a... ".. - SHE'S IS This is to certify that the thesis entitled SKIN AND SKELETAL MUSCLE VASCULAR RESPONSES TO HYPOTHERMIA presented by Terry Charles Major has been accepted towards fulfillment of the requirements for Maledegree in M5119. BY % 16M, 4 flj/ZW'}? gain“ Major prof sor DateNovember 17, 1977 0-7 639 l a . r s . ‘L \ id at i . 4.. 7 _‘ n; s . A vl ,. «L .r- r. n - [L a. a l T. d .. a s .. .1. O Q“ .v. A .l. K. H1 .\n I x 1 III. .H. ,l f Y. .\. V. \ . .- v . . t . . ., SKIN AND SKELETAL MUSCLE VASCULAR RESPONSES TO HYPOTHERMIA BY Terry Charles Major A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF'SCIENCE' ‘ i-“u?tdt l’"‘ ~ -‘:;fl, bin 5 2 Departmentcfl’physiology~ Q, 10333:” ABSTRACT SKIN AND SKELETAL MUSCLE VASCULAR RESPONSES TO HYPOTHERMIA By Terry Charles Major Vascular resistance and capacitance as well as trans- capillary fluid partitioning were studied in innervated or denervated forelimbs of 37 pentobarbitalized dogs. Hypother— mia (38° C to 28° C) was induced systemically, by external cooling of blood which returned to the right heart, or local— ly, by cooling blood perfusing the forelimb. Whole—body cooling to 33° C and then to 28° C elicited significant decreases in limb weight with substantial increases in both skin and skeletal muscle vascular resistances and blood flow decreases. Acute denervation of forelimbs attentuated both the fall in limb weight and increase in skin vascular resistance. Local cooling elicited skin and skeletal muscle vascu- lar dilation at 33° C in both innervated and denervated ,‘wr forelimb whereas a slight increase in skin and skeletal muscle blood vessel resistance resulted upon local cooling to 28° C; perhaps due to a rise in blood viscosity. 0 5 E .éhs}. - Terry Charles Major Cutaneous vasoconstriction during systemic cooling was medi- ated primarily by sympathetic nerves, whereas skeletal 'Muscle vasoconstriction was mediated primarily by circu- lating hormones. The locally—induced vasodilation apparent- ly resulted from a direct inhibition of skin and skeletal muscle vasomotor tone and was attenuated by increases in blood viscosity. Robert - To My Parents reviewing W. "SRSLOn as>‘- ; _ 2 _ . 'iffOtts .re . ~:(,:at A SPF‘. .J .I.{|l ‘- A. . J l. .J’w sacrifice-J .' "-47: u .; . ‘1. . , . my g'uiuui' ' "n- Dr. d.‘ a. A . ..' : : y o) . A t A "0‘1? ~< .. --.... 1‘; ACKNOWLEDGEMENTS I am indebted to my advisor, Dr. James M. Schwinghamer, for his patient assistance in the preparation of this thesis and for his deep support, guidance, and friendship through- out my graduate program. I would like to acknowledge Drs. Jerry B. Scott and Robert P. Pittman for serving on my guidance committee and reviewing this manuscript. Dr. Brian LaLone, Mr. Jeff Musson, and Mr. Stuart Winston assisted with many of the experiments and their efforts are appreciated. A special thanks is due to my parents and friend, Sue, who sacrificed in many ways and gave patient support to complete my graduate training. TABLE OF CONTENTS Page LIST OF TABLES.... ................................. .. vii LIST OF FIGURES ...................................... ix INTRODUCTION ......................................... 1 LITERATURE REVIEW. ............................... .... 4 I. General Considerations ....................... 4 A. Cardiac Output .......................... 4 B. Heart Rate and Stroke Volume ...... . ..... 6 C. Arterial Blood Pressure ................. 8 D. Total Peripheral Resistance ............. 10 II. Control of Peripheral Vascular Resistance During Hypothermia ........................ ... 11 A. Systemic Hypothermia: Neural Control... 12 B. Humoral Control ................. ... 14 C. Local Hypothermia: Direct Effect on Vessel Musculature. ........... ....... 15 D. Effect of Cold on Blood Rheology ........ 16 E. Effect of Cold on Na+ -K ATPase Activity 18 III. Peripheral Vasculature Capacitance During Hypothermia.... .............................. 19 A. Systemic Hypothermia: Neural Control of Capacitance Vessels ........ . ........... . 21 B. Humoral Control ......................... 22 C. Local Hypothermia: Direct Effect on Venous Musculature ...................... 23 D. Passive Venous Responses ............ .... 23 iv TABLE OF CONTENTS--Continued IV. METHODS... Transcapillary Flux During Hypothermia ....... A. Systemic Hypothermia .................... B. Local Hypothermia ....................... Series I: Naturally Perfused, Innervated Forelimbs; Whole-body Cooling and Hypothermia ................................ Series II: Naturally Perfused, Innervated Forelimb; Normothermic Controls ............ Series III: Constant Pressure Perfusion, Innervated Forelimbs; Local and Whole-body Hypothermia ................................ Series IV: Constant Pressure Perfusion, Denervated Forelimb; Local and Whole- body Hypothermia. .............................. DATA ANALYSIS..... ................................... RESULTS. 1' II. ..... OI.I'D.O.IIIUCOOOOOOIOOIOOIOIIIDIOICOIIO Series I and Series II (normothermic con- trols): Naturally Perfused, Innervated Forelimbs; Effects of Whole-body Cooling and Hypothermia .................. . ......... A. Heart Rate ...................... . ....... B. Arterial Hematocrit ..................... C. Intravascular Pressures ....... .......... D. Forelimb Weights ........................ E. Skin Total and Segmental Vascular Resistances ................. F. Muscle Total and Segmental Vascular Resistances. ...... ...................... Series III: Constant Pressure Perfusion, Innervated Forelimbs; Local and Whole-body Hypothermia.. ...... . ............... . ....... A Forelimb Weight ......................... B. Cutaneous Vascular Resistance ........... C Skeletal Muscle Vascular Resistance ..... D. Total Forelimb Vascular Resistance.... Page 23 24 25 27 32 34 34 42 43 4S TABLE OF CONTENTS--continued Page III. Series IV: Constant Pressure Perfusion, Denervated Forelimbs; Local and Whole—body Hypothermia ................................ 87 A Forelimb Weight ......................... 87 B. Cutaneous Vascular Resistance ........... 92 C Skeletal Muscle Vascular Resistance ..... 96 D Total Forelimb Vascular Resistance ...... 100 DISCUSSION.... .......................... .. ........... 105 I. Series I and II: Naturally Perfused, Innervated Forelimbs ....................... 105 A. Forelimb Weight ..................... .... 105 B. Forelimb Vascular Resistance....... ..... 107 II. Series III and Series IV: Constant Pressure Perfusion, Innervated and Denervated Fore— limbs; Local and Whole-body Hypothermia.... 108 A. General Considerations .................. 108 B. Forelimb Weight.. ................... .... 108 C. Forelimb Vascular Resistance ............ 111 SUMMARY AND CONCLUSIONS .............................. 115 BIBLIOGRAPHY ......................................... 118 APPENDICES A. FORELIMB VASCULAR RESISTANCE CALCULATIONS.... 125 B . HEMODYNAMIC RESPONSES TO LOCAL AND SYSTEMIC COOLING (Pressure, Flow, and Resistance Data) TABLES B-l THROUGH B-4 ............. .... 128 B2. HEMODYNAMIC RESPONSES TO LOCAL AND SYSTEMIC COOLING (Pressure, Flow, and Resistance Data) TABLES B-S THROUGH B—8 ..... . ........... 133 C. STATISTICAL METHODS.................. ........ 138 u 3 vi LIST OF TABLES TABLE Page 1. The experimental protocol ....................... 40 2. Effects of whole-body cooling and sustained hypothermia on mean intravascular pressure in innervated skin arteries, small skin vein, and cephalic vein ................................... 53 3. Effects of whole-body cooling and sustained hypothermia on mean intravascular pressures in innervated muscle arteries, small muscle vein, and brachial vein ............ . .................. S4 4. Effects of local and whole-body hypothermia on total skin and muscle vascular resistances as well as total limb vascular resistances in innervated forelimbs ...................... . ..... 88 5. Effects of local and whole-body hypothermia on total skin and muscle vascular resistances as well as total limb vascular resistances in innervated forelimbs ............................ 104 B-l. Systemic arterial blood pressure and brachial artery perfusion pressure ....................... 129 B-2. Cephalic and brachial flows ..................... 