, provided usually by some mixture with carbon dioxide snow and usually ether i n a beaker into which the extremity was dipped f o r a stated length of time. By passing carbon dioxide gas into a small beaker of ether i t was found that a temperature of from -15 GG to -25°C. was r e a d i l y obtainable, and hence this was the temperature range used f o r the bulk of the experiments reviewed by Shumacker. Therefore -20°C. plus or minus 5C.° was accepted as the temperature to be used f o r the standard i n j u r y . I t was not always possible to obtain on any one day a temperature exactly -20°C. but i t was usually possible to maintain throughout each day's work the same temperature, whether -15°C. or -25°C. In a l l experiments the value of -20°G. w i l l be recorded, and where necessary 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 suffered a s l i g h t l y more severe freezing Injury than 193 was usually the case? This might mean a freezing temperature of -23°C. was used. The d i f f i c u l t y i n obtaining the exact temperature each day l a y i n several factors. The discharge nozzle f o r the carbon dioxide would freeze up to a varying extent from day to day. The amount of ether i n the beaker would vary greatly, the warmth of the room would be somewhat of a fact o r , etc. For this reason, the observations of d i f f e r e n t experiments are hardly comparable. The duration of the standard i n j u r y was also-) a problem, but this 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 length of the freezing varied from f i v e seconds through twenty seconds to s%xty seconds. As w i l l be seen l a t e r , the f i v e second injury produced too s l i g h t a reaction f o r standard use, and the s i x t y second Injury produced f a r too severe a reaction. Actually the twenty second i n j u r y (possibly due to minor variations i n degree of coldness, or variations i n the amount of the extremity immersed) was not always i d e a l , but there seemed l i t t l e v irtue i n reducing the time to f i f t e e n seconds. So, with these considerations, a twenty second freezing i n j u r y at -20°C. was accepted as 194 the standard freezing i n j u r y for the purposes of the experiments here performed. Manner of Thawing There always has been a great controversy over whether a frozen extremity should be thawed slowly or r a p i d l y . The time honoured method f o r frozen ears or noses (which more often than not were not "frozen" i n the true sense of the word, but just cooled to the extent that the subcutan- eous f a t s o l i d i f i e d thus blanching the skin) has been to rub the part a c t i v e l y with a handful of snow. This was one of the "certain widely accepted concepts based almost e n t i r e l y upon armchair p h i l - osophy of the past" that Shumacker c r i t i c i s e s so act i v e l y , and he perhaps more than any other person has gather* enough evidence together to show that th i s i s erroneous and can be discarded, at l e a s t on an experimental l e v e l . Most of the experiments reviewed by Shumacker give Immersion of the frozen extremity i n warm water at 42°C. f o r a s p e c i f i e d period ot time, usually two minutes, as the accepted method f o r rapid thawing. These standards were employed In the present experiments, and where possible (providing enough animals were available) both the rapid thawing at 42°C. and ordinary thawing at room temperature was used. This provided 195 additional control series f o r almost every experiment. Apparatus The accompanying photograph i l l u s t r a t e s an actual freezing injury and rapid thawing In progress. The carbon dioxide gas was run i n from an ordinary gas cylinder through maleable metal tubine to the control needle valve ( t h i s was the adapted Spencer Carbon Dioxide Microtome apparatus as adapted f o r the microscopic experiments, but with the expansion cold point needles disconnected and a simple number nineteen spinal anaesthetic needle attached) and then through a long needle into a small 125 cc. beaker h a l f f i l l e d with ether. The gas bubbling i n overflowed the ether, and a larger moat was provided to catch 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 sus- pended i n the freezing mixture and read frequently throughout the performance. The animal was anaesthetized, and the hind l e g was extended and dipped into the mixture almost to the fur l i n e . Generally about ten to twenty seconds were required for the limb to cool down, and freezing was i n d i c - ated by a sudden blanching of the skin and squirming of the animal. Such a limb when frozen was very  197 b r i t t l e , a n d c o u l d b e s n a p p e d a c r o s s m u c h a s a p i e c e o f g l a s s o f s i m i l a r t h i c k n e s s . A s t o p w a t c h , b e c a u s e o f i t s l a r g e s e c o n d h a n d t h a t c o u l d b e s t a r t e d w i t h p r e s s i n g a b u t t o n ^ was g e n e r a l l y u s e d f o r t i m i n g . The t i m i n g was c o u n t e d f r o m t h e moment o f t h i s b l a n c h i n g , a n d t h e l i m b was r e m o v e d f r o m t h e f r e e z i n g m i x t u r e e x a c t l y a t t h e e x p i r y o f t h e t i m e i n t e r v a l ( t w e n t y s e c o n d s ) . Gn t h e r i g h t h a n d s i d e o f t h e p h o t o g r a p h i s d e p i c t e d t h e r a p i d t h a w i n g . The f r o z e n h i n d l e g o f t h e a n i m a l s j u s t f i n i s h e d t h e f r e e z i n g t r e a t m e n t i s s h o w n d i p p e d i n a b e a k e r o f p l a i n t a p w a t e r a t e x a c t l y 4 2 ° C . a s i n d i c a t e d o n t h e t h e r m o m e t e r s u s p e n d e d i n t h e b e a k e r . The l e g was k e p t i n t h e w a t e r f o r a m i n u t e o r two u n t i l t h o r o u g h l y l i m p a n d w a r m . I n a c t u a l p r a c t i c e t h e r a p i d t h a w i n g p a r t o f t h e t r e a t m e n t was p e r f o r m e d b y a h e l p e r , a n d a l a r g e r p r e s e r v i n g k e t t l e was u s e d i n s t e a d o f t h e b e a k e r b e c a u s e t h e l a r g e r v o l u m e o f w a t e r m a i n t a i n e d i t s t e m p e r a t u r e m u c h m o r e c o n s t a n t l y . R e c o r d i n g t h e O b s e r v a t i o n s The c o u r s e o f t h e i n j u r e d l i m b s was n o t r e c o r d e d u n t i l t h e l a p s e o f t w e n t y - f o u r h o u r s a f t e r t h e f r e e z i n g i n j u r y . The a n i m a l s t h a t w e r e t h a w e d a t r o o m t e m p e r a t u r e w e r e m e r e l y p l a c e d b a c k i n t h e i r c a g e s i n q u i e t n e s s , e x c e p t f o r a n y i n j e c t i o n t h e y 198 would be r e c e i v i n g . The f e e t of such animals r would begin to thaw immediately, and any chance contact w i t h the metal of the cage would a i d the warming so that the limb would be limp (thawed) w i t h i n f i v e minutes u s u a l l y . I t would take another ten or f i f t e e n minutes f o r the f o o t to warm up, and w i t h i n three or four hours the l e g would begin 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 , but i t would soon become engorged, p i n k , and warm. The f e e t of the animals thawed r a p i d l y i n the warm water were pink and warm as soon as they were returned to t h e i r cages, and w i t h i n the f o l l o w i n g couple of hours they would be swollen, but s t i l l p ink and warm. On the f o l l o w i n g day, u s u a l l y at the same time as the f r e e z i n g had been done the animals were weighed, and the f e e t examined. Examination was made under four c a t e g o r i e s : oedema, colour change, moisture exudate or slough, and gangrene of toes or other p a r t s of the f o o t . A constant comparison e v a l u a t i o n was attempted, and of course the same person d i d the examining, but when p o s s i b l e some other person present would be asked to corroborate the impressions. Then under each category, an attempt was made to as s i g n a number to each obser- 199 v a t i o n . The number s c a l e u s e d f o r e a c h c a t e g o r y i s o u t l i n e d as f o l l o w s : Oedema C o l o u r E x u d a t e Gangrene None a p p a r e n t 1 M i n i m a l 2 M o d e r a t e 3 I n c o m p l e t e 4 As f u l l y r o u n d e d as p o s s i b l e Normal 1 P i n k e r t h a n n o r m a l 2 Red 3 P u r p l e 4 B l u e , e i t h e r c y a n o s i s o r b e g i n n i n g e s c h a r 5 B l a c k Scab n o t g r a d e d . 0 D r y 1 B a r e l y m o i s t 2 Q u i t e m o i s t o r wet 3 I n f e c t e d u l c e r o r s l o u g h . B l o o d c l o t n o t g r a d e d 0 None a p p a r e n t 1 One t o e g a n g r e n o u s o r m i s s i n g 5 F i v e t o e s g a n g r e n o u s o r m i s s i n g 6 F r o n t h a l f o f paw g a n g r e n o u s 7 Whole paw g a n g r e n o u s o r m i s s i n g 8 Whole i n j u r e d a r e a g a n g r e n o u s o r m i s s i n g 2G0 T h i s c l a s s i f i c a t i o n was not designed completely at the very s t a r t of the experiments. One or two p r e l i m i n a r y t e s t s were performed i n order to determine j u s t exactly what changes to expect, and to c o n s t r u c t a t a b l e t hat would best d e s c r i b e the l e s i o n s . The one adopted as o u t l i n e d was c e r t a i n l y not the o n l y one poss- i b l e , and i n a few r e s p e c t s i t i s not the b e s t . For i n s t a n c e , the degree of s w e l l i n g c o u l d have perhaps been more a c c u r a t e l y measured by a water displacement method. But the method of a s s e s s i n g the changes had to be r e a s o n a b l y accurate only, and the b i o l o g i c a l experiments as performed d i d not r e q u i r e the exactness of an experiment i n p h y s i c s . Such a g r a d i n g system as o u t l i n e d on the p r e c e d i n g page was at a l l times convenient, r a p i d , and accurate w i t h i n the l i m i t s of the e r r o r of the experiment. On the f o l l o w i n g page i s an example of how the i n j u r y changes were graded i n p r a c t i c e . The c o l o u r p r i n t does not g i v e the c o l o u r changes as a c c u r a t e l y as the o r i g i n a l transparency d i d , but I t g i v e s as rough.'.; i l l u s t r a t i o n of what i s meant by the accompanying des- c r i p t i o n . The word "much" under the oedema column i s synonymous w i t h the word "incomplete" used b e f o r e f o r a t h i r d degree oedema change. I n j u r y Index As w i l l be noted on the f o l l o w i n g Example sheet, the 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, I n j u r y 201 Example of Injury Cali b r a t i o n 2 This Is a photograph of the feet of group 5, which were frozen f o r twenty seconds, then thawed slowly at room temperature. The picture was taken on day three, f o r t y - eight hours aft e r the injury occurred. Animal Oedema Colour Exudate Gangrene 1 Much Blue Wet None 2 Much Red Wet None 3 Much Blue Wet None 4 Much Purple Wet None 5 Much Purple Wet None 6 Much Purple Wet None Converting these observations to the arbi t r a r y number code, we have: G Animal 0 C E 1 3 4 2 2 3 2 2 3 3 4 2 4 3 3 2 5 3 3 2 6 3 3 2 Totals 18 19 12 The sum of these four t o t a l s , here 49, i s plotted on a l l subsequent graphs as the "INJURY INDEX". On the sample graph drawn on thi s page, the compon- ents of the ordinate are blocked i n f o r i l l u s t r a t i o n , but the other graphs would be too crowded with these addit- ional d e t a i l s which can be read from the data tables when desired. 50 40 I N J U 30 R Y I • 20 D E X 10 Exu late Col our Oed ema 2 3 D A Y 202 Index i s merely an ar b i t r a r y number invented i n order that the f r o s t b i t e changes, on a time basis, could be represented graphically without too much crowding and hence confusion on the graphs. A sample graph i s indicated with the standard scale used throughout a l l graphs, and the meaning of the ordinate points on the Injury Index l i n e graph explained. Selection of Gross Experiments Gross experiments were performed as outlined i n following pages. The d e t a i l s and purpose of each experiment i s explained under the write-up f o r the experiment. Nine d i f f e r e n t runs were performed as outlined i n the Table of Contents; (1) Duration of Injury and rate of thaw, (2) procaine, (3) repeat pro- caine, together with P r i s c o l i n e , (4) Benadryl, (5) Et- amon and Hydergine, (6) Apresoline and Chlor-Tripolon, (7) r u t i n , (8) ascorbic acid, and (9) histamine. The reason f o r the p a r t i c u l a r grouping of the two drugs Into one experiment was made only f o r convenience. The reasons f o r the dosages selected has already been explained. 203 Record of Gross Observations Here follows a record of observations made grossly on the effects of a standard freezing i n j u r y on the hind l e g of the r a t as outlined previously. The r e s u l t s are grouped by the drugs being tested, according to the order of the table of contents. Duration of Injury and Rate of Thaw Experimental Observations with Time and Rate of Thaw The purpose of the experiment described here was to test the effects of a standard cold i n j u r y as influenced by the duration of the frozen state, and also as influenced by the rate of thawing. Thir t y - six normal adult nonpregnant female Wistar rats were selected, and they were divided into s i x groups of six animals each, with each group designed as follows: Group: 1. A group to receive a freezing i n j u r y of f i v e seconds duration, with rapid thawing In water at 42 GC. 2. A group to receive a freezing Injury ©f twenty seconds duration, with rapid thawing i n water at 42®G. 3. A gr©up t© receive a freezing i n j u r y 204 of 3 i x t y seconds duration, with rapid thawing i n water at 42®C. 4. A group to receive a freezing i n j u r y of f i v e seeonds duration, with slow thawing at room temperature. 5. A group to receive a freezing i n j u r y of twenty seconds duration, with slow thawing at room temperature. 