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Histaminergic vasodilatation in the hindlimb of the dog Graham, Bruce Howard 1974

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HISTAMINERGIC VASODILATATION IN THE HINDLIMB OF THE DOG by BRUCE HOWARD GRAHAM B . S c , U n i v e r s i t y of B r i t i s h Columbia 1 % 9 M.D. U n i v e r s i t y of Calgary 1 9 7 ^ A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the department of PHYSIOLOGY We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 197*+ In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of I ''" 1 — The University of Brit ish Columbia Vancouver 8, Canada Date v j*,y( ^ , ABSTRACT T h i r t y - f o u r dogs were anesthetized w i t h sodium pentothal i . v . and maintained with i . v . a l p h a - c h l o r a l o s e . Neuro-muscular blockade was accomplished w i t h gallamine t r i e t h i o d i d e ( F l a x e d i l ) . R e s p i r a t o r y P P N was monitored continuously while a r t i f i c i a l v e n t i l a t i o n at a r a t e of 1 5 cpm and appropriate t i d a l volume was adjusted to maintain e x p i r a t o r y P Q Q 2 between 3 8 and f^O mm Hg. Blood gas a n a l y s i s (PQQ^I PQ A N ^ P ^ ) allowed maintenance of blood pH between 7 - 3 5 and 7 » ! + 5 by p e r i o d i c a d m i n i s t r a t -ion of i . v . sodium bicarbonate. Blood volume was main-tained with Dextran 7 5 when necessary. Body temperature was monitored continuously with an esophageal t h e r m i s t e r and maintained a u t o m a t i c a l l y with heating elements i n the operating t a b l e . A r t e r i a l v a s c u l a r i s o l a t i o n of the hindlimbs was accomplished by l i g a t i n g a l l major branches of the aorta below the r e n a l a r t e r i e s except the e x t e r n a l i l i a c a r t e r i e s . The dog's own blood, taken from a cannula i n the abdominal aorta j u s t d i s t a l to the r e n a l a r t e r i e s , was perfused at constant f l o w i n t o cannulae i n the extern-a l i l i a c a r t e r i e s through separate pumps. Each e x t e r n a l i l i a c a r t e r y pressure was monitored s e p a r a t e l y ( F i g . 1). A b i l a t e r a l laminectomy allowed access to the L^, ^ and y s p i n a l segments f o r e l e c t r i c a l s t i m u l a t i o n of t h e i r v e n t r a l roots a f t e r s e c t i o n of the corresponding d o r s a l r o o t . In 26 dogs monophasic square wave s t i m u l a t i o n (3 to 10V, 3 msec, 8 to 20 Hz) of the v e n t r a l root of L^, Lg or Ly induced" 1 ) a decrease i n the p e r f u s i o n pres-sure (PP) i n the i p s i l a t e r a l hindlimb (->+1.8 - 2 . 7 mm Hg; mean - SE); 2) a decrease i n the PP i n the contra-l a t e r a l hindlimb ( - 3 2.2 - 2 . 7 ) ; 3 ) a f a l l i n the a o r t i c pressure ( - 1 5-6 - 0 . 7 ) - (Fig.. 3 ) - S i m i l a r e f f e c t s were observed on s t i m u l a t i o n of the p e r i p h e r a l stump of the v e n t r a l r o o t . The above described v a s c u l a r e f f e c t s of v e n t r a l root s t i m u l a t i o n were r e s i s t a n t to i n t r a - a r t e r i a l i n -j e c t i o n s of c h o l i n e r g i c and beta-adrenergic b l o c k i n g agents administered d i r e c t l y i n t o the hindlimb p e r f u s -i o n l i n e s . The e f f e c t i v e n e s s of the blockades was t e s t e d w i t h d i r e c t i n t r a - a r t e r i a l i n j e c t i o n s of the approp-r i a t e a g o n i s t s . A n t i h i s t a m i n i c s (diphenhydramine and mepyramine) s i m i l a r l y administered and t e s t e d d i d a b o l i s h the response i n doses which d i d not suppress vascular-r e a c t i v i t y to a c e t y l c h o l i n e or i s o p r o t e r e n o l . These experiments do not provide a c l e a r explan-a t i o n of the mechanisms r e s p o n s i b l e f o r the contra-l a t e r a l v a s o d i l a t a t i o n or the f a l l i n a o r t i c pressure. The presence of s i g n i f i c a n t anastomotic channels con-necting e i t h e r the two hindlimbs and/or the hindlimbs wi t h the r e s t of-the body was excluded. C o n t r a l a t e r a l v a s o d i l a t a t i o n might perhaps be explained by the pres-ence of nerve f i b r e s c r o s s i n g the m i d l i n e i n . the fused impar ganglion of the dog. The drop i n a o r t i c pressure was not due to the a c t i v a t i o n of a f f e r e n t f i b r e s cours-ing i n the v e n t r a l r o o t s , nor to the p e r i p h e r a l r e l e a s e of a v a s o d i l a t o r substance since the onset of the phen-omenom was too f a s t to be explained on these grounds. The p o s s i b i l i t y e x i s t s that the drop i n a o r t i c pressure i s due to the a c t i v a t i o n by the stimulated e f f e r e n t f i b r e s of some a f f e r e n t nervous pathways c a r r y i n g i n -h i b i t o r y impulses to the vasomotor centers. The present experiments, however, do not provide data supporting or excluding t h i s hypothesis. The experimental r e s u l t s s t r o n g l y suggest that the described v a s o d i l a t a t i o n may be mediated by histamine released d i r e c t l y or i n d i r e c t -l y by the a c t i v a t i o n of f i b r e s coursing i n t o the lower-v e n t r a l r o o t s . Pag'e ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGMENTS' x i PART I. INTRODUCTION 1-16 1) Active and passive vasodilatation 1 2) Evidence supporting the existence 5 of active and passive vasodilatation 3) Nervous pathways involved in active 7 vasodilatation 4) Evidence supporting histamine as a 9 mediator of active r e f l e x vaso-d i l a t a t i o n 5) Evidence against histamine as a 12 mediator of active r e f l e x vaso-d i l a t a t i o n PART I I . METHODS 17-26 1) Animal and anesthesia 17 2) Maintenance of the animal 17 3) Surgical methods 18 4) Methods of hindlimb perfusion 19 ">) 5) Perfusion and pressure recording 20 6) Drugs used 21 7) Experimental protocol 24 8) Interpretation of results 25 9) C r i t e r i a for a viable preparation 26 PART I I I . RESULTS 27-41 1) Stimulation of the intact ventral 27 root 2) Stimulation of the sectioned 29 ventral root 3) Cholinergic blockade 32 4) Beta-adrenergic blockade 32 5) Histaminergic bloakade 32 6) Hexamethonium blockade 32 PART IV. DISCUSSION 42-61 1) Negative cases 42 2) Donald and Ferguson experiments 43 3) Unanswered questions 46 4) Evidence supporting histamine as the 53 mediator of vasodilatation v.iri Page 5) Advantages of theis preparation 57 6) Conclusions 59 PART V. BIBLIOGRAPHY 62-68 LIST OF TABLES Table Page I E f f e c t of ventral root stimulation on the hind- 30 limb perfusion pressure and a o r t i c pressure. II E f f e c t of antihistamines on the vascular effects 36 of ventral root stimulation. Figure Page 1 Schematic representation of the preparation used in most experiments. 22 2 Ef f e c t on perfusion pressures of stopping one 23 perfusion pump at a time. 3 E f f e c t on hindlimb perfusion pressures of intact 28 and sectioned ventral root stimulation. 4 Ef f e c t on hindlimb perfusion pressures of intact 31 and sectioed ventral root stimulation. 5 E f f e c t of ventral root stimulation and i n t r a - 33 a r t e r i a l i n j e c t i o n of acetylcholine on hind-limb perfusion pressure before and aft e r i n t r a -a r t e r i a l atropine. 6 E f f e c t of ventral root stimulation on hindlimb 34 perfusion pressure and blood flow before and after administration of atropine and propran-o l o l . 7 Progressive blockade of hindlimb vascular re- 37 sponse to ventral root stimulation and to i n t r a -a r t e r i a l histamine by repeated Benadryl admin-i s t r a t i o n . 8 Regression l i n e plot of the re l a t i o n s h i p be- 38 tween reduction of vascular response to ventral root stimulation and i n t r a - a r t e r i a l histamine. 9 E f f e c t of ventral root stimulation, i n t r a - 40 a r t e r i a l histamine and Benadryl on hindlimb vasodilatation. 10 E f f e c t of c a r o t i d clamping, ventral root stim- 41 ul a t i o n and hexamethonium on hindlimb perfusion pressures. ACKNOWLEDGMENT I would l i k e to express my g r a t i t u d e and ap p r e c i a t -i o n to Dr. Franco L i o y f o r h i s guidance i n s t r u c t i o n and h e l p f u l c r i t i c i s m i n t h i s work, and my thanks to Mrs. Karen White and Mrs. Jeanette- Sanctuary f o r t h e i r able a s s i s t a n c e . Also, my thanks to Dr. John Ledsome and Dr. John Brown f o r t h e i r time spent i n p e r u s a l of t h i s work. . x i PART I . INTRODUCTION LITERATURE REVIEW 1) A c t i v e and Passive V a s o d i l a t a t i o n Ever since the experiments of Claude Bernard on the r a b b i t ear there has been a consensus amoung p h y s i o l -o g i s t s that the systemic a r t e r i a l blood pressure i s reg-u l a t e d through the t o n i c discharge of the sympathetic v a s o c o n s t r i c t o r nerves to the r e s i s t a n c e v e s s e l s (Bernard, 1 8 5 1; B a y l i s s , 1 9 2 3 ; Beck and Brody, 1 9 6 1 ) . The mechan-ism of p h y s i o l o g i c a l v a s o d i l a t a t i o n , on the other hand, has been the subject of considerable dispute over the years, w i t h respect to both i t s mechanism of occurance and i t s r o l e i n normal blood -pressure homeostasis (Beck and Brody, 1 9 6 1 ) . While Bernard demonstrated the d i l -a t a t i o n of the s a l i v a r y gland vasculature i n response to e l e c t r i c a l s t i m u l a t i o n of the chorda tympani (Bernard, 1 8 5 8 ) , a u t h o r i t i e s i n the f i e l d disagreed on the ques-t i o n of whether d i l a t a t i o n was as a c t i v e response of the v ascular smooth muscle to nerve s t i m u l a t i o n or was a passive response occuring on c e s s a t i o n of f i r i n g of c o n s t r i c t o r f i b r e s to the v e s s e l . The two experiments of Claude Bernard (Bernard, 1 8 5 1 , 1 8 5 8 ) had demonstrat-ed both d i l a t a t i o n s , one occuring on nerve s e c t i o n (passive d i l a t a t i o n ) , and one occuring on s t i m u l a t i o n of the nerve supplying the vas c u l a t u r e ( a c t i v e d i l a t a t -ion) . Acti v e and passive v a s o d i l a t a t i o n have, i n more recent years, been c l a s s i f i e d more e x a c t l y according to t h e i r causes (Beck, 1 9 5 8 b , 1 9 6 1 , 1 9 6 3 ; Beck and Brody, 1 9 6 1; Brody 1 9 6 6 ) . Passive v a s o d i l a t a t i o n has been de-f i n e d as that occuring on i n t e r r u p t i o n of the s e c r e t i o n of a c o n s t r i c t o r substance at the v e s s e l l e v e l . This d e f i n i t i o n , by i t s nature, makes no i m p l i c a t i o n on the mechanism of a c