130 3-3. Cephalic and brachial vein pressures ............ 131 B-4. Heart rate and aortic blood temperature ..... .... 132 B-S. Systemic arterial blood pressure and brachial artery perfusion pressure..... .......... ........ 134 B-6. Cephalic and brachial flows ................ ..... 135 LIST OF TABLES--Continued TABLE Page B-7. Cephalic and brachial vein pressures ............ 136 B-8. Heart rate and aortic blood temperature ......... 137 viii JVMIN FIGURE LIST OF FIGURES l. A schematic of the isolated canine forelimb 10. 11. 12. preparation .................................... . A schematic drawing of the extracorporeal preparation .................................... Schematic of the servosystem used to control brachial artery pressure ....................... . Effects of whole-body hypothermia (28°C) on heart rate ............... . Effects of whole-body hypothermia (28°C) on arterial hematocrit ...... . Effects of whole-body (28°C) on systemic arterial blood pressure ..... Effects of whole-body hypothermia (28°C) on . Effects of whole-body hypothermia (28°C) on resistance ............................. ........ . Effects of whole—body hypothermia (28°C) on resistance. ........... Effects of whole-body hypothermia (28°C) on Effects of whole-body hypothermia (28°C) on resistance .......... ... Effects of whole—body hypothermia (28°C) on resistance ............... . ........ . ............ cooling and sustained cooling and sustained cooling and hypothermia cooling and sustained forelimb weight change... cooling and sustained total skin vascular cooling and sustained skin small vessel cooling and sustained skin venous resistance... cooling and sustained total muscle vascular cooling and sustained muscle small vessel ix Page 30 36 38 47 49 52 S7 59 61 63 66 68 LIST OF FIGURES--continued FIGURE Page 13. Effects of whole-body cooling and sustained hypothermia (28°C) on muscle vanous resistance. 70 14. Effects of local and systemic cooling on weight change in innervated, pump—perfused forelimbs.. 73 15. Resistance response of the cutaneous circula- tion to local and systemic cooling in inner— vated, pump-perfused forelimbs. ............ .... 76 16. Effects of local and systemic cooling on total muscle vascular resistance in innervated, pump- perfused forelimbs ............................. 81 17. Effects of local and systemic cooling on total forelimb vascular resistance in innervated, pump-perfused forelimbs ........ . ........... .... 85 18. Weight changes to local and systemic cooling in denervated, pump-perfused forelimbs ............ 90 19. The resistance response of the cutaneous circu- lation to alterations in local and systemic cooling in denervated, pump-perfused forelimbs. 94 20. Effects of local and systemic cooling on total muscle vascular resistance of denervated pump- perfused forelimbs ............................. 98 21. Effects of local and systemic cooling on total vascular resistance in denervated, pump- perfused forelimbs ............................. 102 U. INTRODUCTION The circulatory responses to whole-body cooling have long interested physiologists and clinicians. Hypothermia is an important tool for studying temperature regulation and hibernation, and recently advances in our understanding of the effects of lowered temperature on metabolic activity and oxygen consumption have led to the use of hypothermia in certain medical and surgical procedures. Metabolic activity is reduced at subnormal temperatures associated with either hypothermia or hibernation. In the case of hypothermia, depression of body temperature is an imposed condition from which the subject may not recover without respiratory and/or circulatory support (2,3,4,7,8). In contrast, animals in deep hibernation maintain homeostasis at body temperatures of 5°C for prolonged periods of time (77). Homeothermic, adult humans and nonhibernating laboratory animals cannot be cooled safely to a body temperature much below 20°C (4). Total metabolic activity as indicated by oxygen con- sumption is reduced at subnormal body temperatures. In dogs, the total body oxygen consumption falls to 50 or 60% of the normothermic control value when rectal temperature is lower- ed to 28°C. According to Thauer (65), oxygen consumption in A nonthermoregulating humans may be lowered by as much as 50% at a rectal temperature of 30°C. When prolonged periods of circulatory or cardiac arrest are desirable, particularly in neuro, cardiac, or transplan- tation surgery (3,16,40,4l,62), mild hypothermia has been used to temporize the effects of ischemia. The reduced oxygen requirement at subnormal temperatures permits inter- ruption of the circulation for longer periods of time than would be possible at normal tissue temperatures. Whole-body hypothermia affects hemodynamics in all organs and vascular beds including skin and skeletal muscle circulations. The latter two beds comprise about 55% of the total body tissue mass. Hypothermia elicits both remote (i.e., neurohumoral) and local (i.e., direct effect of the cold) vasomotor responses which are known to influence the skin and muscle circulations. The characteristics of these two vascular control mechanisms can be examined separately by comparing responses to whole-body (systemic) hypothermia (lS,16,18,19,32) with those of local cooling (58,68—72). Recent investigations have attempted to elucidate more pre- cisely the neural, humoral, and local vascular components of the peripheral circulatory response to whole-body cooling (19). Methods used to induce hypothermia include surface and internally (i.e., blood) cooled procedures (11,39). The study reported in this thesis compares skin and muscle oh no. circulatory response to local and to systemic cooling, induced sequentially and independently, in the same animal. The experimental preparations and protocol described here represent an attempt to identify the neural, humoral, and direct effects of cooling on the skin and skeletal muscle blood vessels. LITERATURE REVIEW 1. General Considerations Tissue perfusion is the primary function of the cardio- vascular system. Since maintenance of an adequate blood supply to systemic organs depends on both systemic arterial pressure and local vascular resistance, it is appropriate to begin this review with an analysis of the cardiac and peripheral vascular responses to hypothermia. It should be noted that the commonly used anesthetics influence overall cardiovascular reSponse to cooling (3,47,50). A. Cardiac Output There is general agreement that anesthetized, non- thermoregulating animals exhibit a large reduction in cardiac output as body temperature decreases (65). Three factors are involved in decreasing the cardiac output: a) the direct effect of cold onto the cardiac excitatory tissue which results in a decreased heart rate, b) the cold-induced decrease in cardiac metabolism which decreases myocardia contractility, and c) the cold-induced increase in blood viscosity (8). Thauer (65) reports that in rats, dogs, and man the cardiac output may be decreased to anywhere from 30 to 70% of normothermic control values when core temperature is lowered to 25°C. The decrease in cardiac output has been reported by some to display a direct linear relationship to temperature (2,8,51). However, Thauer (65) also cites evidence that the decrease in cardiac output may be expon— entially related to temperature because of an exponential decrease in whole-body oxygen consumption. The reason for this variability in the slope of cardiac output curves reported in the literature is most likely the result of dif- ferent types and degrees of anesthesia which cause varying levels of thermoregulatory inhibition process. Rittenhouse £3 21. (52) observe an 82% decrease in the cardiac output of dogs cooled to 20°C. Reissman and Kapoor (57) note a 35% decrease in cardiac output in dogs cooled to 25°C. Hegnauer and D'Amato (12) reduced cardiac output to 11% of control by reducing the rectal temperature of dogs to 17°C. Bullard (8) reduced the body temperature of unanesthetized rats to 16°C and observed that cardiac output fell to 16% of the normothermic control value. He reports a direct, linear relationship between cardiac out- put and rectal temperature (60% of the normothermic cardiac output