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' III: II I II‘IIII I “III 2 I 'II'- II III 2I222IIII22II IIIIIIIIIIIIII IIIII. ' UBRAH MICHIGAN STATES?“ v N0 ERSITY l “'CH. “24 This is to certify that the thesis entitled EFFECT OF HEMORRHAGE, VASOACTIVE AGENTS, ASPHYXIA AND EXERCISE ON THE VASCULATURE OF THE CHICKEN. presented by James Mattes Ploucha has been accepted towards fulfillment of the requirements for Ph.D. Physiology & de ree in . g Poultry Sc1ence f.“ . ’ . KR . (jgfué a‘77fiygfl (la/L) Major professor Date AUQUSt 13, 1982 0-7 639 MSU LIBRAIUES “.- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. LIBRARY Michigan State University EFFECT OF HEHORRHAGE, VASOACTIVE AGENTS, ASPHYXIA AND EXERCISE ON THE VASCULATURE OF THE CHICKEN BY James Mattes Ploucha A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology Department of Animal Science 1982 ABSTRACT THE EFFECT OF HEMORRHAGE, VASOACTIVE AGENTS, ASPHYXIA AND EXERCISE ON THE VASCULATURE OF THE CHICKEN BY James Mattes Ploucha This study examines the vascular response of the chicken to hemorrhage, vasoactive agents, asphyxia, and lexercise. Three studies (using a total of 118 birds) were conducted in domestic chickens (Gallus domesticus). The first study (n=62) examined the effect of hemorrhage to a mean arterial blood pressure (MABP) of 50 mm Hg on various hemotological and vascular parameters. Total peripheral resistance fell slightly or was unaffected and skeletal muscle vascular resistance, judged from changes in perfusion pressure (Pp) in the constantly-perfused hindlimb, was unchanged. Plasma protein concentration was significantly reduced within 30 minutes of hemorrhage indicating that fluid mobilization was immediate and rapid. Plasma osmolality was unchanged by hemorrhage. Secondly, hemorrhage to a MABP of 25 mm Hg (n:28) produced a significant rise in Pp which was unaffected by severence of the sciatic nerve truck or bilateral cervical vagotomy. This vasoconstriction could be completely eliminated by intra-arterial infusion of phentolamine or by pump-perfusing the head with arterial blood during the hypotensive interval. Furthermore, concentrations of serotonin, dopamine, and norepinephrine in plasma were significantly elevated only when the rise in Pp was evident. The vasoconstrictor response to severe hemorrhagic hypotension in the chicken is apparently mediated primarily by an James Mattes Ploucha increase in circulating catecholamines due to cerebral ischemia, rather than a baroreflex. Finally, the change in Pp induced by either a bolus or continuous infusion of vasoactive substances into the extracorporeal perfusion circuit was monitored (n=28). Prostaglandin E (0.5 ug, bolus) produced 1 arteriolar vasodilatation lasting ten minutes, as indicated by a fall in Pp. Histamine (10 ug diphosphate, bolus) or adenosine (5 and 10 ug, bolus) produced vasodilatation of less than 2 min duration. Theophylline infusion (5 mM infused at 1 ml/min, ia) blocked the vasodilatory effect cfi‘ adenosine. Norepinephrine (1 ug, bolus) produced ‘vasoconstriction which was reduced 60% by systemic alpha-adrenergic blockade with phenoxybenzamine (7.5—10 mg/kg, iv). Tracheal occlusion produced intense vasoconstriction which was reduced 70% by alpha-adrenergic blockade. Electrical stimulation of the peripheral end of the cut sciatic nerve (6 Hz) produced an immediate vasodilatation lasting several minutes. These data indicate that the hindlimb vasculature of the chicken responds to vasoactive substances, exercise, and asphyxia in :3 manner similiar to mammals. To Lael ii ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. Robert Ringer, my major advisor, for his support and friendship. I also thank the other members of my doctoral committee; Drs. Jack Hoffert, Ching-Chung Chou, Richard Aulerich, and Steven Bursian. My' most sincere appreciation and debt of eternal gratitude are expressed to the late Dr. Jerry B. Scott, a member of my doctoral committee, whose intellect and compassion I will never forget. I also acknowledge Mr. Thomas and Mrs. Leotta H. Ploucha, my parents, and Dr. Anthony J. and Geraldine A. Miltich, my father and mother-in-law, who have influenced my life in so many ways. Most of all, I thank my wife, Lael, and children, Courtney and Tyler, for the unrelenting love and encouragement that has been my inspiration. TABLE OF LIST OF TABLES . . . . . . . . . LIST OF FIGURES . . . . . . . . LIST OF ABBREVIATIONS . . . . . INTRODUCTION . . . . . . . . . . REVIEW OF LITERATURE I. Hemorrhage in Mammals . II. Hemorrhage in Aves . . . III. Vasoactive Agents in the IV. Vascular Effects of Asphyxia in the Aves V. Vascular Effects of Exercise in the Chicken OBJECTIVES . . . . . . . . . . . MATERIALS AND METHODS Aves. CONTENTS I. Vascular Responses to a Hemorrhage to a Mean Pressure (MABP) of 50 mm Hg. A. Plasma Osmolality and Protein Concentrations Arterial Blood B. Thermodilution Studies During Selective Autonomic Blockade. C. Hindlimb Perfusion Studies 1. Acute Bleed — Phenobarbital Anesthesia . 2. Chronic Bleed - Phenobarbital Anesthesia 3. Acute Bleed - Pentobarbital Anesthesia . A. Isogravimetric Hindlimb Studies - Pentobarbital Anesthesia II. Vascular Response to a Hemorrhage to MABP of 25 mm Hg A. Hindlimb Perfusion Studies 1. Effect of Sciatic Nerve Severance and Alpha-adrenergic Blockade. . . . . . . 2. Effect of Bilateral Vagotomy . iv Page vii ix 11 1a 1a 16 17 18 19 21 21 22 22 25 26 26 3. B. Head Perfusion Studies . Effect on Concentration of Serotonin, Dopamine, and Norepinephrine in Plasma . III. Hindlimb Vascular Response to Vasoactive Agents, asphyxia A. B. C. RESULTS I. and Exercise. Log Dose-response Curves for Vasoactive Agents. Response to Bolus Administration of Vasoactive Agents: the of Adenosine with Theophylline: the Response to Blockade Asphyxia . . . . . Effect of Response Exercise . . . . . Vascular Response to Hemorrhage to MABP of 50 mm to Norepinephrine and Asphyxia; A. Plasma Osmolality and Protein Concentration . B. Thermodilution Studies During Selective Autonomic C. Hindlimb Perfusion Studies Acute Bleed - Phenobarbital Anesthesia Chronic Bleed - Phenobarbital Anesthesia . Acute Bleed - Pentobarbital Anesthesia . Isogravimetric Studies — Pentobarbital Anesthesia Vascular Response to Hemorrhage to MABP of 25 mm Hg Hindlimb Perfusion Studies Alpha-adrenergic Blockade on the Vascular the Effect of Effect of Sciatic Nerve Severance and Alpha—adrenergic Blockade . . . . Effect of Bilateral Vagotomy . Head Perfusion Studies . Effect on Concentration of Serotonin, Norepinephrine, DOpamine in Plasma Hindlimb Vascular ReSponse to Vasoactive Agents, Asphyxia and Exercise Page 27 27 28 29 3O 31 31 3A 36 37 37 NO 1:2 142 1:2 N6 A. Vasoactive Agents 1. Log Dose-response Curves for Vasoactive Agents . . 2. Response to Bolus Administration of Vasoactive Agents 3. Vasodilators . . . . . . . . . . . . . . . . . . . b. vasoconStriCtors O O O O O O O I I O O O O O O O O 3. Inhibition of Adenosine with Theophylline . . . . . A. Inhibition of Norepinephrine with Phenoxybenzamine B. AsthXia O O O O O O O O O O O O O O O O O O O O O O c. ExerCise O O O O O O O O O O O I O O O O O O O O O 0 DISCUSSION I. Hemorrhage to a MABP of 50 mm Hg . . . . . . . . . . II. Hemorrhage to a MABP of 25 mm Hg . . . . . . . . . . III. Vasoactive Agents, Asphyxia, and Exercise . . . . . SUMT4ARY O I I O O O I O O O O O O O O O O O O O O O O O O O 0 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . APPENDICES 1. List of Publications . . . . . . . . . . . . . . . . . vi A6 so 52 52 52 Su 5” 55 61 63 67 69 71 79 Table LIST OF TABLES Effect of hemorrhage on mean arterial blood pressure, hindlimb perfusion pressure, and carotid perfusion pressure in chickens following severance of the sciatic nerve, bilateral vagototomy, intra-arterial phentolamine or artificial perfusion of the head. . . . . . . . . . . Effect of exercise, prostaglandin E , histamine (diphosphate), acetylcholine, phenoxybenzamine, adenosine. theophylline, (n1 perfusion pressure in the constantly-pefused hindlimb of the chicken . . . . . Effect of alpha-adrenergic blockade on skeletal muscle vascular response to norepinephrine and asphyxia in the constantly-perfused hindlimb of the chicken . . . . . . vii Page “3 51 53 LIST OF FIGURES Figure Page 1 Schematic illustration of the isolated hindlimb perfusion technique . . . . . . . . . . . . . . . . . . . . 20 2 Schematic illustration of the isogravimetric isolated hindlimb perfusion technique . . . . . .'. . . . . . . . . 23 3 The effect of hemorrhage on plasma osmolality and total plasma protein concentration in male chickens. Phenobarbital anesthesia . . . . . . . . . . . . . . . . . 32 A The effect of hemorrhage on heart rate, cardiac index, stroke volume, and total peripheral resistance in untreated, alpha-blocked, and beta-blocked male chickens. Pentobarbital anesthesia . . . . . . . . . . . . 33 5 Tracing of the effect of hemorrhage on arterial blood pressure and perfusion pressure in the constantly-perfused hindlimb of a male chicken and the constantly-perfused breast muscle of a female mallard. Phenobarbital anesthesia . . . . . . . .‘. . . . . . . . . 