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Neural and hormonal control of blood pressure and vascular conductance during hemorrhage in hypertensive… Weichert, Gabriele Elizabeth 1995

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NEURAL AND HORMONAL CONTROL OF BLOOD PRESSURE AND VASCULARCONDUCTANCE DURING HEMORRHAGE IN HYPERTENSIVE RABBITSbyGABRIELE ELIZABETH WEICHERTB.Sc., The University of Waterloo, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Physiology)We accept this thesis as conformingjpTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1995© Gabriele Elizabeth Weichert, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Ubrary shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of__________________The University of British ColumbiaVancouver, CanadaDate / /5DE.6 (2)88)IIABSTRACTThe cardiovascular response to hemorrhage has been well characterized inconscious normotensive rabbits. When the autonomic nervous system is intact,baroreflex mechanisms cause peripheral vasoconstriction and tachycardia, therebymaintaining blood pressure. When the autonomic nervous system is blocked, thevasoconstrictor hormones vasopressin (AVP) and angiotensin II (ANG Il) are releasedearly during hemorrhage, and have significant effects on peripheral vascular tone andmaintenance of blood pressure. In hypertension, the reflex control of blood pressurein response to hemorrhage is not well understood. It was the goal of the experimentsperformed in this thesis to characterize the control of blood pressure and vascularconductance during hemorrhage in conscious renal wrap hypertensive rabbits. Inparticular, direct blood flow measurements allowed the control of hindlimb, mesentericand renal vascular conductance to be characterized. In addition, a morphologicalanalysis of hindlimb, mesenteric and renal blood vessels from renal wraphypertensive rabbits was performed in order to further understand functionalalterations to the vasculature in this hypertensive model.During hemorrhage, renal wrap hypertensive rabbits had an impaired ability tocontrol blood pressure. When the autonomic nervous system was intact, this wasassociated with an impaired ability to control heart rate and hindlimb conductance.When the autonomic nervous system was pharmacologically blocked, the impairedability to control blood pressure was associated with an impairement in hormonallymediated (AVP and ANG II) control of hindlimb vascular conductance. There was no111evidence to suggest that neural or hormonal control of renal and mesenteric vascularconductance was impaired in the hypertensive rabbits during hemorrhage.Morphological analysis of hypertensive blood vessels showed that thehindlimb, renal and mesenteric blood vessels had an increased wall-to-lumen ratioindicative of non-specific vascular hyper-responsiveness.The impaired ability of the hypertensive rabbits to control blood pressureduring hemorrhage was associated with an impaired ability to control the hindlimb, butnot the mesenteric and renal vascular beds. These findings suggest that, in renalwrap hypertensive rabbits, there is either a differential control of sympathetic nerveactivity or that there is a differential degree of vascular reactivity to these individualvascular beds.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Figures viList of Tables ixAcknowledgements xINTRODUCTION IHISTORICAL REVIEW IThe Discovery of the Circulation of Blood IThe Discovery of Vasomotor Control 4The Measurement of Blood Pressure 5Hypertension as a Disease 6SHORT-TERM CONTROL OF BLOOD PRESSURE 8Arterial and Cardiopulmonary Baroreceptors 8Angiotensin II 10Vasopressin 13Local Factors 15CONTROL OF BLOOD PRESSURE DURING HEMORRHAGE 17CARDIOVASCULAR ALTERATIONS IN HYPERTENSION 23Vascular Resistance and Blood Pressure 23Functional Alterations to the Vasculature 26Alterations to the Vascular Endothelium 29Alterations in the Reflex Control of Blood Pressure 30RENAL WRAP HYPERTENSION 34RATIONALE AND HYPOTHESES 37GENERAL METHODS 40ANIMALS 40RENAL WRAP SURGERY 40FLOW PROBE SURGERY 40PREPARATION FOR HEMORRHAGE EXPERIMENT 42HEMODYNAMIC MEASUREMENTS 45HEMORRHAGE PROTOCOL 45DESIGN OF HEMORRHAGE EXPERIMENTS 47DRUG TREATMENTS 47DATA ANALYSIS FOR HEMORRHAGE STUDIES 49STATISTICAL ANALYSIS 51VCHAPTER 1 - THE CONTROL OF HINDLIMB VASCULAR CONDUCTANCE ANDBLOOD PRESSURE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITS 52INTRODUCTION 52HYPOTHESES 52PROTOCOL 53EXPLANATION OF DATA ANALYSIS 54RESULTS 65DISCUSSION 86CHAPTER 2- THE CONTROL OF RENAL AND MESENTERIC VASCULARCONDUCTANCE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITS 99INTRODUCTION 99HYPOTHESES 100PROTOCOL 100RESULTS 104DISCUSSION 134GENERAL DISCUSSION - CONTROL OF BLOOD PRESSURE AND VASCULARCONDUCTANCE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITS 156RENAL WRAP HYPERTENSIVE RABBITS 156HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM INTACT 157HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM BLOCKED 160HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM AS WELL AS AVPAND ANG II BLOCKED 162SUMMARY AND FUTURE DIRECTIONS 164REFERENCES 167vLIST OF FIGURESFigure 1 - Surgical Set-up .41Figure 2 - Renal wrap surgery 41Figure 3- Flow probe surgery 43Figure 4 - Preparation of a rabbit for a hemorrhage experiment 44Figure 5 - Experimental setup 46Figure 6 - Mean arterial pressure as % blood volume is removed during hemorrhagewith the autonomic nervous system (ANS) intact and with the ANS blocked in onerepresentative normotensive rabbit 50Figure 7 - Analysis of mean arterial pressure (MAP, mmHg) during hemorrhage. Datais shown for 9 normotensive test rabbits during hemorrhage with the autonomicnervous system (ANS) intact, and with the ANS blocked 55Figure 8 - Analysis of heart rate (HR, beats/mm) during hemorrhage 58Figure 9 - Analysis of hindlimb flow (HLF, mI/mm/kg) and hindlimb conductance (HLC,ml/min/kg/mmHg) during hemorrhage under control conditions 61Figure 10 - Analysis of hindlimb flow (HLF, mI/mm/kg) and hindlimb conductance(HLC, mI/min/kg/mmHg) during hemorrhage with the autonomic nervous system(ANS) blocked 63Figure 11 - Slope of mean arterial pressure (MAP, mmHg) as % blood volume (%BV)is removed during three successive control hemorrhages (Hem) in normotensiveand hypertensive rabbits 68Figure 12 - Slope of heart rate (HR, beats/mm) as % blood volume (%BV) is removedduring three successive control hemorrhages in normotensive and hypertensiverabbits 69Figure 13 - Slope of % hindlimb conductance (%HLC, expressed as %ml/min/kg/mmHg) as % blood volume (%BV) is removed during three successivecontrol hemorrhages (Hem) in normotensive and hypertensive rabbits 70Figure 14 - Regression lines showing the relationship of % blood volume removedand mean arterial pressure (MAP, mmHg), heart rate (HR, beats/mm), andhindlimb conductance (%HLC, mI/min/kg/mmHg expressed as a % of baseline) innormotensive rabbits 75viiFigure 15 - Regression lines showing the relationship of % blood volume removedand (MAP, mmHg), heart rate (HR, beats/mm), and hindlimb conductance(%HLC, ml/min/kg/mmHg expressed as a % of baseline) in hypertensiverabbits 78Figure 16 - Slope of the regression line representing mean arterial pressure (MAP,mmHg) as % blood volume (%BV) was removed in normotensive andhypertensive rabbits during hemorrhage under four conditions of efferentblockade 81Figure 17 - Slope of the regression line representing hind-limb conductance (%HLC;ml/min/kg/mmHg expressed as a % of baseline) as % blood volume (%BV) wasremoved in normotensive and hypertensive rabbits during hemorrhage under fourconditions of efferent blockade 82Figure 18 - Heart rate (HR, beats/mm) response during hemorrhage. A) Slope of theregression line representing HR as % blood volume (%BV) is removed innormotensive and hypertensive rabbits during control (CT) hemorrhage andduring hemorrhage with hormonal blockade of AVP and All (HB) 84Figure 19 - Sampling of blood vessels for morphological study 103Figure 20- Slope of mean arterial pressure (MAP, mmHg) as % blood volume (%BV)is removed during four successive control hemorrhages (Hem) in hypertensiverabbits 106Figure 21 - Slope of heart rate (HR, beats/mm) as % blood volume (%BV) is removedduring four successive control hemorrhages (Hem) in hypertensive rabbits 107Figure 22 - Slope of % hindlimb conductance (%HLC, ml/min/kg/mmHg expressed asa % of baseline) as % blood volume (%BV) is removed during four successivecontrol hemorrhages (Hem) in hypertensive rabbits 108Figure 23 - Regression lines showing the relationship of % blood volume removedand mean arterial pressure (MAP, mmHg), mesenteric conductance (%MESC,ml/min/kg/mmHg expressed as a % of baseline), and renal conductance(%RENC, ml/min/kg/mmHg expressed as a % of baseline) in normotensiverabbits 113Figure 24 - Regression lines showing the relationship of % blood volume removedand mean arterial pressure (MAP, mmHg), mesenteric conductance (%MESC,mllmin/kg/mmHg expressed as a % of baseline), and renal conductance(%RENC, ml/min/kg/mmHg expressed as a % of baseline) in hypertensiverabbits 116Figure 25- Slope of the regression line representing mean arterial pressure (MAP,mmHg) as % blood volume (%BV) was removed in normotensive and 125VuFigure 26 - Slope of the regression line representing mesenteric conductance(%MESC; ml/min/mmHg expressed as a % of baseline) as % blood volume(%BV) was removed in normotensive and hypertensive rabbits duringhemorrhage under four conditions of efferent blockade 122Figure 27 - Slope of the regression line representing renal conductance (%RENC;ml/minlmmHg expressed as a % of baseline) as % blood volume (%BV) wasremoved in normotensive and hypertensive rabbits during hemorrhage under fourconditions of efferent blockade 125Figure 28 - Change in mean arterial pressure (MAP, mmHg) during a two minuteinterval post-hemorrhage 127Figure 29 - Percent change in mesenteric conductance (MESC) during a two minuteinterval post-hemorrhage 128Figure 30 - Percent change in renal conductance (RENC) during a two minute intervalpost-hemorrhage 129Figure 31 - Wall-to-lumen ratio (%) in mesenteric, hindlimb, and renal vessels fromnormotensive and hypertensive rabbits 133xLIST OF TABLESTable I - Control Rabbits: Baseline pre-hemorrhage values for 3 successivehemorrhages 67Table II - Post-mortem measurements of heart weight in normotensive andhypertensive rabbbits 72Table Ill - Baseline pre-hemorrhage hemodynamic variables in normotensive andhypertensive rabbits under four combinations of efferent blockade 73Table IV - Hypertensive control rabbits: Baseline pre-hemorrhage values for 4successive hemorrhages 105Table V - Baseline pre-drug hemodynamic variables in normotensive andhypertensive rabbits during four successive weeks of experiments 110Table VI - Baseline pre-hemorrhage hemodynamic variables in normotensive andhypertensive rabbits under four combinations of efferent blockade 111Table VII - Morphological characteristics of blood vessels in normotensive rabbits andin renal wrap hypertensive rabbits 150xACKNOWLEDGMENTSIn reflecting on the past four years, I feel I have been lucky to have beena part of this department. I am reminded of the support from fellow gradstudents and co-workers. I am indebted to the helpfulness and encouragementthat has been provided to me by various faculty members. Some specificacknowledgments are in order.I would like to thank my supervisory committee, Ken Baimbridge, BillMilsom, Morley Sutter, Norm Kastings, and Ed Moore for their suggestions andinterest at every stage in the development of my research. Thanks to AlisonBuchan for her assistance in the morphology studies.I would like to thank John Sanker and Joe Tay for their technicalassistance and for the production of my slides and posters. I thank Dave Phelanfor his attention to the care of my rabbits, for teaching me about pop culture, andfor always having an opinion. I thank Zaira Khan and Nancy Kilpatrick for theiradministrative assistance and for always being fun to visit.I would like to acknowledge the technical assistance of Birgitta Woodsand Craig Kamimura who, each in their own era, were wholly responsible for themagical organization of the lab, and for being exceptional friends.I would like to thank Ray Pederson for his friendship and advise, foralways having an open door and an empty glass, and for hosting manymemorable events. Even after the surgery, Ray, I will not soon forget you.I would like to thank my supervisor, Carol Ann Courneya, for plucking mesight-unseen out of Ontario to work in her lab. It is through Carol Ann’sencouragement that I accepted the challenge of a Ph.D.; it is through hercommitment to the lab and her attention to detail that my research has run assmoothly as it has. Beyond her role as a supervisor, I thank Carol Ann for herfriendship, for her trust, for counseling me in my decisions for the future, and forinviting me to experience both Hurricanes and Lucky Cheng’s. I hope toremember Carol Ann’s kindness and diplomacy in my interactions with futurecolleagues.I am grateful to my family in Kanata and Ottawa, for being unbelievablysupportive and interested in my work, and for visiting me. Prepare yourselvesfor round three. Finally, I would like to thank VIad for always listening, and fornever doubting in me the ability to achieve this and so much more.IINTRODUCTIONThe research presented in this thesis was aimed at characterizing the controlof peripheral vascular conductance and blood pressure during a hemorrhagicstimulus in renal wrap hypertensive rabbits. The significance of this research canonly be appreciated in the context of our current knowledge of cardiovascularphysiology. This introduction will serve to outline the history behind the physiologicalunderstanding of the circulation and behind the discovery of hypertension as adisease. A modern review of cardiovascular control mechanisms in normotensionand hypertension will follow. Finally, this introduction will conclude with a descriptionof the rationale and hypothesis on which this doctoral research has been based.HISTORICAL REVIEWThe Discovery of the Circulation of BloodThe history of the understanding that blood circulates spans many centuries.The first recorded evidence of observations on the cardiovascular system is to befound in an Egyptian medical papyrus written around 1500 BC. In it, pulse ischaracterized as having a force and frequency, and is indicated to be a marker ofpatient health (Singer, 1956). The Egyptian papyrus. relates the pulse sensation asarising from blood vessels. Around 400 BC, the writings of the Greek physicianHippocrates describe the pulse as being due to the movement of the blood vesselswhich were traced back to the heart (Singer, 1956). At the time, pulse was commonlybeing measured by touch and this drew attention to the assigned connectionsbetween the heart and the vessels. Hippocrates was the first to separate vessels into2arteries and veins, and he was also the first to suggest a connection between the two(Cournand, 1964). In the third century BC, the Athenian philosopher Aristotle (384-322 BC) made many detailed and thoughtful observations of physiology. In hisstudies of the cardiovascular system, he correctly identified the heart as the center ofmotion of blood in the vessels (Cournand, 1964). He also attached an enormous andexaggerated significance to the heart, labeling it as the center of intelligence (Singer,1956) and of bodily heat (Cournand, 1964).Further advancements in the understanding of the cardiovascular system didnot occur until the first century AD with the coming of one of the great physiologists ofantiquity, Galen of Pergamon. Since Roman law strictly forbade the dissection ofhuman bodies, Galen performed detailed studies of the anatomy of different animals(Rapson, 1982). No doubt, his role as physician to the gladiators offered him ampleexperience in trauma surgery and medicine (Cournand, 1964). Galen described thatfood in the intestines was absorbed by veins and transported to the liver, where bloodwas continuously being produced. In his opinion, venous blood from the liversupplied nutrients to the rest of the body, with some of this blood reaching the rightventricle. With particular imagination, Galen theorized that microscopic pores in theventricular septum allowed for blood to move from the right