at a rectal temperature of 27°C and 20% at 15°C). Fisher £3 31. (16) reduced rectal temperature to 23°C for twenty-four hours in anesthetized dogs. They report a 33% decrease in cardiac output after completion of the cooling and a further reduction to 6% of control after twenty-four hours of sustained hypothermia. Zarins and Skinner (77) observed an 83% decrease in the cardiac output of anesthetized dogs cooled to 5°C. This report agrees with Thauer that the cardiac output decreases exponentially with the reduction in body temperature. Evonuk (15) also reported an exponential decrease in cardiac output during cooling in warm-acclimatized, anesthetized dogs. He reports an average 57% reduction in cardiac output from control during the first 3°C dro in rectal tem erature. P P B. Heart Rate and Stroke Volume The two immediate determinants of cardiac output are heart rate and stroke volume. Reissman and Kapoor (51), Rittenhouse gt £1. (52), Hegnauer £3 21. (23), and Thauer (65) agree that there is little, if any change in stroke volume in animals cooled to rectal temperatures as low as 17°C. Therefore, changes in cardiac output are due primarily to changes in heart rate. The previously described linear decreases in cardiac output can be shown to be due to a linear decrease in heart rate (Bullard (8) and Andrews gt El- (2) in unanesthetized rats and Rittenhouse £3 31. (52) in anesthetized dogs). Rittenhouse observes a decrease in heart rate from a mean of 152 beats/min. at 38°C to 60 beats/ min. at 25°C, while Bullard (7) observes that the heart rate of rats decreased from 424 beats/min. at 36°C to 51 beats/ min. at 16°C. Hook and Stormont (26) demonstrated that the ~‘ heart rate fell from 163 beats/min. at 38°C to 22 beats/min. at 18°C. An exponential relationship between heart rate and rectal temperature has been described by Bigelow (4) and Thauer (65). Hegnauer _£ _1. (23) report a sigmoid decrease in heart rate of anesthetized dogs starting at above 170 beats/min. at a rectal temperature of 37°C, decreasing to 120 beats/min. at 28°C and further decreasing to a minimum of 15 beats/min. at 20°C. Evonuk (15) reports that heart rate decreased from 100 beats/min. to 90 beats/min. as rectal temperature was reduced from normal to 32°C in warm- acclimatized, anesthetized dogs. Reissman gt _1. (51) using a canine heart—lung prepara- tion confirmed the parallel decreases in cardiac output and heart rate. In these preparations heart rate decreased from 130 to 80 beats/min. when cardiac tissue temperature was lowered to 27°C. Mohri _t _l. (41) report reductions in heart rate from 175 to 30 beats/min. in human infants cooled from 37°C to between 22 and 19°C while Hicks 23 a1. (24) note a similar progressive decrease in the heart rate of humans, ages three months to thirty—six years; reporting average reductions of heart rate from 120 beat/min. at 37°C to about 45 beats/min at a rectal temperature of 28°C. Thauer (65) reports that the temperature related fall in heart rate is not influenced by vagotomy or atropine. In A ._~.—.~_————.——. This suggests that the bradycardia observed in hypothermia results from a direct effect of lowered temperature on cardiac electrogenic tissue rather than from increased vagal tone. Electrocardiographic evidence (24,26) suggests that hypothermic bradycardia results from a depression of both diastolic depolarization in the S—A node and conduction velocity in the His-Purkinje system. The EKG tracings dur- ing progressive reduction in body temperature (38°C and 25°C) shows gradual prolongation of the P-R interval, QRS complex, and QT segment. Also, marked inversion of the T wave is seen with lowered body temperature (24,40). Hicks £3 £1. (24) reports that during hypotheria a greater portion of the cardiac cycle is spent in systole even though diastole is also lengthened. Hegnauer £5 31. (23) report a 70% increase in systolic duration when core temperature is reduced to 28°C, and a 6-fold increase above normothermic control at 18°C. C. Arterial Blood Pressure The two immediate determinants of mean systemic arterial blood pressure are cardiac output and total peripheral resistance. Thauer (65) has reviewed data which indicate that the reduced blood pressure during hypothermia is almost exclusively due to a bradycardia-induced reduction in cardiac output. As rectal temperature is lowered from normal to approxi- mately 28°C, heart rate and cardiac output fall relatively more than does blood pressure, indicating that total periph- eral resistance is increased. As rectal temperature is reduced below 25°C, heart rate and blood pressure fall approximately equally with mean systemic arterial pressure reaching 40 to 50 mm Hg at 16 to 18°C rectal temperature. Mohri gt gt. (41) report a decrease in mean pressure of human infants during cooling with the pattern of reduction dependent upon the disease being treated. Hegnauer gt gt. (23) and Andrews gt gt. (2) also report that arterial pres- sure falls slightly when rectal temperature is lowered from 37°C to between 29 and 23°C, but when rectal temperature is reduced below 23°C, a proportional reduction in arterial pressure and heart rate is observed. Hook and Stornant (26) report that blood pressure in anesthetized dogs did not de- crease significantly with moderate reductions of body temperature. However, when rectal temperature fell below 26°C, these investigators report large reductions in blood pressure (50 mm Hg at 22°C and 31 mm Hg at 18°C). Evonuk (15) and Rittenhouse gt gt. (52) report no change in mean arterial pressure when rectal temperature was lowered from normal to 32°C. At rectal temperature below 32°C a gradual decline in blood pressure was reported by Rittenhouse gt gt. (112 mm Hg at 25°C and 75 mm Hg at 20°C). Y‘ 10 Hegnauer and D'Amato (12) report that systemic blood pres- sure of rats decreased from 126 mm Hg at 38°C to 54 mm Hg at 17°C (pressure response pattern like that observed in dogs). Bullard's study (8) in unanesthetized rats showed a 10% fall in mean arterial pressure when rectal temperature was re- duced to 22°C, after which the fall began to parallel the decrease in cardiac output. He attributes the slight initial fall in blood pressure to the fact that the decrease in cardiac output was largely counteracted by an increase in total peripheral resistance. Several other investigators also demonstrated that hypothermia causes a prOportionately greater fall in cardiac output than in blood pressure (15, 49). D. Total Pettpheral Resistance According to Thauer's review (65), lowering body temperature to 25°C causes total peripheral resistance to increase anywhere from 2.1 to 2.3 fold. Bullard (8) reports that total peripheral resistance in rats increases three-fold when rectal temperature is lowered from 37 to 16°C. Rittenhouse gt gt. (52) observed a gradual increase from 67 PRU (peripheral resistance units) to 79 PRU as body tempera- ture was reduced from 38 to 30°C; further reductions in rectal temperature produced relatively much larger increases in resistance so that the value for PRU reached 212 at 20°C. Zarin and Skinner (76) lowered rectal temperature of ll anesthetized dogs from 37 to 5°C and observed that total peripheral resistance increased from 80 to 238 mm Hg per liter per minute. Thauer suggests that the inverse relationship between rectal temperature and total peripheral resistance may be due to: l) a passive reduction in cross-sectional area of the vasculature due to the fall in transmural pressure; 2) the anesthesia (especially barbiturate anesthesia) which according to Olmsted gt _t. (46) and to Priano gt gt. (50) increases the peripheral resistance of dogs with normal body temperature. The increasing depth of anesthesia during cooling could then lead to increased peripheral resistance; 3) active vasoconstriction unrelated to changing depth of anesthesia; and 4) increased blood viscosity. 