35 6 Tracing of the effect of hemorrhage on arterial blood pressure and perfusion pressure in the constantly-perfused hindlimb of a male chicken. Pentobarbital anesthesia . . . . . . . . . . . . . . . . . 38 7 The effect of hemorrhage on capillary filtration coefficient in the isogravimetric isolated constantly-perfused hindlimb of male chickens. Pentobarbital anesthesia . . . . . . . . . . . . . . . . . 39 8 Tracing of mean arterial blood pressure, perfusion pressure, venous pressure, and leg weight in the isogravimetric isolated constantly-perfused hindlimb of a male chicken. Pentobarbital anesthesia . . . . . . . . . N1 9 Tracing of the effect of severance of the sciatic nerve and intra-arterial phentolamine infusion on arterial blood pressure and perfusion pressure in the constantly-perfused hindlimb of :3 female chicken during stepwise hemorrhage. Pentobarbital anesthesia .'. . A3 viii Figure Page 10 Tracing of the effect of hemorrhage on arterial blood pressure and perfusion pressure in the constantly-perfused hindlimb of a male chicken with and without artificial perfusion of the head via the carotid arteries. Pentobarbital anesthesia . . . . . . . . . . . . 45 11 The effect of a stepwise hemorrhage on the concentration of norepinephrine, serotonin, and dOpamine in plasma of male chickens. Pentobarbital anesthesia . . . . . . . . . “7 12 The effect of local intra-arterial infusion of histamine (diphosphate), acetylcholine, or norepinephrine on perfusion pressure in the constantly-perfused hindlimb of male chickens. Pentobarbital anesthesia . . . . . . . . A8 13 The effect of local intra—arterial infusion of prostaglandin E1 or adenosine on perfusion pressure in the constantly-perfused hindlimb of male chickens. Pentobarbital anesthesia . . . . . . . . . . . . . . . . . “8 ix ACh ACTH ADO ANOVA CFC CPp DA HCT HIST HPp HR hr Hz ia iv kg MABP MDF min ml msec NE Pa PBZ Pc Pc PE PGA2 PGE1 PGE2 PGI2 pH phentol Pp PPC PROP sec SEM SER TPR Vs LIST OF ABBREVIATIONS Acetylcholine chloride Adrenocorticotrophic hormone Adenosine Analysis-of-variance Capillary filtration coefficient in ml/min/mm Hg/1OO gm Carotid perfusion pressure in mm Hg Dopamine Hematocrit Histamine diphosphate Hindlimb perfusion pressure in mm Hg Heart rate in beats/min Hour Hertz in cycles/sec Intra-arterial Intravenous Kilogram Mean arterial blood pressure in mm Hg Myocardial depressant factor Minute Milliliter Millisecond Norepinephrine Arterial blood pressure in mm Hg Phenoxybenzamine Capillary hydrostatic pressure in mm Hg Isogravimetric capillary pressure in mm Hg Polyethylene Thromboxane Prostaglandin E1 Prostaglandin E2 Prostacyclin Inverse log of hydrogen ion concentration Phentolamine Oxygen partial pressure in mm Hg Hindlimb perfusion pressure in mm Hg Plasma protein concentration in gm% Propranolol Second Standard error of the mean Serotonin Total peripheral resistance in p.r.u. Volt Ventricular stroke volume in ml/beat INTRODUCTION The term "shock" describes a condition in which the circulatory system fails to fulfill its basic function, i.e. to provide the various organs of the body with sufficient blood flow to meet their metabolic demands. This definition implies that shock may occur as a result of either an inappropriate cardiac output and/or peripheral resistance. The latter may be due to widespread peripheral vasodilatation (anaphylactic shock) or vasoconstriction (catecholamine shock) and the former may result from blood loss (hemorrhagic shock), pooling of the blood within the vasculature (septic shock) or from a ldefective myocardium (cardiogenic shock). Regardless of the etiology, if there is prolonged inadequate perfusion of the major organs of the body (brain, heart, splancnic bed and kidney) irreversible cellular damage occurs which eventually leads to the demise of the organism (irreversible shock). The domestic chicken (Gallus domesticus), although intolerant to a: large acute blood loss, can withstand a large slow removal of blood, and reportedly does not exhibit irreversible hemorrhagic shock. The ability of the chicken to tolerate a large slow bleed appears to be related to its ability to rapidly mobilize large volumes of extravascular fluids, whereas its inability to withstand a rapid large blood loss suggests a relatively inefficient sympathico-adrenal system. The latter might help to explain why the chicken does not enter into irreversible hemorrhagic shock, i.e. it. is spared the deleterious effects of prolonged vasoconstriction. Furthermore, most of the drugs and hormones that alter arterial blood pressure in mammals have the same effect in avian species. Previous studies :hi chickens measured heart rate, arterial blood pressure, and cardiac output during drug administration and made assumptions concerning the peripheral vasculature. Likewise, the direct effect of exercise or asphyxia on the hindlimb vasculature has not been reported in the chicken. This study is an attempt to elucidate some of the peculiarities of the response of the chicken to hemorrhage using research techniques and protocols which have been employed for years in mammalian research, but which have not been applied to fowl. These techniques are also utilized to determine the effect of asphyxia, exercise, and vasoactive agents on hindlimb vascular resistance in chickens. LITERATURE REVIEW I. Hemorrhage in Mammals Hypovolemic hypotension in mammals sustained beyond a given duration of time results in myocardial and/or peripheral circulatory failure despite reinfusion of all shed, and even additional blood. Investigations involving the duration of time until the onset of irreversibility are of clinical importance. Certain sympathomimetic agents coupled with volume repletion and correction of the acid/base inbalance are the standard clinical treatment for hemorrhage (Carey, Lowery, and Cloutier, 1971). It is generally understood that primates withstand shock better, vasoconstrict less, and survive longer than dogs (Abel gt _a_l., 1967). Yet, primate tolerance to hemorrhage apparently does not compare to that of avian species where irreversible hemorrhagic shock may not occur. Furthermore, the rate of post-hemorrhagic fluid mobilization in avian species is much greater than that of mammals. Shock is a condition where the circulatory system fails to provide the various organs of the body with sufficient blood flow to meet their metabolic demands. The cardiovascular response of mammals to hemorrhagic hypotension sustained by continuous bleeding is marked by two distinct phases; vascular compensation and vascular decompensation. The initial phase, i.e. compensated shock, involves activation of cardiovascular mechanisms which maintain the mean arterial blood pressure (MABP) in an attempt to maintain blood flow to high priority tissues like the heart and brain. The most prominent and immediate compensatory response is reflex activation cfi‘ the autonomic nervous system (Chien, 1967; Djojosugito, Folkow and Kovach, 1969; Jacobson, 1968; Scott and Eyster, 1979; Shoemaker, 196A). It is this system which elevates vascular resistance, produces positive chronotropic and inotropic cardiac effects, and indirectly promotes the absorption of interstitial fluids (Djojosugito gt al., 1969: Haddy, Scott and Molnar, 1965; Jacobson, 1968; Scott and Eyster, 1979; Shoemaker, 196A; Zweifach, 1974). During compensated hemorrhagic shock small volumes of blood must be continuously removed from the animal to prevent the MABP from rising above a given level of experimental hypotension. The importance of the autonomic system in hemorrhage is attested to by the fact that the ability of an organism to withstand a large acute blood loss is greatly dependent on the effiency of the sympathicoadrenal system (Chien, 1967; Shoemaker, 19614). However, prolonged activation of the latter system has been suggested as one factor that contributes to the phenomenon of "irreversible shock" (Irving, 1968; Zweifach and Fronek, 1975). The compensatory mechanisms begin to fail following several hours of prolonged tissue hypoperfusion due to various metabolic, central nervous, cardiac, and microvascular alterations, and the animal is said to enter "decompensated shock". The MABP begins to wain due to myocardial depression and/or failure of the peripheral vasculature, and shed blood must be returned to the animal to maintain the MABP at a given level of experimental hypotension. The animal is considered to have entered a terminal condition termed "irreversible hemorrhagic shock" when approximately' one—third of the shed volume> has been returned to the animal. The return of all the shed blood, and even additional blood, at this time has only a transient and ever diminishing effect on the MABP. The MABP continues to fall until the animal lapses into peripheral circulatory collapse and death. There are several reports supporting myocardial depression as the cause of decompensated shock. The prolonged intense vasoconstriction produces tissue ischemia and an acidosis which can depress the myocardial response to catecholamines (Darby gt_§l,, 1960). The myocardium can also be depressed by the hyperkalemia which results from cellular exchange of potassium ions for