to the left ventricle, whereit mixed with air and became arterial blood (Rapson, 1982). Galen noted thedifference between arterial and venous blood, recognizing that it was some propertyof air which led to the difference in the colour of blood. He believed that, duringinspiration, air filled the lungs and passed down the pulmonary vein to enter the leftventricle where it altered the blood colour. Galen recognized that the brightly3coloured blood leaving the left ventricle passed through arteries which served todistribute air to the rest of the body.Although Galen identified many important aspects of the circulation, it did notoccur to him that veins and arteries were somehow connected to form a circuit. As aresult of Galen’s educational and political status, his theories were highly esteemed.In fact, Galen’s views on the nature and the action of the heart and blood vesselsremained current for over 1500 years. It was not until the Renaissance in the 15thcentury that a revival in the study of anatomy and physiology occurred.The anatomical studies of Leonardo da Vinci in the late 1400’s were the first toseriously challenge the doctrine of Galen. Among his many observations, da Vincinoted that the pulmonary veins carried blood and not air as Galen had proclaimed(Cournand, 1964). He also showed, by experiment, that cardiac valves operate toallow unidirectional flow and to prevent regurgitation (Rapson, 1982). His work wasnever published in his lifetime, but da Vinci’s many observations served to pique thecuriosity of other physiologists of the time.In the early 1500’s, a Spanish physician by the name of Miguel Servetus madefurther contributions to the study of blood circulation. Servetus became convincedthat the interventricular pores described by Galen did not exist (Rapson, 1982).Servetus went on to describe that blood from the right ventricle was instead pumpedto the lungs to mix with air. He stated that the blood must then pour through to thepulmonary vein to be returned to the heart (Singer, 1956). In essence, he wassuggesting that veins and arteries were somehow connected. Servetus made radicalattempts to relate biblical quotations to his anatomical findings. He did not predict4how his views would affect his contemporaries, and he was burned at the stake underthe charge of heresy (Cournand, 1964).Around the turn of the 17th century, William Harvey, an English physician,presented the first clear and convincing arguments that blood must move in a circuit(Comroe, 1983). Harvey also outlined in detail the nature and function of venousvalves and plainly identified the heart as a muscle.It was the advent of the microscope that finally solved the mystery of bloodcirculation. An Italian physician by the name of Marcello Malpighi is credited with thedevelopment of the early compound microscope in the mid 1600’s (Rapson, 1982). Inhis investigations of lung and bladder tissue, he found that blood passed from arteriesto veins in tiny hair-like tubes. Around the same time Anton van Leeuwenhoek, aDutch cloth merchant with a hobby of lens grinding, was making similar observationsregarding the existence of capillaries by examining the tails of tadpoles (Rapson,1982). Both Malpighi and van Leeuwenhoek published their findings, thussubstantiating Harvey’s theory on blood circulation.The Discovery of Vasomotor ControlIn the early 1700’s, physiologists became particularly curious about the ways inwhich circulatory parameters might be linked to the recent discoveries concerning thenervous system. Pourlois du Petite is credited with the discovery of vasomotornerves in 1727 (Heymans and Folkow, 1964). He reported that sectioning of thecervical sympathetic nerves could induce vasodilation of conjunctival vessels. By the5end of the century, it was speculated that arterial tone must be under some neuralcontrol. In the early 1800’s histologists had become well aware of nerve fibersextending in and around the walls of the blood vessels (Heymans and Folkow, 1964).Around this time, the systematic study of neural vascular control began. In 1851,Claude Bernard described that cutting the cervical sympathetic nerve in a rabbit led tovasodilation of ear vessels (Rapson, 1982). Charles Brown-Sequard added to thisinformation, by showing that stimulation of the cut end of a peripheral sympatheticnerve fiber could induce vasoconstriction (Heymans and Folkow, 1964). BrownSequard also made early observations on the sympatho-adrenal system by notingthat extractions from the adrenal medulla could also induce vasoconstriction. Thestudies performed by these two researchers showed that blood vessels are controlledby tonically active nerve fibers and by circulating substances. By 1863,parasympathetic nerve fibers responsible for some vasodilator effects had also beendiscovered (Heymans and Folkow, 1964). In the late 1800’s, various experiments hadidentified a vasoconstrictor center in the medualla oblongata. It took many years ofresearch to understand the functional significance of these vasomotor fibers. A fullunderstanding of the cardiovascular control centers, as well as the peripheral sensoryreceptors involved in cardiovascular control, was not established until well into thepresent century.The Measurement of Blood PressureThroughout history, it has been well understood that blood in the arteries andveins is under a certain pressure. In 1733, Rev. Stephen Hales made the first6successful measurement of blood pressure by inserting a hollow brass pipe into theneck vessels of a horse. He observed that the rise in blood up a connected glasstube was greater when testing an artery compared to a vein (Cournand, 1964). In1828, Jean-Leonard-Marie Poiseuille improved on Hales’s technique by developingthe less cumbersome mercury manometer. Poiseuille used his manometer to develophis theories on flow resistance in arteries. This work contributed to the formulation ofPoiseuille’s Law of blood flow which states that resisitance is proportional to bloodviscosity and vessel length, but inversely proportional to the fourth power of thevessel radius (Cournand, 1964). The regular measurement of blood pressure inhumans did not occur for many years. In 1896, Scriptoni Riva-Rocci, an Italianphysician, developed the arm sphygmomanometer for the measurement of systolicpressure (Page, 1988). In 1905, a Russian surgeon named Nickolai Korotkoffimproved upon this measurement of blood pressure by advising his students to listento blood flow sounds with a stethoscope instead of feeling for the pulse (Comroe,1983). This allowed for an accurate measurement of both systolic and diastolic bloodpressure. Thus was borne the Riva-Rocci Korotkoff auscultation method formeasurement of blood pressure, a technique widely used and little changed since itsdevelopment.Hypertension as a DiseasePrior to the regular measurement of blood pressure in humans, hypertensionhad already been indirectly identified. In 1836, Richard Bright first described a typeof necrotic kidney disease that was accompanied by an increase in left ventricular7thickness and thickening of the arteries (Pickering, 1964). He characterized theseindividuals as having a hard pulse or an elevation in blood pressure (Comroe, 1983).Physicians formulated the belief that an elevation in blood pressure was essential tomaintain renal perfusion in patients with Bright’s disease. In 1874, FrederickMahomed undertook a study of Bright’s disease only to find that certain patientsexhibited hard pulse and left-ventricular thickening, but displayed no signs of necroticrenal disease (Comroe, 1983). Mahomed went on to make crude measurements ofblood pressure in humans using an early sphygmograph. Based on his findings, hecame to the conclusion that “hypertension is a constant condition in the circulation ofsome individuals” (Pickering, 1964). From that moment on, hypertension became adisease which could by itself be studied. The prevalence of hypertension was onlyrecognized once the pressure cuff auscultation method of blood pressuremeasurement was devised. In 1907, Theodore Janeway published the results of thefirst large-scale study of the history and pathology of human hypertension (Page,1988). His study showed that one in nine patients had a systolic pressure of 165mmHg or higher (Comroe, 1983). At this time, physicians believed that hypertension0should be modified only slightly, lest blood flow to major organs be comprised.Accompanying this belief was the general lack of treatment for hypertension. In 1925,the first surgical sympathectomy was performed on a severely hypertensive patient.The treatment successfully lowered blood pressure while allowing normal urinevolume and composition to be maintained (Comroe, 1983). This surgery opened thedoor for other hypertensive treatments aimed at regulating the autonomic nervoussystem. Other types of treatment soon followed. In 1930, Irving Page performed a8study in which blood pressure was pharmacologically lowered in six patients withvaried hypertensive conditions. In all patients, urea clearance did not change, onceagain showing that an organ perfusion need not be compromised when treatinghypertension (Page, 1988).Since the 1930’s, there has been a boom in hypertension research. We nowrecognize hypertension in many varied forns including essential (with no knowncause), malignant (associated with renal artery stenosis), coarction of the aorta,tumor related, pregnancy related, and others (Comroe, 1983). Major discoveries inphysiology and advancements in chemistry have led to the development of variouspharmacological treatments for the lowering of hypertension. These presently includevasodilators, drugs aimed at dampening the effects of the sympathetic nervoussystem, diuretics, and other agents aimed at hormonal contributors such as the reninangiotensin system.SHORT-TERM CONTROL OF BLOOD PRESSUREArterial and Cardiopulmonary BaroreceptorsThere are two sets of peripheral receptors which play a major role in the shortterm homeostasis of blood pressure. These are the arterial baroreceptors and thecardiopulmonary receptors. The arterial baroreceptors are nerve endings located inthe walls of the aortic arch and the carotid sinus. Arterial baroreceptor nerveafferents are composed mainly of myelinated nerve fibers, with a smaller amount ofnon-myelinated C-fibers (Kirchheim, 1976). When there is a change in bloodpressure, vessels walls stretch accordingly. A deformation of the vessel walls at the9sites of the arterial baroreceptors causes a change in the firing activity of thebaroreceptor afferent nerves (Heymans and Neil, 1958). An increase in bloodpressure has been shown to induce an increase in the frequency of afferent nervefiring, while a decrease in pressure serves to “unload” the baroreceptors, causing adecrease in afferent nerve activity (Bronk and Stella, 1935). However1 the absolutelevel of blood pressure is not the only stimulus for a change in baroreceptor afferentfiring. Baroreceptor nerve firing is also sensitive to the rate of change in bloodpressure (Bronk, 1931). Stretch-sensitive ion channels have been implicated as themechano-electrical transducers of the baroreceptors (Hajduczok et al. 1994). Arterialbaroreceptor afferents synapse within the medulla at the nucleus of the tractussolitarius where modulation of the central cardiovascular drive can occur (Spyer,1982). An elevation in arterial pressure causes a reflex inhibition of sympatheticnerve activity and activation of vagal fibers (Kezdi and Geller, 1968; Kunze, 1972).Conversely, a drop in arterial pressure results in a decrease in vagal activation andloss of sympathetic nerve inhibition. The net result of a drop in pressure is a reflexincrease in sympathetic nerve activity and a withdrawal of vagus nerve tone.Unloading of the arterial baroreceptors therefore causes peripheral vasoconstriction,tachycardia, and a reflex rise in blood pressure.The other set of receptors which contribute to reflex control of blood pressureare the cardiopulmonary receptors. These are a set of diffuse, stretch-sensitivereceptors located in the walls of the atria and the ventricles, and in the pulmonaryvasculature. Innervation of these receptors includes both large myelinated afferentsand small non-myelinated C-fibers. The term “cardiopulmonary baroreceptor” has100become a general term encompassing the reflex actions elicited by both types ofafferent fibers. However, for the most part, it is the activity of the cardiopulmonary C-fibers which have been identified in the reflex control of blood pressure (Thoren,1979). Cardiopulmonary C-fibers are activated by changes in cardiac blood volume(Persson et al. 1989). Discharges from these receptors are sparse, but studies haveshown that they do exert a tonic influence on blood pressure (Bishop et al. 1983).Like the arterial baroreceptors, the cardiopulmonary receptor afferents serve to inhibitsympathetic nerve activity. Decreased cardiopulmonary afferent activity in responseto a decrease in central blood volume causes significant vasoconstriction andtachycardia (Shepherd, 1982). Inputs from both arterial and cardiopulmonaryreceptors have been shown to contribute significantly to renal vasoconstriction duringhemorrhage in anaesthetized animals (Bishop et al. 1983). However, thecardiopulmonary receptors have been shown to contribute very little to renalvasoconstriction in conscious animals during hemorrhage (Courneya and Korner,1991). Cardiopulmonary receptors are not as important as arterial baroreceptors inmediating baroreflex changes to skeletal muscle blood flow in experimental animals(Thoren, 1979). In humans, however, the cardiopulmonary reflexes exert a verypowerful effect on the skeletal muscle vasculature (Zanchetti and Mancia, 1991).Angiotensin IIAngiotensin II is a powerful vasoconstrictive peptide which can contribute tothe control of blood pressure during circulatory stress. This review will focus onangiotensin produced via the classical renin-angiotensin pathway. This pathway11begins with renin release from the kidney and ends with the formation of angiotensinII in the circulation. Tigerstedt and Bergman (1898) first discovered the actions of therenin-angiotensin system when they found that injecting extracts of the kidney couldcause elevations in blood pressure. This extract was later found to contain renin, acatalytic peptide produced and stored by the juxtaglomerular cells of the kidney.When released, renin catalyses the conversion of circulating angiotensinogen toangiotensin I. Angiotensin I is subsequently converted to the physiologically activeangiotensin II by angiotensin converting enzyme produced in the lung. Renin releaseis the rate limiting step in the pathway of angiotensin II production (Skeggs et al.1980).Changes in blood pressure or blood volume can induce the release of renin.One mechanism by which this can occur is through the actions of the renal barostat.Reduced renal perfusion pressure is a powerful local stimulus for lenin release (Davisand Freeman, 1976). Both the cardiopulmonary receptors and the arterialbaroreceptors can influence renin release by varying the sympathetic oufflow to thekidneys (Shepherd, 1982; O’Donnell et al. 1994). A decrease in blood pressure orblood volume detected at these receptors leads to a reflex increase in renalsympathetic nerve activity, thereby causing renin release. Renin release is alsoinduced by a non-cardiovascular stimulus. Systemic sodium depletion leading to adecrease in sodium and chloride ion delivery to the distal tubule can also induce therelease of renin (Zehr et al. 1980).The powerful vasoactive effects of angiotensin II were first described in 1940(Page and Helmer, 1940; Braun-Menedez et al. 1940). In causing contractions in12isolated vessels strips from certain vascular beds, angiotensin II has been shown topossess up to 40 times more potency than noradrenaline (Peach, 1977). In vivostudies have shown angiotenin II to cause the greatest vasocontrictive effects in thevasculature of the gut, skin, and kidney (Mandel and Sapirstein, 1962; Pang, 1983).Angiotensin II can also modulate the actions of the sympathetic nervoussystem. Angiotensin II has been shown to induce catecholamine release from theadrenal medulla (Feldberg and Lewis, 1964), stimulate sympathetic nerve ganglia(Brown et al. 1980), potentiate