11. Control of Peripheral Vascular Resistance Duringlfiipbthermia Peripheral vascular resistance is regulated centrally by neural reflexes and circulating vasoconstrictors and locally by various conditions or stimuli in and around the small arteries and arterioles, precapillary sphincters, and venules. Interplay between these two regulatory systems determines vascular resistance in each of the major, parallel-coupled systemic organs. The study described here focuses on cutaneous and skeletal muscle circulations of the A3 2‘ 12 dog forelimb. These vascular networks were chosen for study because of the relatively large quantity of tissues they supply (skin and skeletal muscle together comprise approxi- mately 60% of the total body mass), because they have been shown to play an important role in reflex compensatory response to systemic hypotension (a condition which accom- panies hypothermia), and because they are responsive to changes in temperature. A. Systemictfiypothermia: Neural Control Neural regulation of the cutaneous and skeletal muscle vasculatures is accomplished primarily by the sympathetic vasoconstrictor fibers which exert prominent a—adrenergic influences on skin and skeletal muscle vessels. Keller (7), concludes that exposure to subnormal environmental tempera- tures brought about increased impulse traffic in vasocon- strictor fiber networks, especially those supplying the cutaneous vasculature. His data suggest that the ”heat loss center" of the anterior hypothalamus is of primary importance in regulating core temperature through vasoconstrictor fiber activation. Haddy gt gt. (12) demonstrate that arterial resistance in the canine forelimb increased significantly when ambient temperature was reduced from 20 to 0°C. Juhasz-Nagy and Kudasz (28), describe an increased total peripheral resistance when the brain is locally cooled to _—..'- a _- -—‘_ 13 25°C. However, below 25°C, they report that blood pressure drops below normothermic values due to a progressive inhibi- tion of the centrally mediated vasomotor tone as well as to decreased cardiac output. Cold elicited vasodilaton in the skin and muscle circu— lations could result either from withdrawal of a-adrenergic neural stimulation or from activation of sympathetic cholen- ergic vasodilator fibers which have been shown to supply resistance vessels in skeletal muscle (5,6). Brooks (7) demonstrated that cooling slows conduction velocity in peripheral nerves and spinal pathways, due to a reduction in the rate of depolarization (spike slope). However, when nerves are cooled to a moderate degree (38 through 25°C) the action potentials evoked are of increased amplitude and duration, a condition which could account for increased central nervous system responsiveness in the cold. These findings show that the central nervous system excita- tion may be established by electrotonic current triggered by cooling (6). According to Thauer (65), the baroreceptor reflex becomes progressively weaker with cooling and is com- pletely abolished between 26 and 20°C. Therefore, cardio- vascular reflexes are probably increased during moderate nerve cooling (38 to 26°C) but when temperatures are reduced from 25 to 20°C, the reflexes (including the baroreceptor reflex) are decreased substantially. 14 B. Humoral Control Holobut (25) reports that the blood plasma levels of both epinephrine and norepinephrine rose significantly dur- ing whole-body cooling to 25°C in anesthetized dogs. Rapid cooling causes small increases in plasma levels of catechol- amines, whereas a slow decrease in body temperature causes relatively larger increases. The effect of cooling on vascular smooth muscle sensi- tivity to catecholamines is somewhat controversial. Rodgers gt gt. (54), using an isolated perfused mesenteric artery preparation, demonstrate that the pressor responses elicited by intraarterially injected catecholamines increased as temperature decreased from 37 to 27°C. The mean pressor responses more than doubled as temperature is reduced from 45 to 29°C in contrast, other investigators have found that resistance vessel sensitivity to plasma catecholamines is reduced with lowered temperatures (33). Despite the in- creased plasma catecholamine levels, Smith (61) demonstrates that the reactivity time (an index of the rate constriction) in dog femoral arteries was greatly prolonged at the reduced temperatures and this may indicate a reduced contractile response to pressor agents. Keatings (30,31) reports that moderate cooling (37 to 28°C) of isolated ulnar arteries usually reduced their contractile response to epinephrine and also showed that the response to epinephrine and .-._..--- . - ...AKL'. ‘. 15 norepinephrine was completely abolished at temperatures of 10°C and below. Resting tension became low with moderate cooling in the isolated arteries but this was not permanent because the control contractile response was re-established when the arteries were warmed. Iar gt gt. (29) show that the pressor responses to norepinephrine and epinephrine fall by 1-7 mm Hg per degree fall in rectal temperature. The effect of cold on vessel responses to other agents has also been considered. According to Smith (61) vascular response time to acetylcholine was prolonged at 27°C. Nayor (43) also reports that cooling decreased vascular sen- sitivity to acetylcholine. Smith (61) reports that cooling to 15°C produced a progressive increase in vascular reactiv- ity time to histamine. Keatinge (31) shows that vasopressin reactivity of ulnar arteries was attenuated at 28°C and near— ly abolished at 17°C. C. Local H othermia: Direct Effect on VesseI Musculature Local cold exposure has been reported to cause vasodi- lation of the skin and skeletal muscle circulations (56). Scott gt gt. (58) reports that resistance in precapillary vessels of the dog forelimb decreased in response to local cooling despite an increased blood viscosity. They suggest that the dilation resulted from a direct effect of cold upon smooth muscle cells. In their study vascular responses to A _.'q—- 16 local cold exposure were not greatly affected by denervation or adrenergic blockade. Brodie gt gt. (6) found that a decrease in temperature reduces the tone of aortic strips. Glover and Wallace (17) also demonstrate that in rabbit ear arteries, smooth muscle tone is reduced by cooling. Finally, Smith (61) found that the rate of cooling influences the response of arterial segments from dogs and pigs. They observed constriction when the cooling rate was 4 to 6°C per minute, and no response at cooling rates of 7°C per minute and above. D. Effect of Cold on Blood Rheology The direct effect of cold on physical characteristics of blood and the resulting influence on resistance to blood flow has been studied recently. The two properties that will be examined here are blood viscosity and red cell con- centration (hematocrit). Hypothermia has been reported to decrease the circulat- ing blood and plasma volumes without producing a significant increase in plasma-protein concentration. In studies on cooled chicks and rabbits, Rodbard gt gt. (53) demonstrated that the circulating plasma volume decreased 31% (41.2 to 25.6°C) in chicks and 36% (39.1 to 29.1°C) in rabbits. Hematocrits increased about 3% in chicks and 3% to 15% in rabbits. The relatively small increases in hematocrit sug- gest that whole blood is being lost from the circulating 17 portion of the cardiovascular system. D'Amato and Hegnauer (12) and Shukla gt gt. (60) have published data which sup- ports Rodbard's conclusion that whole blood is lost from the circulatory system, perhaps by ”locking" or sequestration in the microcirculation (12,53,60). The increased hematocrits described above might cause a small increase in blood viscosity and hence resistance. Shorrock and Hillman (59), however, observed no significant difference in the hematocrit of cooled rats (35 to 10°C) but did observe a 47% increase in hematocrit when the same blood was cooled to 2°C. The relationship of viscosity to hemato- crit has also been described by Marty gt gt. (35). The yield shear stress (an index of viscosity) of whole blood was observed to increase in children who were cooled from 38 to 27°C during an operative procedure. This increase occurred only when the hematocrit value was over 50%. Reissman and Kapoor (48) report increases in viscosity of 15, 42, and 60% at temperatures of 30, 25, and 22°C, respectively when compared to a 37°C control viscosity (hematocrit was 50, 57, and 51.5%, respectively). Merrill gt gt. (37) confirmed the findings of Marty gt gt. that the yield shear stress remains constant at a given hematocrit level, independent of temperature (up to hematocrit 35). At higher hematocrits yield stress increases as temperature drops. 