t i o n of the d i l a t a t i o n , the t r a n s m i t t e r or humoral substance i n v o l v e d , or the p r e c i s e s i t e of vascular r e s i s t a n c e change. I t does however inc l u d e the p r e r e q u i s i t e that a c e r t a i n amount of tone be present i n the va s c u l a r smooth muscle i n order f o r t h i s d i l a t a t -i o n to occur. A c t i v e v a s o d i l a t a t i o n has been defined as that which r e s u l t s from the d i r e c t a c t i o n on th e - v e s s e l of an agent capable of r e l a x i n g v a s c u l a r smooth muscle. Again, no i m p l i c a t i o n about the mechanism of a c t i o n i n -volved i s included i n the d e f i n i t i o n . While the magni-tude of the d i l a t a t i o n must be l i m i t e d by the p r e - e x i s t -ing v a s c u l a r tone, i t i s not d i r e c t l y dependant on the existence of such tone as i n the case of passive d i l -a t a t i o n . The controversy e x i s t i n g before the t u r n of the century, stemming l a r g e l y from Bernard's experiments, was based on two questions ( B a y l i s s , 1 9 2 3 ) . F i r s t , was the tone of the v e s s e l s normally maintained by t o n i c discharge of the c o n s t r i c t o r f i b r e s , and second, was the decrease i n normal tone due to an a c t i v e or passive d i l -a t a t i o n ? I n p a r t i a l answer to t h i s , B a y l i s s ( 1 9 2 3 ) sug-gested t h a t . t h e r e were i n the medulla two centers, a v a s o d i l a t o r center and a v a s o c o n t r i c t o r center. E s s e n t i a l l y , t h i s concept i s s t i l l held by many as v a l i d today. During the 19 l +0 , rs.and early. 1 9 5 0 ' s the theory was developed that normal neurogenic tone to the v e s s e l s arose from the t o n i c discharge of the v a s o c o n s t r i c t o r center of the medulla, that t h i s a c t i v i t y was soely r e s p o n s i b l e f o r the maintenance of systemic a r t e r i a l pressure, and that d i l a t a t i o n of the v e s s e l s occurred when the a c t i v i t y of t h i s center was i n h i b i t e d by input from the baro-receptors of other i n h i b i t o r y a f f e r e n t inputs (Folkow and Uvnas, 19*+8; Frumin et a l , 1 9 5 3 ; Heymans and N e i l , 1 9 5 8 ) . This theory was based on s e v e r a l l i n e s of evidence (Folkow and Uvnas, 1 9 ^ 8 ; L i n g r e n and Uvnas, 195^5 Beck and Brody, 1 9 6 1 ) and was maintained i n s p i t e of the disco v e r y of c h o l i n e r g i c v a s o d i l a t o r f i b r e s i n the e a r l y 1 9 5 0 ' s by.the Scandinavian p h y s i o l o g i s t s ( E l i a s s o n ^ Lingren and Uvnas, 1 9 5 2 , 195^5 Folkow et a l , 1 9 ^ 9 ; Folkow and Uvnas, 1 9 ^ 8 ; Folkow, Haeger and Uvnas, 1 9 ^ 8 ) . Such ev-idence included -the f i n d i n g s that increased a o r t i c arch, or c a r o t i d sinus pressure l e d to a t o t a l or n e a r - t o t a l i n h i b i t i o n of sympathetic discharge (Bronk, P i t t s and Larrabee, 1 9 3 9 ) , and that s t i m u l a t i o n of the baroreceptor nerves l e d to a s i m i l a r i n h i b i t i o n of discharge.(Bronk, Ferguson and Margaria, 1 9 3 6 ) - . The r e f l e x lowering of blood pressure by the pulmonary depressor r e f l e x (chemo-r e f l e x ) and the coronary shemoreflex ( B e n z o l d - J a r i s c h r e f l e x ) have been a t t r i b u t e d to t h i s sort of i n h i b i t i o n (Heymans and N e i l , 1 9 5 8 ) . C e r t a i n l y the f a c t that i n the baroreceptor deafferented animal there i s a r i s e i n systemic blood pressure supports t h i s hypothesis. 2) Evidence Supporting the Existence of Ac t i v e Vaso- d i l a t a t i o n . Notwithstanding the above evidence, there remained the 1 9 5 8 experiment of Bernard i n which s t i m u l a t i o n of the chorda tympani caused d i l a t a t i o n of the v e s s e l s of the submaxillary gland of the r a b b i t . A c t i v e r e f l e x v a s o d i l a t a t i o n was shown i n 19^+7 (Binet and Bernstein) using the c r o s s - c i r c u l a t e d paw of the dog, and the r e f l e x was shown to be mediated by the baroreceptors and to t r a v e l with the sympathetic nerves. The w e l l known c h o l i n e r g i c v a s o d i l a t o r f i b r e s of the Scandinavian authors were d i s - • covered about the same time. C l e a r l y , i t had to be ac-cepted that passive d i l a t a t i o n was not the only method of lowering blood pressure i n the i n t a c t animal, even i f i t were'the most important method. Despite the r e l -a t i v e l y strong evidence.In favor of the existence of an a c t i v e d i l a t o r system there remains strong r e s i s t -ance to the concept. Binet and B e r n s t e i n ( 1 9 ^ 7 a , J\9k7b) demonstrated a c t i v e r e f l e x v a s o d i l a t a t i o n i n an e s s e n t i a l l y cutan-eous p r e p a r a t i o n , the dog paw. I t has been subsequent-l y shown that the r e f l e x occurs i n the skinned hindlimb of the dog as w e l l (Beck, 1 9 6 1 ) . Just as the I n h i b i t i o n of discharge i n the sympathetic nerves i n response to increased c a r o t i d sinus or a o r t i c arch pressure has been used as evidence i n favor of the passive d i l a t a t i o n theory (Heymans and N e i l , 1 9 5 8 ) , increased f i r i n g i n the rab-b i t splanchnic "nerve has been seen i n response to an increase i n systemic a r t e r i a l pressure ( M i l l a r and B r i s c o e , 1 9 6 5 ) 5 suggesting p o s s i b l e a c t i v a t i o n of vaso-d i l a t o r .fibres to the mesenteric v a s c u l a r beds. This was seen i n f i v e of s i x t e e n cases s t u d i e d . More r e c e n t l y , increased f i r i n g i n the sympathetic .nerves of the v e t r a l roots of cats i n response to increased systemic blood pressure has been observed ( T u t t l e , quoted by Brody, 1 9 6 6 ) . One of the pieces of evidence most o f t e n quoted i n favor of an a c t i v e v a s o d i l a t o r system i s that the l e v e l to which v a s c u l a r r e s i s t a n c e f a l l s during a c t i v e r e f l e x d i l a t a t i o n i s lower than the .sustained l e v e l to which i t f a l l s f o l l o w i n g sympathectomy, complete pharmacological g a n g l i o n i c blockade, or complete alpha-adrenergic blockade (Beck, 1 9 6 1 ; Binet and B e r n s t e i n , 19*+7a, 1 9 ^ 7 ^ 5 ' Sakuma and Beck, 1961 ; Frumin, Ngai and Wang, 1 9 5 3 ; Folkow and Uvnas, 1 9 ^ 8 ; Wyse et a l , 1 9 7 D -However, since myogenic a u t o r e g u l a t i o n w i l l r a i s e vascular r e s i s t a n c e a f t e r l o s s of sympathetic tone, i t i s important to compare the v a s c u l a r r e s i s t a n c e observed during the a c t i v e d i l a t a t i o n to the immediate t r a n s i t o r y l e v e l s obtained a f t e r denervation and not to the sustained l e v e l of post-sympathectomy r e s i s t a n c e , which i s p a r t l y due to the non-neurogenic autore.g-u l a t o r y phenomenon. I n accordance w i t h t h i s reasoning, some authors have attempted to e x p l a i n the a c t i v e vaso-d i l a t a t i o n s as t r a n s i e n t responses of the v e s s e l s oc-c u r r i n g upon l o s s of c o n s t r i c t o r tone but before auto-r e g u l a t o r y compensation (Jones and Berne, 1 9 6 3 ; G l i c k , Wechsler and E p s t e i n , 1 9 6 8 ) . Wyse et a l ( 1 9 7 1 ) disagree with t h i s i n t e r p r e t a t i o n of the r e s u l t s seen i n most experiments, demonstrating that the t r a n s i e n t d i l a t a t i o n s seen f o l l o w i n g sympathectomy are a n t i h i s t a m i n e s e n s i t i v e , as are a c t i v e r e f l e x d i l a t a t i o n s , while i n t e n t i o n a l l y induced autoregulatory changes are not. 3 ) Nervous Pathways Involved i n A c t i v e R e f l e x V a s o d i l a t a t i o n . I n searching f o r evidence f o r a depressor r e f l e x c e n t r a l r e l a y p o i n t , Aoki and Brody ( 1 9 6 6 ) have seen increased a c t i v i t y of the medullary v a s o d i l a t o r centers when blood pressure was suddenly r a i s e d by the i n t r a -venous i n j e c t i o n of pressor amines. S t i m u l a t i o n of these same centers causes a p e r i p h e r a l v a s o d i l a t a t i o n (Aoki and Brody, 1 9 6 6 ) . Moreover, i t . h a s been shown that a t r a n s e c t i o n of the medulla j u s t cephalad to the obex abolishes a l l sympathetic c o n s t r i c t o r tone but does not a b o l i s h a c t i v e r e f l e x d i l a t a t i o n invoked by i n t r a -venous i n j e c t i o n of pressor amines (Beck, DuCharme, Gebber, L e v i n and P o l l a r d , 1 9 6 8 ) . In support of the idea that the f i b r e s involved i n t h i s r e f l e x are sympathetic i s the f i n d i n g that the r e f l e x can be abolished by the a d m i n i s t r a t i o n of hexa-methonium i n amounts s u f f i c i e n t to completely block t r a n s m i s s i o n i n the sympathetic ganglion (Wyse et a l , 1 9 7 1 ) - I t has been known f o r some time that the gang-l i o n i c s timulant DMPP i s capable of producing a f a l l i n blood pressure which i s greater than that r e s u l t i n g from sympathectomy CSakuma, 196^-). This has been shown true f o r intravenous v e r a t r i n e (Brody, DuCharme and Beck, 1 9 6 7 ) and f o r the d i l a t a t i o n s produced by In-creased c a r o t i d sinus pressure (Beck and -Brody, 1 9 6 1 ) and by low voltage s t i m u l a t i o n of the medullary de-pressor areas (Aoki and Brody, 1 9 6 6 ) . h) Evidence Supporting Histamine as a Mediator of  Ac t i v e R e f l e x V a s o d i l a t a t i o n . Because of the marked e f f e c t of anti h i s t a m i n e s on a c t i v e r e f l e x d i l a t a t i o n s invoked by se v e r a l methods, i t has been po s t u l a t e d that histamine Is inv o l v e d some-where i n the r e f l e x pathway, very p o s s i b l y as the f i n a l n e u r o t r a n s m i t t e r (Beck, 1 9 5 8 b , 1 9 6 5 ) - The e s s e n t i a l f e a t u r e s of the pharmacological evidence i n support of such a r o l e f o r histamine are as f o l l o w s : 1) Antihistamines of a l l major c l a s s e s a) a b o l i s h a c t i v e r e f l e x d i l a t a t i o n b) a b o l i s h the r e a c t i v i t y of the v e s s e l s to h i s t -amine c) do not a b o l i s h v e s s e l r e a c t i v i t y to other vasoactive agents ( a c e t y l c h o l i n e , Isoproterenol) The above e f f e c t s do not seem to be r e l a t e d to the c e n t r a l depressant a c t i o n of an t i h i s t a m i n e s 2. A c o r r e l a t i o n can be shown between the progres-s i v e r e d u c t i o n of va s c u l a r r e a c t i v i t y to exogen-ous histamine and the progressive r e d u c t i o n of a c t i v e r e f l e x d i l a t a t i o n by an t i h i s t a m i n e s 3 . The a c t i v e r e f l e x d i l a t a t i o n i s both atropine and p r o p r a n o l o l r e s i s t a n t The above f i n d i n g s (Beck, 1 9 6 5 ) are i n agreement with those of others from both dogs and cats (Wellans and Wauters, 1 9 6 6 ; T u t t l e , 1 9 6 5 , 1 9 6 6 ) . An a c t i v e r e f l e x d i l a t a t i o n that i s n o n - c h o l i n e r g i c , although not neces-s a r i l y h i s t a m i n e r g i c , has been seen i n r a t s (Tobia, Miya and Bousquet, 1 9 6 8 ) . While a c t i v e d i l a t a t i o n can be demonstrated i n primates,' i t s pharmacological char-a c t e r i s t i c s have not been e l u c i d a t e d ( L e v i n , B a r t l e t t and Beck, 1 9 6 8 ) . 