release of noradrenaline from nerve terminals (Noshiroet al. 1994), decrease the sensitivity of the baroreflex control of heart rate (Reid,1992), and reset the baroreflex control of heart rate to a higher pressure level (Brookset al. 1993). Fujii and Vatner (1985) studied the role of angiotensin II inducedincreases in sympathetic function in the pressor response to exogenous angiotensinII. They found that half of the pressor response to angiotensin II could be attributedto direct vasoconstrictive effects, while half was due to stimulation of the sympatheticnervous system.Angiotensin II has important effects on fluid regulation. It stimulates therelease of aldosterone, causes thirst, and can increase the secretion of vasopressin(Peach and Levens, 1978; McCaa et al. 1978).Recently, the presence of all components of the renin-angiotensin system havebeen found in many peripheral tissues including the brain, the heart, and vascularsmooth muscles (Lee et al. 1993). The discrete formation of angiotensin II in thevasculature is believed to contribute to local tone as well as having trophic effects viaautocrine and paracrine actions (Schelling et al. 1991). With respect to short term13control of blood pressure, in order to reach vasoactive levels of angiotensin II, largeamounts of renin are required to be released from the kidney (Kato et al. 1993). It isconcievable that the local renin-angiotensin system may contribute to acute control ofblood pressure by using renal renin to produce angiotensin II.VasopressinVasopressin is another powerful vasoconstrictive hormone which cancontribute to control of blood pressure during circulatory stress. It also has animportant effect on conservation of water in the kidney. Vasopressin is produced inthe hypothalamus within neurons of the supraoptic and paraventricular nuclei (Buijs,1987). Axons from these neurons project to the posterior pituitary where, uponstimulation, vasopressin is released to the circulation (Boer, 1987). Release ofvasopressin is controlled by both osmotic and blood volume stimuli. Within thehypothalamus, there are receptors sensitive to changes in osmolality. An increase inblood osmolality of I % or less can increase vasopressin release in vivo (Robertson etal. 1977). Vasopressin release is also controlled by alterations in blood volume. Thereceptors involved in this response include the high pressure receptors (the sinoaorticbaroreceptors) and the low pressure (cardiac) receptors. Briefly, these receptorshave been shown to contribute to the release of vasopressin during hemorrhage inboth anaesthetized (Courneya et al. 1989; Courneya et al. 1988; Courneya et al.1989) and conscious animals (Wehberg et al. 1991; O’Donnell et al. 1992; Courneyaet al. 1992). The low pressure cardiac receptors involved in vasopressin release arestretch sensitive receptors located in the atria (Wilson and Ledsome, 1983) and the14ventricles (Wang et al. 1988). Determination of the relative contribution of the arterialand cardiac receptors in the release of vasopressin has been complicated byvariations in experimental protocol. Both rate and degree of a hemorrhage stimuluscan preferentially stimulate one set of receptors (Courneya et al. 1992). In general, ithas been determined that selective stimulation of the cardiac receptors during nonhypotensive hemorrhage causes vasopressin release in dogs (Wang et al. 1988;Goetz et al. 1984). The cardiac receptors appear to play less of a role in vasopressinrelease in anaesthetized rabbits (Courneya et al. 1989), non-human primates(Gilmore et al. 1980), and humans (Goldsmith et al. 1984).Vasopressin is also called antidiuretic hormone owing to its renal effects.Vasopressin acts to increase the permeability of the collecting ducts, resulting in theconservation of water and the formation of concentrated urine. Vasopressin is also apowerful vasoconstrictive agent. The overall potency of vasopressin is similar to thatof angiotensin II (Hofbauer et al. 1984), although there are some regional differencesin the vasoconstrictive effects of these two peptides. Vasopressin is a less potentrenal vasoconstrictor and a more potent mesenteric vasoconstrictor (Altura andAltura, 1977). The plasma levels of vasopressin required to induce maximumantidiuresis are in the lower range of levels necessary to observe pressor effects(Abboud et al. 1990).Vasopressin dampens its own pressor effects through interactions with theautonomic nervous system. Vasopressin facilitates arterial baroreceptor reflex controlof heart rate, cardiac output, and sympathetic nerve activity (Guo et al. 1986).15There are significant interactions between vasopressin and the reninangiotensin system. Basal levels of vasopressin have been shown to inhibit releaseof renin from the kidney (Share, 1988). It has also been suggested that there areinteractions between vasopressin and angiotensin II with respect to their effects onarterial pressure (Share, 1988). However, much of this evidence is contradictory. Aninteraction of these two peptides has been described to cause an attenuation(Elijovich et at. 1984), and augmentation (Ishikawa et at. 1984), or no change (Cowleyet al. 1986) in the expected pressor response.Local FactorsThere are a variety of local responses which can affect vascular tone such thatlocal blood flow and systemic blood pressure are altered. One such response is theautoregulation of blood flow where blood flow is maintained in the face of changingblood pressure. This mechanism is found in most vascular beds and is particularlydeveloped in the renal, mesenteric, cerebral, and skeletal muscle vascular beds(Jones and Berne, 1964; Stein, 1990; Remak et al. 1994). Autoregulation can occurvia two mechanisms: the myogenic response and the metabolic washout response.The vascular myogenic response to changes in blood pressure was firstdescribed by Sir William Bayliss in 1902. He found that arteries respond to increaseddistention by contracting (Bayliss, 1902). Similarly, a decrease in vessel stretch hasbeen shown to cause a decrease in the active wall tension. In this way, blood flowcan be regulated while blood pressure changes.16Autoregulation of blood flow can also occur through the vasodilator effects oflocal metabolites. When local flow is decreased, there is less washout of metabolicby-products. Under these circumstances, vasodilation can occur due to hypoxia,acidosis, adenosine buildup, increased local osmolality, and increased potassium ionconcentration (Olsson, 1981; Granger and Kvietys, 1981). Adenosine, hypoxia, andacidosis all exert their effects through the release of nitric oxide from the vascularendothelium (Jacobson and Pawlik, 1994; Busse et al. 1993). When blood flowincreases, these metabolic by-products and their vasodilator effects are washed out.There is a variety of other locally produced factors which can influence thestate of vascular constriction and likely contribute to basal tone. They includevascular endothelium derived members of the arachadonic acid metabolic pathwaywhich can induce both vasoconstrictor (thromboxane A2, PGF2a, PGB2, PGD2, andleukotrienes C4 and D4) and vasodilator (PGI2 and PGE) effects (Mullane et al.1979; McGiff et al. 1991). Vascular endothelial cells also produce and release thevasodilator nitric oxide and the potent vasoconstrictor endothelin-1 (Luscher et al.1993). These two vasoactive products have been shown to regulate the vascularactions of many substances (Nerem et al. 1993). In addition, nitric oxide andendothelin-1 may also exert their actions in response to local physical stimuli (Busseetal. 1993).The overall level of smooth muscle tone in any vascular bed is an integrationof inputs from the autonomic nervous system, the circulating hormones such asvasopressin and angiotensin II, as well as the actions of local factors described here.17CONTROL OF BLOOD PRESSURE DURING HEMORRHAGEThe first detailed studies of the cardiovascular response to hemorrhage beganduring World War II (Barcroft et al. 1944). Since that time, a bulk of knowledge hasbeen acquired regarding the mechanisms involved in the response to an acutehypovolemia. Understanding the hemodynamic response to hemorrhage allows us tofurther understand the mechanisms whereby blood pressure is acutely controlled aswell as to gain clinical insight on potential treatments aimed at stabilizing bloodpressure during a hemorrhagic event. This review will focus on the neural andhumoral responses to hemorrhage. As the research performed for this thesis projectused conscious rabbits as a model, the review will emphasize our current knowledgeof hemorrhage in this species. Parallels will be drawn to other species includinghumans.In order to mimic the natural state of cardiovascular control mechanisms, it isadvantageous to perform hemorrhage experiments on conscious animals. Certaintypes of anesthesia have been shown to alter components of the autonomic nervoussystem response to blood loss (Montgomery et al. 1982). Furthermore, hemorrhagein conscious animals is characterized by a biphasic response where there is a periodof well maintained blood pressure followed by an acute loss of sympathetic tone onceblood volume is critically reduced. This biphasic response is either absent or greatlyattenuated in anaesthetized animals (Schadt and Ludbrook, 1991). A more completeunderstanding of the hemodynamic response to hemorrhage in animals and humanswill only result from additional experimentation on conscious animals.18The biphasic response to hemorrhage has been clearly demonstrated inconscious rabbits (Quail et al. 1987), dogs (Schwartz and Reid, 1981), and humans(Barcroft et al. 1944). Briefly, phase I is characterized by an excitation of thesympathetic nervous system. Vascular resistance and heart rate increase while bloodpressure is fairly well maintained. After about a 30% loss of blood volume, a rapidwithdrawal of sympathetic nervous tone signals the beginning of phase II. Thissympathoinhibition results in a relative bradycardia, a sudden decrease in vascularresistance, and a precipitous fall in blood pressure (Evans et al. 1992). Themechanisms involved in this sudden loss of sympathetic activity are not wellunderstood. The signal for sympathoinhibition is believed to originate from afferentfibers in the heart (Evans and Ludbrook, 1991), and has a central ö-opioid receptormechanism within the central nervous system (Evans et al. 1991). The remainder ofthis section will focus on the cardiovascular mechanisms contributing to control ofblood pressure during phase I or sympathoexcitation. A discussion of non-autonomicmechanisms, which are important during phase II or when the autonomic nervoussystem is pharmacologically removed, will ensue.During phase I of hemorrhage, conscious rabbits exhibit a progressivetachycardia and a vasoconstriction of the renal, mesenteric, and hindlimb vascularbeds (Korner et al. 1990; Courneya and Korner, 1991). Mean arterial pressure is wellmaintained. Afferent inputs from both the arterial baroreceptors and thecardiopulmonary receptors are known to cause reflex increases in sympathetic neuraldrive (Courneya et al. 1991). Selective denervation of these fibers in rabbits hasshown that the arterial baroreceptors are mostly responsible for vasoconstriction of19the hindlimb bed, tachycardia, and control of blood pressure during hemorrhage(Courneya et al. 1991; Quail et al. 1987). This has been confirmed in conscious dogsduring hemorrhage (Shen et al. 1990). In humans, hemorrhage simulated by lowerbody negative pressure has shown that afferent information from the cardiopulmonaryreceptors also contributes significantly to sympathetic vasoconstrictor drive (Zoller etal. 1972).Chemoreceptors have also been implicated in the reflex activation ofsympathetic nerve activity during hemorrhage (Heymans and Neil, 1958).Chemoreceptor afferent activity has been shown to cause an increase in peripheralsympathetic nerve activity but not in cardiac sympathetic nerve activity (Chien, 1967).It has been suggested that chemoreceptor afferent activity occurs only after theremoval of a moderate amount of blood volume or after the onset of hypotension(Chien, 1967). Therefore, it is unclear whether chemoreceptor activation contributesto the control of blood pressure during phase I of hemorrhage.The hemodynamic role of epinephrine released from the adrenal medulladuring hemorrhage has also been examined. Plasma epinephrine levels do notincrease during a non-hypotens.ive hemorrhage such as that seen during phase I ofhemorrhage in conscious animals (Schadt and Ludbrook, 1991). Furthermore,adrenalectomy or adrenal denervation has no effect on the rate or magnitude ofdecline in mean arterial pressure during moderate (non-hypotensive) hemorrhage inconscious rabbits (Schadt and Gaddis, 1988).The vasoactive hormones angiotensin II and vasopressin have been implicatedin the control of blood pressure during hemorrhage. In conscious rabbits, plasma20levels of renin and vasopressin begin to rise moderately only after the removal ofabout 15% and 25% blood volume respectively (Quail et al. 1987). The release ofvasopressin is mediated almost entirely by cardiac afferents during slow hemorrhage(2% blood volume per minute) (Quail et al. 1987). A greater role is played by thearterial baroreceptors during a more moderate hemorrhage (3% blood volume perminute) (Courneya et al. 1992). Baroreflexes play no role in the release of renin(Courneya et al. 1992) in conscious rabbits during hemorrhage, indicating thelikelihood of a major contribution by the renal barostat (Davis and Freeman, 1976).Although vasopressin and renin release does occur later in phase I of hemorrhage,these hormones have not been shown to contribute significantly to control of bloodpressure in rabbits. Vasopressin receptor blockade and administration of angiotensinconverting enzyme inhibitor have been shown to have no effect on the rate of renal,mesenteric or hindlimb vasoconstriction in conscious rabbits during hemorrhage(Korner et al. 1990; Courneya and Korner, 1991; Courneya et al. 1991). Autonomicreflex mechanisms are, therefore, entirely responsible for control of blood pressureduring phase I of hemorrhage in conscious rabbits.The role of these hormones in control of blood pressure during phase I ofhemorrhage has also been examined in other species. Vasopressin has been shownto contribute somewhat to control of blood pressure during hemorrhage in consciousdogs (Schwartz and Reid, 1981). Conscious rats exhibit a normal cardiovascularresponse to hemorrhage in the presence of a vasopressin antagonist indicating thatthere is little contribution by vasopressin (Fejes-Toth et al. 1988). Although plasmarenin does increase during hemorrhage in dogs and rats and in humans during21simulated hemorrhage, it is still uncertain as to whether angiotensin II plays asignificant role in blood pressure control (Schadt and Ludbrook, 1991).During phase II of hemorrhage (when sympathoinhibition leads to a profoundloss of blood pressure), both vasopressin and angiotensin II have an importantinfluence on peripheral vascular tone and control of blood pressure. Once consciousrabbits enter the hypotensive phase II of hemorrhage, vasopressin levels risedramatically while renin levels continue to increase steadily (Oliver et al. 1990). Inconscious rabbits, treatment with angiotensin converting enzyme inhibitor andvasopressin antagonist impairs vasoconstriction and the immediate recovery of bloodpressure following the termination of hemorrhage in the hypotensive phase(Courneya and Korner, 1991; Korner et al. 1990). This indicates that vasopressinand angiotensin II achieve vasoactive levels by phase II of hemorrhage andcontribute to control of blood pressure. Vasopressin and angiotensin II have beenshown to have a similar importance during phase II of hemorrhage in rats (Fejes-Tothet al. 1988), monkeys (Arnauld et al. 1977), and humans (Sander-Jensen et a!. 1986).Vasopressin and angiotensin II take on an augmented role in control ofvascular conductance and blood pressure during hemorrhage when efferent inputsfrom the autonomic nervous system are removed. When the response to hemorrhagein conscious rabbits was first studied, it was assumed that the autonomic nervoussystem was responsible for the majority of cardiovascular control mechanisms. It