18 Even a slight increase in blood viscosity due to hypo- thermia can influence the effect of cold on peripheral resistance if the other parameters in the Poiseville's equa- tion are constant. As long as hematocrit and body tempera— ture are normal, the viscous component of resistance to blood flow is quite constant. During moderate hypothermia, blood viscosity increases because of the effects of cold on hematocrit and yield shear stress. These effects result in at least a slight increase in the viscous component of resistance. B. Effect of Cold on Na+-K+ ATPase Activity Na+-K+ dependent adenosine triphosphatase (Na+-K+ ATPase) is an enzyme which resides, among other places, in the membranes of red blood cells and vascular smooth muscle cells and whose activity has been shown to be temperature dependent (14). Ellory gt gt. (14) showed that erythrocyte membrane Na+-K+ ATPase activity was halved by reducing incu- bation temperature from 38 to 30°C. A further dramatic decrease (97.5%) was observed when the temperature was lowered to 10°C. The cold—induced decrease in Na+-K+ ATPase activity in red blood cells might be expected to be associated with a decreased membrane flexibility. Weed gt gt. (73) reduced ATPase activity by substrate deprivation and observed that due to water influx the red cell membrane became turgid and 19 less able to deform. This condition was shown to inhibit smooth movement of the cell through the microcirculation due to the swollen state of the cells. This swollen state also causes an increase in hematocrit. The same effect might occur in red blood cells subjected to cold. Vascular smooth muscle tone has been shown to be de- pendent on the Na+-l(+ ATPase activity (1). As has been previously discussed, cold temperature inhibits Na+-K+ ATPase activity. The lowered activity of the electrogenic Na+—K+ "pump" leads to a decreased net transfer of Na+ ions from the vascular smooth muscle cells into the extracellular fluid and hence cellular depolarization. Ouabain, a well- known inhibitor of Na+-K+ ATPase, substantiates this observa- tion in that upon the addition of ouabain, vascular smooth muscle cells rapidly depolarize while the resistance pro- gressively increases (1). Therefore, the evidence suggests that the response to cold blood perfusion would be a decrease in blood flow via active vasoconstriction. III. Peripheral Vasculature Capacitance During Hipothermia The veins of skin and skeletal muscle contribute rela— tively little to the total vascular resistance in these circuits, but instead play an important role as capac1tance Vessels. Approximately 80 percent of the total blood volume 20 within a given vascular network is contained in the venous system. Furthermore, by actively constricting or dilating, veins are able to mobilize or sequester relatively large amounts of blood. It is a consistent observation that cold exposure, be it local or systemic, elicits venous constric- tion (19,58,68-72). Scott gt gt. (58) found that resistance to flow through small superficial and deep veins of the dog forelimb rose to 313% of control when perfused with 13°C blood. They speculated that increased viscosity contributed to the resistance response. Webb-Peploe and Shepherd (70) reported that locally or centrally cooling the blood perfus- ing superficial dog forelimb veins caused them to constrict. Webb-Peploe (69) reduced central body temperature from 39 to 35°C and observed constriction of saphenous but not of splenic veins. Wood and Echstein (75) observed a 34% de- crease in venous volume of intact human forearms when ambient temperature was reduced to between 21 and 18°C. They also found that local cooling of the forearms caused a 31% decrease in venous volume. According to Webb-Peploe (69), the available evidence concerning superficial venous function during reduced temperatures demonstrates a special- ized thermoregulatory function for these vessels but not for visceral veins. 4.32m- . 21 A. S stemic Hypothermia: Neural Control of Capacitance Vessels According to Ross (55) sympathetic nerve stimulation of veins in the dog's hindpaw produces a relatively slight in- crease in resistance, compared to that of their arterial counterparts. Furthermore, he found that cutaneous veins in the dog displayed intense constriction during sympathetic nerve stimulation, whereas muscle veins in the same animal showed only a very slight constriction. Webb-Peploe and Shepherd (69) also found little or no adrenergic innervation in muscle veins of the hindlimb, but reported that lowering central body temperature from 40 to 33°C elicited intense constriction of canine saphenous veins (70). Since the veins in their preparation were perfused with normothermic blood from a donor dog, they reasoned that the observed venocon- striction was not mediated either humorally or via the direct effect of cold on venous smooth muscle, but rather resulted from a neural reflex triggered by the systemic cooling. In a previous study, Webb—Peploe and Shepherd (70) showed that after sympathectomy, the maximal venoconstriction and thus the maximal blood volume reduction in response to cooling was decreased by about 60%. Webb-Peploe (69) found that reduced body temperature resulted in saphenous venoconstriction which was abolished by lumbar sympathectomy. However, hypothermia did not increase tension in the splenic veins. 22 Therefore, he concluded that the venoconstrictor response to the cooling was dependent on an intact sympathetic nervous system. In contrast, Haddy gt gt. (19) demonstrated that local nerve blockade did not change the venous resist- ance response to cold air exposure. In Haddy's (19) experi- ments cold-induced venous constriction was apparently mediated by circulating catecholamines. His findings differ strikingly from those of Webb-Peploe (69) because of the difference in the type of cooling technique used. One of the objectives of the study described in this thesis is to quantify the role of the sympathetic nervous system in medi- ating venous constriction during systemic cooling. B. Humoral Control Weideman (74) points out that venous smooth muscle shows a great temperature sensitivity in its responsiveness to epinephrine so that a l—2°C fall in temperature causes a 20-fold increase in responsiveness; contrary to that ob— served in arterial smooth muscle. Webb-Peploe and Shephard (70) as mentioned earlier, reduced the maximal saphenous venoconstriction at 27°C by 60% with sympathectomy; subse- quent a-receptor blockade with phenoxybenzamine abolished the remaining 40% of the constriction response. Haddy gt gt. (19) also showed that denervation plus a-adrenergic blockade abolished the venoconstriction normally elicited by cold exposure. 23 C. Local Hypothermia: Direct Effect on Venous Musculature Scott (58) reported that the venous resistance in the canine forelimb increased during local cooling to 13°C. This response was either absent or greatly attenuated after local nerve and a-adrenergic receptor blockade. Similarly, Webb-Peploe (68) abolished the venoconstrictor response associated with local cooling to 27°C by denervation and o-adrenergic receptor blockade. Haddy gt gt. (19) also found, that after nerve block and a-adrenergic blockade, there was no significant venoconstrictor response at an ambient temperature of 0°C. D. Passive Venous Responses Precapillary dilation, such as that elicited by cooling (58), will raise the flow of blood into downstream veins and thereby tend to raise transmural pressure and cause passive venous distention. This passive effect of cold exposure on venous resistance has not been carefully evaluated. IV. Transcapillary Flux During Hypothermia The capillary system is one of the major regulators of body fluid homeostasis. Therefore, it is important to understand how net transcapillary fluid movement is influ- enced by cold-induced hemodynamic changes. A 24 Since capillaries do not contain a smooth muscle layer, active changes in their radii are not possible; blood flow in the microcirculation is dependent on the pre- to post- capillary resistance