hydrogen ions, from inactivation of the electrogenic pump, by the increase in the plasma concentration of circulating gastrointestinal toxins due to the breakdown of the reticulo-endothelial system (Rothe and Selkurt, 1961), and by the release of a myocardial depressant factor (MDF) from the ischemic pancreas (Lefer and Martin, 1970). This combination of effects may produce the myocardial depression indicated by the rightward shift in the ventricular function curve in late shock (Crowell and Guyton, 1962). Other researchers have stated, conversely, that myocardial contractility is enhanced in late shock (Downing, Talner, and Gardner, 1965). Other researchers suggest failure of the peripheral vasculature is the major cause of decompensated shock. When the MABP of the dog is maintained at 50 mm Hg, total peripheral resistance (TPR) (Rothe and Selkurt, 196”) or perfusion pressure in the constantly-perfused hindlimb (Bond gt 31,, 1981) rises initially, then falls significantly within 2 hr. Inadequate cerebral perfusion can depress medullary vasomotor centers resulting in decreased sympathetic vascular tone, which again decreases venous return and cardiac output. The microcirculation may become damaged by .the accumulation of vasoactive substances such as histamine (Galvin, Bunce and Reichard, 1977: Grega, Kinnard and Buckley, 1967), prostaglandins (Bond gt 31., 1981), bradykinin, and others. The blood tends to become hyperosmotic (Jarhult. 1975) and this can depress vascular smooth muscle (Hollenberg and Nickerson, 1970). Cellular swelling and deformaties, and hemoconcentration may reduce the flow velocity resulting in intravascular thrombi causing the blood to demonstrate hypercoagulability (Hardaway st 21., 1962; Shoemaker gt al., 1961). Furthermore, terminal arterioles and precapillary sphincters may lose reactivity to constrictor stimuli (Rothe and Selkurt, 1961). The increase in precapillary resistance immediately following blood loss is important, not only because it maintains the MABP, but also because it tends to shift the Starling equilibrium toward fluid absorption. This raises blood volume, and ultimately, the cardiac output. Interestingly, the plasma oncotic pressure of the chicken is only 11 mm Hg due to a high albumin/globulin ratio. Fluid mobilization after hemorrhage, primarily from the skeletal muscle interstitium, is thought to be brought about in most species by an increase in the pre/post—capillary resistance ratio which effectively lowers the capillary hydrostatic pressure (Pc) (Djojosugito £2.2l-v 1969; Hollenberg and Nickerson, 1970). This is attested to by the fact that the administration of an alpha-adrenergic blocking agent, which prevents the vasoconstriction, results in a reduced rate of post-hemorrhagic fluid mobilization (Grega ‘gt_.al., 1967; Hollenberg ‘gt_.al.. 1970). Fluid mobilization is enhanced by the action of catecholamines on the liver and a decrease in pancreatic insulin secretion, both of which tend to cause an increase in the osmolarity of the plasma which, in turn, favors fluid absorption (Hinshaw, 1976; Strawitz EE,El" 196]; Jarhult, 1975). The reabsorptive process is self-limiting and forces favoring net reabsorption progressively diminish with prolonged hemorrhagic hypotension. The initial stage of fluid mobilization lasts several hours and is capable of replacing about one-half of the shed blood. The subsequent stage of fluid mobilization is slow, requiring 6 to 2A hr for complete blood volume restitution. Renal mechanisms are partially responsible via the renin-angiotensin-aldosterone mechanism. Interestingly, in the chicken angiotensin II produces hypotension followed by hypertension. The initial transient hypotension is thought to be due to vasopressin release, a vasodilator in birds (Moore, Strong, and Buckely, 1981a,b). In mammals, a cortisol mediated mechanism may also be necessary for complete blood volume restitution (Pirkle and Gann, 1975: Swingle and Swingle, 1965). Cortisol facilitates the transfer of cellular water to the interstitium and thereby promotes capillary absorption. :n: has been postulated that cortisol causes this cellular water loss by stimulating active transport of certain electrolytes, presumably sodium, from the cell. II. Hemorrhage in Aves The avian response to hemorrhage differs considerably from the mammalian response in three ways. First, avian species do not demonstrate a phase of vascular decompensation after prolonged hemmorhagic hypotension. Second, avian species have a greater rate of posthemorrhagic fluid mobilization than mammals. Finally, chickens apparently do not demonstrate a phase of shock irreversible to transfusion. Articles published concerning the avian response to hemorrhage are reviewed in the subsequent paragraphs. The first four papers which examined the avian response to hemorrhage were published by the Scandanavian group of physiologists at the University of Goteborg, Sweden, in the late 1960's. The first publication, which examined the vascular and hematologic response of the pigeon to hemorrhage (Kovach and Szasz, 1968), concluded that a large hemorrhage produces only a small fall in MABP and there is immediate, intense, and continuous hemodilution following hemorrhage with no terminal trend towards hemoconcentration. The next publication (Kovach, Szasz and Pilmayer, 1969) examined the effect of graded hemorrhages, i.e. 11 of body weight blood removed/hr, on the mortality of various avian and mamalian species. The study found that flying (pigeons) and diving (ducks) birds survived much longer than mammals. In fact, 30% of the pigeons could survive the loss of 100% of its initial blood volume if bled over a 8 hr duration. Chickens were intermediate in their survival rates during graded blood loss. The third paper (Kovach and Balint, 1969) compared the vascular and hemotologic response of the rat and pigeon to hemorrhage. Rapid hemodilution occurred in the pigeon following hemorrhage (1181 fall in hematocrit (HCT), 7A1 fall in plasma protein concentration (PPC)) compared to the rat (18% fall in HCT, 12% fall in PPC). When NOS of the blood volume was removed in two successive 10 min hemorrhages, separated by 30 min, the MABP of the pigeon fell by only 30 mm Hg. The pigeon demonstrated a distinct pressor response due to cerebral ischemia when both carotid arteries were ligated and the MABP was reduced to 30 mm Hg by hemorrhage. A significant hyperkalemia occurred in both species. Whereas the hemodilution ceased rapidly in the rat (within 30 min), the process continued many hr in the pigeon. The final paper examined the effect of hemorrhage on MABP, cardiac output, and capillary filtration coefficient (CFC) 1J1 the. duck (Djojosugito st 31.. 1969). Mean arterial blood pressure was unchanged by a hemorrhage of 25% of the blood volume. As in the pigeon, this maintainance of the MABP was due to a reflex increase in vascular resistance, i.e. TPR was elevated. It is well documented that the duck, being a natural diver, is capable of demonstrating intense peripheral vasoconstriction (particularly in the skeletal muscle vasculature). The rise in vascular resistance in the duck following hemorrhage was, at least in part, due to sympathetic nerves inasmuch as sciatic nerve block with lidocaine, or treatment with an alpha-adrenergic antagonist, would reduce the rate of fluid mobilization. The nerve block prevented the hemorrhage—induced rise in CFC, i.e. 0.05 to 0.13 ml/min/mm Hg/100 gm. Knowing the isovolumetric capillary pressure, the investigators calculated that this rise in the pre/post—capillary resistance ratio lowered Pc by 11 mm Hg and the nerve block reduced the fall in Pc by 70-75%. The remaining fluid absorption was due simply to a lowering of the MABP which was transmitted to the capillaries. The vascular resistance remained elevated even though the limb became isovolumetric, i.e. it stopped filtering. Djojosugito gt El° (1969) offered two reasons for the rapid rate of post hemorrhagic fluid mobilization in the duck. First, the intense reflex increase in precapillary resistance lowered the mean Pc sufficiently to shift the Starling equilibrium toward fluid absorption. Secondly, the capillary surface area that is available for fluid absorption is reportedly thrice (Folkow fl a_l_., 1966) that of the cat ,(Kjellmer, 1965) and, hence, identical changes in Pc would result in correspondingly greater changes in the rate of blood volume restitution. Folkow _e_t a_l_. (1966) did not state how capillary surface area was extracted from the CFC. Up to this point in time, most avian hemorrhagic 10 research had used flying and diving species and little had been learned about the responses of the chicken. Wyse and Nickerson (1971) examined the effect of prolonged hemorrhagic hypotension on chickens using the standardized hemorrhagic protocol of holding the MABP at 50 mm Hg by continuous small hemorrhages. Plasma volume (via radio-iodinated serum albumin), PPC, HCT, and hemoglobin were determined and vital signs were monitored during a 5 hr hypotensive interval. The results were similiar in some respects to those of flying or diving birds and were considerably different in other respects. The chicken, like the pigeon and duck, demonstrated a high rate of fluid mobilization, i.e. twice that reported for mammals (Hollenberg and Nickerson, 1970). The chickens did not demonstrate a phase of shock irreversible to transfusion following reinfusion of the shed blood after 14 - 5 hr of hypotension. The chickens would survive after being reinfused at the onset of circulatory collapse, as indicated by a sudden fall in the MABP. Unlike the duck or pigeon, the chicken was very sensitive to small hemorrhages. A A ml/kg blood loss produced a 20 mm Hg fall in MABP, and only a 25% reduction in blood volume was required to drop the MABP to 50 mm Hg. This may suggest a reduced capacity for sympathetic activation in this species. Folkow .22..