wasexpected that, if the autonomic nervous system was pharmacologically blocked, thelocal vasodilation response to a hemorrhage stimulus would be unmasked (see LocalFactors). However, under these conditions Qf hemorrhage in conscious rabbits, total22peripheral resistance was found not to change significantly from baseline (Chalmerset al. 1967). Oliver et al (1990) later showed that renin and vasopressin releaseduring hemorrhage in conscious rabbits is enhanced by autonomic blockade.Furthermore, blocking the actions of vasopressin and angiotensin II during autonomicblockade caused a greater loss of blood pressure for the same volume of hemorrhage(Courneya and Korner, 1991; Korner et al. 1990).The likely stimulus for augmented release of these two hormones duringhemorrhage with autonomic blockade is the nature of blood pressure decline. Duringautonomic nervous system blockade, there is only one phase to the hemorrhageresponse; blood pressure declines rapidly and immediately as blood volume isremoved (Korner et al. 1990). Thus, a greater stimulus for release occurs at thecardiac and arterial receptors (inducing vasopressin release) and at the renalbarostat (inducing renin release). Courneya et al (1991) studied the effect of thesehormones on controlling hindlimb conductance during hemorrhage with autonomicblockade. They found that, for the same volume of hemorrhage, there was a similardegree of vasoconstriction in the hindlimb bed when the autonomic nervous systemwas blocked compared to when it was intact. This augmented vasoconstriction wasattributed entirely to the actions of vasopressin and angiotensin II.Despite the powerful vasoactive effects of enhanced release of vasopressinand renin, blood pressure is not as well maintained when the autonomic nervoussystem is blocked. During autonomic blockade, there is no sympathetic drive toincrease heart rate and cardiac contractility. Under these conditions, cardiac outputdeclines more rapidly as blood volume is removed in conscious rabbits (Korner et al.V 231990). The vasoactive effects of vasopressin and angiotensin II cannot balance therapid decline in cardiac output during hemorrhage with autonomic blockade inconscious rabbits. Thus, with respect to blood pressure control during hemorrhage, itappears that augmented hormonal release alone is not an adequate substitute forautonomic support.Another important compensatory response to hemorrhage is the movement offluids from the extravasculature to the circulation (LaForte et al, 1994). Wheninterstitial oncotic pressure exceeds capillary oncotic pressure, there is a passivemovement of fluids into the capillaries (Price et al, 1993). Korner et al (1990) haveshown that fluid shifts can replace up to 40% of the shed blood volume duringhemorrhage in conscious rabbits. Fluid shifts during hemorrhage can cause adecrease in relative viscosity and, therefore, could effect the dynamics of blood flowto various vascular beds. However, a 20% decrease in hematocrit has been shown tocause only marginal changes to relative viscosity in isolated perfused in vivo studies(Whittaker and Winton, 1933).CARDIOVASCULAR ALTERATIONS IN HYPERTENSIONHypertension is characterized by a number of alterations to the cardiovascularsystem. At the local level, there is an increased resistance to blood flow in mostvascular beds. Functionally, hypertensive blood vessels exhibit altered responses toa number of pressor and dilator stimuli. There are also many lines of evidence thatindicate alterations to the reflex control of blood pressure in hypertension. Thissection will review what is known about these changes to the cardiovascular systemin hypertension.24Vascular Resistance and Blood PressureIn hypertension, there is an increase in peripheral vascular resistance andblood pressure. Studies on human essential hypertension and on experimentalmodels of hypertension have shown that increased vascular resistance can occur viastructural changes to the blood vessels, through vessel rarefaction, and, in somemodels, through an increase in basal sympathetic vascular tone. Changes tovascular structure will first be examined. Extensive morphological alterations to large(lumen diameter> 300 pm) and small (lumen diameter < 300 pm) blood vessels havebeen observed in hypertension (Schiffrin, 1992). Since most resistance to blood flowoccurs in small arteries, it is has been of primary importance to characterize structuralchanges in these vessels. The development of the small vessel myograph led to theaccurate and extensive study of small vessel structure in hypertension. Myographstudies clearly confirmed previous observations that hypertensive vessel walls arethickened relative to the lumen diameters (Heagerty et al. 1993). This results in anincrease in the wall to lumen ratio. According to Folkow (1990), an increase in thewall to lumen ratio is an adaptive process which acts to decrease vessel wall stress inhypertension. An increase in the wall to lumen ratio can occur through the addition ofmaterial to the outer or the inner surface of the vessel wall (Mulvany, 1993). Thistype of structural change has been observed in spontaneously hypertensive rats andin renal hypertensive rats (Mulvany et al. 1985; Deng and Schiffrin, 1991). Inspontaneously hypertensive rats, the increase in vessel wall material has beenreported to be due to both hypertrophy and hyperplasia of vascular smooth muscle25cells (Dilley et al. 1994; Owens, 1987; Mulvany et al. 1985). Alternatively, anincrease in the wall to lumen ratio can occur via the redistribution of wall materialsuch that the total wall material has not changed, but the vessel lumen hasdecreased. This has been termed remodeling of the vessel (Baumbach and Heistad,1989). Vessel remodeling has been shown to occur in small gluteal, subcutaneous,and mesenteric vessels in human essential hypertension (Schiffrin et al. 1993;Mulvany, 1993; Short, 1966), and in skeletal muscle arterioles of renal hypertensiverats (Imig and Anderson, 1991). Folkow (1958) was the first to suggest that structuralchanges to hypertensive blood vessels contribute to increased peripheral resistance.This contribution has been clearly demonstrated by the observation that hypertensivevessels have increased resistance even after maximum vasodilation (Wright et at.1987; Baumbach and Heistad, 1989; Folkow et al. 1970).Another mechanism which can contribute to increased peripheral vascularresistance in hypertension is arteriolar rarefaction. This is a phenomenon wherethere is a loss of arteriolar density once hypertension is established. Arteriolarrarefaction has been reported in spontaneously hypertensive rats (Prewitt et al. 1992)and in renal hypertensive rats (Ono et al. 1989). In both of these models ofhypertension, functional rarefaction (vasoconstrictive arteriolar closure) precededanatomical rarefaction (disappearance of arterioles) in the development ofhypertension. The factors serving to induce functional and anatomical rarefaction areunknown. Using a mathematical model, Greene et al (1989) proposed thatrarefaction could be responsible for up to 20 % of the increased arteriolar resistancein hypertension. The overall contribution of arteriolar rarefaction to total peripheral26resistance in human essential and other experimental models of hypertensionremains to be clarified.The autonomic nervous system has also been implicated in contributing toincreased peripheral resistance in some forms of hypertension. In spontaneouslyhypertensive rats, basal sympathetic tone is elevated (Friberg et al. 1988; Thoren,1987). This has a significant effect on resting vascular smooth muscle tone andblood pressure. An increase in sympathetic drive and a decrease in parasympatheticinhibition has been reported in human essential hypertension (Julius, 1991). Patientswith essential hypertension have also been shown to have an increase in regionalnorepinephrine spillover in response to mental stress (Esler et al. 1991). However, ithas been suggested that increased sympathetic activity is more characteristic ofborderline and mildly hypertensive individuals, and that this characteristic maycontribute to the development of established essential hypertension where there ispoor evidence for sympathetic alterations (Panfilov and Reid, 1994).Functional Alterations to the VasculatureAnother important cardiovascular alteration in hypertension is the functionalresponse to pressor stimuli. Increased responsiveness to the pressor actions ofnorepinephrine, vasopressin, and angiotensin II has been reported in renalhypertensive rats (Deng and Schiffrin, 1991; CoIlis and Alps, 1975; Lariviere et at.1988), in renal wrap hypertensive rabbits (Wright et al. 1987), in spontaneouslyhypertensive rats (Folkow et al. 1970; Mulvany et al. 1980), and in human essentialhypertension (Schiffrin et al. 1993). In these studies, different pressor doses were27used to induce changes in the tension of isolated vessels walls in vitro or to inducechanges to either mean arterial blood pressure or to vascular resistance in vivo.Hyperreactivity was defined as a steeper dose-response curve with a greatermaximum response (Folkow et al. 1970). In most cases, this increased reactivity wasnon-specific and could be attributed to hypertensive structural changes of thevasculature. Evidence for this is provided by the fact that there is often a similaramount of shortening in the vascular smooth muscle cells of both normotensive andhypertensive cells exposed to the same pressor dose (Schiffrin et al. 1993; Deng andSchiffrin, 1991). However, owing to the mechanical advantage of an increased wall tolumen ratio in hypertensive vessels, the overall change in vascular resistance andblood pressure is greater (Folkow et al. 1970). Anatomical rarefaction of arterioleshas also been suggested to contribute to nonspecific hyperreactivity of the actions ofpressor agents in hypertension (Schiffrin, 1992).Certain models of hypertension exhibit some specific alterations in smoothmuscle responsiveness to pressor agents. Norepinephrine induces less active vesselwall tension in renal hypertensive rats (Deng and Schiffrin, 1991). A down-regulationof vasopressin receptors has also been reported in renal hypertensive rats (Lariviereet al. 1988). Schiffrin et al (1993) showed decreased wall tension in response toendothelin-1 in vessels from patients with human essential hypertension. In thesestudies, although there were alterations in the degree of smooth muscle shortening orreceptor number, the overall effect of contraction on vascular resistance was notdifferent from the normotensive controls. Again, this was attributed to the amplifyingeffects of the increased wall to lumen ratio in hypertensive vessels.28There is varied evidence concerning specific alterations to norepinephrineinduced vascular contraction in hypertension. Decreased norepinephrine inducedwall tension has been reported in renal hypertensive rats and in human essentialhypertension (Deng and Schiffrin, 1991; Schiffrin et al. 1993). This effect may berelated to alterations in excitation-contraction coupling. However, others havereported an increased sensitivity of vascular smooth muscle cells to norepinephrineinduced contractions in spontaneously hypertensive rats and in renal wraphypertensive rabbits (Mulvany et al. 1980; Hamilton and Reid, 1983; Hamilton et al.1986). In spontaneously hypertensive rats, the increased sensitivity was masked byan increase in norepinephrine uptake at the nerve terminal (Mulvany et al. 1980). Inrenal wrap hypertensive rabbits, the increased sensitivity was attributed to alterationsin the excitation-contraction coupling, as receptor sites for norepinephrine were notchanged (Hamilton et al. 1986). Any specific alteration in norepinephrine inducedsmooth muscle contraction must be viewed as part of the integrated whole-vesselcontraction response. When the whole-vessel reaction is assessed, it has beenshown that the amplifying effects of an increased vessel wall-to-lumen ratio inhypertension can act to normalize a decreased responsiveness to norepinephrine(Schiffrin et al. 1993; Deng and Schiffrin, 1991). Some hypertensive blood vesselshave exhibited an increase in norepinephrine sensitivity. In these cases, thepresence of an increased wall-to-lumen ratio adds increased non-specificresponsiveness to the norepinephrine sensitivity (Hamilton et al. 1986).29Alterations to the Vascular EndotheliumIt is well documented that functional abnormalities in blood vessels are notlimited to smooth muscle cells in hypertension. There is increasing evidence foralterations in the modulation of endothelium-produced vasoactive factors inhypertensive vessels (Schiffrin, 1992). In spontaneously hypertensive rats and renalhypertensive rats, endothel ium-dependent acetyicholme-induced relaxation ofarterioles is impaired (Dohi et al. 1991; Diederich et al. 1990). There is evidence tosuggest that this impairment is due to increased production of endothelium derivedcontractile factors (Shepherd and Katusie, 1991). Both endothelium derivedprostaglandin-H2 and superoxide have been implicated in this role (Fu-Xiang et al.1992). The impairment of endothelium mediated relaxation in hypertension appearsto be pressure dependent and can be reversed with the normalization of bloodpressure (Lockette et al. 1986). Another alteration in vascular endothelial function inhypertension is the impairment of flow induced dilation. This has been demonstratedin spontaneously hypertensive rats (Kroller and Huang, 1994). An attenuated releaseof endothelial nitric oxide in response to increased blood flow has been implicated inthis impairment. The consequences of altered endothelial function in hypertensiveblood vessels are two-fold. First, an increase in endothelium dependent vasculartone could contribute to the overall level of peripheral vascular resistance. Second,modifications in the production and release of endothelium derived factors could alterthe actions of circulating endothelium dependent vasoactive agents. Althoughalterations to the vascular endothelium in hypertension seem to favour endotheliummediated contraction, the overall contribution of this alteration to the development30and maintenance of hypertension remains to be elucidated (Shepherd and Katusie,1991).Alterations in the Reflex Control of Blood PressureCardiovascular reflex mechanisms have been shown to be altered inhypertension. It was originally believed that impaired cardiovascular reflexes couldcontribute to the development of hypertension. It was McCubbin et al (1956) who firstproposed that baroreceptor reflex dysfunction was a cause and not a consequence ofhypertension. Since that time, a number of studies aimed at characterizingalterations to cardiovascular reflex dysfunction in hypertension have been performed.The first impairment of reflex function discovered in hypertension was that ofbaroreflex heart rate control. Bristow et al (1969) was the first to report impairedbaroreflex control of heart rate in response to pressor agents in human essentialhypertension. Korner et al (1974) expanded on this work to show an impairedbaroreflex control of heart rate in response to both pressor and depressor agents. Asimilar impairment has been confirmed in experimental models of hypertensionincluding the spontaneously hypertensive rat (Minami and Head, 1993), and the renalwrap hypertensive rabbit (West and Korner, 1974; Fletcher, 1984). This refleximpairment was related to a loss of the vagal component of heart rate regulation(West and Korner, 1974; Fletcher, 1984). Early studies have indicated an attenuationof baroreceptor afferent firing in hypertension which was related to decreasedvascular distensibility (Angell-James, 1973). This was not supported by later studies.In spontaneously hypertensive rats there is a poor correlation between general31vascular hypertrophy and impaired baroreflex control of heart rate (Minami and Head,1993). Similarity, there is a poor correlation between carotid vascular hypertrophyand impaired baroreflex control of heart rate in hypertensive humans (Lage et al.1993). In spontaneously hypertensive rats, impaired reflex control of heart rate wasmore closely correlated to cardiac hypertrophy, a common consequence of chronichypertension (Frohlich et al. 1992). It has been proposed