6. A group to receive a freezing i n j u r y of s i x t y seconds duration, with slow thawing at room temperature. Although a l l groups are numbered the same throughout a l l other experiments, i n thi s p a r t i c u l a r instance, the numbering i s independent. On Day 1, the day of freezing, each animal i n a group was marked charact- e r i s t i c a l l y on the ear, and i n i t i a l weights were re- corded as on the Data Sheet. A l l animals were given by subcutaneous Injection from 0.1 cc. to 0.125 cc. of 0.6$ nembutal solution. Then, when anaesthetized, one hind l e g was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down t© -20 GG. The foot was immersed up to the hock (just below the f u r l i n e ) , and as so©n as the skin blanched, i n d i c a t i n g the foot was completely frozen (at t h i s moment the r a t in v a r i a b l y squirmed, i n 205 spite of adequate anaesthetic), the time was counted so that the leg was immersed and frozen f o r exactly the required length of time, depending on the group to which the animal belonged. The animals of Groups 4, 5, and 6 were placed i n t h e i r cages and the fe e t were allowed to thaw at room temperature, taking approximately f i f t e e n or twenty minutes to become limp and warm. The animals of groups 1, 2, and 3 were treated by rapid thawing, whereby the frozen feet were immersed immediately at the expiry of the appropriate time i n t e r v a l i n the ether , into water maintained at exactly 42°C. The feet 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 also returned to the i r cages and a l l t h i r t y s i x were l e f t q u ietly alone u n t i l they wakened from the eff e c t s of the nembutal. Observations were made as recorded i n the tables, by the method described above f o r estimating the degree of f r o s t b i t e i n j u r y under the categories of oedema, colour change, exudate and slough, and gangrene. Data Sheets These tables are f a i r l y self-explanatory, being simple t a l l i e s by animals In groups, and by days, Day 1 being the day of the fre e z i n g . They are necessarily as compact as possible. Symbols are: 206 Grp Gm Gm 0 C £ G Gm 0 C E G Gm 0 C E G 1 250 260 1 1 260 * 1 267 * * 174 169 2 1 - 167 1 1 — - 170 1 1 — - 168 171 2 A z - - 172 1 1 — - 172 * A 2 - -232 236 2 1 - 234 1 1 — - 233 1 1 - - 204 201 1 1 — - 202 •|- 1 - - 198 1 1 _ 224 220 1 1 - - 222 2" 1 - - 219 £ 1 - - 2 204 228 3 2 _ — 225 3 2 1 234 3 2 234 237 3 2 1 - 229 3 1 1 228 2 1 — -175 175 3 1 1 - 176 3 1 1 - 177 3 1 - - 231 225 3 1 1 - 227 3 2 1 226 3 2 - 180 182 3 1 1 - 180 3 2 1 - 182 2 2 - -210 207 3 1 1 - 201 3 2 1 202 3 2 - - 3 194 200 4 2 1 191 3 2 2 192 3 3 2 248 251 3 2 1 - 242 3 3 1 - 238 3 4 2 -230 241 3 2 1 1 233 3 2 2 1 230 3 3 2 1 183 186 3 3 1 — 180 3 4 2 - 176 3 4 2 — 238 2o2 3 2 1 — 229 4 2 2 — 224 3 3 2 — 175 173 3 2 1 - 170 3 3 2 - 170 3 3 2 - 4 220 215 3 2 1 216 2 2 2 210 1 1 1 240 235 3 2 1 - 232 3 3 2 - 226 3 3 2 — 164 168 3 1 1 - 162 2 1 - — 165 2 3 _ - 195 196 3 2 1 - 191 2 2 2 - 186 1 2 1 — 205 203 3 3 2 - 199 3 2 2 mm 192 2 2 1 _ 215 210 3 2 1 - 210 3 2 2 - 206 3 3 1 - 5 234 226 3 3 2 225 3 4 2 214 4 4 2 174 170 3 3 2 - 164 3 4 2 - 155 3 3 2 _ 173 168 3 3 2 - 163 3 3 2 mm 157 3 3 2 — 210 210 3 2 1 - 210 3 2 2 — 205 3 3 2 _ 216 215 3 2 1 - 212 o 3 2 — 204 3 3 2 178 178 3 1 - 175 3 3 2 - 176 3 3 1 - 6 235 232 3 4 2 mm 226 3 4 2 1 222 3 4 2 1 215 214 3 4 2 - 207 3 4 2 _ 205 3 5 2 240 242 3 4 2 - 234 3 3 2 — 224 3 4 2 199 199 3 4 1 — 189 3 4 2 - 184 2 5 2 1 238 228 3 4 1 — 224 3 4 2 — 214 1 5 2 1 197 197 1 4 - - 188 3 5 2 8 187 3 5 2 8 Day 1 2 3 4 I 207 Grp Gm O C E G Gm O C E G Gm O C E G 1 260 i £ - - 267 4 4- 163 1 1 - - 160 § § - - 175 4 1 - - 181 225 i i - - 226 & & - - 190 1 1 - - 194 1 1 214 i 1 - - 216 £ - - 2 225 2 2 - - 215 1 1 - - 224 1 1 224 1 1 - - 226 1 1 230 £ 1 - - 172 1 1 1 - 178 1 1 - - 179 1 1 - - 211 2 1 - - 214 1 1 - - 223 1 1 - - 179 1 1 - - 175 1 1 - - 176 1 1 - - 194 2 2 - - 198 1 2 1 - 203 1 2 - - 3 186 3 3 2 - 178 3 3 3 - 184 3 3 3 - 236 3 3 2 - 224 3 3 1 226 3 2 1 - 226 3 3 2 - 231 3 3 3 - 232 2 3 3 - Dead 221 3 3 2 - 226 3 3 3 - 228 3 3 1 - 171 3 3 2 - 172 3 3 3 - 170 3 3 1 - 4 204 2 2 1 - 205 1 2 - - 210 3 3 - 4 218 2 3 1 - 220 1 3 230 1 3 168 1 2 - - 174 1 1 - - 180 1 187 i 1 - - 186 1 1 - - 188 1 1 - - 188 } 1 - - 191 1 1 1 - 195 1 1 - - 204 2 1 - - 203 2 2 203 2 1 5 210 3 4 3 3 211 3 3 - 4 210 3 3 - 4 157 3 3 2 4 158 3 3 3 6 159 3 3 2 6 160 3 3 1 - 163 3 3 3 5 168 3 3 1 5 202 3 2 - - 200 2 3 1 - 204 1 2 1 - 200 3 3 1 - 200 3 3 1 - 204 2 3 - 1 157 3 3 3 4 186 3 4 1 1 189 3 5 - 4 6 214 3 3 2 8 210 3 4 3 8 212 2 4 1 8 195 2 5 2 6 19v7 3 4 3 8 203 2 4 2 8 220 1 5 2 7 216 3 4 3 8 225 2 4 - 8 185 1 4 2 8 184 - 3 - 8 182 8 215 2 4 2 8 210 - 3 - 8 208 8 190 1 2 - 8 190 - 3 - 8 185 8 Day 5 6 7 208 Grp» group, Gm, grams, 0, ©edema, C, colour change, E, exudate, G, gangrene, -, means not present. A l l animals were disposed off with chl©rof©rm fumes on Day 7. Summary Sheet Following 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, gives the t o t a l f o r the injury changes occurring f o r that group that day. As described before, t h i s concept of an Injury Index Is just an arbit r a r y number obtained by adding a l l the pathological changes together f o r each group each day. Note that the values f o r Group 3 on days 5, 6, and 7 are underlined to indicate an extrapolation to make up f o r the animal that died. Graphs The values on the Summary Sheet are plot t e d on two simple l i n e graphs, the f i r s t f o r the Injury Index changes from day to day, and the second f o r the weight changes. These are done In colours f o r easier reading, 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 injury, rather than by group numbers. Condition of the Animals There i s l i t t l e need t© include d e t a i l s of the rats themselves. Within a couple of hours of the freezing, a l l feet were grossly ©edematous, bright 209 Summary sheet Grp Gm Gm I Gm I Gm I 1 209 209 14* 209 209 9- 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 3 4 Grp Gm I 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 7 210 Rapid Thaw Yersu3 Slow Thaw - E f f e c t of Freezing Time \ 60" Freeze * Slow Thaw J 20" Freeze Slow Thaw 60" Freeze Fast Thaw 5" Freeze Slow Thaw 20" Freeze Fast Thaw 5" Freeze Fast Thaw 1 2 3 4 5 6 7 D A Y 211 1 2 3 4 5 6 7 D A Y 212 pink, warm, and essential^nontender, although apparently numb f o r the animals hobbled around t h e i r cages apparently unaware of the positions of t h e i r injured f e e t . The day to day changes i n the appear- ance of the of the fe e t may be r e a d i l y imagined from the values on the Data Sheets. Generally the rats did well, 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 at weight changes mean l i t t l e . Discussion of Observations The Injury Index graph gives a very cl e a r separation of the various groups. An Index readyjng of 80 indicated a very severe reaction, and In thi s case the feet of Group £'s animals a l l sloughed o f f to the l i m i t s of the freezing, whereas, at the other extreme, by Day 7 i t was almost impossible to detect any change In the feet of the animals of Group 1, except f o r a s l i g h t thickening of the skin over the dorsum of the paw. There Is f i r s t of a l l , as would be expected, a worse in j u r y produced the longer the limb i s frozen. The effect of the s i x t y second injury Is worse than f o r the twenty, and the twenty worse than f o r the f i v e . This response was consistent throughout the 213 graph. Then there i s secondly, well demonstrated, the great benefit to be derived from rapid thawing of the frozen limb i n warm water at 42°C. This, e f f e c t was also consistent throughout the graph. Photographs On Day 5, after a repeat dosage of the nembutal anaesthetic, a l l feet were photographed i n colour by groups. The p r i n t s obtained were of to© poor a qua l i t y to merit i n c l u s i o n here. However, the pictureof Group 5, l a b e l l e d "Time 5" has been used e a r l i e r to i l l u s t r a t e how an Injury Index i s calculated. I t would be worthwhile to examine t h i s page again In reference to th i s experiment. Procaine Experimental Observations with Pr©caine Hydrochloride The purpose of the experiment here described was to test the effects of a standard cold i n j u r y as influenced by parenteral i n j e c t i o n s of procaine at a dosage l e v e l ©f 200 mg. per kilogram body weight administered hourly p r i o r to the freezing i n j u r y and f o r forty-eight hours afterwards. Thi r t y normal adult male Wistar rats were selected, and they were divided into f i v e groups ©f s i x animals each, with 214 each group designed as follows: Group: 1. A group to receive a freezing i n j u r y of twenty seconds duration with slow thawing at room temperature. 2. A group to receive only the procaine i n j e c t i o n s as outlined. 3. A group to receive both the procaine inje c t i o n s and a freezing i n j u r y of twenty seconds duration with slow thawing at room temperature. 4. A group to receive a freezing Injury of twenty seconds duration with rap i d thawing i n water at 42°G. 5. A group to receive both the procaine i n j e c t i o n s and a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 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 that Group 1 here corresponds to Group 5 of the l a s t experiment, and Group 4 here with Group 2 there. On day 1 of the experiment, the day of freezing, 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 recorded as on the D*ta Sheet. The animals of Groups 2, 3, and 5 received t h e i r i n i t i a l 215 subcutaneous i n j e c t i o n of procaine solution pre- pared at 20 mg. per cc. f o r ease i n computing doses. Then, approximately twenty minutes l a t e r , the animals of Groups 1, 3, 4, and 5 received by subcutaneous i n j e c t i o n an anaesthetizing dose of 0.6$ nembutal solution ranging from 0.1 ce. to 0.125 cc. Then, when anaesthetized, one hind l e g of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to -20°C. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blanched, Indic- ating the foot was completely frozen (at this moment the r a t in v a r i a b l y squirmed, i n spite of adequate anaesthetic), the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by stop watch. The animals of Groups 1 and 3 were placed i n t h e i r cages and the f e e t were a l l - owed to thaw at room temperature, taking approxim- ately f i f t e e n or twenty minutes to become limp and warm. The animals of groups 4 and 5 were treated by rapid thawing, whereby the frozen feet were imm- ersed immediately at the expiry of the twenty seconds into water maintained at exactly 42®C. The feet 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 also returned to their cages and a l l t h i r t y were l e f t q u i e t l y alone (except f o r the hourly inje c t i o n s of procaine) u n t i l they wakened from the ef f e c t s of the nembutal. Observations were made as recorded i n the tables, by the method described above f o r estimating the degree of f r o s t b i t e i n j u r y under the categories of oedema, colour change, exudate and slough, and gangrene. Data sheets These tables 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 of freezing. They are necessarily as compact as possible. Days 1 and 2 include a record of the hourly i n j e c t i o n s . The symbols used are: Grp, group, Gm, grams, qlh, hourly, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, P, freezing, -, means not present or not administered, N, f u l l dose of procaine, £, one h a l f the dose of procaine, one quarter the dose of procaine (note that £ i n the O C E G columns means ha l f value). A l l animals not already dead were disposed r of with chloroform fumes on Day 4. Summary Sheet Following the Data Sheets i s a Summary Sheet 217 Data Sheet Grp Gm qlh Gm O C E G q lh 1 264 310 180 287 254 318 2 410 295 348 241 267 268 3 279 317 202 306 320 318 4 261 317 292 255 238 244 5 283 242 210 282 341 285 -p ' — 260 3 3 1- -P 304 3 2 1- -p 166 3 3 1- -p 290 3 2 2- -p 246 3 2 2 • -p • 320 3 2 2- N-NNNNN (Died) N-NimNN-----iii|ii£---i--|- 285 N-NNNNN HHiili ?-? 3 3 5 N-NN]Om---^Hfnii ?"? 2 4 8 N-NNNNN llllillll f - f 254 N-NNNNN llllSISi t-t 230 NPNNNNN i-(Died) NPNNNN*(Died). NPNNNNN-- l l l l l l 192 2^2 1 - £|—(Died) NFNNNN& £|£i§3*£H Died) N F N N N N } niiil—i"? 3 0 5 3 3 2 NFNNNNi UHHil 1-1 3 0 8 2 * 3 1 -P 262 3 1 1 -P 312 3 1 1 -F 286 3 1 1 -F 250 3 1 1 -P • 232 3 1 1 -P 240 3 1 1 NPNNNNN l l l l l l l £-£ 272 2 i l 1 NPNNNNN-N §-£ 232 2§1 - NPNNNNN i-llilll §-§ 204 2 1 - NFNNNNN-^--li | | |H| *-# 262 3 1 1 NPNNNNN-N IHIHI £-§ 335 2^1 - NPNNNNN (Died)- ml': Day 1 2 > 218 Data Sheet Grp q l h Gm 0 C E G 1 270 3 3 2 310 3 2 2 165 3 3 2 295 3 3 1 252 3 3 1 323 3 3 2 2 (Dead) i ^ J J J L i i . i 272 i i i t i l l - - - - ts 3 (Dead) (Dead) (Dead) (Dead) - - l i i - i - i j i - : 296 2 3 2 1|— 297 2 4 - 4 --• 266 3 1 - 306 2 1 - 289 3 1 - 249 3 1 1 238 3 1 1 240 3 1 1 5 - i \ 270 1 1 - 230 2 1 - \ 199 2 - - n u i i i m i i i i i . i ocp * -i _ 444444444444444 I 6 0 0 <-> ± - ; (Dead) (Dead) Day 2 (Cont) 3 Gm 0 C E G- 260 3 4 2 303 3 3 3 1 164 3 4 2 - 292 3 3 2 - 249 3 4 2 — 311 3 4 3 - 259 303 221 245 212 (Dying) 281 (Dead wt) 274 2 3 1 - 267 283 289 239 229 244 2 1 3 2 2 2 1 1 - 1 - 262 2 1 - 218 2 1 - 196 2 1 - 237 3 1 - -(Dead) -(Dead) -(Dead) 219 Summary Sheet Grp Gm Gm I Gm I Gm I 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 2 3 4 which gives the average weight f o r each group each day, and under I, or Index, gives the t o t a l f o r the inj u r y changes occurring f o r that group that day. As described before, t h i s concept of an Injury Index i s just.an arb i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. Except f o r Groups 1 and 3 where no animals expired, a l l I values had to be extrapolated up to correspond to six animals per group. As w i l l be explained below, th i s seriously reduces the s i g n i f icance of the data. 220 Graphs The values on the Summary Sheet are pl o t t e d on two simple l i n e graphs, the f i r s t f or the Injury Index changes from day to day, and the second f o r the weight changes. These are done In colours f o r easier reading (except f o r the Time graphs of the l a s t experiment these and a l l succeeding 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 injury and treatment rather than by group numbers. Condition of the Animals I t w i l l be obvious from the Data Sheets that the procaine i n the dosage given took a t e r r i b l e t o l l of the animals. Many, a f t e r the s i x t h dose,were comatose and i n convulsions. This explains why such dra s t i c reductions i n the medication were necessary. Many died, and of those surviving, many sat limply huddled i n t h e i r cages,weakly protesting every d i s - turbance. The day to day changes i n the appearance of the feet may be imagined from the values on the Data Sheets. These d© not r e f l e c t the p i t i f u l cond- i t i o n of the animals. Discussion of Observations Procaine i n the dosage used Is too toxic to be p r a c t i c a l . 221 2 3 4 D A Y 1 2 3 4 D A Y 222 The Injury Index graph confirms the impression that rapid thawing at 42 0G reduces the severity of the in j u r y reaction. This i s consistent both with the procaine treated groups and with the control groups. Procaine treatment administered as outlined apparently resulted i n les s i n j u r y than was shown by the controls. The fac t that the graphs f o r the pro- caine treated animals l i e under the corresponding control graphs supports th i s claim. However, two animals never make a group, and the lack of any s i g - n ificance 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 attest to the toxic effects of the procaine. The procaine treated animals show a steady and rapid weight l e s s , while the controls tend to maintain t h e i r weight more s t e a d i l y . Procaine and P r i s c o l i n e Experiments with Procaine (Repeat) and P r i s c o l i n e The purpose of the experiment here described was to test the effects of a standard cold i n j u r y on the hind legs of rats as influenced by two d i f f e r e n t drugs. Because of the t o x i c i t y and mortality of the 223 procaine at the dosage l e v e l used i n the l a s t exper- iment (200 mg. per kilogram body weight) i t was de- cided necessary to repeat the experiment using the procaine at 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 hourly. The effects of p r i s c o l i n e were also tested at a dosage l e v e l of 50 mg. per kilogram body weight also administered subcutaneously at three hourly i n t e r v a l s . Thirty normal adult nonpregnant female Wistar rats were selected, and they were c l e a r l y divided into two groupings; one of eighteen animals f o r testing with procaine, and the other of twelve animals for. the P r i s c o l i n e tests. Each div- i s i o n was divided into groups of six animals each, with each group designed as follows; Group; 2. A group to receive only the pro- caine inj e c t i o n s as outlined. 