ratio and the pressure gradient. Therefore, cold-induced changes in arterial or venous resist- ance can affect net transcapillary fluid movement by alter- ing capillary blood volume and hence capillary hydrostatic pressure. A. Systemic Hypothermia Systemic cold stress triggers neural and humoral re- sponses which help to buffer the fall in core temperature. The hemodynamic consequences included a reduced microcircu- latory blood flow due to increased precapillary resistance. Precapillary sphincters have adrenergic innervation which is more prominent in cutaneous than in skeletal muscle vascular networks (45). Precapillary sphincters in muscle are regu- lated primarily by tissue metabolites. PaO2 during systemic cooling increased from 64 mm Hg at 38°C to 82.5 mm Hg at 28°C rectal temperature. Increased small vessel resistance during whole-body cooling has been investigated and related to arteriolar and precapillary sphincter constriction (17,58), increased blood viscosity (55,59), and erthrocyte aggregation (59). Haddy gt gt. (19) attributed the increased small vessel resistance which they observed at an air temperature of 0°C to A 25 precapillary constriction caused by increased levels of circulating epinephrine and norepinephrine. In this study, acute denervation does not significantly reduce the precapil- lary constriction. Svanes gt gt. (64) observed that capil- lary flow in the hypothermic rabbit omentum was reduced even though the arterioles were dilated. Mean arterial pressure showed little change despite the fact that body temperature was lowered to 25°C (82 mm Hg at 38°C to 70 mm Hg at 25°C). Therefore, the decreased flow was due to an increase in blood viscosity. When rectal temperature is reduced to 22°C and below, blood flow in the rabbit ear microcirculation is no longer laminer and the red blood cells appear granular. B. Local Hypothermia The effect of local cooling on transcapillary fluid movement has been indirectly studied by Scott gt il- (58), who report that precapillary dilation along with venous constriction results in a decreased pre/post capillary re- sistance ratio which would predictably increase capillary hydrostatic pressure and lead to a net fluid filtration. Svanes gt gt. (64) found significant increases in plasma colloid osmotic pressure in the hypothermic rabbit omentum during active cooling. They suggest that despite elevated plasma protein concentration, fluid movement across single capillaries was about the same as that observed under normothermic conditions. Therefore, capillary hydrostatic 26 pressure was calculated to be somewhat higher at 25 than at 37°C. During a five-hour period of sustained hypothermia, their microcirculation studies showed little or no change in fluid movement across capillary wall (64). Several independent investigations have demonstrated that progressive reductions in heart rate, systemic arterial blood pressure, and circulating blood volume occurs with systemic cooling. This blood volume reduction could result from "locking" or sequestration in microcirculatory channels, expansion of the venous vascular segment, or net transcapil- lary fluid filtration. Contradictory evidence is shown for the responsiveness of arterial and venous smooth muscle to systemic as well as local cooling. The majority of the investigators agree that vascular responsiveness is greatly reduced during either systemic or local cooling to neural and/or humoral inputs. Both systemic and local cooling has been found to influence vascular resistance by means of increased blood viscosity. This increased viscosity is pos- sibly due to swollen red blood cells, net transcapillary fluid filtration and/or the increased yield shear stress (friction between particles of fluid). The present study is designed to examine the relative contributions of neural, humoral, and direct effects of local and systemic cooling on skin and skeletal muscle vascular resistances, capacitances, and net transcapillary fluid fluxes. METHODS Adult mongrel dogs of either sex, weighing 20-25 Kg, were anesthetized with sodium pentobarbital (30 mg/Kg i.v.), intubated and ventilated with a mechanical respirator (Harvard Apparatus Co. model 607). Supplements of sodium pentobarbital were given as necessary during the surgical and experimental procedures. Rectal temperature and in some experiments, central arterial temperature (aortic arch via left common carotid artery) were monitored continuously with thermistor probes connected to a telethermometer (YSI model B-4043l). The animals were maintained normothermic (rectal temperature of 37.5°C to 38.S°C) throughout surgery with a heating pad (The Walker Co., Inc., Middleboro, Mass.). Skin of the right forelimb was sectioned 3-5 cm above the elbow. The brachial artery was isolated as were the brachial and cephalic veins and the muscles and remaining connective tissue were sectionalized by electro-cautery. The forelimb nerves (median, ulnar, radial, and musculo- cutaneous) were isolated and coated with an inert Silicone spray (Antifoam A, Dow Corning, Midland, Michigan) to pre- vent drying. The humerus was cut and the ends of the narrow cavities packed with bone wax (Ethicon, Inc., Sommerville, 27 28 New Jersey). Blood entered the forelimb only through the brachial artery and exited only through the brachial and cephalic veins. When surgery was completed, sodium heparin (The Upjohn Co., Kalamazoo, Michigan) was administered in an initial dose of 1000 USP units/kg body weight followed by hourly supplements of 300 USP units/kg body weight. Intravascular pressures were measured from saline filled, polyethylene (P.E.) cannulae (Intra-medic tubing, Clay Adams) inserted into the following sites (Figure 1): l) brachial artery via the collateral ulnar artery just above the level of the elbow (P.E. 50; outside diameter (o.d.) = 0.038”); 2) skin small artery from the third super— ficial volar metacarpal artery (P.E. 60; o.d. = 0.048")*; 3) muscle small artery from a vessel supplying a flexor muscle on the upper portion of the forelimb (P.E. 50)*; 4) skin small vein from the second superficial dorsal meta- carpal vein (P.E. 60)*; 5) muscle small vein from one of the deep vessels draining a flexor muscle in the middle portion of the forelimb (P.E. 10, o.d. = 0.024")*; 6) skin venous outflow pressure from the cephalic vein via a side branch at the level of the elbow (P.E. 60); and 7) muscle venous outflow pressure from the brachial vein via a side branch at the level of the elbow (P.E. 60). The small vessel cannula- tion techniques originally described by Haddy was used for *In Series I and II. 29 .mmcowueasccmu Hommo> oumaumnaaw nouuon ow mcfizmuu a“ wo>oEou :mev coflumcmmoem nEfiHohom ocflcmo woumaomw 0:» mo oflumEonom < .H ousmfim H ohswfim mmnmmwmm clamp? mmzmwmma z_w> mmzmmwmd >mm._.m< u..._<§m wqumDE 4445mm mJomDE 4132“,... ZEm 30.: z_m>\. 33.18% m. Equ, 354% 304.“. z_m> ofiqzdmo mmnmmmmd . mmbwmwma mmzmmwmn. z_m> z_w> oj 4<_Io oaamgmoo wan amazemen .mucoEouSmmoE eunumuomEou .moasmmopm msoco> was Hmwuouum .cofiumcmmopa Hmohompoomuuxo may mo mcwzmuv oflumsogom < .N ohzmfim 36 12w... quhowm ‘OOOOOOOOOOOOOOOIO. . * ' | l umemwmm .4 AdmO—Zwu fl mEDm N ouswfim mmDm 2.2.1301.- I .> 2.54% Al I L1 v 20.5% ., x. a o \ \ ‘ W 0.0.00. 8 MU on... .6 F ... v a X .r 6 5.553 <5. 0 $355223 :22 2qu ’\ o .... ...“..H . .. .n....... ‘ . o f... , mmnmwwmadd s um >MW¢WWW ...... ...:... 530528 I 3 v... . .35... 2.13% ....x" .u . m a. ....... . m ...”. "a.“ - mwOZpom 0:» mo uaamEmnum .m ouDMfim m ounwflm l02 332:0 42.2.00 ..:: 9:350 a .35; 9.52.2.3 .5330 .523 05.823» rw 2l4 .b.¢.u Ild + . absuahn3504 HOS—30> lo HH< .m: as :H ousmmohm xhouum Hmflaumhn u umcflw gm mmufimumwmmh HMHQUmm> flaws” Hmuou mm HHmS mm w®UQmHmHm0H Hmasumm> oHumSE cam :fixm Hmpou no «fisuonuomx: xwon-oao:3 can Hmooa mo muuommm .v oHan Figure 18. 