that cardiac hypertrophymight alter the cardiopulmonary afferent nerve activity such that there is a centralmodification of the arterial baroreflex control of heart rate (Minami and Head, 1993).This is supported by studies in spontaneously hypertensive rats where the impairedbaroreflex control of heart rate was normalized with regression of ventricularhypertrophy (Head and Minami, 1992). Also, Kingwell et al (1994) have shown thatdogs with ventricular hypertrophy (but not hypertension) have impaired baroreflexcontrol of heart rate. Alternatively, it has been suggested that, in humanhypertension, an increase in basal cardiac sympathetic activity impairs the vagalmediated baroreflex control of heart rate (Munakata et al. 1994). This is supported bystudies in which calcium supplements augmented baroreflex control of heart rate byenhancing parasympathetic activity (Dazai et al. 1994).Reduced baroreflex control of heart rate in hypertension is not, however,representative of an overall baroreflex impairment. Although there is a resetting ofbaroreflex control of vascular resistance and blood pressure in hypertension, there islittle evidence for an impairment. An early study by Mancia et al (1978) used a neckchamber technique to stimulate the carotid baroreceptors in hypertensive humans.No alteration in carotid baroreflex control of blood pressure was observed. In32experimental hypertension, reflex control of blood pressure in response to carotidocclusion was enhanced early in the development of renal wrap hypertension in dogs(Kirby and Vatner, 1987). In this study, the baroreflex response normalized oncehypertension was established. Similarly, there is no change in carotid baroreflexcontrol of hindlimb resistance in established renal wrap hypertension in rabbits(Angell-James and George, 1980). In the same hypertensive model, Guo et al (1983)showed a preservation of pressor induced reflex control of lumbar sympathetic nerveactivity and of hindlimb vascular resistance. In support of these observations,Thames et al (1984) showed that renal wrap hypertensive rabbits have no alterationsin pressor induced aortic baroreceptor afferent nerve activity. This is contrary to workdone by Angell-James et al (1973) who showed impaired aortic nerve firing inresponse to changes in aortic arch perfusion pressures in renal wrap hypertensiverabbits. This impairment was thought to be related to decreased vessel walldistensibility in hypertension.There are some lines of evidence to suggest an impairment of baroreflexcontrol of vascular resistance and blood pressure in hypertension. In renal wraphypertensive rabbits, pressor drugs caused less inhibition of renal sympathetic nerveactivity compared to normotensive rabbits (Thames et aI. 1984). Since no alterationsin afferent firing were detected in this study, central modifications of baroreflexafferents was implicated in the impairment. In human hypertension, an impairment ofmuscle sympathetic nerve activity in response to pressor drugs has been reported(Matsukawa et al. 1991). Evidence for impaired baroreflex control of sympatheticnerve activity is not necessarily evidence for impaired baroreflex control of vascular33resistance. The amplifying effects of structural alterations to hypertensive vesselsmight be expected to normalize an impairement of sympathetic stimulation. Ingeneral, arterial baroreflex control of vascular resistance and blood pressure appearsto be preserved in hypertension.The cardiopulmonary receptors are also known to contribute to reflex control ofsympathetic vasoconstriction and to renin and vasopressin release (Zanchetti andMancia, 1991). Evidence for alterations to the reflexes elicited by these receptors inhypertension is contradictory. The cardiopulmonary reflex has been reported to beunchanged (Trimarco et at. 1986), impaired (Trimarco et al. 1987; Rizzoni et al. 1992;Thames, 1987; Grassi et at. 1988; Grassi et at. 1988), or enhanced (Mark and Kerber,1982; Ricksten and Thoren, 1980) in hypertension. In humans, the cardiopulmonaryreceptors can be selectively studied by applying negative pressure to the lower bodyor by raising the lower body. In humans with essential hypertension, cardiopulmonaryreflex control of forearm vascular resistance, and of renin, vasopressin andnorepinephrine release has been shown to be impaired (Trimarco et al. 1987; Rizzoniet al. 1992; Grassi et al. 1988; Grassi et al. 1988). This is believed to be related toalterations in cardiopulmonary receptor function in the presence of cardiachypertrophy. Support for this theory is given by the observation that treatmentinduced regression of left ventricular hypertrophy improves the attenuatedcardiopulmonary reflex control of forearm vascular resistance (Grassi et al. 1988).This has been contradicted by Trimarco et al (1986) who showed no impairment incardiopulmonary reflex control of forearm resistance in human with ventricularhypertrophy and hypertension. In fact, Mark and Kerber (1982) have shown an34augmented reflex control of forearm vascular resistance in borderline hypertensivehumans. Animal studies show similar contradictions. Renal wrap hypertensiverabbits have an impaired reflex control of renal sympathetic nerve activity duringblood volume expansion (Thames, 1987; Mark and Kerber, 1982). However,spontaneously hypertensive rats exhibit an augmented reflex control of splanchnicsympathetic nerve activity in response to blood volume expansion (Ricksten andThoren, 1980). In general, there is more evidence for an impairment of thecardiopulmonary reflexes in hypertension. When an impairment is present, a role forcardiac hypertrophy has been implicated as a factor leading to reduced sensitivity ofthe cardiopulmonary stretch receptors.RENAL WRAP HYPERTENSIONRenal wrap hypertension was first described by Page in 1939. With an interestin preventing the development of renal cortical collateral circulation in rabbits, Pagewrapped the kidneys in cellophane only to find that hypertension developed 3-4weeks after the procedure (Page, 1939). Renal wrap hypertension has since becomea widely studied model of hypertension. Established renal wrap hypertension ischaracterized by an increase in peripheral resistance and blood pressure (Bolt andSaxena, 1983; Takata et al. 1988; Denton et al. 1983; West et al. 1975). As of yet,the chain of events leading to increased resistance and blood pressure in this modelremains to be fully elucidated. However, a few contributing factors have beenidentified.35Within 3-4 weeks after renal wrap surgery, the presence of cellophane isbelieved to induce an inflammatory reaction leading to the development of a thickfibrous capsule around the kidney (Anderson et al. 1987). This capsule imposes anexternal force on the kidney and presumably induces an increase in intra-renalpressure (Brace et al. 1974; Denton and Anderson, 1989). The mechanical pressureexerted on the kidney by the fibrous capsule would be expected to contribute to anincrease in renal vascular resistance and, hence, total peripheral resistance.However, in established renal wrap hypertension, Takata et al. (1988) showed thatincreased renal resistance makes only about a 10% contribution to the overallincrease in total peripheral resistance. Therefore, additional factors serve to increaseresistance in the renal and other vascular beds.The renin-angiotensin system has also been implicated in the development ofrenal wrap hypertension. Studies aimed at determining such a role have showneither a rise (Denton and Anderson, 1985), or no change in plasma renin levels overthe time of hypertension development (Lewis and Lee, 1971; Campbell et al. 1973)(C.A. Courneya, unpublished observations). Continuous treatment with theconverting enzyme inhibitor Enalapril does blunt the level of hypertension achieved,as well as the degree of change in renal vascular resistance (Denton and Anderson,1985). This treatment may have achieved its effects through the inhibition of the localvascular renin-angiotensin system, and does not necessarily implicate angiotensin IIproduced via the release of renal renin. Although the renin-angiotensin system doesseem to be contributing in some way, it is not wholly responsible for thecardiovascular changes associated with renal wrap hypertension.36Some studies have suggested that an early rise in blood volume and cardiacoutput occurs after the induction of other forms of renal hypertension and that asubsequent vascular autoregulatory response leads to an increase in total peripheralresistance (Guyton et al. 1974). A rise in cardiac output does occur after wrapsurgery (Fletcher et al. 1976). However, this effect occurs equally in wrapped and incontrol rabbits, and is likely a non-specific result of surgery. In this model ofhypertension, there is no evidence to support a role for volume factors duringhypertension development.Despite an incomplete understanding of the factors contributing to thedevelopment of renal wrap hypertension, this model is nonetheless an excellentchoice for studies aimed at characterizing established hypertension. Renal wraphypertension in rabbits is a very stable model of hypertension. The longest reportedstudy on renal wrap hypertensive rabbits describes a stable level of blood pressurefor 10 weeks after the hypertension is established (3-4 weeks after wrap surgery)(Denton et al. 1983). We have confirmed this in our laboratory (Courneya andWeichert, 1995). In the established phase, there are no volume effects contributingto the increase in blood pressure. Established renal wrap hypertension is aresistance mediated form of hypertension. Increased total peripheral resistance aswell as increased resistance of the renal and hindlimb vascular beds has beenreported (Bolt and Saxena, 1983; Takata et al. 1988; Denton et al. 1983; West et al.1975). Vessels from this model of hypertension exhibit properties which indicatestructural changes such as an increased wall to lumen ratio (West et al. 1975; Wrightet al. 1987). In essence, these characteristics are very similar to human essential37hypertension. Therefore, renal wrap hypertension is an appropriate model which canbe used to further our knowledge of cardiovascular alterations in hypertension.RATIONALE AND HYPOTHESESMost studies aimed at examining reflex control mechanism in hypertensionhave selectively stimulated either the arterial baroreceptors or the cardiopulmonarybaroreceptors. Although this has provided us with valuable information on specificalterations to these reflex mechanisms, it has not provided us with any insight on thecombined effects of both arterial and cardiopulmonary baroreflex stimulation. Also,most studies have made isolated observations of the efferent results of baroreceptorstimulation. Observations have included heart rate, efferent sympathetic nerveactivity, and, in some cases, skeletal blood flow. The differences in experimentaldesign between these studies has made it difficult to draw conclusions about theoverall systemic response to baroreflex stimulation in hypertension. Therefore, whatis lacking in our current knowledge is a comr,rehensive understanding of howvascular conductance and blood pressure is regulated in hypertensive animals andhumans. The questions which require answering are 1) What is the net effect ofcombined stimulation of the arterial and the cardiopulmonary receptors? and 2) Howdoes this integrated response serve to alter vascular conductance in the variousperipheral beds? It was the aim of the research presented in this thesis to providesome answers to these questions.There were two main goals to the experiments performed. First to characterizethe control of blood pressure and peripheral vascular conductance duringhemorrhage in conscious renal wrap hypertensive rabbits. Hemorrhage was used as38a tool to stimulate reflex control of blood pressure. In conscious hypertensive rabbits,hemorrhage has been shown to stimulate both the arterial and the cardiopulmonarybaroreceptors (Courneya et al. 1991). In two series of experiments, flow probes wereused to gather information on vascular conductance in the renal, mesenteric andhindlimb (skeletal) vascular beds. Thus, a more comprehensive understanding of theoverall control of peripheral vascular conductance could be obtained. It washypothesized that the integration of structural alterations to the vasculature andalterations to baroreflex control mechanisms would result in an augmented control ofperipheral vascular conductance and blood pressure during hemorrhage in consciousrenal wrap hypertensive rabbits.The second research goal was to characterize the roles of the autonomicnervous system as well the vasoactive hormones vasopressin and angiotensin II inthe control of peripheral vascular conductance and blood pressure duringhemorrhage in conscious renal wrap hypertensive rabbits. These roles have beenpreviously characterized in conscious normotensive rabbits (Courneya and Korner,1991; Korner et al. 1990). Conscious normotensive and renal wrap hypertensiverabbits were hemorrhaged under treatment conditions aimed at isolating the pressoreffects of the autonomic nervous system and of the hormones vasopressin andangiotensin II. In renal wrap hypertensive rabbits, it was expected that peripheralvasoconstriction during hemorrhage would be non-specific and occurring under alltreatment conditions during which pressor inputs were intact. Therefore, it washypothesized that the roles of the autonomic nervous system as well as vasopressin39and angiotensin II in contributing to peripheral vasoconstriction and control of bloodpressure would not change in the renal wrap hypertensive rabbits during hemorrhage.40GENERAL METHODSANIMALSAll experimental procedures were performed on female New Zealand Whiterabbits with an initial weight of 2.0-2.5 kg. Treatment of rabbits was in accordancewith The Guide to Animal Care and Use of Experimental Animals, vol. 1-2, CanadianCouncil of Animal Care, 1980. Throughout the experimental period, rabbits weregiven weekly examinations to ensure that they were healthy, gaining weight steadily,and that body temperature was normal.RENAL WRAP SURGERYSurgery was performed under general Halothane (M.T.C.) anaesthesia withKetamine (Ayerst) induction. The rabbits were prepared for surgery using steriletechniques (Figure 1). A retroperitoneal flank incision was made to expose eachkidney. The kidneys were gently cleared of surrounding fat and wrapped with a 17cmx 13 cm piece of cellophane (Flexell) (Figure 2). The cellophane was held in placewith a loose silk ligature at the base of the kidney. This procedure has been shown toinduce stable hypertension after 3-4 weeks (Bolt and Saxena, 1983). Sham wraprabbits underwent a similar procedure where the kidneys were surgically exposed butnot otherwise disturbed.FLOW PROBE SURGERYOne group of rabbits underwent surgery for the implantation of a hindlimb flowprobe (Transonic Systems Inc.), while another group underwent surgery for the41Figure 1 - Surgical setup. All surgeries were performed under sterile conditions.Figure 2 - Renal wrap surgery. A sheet of cellophane is placed around the bothkidneys and secured with a loose silk ligature.42implantation of both a renal and a mesenteric flow probe (Transonic Systems Inc.).Flow probe surgery was performed under general Halothane (M.T.C.) anaesthesiawith Ketamine (35 mg kg1, Ayerst) and Robinul (0.02 mg kg1, Ayerst) induction.Robinul was used to limit bronchial secretions during surgery. A midline abdominalincision was made and the hindlimb flow probes were placed on the descending aortajust proximal to the iliac bifurcation (Figure 3). Renal flow probes were placed aroundthe left renal artery, and mesenteric flow probes were placed around the celiacmesenteric artery. Flow probe wires were tunneled under the skin and securedsubcutaneously in the neck region.PREPARATION FOR HEMORRHAGE EXPERIMENTHemorrhage experiments began at least 4 weeks after renal wrap or shamwrap surgery. On the day of each hemorrhage experiment, conscious rabbits wereweighed and placed in a restraining box which allowed them to rest comfortably butnot turn around. The preparation of the rabbit is shown in Figure 4. Under localanesthesia, a small incision was made in the neck region to retrieve the flow probewire. For those experiments requiring drug infusions, an ear vein was cannulatedusing a Jelco medical catheter (Johnson and Johnson). For all experiments, bothcentral ear arteries were also cannulated with a Jelco medical catheter. One earartery was catheterized for the measurement of arterial pressure. The second earartery catheter (the bleed catheter) was used for blood removal during hemorrhage.The bleed catheter was attached to heparin rinsed syringes on a Harvard ApparatusInfusion-withdrawal pump (model 22). The above preparations were generally carriedout in 10-15 minutes and with minimal stress to the animal. Following these43Figure 3 - Flow probe surgery. This photo shows the placement of a flow probe(Transonic Systems) around the renal artery. In other procedures, flow probes werealso placed around the descending aorta and around the celiac mesenteric artery.44central ear arteries (cannuiations IFigure 4 - Preparation of a rabbit for a hemorrhage experiment. Under localanesthesia, the flow probe wire is retrieved from the skin flap behind the ears. Bothcentral ear arteries and one peripheral ear vein are cannulated.