1 h Radioisotope work has shown the re l e a s e of C l a b e l l e d histamine i n t o the venous e f f l u e n t of a per-fused limb concurrent w i t h the increased blood f l o w of a c t i v e r e f l e x d i l a t a t i o n (Brody, 1 9 6 6). S i m i l a r l y , stim-u l a t i o n of the sympathetic trunk w i t h parameters that produce a c t i v e d i l a t a t i o n i s followed by the increased r a t e of i n c o r p o r a t i o n of l a b e l l e d h i s t i d i n e i n t o h i s t -amine, p o s s i b l y by the a c t i v a t i o n of h i s t i d i n e decarbox-y l a s e (Schayer, 1 9 6 0 ; T u t t l e , 1 9 6 7 ) -There are numerous r e p o r t s i n the l i t e r a t u r e of increased blood histamine l e v e l s during increased baro-receptor a c t i v i t y (Brody, 1 9 6 6 5 Tobia, Adams, Miya and Bousquet, 1 9 6 9 ; T u t t l e , 1 9 6 7 ) 5 while s t a b l e histamine l e v e l s are reported i n chronic b u f f e r nerve deafferented preparations (Went and Varga, 1 9 5 2 ) . S t i m u l a t i o n of the c e n t r a l nervous system can re l e a s e histamine i n t o the blood at p e r i p h e r a l s i t e s ( T u t t l e , 1 9 6 6 ; T u t t l e and McLeary, 1 9 7 0 ) . Examination of the d i s t r i b u t i o n of histamine i n the nervous system shows the highest concentrations i n the p o s t - g a n g l i o n i c sympathetic nerves while the lowest i s i n the s p i n a l cord (Green, 1 9 6 ^ ; Schayer, 1 9 6 2 ; E a r l e and Palm," 1 9 5 0 ) . Appreciable concentrations of histamine-N-methyl t r a n s f e r a s e ( r e s p o n s i b l e f o r the degradation of histamine to methyl-histamine) have been found i n the s c i a t i c nerve i n a s s o c i a t i o n with sympathetic neurons (Brown, Tomchick and Axelrod, 1 9 5 9 ) . 5) Evidence Against Histamine as a Mediator of A c t i v e  R e f l e x V a s o d i l a t a t i o n C r i t i c i s m of the hypothesis that histamine i s a neurotransmitter at p e r i p h e r a l s i t e s has taken the form that the anti h i s t a m i n e s used i n such stud i e s i n t e r f e r e w i t h normal p y s i o l o g i c a l processes of the perfused area. The strongest argument of t h i s type i s the c l a i m that c e r t a i n a n t i h i s t a m i n e s block the reup-take of norepinephrine i n t o the nerve t e r m i n a l s (Isaac and Goth, 1 9 6 7 ) and may thus decrease the magnitude of a p u r e l y passive d i l a t a t i o n by.prolonging the e f f e c t of endogenously released amine. On the other hand, i t has been claimed that some drugs which I n h i b i t adren-e r g i c t r a n s m i s s i o n also i n h i b i t a c t i v e r e f l e x d i l a t a t -i o n ( G l i c k et a l , 1 9 6 8 ) . However, i t has r e c e n t l y been reported that one such drug, phenoxybenzamine, not only blocks alpha r e c e p t o r s , as shown by an i n -crease i n blood norepinephrine l e v e l s on s t i m u l a t i o n of the sympathetic nerves to the area, but also i n h i b -i t s the r e l e a s e of l a b e l l e d histamine during a c t i v e r e f l e x d i l a t a t i o n (Boerth, Ryan and Brody, 1 9 7 0 ) . Hence the r e d u c t i o n i n magnitude of r e f l e x d i l a t a t i o n f o l l o w i n g the a d m i n i s t r a t i o n of t h i s drug might p o s s i b l y be due not to i t s alpha b l o c k i n g c h a r a c t e r i s t i c s but to i t s a b i l i t y to prevent r e l e a s e of histamine at the v e s s e l l e v e l . Moreover, apart from i t s a n t i h i s t a m i n i c p r o p e r t i e s , the f a c t that phenoxybenzamine decreases the normal c o n s t r i c t o r tone to the v e s s e l s would de-crease the magnitude of a r e f l e x d i l a t a t i o n . I t has s i m i l a r l y been shown (Boerth et a l , 1 9 7 0 ) that other pharmacological agents which have been claimed to decrease the magnitude of r e f l e x d i l a t a t i o n have a n t i h i s t a m i n i c p r o p e r t i e s . Included amoung these are tripelenamine and cocaine. A p o s s i b l e a l t e r n a t i v e e x p l a n a t i o n f o r the i n -creased l e v e l of l a b e l l e d histamine shown i n the per-fusate of a s k e l e t a l muscle va s c u l a r bed during a c t i v e r e f l e x d i l a t a t i o n i s that the r e d i s r i b u t i o n of blood flow' i n that r e g i o n r e s u l t s i n an increased washout of histarine without an increased amount of histamine n e c a s s a r i l y being r e l e a s e d from the nerves i n the area ( G l i c k et a l , 1 9 6 7 )• T h i s argument has been r e f u t e d by the ob-s e r v a t i o n that the decrease of the d i l a t a t i o n by block-ing agents does not a f f e c t the e l e v a t i o n of blood histamine during the r e f l e x (Boerth et a l , 1 9 7 0 ) . I f the increase i n blood histamine at that time were due to a washout e f f e c t then any dimin u t i o n of the r e f l e x should be accompanied by a dim i n u t i o n of the washout. This was shown to be untrue .by Boerth et a l ( 1 9 7 0 ) . A complicating f a c t o r i n the examination of the p o s t u l a t e d h i s t a m i n e r g i c d i l a t o r f i b r e s to the extrem-i t i e s has been the a c t i o n of agents used to block • c o n s t r i c t o r tone to these regions. Thus b r e t y l i u m , guanethidine and x y l o c a i n e have been shown to i n h i b i t h i s t a m i n e r g i c v a s o d i l a t a t i o n as w e l l as adrenergic t r a n s m i s s i o n . Chronic r e s e r p i n e treatment abolishes both c o n s t r i c t i o n and r e f l e x d i l a t a t i o n , while acute res e r p i n e treatment r e s u l t s i n a b o l u t i o n of r e f l e x d i l -a t a t i o n befroe a b o l i t i o n of r e f l e x c o n s t r i c t i o n (Sakuma and Beck, 1 9 6 1 ) . Ergot a l k a l o i d s are capable of a b o l i s h i n g h i s t a m i n e r g i c d i l a t a t i o n at doses that do not a f f e c t the c o n s t r i c t o r f i b r e s (Beck, 1 9 6 1 ; Wellans, 196k). more r e c e n t l y , work by Wyse, Beck, Burks and Spalding ( 1 9 7 1 ) has shown that many drugs i n t e r f e r e w i t h the r e l e a s e of histamine i n the perfused hindlimb of dogs. Ryan and Brody ( 1 9 7 2 ) have r e c e n t l y challenged the concept of a h i s t a m i n e r g i c i n n e r v a t i o n f u n c t i o n i n g i n a c l a s s i c a l way, that i s , w i t h histamine being d i r e c t l y r eleased by p o s t - g a n g l i o n i c t e r m i n a l s . They suggest that histamine i s probably rel e a s e d from non-neurogenic storage s i t e , p o s s i b l y the mast c e l l s , under the a c t i o n of e i t h e r a c o l l a t e r a l branch of a c o n s t r i c t o r f i b r e or of a separate adrenergic neuron. In a monograph on the subject of smooth muscle, Campbell makes the f o l l o w i n g statement: "In view of the d i f f i c u l t y i n f i n d i n g drugs which s e l e c t i v e l y e l i m i n -ate the v a s o c o n s t r i c t o r e f f e c t s of adrenergic nerves to r e v e a l h i s t a m i n e r g i c v a s o d i l a t o r responses, i t i s c l e a r that the most e f f e c t i v e way to study the h i s t -aminergic' v a s o d i l a t o r n e r v e s , i s by c r e a t i n g a s i t -u a t i o n i n which only these nerves are s t i m u l a t e d , a r e s u l t which cannot be obtained by s t i m u l a t i o n of mixed nerve trunks i n the p'eriphery." (Campbell, 1 9 7 0 ) . While s e l e c t i v e s t i m u l a t i o n of h i s t a m i n e r g i c pathways has now been obtained by medullary s t i m u l a t i o n ( T u t t l e , 1 9 6 6 ) there i s no repo r t i n the l i t e r a t u r e d e s c r i b i n g a pure v a s o d i l a t a t i o n obtained by s t i m u l a t i o n of nerves outside the c e n t r a l nervous system. Donald and Ferguson ( 1 9 7 0 ) , i n a study of the l e v e l of e x i t of sympathetic c o n s t r i c t o r f i b r e s from the s p i n a l cord i n dogs, have demonstrated that the lowest l e v e l of e x i t of such f i b r e s i s or and that s t i m u l a t i o n of the v e n t r a l r o o t s of L ^ , o r r e s u l t s i n a pure hindlimb v a s o d i l a t a t i o n , uncontam-inated by v a s o c o n s t r i c t o r responses. They found that t h i s d i l a t a t i o n was r e s i s t a n t to the intravenous i n -j e c t i o n of a t r o p i n e , p r o p r a n o l o l or Benadryl, an a n t i h i s t a m i n i c . Since no t e s t s of the e f f e c t i v e n e s s of any of the blockades were performed by these authors, we decided to i n v e s t i g a t e ' t h i s p e c u l i a r v a s o d i l a t a t i o n further,, i n j e c t i n g the b l o c k i n g agents d i r e c t l y i n t o the perfused limbs and checking the extent of the blockades by i n j e c t i n g the appropriate agonists d i r e c t l y into the p e r f u s i o n l i n e s as w e l l . METHODS 1) Animals and Anesthesia F o r t y - e i g h t adult mongrel dogs weighing from 1 0 . 0 to 3 0 . 0 kg and eight cats weighing from 2 . 3 to h.2 hg were used. Anesthesia was induced w i t h sodium pentothal ( 2 0 mg/kg i . v . ) i n the dogs and wit h fluothane (Ayerst) i n the. cat s . A l l animals were maintained at a steady l e v e l of anesthesia w i t h a l p h a - c h l o r a l o s e • • ( 6 0 . 