4. A group to receive a freezing i n j u r y of twenty seconds duration with o rapid thawing i n water at 42 C. 5. A group to receive both the procaine i n j e c t i o n s and a freezing Injury of twenty seconds duration with ra p i d thawing i n water at 42®C. Group; 4. A group to receive a freezing i n j u r y 224 of twenty seconds duration with o rapid thawing i n water at 42 C. (Group) 5. A group to receive both P r i s c o l i n e i n j e c t i o n s as outlined and a freezing injury of twenty seconds duration with rapid 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 ) . On the day the freezing was done, Day 1, the animals of 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 recorded as on the Data Sheet. The animals of Group 2 and both Groups 5 received two i n i t i a l i n j e c t i o n s of th e i r appropriate drug. The procaine solution was i n a concentration of 10 mg. per cc. and the P r i s c o l i n e was 2 mg. per cc. Then one hour approximately aft e r the second dose of the drugs the animals of both Groups 4 and both Groups 5 were anaesthetized with 0.1 to 0J25 cc. of 0.6$ nembutal solution administered subcutaneously. When anaesth- etized, one hind leg of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down o to -20 C. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blanched, 225 in d i c a t i n g the foot was completely frozen (at t h i s moment the r a t in v a r i a b l y squirmed, i n spite of adequate anaesthetic), the time was counted so that the leg was immersed and frozen f o r exactly twenty seconds by stop watch. A l l animals were treated by rapid thawing, whereby the frozen feet were Immersed immediately at the expiry of the twenty seconds into water maintained at exactly 42°C. The fe 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 returned to t h e i r cages and l e f t quietly alone (except f o r the three hourly injections) u n t i l they wakenSd from the effects of the nembutal. Observ- ations were made as recorded i n the tables, byt the method described above f o r estimating the degree of f r o s t b i t e injury under the categories of oedema, colour change, exudate and slough, and gangrene. Data sheets These tables 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 of freezing. They are necessarily as compact as possible. Days 1, 2, and 3 include a record of the three hourly i n j e c t i o n s . The symbols used are: G-rp, group, Gm, grams, 226 Grp Gm 2 197 184 194 178 178 181 4 188 195 170 186 173 191 5 201 189 170 207 201 194 Day 1 Grp Gm 4 192 146 182 195 198 195 5 185 204 197 184 205 206 Day 1 Data Sheet Procaine q3h 0 C E G q3h Gm 0 C E G q3h NN-NNNNNNNNN NNNNNN 182 NNNN NN-mOTONNNNN NNNNNN 175 NNNN NN-NNNNNNNNN NNNNNN 175 NNNN NN-10JNNNNNNN NNNNNN 168 NNNN NN-NNNNNNNNN NNNNNN 165 NNNN NN-NNNNNNNNN NNNNNN 168 NNNN _ _ p  3 2 1 — 177 3 1 1 <*• tm p 187 3 4 2 _ ~ F (Died) o o Ct p--- — 3 2 1 - 175 3 2 1 - -.-p—-- 3 3 — 161 3 3 1 - ca mm 3 2 - mm 181 2 1 - - ———— NNFNNNNNNNNN 3 1 _ NNNNNN 187 3 1 _ _ NNNN NNFNNNNNNNNN 3 1 1 - NNNNNN 174 2 1 - - NNNN NNF (Died) NNFNNNNNNNNN 3 2 - - NNNNNN 190 3 3 - - NNNN NNFNNNNNNNNN 3 2 1 - NNNNNN 187 3 2 - - NNNN NNFNNNNNNNNN 3 1 1 mm NNNNNN 182 3 1 - - NNNN 2 3 Pr i s c o l i n e q3h O C E G q3h •P 3 3 1 •P 3 3 1 •P 3 3 2 -P 3 2 2 -P 3 1 2 -P 3 2 2 Gm O C E G q3h 187 3 2 1 150 3 3 1 175 3 3 1 192 3 2 1 194 3 1 - 180 3 2 - PPPPPPPPPPPP 3 3 - - PPPPPP 181 3 3 PPPPPPPPPPPP 3 1 - - PPPPPP 198 3 2 PPPPPPPPPPPP 3 2 1 - PPPPPP 190 3 1 PPPPPPPPPPPP 3 3 1 - PPPPPP 179 3 3 PPPPPPPPPPPP 3 2 2 - PPPPPP 198 3 2 PPPPPPPPPPPP 3 2 1 - PPPPPP 200 3 1 PPPP PPPP PPPP PPPP PPPP PPPP 227 Data Sheet Procaine Gm 0 C E G Gm 0 C E G Gm 0 C E G Gm 0 C E G 175 188 176 217 168 182 170 216 165 178 170 212 163 178 177 217 155 174 174 208 159 174 175 211 168 2 2 1 174 2 2 2 «. 173 2 2 2 _ 175 1 2 3 - 178 3 3 2 — 176 2 4 3 6 184 2 3 — 6 186 1 2 - 6 (Dead) 168 2.2 1 - 176 1 2 1 - 162 1 1 - - 187 1 2 2 - 155 2 3 1 - 164 1 3 - — 168 1 2 — 184 1 4 1 -176 1 1 - - 180 1 1 - - 186 1 1 - - 202 1 1 179 2 1 190 2 1 198 1 1 mm 228 2 3 - - 167 2 1 _ 174 2 2 _ — 178 1 2 — _ 198 2 2 1 - (Dead) 182 3 3 1 - 190 2 1 - - 194 2 3 - - 208 2 3 181 2 1 1 - 190 3 3 2 - 192 1 1 — _ 212 2 2 1 • 176 2 2 1 - 190 2 1 - - 197 1 1 - 214 2 2 3 - 4 6 8 10 P r i s c o l i n e i — - — — — - — ^ — — Grp Gm 0 C E G Gm 0 C E G Gm 0 C E G Gm 0 G E G 4 184 2 2 a. 196 1 3 1 _ 205 1 2 228 1 2 146 3 3 - - 131 2 3 - - 132 1 2 1 - 140 1 1 — 1 171 3 4 2 - 172 3 4 2 — 176 3 3 1 - 193 2 3 3 — 188 1 2 - - 192 - 1 - mm 185 1 1 - - 214 1 1 — — 184 2 2 - 202 _ 2 — — 204 1 2 1 — 235 1 2 — — 178 2 2 - - 180 2 2 - - 181 1 1 • -- 200 1 2 - mm 5 175 3 3 1 178 3 3 1 _ 185 2 3 202 2 2 mm 188 2 1 - - 190 1 1 — - 197 1 1 - - 207 1 2 - - 184 1 1 1 - 181 2 3 2 mm 188 1 3 1 — 210 1 2 3 — 172 3 2 1 - 172 2 2 - — 170 1 2 - - 194 1 2 2 a* 194 3 2 1 - 195 2 2 2 - 196 2 2 - - 210 2 3 3 1 197 3 2 1 — 187 2 2 1 - 194 1 2 1 — 205 1 3 2 — Day 4 6 8 10 228 Data Sheet Procaine Grp Gm 0 C E G Gm 0 C E G Gm 0 C E G 2 201 207 220 203 205 210 198 201 213 207 199 205 194 201 200 193 194 200 4 172 1 2 3 180 1 2 3 mm 192 mm mm 181 1 1 - 6 186 1 1 - 6 189 1 1 - 6 (Dead) 169 1 - 3 - 173 1 1 2 - 190 1 - - mm 167 1 3 3 - 175 - 2 - 3 183 — - — 1 189 - 1 - - 197 - - - - 200 - - - - 5 210 1 _ 219 1 1 219 1 180 1 3 - — 184 1 2 1 — 197 1 1 2 -(Dead) 190 2. 3 - 192 1 3 1 - 207 2 2 3 5 202 2 2 1 - 204 2 2 1 — 220 1 2 1 - 199 2 2 2 - 205 3 2 3 - 206 2 2 1 5 Day 11 12 13 P r i s c o l i n e Grp Gm 0 C E G Gm 0 C E G Gm 0 C E G 4 212 1 2 _ _ 214 1 2 210 1 1 mm 133 1 2 1 - 134 1 2 - - 149 1 2 1 1 178 1 3 1 - 182 2 3 2 - 186 2 2 3 2 193 - 1 - - 193 — 1 — 210 - _ — mm 221 - 2 - - 230 - 1 - - 214 — - - - 184 - 2 - - 191 1 1 - mm 210 - - mm - 5 180 1 2 1 180 1 2 2 _ 200 1 194 - 1 - - 194 - - — - 206 1 1 - — 197 1 2 - mm 197 1 2 1 - 214 — 1 — - 184 - 2 2 - 189 1 2 1 - 180 1 1 1 — 195 2 2 3 - 202 2 2 3 210 1 2 3 2 194 1 2 2 - 198 1 2 1 - 200 — 1 - — Day 11 12 13 2 2 9 q3h, every three hours, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, N, i n j e c t i o n of procaine, P, freezing, -, means not present or not administered, P, i n j e c t i o n of P r i s c o l i n e . A l l surviving animals were disposed of on Day 13 with chloroform fumes. Summary Sheet Following 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, gives the t o t a l f o r the Injury changes occurring f o r that group that day. As described before, this concept of an Injury Index i s just an ar b i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. Where an animal died, the Injury Index was extrapolated to raise the value to what could be expected f o r a complete group. Graphs The values on the Summary Sheet are plot t e d on two simple l i n e graphs, the f i r s t f o r procaine and the second f o r P r i s c o l i n e . The weight curves and the Injury curves are put onto the same corresponding graph, and plotted against time. The X marks on the graphs Indicate missed days to permit the graph to 230 Summary Sheet Procaine Grp Gm I Gm I Gm I Gm I Gm I 2 185 172 164 179 174 4 187 37 175 36 168 31 174 37 175 29 5 198 30 184 26 177 27 187 25 192 17 Day 1 2 3 4 6 8 Grp Gm I Gm I Gm I Gm I 2 177 199 201 208 4 187 34 176 31 182 28 191 12 5 212 28 198 25 201 29 210 37 Day 10 11 12 13 P r i s c o l i n e Grp Gm I Gm I Gm I Gm I Gm I 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 Gm I Gm I 4 210 22 185 17 191 17 196 16 5 205 33 191 24 193 24 202 15 Day 10 11 12 13 231 10 1 2 3 4 X 6 X 8 X 10 11 12 13 D A Y 2 3 2 2 1 0 1 2 3 4 X 6 X 8 X 10 11 12 13 D A Y 233 be contained within one page. These graphs are done i n colours f o r easier reading ( a l l graphs have i d e n t i c a l colours except f o r the Time graphs of the f i r s t experiment), 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 rather than by group numbers. Condition of the Animals The animals receiving procaine were i n much better condition on the reduced dosage received l e s s often. However, they were somewhat affected, appearing rather out or sorts, and l e s s active than healthy rats should be. The P i r s c o l i n e had no observable i l l e f f e c t on the group of animals receiv- ing i t . Discussion of Observations A casual inspection of the Injury Index graphs for procaine and P r i s c o l i n e would give the impression that procaine protected the r a t legs and that the P r i s c o l i n e permitted a worse injury. The key to the question l i e s i n the f a c t that Injury Index l i n e s f o r the control groups, Groups 4, on both the procaine and 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 Identical groups receiving the standard freezing injury, follow- 234 ed by rapid thawing, without any medication. Act- u a l l y there was l i t t l e s i g n i f i c a n t difference i n the appearance of the various groups of animals. Weight changes were compa^rable between the various groups, i n d i c a t i n g that none were adversely affected by the drugs. Benadryl Experimental Observations with Benadryl The purpose of the experiment here described was to tes t the effects of a standard cold injury as Influenced by parenteral in j e c t i o n s of Benadryl at a dosage l e v e l of 2 mg. per kilogram body weight admin- 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 freezing i n j u r y and f o r forty-eight hours a f t - erwards. T h i r t y - s i x normal adult nonpregnant female Wistar rats were selected, and they were divided into f i v e groups with each group designed as follows: Group: 1. A group of eight animals to serve as a .control group and to receive a freezing i n j u r y of twenty seconds duration with slow thawing at room temperature. 2. A group of f i v e animals to serve as a 235 control also, and to receive only the Benadryl inje c t i o n s as outlined. 3. A group of eight animals to receive both the Benadryl in j e c t i o n s and a freezing injury of twenty seconds with slow thawing at room temperature. 4. A group of eight animals to serve as a control, to receive a freezing I n j - ury of twenty seconds duration with rapid thawing i n water at 42°G. 5. A group of seven animals (one of the eight o r i g i n a l l y set aside died when being anaesthetized withnembutal, and was eliminated completely from the records) to receive both the Benadryl inj e c t i o n s and a freezing i n j u r y of twenty seconds duration with<> rapid thawing i n water at 42°C. The larger number of animals being used was decided on because several other animals i n the same room had chest conditions and i t was feared a few of the animals i n this experiment might l a t e r develop i n f e c t i o n and have to be discarded, hence two extra rats were added to each group as spares. Group 2 i s small because there were not enough animals to f i l l the desired quota. These 236 groups are numbered i d e n t i c a l l y throughout a l l sub- sequent gross experiments. On Day 1 of the exper- iment, the day of freezing, 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 recorded as on the Data Sheet. The animals of Groups 2, 3, and 5 received t h e i r i n i t i a l subcutan- eous i n j e c t i o n of Benadryl solution prepared by ©f d i l u t l o n A t h e Parke-Davis product containing 10 mg. per cc. to a more convenient strength of 2 mg. per cc. and each animal was given 0.1 cc. f o r each 100 grams of body weight. By using a tuberculin syringe graduated i n hundredths of a cc. i t was a simple matter to i n j e c t the correct amount just by giving the animal's weight i n 1/100's of a cc. The in j e c t i o n s were given every three hours f o r a t o t a l of eighteen i n j e c t i o n s , l a s t i n g from h a l f past ten o'clock of Day 1 to h a l f past one o'clock 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, at h a l f past seven o'clock on the evening of Day 2 because of a shortage of the drug. The animals were kept i n f i v e cages, a l l the animals of one group i n the same cage. At l e a s t one hour aft 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 given subcutaneously from 0.07 cc. to 0.1 cc. of 0,6% nembutal solution. Then, when anaesthetized, one hind leg of each animal i n these groups was dipped i n a 237 beaker ©f ether through which carbon dioxide gas was © bubbling to bring the temperature down to -20 C. The foot was Immersed up to the hock (just below the f u r l i n e ) , and as soon as the skin blanched, i n d i c a t i n g the foot was completely frozen (at t h i s moment the rat i n v a r i a b l y squirmed, i n spite of adequate anaesth- esia) , the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by stop watch. The animals Groups 1 and 3 were placed i n t h e i r cages and the feet were allowed t© thaw at room temperature, taking approximately f i f t e e n or twenty minutes to become limp and warm. The animals of groups 4 and 5 were treated by rapid thawing, where- by the frozen feet were immersed Immediately at the expiry of the twenty seconds into water maintained at exactly 42°C. The feet 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 also returned to thei 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 hourly inj e c t i o n s of Benadryl) u n t i l they wakened from the effects of the nembutal. Observations were made as recorded on the Data Sheets, by the method described above f o r estimating the degree of f r o s t b i t e i n j u r y under the categories of oedema, 238 colour change, exudate and slough, and gangrene. Data Sheets These tables 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 of freezing. They are necessarily as compact as possible. Days 1 and 2 include a record of the three hourly i n j e c t i o n s . The symbols used are: Grp, group, Gm, grams, q3h, every three hours, 0, oedema, G, colour change, E, exud- ate and slough, G, gangrene, P, freezing, -, means not present or not administered, N, anaesthetic, B, Benadryl i n j e c t i o n . A l l surviving animals were disposed of on Day 10 with chloroform fumes. Summary Sheet Following the Data Sheets i s a Summary Sheet which i n thi s experiment does two things. Because of the unusually large number of animals per group, the Summary Sheet f i r s t summates the values each day by groups, and under I, or Index, gives the t o t a l for the injury changes occurring f o r that group that day. As described before, this concept of an Injury Index i s just an ar b i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. In the lower h a l f of the Summary Sheet the values are interpolated down f o r a 239 Data Sheet Grp 1 Gm q3h 0 C E G q3h 0 C E G 233 -NP 3 3 - - 288 -NP 3 2 - - 284 -NP 3 4 1 - 298 -NP 3 2 - - 250 -NP 3 1 317 -NP 3 3 276 -NP 3 1 1 - 276 -NP 3 3 3 3 3 3 3 3 3 3 2 1 2 1 2 2 1 280 B — BBBBBBBB BB-BBBBB 325 B—BBBBBBBB BB-BBBBB 272 B—BBBBBBBB BB-BBBBB 272 B—BBBBBBBB BB-BBBBB 253 B--BBBBBBBB BB-BBBBB 218 BNPBBBBBBBB 3 1 BBBBBBBB 3 3 2 _ 326 BNPBBBBBBBB 3 1 - - BBBBBBBB 3 4 2 1 305 BNPBBBBBBBB 3 3 - - BBBBBBBB 3 4 2 1 308 BNPBBBBBBBB 3 3 - - BBBBBBBB 3 4 2 1 272 BNPBBBBBBBB 3 3 - - BBBBBBBB 3 4 2 1 190 BNPBBBBBBBB 3 2 - - BBBBBBBB 3 4 2 1 280 BNPBBBBBBBB 3 3 - 2 BBBBBBBB 3 5 3 5 188 BNPBBBBBBBB 3 2 - - BBBBBBBB 3 5 2 2 302 -NP 3 1 3 1 1 260 -NP 3 1 - — 3 1 1 - 308 -NP 3 1 — - 3 3 1 — 250 -NP 3 1 - - 3 1 1 — 315 -NP 3 1 - — 3 1 1 1 274 3 1 - mm 3 1 1 _ 280 -NP 3 1 - - - — - 3 1 — — 280 -NP 3 1 - - 3 1 1 - 232 BNP Died 275 BNPBBBBBBBB 3 1 - - BB-BBBBB 3 2 1 - 254 BNPBBBBBBBB 3 1 - - BB-BBBBB 3 1 1 - 214 BNPBBBBBBBB 3 1 - - BB-BBBBB 3 2 1 mm 268 BNPBBBBBBBB 3 2 - - BB-BBBBB 3 3 1 -246 BNPBBBBBBBB 3 1 - — BB-BBBBB 3 2 2 -242 BNPBBBBBBBB 3 1 - - BB-BBBBB 3 1 — - 196 BNPBBBBBBBB 3 1 - - BB-BBBBB 3 2 2 - Day 1 2 3 240 rrp Gm q3h 0 C E G 1 216 B 3 4 2 280 B 3 3 1 - 280 B 3 5 2 280 B 2 3 2 - 240 B 3 3 1 - •305 B 3 3 2 - 265 B 3 4 2 — 270 B 3 3 1 - 2 290 B 330 B 260 B 270 B, 250 B 3 210 B 3 5 2 4 305 B 3 5 3 1 290 B 3 5 2 - 300 B 3 5 2 2 260 B 3 5 3 - 175 B 2 3 3 — 260 B 3 5 3 5 165 B 2 5 2 2 4 300 2 1 M 250 — 1 1 - - 310 - 3 3 2 - 240 — 2 3 — - 295 — 2 1 - 1 275 - 3 3 1 - 275 _ 2 1 — — 270 - 2 2 - - 5 (Dead) 265 B„ 3 2 1 - 240 B 3 2 1 220 B 3 3 1 -260 B 2 3 - - 240 B 2 1 1 - 230 B 3 1 - - 180 B Died Day 3(cont) 4 Data Sheet Gm 0 C E G Gm 215 3 3 2 1 210 275 3 3 1 - 250 280 2 5 1 - 270 260 1 5 - 3 270 230 3 3 2 mm 230 290 3 4 1 - 285 260 3 3 2 - 250 270 3 3 1 mm 260 280 280 320 320 265 260 270 270 250 245 205 2 3 4 200 325 2 3 1 1 280 280 2 3 2 1 280 290 2 5 - 4 285 250 2 3 2 — 250 175 2 3 1 - 180 260 2 4 - 5 260 160 1 5 1 4 170 295 1 1 280 245 1 1 - - 250 300 2 3 1 — 265 230 1 3 2 mm 235 280 1 - - 1 290 265 2 2 2 - 260 275 1 1 - — 260 255 1 1 1 mm 250 260 2 2 _ 240 230 2 3 1 - 235 220 2 3 1 - 220 250 1 4 - - 250 230 1 1 — - 235 235 2 1 — - 230 0 C E G Gm 0 C E G 3 5 — 3 205 3 3 2 5 2 5 — 4 260 2 1 - 4 3 5 — 5 270 3 1 2 5 2 5 — 5 280 2 1 - 5 3 5 5 225 2 1 - 5 3 5 — 5 280 3 2 - 4 2 5 — 4 245 2 1 0 5 3 3 1 mm 255 3 3 - 5 275 310 265 270 250 2 3 1 5 200 3 1 4 2 5 - 4 285 2 2 — 4 2 3 2 2 285 2 2 1 3 3 5 - 5 265 3 3 2 5 3 4 3 4 245 3 2 2 5 1 2 2 — 170 1 2 — - 2 5 5 260 3 2 — 5 2 2 1 - 180 3 2 1 5 _ 1 1 280 1 — 1 2 1 - 250 - 1 - - 1 2 2 - 280 1 1 - - 1 3 1 - 225 - 1 1 — 1 1 - 1 305 1 1 - 1 1 2 1 — 250 — 1 — - 1 1 - - 260 - 1 — — 1 1 — - 270 1 1 — — 1 2 1 mm 235 1 2 1 1 1 - 225 1 1 - - Qj 2 2 .