\ peripheral ear veinprobe wire(s) retrieved45preparations, the rabbits received a bolus injection of Heparin (1000 IU, Sigma) andwere allowed to stabilize for 30 minutes prior to drug infusions and hemorrhage.HEMODYNAMIC MEASUREMENTSThe experimental setup is shown in Figure 5. Phasic arterial pressure wasmeasured through a catheter inserted into a central ear artery and connected to aCoulbourn pressure transducer. Mean arterial pressure (MAP) was calculated usinga Coulbourn blood pressure processor (S-7225). Heart rate (HR) was measured fromthe phasic arterial pressure using a Coulbourn tachograph (S7726). Hind-limb bloodflow (HLF), renal blood flow (RF), and mesenteric blood flow (MF) were eachmeasured in ml min using a Transonic flow meter (T206) attached to the chronicallyimplanted flow probe. All hemodynamic variables were recorded on a Thermal ArrayRecorder (Astro-Med MT-95000). All variables were averaged and recorded using asoftware package from the Baker Institute, Melbourne Australia. Baseline vascularconductance values were calculated as ml min kg mmHg1. During hemorrhage,hindlimb conductance (HLC), renal conductance (RC), and mesenteric conductance(MC) were calculated as a % of baseline value.HEMORRHAGE PROTOCOL0Each hemorrhage experiment consisted of data collection during 1) a 5 minutecontrol period, 2) a hemorrhage period where blood was withdrawn at 4 ml min untilMAP was reduced to 70% of the control value (5-20 minutes depending on drugtreatment), 3) a 2 minute interval period with no further change in blood volume, 4) areinfusion period where blood was returned at 4 ml min1, and 5) a 15 minute period46Figure 5 - Experimental setup. This photo shows the equipment used during ahemorrhage experiment. On the right is the restraining box for the conscious rabbits.The data collection bank is shown in the middle, and the on-line computer system ison the left.47of data collection after reinfusion. Rabbits tolerated this hemorrhage protocol welland showed no signs of discomfort. A hematocrit sample was taken prior to eachhemorrhage experiment.DESIGN OF HEMORRHAGE EXPERIMENTSEach experimental rabbit underwent 4 hemorrhage experiments at least oneweek apart. Each hemorrhage experiment was performed under a differentpharmacological treatment condition to eliminate selectively the pressor systemsinvolved in blood pressure control during hemorrhage. These condition were: 1)Control (CT), no treatment; 2) Hormonal blockade (HB), actions of AVP and synthesisof All blocked; 3) Autonomic nervous system blockade (ANSB); 4) Hormonal andautonomic nervous system blockade (HB + ANSB). To avoid bias, a Latin squareexperimental design was used where different rabbits underwent the four treatmenthemorrhages in different orders. At the end of the experimental period, rabbits wereeuthanized with 480 mg of Euthanol (M.T.C.)DRUG TREATMENTS1) Hormonal Blockade (HB). Rabbits were given Captopril (GEl, Sqibb) at 10pg kg1 min1 iv and an antagonist of the vascular pressor effects of AVP,penicillamine 0-methyltyrosine AVP (AVPA, Sigma) at 30 pg hr iv. These infusionswere carried out for at least 30 minutes prior to hemorrhage. This dose of CEI caninduce a 100-fold shift in the response curve of angiotensin-l induced blood pressureincreases, while this dose of AVPA abolishes the pressor response to 75 ng of AVP(G. Weichert and C.A. Courneya, unpublished observations). 2) Autonomic48Nervous System Blockade (ANSB). In the first series of hemorrhage experiments(hindlimb flow probes), blockade of the autonomic nervous system was induced withthe ganglionic blocker mecamylamine (Sigma) at 10 mg kg iv given over 15minutes. In the second series of experiments (renal and mesenteric flow probes),blockade of the autonomic nervous system was induced by an iv bolus injection ofpentolinium (Sigma) at 5 mg kg1. Blood pressure reductions caused bymecamylamine or pentolinium were counteracted by iv infusion of noradrenaline (NA,Levophed, Winthrop) starting at an infusion rate of 0.17 pg min1 kg. This infusionrate was adjusted every five mm until MAP was restored to ±10 mmHg of the pre-drugvalue. Once MAP was stable, the rate of NA infusion was held constant for theduration of the experiment. The dose of NA used to stabilize MAP ranged from 0.1 to.34 pg min kg and was similar between normotensive and hypertensive rabbits.The effectiveness of autonomic nervous system blockade was tested in each rabbitby eliciting the nasopharyngeal reflex where a puff of cigarette smoke induces reflexvasoconstriction and bradycardia (Wright et al. 1975). Mecamylamine or pentoliniumtreatment each completely abolished these reflex effects. 3) Autonomic NervousSystem Blockade + Hormonal Blockade (ANSB+HB). Mecamylamine orpentolinium infusion was followed by a period of NA adjustment. The rate of NAinfusion was held constant for the duration of the experiment. GEl and AVPAinfusions then began and were allowed to run for at least 30 minutes prior tohemorrhage.49DATA ANALYSIS FOR HEMORRHAGE STUDIESThe portion of hemorrhage data analyzed differed depending on the treatment.When the autonomic nervous system (ANS) was intact (CT or HB), blood pressurewas well maintained and then suddenly declined (Figure 6). This corresponded tophase I and phase II of hemorrhage (see Control of Blood Pressure DuringHemorrhage). The period of well maintained blood pressure was defined bycomputer analysis. The last data point pertaining to this phase was determined byanalyzing the data points in sequence. When three data point in a row differed bytwo or more standard deviations from the regression line, the first data point of thesequence was flagged as the hemorrhage “breakpoint” (Korner et al. 1990). All datapoints from the beginning of hemorrhage to the breakpoint were used to represent thephase of well maintained blood pressure. When the ANS was blocked (ANSB orANSB+HB) blood pressure began to decline rapidly and immediately as blood wasremoved. Linear regression analysis was performed on data pertaining to the periodof well maintained blood pressure (phase I) during CT and HB hemorrhage and on alldata points during ANSB or ANSB+HB hemorrhage. Linear regression analysis wasperformed on the hemodynamics for each separate hemorrhage experiment. A slopevalue and Y-intercept was obtained for each linear regression curve. With thisanalysis, the relationship between the % of blood volume (BV) removed and each ofthe hemodynamic variables measured (MAP, HR, and % change in HLC, RC or MC)was characterized. Blood volume was calculated as 60 ml kg for both normotensive(Courneya et at. 1989) and hypertensive rabbits (C.A. Courneya and G. Weichert,unpublished observations).50100-80-80-40-ANS INTACT0 10-- I I20 30phaBe II40% BLOOD VOLUME REMOVEDFigure 6 - Mean arterial pressureas % blood volume is removed during hemorrhagewith the autonomic nervous system (ANS) intact and with the ANS blocked in onerepresentative conscious, normotensive rabbit. Data were collected at 20 secondintervals. When the ANS was intact, hemorrhage was characterized by phase I andphase Il of hemorrhage. The “breakpoint” defined the transition between these twophases. Linear regression (solid lines) was performed on data points pertaining tophase I of hemorrhage with the ANS intact, and on all data points when the ANS wasblocked.“breakpoint”phaae IANS BLOCKED51STATISTICAL ANALYSISHemorrhage data was evaluated using the slope values obtained from theregression equation. Baseline and hemorrhage data was assessed for normalityusing a Kolmogrov-Smirnov test. Data across experimental treatment and betweennormotensive and hypertensive groups was first analyzed by a repeated measuresANOVA. When appropriate, experimental treatments were analyzed across a group(normotensive or hypertensive) using a randomized block ANOVA followed byDuncans a-posteriori test. Similarly, significance between normotensive andhypertensive groups within treatment conditions (i.e. control hemorrhage) wasanalyzed by an unpaired t-test. All statistical tests were performed at a significancelevel of 5%.052CHAPTER 1 - THE CONTROL OF HINDLIMB VASCULAR CONDUCTANCE ANDBLOOD PRESSURE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITSINTRODUCTIONThere were three aims for the experiments described out in Chapter 1. First, toensure that the hemodynamic response to successive hemorrhage is stable inconscious rabbits. Second to determine the roles of: a) the autonomic nervoussystem, as well as b) vasopressin (AVP) and angiotensin II (ANG II) in the control ofhindlimb conductance (HLC) and mean arterial pressure (MAP) during hemorrhage inhypertensive rabbits. Third, to compare the ability of normotensive and hypertensiverabbits to control heart rate (HR), HLC, and MAP during hemorrhage.HYPOTHESIS1) Baseline hemodynamics and the hemodynamic response to control (no treatment)hemorrhage will be stable for three consecutive weeks of study in both normotensiveand hypertensive rabbits.2) The relative roles of the autonomic nervous system as well as AVP and ANG II inthe control of HLC and MAP will be similar between normotensive and hypertensiverabbits.3) Hypertensive rabbits will have a greater ability to control HLC and MAP duringhemorrhage compared to normotensive rabbits.53PROTOCOLExperiments were carried out on 12 control rabbits (6 normotensive and 6hypertensive) and on 18 test rabbits (9 normotensive and 9 hypertensive). Details ofsurgical methods are described in the General Methods. Two weeks after eithersham or wrap surgery, all rabbits underwent surgery for the implantation of a hindlimbflow probe. Two weeks later, (4 weeks after sham/wrap surgery), experiments began.The protocol was originally designed so that each rabbit underwent 3hemorrhages at least one week apart. This was a conservative protocol based thenumber of weeks the hypertensive rabbits could be expected to remain in goodhealth. The control experiments were based on this design. Once data collection onthe control rabbits began, it was found that the hypertensive rabbits maintained verygood health until the end of the experimental period. Since the test experimentsinvolved four different hemorrhage treatment conditions, the number of hemorrhagesper test rabbit was extended to four.The final protocol for experiments presented in this chapter is as follows.Control rabbits underwent 3 control hemorrhages with at least one week betweeneach successive hemorrhage. Test rabbits underwent 4 successive hemorrhages atleast one week apart. Drug treatments are outlined in General Methods. Briefly,hemorrhage treatments were: 1) Control (CT), 2) Hormonal blockade of vasopressinand angiotensin II (HB), 3) Autonomic nervous system blockade with mecamylamine(ANSB), and 4) Combined autonomic and hormonal blockade (ANSB+HB).54After the test rabbits were euthanized, the hearts were removed, cut into thefour separate chambers, and weighed. The septal walls were included with the leftatrial and left ventricular segments.EXPLANATION OF DATA ANALYSISFigures 7-10 outline the hemodynamic response to hemorrhage in 9normotensive test rabbits. These figures have been included at this point to illustrateclearly the hemorrhagic response as well as to outline the method of data analysis.The complete set of results for the control rabbits and the test rabbits will bepresented following this section. Figure 7 shows the profile of MAP duringhemorrhage under control conditions (CT) and when the autonomic nervous system isblocked (ANSB). Figure 7a demonstrates the biphasic response to hemorrhage whenthe autonomic nervous system is intact. In all rabbits, there is a rapid drop in MAPafter the removal of approximately 30 % blood volume (By). This response wascharacteristic of both control (CT) and hormonal blockade (HB) conditions.Regression analysis was performed on each rabbit’s individual response. Figure 7brepresents the average regression line and its slope value. The analysis onlyincluded data points within phase I of the hemorrhage, where MAP was wellmaintained until the removal of about 30% blood volume. Determination of phase I ofhemorrhage is described in detail in General Methods. Figure 7c illustrates the rapidand immediate decline in MAP as blood volume was removed with the autonomicnervous system blocked (ANSB). Since hemorrhage was terminated once MAPdeclined by 30 %, less blood removal was required under conditions of the55Figure 7 - Analysis of mean arterial pressure (MAP, mmHg) during hemorrhage. Datais shown for 9 normotensive test rabbits during hemorrhage with the autonomicnervous system (ANS) intact, and with the ANS blocked. A) shows raw data for theMAP response during hemorrhage with the ANS intact. B) shows the average linearregression line and its slope value for hemorrhage with the ANS intact. Under theseconditions, linear regression analysis was performed only on data points pertaining tophase I of hemorrhage. C) shows raw data for the MAP response during hemorrhagewith the ANS blocked. D) shows the average linear regression line and its slope valuefor hemorrhage with the ANS blocked. Under these conditions, linear regressionanalysis was performed on all data points. Slope values are mean ± SE.056ANS INTACTA B100 10080 8080 -40801ope= —0.13 +/— 0.440 -20- 20-0- I I I 0-0 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED% BLOOD VOLUME REMOVEDANS BLOCKEDC D100- 1008080-80.60-40- 40- lope= —1.34 +/— 0.2420- 20-0- I I I I 00 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVED57autonomic nervous system blockade (ANSB) compared to control (CT) conditions.This rapid decline in MAP during hemorrhage was characteristic of both autonomicnervous system blockade (ANSB) and combined autonomic nervous system andhormonal blockade (ANSB4-HB). Regression analysis was performed on all datapoints when the autonomic nervous system was blocked. Figure 7d represents theaverage regression line and its slope value. The difference between the slope valuesshown in Figures 7b and 7d demonstrates the significant contribution made by theautonomic nervous system in the control of MAP during hemorrhage.Figure 8 represents the HR response to hemorrhage. When the autonomicnervous system was intact (Figure 8a), HR increased as blood volume was removed.This represented the reflex activation of the autonomic nervous system in response tohemorrhage. At the removal of about 30% blood volume, rabbits entered phase II ofhemorrhage (sympathoinhibition) which was characterized by a sharp drop in HR.This response was characteristic of both control (CT) and hormonal blockade (HB)conditions. Linear regression analysis was performed on data points representativeof phase I of hemorrhage when the autonomic nervous system was intact. Figure 8brepresents the average regression line and its slope value. When the autonomicnervous system was blocked (ANSB), HR remained relatively stable over thehemorrhage period (Figure 8c). This response was also characteristic of combinedautonomic nervous system and hormonal blockade (ANSB+HB). Linear regressionwas performed on all data points when the autonomic nervous system was blocked.The average regression line and its slope value is shown in Figure 8d. Linearregression of the HR response when the