89 Weight changes (grams) to local and systemic cooling in denervated, pump-perfused forelimbs. Ordinate shows limb weight changes (grams) and abcissae show rectal and forelimb temperatures (°C). Data represent mean values 1 standard errors in 9 experiments. a* is significant from a at 5% level. b* is significant from b at 5% level. c* is significant from c at 5% level. grams l2.0 l0.0 8.0 6.0 4.0 2.0 -2.0 -4£) ~6C) -8.0 -K10 T I I 90 Limb Weight Change n=9 3* 3* l b* a*c 38 38383838383338 38383837363533333333323302921858284 Rectal Temperature (°C) L$L L A 38 38 38 38 Forelimb Temperature (°C) 38 28 323T Figure 18 91 3.4 1 1.0 grams and was not statistically significant. Rewarming brachial artery blood to 38°C caused limb weight to return toward but stabilize at a value significantly (P‘<0.05) above the initial control weights (venous pres- sures and blood flows were significantly elevated at this point as shown in Appendix B2)- Whole-body cooling with brachial artery blood main- tained normothermic elicited a progressive fall in limb weight. When rectal temperature reached 33°C, limb weight had fallen 5.5 1 0.3 grams and was significantly (P<:0.05) below the value recorded immediately before the onset of whole-body cooling. It was not, however, different from the original normothermic control weight. Rectal temperature was maintained at 33°C and forelimb perfusion temperature was reduced from 38 to 33°C. This maneuver caused a signi- ficant (P<:0.05) weight gain which averaged 3.7 1 0.5 grams. Rewarming brachial artery blood to 38°C while rectal tempera- ture remained at 33°C caused forelimb weight to fall 2.4 1 0.7 grams to a value not significantly different from that immediately before local cooling. Forelimb perfusion temperature was maintained at 38°C and the animals were subjected to further systemic cooling which elicited an additional, progressive weight loss. When rectal temperature reached 28°C, weight of the normothermic limbs was significantly (3.9 grams; P< 0.05) below the value 92 recorded immediately before the onset of whole-body cooling. Limb weight at rectal temperature 28°C and limb temperature 38°C was not, however, different from the original normo- thermic control value. Rectal temperature was maintained at 28°C and forelimb perfusion temperature was reduced from 38 to 28°C. This maneuver caused a significant increase (P‘<0.05) in limb weight which averaged 2.6 1 0.9 grams. Rewarming brachial artery blood to 38°C while rectal tempera- ture remained at 28°C caused limb weight to fall an average of 2.4 1 0.8 grams, to a value not different from that imme- diately before the local cooling procedure. Therefore, at the end of the experiment, rectal temperature was 28°C, forelimb perfusion temperature 28°C and limb weight was significantly below that recorded immediately before the on- set of whole-body cooling, but was not significantly differ- ent from the original normothermic control weight. B. Cutaneous Vascular Resistance Figure 19 reports the effects of local and whole—body hypothermia on vascular resistance in the segment supplied by the brachail artery and drained by the cephalic vein. Cutaneous vascular resistance was calculated by dividing cephalic vein flow in ml per min per 100 gm forelimb weight, into the pressure head (brachial artery minus cephalic vein pressure). Data are expressed as logarithmic means 1 standard errors (see Appendix C). In normothermic dogs, Figure 19. 93 The resistance response of the cutaneous circu- lation to alterations in local and systemic cooling in denervated, pump-perfused forelimbs. Ordinate shows In total skin vascular resistance and abcissae show rectal and forelimb tempera- ture (°C). Data represent mean logarithmic values 1 standard errors from 9 experiments. b* is significant from b at 5% level. 1." (Total Skin Vascular Resistance (mm Hg-min/cc/IOO gms.» 94 F” C) , Cutaneous Vascular Resistance n=9 :5 C) S” (D I“ C) ééfifiiifiééééfiméfifim Rectal Temperature (°C I 3834 28 383838383838 333838383838382‘83‘8T ForelImb Temperature (°C) .J 8r Figure 19 95 cooling brachial artery blood to 33 and then to 28°C elicited a slight fall in cutaneous vascular resistance which was not statistically significant and skin blood flow increased from 7.95 1 1.12 to 11.67 1 1.43 at 33°C and then to 10.40 1 1.57 ml/min/lOO gms at 25°C. Rewarming brachial artery blood to 38°C caused a correspondingly small in- crease in cutaneous vascular resistance. Whole-body cooling with brachial artery blood main- tained normothermic elicited a slight increase in cutaneous vascular resistance which was not statistically significant at any point in the hypothermia protocol. While rectal temperature was maintained at 33°C, forelimb perfusion temperature was reduced from 38 to 33°C. This maneuver caused cutaneous vascular resistance to decrease by an average 16.7% and blood flow increased from 3.17 1 0.38 to 5.95 1 1.27 ml/min/100 gms. This response, although quite small, was statistically Significant (P< 0.05). Rewarming brachial artery blood to 38°C while rectal temperature remained at 33°C caused Skin vascular resistance to return to an average value not signifidantly different from that immediately before local cooling. Forelimb perfusion temperature was maintained at 38°C and the animals were subjected to further systemic cooling. Rectal temperature was then maintained at 28°C and forelimb perfusion temperature was reduced from 38 to 28°C. 96 This maneuver caused a slight decrease in cutaneous vascular resistance which was not statistically significant and skin blood flow increased slightly from 2.21 1 0.30 to 2.92 1 0.53 ml/min/lOO gms. Rewarming brachial artery blood to 38°C while rectal temperature remained at 28°C elicited a corresponding small increase in cutaneous Vascular resist- ance. Therefore, local cold eXposure appears to elicit a slight dilation of the cutaneous vascular segment in denerv- ated forelimbs. This response is appreciably less than that observed for innervated limbs subjected to the same stimulus. Data reported in Figure 19 also indicate that acute denerva- tion eliminates the cutaneous vasoconstriction observed in innervated limbs during whole-body hypothermia. C. Skeletal Muscle Vascular Resistance Figure 20 reports the effects of local and whole-body hypothermia on vascular resistance in the segment supplied by the brachial artery and drained by the brachial vein. Procedures for calculating, normalizing, and expressing data are identical to those described for Figure 19. In normo- thermic dogs, cooling brachial artery blood to 33 and then to 28°C elicited a slight fall in skeletal muscle vascular resistance which was not statistically significant and muscle blood flow increased from 5.21 1 0.68 to 8.14 1 1.33 at 23°C and then to 7.64 1 7.17 ml/min/lOO gms at 28°C. Rewarming brachial artery blood to 38°C caused a correspond- ing small increase in skeletal muscle vascular resistance. Figure 20. 97 Effects of local and systemic cooling on total muscle vascular resistance of denervated pump- perfused forelimbs. Ordinate shows in total muscle vascular resistance and abcissae show rectal and forelimb temperature (°C). Data represent mean logarithmic values 1 standard errors from 9 experiments. a* is significant from a at 5% level. In (Total Muscle Vascular Resistance(mm Hg.min/cc/IOO gmsll 5.0 - 4,0» 3*32 3* 3* 3*C 2.0 ~ b 98 Skeletal Muscle Vascular Resistance n=9 3* l L L + J 1 L J 1 L L k A 1 L A 41 J 383838383736353433333332 3I 3029282828 Rectal Temperature (°C) fififiéméfifiéfiflfiéfifimém Forelimb Temperature (°C) Figure 20 99 Lowering rectal temperature to 33°C while brachial artery blood was maintained at 38°C elicited a slight in- crease in skeletal muscle vascular resistance which was not statistically significant. Rectal temperature was main- tained at 33°C and forelimb perfusion temperature was re- duced from 38 to 33°C. This procedure caused a 10.5% decrease in muscle vascular resistance and increased blood flow from 2.35 1 0.30 to 3.64 1 0.66 ml/min/lOO gms. This change was not statistically significant and rewarming brachial artery blood to 38°C caused a correspondingly small increase in resistance. Core temperature was reduced further while brachial artery blood was maintained at 38°C. This procedure elicited an increase in skeletal muscle vascular resistance which was statistically significant (P< 0.05) at a rectal temperature of 32°C and below (i.e., skeletal muscle vascular resistance was above the original normothermic control value) and decreased blood flow to 1.56 1 0.23 ml/min/100 gms. Rectal temperature was maintained at 28°C and forelimb perfusion temperature was reduced from 38 to 28°C. This maneuver caused a slight decrease in muscle vascular resistance which was not statistically significant and muscle blood flow in- creased to 2.16 1 0.49 ml/min/100 gms. Rewarming brachial artery blood to 38°C while rectal temperature remained at 28°C caused a correspondingly small increase in muscle vascular resistance. 