§l' (1966) has demonstrated more dense adrenergic innervation in the adventitia of the duck femoral arterial vasculature than that occurring in the turkey or cat. Recent research has also indicated that reactivity of mesenteric and skeletal muscle vasculature of ducklings to exogenous norepinephrine or electrical stimulation is considerably greater than in chicks (Gooden, 1978). Recent research has indicated that following hemorrhage in the hen 11 the intraerythrocytic concentration of 1,3,”,5,6 myoinsitol pentophosphate concentratioon increased, shifting the oxygen dissociation curve far right and enhanced oxygen delivery to the tissues (Jones, Smith and Board, 1978). This is different from mammals, where the main controller of oxygen transport is intracellular concentrations of 2,3 diphosphoglycerate. These researchers also found the rate of erythrogenesis in the hen was comparable to that of the dog. Further research concerning the response of the chicken to systemic hypotension was given by Ploucha (1979) and Ploucha, Scott and Ringer (1981). In these studies, chickens were held at a MABP of 50 mm Hg for 225 min by continuous small bleedings while various hemodynamic and hematological parameters were measured. .A hemorrhage of approximately 25% of the initial blood volume would reduce the MABP to 50 mm Hg (similiar to Wyse and Nickerson, 1971), and this was followed by intense, immediate, and continuing hemodilution with no terminal trend toward hemoconcentration. A progressive hyperkalemia and hyperglycemia occurred. The chickens were not acidotic after A hr of sustained hemorrhagic hypotension. Interestingly, cardiac output (measured by dye dilution) indicated that TPR fell with hemorrhage. It is possible that the chicken is sensitive to a small blood loss due to a lack of peripheral baroreceptor activity. Although avian researchers have recorded afferent. impulse traffic along the cardiac depressor nerve of ducks (Jones and West, 1978) and chickens (Estravillo and Burger, 197Aa,b) which correspond with arterial systole, little physiological evidence exists demonstrating the existance of functional peripheral-vascular baroreceptors in the chicken. Harvey 32 El' (195“) reported that during the course of pharmacological studies in the chicken 12 11 birds were used before one would demonstrate a rise in MABP greater than 10 mm Hg in response to bilateral carotid occlusion in the cervical area, a response readily seen in mammalian studies. Durfee (196A) also found no pressure reflexogenic areas in association with the carotid arteries in the chicken. McGinnis (196A) and McGinnis and Ringer (1965, 1967) found that bilateral occlusion of the carotid and vertebral arteries of the chicken did not produoe a baroreceptor-induced rise in MABP. The chicken did demonstrate a general cerebral ischemic response when cerebral perfusion pressure, measured through a carotid cannula introduced in a cranial direction, fell to about 26 mm Hg following bilateral carotid and vertebral artery occlusion (McGinnis, 196A). Two recent morphological reports have been published dealing with birds which site the existence of possible cardiac stretch receptors in the conducting system of the avian heart (Bogusch, 197Aa,b) and possible baroreceptor—like endings exist in the subendocardium of the pigeon (Mather and Mather, 1979). Other researchers have suggested that circulating adrenal-medullary hormones, rather than sympathetic vascular innervation, play a major role in regulating cardiac performance and blood pressure in the chicken (Karg and Scrams, 1966; DeSantis gt 3&3, 1975). The simple stress of being hand-held will double plasma corticosterone concentration of a chicken, presumably due to the release of adrenocorticotrophic hormone (ACTH) (Beuving and Vonder, 1978). Interestingly, in chickens ACTH stimulates not only adrenal cortical tissue, but adrenalmudullary tissue as well (Newcomer, Gephardt, and Hurst, 1972). 13 III. Vasoactive Agents in Aves Most of the drugs and hormones that alter blood pressure in mammals are reported to have the same effect in avian species. The effect of various sympathomimetics (Akers and Peiss, 1963; Szeto e_t 31., 1977), parasympathomimetics (Peterson and Ringer, 1968; Rodbard and Fink, 19N8). histamine (Natoff and Lockett, 1957: E1 Ackad, 1972: Knight and McGreggor, 19711), angiotensin (Moore, Strong and Buckley, 1981a,b) and prostaglandins (Horton, 1971; Bult 32 31., 1981) have been examined in chickens. The direct action of the drugs in these studies is difficult to ascertain since they all employed intravenous drug administration. Recent research has indicated that PGE1 and PGE strongly inhibit 2 thrombocyte aggregation in whole chicken blood. Prostacyclin (PGIZ) does not exhibit anti-aggregratory activity in chicken blood, nor is it produced by aortic tissue (Claeys gt 31.. 1981a: Bult 3t a_l_., 1981). Furthermore, the metabolism of arachadonic acid in chicken aortic tissue is geared mainly toward the formation of PCB and, in contrast to 2 mammals, virtually no prostacyclin synthetase is present. However, the capacity of chicken thrombocytes to generate thromboxane (PGA2) is similiar to that observed for mammalian platelets (Claeys 3£_31,, 1981b). In birds, it appears that PGE not PGI is the antithrombotic factor. 2' 2' Since E type prostaglandins are formed in the chicken vascular tissue, a possible role of PGE1 or PGE2 in avian hemostasis and/or in the developement of spontaneous avian atherosclerotic vascular lesions deserves further investigation. Knight and McGreggor (197A) and McGreggor (1979) pump perfused the amputated feet of chickens and ducks with a Krebs solution and monitored perfusion pressure upon intra-arterial (ia) administration of vasoactive 1” drugs and exercise. They reported that there is a noncholinergic nonadrenergic vasodilator released in the feet of the bird. They were perfusing amputated feet, which consist mostly of bone, skin, and connective tissue, with very little skeletal muscle. IV. Vascular Effect of Asphyxia in Aves The mammalian response to asphyxia is intense vasoconstriction (Weissman, Sonnenschein and Rubinstein, 1978). Asphyxia in the chicken, induced by tracheal occlusion, produces hypertension subsequent to a transient period of hypotension (Harvey 31U31., 195A; Richards and Sykes, 1967). The initial hypotension is likely due to the local effect of systemic hypoxia, since hypoxia has been shown to produce a 35% fall in TPR and systemic hypotension in chickens (Besche and Kadono, 1978). Asphyxia, induced by’ submersion, is poorly tolerated by the chicken compared to a natural avian diver like the «duck (Bond, Douglas and Gilbert, 1961). Furthermore, the chicken myocardium is depressed more by hypoxia than the duck heart (Sturkie and Abati, 1978) and levels of reduced nicotinamide-adenine dinucleotide accumulate much more quickly in the chicken brain during asphyxia than in the duck (Jones and West, 1978). V. Vascular Effect of Exercise in the Chicken Skeletal muscle activity produces a local vasodilatation, i.e. active hyperemia, in mammalian skeletal muscle vasculature. Perfusion pressure in the constantly-perfused canine hindlimb will fall with the onset of muscular activity and will remain lowered for several min following cessation of muscular activity (Tabaie, Scott and Haddy, 1977). 15 This is a local phenomenon which may be mediated by the release of local vasodilator metabolites, including potassium :hma, hydrogen ion, adenosine-diphosphate, adenosine-triphosphate, blood gas tension, adenosine, and/or others (Haddy and Scott, 1968). This local vasodilatation in mammals will overide a neurogenic (remote) vasoconstriction induced by hemorrhage (Kjellmer, 1965). Active hyperemia in the duck will not "break through" an intense hemorrhage-induced neurogenic vasoconstriction (Folkow 33 fl” 1966). The effect of exercise on blood flow through the skeletal muscle vasculature of the chicken has not been investigated. However, ischemic (reactive) hyperemia has been demonstrated in the skeletelal muscle vasculature of the chicken (Klabunde and Johnson, 1977) and an active hyperemia occurs in the duck, turkey (Folkow 31‘31., 1966), and amputated Krebs-perfused chicken foot (McGreggor, 1979). OBJECTIVES 1. To determine the effect of various degrees of hemorrhagic hypotension on hematologic and hemodynamic parameters in the chicken, particularly in regard to activation of the sympathico-adrenal axis. 2. To determine the response of the skeletal muscle vasculature to vasoactive agents, asphyxia, and exercise. 