-1 a 220 1 3 mm 1 4 1 - 240 1 2 - - — 2 1 - 230 1 2 - -- 1 - mm 230 _ 1 — - 10 241 Totals Sheet Grp Gm I Gm I I Gm I Gm I 1 2222 45 2136 66 64 2080 59 2025 91 2 1402 1400 1385 1375 3 2087 44 1965 86 95 1945 70 1905 85 4 2269 32 2215 42 36 2145 29 2090 27 5 1526 29 1455 42 32 1425 26 1410 22 Day 1 2 3 4 5 7 Grp Gm I 1 2020 75 2 1370 3 1890 73 4 2110 13 5 1380 16 Day 10 Summary Sheet Grp Gm I Gm I I Gm I Gm I Gm I 1 277 33 267 49 48 260 45 253 69 252 57 2 280 280 277 275 274 3 261 33 246 64 72 243 52 238 63 236 54 4 284 24 277 30 27 268 21 261 21 264 9 5 254 25 242 37 32 237 26 235 22 230 16 Day 1 2 3 4 5 7 10 242 theoretical s i x animal group, and average weights were obtained by div i d i n g the t o t a l weight f o r the group by the number of animals i n the group. Photographs On Day 3, at approximately three o'clock i n the afternoon, a l l animals were again anaesthetized with the same dosage of 0.6$ nembutal solution, and were photographed i n colour. Colour p r i n t s of Groups 1, 2, 3, and 4 are attached below, but unfort- unately the picture of Group 5 was improperly ex- posed and did not develop. The feet can be com- pared with the corresponding entries on the Data Sheets on Day 3. 245 244 Graphs The f i n a l interpolated values on the Summary Sheet are plotted on two simple l i n e graphs, the f i r s t f o r the Injury Index changes from day to day, and the second- f o r the corresponding weight changes. These are done i n colours f o r easier reading (except f o r the Time graphs of the f i r s t experiment, these and a l l other gross graphs have Identical 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 injury and treatment rather than by group numbers. Condition of the Animals The animals remained i n general good health throughout the duration of the experiment. As w i l l be apparent from the Weight graph, the Benadryl treated animals did not lose weight any more than the control animals. Th^e behaxior and appearance of the animals did not suffer much. Discussion of the Observations I t was f e l t that Benadryl as administered, had a consistently harmful e f f e c t on the c l i n i c a l course of the f r o ^ s t b l t e lesions as produced In t h i s experiment. This was r e a d i l y apparent by casual observation of the animals i n the various groups, as well as apparent from a study of the data obtained and the graph drawn from i t . Although the colour 245 1 2 3 4 5 D A T 7 10 246 J 5 5 7 D A Y 10 247 photographs are incomplete f o r group 5, groups 1 and 3 may be compared, and t h e i r positions on the Injury graph f o r day 3 placed (dotted l i n e s ) . The previously noted b e n e f i c i a l effects of the rapid thawing at 42°G. was again confirmed, and with Groups 4 and 5, although group 5 was ad- versely affected by the Benadryl, both groups were i n a l l respects less affected by the f r o s t b i t e after rapid thawing than wereethe animals i n Groups 1 and 3 which were thawed at room temperature. A l l the groups showed a s i g n i f i c a n t average weight l o s s , except the control group receiving the Benadryl only. This loss was attributed s o l e l y to the effects of the freezing injury, both through probably pain, and toxic effects l o c a l l y . The f a c t that an antihistaminic drug adversely affected the course of the lesions produced by a stand- ard f r o s t b i t e i n j u r y suggested both the need f o r re- peating the experiment with another antihistaminic (Chlor-Tripolon) and also the p o s s i b i l i t y that, i f Benadryl antagonizing histamine or a histamine-like substance l i b e r a t e d by the trauma of freezing, a drug l i k e histamine should be investigated f o r i t s r o l e i n the tissue reaction, and f o r any possible influence i t could 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 Observations with Etamon and Hydergine The purpose of the experiment here described was to test the effects of a standard cold i n j u r y as influenced by parenteral Injections of two d i f f e r e n t drugs. Etamon chloride (tetra-ethyl ammonium chlor- ide) was to be tested at a dosage l e v e l of 50 mg. per kilogram body weight administered subcutaneously at three hour i n t e r v a l s , p r i o r to the freezing i n j u r y and f o r forty-eight hours afterwards. Hydergine was to be tested i n d i f f e r e n t animals at a dosage l e v e l of 1 cc. per animal administered also subcutaneously at three hour Intervals, p r i o r to'the freezing i n j u r y and f o r forty-eight hours afterwards. Twenty-four normal adult male Wistar rats were selected, and they were divided into four groups with each group designed as follows: Group: 4. A group of six animals to serve as a control, to receive a freezing i n j - ury of twenty seconds duration with rapid thawing i n water at 42©C. 5. A group of six animals to receive both the Etamon Injections and a freezing i n j u r y of twenty seconds dur- ation with rapid thawing i n water 249 at 42°G. 4. Another group of s i x animals to serve as a control, to receive a freezing i n j u r y of twenty seconds duration with rapid thawing at 42°C. 5. A group of six animals to receive both the Hydergine i n j e c t i o n s and a freezing injury of twenty seconds duration with rapid thawing i n water at 42°C. The group numbers are kept uniform throughout a l l the gross experiments, hence explaining the duplication. There w i l l be no ambiguity because the group being discussed w i l l be exactly referred to. On the t h i r d day p r i o r to the freezing (Day -3 on the Data Sheets), 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 recorded. On Day 1, the day of the freezing, weights were again recorded and then the animals of both Groups 5 received t h e i r i n i t i a l subcutaneous i n j e c t i o n s of either Etamon of Hydergine. - This was at a quarter of ten o'clock. The respective in j e c t i o n s were continued every three hours u n t i l a quarter after ten o'clock on Day 3, with no doses midsed. The record of these i n j e c t i o n s was not put onto the Data Sheets. The animals were 250 kept i n four cages, a l l 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 i n j e c t i o n s the animals were given ( a l l animals, that i s ) subcut- aneously from 0.07 to 0.12 cc. of 0.6$ nembutal solution. Then when anaesthetized, one hind l e g of each animal was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to -20°C. The foot was immersed up to the hock (just below the f u r line),- and as soon as the skin blanched, i n d i c a t i n g the foot was complete l y frozen (at thi s moment the rat in v a r i a b l y squirmed, i n spite of adequate anaesthesia), the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by stop watch. The animals were then a l l treated by rapid thawing, whereby the frozen feet were immersed immediately at the expiry of the twenty seconds into water maintained at exactly 42°C. The feet 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 the animals were returned to th e i r cages and were l e f t q u i e t l y alone (except f o r the three hourly injections) u n t i l they wakened from the effects of the nembutal. Observations were made as recorded on the Data Sheets, by the method described above f o r estimating the degree of f r o s t b i t e intiury 251 under the categories of oedema, colour change, exudate and slough, and gangrene. Data Sheets These tables 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 of freezing. They are necessarily as compact as possible. A l l the data fo r Etamon i s placed on the f i r s t , and the data f o r Hydergine i s placed on the second. The symbols used are: G-rp, group, Gm, grams, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, -, means not present. A l l surviving animals were disposed of on Day 13 with chloroform fumes. Summary Sheet Following 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, gives the t o t a l f o r the injury changes occurring f o r that group that day. As described before, t h i s concept of an Injury Index i s just an a r b i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. In view of the f a c t none of the animals died, no extrapolation was necessary. As noted on the Summary Sheet, the top h a l f applies to Etamon, and the remainder to Hydergine. 252 Data Sheet - Etamon Grp Gm Gm Gm 0 C E G Gm 0 C E G 4 168 170 163 3 1 165 3 2 1 184 193 180 3 2 — - 186 2 1 - - 171 176 166 3 1 _ — 167 3 2 1 - 175 183 172 3 2 1 _ 176 2 2 - - | 173 191 178 2 2 1 182 1 1 - -f 156 157 151 3 2 - 156 3 2 1 — 5 168 180 172 3 2 1 173 3 2 1 _ 198 197 199 3 2 1 204 3 2 - - 180 184 178 3 2 - 181 3 2 - 167 169 166 3 1 1 mm 168 3 1 - - 176 172 170 3 2 1 - 171 3 1 1 - 174 177 174 3 3 1 mm 176 3 2 1 — Day -3 1 2 3 Grp Gm 0 C E G Gm 0 C E G Gm 0 C E 4 161 2 2 2 — 161 2 2 2 178 1 2 3 184 - 1 - - 190 - 1 — - 206 — — — 163 2 2 2 - 166 2 2 1 - 175 2 2 3 180 1 2 - - 182 1 2 - — 205 — _ _ 179 1 1 - - 188 - 1 - - 200 — — _ 150 2 1 - - 159 1 2 - - 182 - - mm 5 168 2 2 - 172 2 2 190 198 2 1 - - 203 1 2 — — 218 - _ — 180 2 2 - - 182 1 2 _ - 194 1 — — 163 2 2 1 - 169 1 2 - — 184 1 _ - 167 2 2 2 1 167 2 2 1 1 189 1 2 — 173 2 3 2 - 176 2 2 - - 195 — 2 2 Day 13 253 Data Sheet - Hydergine Grp Gm Gm Gm 0 C E G Gm 0 C E G 4 330 308 316 3 2 _ 315 3 2 1 362 342 350 3 2 1 - 352 3 2 1 - 336 320 329 3 1 - - 329 3 2 - - 268 273 281 3 2 — - 284 2 2 1 - 264 257 259 3 2 1 - 260 3 2 - - 272 270 275 3 2 1 - 273 3 2 1 - 5 289 282 283 3 2 1 mm 281 3 2 1 295 296 295 3 2 1 - 291 3 1 1 - 279 285 279 3 2 1 — 273 3 1 1 — 208 203 204 3 3 1 - 202 3 3 - - 267 266 263 3 3 - — 254 3 3 1 - 297 296 290 3 1 - - 281 3 2 1 - Day -3 1 2 3 Grp Gm 0 C E G Gm 0 C E G Gm 0 C E G 4 311 3 3 1 311 2 3 1 _ 322 2 2 2 346 3 3 2 _ 344 3 3 2 - 343 1 2 — - 325 2 3 - — 326 2 2 - - 343 - — — - 286 2 2 3 - 282 2 2 3 — 329 1 1 2 - 258 2 1 1 _ 258 2 2 - - 275 - - — - 275 2 3 1 - 275 2 3 1 - 285 1 1 - - 5 273 3 3 1 279 2 3 2 _ 293 1 2 2 287 3 2 - - 290 3 1 - - 298 2, _ mm 268 3 2 2 _ 269 3 3 2 - 272 2 2 3 _ 193 3 3 1 — 201 3 3 1 - 221 1 2 1 1 261 3 2 1 - 257 3 3 2 - 285 1 2 2 1 275 8 1 ± - 279 3 2 2 - 303 1 2 - - Day 4 5 13 254 Summary Sheet Etamon Grp 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 3 4 Grp Gm 1 Gm 1 4 174 19 191 13 5 178 23 196 9 Day 5 13 Hydergine Grp 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 3 4 Grp Gm 1 Gm 1 4 299 35 316 15 5 263 41 279 26 Day 5 13 255 Graphs The values on the Summary Sheet are plotted d i r e c t l y on the two simple l i n e graphs, the f i r s t f o r Etamon and the second f o r Hydergine, Weight and Injury Index changes are plo t t e d against the days. The graphs had to be foreshortened between Days 5 and 13 i n order to contain them within one page at the scale chosen. The graphs are done i n colours f o r easier reading (except f o r the Time graphs of the f i r s t experiment, these and a l l other 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 injury and treatment rather than by group numbers. Condition of the Animals The animals remained well throughout the duration of the experiment. I n i t i a l l y 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 normally active. The Etamon inj e c t i o n s were somewhat p a i n f u l , causing squealing each time the material was injected. The Hydergine had no apparent ef f e c t on the animals receiving i t . Discussion of the Observations The impression c l i n i c a l l y was that Etamon had no influence on the course of the f r o s t b i t e i n j - ury, 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 Injury Index l i n e f o r the Etamon treated group remained s l i g h t l y above the control l i n e , l a t e r crossing i t . However with the Hydergine treated Index l i n e there i s a l a t e r divergence. Noteworthy i s the f a c t that both control injury Index l i n e s are s imilar, which they should be i n that s i m i l a r groups, Groups 4, were both treated i d e n t i c a l l y . I f the mean of these two red l i n e s were drawn i n , the r e s u l t i n g l i n e would only serve to accentuate the differences 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 and Chlor-Tripolon Experiments with Apresoline and Chlor-Tripolon The purpose of the experiment here described was to t e s t the effects of a standard cold i n j u r y as influenced by parenteral Injections of two d i f f e r e n t drugs. Apresoline was to be tested at a dosage l e v e l of 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 freezing i n j u r y and f o r forty-eight hours afterwards. Chlor-Tirpolon was to be tested 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 also subcutaneously at three hour i n t e r v a l s , p r i o r to the freezing i n j u r y and f o r forty-eight hours afterwards. Eighteen normal adult nonpregnant female Wistar rats were selected, and they were divided into three groups of six animals each, with each group designed as follows: Group: 4. A group of s i x animals to serve as a control, to receive a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 42°C. 5. A group of six animals to receive both the Apresoline i n j e c t i o n s and a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 42oc. 5. A group of six animals to receive both the Chlor-Tripolon i n j e c t i o n s and a freezing injury of twenty seconds duration with rapid thawing i n water at 42°C. The group numbers are kept uniform throughout a l l the gross experiments, hence explaining the duplication. There w i l l be no ambiguity because the group being discussed w i l l be exactly referred to. On Day 1 of the experiment, the day of freezing, 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 weights were recorded as on the Data Sheet. The animals of both Groups 5 received t h e i r appropriate i n i t i a l i n j e c t i o n s at ten o'clock and again at one o'clock. At three o'clock a l l animals received by in t r a p e r i t o n - eal i n j e c t i o n an anaesthetizing dose of 0 . 6 $ nembutal solution of 0 . 0 7 5 cc. Then when anaesthetized, one hind leg of each animal was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to - 2 0 C. The foot was immersed up to the hock (just below the f u r l i n e ) , and as soon as the skin blanched, i n d i c a t i n g the foot was completely frozen (at this moment the rat i n v a r i a b l y squirmed, i n spite of adequate anaesthesia), the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by stop watch. The animals were then a l l treated by rapid thawing, whereby the frozen feet were immersed Immediately at the expiry of the twenty seconds into water maintained at exactly 42©c. The feet were l e f t i n the warm water f o r approx- imately one minute,- u n t i l thoroughly thawed and warm. Then the animals were returned to th e i r cages and were l e f t q u ietly alone (except f o r the three hourly i n j e c t - ions) u n t i l they wakened from the effects of the nembutal. Observations were made as recorded on the 261 Data Sheets, by the method described above f o r estimating the degree of f r o s t b i t e injury under the categories of oedema, colour change, exudate and slough, and gangrene. The animals were kept i n three cages, a l l animals of one group i n the same cage. On Day 1̂ _4 a l l animals were disposed of with chloroform fumes. Data Sheets These tables 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 of freezing. They are n ~ •?