autonomic nervous system was58Figure 8 - Analysis of heart rate (HR, beats/mm) during hemorrhage. Data is shownfor 9 normotensive test rabbits during hemorrhage with the autonomic nervous system(ANS) intact, and with the ANS blocked. A) shows raw data for the HR responseduring hemorrhage with the ANS intact. B) shows the average linear regression lineand its slope value for hemorrhage with the ANS intact. Under these conditions, linearregression analysis was performed only on data points pertaining to phase I ofhemorrhage. Phase I was defined from an analysis of the MAP -% blood volumecurve (see Figure 6). C) shows raw data for the HR response during hemorrhagewith the ANS blocked. D) shows the average linear regression line and its slope valuefor hemorrhage with the ANS blocked. Under these conditions, linear regressionanalysis was performed on all data points. Slope values are mean ± S.E.59ANS INTACTA B300- 300-250 -250 -20O• 200150 150.iope=1.3O +/— o.ie100- I I I I IO 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVEDANS BLOCKEDC D300 300 -250- 250-200 -200 -• 1ope0.23 +/— 0.2015O- 150-100- I1000 10 20 SO 40 0 10 20 SO 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVED60pharmacologically blocked was performed only to ensure that HR remained stableduring hemorrhage, indicating a complete block. The slope of HR under theseconditions was not used for statistical analysis.Figure 9 summarizes the hindlimb blood flow (HLF) and HLC changes duringhemorrhage under control (CT) conditions. Absolute values of HLF and HLCdecreased as blood volume was removed (Figures 9a and 9b). In Figure 9c, thevalues for HLC were normalized to pre-hemorrhage baseline values and expressedas a %. Since pre-hemorrhage values for HLC differed between normotensive andhypertensive rabbits, this data transformation allowed us to make relevantcomparisons between the normotensive and hypertensive response. During phase Iof hemorrhage, %HLC declined, indicating a vasoconstriction response. As rabbitsentered phase II of hemorrhage (sympathoinhibition), a sharp increase in %HLC wasobserved in five of the rabbits. This indicated a vasodilation response in parallel withthe known loss of sympathetic tone. The response shown for CT conditions was alsocharacteristic of hormonal blockade (HB). Linear regression was performed on allindividual %HLC data points pertaining to phase I of hemorrhage. The average linearregression line with the average slope value for %HLC during CT hemorrhage isshown in Figure 9d.Figure 10 shows the HLF, HLC and %HLC response to hemorrhage when theautonomic nervous system was blocked (ANSB). All three hindlimb variablesdeclined as blood volume was removed. A decline in %HLC during hemorrhage(Figure 1 Oc) represented a vasoconstriction response. Linear regression wasperformed on all %HLC data points during hemorrhage with the autonomic nervous61Figure 9 - Analysis of hindlimb flow (HLF, mi/mm/kg) and hindlimb conductance (HLC,ml/min/kg/mmHg) during hemorrhage with the autonomic nervous system (ANS)intact. Data is shown for 9 normotensive test rabbits. A) shows raw data for the HLFresponse during hemorrhage. B) shows the calculated data for HLC duringhemorrhage. C) shows HLC normalized to the pre-hemorrhage baseline value(expressed as %HLC). D) shows the average linear regression line and its slopevalue for %HLC during hemorrhage. Under these conditions, linear regressionanalysis was performed only on data points pertaining to phase I of hemorrhage.Phase I was defined from an analysis of the MAP -% blood volume curve. The slopevalue is expressed as mean ± S.E.62ANS INTACTA B0.5 -•30 -Q—. 20 - 0.2-10 0.10.0I I I I I0 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED Z BLOOD VOLUME REMOVEDCo D0.4120-UUI’-I.80- Q 80-100. % 100-: eo-40- 40-______________________.lope—1.5 +/— 0.520- 20-I I I I I I I I I0 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVED63Figure 10 - Analysis of hindlimb flow (HLF, mI/mm/kg) and hindlimb conductance(HLC, mI/min/kg/mmHg) during hemorrhage with the autonomic nervous system(ANS) blocked. Data is shown for 9 normotensive test rabbits. A) shows raw data forthe HLF response during hemorrhage. B) shows the calculated data for HLC duringhemorrhage. C) shows HLC normalized to the pre-hemorrhage baseline value(expressed as %HLC). D) shows the average linear regression line and its slopevalue for %HLC during hemorrhage. Under these conditions, linear regressionanalysis was performed on all data points. The slope value is expressed as mean ±S.E.64ANS BLOCKEDBA0.6 -0.5 -40 -B 0.4-B0.3-b3O.2-—10- B 0.1 -‘-4I I I I I 0.0—0-0 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVEDC D° 120- ° 120C 04 100- < 100.60-80-I_I. I-60- 60-• Cm 40- 40- ilope —1.0 +1— 0.14%C.)20- i 20-0 10 20 30 40 0 10 20 30 40% BLOOD VOLUME REMOVED % BLOOD VOLUME REMOVED65system blocked (ANSB). The average linear regression line with the average slopevalue for %HLC during ANSB hemorrhage is shown in Figure lOd. If Figures 9d andlOd are compared, it is clear that the average slope values for %HLC duringhemorrhage were not significantly different between CT and ANSB hemorrhage. Thiscomparison will be further explored in the Results section (Test Rabbits). Although itis not shown in this series of figures, the hindlimb response to hemorrhage wasdifferent under conditions of combined autonomic nervous system and hormonalblockade (ANSB+HB) compared to ANSB alone(see Slope of HemodynamicVariables During Hemorrhage). During ANSB+HB hemorrhage, %HLC increasedimmediately as blood volume was removed during hemorrhage. This indicated avasodilation response during the hemorrhage.Having presented a general description of the hemodynamic response tohemorrhage and the methods of hemorrhage data analysis, the following Resultssections will examine the actual experimental data including statistical analysis.RESULTSControl RabbitsBoth normotensive and hypertensive rabbits increased in weight over the threeweeks of experiments. From week I to week 3 of experiments, normotensive rabbitsincreased in weight from 3.12±0.18 to 3.32±0.33 kg. Over the same period, theweight gain in the hypertensive rabbits was not different from the normotensiverabbits (3.04±0.13 to 3.43±0.16 kg). One rabbit in each of the normotensive andhypertensive groups had a signal failure of the hindlimb flow probe. Therefore, all66results referring to HLC in the control rabbits are based on n=5. Pre-hemorrhagebaseline values are shown in Table I. In the normotensive group, MAP, HR, and HLCwere unchanged over the three experiments. Hypertensive rabbits had a significantand consistent elevation in pre-hemorrhage MAP compared to normotensive rabbits.HR was not significantly different between normotensive and hypertensive rabbits.However, the pre-hemorrhage hypertensive HR value in the third week was lowerthan the value for the first week. In the hypertensive rabbits, pre-hemorrhage HLCdeclined over the three week period. At week three, hypertensive HLC wassignificantly different from the normotensive value, and from the hypertensive value atweek one. These results indicate the presence of some time-related effects on prehemorrhage hemodynamics in the hypertensive rabbits.Figure 11 depicts the slope of MAP during hemorrhage in normotensive andhypertensive rabbits over three successive hemorrhages, at least one week apart.The normotensive slope values for MAP during hemorrhage did not change over thethree weeks of experiments. In hemorrhages 2 and 3, hypertensive rabbits had asignificantly faster rate of MAP decline compared to normotensive rabbits andcompared to the hypertensive response in hemorrhage 1. Figure 12 shows the slopeof HR increase as % blood volume is removed. The response was stable over threeweeks of experiments and there was no significant difference between normotensiveand hypertensive rabbits. The slope of %HLC during hemorrhage is shown in Figure13. The normotensive slope values for %HLC during hemorrhage did not changeover the three weeks of experiments. Hypertensive rabbits had a slower rate ofdecline (slower vasoconstriction) in %HLC which was67Table I - Control Rabbits: Baseline pre-hemorrhage values for 3 successivehemorrhagesHEMORRHAGE I HEMORRHAGE 2 HEMORRHAGE 3MAP(mmHg)Normotensive (n=6) 81.3 ± 2.8 78.4 ± 2.4 81.8 ± 2.0Hypertensive (n=6) 111.1 ± 4.2 * 114.0 ± 2.7 * 122.3 ± 4.4 *HR(beats/mm)Normotensive (n=6) 189.9 ± 6.5 178.4 ± 9.9 180.9 ± 5.7Hypertensive (n=6) 176.6 ± 4.5 171.6 ± 7.4 152.6 ±9.5 §HLC(ml!minlkglmmHg)Normotensive (n=5) 0.326 ± 0.021 0.284 ± 0.010 0.292 ± 0.002Hypertensive (n=5) 0.256 ± 0.030 0.208 ± 0.034 0.166 ± 0.020All values expressed as mean ± S.E.. Variables recorded are MAP (mean arterialpressure), HR (heart rate), and HLC (hindlimb conductance). * represents p<0.05 ascompared to normotensive value. § represents p<0.05 as compared to hemorrhage 1.680.20.0 fl;—0.2SLOPE OFMAP/%BV 041*0 1—1.0Heml HeinZ Hem3I I Normoteniive[SS4 HyperteniiveFigure 11 - Slope of mean arterial pressure (MAP, mmHg) as % blood volume (%BV)is removed during three successive control hemorrhages (Hem) in normotensive andhypertensive rabbits. Negative slope values indicate a decline in MAP as %BV isremoved. Hemorrhages were performed once a week for 3 weeks. * p<O.05 ascompared to normotensive. @ p<O.05 as compared to Hem 1.693.02.5T T2.0 JSLOPEOF THR/%BV1.51.00.50.0—0.5Heml Hem2 HemSI I NormotenBivetsX4 HypertenaiveFigure 12 - Slope of heart rate (HR, beats/mm) as % blood volume (%BV) is removedduring three successive control hemorrhages in normotensive and hypertensiverabbits. All slope values are expressed as mean ± SE. Positive slope values indicatean increase in HR as %BV is removed. Hemorrhages were performed once a weekfor 3 weeks.0700.50.0SLOPE OF —0.5%HLC/%BV—1.0—1.5 1—2.0Heml Hem2 HemSI I NormotenaiveL\\ HypertenaiveFigure 13 - Slope of % hindlimb conductance (%HLC, expressed as %ml/min/kg/mmHg) as % blood volume (%BV) is removed during three successivecontrol hemorrhages (Hem) in normotensive and hypertensive rabbits. All slopevalues are expressed as mean ± SE. Negative slope values indicate a decline in%HLC (vasoconstriction) as %BV is removed. Hemorrhages were performed once aweek for 3 weeks. * p<0.05 as compared to normotensive71significantly different from the normotensive rabbits for hemorrhages 2 and 3. Again,these results indicate the presence of some time-related effects on the hypertensiveresponse to hemorrhage.Test RabbitsNormotensive test rabbits gained an average of 0.5 kg over the 4 weeks ofhemorrhage experiments. Since the order of experimental treatments wererandomized, the average weight for each treatment was unchanged. Hypertensiverabbits did not grow as quickly and generally weighed 10% less than thenormotensive group. At the end of the study period normotensive rabbits had a leftventricle-to-body weight ratio of 1.18 while hypertensive rabbits had a significantlygreater ratio of 1.83 (55% increased) (Table II). One rabbit in each of thenormotensive and hypertensive groups had a signal failure of the hindlimb flow probe.Therefore, all results referring to HLC in the test rabbits are based on n=8.Baseline hemodynamic dataThe baseline pre-hemorrhage values of MAP, HR, and HLC in bothnormotensive and hypertensive rabbits are summarized in Table Ill. Hypertensiverabbits had a higher resting MAP and lower resting HLC compared to normotensiverabbits. This was independent of treatment groups. Decreased HLC is indicative ofeither an increased resting vascular tone or a structural change to the hindlimbvasculature. In general, HR was also lower in the hypertensive rabbits. Hormonalblockade with the autonomic nervous system intact (HB) caused a decrease in MAPin the hypertensive rabbits. HLC was not changed in either group indicating that72HYPERTENSIVE8.64 ± 0.31 *6.25 ± 0.30 *3.41 ±0.17*2.55 ± 0.09 *1.83 ± 0.07 *All measurements are mean ± S.E.. * represents p<0.05 for normotensive compared tohypertensive.Table II - Post-mortem measurements of heart weight in normotensive andhypertensive rabbbitswhole heart (g)left ventricle (g)body weight (kg)whole heart:body weight (g/kg)left ventricle:body weight (g/kg)NORMOTENSIVE6.85 ± 0.224.42 ± 0.213.82 ± 0.111.81 ± 0.051.18±0.04TABLEIll-Baselinepre-hemorrhagehemodynamicvariablesinnormotensiveandhypertensiverabbitsunderfourcombinationsof efferentblockadeNormotensiveCTHBANSBANSB+HBMAP(mmHg)78.3±0.877.0±2.274.5±2.979.4±1.8HR(beats/mm)188.9±7.0177.4±5.9230.0±8.5*229.2±6.7*HLC(mllminlkglmmHg)0.313±0.0240.280±0.0320.342±0.0330.259±0.020tHypertensiveMAP(mmHg)124.5±3.8§112.0±3.0*114.9±3.0*114.7±4.6HR(beats/min)161.0±3.1§158.1±7.3212.4±7.3*198.0±6.6*I-ILC(ml/min/kg/mmHg)0.140±0.012§0.156±0.010§0.214±0.021*0.160±0.011§tValuesareexpressedasmean±SE.VariablesrecordedareMAP(meanarterial pressure),HR(heartrate),andHLC(hind-limbvascularconductance).MAPandHR,n=9.HLC,n=8.TreatmentconditionsareCT(control),HB(hormonalblockade),ANSB(autonomicblockade),andANSB+HB(combinedautonomicandhormonalblockade).*representsp<0.O5ascomparedtoCT; trepresentsp<O.05forANSBcomparedtoANSB+HB;§ representsP<0.05ascomparedtonormotensivevalue.-.474resting levels of ANG II and AVP did not effect resting vascular tone in the hind-limbbed. A hormone dependent vasodilation in other vascular beds may have contributedto the drop in MAP in the hypertensive rabbits. After blockade of the autonomicnervous system (ANSB), HR increased in normotensive rabbits while MAP wasmaintained at control levels by a constant infusion of NA. ANSB caused baseline HRto increase in hypertensive rabbits but, in contrast to the normotensive rabbits, MAPwas lower than control levels possibly due to an insufficient rate of NE infusion (seeGenera! Methods). ANSB also caused HLC to increase in hypertensive rabbits whichmay be evidence for an increased level of sympathetic tone under normal restingconditions. Blocking the hormones ANG II and AVP in addition to the autonomicnervous system (ANSB+HB) caused no further change in HR or MAP in either groupof rabbits. However, HLC was unexpectedly decreased compared to blockade of theautonomic nervous system alone (ANSB).HemorrhageFigures 14 and 15 serve as a visual representation of the response tohemorrhage in normotensive and hypertensive rabbits. The pattern of hemodynamicchange associated with hemorhage will be described. Following this section, Figures16-18 will outline the statistical differences between the normotensive andhypertensive response to hemorrhage, and between the four different treatmentconditions.Normotensive Response to HemorrhageThe average linear regression curves of the normotensive rabbits’ response tohemorrhage under the four different treatments are shown in Figure 14. As blood75Figure 14 - The relationship of % blood volume removed versus mean arterialpressure (MAP, mmHg), heart rate (HR, beats/mm), and hindlimb conductance(%HLC, ml/min/kglmmHg expressed as a % of baseline) in normotensive rabbits.Hemorrhage conditions were: control (CT; solid lines), hormonal blockade of AVPand All (HB; long dashed lines), autonomic nervous system blocked (ANSB; shortdashed lines), and combined autonomic and hormonal blockade (ANSB-i-HB; dottedlines). Lines are drawn using the regression equations (see Results).76NORMOTENSIVE RABBITS12010080RB60ANSB+HB . ANSB0 10 20 30240• ANSB•220 ANSB+HB CT200,180 /,1600 10 20 30o 120SS ANSB+HB80 ANSB“. RBCTK________________‘ 400 10 20 30% BLOOD VOLUME REMOVED77volume (BV) was removed during control (CT) hemorrhage, MAP declined slowlywhile HR increased and %HLC decreased, indicating vasoconstriction. Hormonalblockade alone (HB) did not appreciably change the pattern of cardiovascularvariables during hemorrhage. When the autonomic nervous system was blocked(ANSB), MAP declined rapidly and %HLC decreased at a rate similar to when theautonomic nervous system was intact indicating the presence of non-neuralvasoconstriction. HR remained relatively stable during the hemorrhages with theautonomic nervous system blocked (ANSB or ANSB+HB). Hormonal blockade inaddition to blockade of the autonomic nervous system (ANSB+HB) resulted in aneven greater rate of MAP decline, while %HLC was seen to increase slightly as BVwas removed, indicating vasodilation of the hind-limb bed.Hypertensive Response to HemorrhageThe average linear regression curves corresponding to the hemorrhagicresponse in hypertensive rabbits are shown in Figure 15. Hypertensive rabbitsgenerally showed a pattern of hemodynamic change similar to the normotensiverabbits during the four hemorrhage treatments. There were however some specificalterations (Figure 14 compared to Figure 15). During all conditions of hemorrhage,hypertensive rabbits had a significantly faster rate of MAP decline as BV wasremoved. The rate of %HLC decline during hemorrhage was attenuated in thehypertensive (CT, HB and ANSB hemorrhages) as compared to the normotensiverabbits. When the autonomic nervous system was intact, HR increased at a fasterrate compared to the normotensive group.78Figure 15 - The relationship of % blood volume removed versus (MAP, mmHg),heart rate (HR, beats/mm), and hindlimb conductance (%HLC, ml/min/kg/mmHgexpressed as a % of baseline) in hypertensive rabbits. Hemorrhage conditions were:control (CT; solid lines), hormonal blockade of AVP and All (HB; long dashed lines),autonomic nervous system blocked (ANSB; short dashed lines), and combinedautonomic and hormonal blockade (ANSB+HB; dotted lines). Lines are drawn usingthe regression equations (see Results).79HYPERTENSIVE RABBITS120CTS100RB8O‘. ‘.ANSBANSB +1113600 10 20 30240CT._. 