16 MATERIALS AND METHODS I. VASCULAR RESPONSE TO HEMORRHAGE TO HEAR ARTERIAL BLOOD PRESSURE OF 50 mm Hg Unless otherwise stated, the MABP was measured in all chickens (1.5 - 3.9 kg) used in this study via a cannula inserted into a brachial or carotid artery connected to a Statham (PA-23AC) pressure transducer and either a Grass (7A) or Hewlett-Packard (956-100W) polygraph. Various breeds were used. All animals were tracheotomized and heparinized systemically (390 IU/kg, iv) and, unless otherwise indicated, were anesthetized with sodium pentobarbital (25 mg/kg, iv). The animals were artificially ventilated at a tidal volume of 35 cc and a rate of 25 strokes per min (Harvard small animal respirator). Unless otherwise indicated, all statistical analysis was via a one-way analysis-of-variance (ANOVA) with a Dunnett test, a P<0.05 was considered significant. All values are given as mean :SEM. A. Plasma Osmolality and Protein Concentrations This experiment investigated the effect of hemorrhage on plasma osmolality and total protein in 12 phenobarbital anesthetized male birds (1.91 30.08 kg) bled via a cannula (PE-90) in the right ischiadic artery. The MABP was held at 50 mm Hg for 150 min by continuous small bleedings, after this time the shed blood (A1OC) was returned to the animal via an ischiadic vein. The birds were monitored for 1 hr after reinfusion. Plasma osmolality (Advanced instruments) and plasma protein concentration (PPC) (Acustat, Clay Adams) were measured initially, every 30 min after hemorrhage, and then 30 min after reinfusion of shed blood. 17 18 B. Thermodilution Studies During Selective Autonomic Blockade This experiment used birds anesthetized with sodium pentobarbital (25 mg/kg, iv) and cardiac output was determined by the thermodilution technique during hemorrhagic hypotension. This cardiac output was determined by advancing a pediatric (11 French) Swan-Ganz catheter down the right jugular vein such that the thermistor was either in or near the pulmonary outflow tract. The position of the catheter was determined at autopsy. A 1 ma bolus of cold saline (0.1OC) was rapidly injected into the vena cava through the catheter side port. The resultant temperature deflection curve (sensed by a catheter-tip thermistor) was indicated on an analog meter and integrated by a Cardiotherm—SOO computer (Columbus Instruments) giving a digital display of cardiac output. A minimum of four cardiac output determinations were obtained at each sampling time. Cardiac output was converted to cardiac index by dividing cardiac output by the body weight in kg raised to the 0.734 power (Speckmann and Ringer, 1963). Twenty-six (3.22 :0.06) male chickens were divided into three unequal groups. Eight were untreated, eight received the alpha-adrenergic antagonist, phenoxybenzamine (PBZ, 5 mg/kg, iv), and ten received the beta-adrenergic antagonist, propranolol (PROP, 0.25 mg/kg iv bolus followed by 5 ug/kg/min infusion into the brachial vein). The cardiac output was determined initially and the blocking agents were then administered. Cardiac output was determined 30 min later. The animal was then immediately hemorrhaged to a MABP of 50 mm Hg and was held at that level of hypotension by continuous small bleedings. Cardiac output was then determined at 5, 30, 60, 90, and 120 min after the onset of hemorrhage. 19 C. Hindlimb Perfusion Studies The blood supply to the gastrocnemius muscle was isolated in male chickens anesthetized with sodium phenobarbital or sodium pentobarbital and the muscle was pump perfused (Masterflex pump) with arterial blood at a constant flow (Figure 1). Since flow was held constant, the perfusion pressure will vary' directly with vascular resistance. Hence, these experiments permitted the direct determination of the response of the skeletal muscle vasculature. Vascular isolation of this muscle was accomplished by ligating the external iliac artery and placing a tourniquet above the tibial-metatarsus junction. The muscle was then pump perfused via the ischiadic artery. The tourniquet prevented blood from flowing to the lower leg and toes through the anterior tibialis artery, thus shunting the perfusing blood to the femoral caudalis artery and medial and posterior tibialis arteries. The medial tibialis artery supplies the gastrocnemius muscle and skin (Koch, 1973: Nishida, 1963; ‘Westpfahl, 1961). The posterior tibialis artery supplies the gastrocnemius muscle and also sends poorly developed branches to the flexor perforans muscles and skin. The caudal femoralis artery supplies blood primarily to skin. Thus, this is not an entirely isolated muscle preparation but the amount of skin perfused relative to muscle is small. To perfuse the muscle, a polyethylene cannula (PE-160) was inserted midthigh in a cranial direction several centimeters into the left ischiadic artery. Blood was then shunted to the perfusion pump and returned to the same artery through a cannula (PE-160) inserted caudally. The perfusion pressure (Pp) was measured in the pump outflow line with a Statham pressure transducer (PA-23AC). Blood flow was adjusted to 20 (9 ll]: ARTERIAL WE 'ACMIAI. A. 1 mac A. nuusoow caisson (\J \ y —. \ . scum A. ,a‘ ‘i\ 1” ‘ as GRASS STIMAAATOI SCIAIIC N. GASTIOCNIMIUS M. A Figure 1. Diagramatic illustration of the isolated hindlimb perfusion technique. 21 produce a control Pp approximately equal to systemic pressure and then flow was held constant throughout the experiment. The pressure waveform generated by the perfusion pump had a pulse pressure similiar to that of the bird, while the pump rate was somewhat less than the ‘bird (see Figures 5.6.9). Prior to hemorrhage, steps were taken to assure that the perfused muscle was a valid assay organ. First, the perfusion pump was turned off and a Pp less than 20 mm Hg indicated adequate vascular isolation. A Pp greater that this with the pump off would indicate collateral circulation to the leg. Next, 1.0 ug acetylcholine (ACh) and then 1.0 ug norepinephrine (NE) were injected into the extracorporal perfusion line prior to the pump to determine if the vascular bed would respond. Statistical analysis in study IC was via a Students t test, and 3 P<0.05 was considered significant. 1. Acute Bleed - Phenobarbital Anesthesia Six male chickens (2.50 :0.17 kg) were anesthetized with phenobarbital and blood was removed rapidly (2 thin) from the right ischiadic artery until the peak systolic arterial pressure fell to approximately 50 mm Hg. The Pp was monitored during a 15 min hypovolemic interval after which the shed blood was reinfused and the bird monitored for 30 min. The experiment was then repeated yielding two sets of data for each animal. Flow averaged 8.3 :0.9 m1/min/100 gm leg weight. 2. Chronic Bleed - Phenobarbital Anesthesia Nine male chickens (2-80 :0.12 kg) were anesthetized with phenobarbital (100 mg/kg). bled at a rate of 2 ml/kg/min to a MABP of 50 22 mm Hg, and maintained at that level of hypotension for 60 min by subequent bleeding. The hindlimb Pp was continuously monitored. 3. Acute Bleed - Pentobarbital Anesthesia Nine male chickens (2.80 :0.1ll kg) were anesthetized with sodium pentobarbital (25 mg/kg) and then blood was removed rapidly (2 min) from the right ischiadic artery until systolic pressure fell to approximately 50 mm Hg. Hindlimb Pp was monitored before and during a 10 to 15 min hypovolemic interval after which the shed blood ‘was returned to the animal. This series of experiments was designed to determine if phenobarbital influenced the responses seen under phenobarbital anesthesia. Pentobarbital was selected because perfusion studies have demonstrated that this agent does not preclude vascular constriction in the dog skeletal muscle during hemorrhage (Haddy.31'31., 1965). u. Isogravimetric Hindlimb Studies - Pentobarbital Anesthesia The effect of hemorrhagic hypotension on capillary filtration coefficient (CFC) was determined in three experiments using 18 Inale chickens (2.65 :0.05 kg). A schematic illustration of the experimental preparation is shown in Figure 2. The birds were anesthetized with pentobarbital (251 mg/kg), tracheotomized, and artificially ventilated (Harvard small animal respirator). Body temperature was maintained at ”1°C with a heating pad placed under the animal. Arterial blood pressure was monitored via a cannula inserted into a carotid artery attached to a Statham transducer (PA-23AC) and a Grass 7A polygraph. A cannula (PE-160) was inserted into the left ischiadic artery in a cranial direction for hemorrhaging the animal into an elevated and pressurized 23 was"! rams ow/orr FLUID 0: VOL. new. PUMP § ‘ W / ‘0' VALVE fl \ / cm" wnmurwn" ""090! \ nuwu PUMP . (D Figure 2. Diagramatic illustration of the isogravimetric isolated hindlimb perfusion technique . 