100 Therefore, acute denervation essentially eliminates the vasodilatory effect of local cold exposure to forelimb skeletal muscle. The data described in the preceding para- graphs and reported in Figure 20 also suggest that systemic cooling elicits humorally mediated vasoconstriction since resistance in the denervated skeletal muscle vasculature increased significantly at rectal temperatures below 33°C. D. Total Forelimb Vascular Resistance Figure 21 reports the effects of local and whole-body hypothermia on total forelimb vascular resistance; procedures for normalizing and expressing data are identical to those described for Figure 19. Locally cooling brachial artery blood 1x1 33°C and then to 28°C elicited a slight decrease in total forelimb vascular resistance which was not statis- tically significant. Rewarming brachial artery blood to 38°C caused a correspondingly small increase in total fore- limb vascular resistance. Lowering rectal temperature from 38 to 33°C while brachial artery blood was maintained normothermic failed to elicit a significant change in total forelimb vascular resistance. Rectal temperature was maintained at 33°C and forelimb perfusion temperature reduced from 38 to 33°C. This procedure produced an18.9% decrease in total forelimb vascular resistance (P<<0.05). Rewarming of brachial artery Figure 21. 101 Effects of local and systemic cooling on total vascular resistance in denervated, pump-perfused forelimbs. Ordinate shows in total forelimb vascular resistance and abscissae show rectal and forelimb temperatures (°C). Data represent mean logarithmic values 1 standard errors from 9 experiments. a* is significant from a at 5% level. b* is significant from b at 5% level. Ln (Total Limb Vascular Resistance (mm Hg-min lcc/IOO gms.» 4.0. 3.0 2.0 102 Total Forelimb Vascular Resistance n=9 a*b , L If bt I A .4 3433333332 93029282828 atITe mperature(°C) if 1 1 . . awwmwwwéfig 6 Temperature (°C) 33‘ 91 § 25 Figure 21 103 blood while rectal temperature was maintained at 33°C caused total forelimb vascular resistance to return to a value not significantly different from that immediately prior to local cooling. Further systemic cooling with brachial artery blood temperature maintained at 38°C elicited a significant (P‘<0.05) increase in total forelimb vascular resistance. Rectal temperature was maintained at 28°C and brachial artery blood was cooled from 38 to 28°C. This procedure caused a slight decrease in total forelimb vascular resist- ance which was not statistically significant. Rewarming brachial artery blood to 38°C while rectal temperature remained at 28°C caused a correspondingly small increase in vascular resistance of the circuit supplied by the brachial artery and drained by the brachial and cephalic veins. 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Series I and II: Naturally Perfused, Innervated’Forelimbs A. Forelimb Weight The fall in forelimb weight that accompanied hypother- mia appears to be due primarily to decreases in skin and skeletal muscle vascular capacity. This conclusion is sup- ported by the concomitant increase in forelimb venous resistance. An additional, slow reduction in forelimb weight occurred throughout the two-hour hypothermia period (28°C rectal temperature) and can likely be attributed to net transvascular fluid reabsorption caused by a fall in capil- lary hydrostatic pressure. This interpretation is supported by the reduced small vein pressures observed throughout the two-hour hypothermia period. Furthermore, the pre- to post- capillary resistance ratio almost certainly was increased during this period as evidenced by a marked elevation in small vessel segment resistance as shown in Figures 9 and 12. Haddy 33 El- (19) report that whole-body cold exposure elicits venous constriction. However, they observed that cooling caused a net gain in limb weight which apparently resulted from the mode of forelimb perfusion. Haddy 33 31. 105 106 (19) studied limbs which were pump-perfused at constant flow so that any increase in venous resistance would cause capil- lary hydrostatic pressure to rise even though precapillary resistance also increased. The weight gain which they observed appears, therefore, to have been an experimental artifact. In our study (Series I), the limbs were naturally perfused so that capillary hydrostatic pressure was deter- mined by arterial and venous pressure and by the pre- to postcapillary resistance ratio as described by Pappenheimer and Soto-Rivera (48): TV a 0 Pa + PV Pc = 1+:l ra Where: pressure resistance precapillary postcapillary mo maggom 144 where: yi j = observation on ith dog and jth temperature. 9 variation, nd - number of dogs, n = number of temperature t variations, 7 j = mean of all observations on jth tempera- ture variation, {yl,j’°"’ynd}’ yi. = mean of all observa- tions on ith dog, {yi 1,...,ynt}, and 7 = mean of all ’ observations. The null hypothesis for the ANOVA test is that the expected parameter, KAZ, be H0 : KA2 = 0. The alternative H1 would be H1 : KA2 f 0. If the F-test (TrgatmenfisMS) at P 5 .05 supports H1, then there are significant vascular responses to the 18 experimental temperature variations (systemic and local hypothermia). A. Comparison of Means of Total Skin and Muscle Vascular Resistances, Systemic Blood Pressure andChangein Forelimb Wéight During Iocal and Systemic Hypothermia If the F-test from the ANOVA examination was signifi- cant for those vascular parameters tested, Dunnett's one- sided t-test (P:i0.05) was used to test significance on a paired basis of the individual controls and experimental values across the 18 temperature treatments. The Dunnett test was selected because it holds the error rate on any experimentwise basis. It uses a single range value, calcu- lated as follows: 3753 where 33 =¢/2 (Error M81 145 for each particular seat of controls and experimental values compared, regardless of how many means are to be in a subset. Significance between the control and experimental treatment pairs was found when the calculated Dunnett's value was greater than the tabulated Dunnett's value (tD, 0.05, k, v where k = no. of means less control and v = degress of freedom). B. Comparison of the Resistance ReSponses of Inner- vated and Denervated Limbs During Hypothermia The systemic blood pressure responses in the innerva- ted and denervated forelimbs during hypothermia followed one another relatively close (statistically the same), which is a requirement for comparing resistance changes across the two series (appendix B1 and B2 reports pressure, flow, and temperature data for series III and IV, respectively). Given populations (innervated and denervated forelimbs) with unequal variances, an approximation of t was calculated for the independent samples. The test statistic (ts) for each comparison at the corresponding experimental temperature variation was: xl-x ts= O 512 + SD2 n1 n1) 146 where XI and X0 = mean resistance in corresponding vascular segments of innervated and denervated forelimbs at corre- sponding temperature variation; 512 and SD2 = variances of mean resistances in innervated and denervated forelimbs; nI and 11D = number of observations. This statistic (ts) is not distributed as Student's t. However, the probability for ts can be approximated by treating it as t, but with degrees of freedom: [(SIZ/nl) + (SDZ/nDll2 [(SIZ/DI)2/(HI'1)] + [SDZ/HD)2/(HD'1)] d.f. = If ts exceeded the tabulated t value at 5% level, the null hypothesis (“I = uD) was rejected and the alternative hypothesis (uI # uD) was accepted. REFERENCES FOR STATISTICAL APPENDIX Kirk, R.E. Experimental Design: Procedures for the Behavioral Sciences. Belmont, Calif.: Wadsworth, 1968, p. 94-98, and pp. 191-503. Snedecor, G.W. and W.G. Cochran. Statistical Methods. The Iowa State University Press. Ames, Iowa, 1967, p. 299-337. 11111111111 3 03145 0681 S“ R" VI Nm U" 3 129 ll ilHllHllefllll