0 mg/kg i . v . I n i t i a l l y f o l lowed by 5*0 mg/kg i . v . every 3 0 min., given as a \% s o l u t i o n In s a l i n e ) . A l l i . v . ' i n j e c t i o n s were made v i a a polyethylene cannula i n s e r t e d i n the l e f t saphenous v e i n and advanced as f a r as the i n f e r i o r vena cava. 2 ) Maintenance of the Animal R e s p i r a t o r y P Q Q^ w a s monitered continuously w i t h a Beckman model Lb - 1 medical gas analyser. The animals were a r t i f i c i a l l y v e n t i l a t e d w i t h a harvard model 6 1^ resp-i r a t o r . V e n t i l a t o r y r a t e was set i n i t i a l l y at 15 cpm with appropriate t i d a l volume to give an end-epir-atory PQQ of 38 to M-0 mm Hg and was adjusted as nec-essary to maintain t h i s value. A 5 0 % Og? 50% N 2 mix-ture was administered i n open c i r c u i t . End e x p i r a t o r y r e s i s t a n c e was set at 3 cm of water. A r t e r i a l blood samples were taken p e r i o d i c a l l y and tes t e d f o r P ,P and pH values with an Instrumentation Laboratory blood gas analyser. A r t e r i a l pH was maintained near 7- l+ by a d m i n i s t r a t i o n of appropriate amounts of sodium b i c a r b -onate ( 9 0 mM.% i n s a l i n e ) when necessary. Blood volume was maintained during the experiment by the slow i . v . i n f u s i o n of Dextran 7 5 - Heparin ( 3 mg/kg i n i t i a l l y f o llowed by 0 . 7 5 mg/kg every 3 0 min. throughout the experiment) was given i . v . j u s t before the cann u l a t i o n of the hindlimb v e s s e l s . Esophageal temperature was monitered continuously w i t h a thermal probe (Yellow Springs Instrument Company) and maintained near 3 7 degrees C by heating elements i n the t a b l e and a 0 to 2 0 0 0 watt v a r i a b l e i n f r a - r e d lamp above the t a b l e . 3 ) S u r g i c a l Methods A t r a c h e a l cannula was i n s e r t e d and f i t t e d w i t h a side needle f o r monitering end-expiratory Prn . Both c u 2 r i g h t and l e f t c a r o t i d a r t e r i e s were i s o l a t e d and a loose l i g a t u r e of u m b i l i c a l tape was placed around them to allow l a t e r p e r i o d i c o c c l u s i o n as a t e s t of baroreceptor respnse. A short s t i f f polyethylene cannula was placed i n the r i g h t b r a c h i a l a r t e r y and used f o r monitoring c e n t r a l a r t e r i a l pressure and f o r o b t a i n i n g a r t e r i a l blood samples f o r blood gas and pH determinat-i o n s . A b i l a t e r a l laminectomy was performed between L1+ and i n c l u s i v e to all o w access to the 5 t h , 6 t h and 7 t h lumbar s p i n a l nerve r o o t s on both s i d e s . A f t e r removal of the lamina the dura was l e f t i n t a c t u n t i l a l l other surgery was completed i n order to minimize trauma to and drying of the s p i n a l cord. *+) Methods of Hindlimb P e r f u s i o n The abdominal aorta was approached through a mid-l i n e abdominal i n c i s i o n and a polyethylene cannula, as l a r g e as could be i n s e r t e d , was placed i n the aorta and advanced to a point j u s t d i s t a l to the r e n a l a r t e r i e s . This cannula was f i t t e d w i t h a "Y" connector attached to the two d i s t a l cannulae by two lengths of Tygon tubing. I n a l l cats and some dogs the d i s t a l cannulae were Inserted i n t o the e x t e r n a l i l i a c a r t e r i e s . I n most dogs the d i s t a l cannulae were i n s e r t e d i n t o the femoral a r t e r i e s . To minimize c o l l a t e r a l f l o w between the hind-limbs and other areas, a l l major branches of the aorta below the r e n a l a r t e r i e s were l i g a t e d and the abdominal aorta was l i g a t e d around the c e n t r a l cannula ( F i g . 1 ) . Adequate va s c u l a r i s o l a t i o n of a hindlimb was assum-ed I f , on stoping the p e r f u s i o n pump to that limb, the p e r f u s i o n pressure f e l l to about 3 0 mm Hg, w e l l below both the systemic a r t e r i a l pressure and the p e r f u s i o n pressure of the c o n t r a l a t e r a l limb ( F i g . 2 ) . 5) P e r f u s i o n Pressure Recording Each hindlimb was perfused w i t h a peparate Watson-Marlowe pump. Both of these r o l l e r pumps d e l i v e r e d constant flows over a pressure range of 0 to 3 0 0 mm Hg p e r f u s i o n l i n e pressure. I n some experiments a cannulat-ing electromagnetic f l o w meter probe was placed i n the p e r f u s i o n l i n e d i s t a l to the pump and flows were mon-i t o r e d continuously throughout the experiment w i t h a B i o t r o n i x flowmeter. The flowmeter was c a l i b r a t e d at the end of the experiment using the same cannulae used during the experiment. P e r f u s i o n pressures were measured d i s t a l to the pumps using Statham P 23 Db transducers and recorded on e i t h e r E l e c t r o n i c s f o r Medicine or S.E. La b o r a t o r i e s o p t i c a l r e c o r d e r s . Pump speed was adjusted at the be-ginning of the experiment to provide a p e r f u s i o n pres-sure equal to that i n the b r a c h i a l a r t e r y , which was also recorded continuously (Fig.. 1 ) . 6) Drugs Used Drugs were i n j e c t e d i n t o the p e r f u s i o n l i n e im-mediately proximal to the pumps ( F i g . 1 ) . The f o l l o w -ing drugs were used: 1. Pentothal sodium (Abott, Montreal) 3 0 mg/kg 2 . alpha-Chloralose (BDH, Toronto) 60 mg/kg i n i t i a l l y , 5 mg/kg every 3 0 min maintenance 3 . Heparin sodium ( N u t r i t i o n a l Biochemicals, Cleveland) 3 mg/kg i n i t i a l l y , 0 . 7 5 mg/kg every 3 0 min maintenance k. Gallamine t r i e t h i o d i d e ( F l a x e d i l , Poulenc, Montreal) 50 mg every hour or more o f t e n i f r e q u i r e d 5 . P r o p r a n o l o l ( I n d e r a l , Ayerst, Montreal) 1 . 0 mg/kg 6. I s o p r o t e r e n o l ( I s u p r e l , K & K L a b o r a t o r i e s , N.Y.) 1 ug 7 . Atropine s u l f a t e ( N u t r i t i o n a l Biochemicals, Cleveland) 0 . 2 mg/kg Figure 1. Schematic representation of the preparation used in most experiments. PT = perfusion pressure; P = perfusion pump; L 5 , 5 , 7 = lumbar ventral ro 5 , 6 or 7 . Drug administration was into the per fusion l i n e proximal to the perfusion pump„ Figure 2. Eff e c t on perfusion pressures of stopping one perfusion pump at a time. From above: a o r t i c pressure, right hindlimb perfusion pressure, l e f t hindlimb perfusion pressure. 8 . A c e t y l c h o l i n e (Calbiochem, Los Angeles) 2 u g 9 . Diphenhydramine (Benadryl, Parke-Davis, B r o c k v i l l e , Ontario) 1 . 5 to 3 - 0 mg/kg 1 0 . Histamine (BDH, Toronto) 1 to k ug 1 1 . Mepyramine maleate (Neo-Antergan, Poulenc, Montreal) 5 . 0 mg/kg 1 2 . 6% Gentran 7 5 (Dextran 7 5 ) (Travenol L a b o r a t o r i e s , Morton Grove 111.) 1-3. Hexamethonium bromide (K & K L a b o r a t o r i e s , N.Y.) 10 mg/kg 7 ) Experimental P r o t o c o l A f t e r a d m i n i s t r a t i o n of F l a x e d i l ( 5 0 mg/kg) the dura was opened and a s p i n a l nerve d o r s a l root (L^, 5 or 7 ) was sectioned and p u l l e d c l e a r of the f i e l d . The cor-responding v e n t r a l root was then placed on platinum wire e l e c t r o d e s and bathed i n a pool of warm p a r a f f i n o i l . Monophasic square wave pulses ( 1 0 V, 8 - 2 0 Hz, 3 msec) were used f o r s t i m u l a t i o n . At l e a s t three minutes were allowed between successive s t i m u l a t i o n s . In the pharmacological block experiments the f o l l o w -ing procedure was observed: the va s c u l a r response to vent-r a l root s t i m u l a t i o n was recorded, the the v a s c u l a r r e -sponse to a t e s t dose of the agonist drug was recorded as the agonist was i n j e c t e d i n t o the p e r f u s i o n l i n e . The appropriate b l o c k i n g agent was i n j e c t e d slowly i n t o the p e r f u s i o n l i n e and i t s e f f e c t s t e s t e d w i t h another i n j e c t -i o n of agonist. The degree of block of the v a s c u l a r r e -sponse to the agonist was c a l c u l a t e d as a percentage of the o r i g i n a l response. A second s t i m u l a t i o n of the vent-r a l root was made to evaluate the degree of block of neurogenic v a s o d i l a t a t i o n . The degree of block of the v e n t r a l root response was c a l c u l a t e d as a percentage of the f i r s t ( p r e-blocking agent) s t i m u l a t i o n response. 8) I n t e r p r e t a t i o n of R e s u l t s Because both blood flow r a t e and cannula r e s i s t a n c e were constant, a change i n v a s c u l a r r e s i s t a n c e i n the hind-limb was r e f l e c t e d i n a change i n p e r f u s i o n pressure to that hindlimb. Both the absolute magnitude of pressure change and percentage (of t o t a l p e r f u s i o n pressure) change were c a l c u l a t e d , r e s u l t s are expressed as the absolute change i n p e r f u s i o n pressure. Responses from each animal were averaged and the r e s u l t i n g value was then used to c a l c u l a t e the mean and standard e r r o r presented i n the t a b l e s . Duration of the stimulus r e -sponse was measured from the onset of v a s c u l a r response to the point at which the p e r f u s i o n pressure returned to pre-stimulus l e v e l s . S t a t i s t i c a l a n a l y s i s was done using the T r i a n g u l a r Regression Package (TRIP) program on the U.B.C. IBM 36O/67 computer. 9) C r i t e r i a f o r a V i a b l e Preparation. Only preparations which'met the f o l l o w i n g c r i t e r i a were considered v i a b l e : 1) a small blood l o s s during surgery and an a o r t i c mean pressure above 100 mm Hg; 2) a r t e r i a l blood gases and pH and body temperature w i t h i n normal l i m i t s ; 3) r a p i d cannulation of the v e s s e l s and no evidence of blood c l o t t i n g i n the p e r f u s i o n l i n e s ; h) s a t i s f a c t o r y v a s c u l a r I s o l a t i o n of the hind-limbs; 5) absence of drying of or trauma to the s p i n a l nerve ro o t s or s p i n a l cord; 6) good v a s c u l a r r e a c t i v i t y of the hindlimb v e s s e l s to c a r o t i d clamping or stim-u l a t i o n of the proximal stump of the d o r s a l r o o t . T h i r t y -four dogs and eight eats met these requirements. RESULTS 1) S t i m u l a t i o n of the I n t a c t V e n t r a l Root In 26 of 3*+ v i a b l e dog pr e p a r a t i o n s , 128 stim-u l a t i o n s of the v e n t r a l r o o t s of L^, 5 or 7 , a f t e r s e c t i o n of the corresponding d o r s a l r o o t s , produced a f a l l i n i p s i l a t e r a l hindlimb p e r f u s i o n pressure of 15 to 6 0 mm Hg .('^2 . 9 + ^.1 SE). The rat e of p e r f u s i o n pressure decrease was i n the order of 5 mm Hg/sec, such that the d i l a t a t i o n reached a maximum approximately 10 to 12 seconds a f t e r onset. The d i l a t a t i o n showed an "escape" a f t e r reaching i t s peak magnitude and per-f u s i o n pressure then returned to c o n t r o l l e v e l s d e s p ite continued s t i m u l a t i o n . I n most experiments the s t i m u l a t -i o n was the r e f o r e discontinued when maximum d i l a t a t i o n had been achieved. Recovery time f o r the d i l a t a t i o n was i n the order of 10 to .13 seconds and ther e f o r e the e n t i r e response had a d u r a t i o n of about 2 5 seconds ( F i g . 