• c * .=? a necessarily as compact as possible. Days 1, 2, and 3 include a record of the three hourly i n j e c t i o n s . The symbols used are: Grp, group, Gm, grams, q3h, every three hours, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, -, means not present or not administered, P, freezing injury, A, Apresoline i n j - ection, G, Chlor-Tripolon i n j e c t i o n . Summary Sheet Following 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, gives the t o t a l f o r the injury changes occurring f o r that group that day. As described before, this concept of an Injury Index i s just an a r b i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group 262 Data Sheet Apresoline Grp Gin q3h Gm O C E G q3h Gm O C E G q3h 4 155 — P 141 3 2 - - 145 3 1 © - — 158 — p 143 3 1 - - 141 2 1 - - — 153 — p 134 3 2 - - 131 3 2 - - — 133 — P 145 3 4 - - 146 3 4 - - — 131 — p 140 3 4 1 - 143 3 4 1 - — 132 — p 145 3 2 - - 150 3 2 1 - — 5 163 AAPAAAAAAAAAA 153 3 1 - - AAAAA 152 3 1 AA 159 AAPAAAAAAAAAA 146 3 1 AAAAA 146 3 1 AA 162 AAPAAAAAAAAAA 148 3 1 - - AAAAA 151 3 1 - - AA 167 AAPAAAAAAAAAA 157 3 1 - - AAAAA 156 2 1 - - AA 163 AAPAAAAAAAAAA 151 3 1 - - AAAAA 147 3 1 - - AA 161 AAPAAAAAAAAAA 144 3 1 AAAAA 149 3 1 AA Day 1 2 3 Chlor-Tripolon Grp Gm q3h Gm O C E G q3h Gm O C E G q3h 5 140 CCPCC—CCCCC 132 2 3 - - C 125 1 5 CCh 132 CCPCC—CCCCC 121 3 3 C 115 3 3 - 1 CC 175 CCPCC—CCCCC 161 3 3 - - C 155 3 4 - - CC 189 CCPCC—CCCCC 171 3 2 1 - C 162 3 2 2 - CC 151 CCPCC CCCCC 142 3 2 C 137 3 3 1 - CC 136 CCPCC—CCCCC 126 3 2 - - G 120 3 3 CC Day 1 2 3 263 Data Sheet Apresoline Grp Gm 0 C E G GM 0 C E G Gm 0 C E G Gm 0 C E G 4 155 2 1 _ 158 1 1 160 2 mm 163 _ 1 _ 153 1 1 - - 157 - 1 - - 164 - 1 - - 167 - - - - 148 2 2 - — 153 1 2 - 159 1 2 mm - 159 1 1 - - 146 3 4 — 152 3 4 - - 158 2 2 2 6 158 1 1 1 6 147 3 3 - _ 153 3 3 1 - 155 2 2 - 2 159 1 2 - 2 150 3 1 1 - 157 2 1 - - 162 1 2 2 - 162 2 2 4 - 5 155 2 1 _ 162 1 1 174 1 2 2 181 1 2 1 *• 145 2 1 - - 150 1 2 2 — 169 1 2 1 - 159 1 2 2 - 156 2 1 - - 158 2 2 — - 173 1 2 1 - 174 1 1 1 - 161 1 1 - - 163 - 1 - — 180 - 1 - - 180 - - - - 159 1 1 - - 153 1 - - - 165 - 1 — - 163 - 1 - - 149 1 1 2 - 155 1 1 2 - 168 1 2 2 - 168 1, 2 2 - Day 4 5 9 11 Chi or •-Tripolon Grp Gm 0 C E G Gm 0 C E G Gm 0 C E G Gm 0 C E G 5 121 _ 5 _ 123 _ 5 mm 5 125 1 1 7 130 mm 7 107 2 1 - 6 108 2 1 2 7 122 2 1 mm 7 123 - — 7 145 3 2 — 1 148 2 1 — 4 152 1 1 2 6 154 1 _ - 6 152 2 2 1 5 156 2 1 2 5 175 1 1 - 6 182 - — — 6 128 2 3 2 1 134 3 4 1 1 150 1 1 — 6 153 1 1 — 6 112 1 5 2 3 112 1 5 1 6 113 1 1 - 7 122 - - - 7 Day 4 5 9 11 264 Data Sheet Apresoline Grp 4 Gm 0 C E G 166 _ 1 - 1 164 - - - - 164 — 1 — — 143 1 1 - 6 142 2 2 - 2 135 1 2 — - 198 1 1 166 1 2 187 1 2 186 - - 169 - - 178 1 2 Day 14 Chlor-Tripolon Grp Gm 0 C E G 5 134 1 1 7 135 2 1 — 7 175 2 1 2 6 194 1 1 — 6 162 2 1 1 6 125 2 2 — 7 Day 14 265 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 5 9 Apresoline Grp Gm I Gm I 4 161 25 152 20 5 171 18 181 13 Day 11 14 Grp Gm I Gm I 5 144 42 154 59 Day 11 14 Chior-Tripolon 266 each day. In view of the f a c t that none of the animals died, no extrapolation was necessary. Graphs The values on the Summary Sheet are d i r e c t l y plotted onto the two simple l i n e graphs, the f i r s t being f o r Injury Index changes by days, and the second f o r weight changes also by days. The graphs had to be foreshortened between Days 5 and 14 i n order to contain them within one page at the scale chosen. The graphs are done i n colours f o r easier reading (except f o r the Time graphs of the f i r s t experiment, these and a l l other 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 injury and treatment rather than by group numbers. Condition of the Animals The animals receiving Apresoline were apparently quite unaffected by the drug. However, the Chlor- Tripolon quite adversely affected the rats receiving i t . As w i l l be noted from the Data Sheets, quite a few of the inje c t i o n s with Chlor-Tripolon had to be missed because of serious I l l n e s s i n the animals. However none expired, and a l l recovered to an apparently normal state a f t e r the Chlor-Tripolon was discontinued. 267 1 0 1 2 3 4 5 9 - 11 - - 14 D A T 268 1 2 3 4 5 9 - 11 - - 14 D A Y 269 Discussion of the Observations There was only one untreated control 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 Injury Index l i n e graph f o r t h i s group Is comparable with the si m i l a r graph i n other experiments. Generally speaking Apresoline favour- ably influenced the behaviour of the injured feet of those animals receiving i t . This was the general impression c l i n i c a l l y , and the Apresoline Injury Index graph bears this out. However, the Chlor- Tripolon treated animals were made very much worse after the freezing i n j u r y . This reaction i s much greater than I t was fo r Benadryl. I t i s possible the general effects the Chlor-Tripolon had on the animals could have influenced the healing of the injured feet. The weight graph confirms the f a c t that the Chlor-Tripolon had an adverse effect generally on the animals receiving i t . Rutin Experimental Observations with Rutftn The purpose of the experiment here described was to test the effects of a standard cold i n j u r y as 270 influenced by r u t i n administered o r a l l y . Thirty normal adult male Wistar rats were selected f o r the test and they were divided into f i v e groups of s i x animals each, with each group designed as follows: Group: 1. A control group to receive a f r e e z - ing injury of twenty seconds duration with slow thawing at room temperature. 2. Another control group to receive only the o r a l r u t i n medication. 3. A group to receive both the r u t i n and a freezing injury of twenty sec- onds duration with slow thawing at room temperature. 4. A control group to receive only a freezing injury of twenty seconds duration with rapid thawing i n water at 42°C. 5. A group to receive both the r u t i n and a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 42°C. As noted previously, these groupings are the same throughout a l l gross experiments. On Days -9, -7, and 271 -5, being the ninth, seventh, and f i f t h days p r i o r to freezing, a l l animals i n groups 2, 3, and 5 re- ceived 100 cc. of water containing 100 mg of r u t i n (two ground up 50 mg. compressed r u t i n t a b l e t s ) . Each animal was i n a separate cage, eaeh receiving i t s own in d i v i d u a l water supply containing the r u t i n . On Day -4, being the fourth day before the freezing, the concentration of the r u t i n solution was increased so that each animal of Groups 2, 3, and 5 received each day 100 mg. of r u t i n contained i n 35 cc. This was administered on eaeh of Days -4, -3, -2, and -1, and of course the same solution was given d a i l y afterwards u n t i l the animals were disposed of on day 6. No record was kept of the precise amount of r u t i n consumed by the animals receiving i t . There was a l i t t l e wastage, and several of the animals l e f t some of th e i r solution each day. However, i t was f e l t that i t could be safely claimed each animal took at l e a s t 50 mg. of r u t i n per day, and many took as much as 90 or 95 mg. Although no record was kept of the f a c t , i t was noted that the animals were some- what consistent i n whether or not they drank up a l l t h e i r r u t i n solution each day. A l l animals i n Groups 1 and 4 were kept s i x to a cage, that i s , one cage f o r each of these two groups. A l l animals were weighed on Days 1, 3, and 5. 272 On Day 1, the day of the freezing, the animals of Groups 1, 3, 4, and 5 were given subcutaneously from 0.1 to 0.125 cc. of 0.6$ nembutal solution. Then when the animals were anaesthetized, one hind leg of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to -20°C. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blanched, i n d i c a t - ing the foot was completely frozen (at t h i s moment the rat invar i a b l y squirmed, i n spite of adequate anaesthetic), the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by o stop watch. The animals of Groups 1 and 3 were placed i n the i r cages and the feet were allowed te thaw at room temperature, taking approximately f i f t e e n or twenty minutes to become limp and warm. The animals of groups 4 and 5 were treated by rapid thaw- ing, whereby the frozen feet were immersed immediately at the expiry of the twenty seconds into water maintained at exactly 42°C. The feet were l e f t i n the warm water fo r approximately one minute, u n t i l thoroughly thawed and warm. Then these animals were also returned t© the 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 effects of the nembutal. 273 Observations were made as recorded i n the Data Sheet, by the method described before f o r estimating the degree of f r o s t b i t e injury under the categories of oedema, colour 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 of freezing. I t i s necessarily as compact as possible. The symbols used are; Grp, group, Gm, grams, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, P, freezing, -, means not present. Summary Sheet Following the Data Sheet i s a Summary Sheet which gives the average weight f o r each group each day, and under I, or Index, gives the t o t a l f o r the injury changes occurring f o r that group that day. As described before, this concept of an Injury Index i s just an ar b i t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. Except f o r Group 2, where of course no f r eezing was performed, and no Index number--wilife appear. Because no animals expired no extrapolation was nec- essary. 274 Data Sheet Grp Gm 0 C E G Gm 0 G E G 0 C E G Gm 0 C E G 0 C E G 1 230 3 2 2 210 2 2 2 3 3 3 1 220 3 3 2 1 3 2 _ 1 270 3 1 1 - 255 3 1 2 - 3 3 3 - 245 2 3 3 - 2 2 3 mm 280 2 3 2 2 250 3 2 1 2 3 3 3 1 250 3 3 3 1 3 3 1 2 255 3 2 2 — 230 3 1 2 - 3 3 3 — 210 2 1 1 2 2 1 - 3 245 3 2 2 - 225 2 1 2 - 3 3 3 - 220 3 3 2 — 3 3 — - 260 3 2 2 _ 240 3 1 1 — 2 2 2 - 240 2 2 2 1 2 - - 2 240 235 240 235 200 225 250 205 235 240 230 235 255 245 255 200 200 215 3 215 222 2 — 210 2 1 2 - 3 2 3 - 210 2 2 2 - 2 2 1 - 240 2 2 2 — 230 2 2 2 1 3 3 2 1 235 2 3 3 2 2 3 2 3 235 3 2 1 — 200 2 2 2 1 3 2 1 1 210 3 2 2 1 1 1 1 — 195 2 1 1 — 190 2 1 2 - 2 2 2 - 180 1 1 1 - 1 1 1 - 210 2 1 1 mm 215 2 1 1 - 1 2 2 - 215 1 1 1 - 1 1 1 mm 235 2 1 2 - 205 2 2 2 - 2 2 2 - 225 1 1 1 - 1 1 1 - 4 190 3 2 2 185 111 1 2 2 2 mm 190 2 2 2 2 2 _ 215 2 2 2 2 205 1 1 — 2 1 1 - 2 210 1 1 — 2 1 1 - 2 240 3 1 1 — 230 2 1 2 - 2 1 2 - 220 2 2 1 - 1 2 2 - 265 3 1 - — 260 1 1 — - 1 2 2 — 270 - 1 — — 1 1 — - 230 2 2 2 - 215 1 2 2 — 1 1 — - 220 1 2 1 - 1 2 — - 245 3 1 1 240 1 1 - - 1 1 1 - 245 - 2 2 - 1 2 1 - 5 250 2 1 225 1 1 230 1 _ _ _ 235 1 1 — - 210 - - — 1 - 1 — 1 205 — - - 1 - - - 1 230 2 1 - — 210 2 2 2 — 2 2 2 - 210 1 2 2 - 1 2 2 - 245 2 1 - - 215 215 205 2 1 1 - 180 2 1 1 — 2 1 2 - 175 2 2 2 — 1 1 2 - 245 1 1 - 230 1 - - - 1 1 - - 250 Day 1 2 ;3 5 6 275 Summary Sheet Grp Gm I Gm I I Gm I I 1 257 42 235 36 53 231 47 37 2 237 237 234 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 Day 1 2 3 4 5 6 Graphs The values appearing on the Summary Sheet above were plotted 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 Injury Index changes from day to day, and the second f o r the weight changes. These are done i n colours f o r easier reading (except f o r the Time graphs of the f i r s t experiment, these and a l l succeeding gross graphs have i d e n t i c a l colours, and of course, a l l are on the same scale), 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 jury and treatment rather than by group numbers. Condition of the Animals A l l animals remained i n excellent condition f o r 276 1 2 3 4 5 6 D A Y 277 260 1 2 5 4 5 D A Y 278 t h e d u r a t i o n o f t h e e x p e r i m e n t . T h e y d r a n k t h e i r r u t i n s o l u t i o n r e a d i l y . P h o t o g r a p h s On D a y 4, a f t e r a g a i n a n a e s t h e t i z i n g t h e a n i m a l s w i t h 0 . 1 t o 0 . 1 2 5 c c o f 0 . 6 $ n e m b u t a l s o l u t i o n , a l l t h e f e e t w e r e p h o t o g r a p h e d i n c o l o u r a s i n d i c a t e d b y t h e c o l o u r p r i n t s b e l o w . I t i s i n t e r e s t i n g t o c o m p a r e t h e a p p e a r a n c e o f t h e s e f e e t w i t h t h e v a l u e s r e c o r d e d f o r D a y 4 o n t h e D a t a S h e e t , a n d a l s o w i t h t h e I n j u r y I n d e x g r a p h f o r Day 4. 279 280 Discussion of Observations I t was f e l t both c l i n i c a l l y and through observation of the Data Sheet and graphs that r u t i n as administered had a consistently b e n e f i c i a l e f f e c t on the c l i n i c a l course of f r o s t b i t e lesions as pro- duced i n this experiment. This consisted i n decreased development of oedema, less colour change, less skin slough, and less gangrene. The Injury Index graph l i n e s are well and consistently separated. The previously noted b e n e f i c i a l e f f e c t of rapid thawing i n water at 42 QG. was again confirmed,* and with r u t i n , the animals receiving the drug were i n a l l respects lefis affected by the Injury than those not receiving the drug, although both groups were rap i d l y thawed. Rutin as administered appeared to have no ad- verse effects on the animals receiving i t . A l l of 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 control group receiving r u t i n only. The r o l e that l i m i t e d f l u i d intake played i n this weight loss was not assessed. Note; The senior medical student who was helping with the project part time performed the actual freezing ©f 281 the feet, and while he froze them more or less uniformly throughtout the r u t i n series, h i s degree of injury was only about eighty percent as severe as the injury used elsewhere In the experiments. Although i t i s easy to write about a standard cold injury, the actual performance of the same i s a f f l i c t e d with many minor v a r i a t i o n s . Ascorbic Acid Experimental Observations with Ascorbic Acid The purpose of the experiment here described was to tes t the effects of a standard cold i n j u r y as influenced by parenteral in j e c t i o n s of ascorbic acid at a dosage l e v e l of 50 mg. administered sub- cutaneously i n 1 cc. amounts twice a day f o r f i v e days preceding the freezing injury and f o r f i v e days afterwards. Forty-two normal adult nonpregnant female Wistar rats were selected, and they were d i v i d - ed into seven groups of six animals each, with each group designed as follows: Group: 1. A control group to receive a freezing i n j u r y of twenty seconds duration with slow thawing at room temperature. 2. A group to receive only the ascorbic 282 acid i n j e c t i o n s as outlined. 3. A group to receive both the ascorbic acid i n j e c t i o n s and a freezing i n j - ury of twenty seconds duration with slow thawing at room temperature. 