21: glass reservior. Skin was removed from the leg below the thigh by electrocautery. Three heavy string tourniquets were applied to the mid-thigh musculature and the muscle was then severed by electrocautery proximal to the tourniquets. The first tourniquet bound the gracillis, adductor, and quadricepts femoris muscles. The second tourniquet bound the semimembranosus and semitendonosis muscles. The third tourniquet bound the sartorium with glutaeus superficial muscle, and the tensor fasciae latae with the glutaeus superficial and bicepts femoris muscles. The animals were then heparinized systemically (390 IU/kg). The limb was pump perfused (Masterflex pump) at constant flow via the ischiadic artery with autologous blood drawn from the same artery. The limb venous outflow was directed via a cannula (PE-2110) through a 1/11 inch needle valve to a reservoir (A0 ml beaker) in a ”1°C water bath. Venous pressure was measured in this cannula immediately in front of the needle valve via a Statham transducer (PA-23BC). The reservoir volume was held constant at approximately 20 ml by a fluid volume regulator which activated the venous return pump (Holter roller pump). Blood in the reservoir was then returned to the animal through a long cannula which was coiled in the water bath before entering the ischiadic vein. The femur was severed mid-thigh and sealed with bone wax, the limb was placed. on a ‘wire mesh, and suspended from a sensitive strain gauge (Unimeasure/BO force displacement transducer). The sensitivity of the strain gauge was adjusted so that a 2 gm weight on the grid would produce a 20 mm pen deflection on the recording paper. Capillary filtration coefficient was calculated as filtration rate, i.e. the rate of gain in leg weight with a given rise in venous pressure 25 in the leg (in gm/min/100 gm), divided by the change in venous pressure (in mm Hg). Isogravimetric capillary pressure (Pei) was estimated by the stop flow technique described by Johnson (1965). This technique involves shutting off the perfusion pump, clamping venous outflow, and then elevating venous outflow pressure (by raising and lowering a static fluid column connected to the venous outflow cannula) such that the limb remains isogravimetric. The venous pressure and Pp would then equilibrate at the Pci. Eighteen animals were divided into three equal groups. In all three groups, CFC was determined at 5, 15, 30, A0, 50, and 60 min. The Pc was 1 determined immediately after the 5, 30, and 50 min CFC determinations in all groups. Group 1 was a control (non-hemorrhaged, nerve cut) group to determine the effect of time on CFC and Pci. The animals in groups 2 and 3 were hemorrhaged to a MABP of 50 mmHg immediately after the 15 min CFC determination and were maintained at that level of hypotension by continuous small hemorrhages from the contralateral ischiadic artery. The sciatic nerve trunk was cut initially in groups 1 and 2, whereas the nerve remained intact in group 3 by suspending the limb in close juxtaposition to the body of the animal. II. VASCULAR RESPONSE TO HEMORRHAGE TO A MEAN ARTERIAL BLOOD PRESSURE 0F 25 mm Hg A. Hindlimb Perfusion Studies Four experimental series were performed using 26 chickens of both sexes. The chickens were anesthetized with sodium pentobarbital (25 mg/kg, iv), tracheotomized, artificially ventilated, and heparinized systemically (390 IU/kg). Arterial blood pressure was monitored from a 26 cannula inserted into a carotid or brachial artery. Body temperature was maintained at “1°C by a heating pad under the animal. The right ischiadic artery was cannulated for hemorrhaging and reinfusing the animals. In some animals, the sciatic nerve of the perfused limb or the vagi were isolated and looped with suture for subsequent sectioning. In study IIA, the blood supply to the left leg was isolated, and the limb was pump perfused as described previously. All statistical analysis in study IIA was by a Students t test, and a P<0.05 was considered significant. 1. Effect of Sciatic Nerve Severance and Alpha-Adrenergic Blockade Six female chickens (1.84 :0.07 kg) were hemorrhaged to a MABP of 50 mm Hg and the Pp was recorded. The MABP was held at 50 mm Hg by continuous small bleedings. When all values had stabilized the animal was further hemorrhaged to a MABP of 25 mm Hg and was maintained at that level of hypotension by subsequent small bleedings and the Pp was again recorded. When the Pp stabilized the sciatic nerve trunk was severed and the Pp was monitored. Next, the alpha-adrenergic antagonist phentolamine was infused into the perfusion line prior to the perfusion pump (50 ug/min). The Pp was monitored until it stabilized at which time the phentolamine infusion was discontinued and the shed blood was returned to the animal. 2. Effect of Bilateral Vagotomy Seven female chickens (1-82 10.07 kg) were hemorrhaged to a MABP of 50 mm Hg and then were further hemorrhaged to a MABP of 25 mm Hg while Pp was monitored. The shed blood was returned, a bilateral cervical 27 vagotomy was performed and the animals were again hemorrhaged to a MABP of 25 mm Hg while the Pp was recorded. 3. Head Perfusion Studies Eight male chickens (2.33 30.06 kg) were hemorrhaged and the hindlimb Pp was monitored as in the previous studies. However, in this study, the head of the bird was also pump (Sigma motor pump) perfused. This was done by pumping blood from the contralateral ischiadic artery through a bifurcated cannula (PE-90) inserted in a cranial direction into both carotid arteries. The carotid perfusion pressure was monitored in the pump outflow line. Mean arterial blood pressure was monitored from a brachial artery and the animal was hemorrhaged from a cannula inserted caudally into a carotid artery. The shed blood was reinfused through a cannula inserted caudally into an ischiadic artery. The carotid perfusion pump was turned off and the animal was hemorrhaged to a MABP of about 35 mm Hg and was held at that pressure several minutes by continuous small bleedings. The shed blood was then reinfused, the carotid perfusion pump was turned back on, and the animal was allowed to stabilize. The animal was then again hemorrhaged to the same MABP as in the initial hemorrhage while the carotid perfusion pump continued to perfuse the head. The carotid perfusion pressure and hindlimb Pp were monitored during both hemorrhages. B. Effect on Concentration of Serotonin, Dopamine, and Norepinephrine in Plasma Five male chickens (1.96 :0.1A kg) were held at a MABP of 50 mm Hg for 30 min and then at a MABP of 25 mm Hg for 30 min by continuous small 28 bleedings. Arterial blood samples (1 ml) were drawn prior to hemorrhage, at 5, 15, and 30 min of each level of hypotension, and at 10 inin following reinfusion of the bled volume. The blood was centrifuged and the plasma was frozen for subsequent analysis for serotonin, dopamine, and norepinephrine concentrations by a modification of the method of Jacobowitz and Richardson (1978). Instead of brain tissue and an amount of 0.01N HCl which is dependent on tissue weight being added to 5 ml of butanol in the first step of the assay, 0.5 ml plasma and 0.5 ml 0.01N HCl were added to 5 ml butanol. The rest of the assay was performed according to Jacobowitz and Richardson (1978). The changes in plasma hormone concentration over time were compared with the initial value by a one-way ANOVA with a Student-Newman-Keuls test. III. HINDLIMB VASCULAR RESPONSE TO VASOACTIVE AGENTS, ASPHYXIA, AND' EXERCISE A. Log Dose-response Curves for Vasoactive Agents In eight male chickens (2.51 :0.11 kg) the perfusion pump flow rate was adjusted to produce a Pp of 125 mm Hg and flow was maintained constant for the duration of the experiment. Acetylcholine (ACh, 50 ug/ml), adenosine (ADO, 500 ug/ml), histamine diphosphate (HIST, 55 ug/ml), prostaglandin E1 (PGE1, S ug/ml), and norepinephrine (NE, 10 and 100 ug/ml) were individually infused into the emtracorporeal perfusion circuit prior to the perfusion pump by a infusion/withdrawal pump (Harvard Model 950). The infusion rate of each drug was incrementally increased starting from a subminimal rate until a further increase in infusion rate failed to produce a change in Pp. This technique produced 5 - 10 points per dose-response curve per animal. The saline vehicle was 29 also infused so that Pp could be corrected for dilution effects. The effective blood concentration of each agent could be calculated knowing the infusion rate, the concentration of the infusate, and the limb blood flow. Log dose-response curves were drawn from these data. B. Response to Bolus Administration of Vasoactive Agents: Blockade of Adenosine with Theophylline: the Response to Asphyxia In nine male chickens (2.80 30.111 kg), vasopressor and depressor agents were delivered in a 0.1 ml saline vehicle via bolus administration into the extracorporal muscle perfusion circuit prior to the pump. A control 0.1 ml bolus of physiological saline was administered at the start of each experiment. The Pp was monitored following each drug injection until it returned to the control level at which time the next drug was administered. The maximal change in Pp, the duration of change, and the integral of the curve were recorded. The curves were integrated with a digitizer. The drugs tested were ACh (1 ug). ADO (5 and 10 ug). PGE1 (0.5 ug), and NE (1 ug). Five of these animals also received HIST (10 ug). The effect of two