3 ) . C o i n c i d e n t a l w i t h the i p s i l a t e r a l hindlimb response was a f a l l i n c o n t r a l a t e r a l hindlimb p e r f u s i o n pressure ( F i g . h). While the time of onset and r a t e of f a l l were roughly s i m i l a r to those of the i p s i l a t e r a l l imb, the r 100 R.L.g R.L.g Intact Periph.Stump Figure 3. Effec t on hindlimb perfusion pressures of intact and sectioned ventral root stimulation. From above, a o r t i c pressure, right hindlimb perfusion pressure, l e f t hindlimb perfusion pressure. absolute magnitude of the d i l a t a t i o n was approximately 7 5 to 80% of that on the i p s i l a t e r a l s i d e . This was seen i n 76 t r i a l s i n the 17 animals i n which the p e r f u s i o n pressure of the c o n t r a l a t e r a l limb was measured ( - 3 6 . l + + 5 « 1 5 Table 1).•The d i f f e r e n c e between the i p s i l a t e r a l and c o n t r a l a t e r a l d i l a t a t i o n s was not s i g n i f i c a n t (P 0 . 2 ; unpaired t - t e s t ) . The f i b r e s r e s p o n s i b l e f o r these e f f e c t s appear to have a th r e s h o l d of 3 - 5 to h.O V i n the unsheathed v e n t r a l r o o t . Also c o i n c i d e n t a l w i t h the onset of the i p s i l a t e r a l t d i l a t a t i o n was a f a l l i n the systemic a r t e r i a l pressure of 5 to 2 5 mm Hg ( - 1 ! +.3 ± 1*5) ? without appreciable changes i n heart r a t e or pulse pressure ( F i g . l+, Tabke 1 ) . The time l a g between the s t a r t of e l e c t r i c a l stim-u l a t i o n and the onset of the above described e f f e c t s was g e n e r a l l y 1 to 2 seconds.. 2 ) S t i m u l a t i o n of the Sectioned V e n t r a l Root P e r i p h e r a l s t i m u l a t i o n of the sectioned v e n t r a l root of Lpj, 5 or y produced the same e f f e c t s described above on s t i m u l a t i o n of the i n t a c t r o o t s . C e n t r a l s t i m -u l a t i o n of the sectioned v e n t r a l root d i d not produce any e f f e c t . I n 5 animals used there was no s i g n i f i c a n t difference Table I Eff e c t of VR stimulation on the hindlimb PP and A o r t i c Pressure ventral root i p s i l a t e r a l P c o n t r a l a t e r a l P • a o r t i c pressure P intact root -42.9 + 4,il (26) -36.4 ± 5.1 (17) -14.3 •+ 1.5 (26) cut root -37.6 + 7.2 (5) -30.4 ± 4.9 (4) -11.2 + 1.6 (4) )'-' P 0.2 0.2 0.5 Table I. P = pressure change. Values are expressed as mean + S.E. Number i n brackets = number of t r i a l s . P = paired t - t e s t . between the pre and post s e c t i o n r e s u l t s (Table 1); i p s i -l a t e r a l v a s o d i l a t a t i o n - 3 7 - 6 + 7 - 2 , c o n t r a l a t e r a l - 3 0 . ^ + a o r t i c pressure change - 1 1 . 2 + 1 . 6 3 ) C h o l i n e r g i c Blockade Atropine was i n j e c t e d i . a . i n 6 animals and te s t e d with a c e t y l c h o l i n e as described i n the Methods. The r e s u l t i n g c h o l i n e r g i c blocks were between 7 0 and 1 0 0 % . However, there was no s i g n i f i c a n t decrease i n the i p s i -l a t e r a l (P 0 . 1 0 ) , c o n t r a l a t e r a l (P 0 . 1 0 ) - o r a o r t i c (P 0 . 9 0 ) responses to v e n t r a l root s t i m u l a t i o n ( F i g . 5) • In 3 dogs the intravenous i n j e c t i o n of atropine was test e d and not found to a l t e r v e n t r a l root s t i m u l a t i o n e f f e c t s . i+) Beta-adrenergic Blockade I n k- animals the i . a . i n j e c t i o n of p r o p r a n o l o l (1 mg/kg) was e f f e c t i v e i n producing an 8 0 % blockade of i . a . i s o p r o t e r e n o l (1 ug). Again, v e n t r a l root stim-u l a t i o n e f f e c t s on the I p s i l a t e r a l (P 0 . 1 0 , cantra-l a t e r a l (P 0 . 2 0 ) p r a o r t i c (P 0 A 0 ) pressures were not s i g n i f i c a n t l y a l t e r e d . The intravenous i n j e c t i o n of the same dose of p r o p r a n o l o l i n 2 dogs was without e f f e c t i n b l o c k i n g the v e n t r a l root responses ( F i g . 6 ) . . s < 100 L ISO t 100 a. R.L.6 20 sec. Ach 3 jug. i.a. Ach 3 jug. I.a. R.L.6 Atropine 0.2 mg./kg. I.a. Figure 5 . Effect of ventral root stimulation and i n t r a -a r t e r i a l i n j e c t i o n of acetylcholine on hindlimb perfusion pressure before and after i n t r a -a r t e r i a l atropine. From above: a o r t i c pressure, l e f t hindlimb perfusion pressure, right hindlimb perfusion pressure. I 50r 100 5 0 S 2rao x £ £ V I A V V W W V W A / V V A A A A W A / / \ M A A A / u V V V W \ A M A / W V V V ^ o < 100 50 2 0 sec R . L . R.L., R . L . At rop ine 0.2 mg./kg. i.a. Proprano lo l I m g . / kg. i.a. Figure 6. Effect of ventral root stimulation on hind-limb perfusion pressure and blood flow before and a f t e r administration of atropine or pro-pranolol. From above: hindlimb perfusion pressure, hindlimb blood flow, a o r t i c pressure. 5) H i s t a m i n e r g i c Blockade Intravenous a d m i n i s t r a t i o n of Benadryl. ( 2 . 5 mg/kg) i n 3 animals d i d not a l t e r the vas c u l a r responses to v e n t r a l root s t i m u l a t i o n . When, however, Benadryl ( 2 animals, 2 . 5 mg/kg) or mepyramine maleate (k- animals, 5 . 0 mg/kg) were i n j e c t e d i . a . blockades of between 6 5 and 1 0 0 % were obtained to t e s t doses of histamine, and the i p s i l a t e r a l v e n t r a l root response was e i t h e r d r a s t i c -a l l y reduced or e l i m i n a t e d . C o n t r a l a t e r a l response was reduced a f t e r a time l a g of se v e r a l minutes. The a o r t i c e f f e c t was sometimes reduced i n the face of l a r g e doses of a n t i h i s t a m i n e but at times could not be abolished at a l l ( F i g . 7 , Table 2 ) . . , I n 5 experiments smaller doses of ant i h i s t a m i n e s were i n j e c t e d i . a . i n a stepwise f a s h i o n u n t i l the t o t a l dose had been i n j e c t e d . A progressive and p a r a l l e l r e -duction of the vasc u l a r responses to v e n t r a l root stim-u l a t i o n and to t e s t doses of i . a . histamine (1 to 2 ug) was observed ( F i g . 7)- Figure 8 shows that there i s a d i r e c t r e l a t i o n s h i p between the percent r e d u c t i o n of the vascu l a r response to v e n t r a l root s t i m u l a t i o n and to i . a . histamine. The r e g r e s s i o n c o e f f i c i e n t f o r t h i s set Table I Effect of Anti-Histamines on the Vascular Effects of VR Stimulation Group Ipsi Lateral (A P) Contra Lateral (AP) Aortic Pressure (AP) Control - 54 .8 ± 9 . 6 (6 ) - 50.3 ± 14.0 (4) - 18.9 ± 3.8 (6) Antihistamines - 6.46 ±3 .1 - 2 7.4 ± 17.0 - 9.0 ± 4.2 P< O.0O5 0.05 0.10 Table II. Abbreviations as in Table I. Values are expressed as mean + S.E. Number in brackets = paired t - t e s t . 150 j-100 -— ' < o> 50 |-X E E 3 (/> £ 150 r j j jUUlMU . U i U i y i | A A y i | | ^ f a ^ j i l ^ ^ ^ ^ ^ ^ ^ « 100 . 5ok I min. R.L.g Hist. | R.L.g Hist. I;ug. i.a. f R.L.g Hist. Ijug. i.o. I jug. i.a. Ben. Ben. I mg/kg. i.a. I mg./kg. i.a. Figure 7. Progressive blockade of hindlimb vascular response to ventral root stimulation and to i n t r a - a r t e r i a l histamine by repeated Benadryl administration. Figure 8. Regression l i n e p l o t of the relationship between reduction of vascular response to ventral root stimulation and to i n t r a - a r t e r i a l histamine. Dotted lines represent 95% confidence l i m i t s , r = regression c o e f f i c i e n t . of data i s 0 . 8 8 . During complete blockade of v a s c u l a r response to v e n t r a l root s t i m u l a t i o n the v e s s e l s were s t i l l responsive to i . a . a c e t y l c h o l i n e ( 2 to 3 ug) or i s o p r o t e r e n o l (1 ug) ( F i g . 9 ) 5 and v a s o c o n s t r i c t i o n was' s t i l l present f o l -lowing clamping of the c a r o t i d a r t e r i e s ( F i g . 1 0 ) . 6 ) Hexamthbnium Blockade I n 3 experiments the intravenous a d m i n i s t r a t i o n of hexamthonium ( 5 - 1 0 mg/kg) caused a f a l l in- both a o r t i c and p e r f u s i o n pressures and the complete a b o l i t i o n of of a l l the vas c u l a r e f f e c t s induced by v e n t r a l root stim-u l a t i o n . The completeness of g a n g l i o n i c blockade was demonstrated by the disappearance of the v a s c u l a r responses to c a r o t i d clamping or to d o r s a l root c e n t r a l s t i m u l a t i o n . However, the v e s s e l s could s t i l l respond to t e s t doses of i . a . a c e t y l c h o l i n e , i s o p r o t e r e n o l or histamine ( F i g . 1 0 ) . In only one of the 8 cats used, s t i m u l a t i o n of the v e n t r a l root induced the above described e f f e c t s . A l s o , i n t h i s animal the d i l a t a t i o n was atropine and p r o r a n o l o l r e s i s t a n t but antihistamine s e n s i t i v e . The r e s u l t s of t h i s ' s i n g l e experiment were not pooled w i t h those obtained i n dogs. 150 r o d. < I £ £ 100 • 5 0 gym M^iUu* g ^ | | | ^ ^ ^ i ^ ^ m m m***m •••• PBH 0 I-3 in 0_ c o Q_ 150 100 5 0 • R.U.g Hist. I jag. i.a. R . L 6 Hist. I jug. i.a. Ben. 2 mg./kg. I min. A c h 3 / i g . Isop. I jug. i.a. Figure 9. Effec t of ventral' root stimulation, i n t r a -a r t e r i a l histamine and Benadryl on hindlimb vasodilatation. H i s t . = histamine, Ben. = Benadryl. 20Or C.C. L L - 5 I L - ' - ' 5 c c A o h 3 / j g . \a. Isopr. I jug. ia. 10 mg . / kg . Figure 10. E f f e c t of carotid clamping, ventral root stimulation and hexamethonium on hindlimb perfusion pressures. From above: a o r t i c pressure, right hindlimb per-fusion pressure, l e f t hindlimb perfusion pressure. C.C. = carotid clamping, C^ = hexamethonium, Ach, = acetylcholine, isopr. = isoproterenol. h2 1) Negative cases Of the 3*+ dogs which s a t i s f i e d the c r i t e r i a f o r a v i a b l e p r e p a r a t i o n o u t l i n e d i n the Methods, 8 showed no d i l a t a t i o n . o f the hindlimb v a s c u l a t u r e on s t i m u l a t i o n of the v e n t r a l r o o t s of L,-., or H. and no s u b s t a n t i a l 5 ' 6 1 explanation can be given f o r these cases, although sev-e r a l p o s s i b i l i t i e s might.be considered. I n the dog the two p a r a v e r t e b r a l sympathetic chains fuse i n the r e g i o n of the sacrum or as high as (Mehler et a l , 1 9 5 2 ) . I t i s conceivable t h a t ' d u r i n g the cannulation procedure t h i s g a n g l i o n i c chain or perhaps the p e r i v a s c u l a r nerve plexuses were damaged, destroying the v a s o d i l a t o r y i n n e r v a t i o n of the hindlimb v a s c u l a t u r e . .Indeed, a higher frequency of f a i l u r e s was observed i n those experiments i n which d i s t a l c a nnulation was at the l e v e l of the i l i a c a r t e r i e s r a t h e r than the femoral a r t e r i e s . A l t e r n a t i v e l y , ischemia of the sympathetic g a n g l i a i n the lumbar r e g i o n as a r e s u l t of l i g a t i o n of the 5 t h , 6 t h or 7 t h lumbar a r t e r i e s could have co n t r i b u t e d to the negative r e s u l t s . F i n a l l y , i t Is p o s s i b l e that some damage of the v e n t r a l r o o t s may have occurred i n s p i t e of the e f f o r t s to maintain them i n Q>2_ saturated warm p a r a f f i n o i l , w i t h the r e s u l t that p e r i p h e r a l ( e f f e r e n t ) conduction was impaired. 