4. A group to receive a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 42°G. 5. A group to receive both the ascorbic acid i n j e c t i o n s and a freezing i n j - ury of twenty seconds duration with rapid thawing i n water at 42°C. 6. A control group f o r microscopic obs- ervation to receive no ascorbic acid. 7. A group f o r microscopic observation to receive the ascorbic Injections as outlined. These groups, with the exception of 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 microscopic work was not performed as planned on these l a s t two groups, but they were carried along u n t i l Day 3, and t h e i r weights were included i n the Data Sheets as controls. On the f i f t h day before the freezing, Day -5, each animal was marked characterist- 283 i c a l l y on the ear, and i n i t i a l weights were recorded as on the Data Sheets. The animals of Groups 2, 3, 5, and 7 a l l received t h e i r i n i t i a l subcutaneous injec t i o n s of ascorbic acid at nine o'clock. The inject i o n s were continued at nine o'clock both morning and evening u n t i l nine o'clock i n the morning of Day 3, with the injec t i o n s being missed once, the evening dose on Day -1. Daily weights were recorded as on the Data Sheets. On Day 1, the day of the freezing, the animals of Groups 1, 3, 4, and 5, at a quarter past ten o'clock, were anaesthetized with an intr a p e r i t o n - eal i n j e c t i o n of 0.075 to 0.1 cc of 0.6$ nembutal solution. Then, when anaesthetized, one hind l e g of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to -20®G. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blanched, Indic- ating the foot was completely frozen (at t h i s moment the rat inv a r i a b l y squirmed, i n spite of adequate anaesthetic), the time was counted so that the leg was immersed and frozen f o r exactly twenty seconds by stop watch. The animals of Groups 1 and 3 were placed i n t h e i r cages and the fe e t were allowed to thaw at room temperature, taking approximately 284 f i f t e e n or twenty minutes to become limp and warm. The animals of groups 4 and 5 were treated by rapid thawing, whereby the frozen feet were immersed immediately at the expiry of the twenty seconds into water maintained at exactly 42 GC. The feet 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© returned 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 effects of the nembutal. Observations were made as recorded i n the tables, by the method describ- ed above f o r estimating the degree of f r o s t b i t e from the categories of oedema, colour change, exudate and slough, and gangrene. The animals of Groups 6 and 7 were destroyed ©n Day 3 and the remainder on Day 8, a l l by chloroform fumes. Data Sheets These tables 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 of freezing. They are nec- e s s a r i l y as compact as possible. There i s included a record of a l l ascorbic acid i n j e c t i o n s . The symbols used are: Grp, group, Gm, grams, bid, twice a day, 285 Grp Gm Gm b i d 1 190 191 _ _ 192 195 — 172 176 — 219 217 — 195 199 — 206 208 — 2 200 197 CC 199 194 CC 208 209 cc 203 205 c c 190 198 c c 208 214 c c 3 215 225 c c 220 220 c c 206 209 cc 212 217 c c 220 222 c c 203 209 c c 4 204 204 218 215 — 200 201 185 187 — 208 211 — 219 219 — 5 201 201 c c 209 214 c c 204 204 c c 198 194 c c 200 206 c c 190 195 c c 6 219 220 _ _ 200 200 216 214 — 185 186 174 174 — 182 186 — 7 210 214 c c 206 210 c c 200 205 c c 195 198 c c 193 195 c c 192 196 c c Day -5 -4 Data Sheet Gm b i d Gm b i d 193 _ mm 193 _ mm 198 198 -. — 177 180 221 221 204 204 mm mm 212 ~ - 210 — 200 CC 201 CC 189 CC 195 c c 215 c c 220 c c 212 c c 212 c c 198 c c 201 cc 219 c c 217 c c 225 c c 232 c c 224 c c 231 c c 212 c c 210 cc 221 c c 225 c c 229 cc 233 c c 212 c c 220 c c 208 -mmm 210 220 — 222 201 —_ 202 190 — 195 209 —— 210 221 - - 220 - - 206 CC 205 GG 221 c c 229 c c 206 c c 200 c c 199 c c 202 c c 210 c c 211 c c 192 c c 194 c c 216 _ _ 226 _ _ 200 200 211 — 211 182 191 178 179 187 — 191 — 217 c c 214 c c 207 c c 219 c c 206 c c 203 c c 201 c c 212 c c 196 c c 196 c c 199 c c 207 c c -3 -2 Gm b i d Gm b i d 193 196 - P - 198 — 198 —P<— 181 — 180 - F -227 — 230 - F -205 -- 201 - F -212 — 212 - F - 205 C- 201 C-C 201 c - 196 c - c 218 c - 217 c - c 216 c - 205 c - c 203 c - 194 c - c 219 c - 206 c - c 233 c - 227 CPC 225 c - 226 CFC 210 c - 212 CFC 229 c - 229 CPC 229 c - 233 CFC 224 c - 225 CFC 213 218 - F - 224 — 226 - F -201 mm mm 203 - F -193 — 193 - F -212 mm mm 215 - F -227 -- 232 - F - 206 c - 206 CFC 233 c - 230 CFC 203 c - 205 CPC 200 c - 199 CPC 214 c - 210 CPC 192 c - 196 CPC 226 _ _ 230 _ _ _ 199 — 203 217 — 218 189 — 189 186 — 186 186 -- 188 213 c - 212 G-C 218 c - 217 C-C 196 c - 199 C-C 207 c - 210 C-C 196 c - 196 C-C 203 c - 196 C-C -1 1 286 Data Sheet Grp Gm 0 C E G b i d Gm 0 C E G b i d Gm 0 C E G Gm 1 199 4 3 1 _ _ _ 196 4 3 2 _ _ 194 3 3 2 _ 185 199 4 3 1 - — 199 4 3 2 — 202 3 4 2 — 195 180 4 3 2 - — 181 4 4 1 - —— 179 3 4 1 - 175 228 4 3 2 - 223 4 3 2 — 221 3 2 1 220 200 4 3 2 — 201 4 3 2 - 196 3 3 1 - 190 217 4 3 2 - — 218 4 3 2 - — 211 3 3 2 - 206 2 203 CC 204 CC 206 208 199 CC 200 CC 202 202 223 cc 214 cc 215 210 199 cc 187 cc 183 176 186 cc 177 cc 171 164 198 cc 189 cc 181 174 3 227 4 3 2 cc 220 4 4 2 _ cc 217 3 3 2 _ 214 230 4 3 2 cc 221 3 4 2 cc 222 1 4 — 1 221 217 4 3 2 - cc 213 3 4 2 - cc 213 1 5 1 2 216 228 4 3 2 - cc 219 3 4 2 — cc 221 2 5 1 1 218 235 4 3 2 - cc 223 4 4 2 - cc 226 4 3 1 - 224 227 4 3 2 - cc 217 4 3 2 - cc 211 3 3 2 - 205 4 217 4 1 2 mm mm 211 4 1 — ,211 3 2 1 212 225 4 1 2 - 219 4 1 1 mm mm 220 3 2 2 - 215 203 4 1 2 195 3 2 - — — 193 3 1 2 - 193 187 3 3 — - - - 183 3 3 — — 176 3 3 — mm 184 214 4 1 1 •* 206 4 1 1 - _ — 195 2 1 — — 199 232 4 3 1 - mm mm 226 4 3 1 — 204 3 3 1 217 5 204 4 2 2 CC 202 3 3 3 cc 201 3 3 2 198 236 4 2 1 - cc 226 3 2 1 - cc 223 3 3 2 — 220 209 4 3 1 - cc 202 3 2 — - cc 203 3 3 — — 198 204 4 1 2 - cc 203 3 2 — — cc 203 3 2 1 - 208 215 4 1 1 - cc 210 3 1 1 cc 214 2 2 1 - 211 202 3 2 - - cc 198 3 2 2 cc 199 2 2 2 205 6 235 231 — mm 204 — 204 mmmm 208 — 221 mm mm 195 — 188 - - 192 191 - — 194 - - 196 7 214 cc 211 CC 223 cc 223 CC 202 cc 200 CC 216 cc 222 CC 202 cc 196 CC 202 cc 202 cc Day 2 3 4 5 287 Data Sheet Grp 0 C E G Gm 0 C E G 1 3 4 2 195 3 3 1 4 3 4 2 — 199 3 4 2 4 3 4 2 - 178 2 4 1 2 3 4 2 1 219 3 4 2 5 3 4 1 - 192 3 4 2 1 3 4 2 - 199 3 4 2 5 2 214 205 219 167 156 164 3 3 4 2 210 2 5 1 4 1 4 - 7 211 - - - 9 1 4 1 6 218 1 5 1 7 2 4 - 3 214 2 3 2 7 3 4 2 - 221 3 3 2 4 3 4 2 - 203 3 3 3 6 4 3 3 2 215 2 2 1 M 3 3 3 - 221 3 3 2 3 1 2 - 202 2 — 2 — 3 4 1 - 193 2 4 2 2 2 1 1 204 1 1 2 - 3 3 3 - 219 3 3 3 - 5 3 2 3 202 3 2 3 3 2 3 - 217 3 3 3 — 3 2 - 3 208 3 2 — 3 2 2 1 - 209 2 2 — - 2 2 1 - 218 2 2 — - 2 2 2 - 211 1 2 2 Gm 0 C E G Gm 0 C E G 196 3 3 3 3 194 3 3 1 4 198 2 2 3 6 196 2 3 2 6 174 2 4 3 3 171 2 3 3 6 219 2 3 5 5 212 2 3 2 6 194 3 5 3 5 193 2 3 3 6 201 3 4 3 5 204 3 3 3 5 220 215 205 200 220 210 161 148 154 154 157 151 207 2 5 2 5 210 2 2 3 8 210 1 3 2 9 212 2 2 1 9 214 3 3 1 8 214 2 2 0 9 208 3 3 2 8 214 3 3 2 9 217 3 3 3 5 220 3 3 3 5 203 3 3 3 6 200 3 3 2 7 215 2 3 3 « , 213 1 3 2 mm 220 3 3 2 - 211 2 3 3 - 199 2 1 2 - 197 2 2 2 «M 189 3 3 2 2 188 2 3 2 3 202 2 2 2 - 202 2 2 1 - 213 3 3 3 - 211 3 3 3 - 205 3 2 3 mm 196 3 3 3 216 3 3 3 - 214 3 3 3 - 208 3 3 2 3 210 2 2 1 3 207 2 2 1 an 212 1 2 1 — 219 1 2 1 - 214 2 2 2 — 203 1 3 2 - 208 1 3 2 — Day 5(Cont) 6 7 8 288 0 , oedema, G, colour change, E, exudate and slough, G, gangrene, C, ascorbic acid i n j e c t i o n , P, freezing injury, -, means not present or not administered. Summary Sheet Following the Data Sheets Is a Summary Sheet which gives the average weight f o r each group each day, and under I, or Injury Index, gives the t o t a l f o r the injury changes occurring f o r that group that day. As described before, t h i s concept of an Injury Index i s just an arbi t r a r y number obtained by adding a l l the pathological changes apparent together f o r each group each day. Since no animals expired there was no extrapolating required. Graphs The values 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 Injury Index changes, and the second for the corresponding weight changes. These are done i n colours f o r easier reading (the same colours are used throughout a l l gross graphs), 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 Injury and treatment rather than by group numbers. In the case of the weight graph, two shorter l i n e s are included f o r Groups 6 and 7 . I t haSjbecause of crowding,been necessary to Indicate Group numbers on a few of the crossing l i n e s . 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 Day -5 -4 -3 -2 -1 1 2 3 Grp Gm I Gm I 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 I I J U I Y I H D £ X 8C 70 6C 50 40 \ ' A / / v Ascorbic j / \ Slow Tnaw / v I I v / / Control / / Slow Thaw / / ' / / / / / W / \ / / Control Past Thaw Ascorbic Past Thaw 30 20 10 •4 -2 1 2 3 4 5 6 7 8 D A Y 291 -4 -2 1 2 3 4 5 6 7 8 292 Condition of the Animals The ascorbic acid i n j e c t i o n s as performed with the dosage used caused a skin slough at the s i t e of i n j e c t i o n . The skin over the deposit of the drug would turn black within 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 of skin and f u r would drop out, leaving a clean c i r c u l a r defect. This must be attributable to a s t r i c t l y l o c a l e f f e c t , with no generalized reactions. Except f o r the pain of the i n j e c t i o n material, the animals seemed other- wise unaffected by the drug. There i s no clear-cut tendency toward weight changes apparent from the graphs that would Indicate a general adverse e f f e c t . The control group, Group 2, receiving only ascorbic acid i n j e c t i o n s does show a marked weight loss that could be attributed ©nly to the Injections. Biscussion of Observations Ascorbic acid as administered caused l i t t l e influence on the course of the lesions a f t e r the standard cold i n j u r y as performed. In the case of slow thawing groups, the ascorbic acid l i n e i s at a l l times s l i g h t l y higher than the control, i\is als© quite p a r a l l e l to i t except on Day 8 when there i s diversion. I t i s doubtful i f th i s difference i s s i g n i f i c a n t . In the case of the rapid thaw groups 293 the l i n e s cross, but generally remain close together. So l i t t l e e f f e c t did the ascorbic acid have c l i n i c a l l y that i t was decided useless to perform the microscopic experiments, and as mentioned the animals of Groups 6 and 7 were destroyed on Day 3. The Injury graph gives a dramatic demonstrat- 0 ion of the value of rapid thawing i n water at 42 C. as contrasted with thawing at room temperature. The control graphs are not quite t y p i c a l of what usually happend. This would indicate a s l i g h t l y more severe freezing injury than i s usually performed. Histamine gxperimental Observations with Histamine The purpose of the experiment here described was to test the e f f e c t s of a standard cold i n j u r y as influenced by parenteral in j e c t i o n s of histamine at a dosage l e v e l of 10 mg. per kilogram body weight administered subcutaneously at three hour Intervals beginning immediately p r i o r to the freezing i n j u r y and continuing f o r forty-eight hours afterwards. In view of the f a c t an e f f e c t i v e dosage of histamine was unknown, thi s experiment was i n the nature of a t r i a l run, and accordingly, because animals were scarce, 294 a varied c o l l e c t i o n of rats were used, with almost no selection possible. There were three groups of six animals, each group containing an adult normal male Wistar, two normal adult male Hoodeds, and three normal adult nonpregnant female Hoodeds. Although each group was a mixture of breeds, each group was f a i r l y s i milar to each other. The groups were designed as follows: Group: 2. A control group to receive only the histamine inj e c t i o n s as outlined. 4. A control group also to receive a freezing injury of twenty seconds duration with rapid thawing i n water at 42°C. 5. A test group to receive both the histamine inj e c t i o n s as outlined, and a freezing i n j u r y of twenty seconds duration with rapid thawing i n water at 42°G. These groups have the same numbering as s i m i l a r groups throughout a l l the gross experiments. Daily weights were taken after 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 recorded on the Data Sheets. On Day 1, the day of the freezing, the animals of 295 Groups 2 and 5 received t h e i r i n i t i a l subcutaneous i n j e c t i o n of histamine, and an hour and a h a l f l a t e r , at h a l f past ten o'clock, thesanimals of Groups 4 and 5 were anaesthetized with 0.1 cc. of 0.6$ nembutal solution administered subcutaneously. When anaesth- etized, one hind leg of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down to -20°C. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blan- ched, i n d i c a t i n g the foot was completely frozen, the time was counted so that the l e g was immersed and frozen f o r exactly twenty seconds by stop watch. The animals were then treated by rapid thawing, whereby the frozen feet were immersed immediately at the ex- p i r y of the twenty seconds into water maintained at exactly 42°G. The fe e t were l e f t In the warm water f o r approximately one minute, u n t i l thoroughly thawed and warm. Then the animals were returned 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 hourly i n j e c t i o n s of histamine) u n t i l they wakened from the effects of the nembutal. Observations were made as recorded on the Data Sheets, by the method described above f o r estimating the degree of f r o s t b i t e 296 injury under the categories of oedema, colour change, exudate and slough, and gangrene. Data Sheets These tables 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 of freezing. They are necessarily as compact as possible. Days 1 and 2 include a record of the three hourly i n j e c t i o n s . The symbols used are; Grp, group, Gm, grams, q3h, every three hours, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, W, Wistar, H, Hooded, m, male, f, female, H, histamine i n j e c t i o n , -, means not present or not administered, P, freezing i n j u r y . A l l surviving animals were disposed of on Day 8 with chloroform fumes. Summary Sheet Following the Data Sheets i s a Summary Sheet which gives the average weight f o r each group each day, and under I, or Injury Index, gives the t o t a l f o r the i n jury changes occurring f o r that group that day. As described before, t h i s concept of an Injury Index i s just an a r b i t r a r y number obtained by adding a l l the pathological changes together f o r each group each day. Note that several values had to be obtained by extrap- 9 2 (q-troo) 3 ted — I s s *2T - 3 3 2 £21 - T i 2 *2T HHHHHHHH - e z 2 391 - 3 I 2 i H - 3 T 2 9vT HHHHHHHH - e 3 391 mm 2 2 3 191 - 3 2 3 091 HHHHHHHH - 2 s 2 8£T mm S £ e T i l - T 2 2 991 HHHHHHHH (pBecr) — e s 2 r8T — 3 3 2 28T - T 3 2 9£T HHHHHHHH 2 - 2 s 2 v9T - 1 3 2 99T - T 3 f 09T - S z 2 9ST - 1 3 2 29T - 3 T V 99T - 2 z 2 £91 - 3 2 2 091 - 3 2 2 £91 - I I I 203 - TfTfT 303 - T 3 2 061 — mm s S 933 — I 3 2 133 - 3 3 p 033 — 3 I 2 603 — 3 1 2 ITS — 3 T * 8T3 r£T 69T 691 HHHHHHHH LftT £*T If! HHHHHHHH *6T 26T 98T HHHHHHHH 623 223 v33 HHHHHHHH 623 023 £33 HHHHHHHH f-23 223 323 HHHHHHHH 3 0 a 0 0 a o o «D 0 a 0 0 TOO 3 T T- 2- v- Asa 32T 02T 09T JH 69T 221 m ££T 9 - T T V 89T d- 691 £21 09T £91 - 3 T 29T £91 OvT 29T 69T - 3 2 29T £91 921 09T 89T JH - T T £81 86 T 26 T 08T 081 - 3 3 6T3 933 933 9T3 233 mn — 3 T * 033 233 6T3 6T3 frT3 mm * 99T HHHHHHHHH-H 0£T 991 T9T 39T 9frT HHHHHHHHH-H 9*1 6f-T 62T 39T 261 HHHHHHHHH-H 261 06 T ££T 261 JH 933 HHHHHHHHH-H 223 223 023 323 023 HHHHHHHHH-H 923 823 923 323 m 933 HHHHHHHHH-H 633 633 6T3 0T3 3 0 a 0 0 ras t * 2 D ra© TOD mo to© — - T 82T HHHHHHHffiMH 9vT TvT £21 - 3 I LfZ HHHHHHHHHdH r9T v9T 091 - 3 3 3 09 T HHHHHHHHHiffl 99T 99T £9T - 3 3 £9T HHHHHHHHHiSH T£T 691 891 I,.'