blocking agents was then determined. Adenosine (5 and 10 ug, bolus) was readministered during a local ia infusion of theophylline (5 mM infused at 1 ml/min), a competitive inhibitor of adenosine (Bunger, Haddy, and Gerlach, 1975). The effect of saline vehicle infusion was also determined. In 11 additional animals (2.89 :0.06 kg), NE (1 and 5 ug, bolus) was administered before and 20 min after systemic alpha-adrenergic blockade with phenoxybenzamine (PBZ, 7.5 - 10 mg/kg, iv). Significance of the differences between means during the control and experimental periods was evaluated by paired Students t test, and a P < 0.05 was considered '11:: 30 significant. C. Effect of Alpha-adrenergic Blockade on the Vascular Response to Norepinephrine and Asphyxia; the Effect of Exercise The effect of asphyxia induced by tracheal occlusion was examined in the above mentioned 11 birds before and after PBZ administration. Finally, the effects of exercise and asphyxia were examined. Exercise was produced in the perfused leg in S of the 11 animals by electrical stimulation of the peripheral end of the cut sciatic nerve (1.6 msec duration, 6V, and 6 Hz for 15 sec). The maximal change in Pp, the duration of change, and the integral (via digitization) of the curves were recorded. Significance of the differences between means during the control and experimental periods was evaluated by paired Students t test, and a P < 0.05 was considered significant. RESULTS I. VASCULAR RESPONSE TO HEMORRHAGE T0 MEAN ARTERIAL BLOOD PRESSURE 0F 50 mm Hg A. Plasma Osmolality and Protein Concentration The effects of hemorrhage on plasma osmolality and plasma protein concentration (PPC) are shown in Figure 3. While there was a tendency for osmolality to increase during hemorrhage, osmolality never was significantly elevated above the prehemorrhage value. 0f 11 birds studied, osmolality rose in 9, remained unchanged in one, and fell in one. The PPC was determined in seven of the 11 roosters. There was a significant fall in PPC within 30 min of hemorrhage and PPC fell to 514% of the initial value after 150 min. B. Thermodilution Studies During Selective Autonomic Blockade The effects of acute blood loss sufficient to reduce and maintain MABP at approximately 50 mm Hg for 120 min on cardiac index, stroke volume (Vs), heart rate (HR), and TPR are presented in Figure A. Cardiac index in the untreated (control) birds was significantly reduced only at 5 and 120 min of hemorrhage and TPR was reduced only at the 30 min of hemorrhage. Heart rate was not affected by hemorrhage in the control group. Propranolol (PROP), 231: g, significantly reduced MABP from 156 :9 to 130 :9 mm Hg, HR from 2911 :21 to 201 323 beats/min, and cardiac .index from 297 1311 to 177 :20 ml/min/kg0'73u (P<0.05, analysis by a paired Students t test). Total peripheral resistance and Vs were not altered by PROP. During hemorrhage the only cardiovascular parameter that changed in the PROP group was TPR, which was significantly reduced 31 32 o 30 60 90 120150" :5 I11... TIME (mln.) '3‘ Figure 3. The effect of hemorrhage on plasma osmolality (OSM) (N=11) and plasma protein (PPC) (N=7) concentrations in male chickens. hem: mean arterial blood pressure reduced to, and sustained at, 50 mm Hg. reinf: reinfusion of the shed blood. * indicates significant change from initial value (P<0.05). 33 a , . l ‘I(\ /{>\I x .———. E E “L/‘ijji\i i/i g: Kl I i__; g i: I < .E >‘g/ I \ixf/i\i—f—f Si I\ /T I\l 31;; 1 l I 55391 F «7?: ‘" T\\--—é l» g. 1 g 1 l m x\\;/1/1 2: F -—' r 1,.—-”/ r‘ :2 >1” N U “1'1 -33 hi): 3'0 ob o'o 150 an; -33 it: 3'0 6'0 9'0 150 Figure A. The effect of hemorrhage on heart rate, cardiac index, stroke volume, and total peripheral resistance in untreated, n:5 (*---*). alpha-blocked, n=6 (O-—-—O), and beta—blocked, N=5 (.--——.), male chickens. Pentobarbital anesthesia. The drugs were administered 30 min prior to hemorrhage and the first cardiac output determination was taken immediately prior to drug administration. hem: mean arterial blood pressure reduced to, and sustained at, 50 mm Hg. Small star designates significant change from the value at time = 0 (P<0.05). 34 at 60 min of hemorrhage (Figure A) (statistics via one-way ANOVA with a Dunnett test). However, of the five PROP-treated chickens in which results are shown in Figure A cardiac index and Vs were reduced below the O-time value at 5 and 30 min of hemorrhage and both were reduced in four of the five birds throughout the hemorrhagic period. Phenoxybenzamine (PBZ), ‘235_.33, did not affect any' measured or calculated parameter, however there was a tendency for the MABP to fall. The birds pretreated with PBZ responded to hemorrhage similiarly to the untreated birds except TPR remained significantly reduced throughout the hemorrhagic period (Figure ll). The results shown in Figure A are only from birds that survived the entire experiment. This study concurs with the results of Ploucha (1979) which showed that TPR in the chicken is unaffected by hemorrhage. C. Hindlimb Perfusion Studies 1. Acute Bleed - Phenobarbital Anesthesia In six male chickens (total of 12 separate hemorrhages) Pp averaged 146 :11 mm Hg initially, 159 :7 mm Hg at 5 min, 1'17 :7 mm Hg at 10 min, and 198 :5 mm Hg at 15 min of hemorrhage. None of the pressures during hemorrhage were significantly different from the prehemorrhage value. Corresponding values for MABP were 113 :6 (initially). 38 in (5 min), 55 in (10 min), and 6A in mm Hg (15 min). Typical vascular responses of the i_n_ si_tu_ constantly-perfused chicken hindlimb to acute volume depletion are shown in Figure 5. Panel A of Figure 5 is a tracing from a chicken in which the estimated blood volume was rapidly reduced by 26%. This was associated with an immediate drop in arterial blood pressure (Pa in Figure 5) from 199 mm Hg to about 50 mm Hg. Over the subsequent 15 min, 35 32 .2: I h “‘ J “ ‘\\\\“\. v w haw --.~.-.IIIIIIIIIIIII , 0" ‘0' ,__"‘_’_"_____. ABEL. ‘0' "‘ ‘\ p. ,. , , - ”‘3. .._ IV/ '3? n __ o to _ do 3 0° _ 1 I 1 .c ; , h ‘ l Ian-Nu 3 new .‘ an. air. on Figure 5. Tracing of the effect of hemorrhage on arterial blood pressure (Pa) and perfusion pressure (Pp) in the constantly-perfused hindlimb of a male chicken (panels A and B) and the constantly—perfused breast muscle of a female mallard (panel C). Phenobarbital anesthesia. Panel A shows the effect of a rapid hemorrhage and reinfusion after a 15 min hypovolemic interval (NE=1.25 ug norepinephrine, ACH=1.0 ug acetylcholine, 0FF=perfusion pump off). Panel B shows the effects of sustained hemorrhagic hypotension (MABP = 50 mm Hg) for one hour followed by reinfusion. Numbers indicate minutes after hemorrhage. Panel C shows the effect of vasoactive agents (NE=0.25 ug) and rapid hemorrhage on Pa and Pp in the mallard. 36 arterial pressure rose by approximately 25 mm Hg and returned to near the prehemorrhage level after reinfusion of the shed blood. In contrast, Pp did not change during the entire period of hypotension nor was it affected by reinfusion of the bled volume. The hindlimb vasculature responded to NE and ACh before and after hemorrhage and Pp fell to approximately 10 mm Hg when the perfusion pump was turned off. The latter indicates adequate vascular isolation, i.e. no collateral circulation to the assay limb. Reactive dilatation, as indicated by the slow' return of‘ Pp on restarting flow, was seen in the majority of experiments. These results demonstrate that an acute hemorrhage to a MABP of 50 mm Hg in the phenobarbitalized chicken does not produce a rise in hindlimb vascular resistance. 2. Chronic Bleed - Phenobarbital Anesthesia In this series, consisting of nine male chickens, the initial Pp was 1111 :11 mm Hg and it reached a maximal value of 121 :6 mm Hg (nonsignificant change) at 90 min of hemorrhage. Panel B of Figure 5 is a tracing from an experiment in which blood was withdrawn to lower and maintain MABP at 50 mm Hg for 60 min. Again, there was no evidence of vasoconstriction at any time during the hypovolemic period. Panel C of Figure 5 shows the vascular response in an isolated constantly-perfused breast muscle of a phenobarbital anesthetized mallard duck during hemorrhage and reinfusion of the shed blood. Mean arterial blood pressure was reduced by hemorrhage from 110 to 50 mm Hg. The bottom tracing of Pp shows that the vasculature responded to NE (0.25 ug) and ACh (1.0 ug) and fell to less than 20 mm Hg when the pump was turned off, again indicating adequate vascular isolation. Also, there is evidence of 1'. 37 reactive dilatation. Perfusion pressure began to rise immediately following the onset of bleeding, remained elevated during the hypotensive period, and returned to the prehemorrhage level after reinfusion of the shed blood. This increase in skeletal muscle vascular resistance is similiar to that seen by Jones and West (1978) in the constantly-perfused duck hindlimb during submersion. These results demonstrate that holding the MABP at 50 mm Hg for an hour in the phenobarbitalized chicken does not produce a rise in hindlimb vascular resistance. However, an acute hemorrhage in mallards may produce a sharp rise in hindlimb vascular resistance. 