2 ) Donald and Ferguson Experiments I n our experiments the r e s u l t of u n i l a t e r a l v e n t r a l root s t i m u l a t i o n ( L 5 , 6 , 7 ) was, i n the m a j o r i t y of cases, a b i l a t e r a l v a s o d i l a t a t i o n i n the hindllmbs and a f a l l i n a o r t i c pressure w i t h i n 1 or 2 seconds from the onset of the s t i m u l a t i o n , and l a s t i n g no more than 2 5 or 3 0 seconds despite maintained s t i m u l a t i o n ( F i g . F i g . 1 0 ) . Donald and Ferguson ( 1 9 7 0 ) observed i p s i l a t e r a l hindlimb v a s o d i l a t a t i o n i n dogs induced by s t i m u l a t i o n of the v e n t r a l r o o t s of L5 to Ly. The major d i f f e r e n c e with the present experiments was that i n t h e i r prepara-t i o n s the s p i n a l cord had been sectioned and the s p i n a l cord segments corresponding to the stimulated r o o t s removed. I n our experiments the s p i n a l cord was l e f t i n t a c t , although i n some cases the v e n t r a l root was i s o l a t e d from the CNS by s e c t i o n . Perhaps as a consequence of these d i f f e r e n c e s i n pr e p a r a t i o n , s e v e r a l major d i f f e r e n c e s appear between the r e s u l t s of those workers and those reported here: kk i ) the d u r a t i o n of the response reported here was i n the order of 2 5 seconds while that of Donald and Ferguson was i n the order of s e v e r a l minutes; i i ) the c o n t r a l a t e r a l v a s o d i l a t a t i o n seen i n these experiments was not observed by Donald and Ferguson; i i i ) the systemic a r t e r i a l pressure change seen here was not a part of the response reported by Donald and Ferguson; i v ) while those workers found no a b o l i t i o n of the response by a t r o p i n e , p r o p r a n o l o l or Benadryl when administered i . v . , our experiments show that Benadryl, when'administered i n t r a -a r t e r i a l l y does a b o l i s h the response, as does mepyramine administered by the same rou t e ; v) although sympathect-omy from L 2 through I, does not a b o l i s h the v a s o d i l -a t a t i o n observed by Donald and Ferguson, these e x p e r i -ments demonstrate the a b o l i t i o n of the v a s o d i l a t a t i o n by hexamethonium. In short, these two s t u d i e s d i f f e r i n p r e p a r a t i o n by the presence or absence of an i n t a c t s p i n a l cord and the route of a d m i n i s t r a t i o n and dosage of the drugs given. The r e s u l t s d i f f e r i n d u r a t i o n of the response, involvement of the systemic a r t e r i a l pressure, i n v o l v e -merit of the c o n t r a l a t e r a l v a s c u l a t u r e , e f f e c t s of a n t i -h i s t a m i n e s , and e f f e c t of sympathectomy (whether surg-i c a l or chemical). I n view of these d i f f e r e n c e s , most p a r t i c u l a r l y the systemic a r t e r i a l pressure changes, the hindlimb c o n t r a l a t e r a l v a s o d i l a t a t i o n and the d u r a t i o n of the response, the question of whether these two phenom-enon are perhaps d i f f e r e n t must be considered. C e r t a i n l y , the responses seen i n the two studi e s were both e l i c i t e d by the s t i m u l a t i o n of L^, ^ or y v e n t r a l r o o t s , the greatest magnitude of response being e l i c i t e d from L^, and both occurred w i t h i n 2 seconds of onset of s t i m u l a t i o n , suggesting a neurogenic v a s o d i l -a t a t i o n . However, i n almost a l l other parameters the phenomenon d i f f e r e d . The most convincing evidence that these two phenomenon are indeed the same would be the a b o l i t i o n of the c e n t r a l and c o n t r a l a t e r a l e f f e c t s and . an increase i n the d u r a t i o n of the response seen i n our experiments a f t e r s e c t i o n of the s p i n a l cord. The p e r s i s -tance of the Donald and Ferguson d i l a t a t i o n a f t e r symp-athectomy and the a b o l i t i o n of our d i l a t a t i o n a f t e r symp-athectomy and the a b o l i t i o n of our d i l a t a t i o n a f t e r hexa-if6 methonium'administration could perhaps be explained since i t i s p o s s i b l e that the f i b r e s r e s p o n s i b l e f o r these e f f e c t s synapse i n the parasympathetic g a n g l i a which would be blocked by the hexamethonium used i n our experiments but would not be removed by the sympathectomy c a r r i e d out by Donald and F erguson. On the b a s i s of the p r e s e n t l y a v a i l -able evidence the assumption that these two d i l a t a t i o n s are mediated by the same pathway may only be made wit h considerable caution. 3) Unanswered Questions The observations reported here leave s e v e r a l import-ant questions to be answered. F i r s t , i s the hindlimb v a s o d i l a t a t i o n a c t i v e or passive as those terms have been defined by Beck and Brody ( 1 9 6 1 ) ? Second, i s the hind-limb d i l a t a t i o n the cause or the r e s u l t of the c e n t r a l pressure change, or i s there any a s s o c i a t i o n between the two? T h i r d , what i s the - r e l a t i o n s h i p between the i p s i -l a t e r a l and c o n t r a l a t e r a l v a s o d i l a t a t i o n s , And f o u r t h , what i s the mechanism r e s p o n s i b l e f o r the e f f e c t on a o r t -i c pressure? These questions w i l l be considered seperately below. i ) A c t i v e v a s o d i l a t a t i o n as defined here i s that h7 which r e s u l t s d i r e c t l y from the increased c o n c e n t r a t i o n at the v e s s e l l e v e l of a d i l a t o r substance of e i t h e r neural of exogenous o r i g i n (Beck and Brody, 1 9 6 1 ) . This study i n d i c a t e s that the d i l a t a t i o n i s abolished by a n t i -histamines, an a b o l i t i o n that p a r a l l e l s the progressive l o s s of va s c u l a r r e a c t i v i t y to histamine, which i s con-s i s t e n t w i t h the f i n d i n g s of Beck ( 1 9 6 5 ) , T u t t l e ( 1 9 6 5 , 1 9 6 6 ) and Wellans and Wauters ( 1 9 6 6 ) , a l l of whom describe a c t i v e r e f l e x d i l a t a t i o n s . While the speed of onset of the d i l a t a t i o n f o l l o w i n g s t i m u l a t i o n i n these experiments supports the assumption that the d i l a t a t i o n i s neurogenic, and while the s e n s i t i v i t y to ant i h i s t a m i n e s supports the p o s s i b i l i t y that i t i s analagous to the anti h i s t a m i n e s e n s i t i v e a c t i v e r e f l e x d i l a t a t i o n s described by others, a d e f i n i t e c o n c l u s i o n to t h i s e f f e c t must await the dem-o n s t r a t i o n of neurogenic r e l e a s e at the v e s s e l l e v e l of histamine. i i ) The p o s s i b i l i t y of the i p s i l a t e r a l v a s o d i l a t a t i o n being secondary to a drop i n c e n t r a l a r t e r i a l pressure is u n l i k e l y f o r several reasons. The constant f l o w p e r f u s i o n pumps i s o l a t e the hindlimbs from changes i n c e n t r a l 1+8 a r t e r i a l pressure of the magnitudes seen here. Moreover, the d i f f e r e n c e i n magnitude of the d i l a t a t i o n s of the two hindlimbs would be d i f f i c u l t to e x p l a i n on the basis of a common c e n t r a l cause. And f i n a l l y , the d i f f e r e n t sens-i t i v i t i e s of the p e r i p h e r a l pressure changes and the a o r t i c pressure change to ant i h i s t a m i n e s weigh against a common cause. i i i ) C o n t r a l a t e r a l v a s o d i l a t a t i o n i s observed here In response to s t i m u l a t i o n of the v e n t r a l r o o t s of L5, g and rj. I t i s p o s s i b l e that t h i s i s due to the pe r f u s -i o n by both pumps of a common vasc u l a r bed despite a l l e f f o r t s to i s o l a t e the hindlimbs s u r g i c a l l y . . However, the experiments included i n t h e i r p r o t o c o l the stoppage of one pump at a time ( F i g . 2 ) to ensure that p e r f u s i o n pressure i n that limb f e l l to l e s s than 3 0 mm Hg i n d i c a t i n g that the limb was adequately i s o l a t e d from the c o n t r a l a t e r a l limb and from the c e n t r a l a r t e r i a l pressure. Persistance of a p e r f u s i o n pressure c l o s e r to that of the other limb or the c e n t r a l a r t e r i a l pressure could have i n d i c a t e d a c o l a t e r a l a r t e r i a l p e r f u s i o n to the hindlimb. A b i l a t e r a l vasomotor response to a u n i l a t e r a l pre-g a n g l i o n i c nerve s t i m u l a t i o n i s not described i n the l i t -e r a t u r e . However, apart from the d e s c r i p t i o n by Donald and Ferguson ( 1 9 7 0 ) , n e i t h e r i s there i n the l i t e r a t u r e a d e s c r i p t i o n of vasomotor f i b r e s l e a v i n g the s p i n a l cord below the l e v e l of L3 - of dogs and t h e r e f o r e c r o s s - i n n e r v a t i o n should not be r u l e d out as the p o s s i b l e cause of b i l a t e r a l vasomotor response i n these experiments. On the contrary, Mehler et a l ( 1 9 5 2 ) demonstrated that i n at l e a s t 3 5 % of a s e r i e s of 100 d i s s e c t i o n s i n dogs there was a p r e g a n g l i o n i c outflow from the s p i n a l cord below the l e v e l and that these f i b r e s coursed d i a g o n a l l y caudad to j o i n the L5 or Ly sympathetic ganglion. Moreover, the L7 ganglion could o f t e n be shown to be a fused ganglion composed of both r i g h t and l e f t counterparts, and that the f i b r e s d i d cross the m i d l i n e as shown by osmium s t a i n -i n g . However, these workers were not able to show contra-l a t e r a l vasomotor responses by e l e c t r i c a l s t i m u l a t i o n of the g a n g l i a j u s t r o r s t r a l to the poi n t of f u s i o n , but i t should