.;'(..R'8iTCI.0.n«3H £31 63T 931 - T 3 381 HHHHHHHHHiffl *8T 6£T ££T £63 298 Grp Gm 0 C E G 2 239 233 239 197 151 172 4 205 2 1 2 222 2 2 3 - 208 1 2 2 - 167 2 3 2 - 157 2 3 2 - 160 3 3 3 - 5 186 2 3 — (Dead) 181 2 2 2 - 153 2 3 2 6 157 3 2 2 144 2 2 2 — Day 6 Grp Gm Gm Gm 2 197 192 201 4 182 183 175 5 156 156 160 Day -4 -3 -1 Grp Gm I Gm I 2 205 205 4 186 36 187 40 5 160 46 164 44 Day 5 6 Data Sheet Gm 0 C E. G Gm 0 C E G 242 245 237 240 244 244 195 200 150 150 175 180 203 2 2 3 266 2 2 2 _ 221 2 2 3 - 222 2 2 3 - 207 1 2 3 - 215 1 2 1 - 163 2 3 2 - 167 2 3 2 - 159 2 2 - — 161 3 2 3 - 165 3 3 3 - 167 3 3 3 - 187 2 3 1 - 187 2 3 2 - 188 2 3 1 _ 193 2 3 2 157 2 3 3 6 152 2 3 2 7 147 3 3 3 - 134 2 3 3 141 2 3 3 - (Died) 7 8 Summary Sheet Gm Gm I Gm I Gm I 201 198 197 201 187 182 43 184 43 187 36 156 157 40 154 37 158 43 1 2 3 4 Gm I Gm I 207 210 186 40 200 41 164 52 161 54 7 8 299 ol a t i o n i n order to make up f o r the animals that expired. There i s , of course, no Injury Index f o r Group 2. j&raphsC • The values on the Summary Sheet are plotted d i r e c t l y on the simple l i n e graph. The upper part of the graph deals with the weight changes from day to day, and there i s f o r t h i s part three l i n e s . The lower part contains the Injury Index changes. These are done i n colours f o r easier reading, 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 injury and treatment rather than by group numbers. The same colours and scale hold f o r a l l gross graphs. Condition of the Animals Two of the female Hooded rats of Group 5 were puny and did not survive the f u l l course of the exper- iment. Several of the other animals appeared s i c k l y , but t h i s was considered quite t r u t h f u l l y to be the f a u l t of the animals themselves, rather than caused by the demands of the experiment. Discussion of Observations As just mentioned the animals used were rather a scrubby l o t . They did so poorly generally that i t would not be s i g n i f i c a n t to derive any p o s i t i v e conclus- ions from them. However, i t w i l l be noted that the 3 0 0 301 Injury Index l i n e f o r histamine ( s o l i d green) i s higher than f o r the control. I t may not be possible to state the histamine influenced the course of the f r o s t b i t e lesions adversely, but i t i s quite true that there was no b e n e f i c i a l e f f e c t . As mentioned at the s t a r t , t h i s experiment was i n the nature of a t r i a l run. I t obviously needed repeating with a more suitable group of animals. Histamine Experimental Observations with Histamine (repeat) Tbe purpose of the experiment here outlined was to repeat the effects of a standard cold i n j u r y as influenced by parenteral injections of histamine at a higher dosage l e v e l of 50 mg. per kilogram body weight administered subcutaneously at three hour in t e r v a l s beginning immediately p r i o r to the freezing Injury and continuing f o r forty-eight hours afterwards. Eightsen normal adult maleWistar rats were selected and they were divided into three groups of six animals designed as follows: Group: 2. A control group to receive only the histamine inj e c t i o n s as outlined. 4. A control group also to receive a 302 freezing injury of twenty seconds duration with rapid thawing i n water at 42°C. 5. A test group to receive both the histamine injections as outlined, and a freezing Injury of twenty seconds duration with rapid thawing i n water at 42°C. These groups have the same numbering as si 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 i n i t i a l weights were recorded as on the Data Sheet. The animals of Groups 2 and 5 received t h e i r i n i t i a l subcutaneous i n j e c t i o n of histamine, and an hour and a h a l f l a t e r , at h a l f past nine o'clock i n the evening, the animals of Groups 4 and 5 were anaesthetized with 0.1 cc of 0.6$ nembutal solution administered i n t r a p e r i t o n e a l l y . When anaesthetized, one hind l e g of each animal i n these groups was dipped i n a beaker of ether through which carbon dioxide gas was bubbling to bring the temperature down t© -20 GG. The foot was immersed up to the hock (just below the fur l i n e ) , and as soon as the skin blanched, i n d i c a t i n g the foot was completely frozen 303 the time was counted so that the leg was immersed and frozen f o r exactly twenty seconds by stop watch. The animals were then treated by rapid thawing, whereby the frozen feet were immersed immediately at the ex- p i r y of the twenty seconds into water maintained at exactly 42°C. The feet were l e f t i n the warm water for approximately one minute, u n t i l thoroughly thawed and warm. Then the animals were returned to th e i r cages, and were l e f t q u i e t l y alone (except f o r the three hourly injections of histamine) u n t i l they wakened from the effects of the nembutal. Observations were made as recorded on the Data Sheet, by the method described above f o r estimating the degree of f r o s t b i t e Injury under the categories of oedema, colour change, exudate and slough, and gangrene. Data Sheets These tables are f a i r l y s e l f explanatory, being simple t a l l i e s by animals i n groups, and by days, Day 1 being the day of freeaing. They are nec- e s s a r i l y as compact as possible. Days 1, 2, and 3 include a record of the three hourly i n j e c t i o n s . The symbols used are: Grp, group, Gm, grams, q3h, every three hours, 0, oedema, C, colour change, E, exudate and slough, G, gangrene, H, histamine i n j e c t i o n , 9 — I 3 3 861 - 2 2 3 Z,*3 - mm 2 3 023 - 2 2 2 * 2 3 - 3 3 2 9T3 — 2 2 2 681 - 3 2 2 * 3 3 - - I I 163 - I 3 3 961 - - 3 T * * 3 mm - I T 693 — I I 323 * 3 3 * * 3 233 023 £63 T * 3 a 0 0 XBQ s - 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 - 3 2 2 833 - - I I 002 - 3 3 3 103 - 1 3 3 923 - - I I i9S - I I I 833 *33 2*3 733 LZZ f6Z 1*3 0 3 0 0 - 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 - 3 2 2 933 3 3 063 - 1 2 2 203 - 1 3 2 1 * 3 - - I T TA3 - - I I £23 * 213 623 813 233 963 A23 3 O 3 0 0 rao decs 2 3 1 £B<I HH - T T 2 361 HHHHHHHHH I T 3 * 003 HHHHiffl 603 HH - I 3 2 3 * 3 HHHHHHHHH - - I * 253 HHHHi3H 193 HH — I 3 2 233 HHHHHHHHH *• I 3 * * 2 3 HHHHiSH 633 HH — I 3 * 023 HHHHHHHHH mm I I * A * 3 HHHH3H 623 HH - I 3 * 603 HHHHHHHHH mm I I * Z.I3 HHHHiSH 6T3 HH — I 3 2 * 8 I HHHHHHHHH — T 3 2 161 HHHHdH 88T 5 - - - I 3 2 023 - I I * LZZ d- * 2 3 - - 3 2 102 I I * 563 a - 502 mm mm — I 3 2 261 - I 3 * 803 a - 503 — - T 3 2 223 - I 3 * Z,*3 a - 6 * 3 •» mm - - 3 2 293 - I I * 6LZ - - — a - 9LZ mm mm mm — 3 3 * 3 3 — — — I * * 2 3 a - 2 * 3 * HH Z.03 HHHHHHHHH 113 HHHH-H 903 HH Z.33 HHHHHHHHH 523 HHHH-H * * 3 HH 903 HHHHHHHHH * T 3 HHHH-H 213 HH 613 HHHHHHHHH 633 HHHH-H 323 HH 163 HHHHHHHHH 963 HHHH-H 212 HH 323 HHHHHHHHH 1 * 3 HHHH-H 8 * 3 3 t*2b £) a 0 0 it© 0 a 0 0 rao qgb TOO d«i£) *02 305 means not present or not administered, P, freezing injury. A l l animals were disposed of on Day 6 with chloroform fumes. Summary Sheet Following the Data Sheet i s a Summary Sheet which gives the average weight f o r each group each day, and under I, or Injury Index, gives the t o t a l f o r the Injury changes occurring f o r that gropp that day. As described before, t h i s concept of an Injury Index i s just an a r b i t r a r y number obtained by adding a l l the pathological changes together f o r each group each day. In as much as no animals died, no extrapolation was required to complete the groups f o r six animals. Graph.3 The values on the Summary Sheet are plo t t e d d i r e c t l y on the simple l i n e graph. The upper part of the graph deals with the weight changes from day to day, and there i s f o r th i s part three l i n e s . The lower part contains the Injury Index changes. These are drawn i n colours f o r easier reading, 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 injury and treatment rather than by group numbers. The same colours and scale hold f o r a l l gross graphs. 306 Grp Gm Gm I 2 243 238 4 252 248 37 5 224 223 38 Day 1 2 Summary Sheet Gm I Gm I 230 238 241 32 244 29 214 37 217 39 3 4 Gm I Gm I 242 243 243 26 243 22 212 43 219 43 5 6 Condition of the Animals The present s e l e c t i o n of rats, feeing a more mature group than the animals used i n the l a s t experi- ment survived excellently, and were i n no way adversely affected by the histamine i n j e c t i o n s . Discussion of Observations In view of the f a c t a further experiment with histamine with a better group of animals continued to give diverging Injury Index graph l i n e s , with the l i n e f o r the histamine on top, a l l adds significance to the contention that histamine, as administered^adversely influences the course of f r o s t b i t e lesions as produced i n this experiment. 307 D E X 10 I 1 2 3 4 5 6 D A Y 308 VTI Significance of Data There i s no intention of analyzing s t a t i s t i c - a l l y i n this section of the thesis a l l of the data ob- tained so f a r . Such would be a tremendous task, quite beyond the c a p a b i l i t i e s of the writer, and c e r t a i n l y not j u s t i f i e d because of the very subjective nature of the observations, especially of the microscopic part. However, conclusions have been made from the data which do not have obvious j u s t i f i c a t i o n , and i t i s proposed to study a few selections of the data to determine whether or not thiy&re s i g n i f i c a n t . In t h i s way the remainder of the data may be related to the analyzed material. Threshold Epinephrine Response The main purpose behind the preliminary micro- scopic experiments i n Part III was to provide a standard fo r microscopic observation of the mesoappendix vascul- ature that could be related to a l l subsequent observations. Accordingly, the data obtained must be considered and the Standard Error (S.E.) calculated. On page.49 we have a summary of data which i s here recorded, to the nearest m i l l i o n t h s d i l u t i o n . X r e f e r s to each i n d i v i d u a l number, X i s the mean, WZ, the sum, — — 2 x-x the deviation of each number from the mean, (x-x) the square of th i s deviation. 309 Data x x-x (x-x) 5 .4 .16 4 .6 .36 5 .4 .16 5 .4 .16 5 .4 .16 4 .6 .36 4 .6 .36 5 .4 .16 x 4.6 £1.88 S . E . --JWE? - !lM V n(n-l) This being less than 2.0 suggests a very close relation- ship between the values, and the fact that twice the Standard Error is only .366, also far less than 2.0 i n - 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 animals made hypertensive with compound F. Here we have two sets of figures recorded to the nearest millionths of dilution; Control 5 Test 18 5 18 5 18 7 18 4 20 10 5 The difference between these sets is highly significant because there Is absolutely no overlap of any of the numbers, and i f none of the data overlap, the values 310 fo r twice the s i g n i f i c a n t error ©n either side of the mean f o r both groups could not possibly overlap ei t h e r . Procaine MlcroacQpieall^ In the $ile-roeeoplc ©xperiiwats using t o p i c a l procaine,, the f i r s t data obtained provides two sets of fig u r e s , as copied below from peg© 70, the numbers ind i c a t i n g the time i n seconds f o r the onset of vascular s t a s i s ; X x-x Control 4 § 49 210 116 30 64 90 4 i is; 375 94 £ 19969 S.33. 5 40,8 Tost 20 210 90 ®Q •:14S0 152 38 82 82 278 23104 1444 8724 6724 77284 < 860 172 £ 116280 S.E. " 75.9 These Standard Errors ar© exceedingly large, and the spread on either side of the mean f o r the control group would be from 53.2 to 130.3, and f o r the test group f r o a 96.1 to 247.9 There i s too much overlap f o r there to b© any r e a l s i g n i f i c a n t difference between th© two groups of data, and any significanee i s only s l i g h t l y p o s s i b l e . However* we f i n d more favourable data f o r procaine (x-x) 8401 13466 4096 10 311 parenterally. The summary table on pages 82 and 83 gives the following figures, also f o r time f o r onset of s t a s i s i n seconds: Control Test x X x - x ( x - x ) 45 10 100 30 5 25 45 10 100 30 5 25 30 5 25 30 5 25 210 r 300 35 60 10 100 30 40 400 180 110 12100 60 10 100 40 30 900 50 20 400 420 14000 S.E. - 3.16 70 S.E. = 21.6 The spread of the Standard Error about the mean f o r the control group i s 31.84 to 38.16, and f o r the test group i t i s from 48.4 to 91.6. I t w i l l "be noted that there i s no overlapping In these two ranges, which suggests a probable s i g n i f i c a n t difference between the two groups. However, the same spread using twice the Standard Error i s for the control groups from 28.68 to 41.32 and f o r the test group I t i s from 26.8 to 113.2. Here we notice the range f o r the test values embraces the spread f o r the controls. This greatly reduces the chances of the difference being s i g n i f i c a n t , although a d e f i n i t e difference i s s t i l l p o ssible ; 312 The data thus f a r examined indicates the trend i n the experiments involving the microscopic observation. Time and Rate of Thawing Concerning the gross experiments, we have f i r s t the time and rate of thawing data to consider. The graphs and the summary sheet are but two expressions of the same summarized data (see pages 209 and 210). What 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 other. To s e t t l e t h i s we must f i r s t e s t a b l i s h whether any two points on the graph l i n e s i n question on the same day are s i g n i f i c a n t l y d i f f e r e n t . On Day 2, the Injury Index value f o r Group 3 was 39 and f o r Group 6 i t was 48 (page 209). These are two points r e l a t i n g the influence of the rate of thaw i n a freezing injury of sixty seconds (broken blue l i n e s ) . The data sheet f o r Day 2 (page 206) r e l a t i v e to the animals of these groups gives us, by summing the i n j u r y values f o r each animaln i n turn the following data; - .2 x x-x (x-x) Group 3 7 .5 .25 6 .5 .25 7 .5 .25 7 .5 .25 6 .5 .25 6 .5 .25 39 1 » 5 0 6.5 S.E^r .7 3 1 3 x x - x G r o u p 6 9 9 9 8 8 5 1 1 1 0 0 3 1 1 1 0 0 9 z 4 8 8 £ 1 2 S . E . = . 6 3 The s p r e a d f o r t w i c e t h e S t a n d a r d E r r o r a b o u t t h e mean g i v e s a r a n g e f o r G r o u p 3 o f f r o m 5 . 8 t o 7 . 2 a n d f o r G r o u p 6 o f f r o m 7 . 3 7 t o 8 . 