3. Acute Bleed - Pentobarbital Anesthesia Perfusion pressure in nine males did not change significantly during a 10 to 15 min hemorrhagic period. The initial MABP in this group was 175 :7 mm Hg and initial Pp was 165 :6 mm Hg. The latter increased to 171 :8 mm Hg during the hemorrhagic period. A tracing from a typical experiment is shown in Figure 6. These results demonstrate that acute hemorrhage to a MABP of 50 mm Hg in the pentobarbitalized chicken does not produce a rise in hindlimb vascular resistance. A. Isogravimetric Studies - Pentobarbital Anesthesia The MABP immediately prior to hemorrhage, leg weight, and leg blood flow did not differ significantly between the three group. The overall mean values (n = 18) were: MABP = 95.8 :1.2 mm Hg, leg weight = 217 :12 am. and leg blood flow = 1“-9 30.6 ml/min/lOOgm. Figure 7 summarizes the results of the first three experimental series. The mean CFC values of the three groups were compared statistically at each determination time 38 .33 W told. h—I dice-hue hum. u——a PP‘WW (nu-u.) ._¢_- '3aif 2&5: Figure 6. Tracing of the effect of hemorrhage on arterial blood pressure (Pa) and perfusion pressure (Pp) in the constantly-perfused hindlimb of a male chicken. Pentobarbital anesthesia. off = 1 minute perfusion pump off; ach = 1.0 ug acetylcholine delivered behind the pump; ne = 1.0 ug norepinephrine; hem.= rapid removal of about 50 ml of blood; reinf. = reinfusion of the shed blood. PM” c P if E: CC F \FNIEE\ :uE\ -E\ .bvzm-O-NnNnmoo zo-l-Il‘EIPI-nhh >NH§<|~I-AK<0 CAPILLARY FILTRATION COEFFICIENT 39 9 .I A l. CONTROL (NON-HEM) c . "é 7' \ 23:23:31.: : 26' 05' l \ 94' \l\l\l/l 32' I i/I 1. hem o . l . . . . . O 10 20 30 4O 50 60 TIME (minutes) Figure 7. The effect of hemorrhage on capillary filtration coefficient in the isogravimetric isolated constantly—perfused hindlimb of male chickens. Pentobarbital anesthesia. Six animals per group. hem: mean arterial blood pressure reduced to, and sustained at, 50 mm Hg. "a" indicates significantly different from the control group at that time (P<0.05). NO by a one-way ANOVA using a Dunnett test. The initial (5 min) CFC determination in the nerve intact group was significantly different from the other two groups at that time. This was the only instance of a significant difference between the three experimental groups. The 5 min CFC of each group was not significantly different from the 60 min CFC (Students t test). A representative tracing from this study is shown in Figure 8. As the venous pressure is increased to approximately A0 mm Hg, there was a rapid increase in limb weight due to distention of the capacitance vessels followed by a steady slope which represents capillary filtration. The Pp increased due to the increase in venous outflow resistance and the MABP (Pa in Figure 8) fell due to the volume shift into the limb. The Pei values ranged from 13.2 :2.8 to 18.3 :1.5, and were unchanged by hemorrhage. This study demonstrates that hemorrhage in the chicken does not affect CFC, whether the sciatic nerve trunk is intact or severed. II. VASCUALAR RESPONSE TO HEMORRHAGE T0 MEAN ARTERIAL BLOOD PRESSURE 0F 25 mm Hg A. Hindlimb Perfusion Studies In all animals, a hemorrhage to a MABP of 50 mm Hg did not produce a change in Pp, whereas a further hemorrhage to 25 mm Hg produced a significant increase in Pp. The Pp immediately began to increase as the MABP fell near 25 mm Hg and it remained elevated until the shed blood was reinfused. During reinfusion of the shed blood, the Pp would decrease to the control value following the return of enough blood to raise the MABP to approximately 50 mm Hg. l11 200, Pp 100» (MM H6) 0 1 A L 1 1 A 1 1 1 40 f Pv (MM MG) 0. . - . Figure 8. Tracing of mean arterial blood pressure (Pa), perfusion pressure (Pp), venous pressure (Pv), and leg weight (Wgt.) in the isogravimetric isolated constantly-perfused hindlimb of a male chicken. Pentobarbital anesthesia. N2 1. Effect of Sciatic Nerve Severance and Alpha-adrenergic Blockade Severance of the sciatic nerve during the elevated Pp would generally produce only a transient (30 sec) 25 - 30 mm Hg fall in Pp, other than this the Pp was unaffected (Table 1). In contrast, an intra-arterial infusion of phentolamine would promptly return the Pp to the prehemorrhage level, where it was maintained as long as the alpha-blocker was infused (Figure 9). This study demonstrates that severence of the sciatic nerve trunk does not attenuate the rise in hindlimb vascular reisitance seen during severe hemorrhagic hypotension. 2. Effect of Bilateral Vagotomy The Pp again was unaffected by a hemorrhage to 50 mm Hg and increased significantly as the MABP was reduced to 25 mm Hg (Table 1). Mean arterial blood pressure and Pp returned to control values following reinfusion of the shed blood. The rise in Pp in response to severe hemorrhage was unaltered by bilateral vagotomy. This study demonstrates that the rise in hindlimb vascular resistance during severe hemorrhagic hypotension is not mediated through the vagi. 3. Head Perfusion Studies A hemorrhage to a MABP of 25 mm Hg was not required to produce a rise in limb Pp in this study (Table 1). Some birds demonstrated intense vasoconstriction in the limb at a MABP of A5 mm Hg, however, at this time carotid perfusion pressure was less than 25 mm Hg. When head blood flow was artificially maintained during hemorrhage, no rise in hindlimb Pp occurred (Table 1). Figure 10 is.aa representative tracing showing the effect of hemorrhage on limb Pp with and without head perfusion. It is “3 Table 1. The effect of hemorrhage on mean arterial blood pressure (MABP), hindlimb perfusion pressure (HPp), and carotid perfusion pressure (CPp) in chickens following severence of the sciatic nerve, bilateral vagotomy, intra-arterial phentolamine, or artificial perfusion of the head. All values expressed as mean +SEM. * designates significant change in INN) from the Ereceding control (P<0.05). CONDITION MABP HPp CPp (mmHg) (mmHg) (mmHg) SERIES 1 (n = 6) control 12a :6 97 :5 ---a hem.b 50 :2 97 :5 --- a hem. 25 :2 17M :10 --- . . c * hem. + sc1atic 25 :2 172 :11 --- hem. + phentol.d 25 :2 92 :10 --- SERIES 2 (n = 7) control 109 :6 77 :8 --_ hem. 50 :2 77 :8 --_ a hem. 25 :2 189 :9 --- control 111 :u 79 :9 _-- e i» hem. + vagot. 25 :2 18M :10 --- SERIES 3 (n = 8) control 109 :6 1AA :7 1H1 :7 hem. + CPPf off 37 :2 335 :20' 19 :2 control 121 :9 153 :12 157 :8 hem. + CPP on 35 :2 17A :15 96 112 a CPp not measured in series 1 and 2. b rapid hemorrhage c severance of the sciatic nerve trunk during hemorrhage d intra-arterial infusion of phentolamine (50 ug/min) during hemorrhage bilateral cervical vagotomy prior to hemorrhage CPP designates carotid perfusion pump ”3‘3 NM Figure 9. Continuous tracing of the effect of severence of the sciatic nerve and intra-arterial phentolamine infusion on mean arterial blood pressure (Pa) and hindlimb perfusion pressure (Pp) in the chicken after stepwise hemorrhage. off, perfusion pump off; ach, 1.0 ug acetylcholine: ne, 1.0 ug norepinephrine; hem., hemorrhage; reinf., reinfusion of 10 ml shed blood; phentol., phentolamine infused at 50 ug/min, ia. (mmHg) (mmHg) (3:83;) Hit? A E l 7‘3 l HPp C Pp 45 § hem. reinf. he'm. reinf. Figure 10. Tracing of the effect of hemorrhage on mean arterial blood pressure (MABP) and perfusion pressure (HPp) in the constantly-perfused hindlimb of a male chicken with and without artificial perfusion of the head via the carotid arteries. Pentobarbital anesthesia. CPp, carotid perfusion pressure; off, carotid perfusion pump turned off: hem, MABP reduced to, and sustained at, 50 mm Hg: reinf. reinfusion of shed blood; on, carotid perfusion pump turned on. N6 evident that Pp increased markedly, i.e. 150 mm Hg, when carotid perfusion pressure fell below 25 mm Hg even though MABP was 110 mm Hg. However, when head flow was maintained, the limb Pp rose only 20 mm Hg during the hypotensive period. This study demonstrates that the rise in hindlimb vascular resistance during severe hemorrhagic hypotension can be completely eliminated by artificially maintaining blood flow to the head. B. Effect on Concentration of Serotonin, Norepinephrine and Dopamine in Plasma The initial MABP in this series was 127 :15 mm Hg. While a hemorrhage to a MABP of 50 mm Hg did not affect the concentration of serotonin (SER), dopamine (DA), or NE in plasma, the concentrations of all three hormones increased significantly when the MABP was reduced 25 mm Hg (Figure 11). This study demonstrates that a moderate hemorrhage (MABP = 50 mm Hg) does not significantly effect the concentration of SER, DA, and NE in plasma, while a more severe hemorrhage (MABP = 25 mm Hg) produces a significant increase in the concentration of all three hormones in plasma. III. HINDLIMB VASCULAR RESPONSE TO VASOACTIVE AGENTS, ASPHYXIA, AND EXERCISE A. Vasoactive Agents 1. Log Dose-response Curves for Vasoactive Agents The initial MABP and Pp for the eight animmals in this study were 13A :11 and 125 mm Hg, respectively. Figures 12 and 13 show log dose-response curves for the agents tested. The drug infusion rate in this series ranged from 0.002 to 2u0 nfl/min. Though five dose-response 117 ° SEflo—o . l'\ . ‘5 52 .. ._. i