be noted that t h e i r c r i t e r i a f o r vasomotor a c t i v i t y was a v a s c u l a r response i n the dog paw, which i s an.almost pu r e l y cutaneous vascular t e r r i t o r y . The a b o l i t i o n of the responses seen i n our experiments by hexamethonium sup-ports the p o s s i b i l i t y of a p r e g a n g l i o n i c pathway that could well synapse in the fused L sympathetic ganglion 7 and cross the midline to innervate some contra l a t e r a l vascular beds. iv) Most puzzling i s that the stimulation of a sup-posedly purely efferent pathway, (the ventral root) i n -nervating a vascularly i s o l a t e d t e r r i t o r y should cause a central a r t e r i a l pressure drop. Sherrington (1894) describ-ed afferent f i b r e s within the ventral roots of cats and monkeys as have others more recently i n cats and rats (Kato and Hirata, 1965; Dimsdale and Kemp, 1966). The "central e f f e c t " on a o r t i c pressure i n our experiments occurs also on stimulation I f the peripheral stump of the sectioned ventral root while 'it was absent when the central stump of.the ventral root was stimulated. These findings should rule out the p o s s i b i l i t y of these f i b r e s contributing to the central e f f e c t . Previous section of the dorsal roots rules out the p o s s i b i l i t y of a r e f l e x arc at t h i s spinal l e v e l causing afferent stimulation of CNS centers. B i l a t e r a l section of the dorsal roots from Lr through L 7 i n three experiments did not abolish the central effect so presumably:it is not mediated by a re f l e x arc within one or two spinal segments of the periph-e r a l l y stimulated ventral root. It would be possible to produce the central effect by ventral root stimulation i f there were i n the root vasodilator f i b r e s which were innervating a large vasc-ular bed not controlled by the pumps. This i s not l i k e l y the case i n these experiments however for two reasons. The major branches-;of the aorta below the renal a r t e r i e s (including the i n f e r i o r mesenteric artery) had a l l been ligated i n these experiments. Furthermore, the innervation of the splanchnic vascular beds i s derived from much higher spinal segments (M i l l e r , 1967). It is unlikely that venous d i l a t a t i o n in the hindlimb i n response to ventral root stimulation with consequent trapping of blood i n the periphery r e s u l t i n g in decreased, venous return is responsible for the f a l l i n a o r t i c pressure. Not only does the central effect occur too soon after the onset of stimulation, but the lack of changed i n pulse pressure or heart rate suggests a change i n peripheral resistance rather than a change in cardiac output as the cause of the a o r t i c pressure change. Anastomotic channels between central a r t e r i a l sup-ply and the hindlimb vascular beds could t h e o r e t i c a l l y allow a r e f l e c t i o n of massive peripheral vasodilatation in the central a r t e r i a l pressure. However, i n these ex-periments great care was. taken to eliminate such channels by l i g a t i n g a l l a r t e r i e s below the renal..arteries and above the point of cannulation. The success of these precautions is r e f l e c t e d i n the lack of sustained a r t e r i a l pressure in either hindlimb during the stoppage of i t s respective perfusion pump (Fig. 2). . Another p o s s i b i l i t y i s that the-^efferent f i b r e s which are being stimulated in the ventral roots t r a v e l eephalad within the sympathetic ganglia from which afferent f i b r e s then reach the CNS through some as yet undefined pathway. Absence of the central effect (and the co n t r a l a t e r a l effect) in the Donald and Ferguson experiments, which included b i l a t e r a l sympathectomy sup-ports t h i s p o s s i b i l i t y , since, i f such were the case, no central effect would appear i n their experiments i n which segments of the spinal cord had been removed. A b o l i t i o n of the central pressure changes in our experiments by the administration of hexamethonium i s consistent with this hypothesis. The central effect cannot be explained by postulating the release of a vasodilator substance i n the hindlimbs, which would then reach more central vascular beds through the venous return from the hindlimbs, causing a more gen-e r a l i z e d vasodilatation because the time course of the effect i s too short. The immediate onset of the central effect suggests a neurogenic mediation, either by active d i l a t a t i o n or by central i n h i b i t i o n of vascular tone such as i s seen in the vaso-vagal and baroreceptor ref l e x e s . 4) Evidence Supporting Histamine as the Mediator of  Vasodilatation Evidence presented here supports histamine as the d i l a t o r substance responsible for at least the peripher a l e f f e c t s . The time course of the vasodilatation and the observation that the histamine content of post-gang l i o n i c sympathetic neurons i s the highest found any-where in the nervous system (Green, 1964; Schayer, 1962; Werle and Palm, 1950) make i t l i k e l y that these d i l a t a t i o n s are mediated by histamine released from post-ganglionic sympathetic neurons at the vessel l e v e l . The hexamethonium block of the response to ventral root stimulation suggests that preganglionic f i b r e s are being stimulated within the ventral root. It can be seen (Fig. 6) that the antihistamineiblock of vasodilatation does not block the vessel response to acetylcholine or isoproterenol. Also, i t i s seen that the progressive loss of vessel r e a c t i v i t y to exogenous histamine following repeated additions of antihistamine (Benadryl) i s accompanied by progressive and p a r a l l e l loss of vasodilator response to ventral root stimulation (Fig. 7, F i g . 11). A similar a b o l i t i o n of vasodilatation can be demonstrated following i n t r a -a r t e r i a l i n j e c t i o n of mepyramine. While these observations support the p o s s i b i l i t y that histamine i s responsible for the d i l a t a t i o n s they do not provide complete proof. It remains to be demonstrat-ed that histamine i s released in response to nerve stim-u l a t i o n . Others have demonstrated that l a b e l l e d histamine is released in vascular beds undergoing active vaso-d i l a t a t i o n (Brody, 1966; Tu t t l e , 1967), and the con-comitant increase i n the rate of conversion of l a b e l l e d h i s t i d i n e to histamine (Schayer, 1967). Stimulation of the CNS or the sympathetic trunk can cause the release of histamine peripherally (Tuttle, 1966; T u t t l e and McLeary, 1970). S i g n i f i c a n t levels of the ..degrading enzyme necessary for the removal of active histamine from the region of the nerve terminal, histamine-N-methyl transferase, has also been demonstrated in post-ganglionic sympathetic nerves (Brown, Tomchick and Axelrod, 1959), as has histamine i t s e l f (Green, 1964). The accumulated evidence cited here would appear to f u l f i l l the requirements generally recognized as nec-essary for acceptance of a substance as a neurotrans-mitter (see McLennan, 1969). However, there remain at least three objections to such a conclusion, most of which have been discussed in the l i t e r a t u r e review (see Introduction above) . The ubiquity of histamine i n the vascular system and the perivascular tissues allows postulations of histamine release from tissues other than nerve terminals (Ryan and Brddy, 1972). The i n -creased tissue perfusion r e s u l t i n g from vasodilatation suggests the p o s s i b i l i t y that histamine appearing i n the perfusate may merely represent histamine that i s being washed out of previously unperfused areas, although this p o s s i b i l i t y has been largely eliminated by the use of l a b e l l e d h i s t i d i n e as already discussed (see Introduction). F i n a l l y , the necessity i n most experimental preparations of providing cholinergic and adrenergic blockade i n order to unmask the histaminergic vasodilatation introduces the complication of interference of these agents with the release or action of histamine on the vascular smooth muscle. Antihistamines themselves have been known to i n t e r f e r e with the reuptake of norepinephrine into the preganglionic nerve terminals (Isaac and Goth, 1967). Wyse et a l (1971) have shown that many drugs used i n thi s f i e l d have some histamine releasing a c t i v i t y . 5) Advantages of thi s Preparation An advantage of the preparation used in this study is the a b i l i t y to e l i c i t an antihistamine sensitive vaso-d i l a t a t i o n in the absence of cholinergic and adrenergic blockade, thus avoiding some of the above mentioned pharmacological complications. The p o s s i b i l i t y of interference with the reuptake of norepinephrine by antihistaminics, which would decrease the magnitude of a d i l a t a t i o n by prolonging the presence of a constrictor substance ( i . e . "norepinephrine) i n the v i c i n i t y of the vessels applies to the vasodilatations reported here only i f they are passive i n nature. Without demonstrating histamine release during ventral root stimulation this d i l a t a t i o n cannot be d i f f e r e n t i a t e d as either a c t i v e or passive, although the s t r i k i n g s i m i l a r i t y of these findings with those of Beck, T u t t l e and others make i t reasonably safe to assume that i t is an active histaminergic d i l a t a t i o n . Beck (1965, 1966) has outlined the conditions which should be s a t i s f i e d in order to demonstrate an active histaminergic vasodilatation (see Introduction above). The results described here s a t i s f y a l l those conditions as they apply; i ) the vascular responses to nerve stim-u l a t i o n are abolished by antihistamines of the two major classes that were used; i i ) the r e a c t i v i t y of the vessels to other vasoactive agents i s maintained during histamine blockade; i i i ) a c o r r e l a t i o n is shown between the re-duction of vascular response to exogenously released histamine and to neurogenically induced d i l a t a t i o n ; iv) the d i l a t a t i o n i s both atropine and propranolol r e s i s t a n t . In view of these s i m i l a r i t i e s i s seems possible that the efferent nervous pathway responsible for the a c t i v e r e f l e x d i l a t a t i o n described by Beck and that respons-i b l e for t h i s d i l a t a t i o n are the same. The pathway respons-i b l e for the active r e f l e x d i l a t a t i o n of Beck apparently synapses in the