6 3 . The f a c t t h a t t h e r e I s no o v e r l a p o f e v e n t w i c e t h e S t a n d a r d E r r o r i s v e r y s t r o n g l y s u g g e s t i v e t h a t t h e r e I s a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two g r o u p s . S u c h a c o n c l u s i o n w o u l d a p p l y t o a l l t h e d a t a o f t h i s e x p e r i m e n t p r o v i d e d we c o m p a r e o n l y t h e l i n e s o f t h e same c o l o u r o r o n l y s o l i d o r d o t t e d l i n e s . C o m p a r i n g a s o l i d l i n e o f one c o l o u r w i t h a d o t t e d l i n e o f a n o t h e r c o l o u r w o u l d l e a d t o m e a n i n g l e s s c o m p a r i s o n s . F o r i n s t a n c e , a c o m p a r i s o n o f t h e f i v e s e c o n d f r e e z e g r a p h w i t h s l o w t h a w a g a i n s t t h e t w e n t y s e c o n d f r e e z e g r a p h w i t h f a s t t h a w ( b l u e b r o k e n a n d r e d s o l i d l i n e s o n p a g e 2 1 0 ) w o u l d i n v o l v e two v a r i a b l e s , d u r a t i o n o f f r e e z i n g a n d r a t e o f t h a w . R u t i n g r a p h s f o r r u t i n w i l l b e c o n s i d e r e d . On t h e D a t a S h e e t , p a g e 2 7 4 , i t w i l l b e s e e n t h a t n o a n i m a l s d i e d . The d a t a A s a f u r t h e r e x a m p l e o f s i g n i f i c a n t d a t a , t h e o 314 f o r Groups 1 and 3 on Day 6 gives two sets of inj u r y values by animals as below: Group 1 X x-x (x-x) 6 0 0 7 1 1 9 3 9 6 0 0 6 0 0 3 3 9 I 37 Z 19 X 6 3 5 .5 .25 10 5.5 30.25 3 1.5 2.25 3 1.5 2.25 3 1.5 2.25 3 1.5 2.25 I 27 £ 39.50 X 4.5 S.E. — .8 S.E. - 1.2 The spread about the mean f o r Group 1 given by the Stand- ard error gives a range of from 5.2 to 6.8 and f o r Group 3 the corresponding range would be from 3.3 to 5.7. Here there i s some overlap which does reduce the significance of the difference considerably, but s t i l l a difference i n the behaviour of 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 . Only one aspect of the complete graph has been examined, and thi 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 question were f a i r l y close together. On Day 4 the same two graphs are furthest apart. Taking the data from page 274 for these two groups, Groups 1 and 3 on Day 4 we have again in j u r y t o t a l s by animals as follows: 315 Group 1 Group 3 X x - x ( x - x ) 10 1 1 9 0 0 10 1 1 9 0 0 9 0 0 6 3 3 r 53 X 9 8 1 1 9 2 4 7 0 0 6 1 1 5 2 4 6 1 1 I 41 X 7 S.E. = .6 S.E. - .6 Here the spread on either side of the mean given by the Standard Error gives f o r Group 1 a range of 8.4 to 9.6 and f o r Group 3 a range of from 6.4 to 7.6. Here we see no overlap which indicates a s i g n i f i c a n t difference although examination of the same spread provided through the more exacting standard of using twice the Standard Error gives a range f o r Group 1 of 7.8 to 10.2 and f o r Group 3 of from 5.8 to 8.2. This reduces the p r o b a b i l i t y of the difference being s i g n i f i c a n t , but does not necess- a r i l y cancel out the s i g n i f i c a n c e . Hence, incorporating both these analyses into the graphs for slow thaw f r o s t b i t e lesions permits us to say i t i s l i k e l y but not e n t i r e l y 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 severe course over the time i n t e r v a l studied. 316 Ascorbic Acid Then, at the other extreme, as i n the case with ascorbic acie, we have to consider whether the corresp- onding graphs are sim i l a r or not. The dotted l i n e s on page 290, as are the s o l i d l i n e s , are running quite p a r a l l e l to each other. I t i s almost apparent, without ca l c u l a t i n g standard error that the medication has not made the s l i g h t e s t difference on the course of the f r o s t b i t e l e s i o n s . However, the other comparison i s made even more s i g n i f i c a n t , that rapid thawing re s u l t s i n an obviously decreased severity i n the course of f r o s t b i t e l e s i o n s . Conclusion This b r i e f examination of the data as has been done w i l l serve to strengthen the v a l i d i t y of the conclus- ions arrived at i n the f i n a l section of this thesis. Al^.1 the data c e r t a i n l y could not have been analyzed, nor could the more elaborate s t a t i s t i c a l methods dealing with the degree of p r o b a b i l i t y be applied. At best we can only speak with rough g e n e r a l i t i e s . 317 VIII Conclusions Reached This investigation was an i n d i r e c t attempt to determine whether a s p e c i f i c selection of ten medic- ations would or would not influence favourably the vascular responses i n the frozen extremities of the r a t . An i n d i r e c t approach was considered necessary because i t was not considered p r a c t i c a l to observe the vessels i n the extremities. A„s has been outlined at the beginning, the vascular bed of the mesoappendix was selected as the s i t e f o r the microscopic studies. Any observations made at the mesoappendix portion of the vascular system had to be compared with the gross effects at the s i t e of o r i g i n a l i n t e r e s t , the extremities. This whole invest i g a t i o n was at a l l times a most int e r e s t i n g one to be working on. Much experience was gained i n the microscopic examination of vascular react- ions. Each of the preparations studied required at l e a s t h a l f an hour. Such experience c e r t a i n l y provided a d i f f e r e n t concept of the vascular system than had been entertained previously by the writer. The threshold epinephrine response test of Zwei- fach was duplicated with some high degree of r e l i a b i l i t y , which was very g r a t i f y i n g i n view of the f a c t such work 318 was the writer's f i r s t experience with microtechnique of this nature, and also the vasotropic e f f e c t of compound F was corroborated with Warner's findings, which was a source of keen s a t i s f a c t i o n to us both. As f a r as the influence that the various medic- ations had on the vascular system of the mesoappendix after l o c a l i z e d freezing was concerned, i t i s f i r s t l y important to remember that a l l the drugs tested were ex- pected to have some influence. Such influence as was noted was observed according to very highly subjective impressions. These impressions could not be proven to be s t a t i s t i c a l l y s i g n i f i c a n t . A l l the microscopic experiments resulted i n the same conclusion; any differ:ences noted i n behavior of the c i r c u l a t i o n due to treatment by one of the ten drug agents were of doubtful s i g n i f i c a n c e . Because of this i t was decided that the technique as emp- loyed for observing the ef f e c t of l o c a l i z e d cold i n j u r y on the vascular bed of the mesoappendix was a very poor tool f o r investigating cold i n j u r y . In regard to the gross experiments more d e f i n i t e findings were found; 1. As expectedjthe rate of thawing a frozen extremity was a s i g n i f i c a n t factor i n determining the degree of inj u r y that freezing would produce. Warm thawing i n water at 42°C. was the method of rapid thawing selected. 319 2. The duration of the frozen state also bore a d i r e c t relationship to the severity of the r e s u l t i n g lesions, up to a maximum, of course, of t o t a l gangrene and subsequent amputation by the animal. 3. Procaine was found to be very toxic i n the dosage l e v e l administered, and on a repeat experiment i t was found to provide no s i g n i f i c a n t benefit. 4. P r i s c o l i n e provided no s i g n i f i c a n t benefit grossly. 5. Benadryl provided no s i g n i f i c a n t benefit. I t was suggested but not proved that Benadryl might ac t u a l l y have a detrimental e f f e c t on the course of f r o s t b i t e l e s i o n s . •••! 6. Etamon and Hydergine provided no s i g n i f i c a n t beflefit. 7. Apresoline provided some possibly s i g n i f i c a n t benefit, but Chlor-Tripolon as administered made the course of the f r o s t b i t e i n j u r y s i g n i f i c a n t l y more severe. 8. Rutin has been considered already on page 313, and i t was f e l t that r u t i n had a probably s i g n i f i c a n t benefit on the course of the f r o s t b i t e l e s i o n s . 9. Ascorbic acid as administered had absolutely no influence whatever on the course of the f r o s t b i t e i n j u r y . 10. Histamine was found to have a probably s i g n i f - icant harmful e f f e c t . 320 The attempt to follow the course of a f r o s t b i t e Injury on a time basis was made throughout a l l the gross experiments, and i t was found quite p r a c t i c a l to present the day by day changes on a simple l i n e graph. However to accomplish this one a numbrical basis, the a r b i t r a r y concept of an Injury Index was introduced. The actual determination of an a l l i n c l u s i v e Index evaluation pre- sented some d i f f i c u l t i e s , but a workable solution was found and this was employed throughout a l l the gross ex- periments. By way of apparatus, the equipment f o r the micro- scopic examination of the r a t mesoappendix was constructed according to Zweifach's direc t i o n s , and the cold point f o r controlled hypothermia of Hass and Taylor was made. This equipment was found to work quite s a t i s f a c t o r i l y . Summary of Conclusions 1 . Usefulness of the threshold epinephrine response test for vasotropic substances was confirmed. 2. The vasculature of the r a t mesoappendix was observed i n respect to i t s reactions to a standard l o c a l i z e d freezing i n j u r y . Procaine, P r i s c o l i n e , Benadryl, Etamon, Hydergine, Apresoline, Chlor-Tripolon, Rutin, Ascorbic acid, and Histamine f a i l e d to influence these reactions s i g n i f i c a n t l y . 3. A dynamic graphical method was described 321 f o r recording gross f r o s t b i t e lesions grossly, and the a r b i t r a r y concept of an Injury Index was introduced. 4. Procaine, P r i s c o l i n e , Benadryl, Etamon, Hydergine, and Ascorbic acid were found to have no s i g - n i f i c a n t e f f e c t on the course of f r o s t b i t e lesions grossly. 5. Chlor-Tripolon and Histamine probably had a s i g n i f i c a n t adverse e f f e c t . 6. Apresoline, and e s p e c i a l l y Rutin probably had a b e n e f i c i a l e f f e c t . 7. The value of rapid thawing i n water at 42°C. was consistantly confirmed. 322 APPENDIX Equipment for Hass and Taylor's Cold Point Apparatus 1. Cylinder of carbon dioxide gas at 1,000 pounds pressure. 2. Freezing attachment f o r Spencer carbon d i - oxide microtome, complete with copper tube connection. S c i e n t i f i c Supplies Co. Ltd., Cat. No. 50, p.630, Item No. 38756. 3. Hypodermic needles as i n Becton, Dickinson and Company (B.-D.) Cataloque f o r May, 1951. Item LNR 19 G 3" page 26. Item LNRS 15 G 1*" page 26. 4. Connectors; Luer-Lok hose end connector, H/468L, Page 33 Adams Luer-Lok connector with side arm adapter, 429A, page 36. 5. Labour required; (1) Attach H/468L to freezing attachment (2) Solder 429A at proper s i t e on shaft of No.19 needle. Chemicals Used Water, ordinary laboratory single d i s t i l l e d from c i t y supply (chlorinated), sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium 323 dihydrogen phosphate, glucose, ash-free g e l a t i n , a l l as ordinary C P . laboratory reagents as available without special ordering. Procaine hydrochloride, 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 Limited brand of 2- benzyl-imidazoline hydrochloride (benzazoline) available i n rubber capped v i a l s of 10 c c , each cc. containing 25 mg. Benadryl hydrochloride, Parke, Davis and Co. Ltd. brand of diphenhydramine hydrochloride available i n 10 cc. S t e r i - v i a l s each c c containing 10 mg. Etamon Chloride, Parke, Davis and Co. Ltd. brand of tetraethylammonium chloride available i n 20 cc. S t e r i - v i a l s with 100 mg. per cc. Hydergine, Sandoz (Canada) Ltd. brand of equal proportions each of dihydroergocornine, dihydroergocris- tine, and dihydroergokryptine, available i n 1 cc. ampoules containing 0.3 mg. of the mixture. Apresoline Hydrochloride? Ciba Company Ltd. brand of 1-hydrazinophthalazine hydrochloride, available i n 1 cc. ampoules containing 20 mg. i n boxes of 5 ampoules. Chlor-Tripolon, Schering Corporation Limited brand of chlorprohpenpyridamine maleate, obtained as the pure powder. 324 Rutin, Parke, Davis and Co. Ltd., obtained as 50 mg. compressed tablets i n bottle of 500. Ascorbic acid 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. Ltd. brand of hydrocortisone acetate (compound F) available as the saline suspension i n s t e r i l e rubber-capped v i a l s cont- aining 25 mg. per cc. Epinephrine, Connaught Medical Research Lab- oratories 1:1000 solution of epinephrine hydrochloride, available i n 30 cc. s t e r i l e rubber-capped v i a l s . Nembutal, Abbott Laboratories Limited, Item No. 8612. 100 cc. rubber diaphragm capped v i a l f o r veterinary use containing 60 mg. per cc. Ringer-Locke Solution Sodium chloride 36 gms. Potassium chloride 1.68 Calcium chloride .96 Sodium bicarbonate .80 Water 4000 cc. Glucose 8 gms. Gelatin 40 A l l s o l i d ingredients except g e l a t i n were weighed out c a r e f u l l y and dissolved i n the water. The g e l a t i n was dissolved i n a 1000 cc only, heated u n t i l completely dissolved, then f i l t e r e d under suction and added to the rest of the solution. Usually the sodium bicarbonate was notweighed out, but when the solution was mixed, a 3 2 5 dropper f u l l of methyl red indicator was added to the completed solution, and then sodium bicarbonate was slowly added u n t i l the f i r s t change to red. 326 References Cited (1) Orr, K.D. and Fainer, D.C, Cold.Injury i n Korea During Winter of 1950—51 Medicine 31:177-220,(may)1952 (2) Webster, D.R. and Bigelow, W.G., Injuries Dueto Cold, Frostbite, Immersion Foot, and Hypothermia, Canad.M.A.ffi., C i v i l Defence Issue, 67: 534-538,(Dec)1952. (3) Ferrer, M.Irene, ed., Trans. F i r s t Conference, Cold Injury, June 4-5, 1951, Josiah Macy J r . Foundation, New York, 1952. (4) Shumacker, H.B.Jr., and Lempke, R.E., Recent Advances i n Frostbite, Surgery 30:873-904,(Nov)1951 (4a) E d i t o r i a l , F r o s t b i t e , J.A.M.A. 148:940-941,(Mar.15)1952. (5) Laufman, H., . _ Profound Accidental Hypothermia, J.A.M.A. 147-.1201-1212, (Nov.24)1951. (6) Larrey, D.J., . Memoirs of M i l i t a r y Surgery and Campaigns of the French Armies, Trans, by H a l l , R.W. Baltimore, J.Cushing, 1914, vol.11, 156-164. (7) Warner, D.L., , .. Ca p i l l a r y Changes i n Rats Rendered Hyper- Tensive by the Administration of Compound F. Unpublished Graduating Thesis, Faculty of . Medicine, University of British.Columbia, A p r i l , 1954. (8) Hass, G.M. and Taylor, C.V., A Quantitative Hypothermal Method f o r the Production of Local Injury of Tissue, Arch. Path. 145:563-580, (May)1948. 327 (9) G r i f f i t h , J.Q. and F a r r i s , E.J., eds., The Rat i n Laboratory Investigation, J.B. Lippincott, Philadelphia, 1942. (10) Beckman, H., Pharmacology In C l i n i c a l Practice, W.B. Saunders, Philadelphia, 1953, p.208. (11) Hurley, L. A., ^-Roberts, J.E., Buchanan, A.R., and T i l l q u i s t , G., Preliminary Investigations of the Value of the Dihydrogenated Alkaloids of Ergot i n the Treatment of Experimental Frostbite, Surgery, Gynecology, and Obstetrics, 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 Derivatives on the C i r c u l a t i o n i n Man With Special Reference to Two New Hydrogenated Compounds (Dihydro- ergotamine and Dihydroergocornine), Am. Heart J., 35:873-894,1948. (13) Rothlin, E., Pharmacology of the Natural and Dihydro- genated Alkaloids of Ergot. B u l l . Acad. Suiss. Sc.M., 2:249-273,1946-47. (14) Zweifach, B.W., Indirect Methods f o r Regional Blood Plow: I, Microscopic 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 Vasotropic Substances, i n Potter, V.R., ed., Methods i n Medical Re- search, 1:131-139,1948, Year Book Pub. Chicago, (15) Knisely, M.H., Method of Illuminating Internal Organs, Anat. Record, 64;499,1936. (16) Knisely, M.H., Improved Fused Quartz Illumination, Anat. Record, 58;73,1934, and 71:503,1938. (17) Knisely, M.H., Microscopic Observations on Transillumined Spleens, Proc.Soc.Exp.Biol.&.Med., 32:212,1934. 328 (18) Knisely, M.H., i n Handbook of Microscopical Technique, McClung, C.E., ed., p.632-642. (19) Chambers, R., Vasomotion i n Hemodynamics of Blood Ca p i l l a r y C i r c u l a t i o n . Ann. N.Y. Acad. Sc., 49:549-552,(May)1948. (20) Chambers,, R.,/.and Zweifach, B.W., Topography and Function of Mesenteric Ca p i l l a r y C i r c u l a t i o n . , 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 Conditions and i n Traumatic Shock, Nature, London, 162:835-837,(Nov)1948. (22) Chambers, R. and Zweifach, B.W., Functional A c t i v i t y of the Blood C a p i l l a r y Bed, With Special 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 Outline of General Physiology, Heilbrunn, L.V., Saunders, Philadelphia, 3rd ed., 1952, p.491.

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