sympathetic ganglion since the r e f l e x i s abolished by doses of hexamethonium s u f f i c i e n t to block transmossion in the sympathetic ganglion (Wyse et a l , 1971). The same dose of hexamethonium i s found to block the vasodilatation reported here. 6) Conclusions The i p s i l a t e r a l , neurogenically mediated vasodil-atation seen here i s s e n s i t i v e to antihistamines but resis t a n t to cholinergic and adrenergic blocking agents. It appears, i n most parameters, similar to the active r e f l e x vasodilatation induced by sudden increases i n ar-t e r i a l pressure as demonstrated by Beck and his coworkers (Beck, 1958) . There are several points remaining un-explained in these observations. No explanation can be offered for the c o n t r a l a t e r a l vasodilatation,.nor for the concomitant f a l l in central .a r t e r i a l pressure. The i p s i l a t e r a l vasodilatation i s abolished by antihistamines of two major classes but there has been no demonstration in these experiments of histamine release from the area of vasodilatation. The cause of "escape" of the vaso-d i l a t a t i o n despite continued stimulation seen in these experiments has not been explained, although depletion of the pools of a v a i l a b l e histamine is a p o s s i b i l i t y . These experiments demonstrate a neural output to the vessels via ventral roots which were not known to have one before the Donald and E.erguson work, and further, they demonstrate an efferent preganglionic pathway of "pure" vasodilatory f i b r e s when none was previously known. If t h i s vasodilatation i s confirmed to be histamine mediated, either through demonstration of l a b e l l e d histamine release, or any other method, th i s preparation would offer a d i s t i n c t advantage over other preparations in use for the study of histaminergic vasodilator f i b r e s since no adrenergic or cholinergic blockade i s required before the histaminergic component of the d i l a t a t i o n i s seen, thus avoiding the uncertainty caused by the use of multiple drugs. LITERATURE CITED Aoki, V.S., Brody, M.J. Medullary control of vascular resistance: An electrophysiological analysis. C i r c . Res. 18 Suppl.l: 75-85, 1966. Bayliss, W.M. The vasomotor system. Longmans, Green and Co. London, 1923. B&ck, L. E f f e c t of the autonomic nervous system on a r t e r i o l a r tone i n the experimental animal. C i r c u l a t i o n 17: 798-806, 1958a. Beck, L. The action of antihistamines on r e f l e x d i l a t a t -ion i n the hindlimb. J . Pharmacol. Exper. Therap. 122: 4A, 1958b. Beck, L. Active r e f l e x d i l a t a t i o n i n the innervated perfused hind limb of the dog. Amer. J. Physiol. 201: 123-128, 1961. Beck, L. A new concept i n autonomic control: Peripheral i n h i b i t i o n and d i s i n h i b i t i o n . Univ. Mich. Med. B u l l . 29: 292-296, 1963. Beck, L. Histamine as the p o t e n t i a l mediator of active r e f l e x d i l a t a t i o n . Fed. Proc. 24: 1298-1310, 1965. Beck, L., Brody, M.J. The physiology of vasodilatation. Angiology 12: 202-222, 1961. Beck, L., Ducharme, D.W., Gerber, G.L., Levin, L.A., Pollard, A.A. In h i b i t i o n of adrenergic a c t i v i t y at a locus peripheral to the brain and spinal cord. C i r c . Res. 18: Suppl.l 55-72, 1966. Bernard, C. Influence du grand ;sympathetique sur l a s e n s i b i l i t e et sur l a c a l o r i f i c a t i o n . Compt. Rend. Soc. B i o l . 3: 163, 1851. Bernard, C. Sur les variations de couleur dans l a sang veineux des organs glandulaire suivant leur etat de function ou de repos. J . Physiol. Paris 1: 233, 1858. Binet, L., Bernstein, M. Sur les reactions vasculaires d'origine sinusale au niveau de l a patte perfusee Compt. Rend. Soc. B i o l . 141: 248-250, 1947a. Binet, L., Bernstein, L. Sur l a vasodilatation periph-erique d'origine adrenaUnique. Compt. Rend. Soc. B i o l . 141: 623-630, 1947b. Boerth, R .C ., Ryan, M. J . . tPharmacologic blockade of r e f l v asodilatation: e f f e c t s , postulated neurohumoral mechanisms. J . Pharmacol. Exp. Ther. 172: 52-61, 1970. Brody, M.J. Neurohumoral mediation of active r e f l e x vasodilatation. Fed. Proc. 25: 1583-1592, 1966. Brody, M.J. Mechanisms of blockade of r e f l e x vasodil-atation. Fed. Proc. 27: 756-757, 1968. Brody, M.J., Dorr, L.D., Schaffer, R.A. Reflex vaso-d i l a t a t i o n and sympathetic transmission i n the renal hypertensive dog. Am. J . Physiol. 219: 1746-1750, 1970. Brody, M.J., DuCharme, D.W., Beck, L. Active r e f l e x vasodilatation induced by veratrine and dopamine. J . Pharmacol. Exp. Therap. 155: 84-90, 1967. Brody, M.J., Schaffer, R.A. D i s t r i b u t i o n of vasodilator nerves in the canine hidlimb. Am. J . Physiol. 218: 470-474,1970. Bronk, D.W., Ferguson, L.K., Margaria, R., Solandt, D.Y. Am. J . Physiol. 117: 237-249, 1936. Bronk, D .W., P i t t s , R.F., Larrabee, M.G. Role of the hypothalamus in cardiovascular regulation. A. Research Ment. Dis. Proc. 20: 323-341, 1940. Brown, D.D., Tomchick, R., Axelrod, J . The d i s t r i b u t i o n and properties of a histamine methylating enzyme. J . B i o l . Chem. 234: 2948-2950, 1959. Campbell, G. Autonomic nervous supply to effector tissues. In Smooth Muscle Bulbring, E., Brading, A.F., Jones, A.W., Tomita, T. Ed i t o r s . Edward Arnold, London, 1970. Dimsdale, J.A., Kemp, J.M. Afferent f i b r e s in the ventral roots in the r a t . J . Physiol. 23-24: 25P, 1966. Donald, D.E., Ferguson, D.A. Study of the sympathetic vasoconstrictor nerves to the vessels of the dog hindlimb. C i r c . Res. 26: 171-184, 1970. Eliasson, S., Lindgren, P.-V Uvnas, B. Representation in the hypothalamus and motor cortex i n the dog of sympathetic vasodilator outflow to s k e l e t a l muscle. Acta. Physiol. Scandinav. 27: 18-37, 1952. Eliasson, S., Lindgren, P., Uvnas, B. The sympathetic vasomotor innervation of the skin of the dog. Acta Physiol. Scandinav. 17: 195-207, 1949. Folkow, B., Haeger, K., Uvnas, B. Cholinergic vaso-d i l a t o r nerves i n the sympathetic outflow to the muscles of the hindlimb of the cat. Acta Physiol. Scandinav. 15: 401-411, 1948. Folkow, B., Frost, J., Haeger, K., Uvnas, B. The sympath-e t i c vasomotor innervation of the skin of the dog. Acta Physiol. Scandinav. 17: 195-200, 1949. Folkow, B., Uvnas, B. D i s t r i b u t i o n and functional s i g -n ificance of the sympathetic vasodilators to the hindlimbs of the cat. Acta Physiol. Scandinav. 15: 389-400, 1948. Frumin, N.G., Ngai, S.H., Wang, S.C. Evaluation of the vasodilator mechanisms in the canine hindlimb: question of dorsal root p a r t i c i p a t i o n . Am. J . Physiol. 173: 428-436, 1953. Glick, G., Epstein, S.E., Wechsler, A.S., Braunwald, E. Physiological differences between the effects of neuronally released and bloodborne norepineph-rine on beta adrenergic receptors in the a r t e r i a l bed of the dog. C i r c . Res. 21: 217-227, 1967. Glick, G.Wechsler, A.S., Epstein, S .E. Mechanisms of r e f l e x d i l a t a t i o n : assessment of the role neural reuptake of norepinephrine and release of histamine. J . C l i n . Invest. 47: 511-520, 1968. Green, J.P. Histamine and the nervous system. Fed. Proc. 23: 1059-1102, 1964. Heymans, C , N e i l , E. Reflexogenic areas of the cardiovascular system. L i t t l e , Brown and Company, Boston, 19-5-8. Isaac, L., Goth, A. The mechanisms of the potentiation of norepinephrine by antihistaminics. J . Pharm-ac o l . Exper. Therap. 156: 463-468, 1967. Jones, R.D., Berne, R.M. Vasodilatation in s k e l e t a l muscle. Am. J . Physiol. 204: 461-466, 1963. Kato, M., Hirata, Y. Sensory neurons i n the ventral roots of the cat. Brain Res. 7: 479-482, 1968. Lindgren, P., Uvnas, B. Postulated vasodilator centers in the medulla oblongata. Am. J . Physiol. 176: 68-76, 1954. Levin, J ., B a r t l e t t , J.D., Beck, L. Active r e f l e x vaso-d i l a t a t i o n induced by epinephrine or norepinephrine in primates. J . Pharmacol. Exper. Therap. 174:262-270, 1968. M i l l a r , R.A., Briscoe, T.J. Preganglionic sympathetic a c t i v i t y and the effects of aneasthetics. B r i t . J . Anaesthesia 37: 804-832, 1965. M i l l e r , M.E. Anatomy of the dog. Saunders, 1964. Mehler, W,R., Fischer, J.C., Alexander, W.F. The anat-omy and variations of the lumbosacral sympathetic trunk in the dog. Anat Rec. 113: 421-435, 1952. Ryan, M.J., Brody, M.J. Neurogenic and vascular stores of histamine in the dog. J . Pharmacol. Exp. Therap. 181: 83-91, 1972. Sakuma, A. Three d i s t i n c t vasomotor mechanisms a c t i v -ated by DMPP. Japan J . Pharmacol. 14: 421-435, 1952. Sakuma, A., Beck, L.,Pharmacologic evidence for active r e f l e x vasodilatation. Am. J. Physiol. 201-: 129-133, 1961. Schayer, R.W. Relationship of induced histamine decarb-oxylase a c t i v i t y and histamine synthesis in shock from stress and endotoxin. Am. J . Physiol. 198: 1187-1192, 1960. Schayer, R.W. Evidence that induced histamine is an i n t r i n s i c regulator of the microcirculatory system. Am. J . Physiol. 202: 66-72, 1962. Tobia, A.J., Adams, M.D., Miya, T.S., Bousquet, W.F. Histamine and r e f l e x vasodilatation in the r a t . L i f e S c i . 8: 745-750, 1969. Tuttle,\R.S. Relationship between blood histamine and cen t r a l l y evoked hypotensive response. Am. J . Physiol. 209: 745-750, 1969. Tut tie,.: R .S . Histaminergic component i n the barore-ceptor r e f l e x of the pyramidal cat. Fed. Proc. 25: 1593-1595, 1966. Tut t l e , R.S. Physiological release of histamine in the pyramidal cat. Am. J . Physiol. 213: 620-624, 1967. Tutt l e , R.S., McLeary, M. E f f e c t of sympathetic nerve a c t i v i t y on l a b e l l i n g and release of histamine i n the cat. Am. J . Physiol. 218: 143-148, 1970. Tuttle, R ,S ., McLeary, M. Hemodynamic c h a r a c t e r i s t i c s of histaminergic vasodilatation i n the pyramidal cat. Am. J . Physiol. 220: 337-346, 1971. Wellans, D. Inh i b i t i o n of norepinephrine induced vaso-d i l a t a t i o n i n the hindlimb. Arch. Int. Pharmaco-dyn. 151: 281-285, 1964. Wellans, D., Wauters, E. Norepinephrine induced r e f l e x vasodilatation and adrenergic beta-receptors. Arch Int. Pharmacodyn. 159: 401-404, 1966. Went, I., Varga, E. Experimentelle untersuchungen uber die chemiche gegenregulation des blutdruckes. Acta Physiol. Acad. S c i . Hung. 3: 337, 1952. Werle, E., Palm, D. Histamin in Nervenn. Biochem. Z. 320: 322-334, 1950. Wyse, D.G., Beck, L., Burks, T.F., Spalding, C.T. Further evidence for active r e f l e x vasodilatation. Can. J . Physiol. Pharmacol. 49: 147-159, 1970. 

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