220•.. ANSB /S 200.... /ANSB+H,/’180a)A‘ 1600 10 20 30120.ANSB+HBS100 ANSBRB80Kj60SK400 10 20 30% BLOOD VOLUME REMOVED80Statistical Analysis of Hemodynamic Slope Values During HemorrhageStatistical comparisons were obtained from an evaluation of the slope valuesof the hemodynamic variables measured during the hemorrhage period (Figures 14and 15). Comparisons were made across the treatment conditions, and between thenormotensive and hypertensive rabbits. The following results specifically addressthese slope value comparisons.Mean arterial pressure (MAP)Figure 16 emphasizes the differences in slope values of MAP betweennormotensive and hypertensive rabbits during hemorrhage under control conditionsand under the 3 conditions of efferent blockade. Under all conditions, the slope ofMAP vs. %BV removed indicated a faster rate of MAP decline in the hypertensive ascompared to the normotensive rabbits. When the autonomic nervous system wasintact, blockade of the hormones AVP and ANG II (HB) had no effect on slope of MAPvs. %BV removed in either normotensive or hypertensive rabbits. When theautonomic nervous system was blocked (ANSB), the slope value for MAP vs. %BVremoved became more negative as compared to the when the autonomic nervoussystem was intact, indicating a faster rate of MAP decline. In both normotensive andhypertensive rabbits, blocking the hormones in addition to the autonomic nervoussystem (ANSB+HB) caused a further increase in the rate of MAP decline. Thisindicated that AVP and ANG II made a significant contribution to maintenance of MAPduring hemorrhage only when inputs from the autonomic nervous system wereremoved.81/ // /4O_3 #/CT HB ANSB ANSB+HBI I NORMOTENSIVEV/A HYPERTENSIVEFigure 16 - Slope of the regression line representing mean arterial pressure (MAP,mmHg) as % blood volume (%BV) was removed in normotensive and hypertensiverabbits during hemorrhage under four conditions of efferent blockade. All slopevalues are expressed as mean ± SE. Treatment conditions were control (CT),hormonal blockade (HB), autonomic nervous system blockade (ANSB) and combinedautonomic and hormonal blockade (ANSB+HB). Negative slope values indicateddeclining MAP as % blood volume was removed. * p<O.05 for normotensive versushypertensive; p<O.05 for CT versus ANSB; # p<O.05 for ANSB versus ANSB+HB.823.E:1CT HB ANSB ANSB+HBI I NORMOTENSIVE/A HYPERTENSIVEFigure 17- Slope of the regression line representing hind-limb conductance (%HLC;ml/min/kg/mmHg expressed as a % of baseline) as % blood volume (%BV) wasremoved in normotensive and hypertensive rabbits during hemorrhage under fourconditions of efferent blockade. All slope values are expressed as mean ± SE.Treatment conditions were control (CT), hormonal blockade (HB), autonomic nervoussystem blockade (ANSB) and combined autonomic and hormonal blockade(ANSB+HB). Negative slope values indicated a decline in %HLC as % blood volumewas removed (vasoconstriction) while positive slope values represented an increasein %HLC as % blood volume was removed (vasodilation). * p<O.05 for normotensiveversus hypertensive; # p<O.05 for ANSB versus ANSB÷HB.83Hind-limb Conductance (HLC)The slope values for the relationship between %HLC as %BV was removed areshown in Figure 17. When the autonomic nervous system was intact (CT or HB),hypertensive rabbits had reduced slope values for %HLC compared to normotensiverabbits during hemorrhage. This indicated less of a hind-limb vasoconstrictiveresponse in the hypertensive compared to the normotensive group. In bothnormotensive and hypertensive rabbits, blockade of the hormones AVP and ANG II(HB) did not significantly alter the slope of the HLC response. After the autonomicnervous system was blocked (ANSB), hypertensive rabbits still exhibited less of avasoconstrictive response during hemorrhage. Under these conditions however, thedifference between the normotensive and hypertensive %HLC response was notsignificant. During ANSB, both groups had a similar rate of fall in %HLC as that seenwhen the autonomic nervous system was intact. This indicated that the actions ofAVP and ANG II alone could maintain hind-limb vascular tone during hemorrhage.This was further evidenced by the fact that removing the inputs from these hormoneswhen the autonomic nervous system was blocked (ANSB+HB) caused the slopevalues of %HLC vs. % BV removed to become positive, indicating vasodilation.Heart Rate (HR)With the autonomic nervous system intact, HR increased as BV was removedas indicated by a positive slope value for the relationship of HR vs. %BV removed(Figure 18a). During control hemorrhage (CT), hypertensive rabbits had a greaterincrease in heart rate for the same %BV removed compared normotensive rabbits.Although this trend was apparent during hormonal blockade (HB), the difference84Figure 18 - Heart rate (HR, beats/mm) response during hemorrhage. A) Slope of theregression line representing HR as % blood volume (%BV) is removed innormotensive and hypertensive rabbits during control (CT) hemorrhage and duringhemorrhage with hormonal blockade of AVP and All (HB). All slope values areexpressed as mean ± SE. Positive slope values indicated an increase in HR as %BVwas removed. B) Ratio of change in HR versus change in mean arterial pressure(MAP, mmHg) at the end of phase I of hemorrhage during control (CT) hemorrhage,and during hemorrhage with hormonal blockade of AVP and All (HB). * p<O.05 fornormotensive versus hypertensive.CHANGEINHR/CHANGEINMAPSLOPEOFHR/XBVoo••••••aa’aa’aaa’aa’aa’a___HN_(Ti86between groups was not significant. The overall change in HR was also related to thechange in MAP. In Figure 18b, MAP and HR were calculated as a change frombaseline to breakpoint BV. During control hemorrhage, a decrease in MAP of ImmHg was related to an average increase in HR of 10.1 beats/minute in thenormotensive rabbits, but only 5.2 beats/minute in the hypertensive rabbits. Duringhormonal blockade hemorrhage, a I mmHg reduction in MAP was associated with aHR increase of 14.1 beats/minute in the normotensive rabbits and 3.7 beats/minute inthe hypertensive rabbits. When HR was therefore related to MAP instead of %BV, animpairment of the HR response in hypertensive rabbits was suggested.Due to ganglionic blockade during ANSB and ANSBi-HB hemorrhage, HRremained stable as %BV was removed. Slope values for HR during hemorrhageunder these conditions were therefore, not presented graphically.DISCUSSIONControl RabbitsThe control group of experiments was designed to test two hypotheses. First,that pre-hemorrhage hemodynamic values would remain constant for bothnormotensive and hypertensive rabbits over three successive hemorrhagesexperiments. Second, that the slope values for MAP, HR, and HLC duringhemorrhage would remain constant in each group of rabbits. In the normotensiverabbits, pre-hemorrhage hemodynamics were unchanged over three consecutiveweeks. Similarly, the slope values for MAP, HR, and HLC during hemorrhage were87also stable for three hemorrhage experiments, one week apart. The results indicatedthat normotensive rabbits tolerated this protocol well and gave the samehemodynamic response for three consecutive weeks. Hemodynamics in thehypertensive rabbits were not stable over the three week period. In this study,hypertensive pre-hemorrhage MAP was elevated and unchanged for the three weekperiod. However, baseline HLC and HR were significantly lower by the third week ofexperiments. With respect to hemorrhage, the MAP and %HLC response wassignificantly different from the normotensive rabbits in weeks two and three. Sincethe normotensive rabbits exhibited no changes in the response over the threeexperiments, it is unlikely that exposure to hemorrhage alone was inducing a changein the response over three weeks in the hypertensive rabbits. The hemodynamicchanges over the three week period in the hypertensive group likely reflectdevelopmental changes related to the hypertension.Denton et al (1983) previously reported stable hypertension in renal wraprabbits for weeks four through eight after the wrap surgery. HR has previously beenshown not to change with the development of renal wrap hypertension in rabbits (Boltand Saxena, 1983; Fletcher et al. 1976). Other studies examining HLC in renal wraphypertensive rabbits have reported a 55% decrease 4% to 5 weeks after surgery(Wright et al. 1987; West et al. 1975). These studies did not, however, evaluatewhether the change in HLC was stable by this period. By week 6 after wrap surgery,the hypertensive control rabbits in the present study achieved a decrease in prehemorrhage HLC of only 43%. It is possible that developmental changes to our renalwrap rabbits occurred slower than others have described. Our experiments began at88four weeks after the wrap surgery. Based on the stable hypertension reported byDenton et al (1983) at four weeks after renal wrap surgery, we had hypothesized thatall hemodynamic variables including the response to hemorrhage would be stable bythis period. Our results indicate that, over the weeks of study (weeks four, five andsix post renal wrap surgery), developmental changes occurred to the hindlimb bedand possibly to the mechanisms governing control of hindlimb conductance and bloodpressure during hemorrhage. We therefore modified the experimental protocol for thestudy performed in Chapter 2. Specifically, we began the hemorrhage experiments atweek five after renal wrap surgery instead of week four. We hypothesized thatdevelopmental changes to the hypertensive rabbits might be stable by this time.The finding that the hypertensive control rabbits were not hemodynamicallystable by week four after wrap surgery had some implications on the results obtainedin the test rabbits. The test rabbits underwent four hemorrhage experiments underthe following treatment conditions: CT, HB, ANSB, ANSB+HB. However, each rabbitbegan the series with a treatment condition which was chosen at random. Therefore,within each hypertensive treatment condition, there was a grouping of rabbits whichwere anywhere from week four to week seven post-renal wrap surgery. A few rabbitsin each hypertensive treatment group were likely hemodynamically unstable. Thiswould introduce a certain degree of variability into the average hemodynamic valuesobtained in the hypertensive rabbits of the test study.89Test RabbitsBaseline valuesBaseline hemodynamic values showed that, under control condition, the renalwrap hypertensive rabbits had a 46 mmHg elevation in MAP which was accompaniedby a 55% reduction in HLC as compared to the normotensive rabbits. This decreasein HLC is greater than that found in the hypertensive control rabbits, most likelybecause of the randomized grouping of rabbits with respect to the number of weeksafter renal wrap surgery. Consistent with the prese°nt study, others have describedestablished renal wrap hypertension as a resistance mediated form of hypertensionwhere cardiac output was normal or decreased while total peripheral conductancewas decreased up to 43% (Bolt and Saxena, 1983; Courneya and Korner, 1991;Fletcher et al. 1976; Takata et al. 1988). In the present study basal levels of AVP andANG II did not have significant effects on vascular tone as indicated by the lack ofchange in HLC once hormonal blockade was established. AVP and ANG II blockadedid cause MAP to decline in the hypertensive rabbits suggesting the possibility ofother vascular beds with tonic sensitivity to AVP and ANG II. Blocking the autonomicnervous system (ANSB) caused a significant increase in hind limb conductance in thehypertensive group suggesting a greater degree of resting sympathetic tone in thehypertensive as compared to the normotensive rabbits. This result must be viewedwith caution however, since as described by West et al (1975), conductance changesmust be compared at a similar MAP before and after autonomic blockade so that theautonomic component of vascular tone is not overestimated. In our results, MAPduring autonomic blockade was slightly lower than control MAP in the hypertensive90group due to insufficient infusion of noradrenaline. In their study of iliac vascular tonein renal wrap hypertensive rabbits, West and Korner (1974) concluded that nonautonomic factors contributed entirely to the reduction in iliac conductance whenmeasurements were made at similar levels of MAP before and after autonomicblockade. In these results, HLC was still 37% less than the normotensive group afterautonomic nervous system blockade, indicating that structural and other nonautonomic factors were contributing to the reduced conductance.HemorrhageHemorrhage in conscious mammals is characterized by two distinct phases asdescribed previously by Schadt and Ludbrook (1991). During phase I, sympatheticneural outputs increased as cardiac output declined with hemorrhage. Once bloodvolume (BV) decreased by about 30%, there was an abrupt shift (breakpoint) tophase II of hemorrhage. Phase II was characterized by a withdrawal of sympatheticvasoconstrictor drive, relative bradycardia and a rapid decline in MAP. There wasspeculation that, in rabbits, the signal initiating the shift from phase I to phase II ofhemorrhage originated in cardiopulmonary afferents (Schadt and Ludbrook, 1991). Inthe present study, the shift between phase I and phase II of hemorrhage occurred atabout 30% of BV removed in both normotensive and hypertensive rabbits duringcontrol hemorrhage. Breakpoint occurred at about 25 % BV removed duringhemorrhage with the hormones AVP and ANG II blocked. This may have beenrelated to a slightly faster (but non-significant) rate of MAP decline during hormonalblockade hemorrhage in both normotensive and hypertensive rabbits. The discussionof the hemorrhagic response under control and hormonal blockade conditions has91been limited to phase I where sympathetic drive was active. When the autonomicnervous system was pharmacologically blocked (ANSB or ANSB+HB) thehemodynamic response to hemorrhage had only one phase where MAP declinedrapidly and immediately as BV was removed.Under control conditions, hemorrhage in normotensive rabbits wascharacterized by hind-limb vasoconstriction, an increase in heart rate and wellmaintained MAP up to the breakpoint. Blocking the actions of AVP and ANG II hadno effect on the slope values for %HLC, HR or MAP in both normotensive andhypertensive rabbits. Therefore, AVP and ANG II played no significant role ininducing hind-limb vasoconstriction or maintaining MAP during hemorrhage when theautonomic nervous system was intact as described previously in consciousnormotensive rabbits (Korner et al. 1990). This was also true for the conscioushypertensive rabbits. When all systems were fully intact, it was the reflex actions ofthe autonomic nervous system that were responsible for hind-limb vasoconstriction,tachycardia and maintenance of MAP during hemorrhage in conscious rabbits.When the autonomic nervous system was blocked but the hormones were stillintact (ANSB), the rate of MAP decline was much greater than the hemorrhageconditions with the autonomic nervous system intact. In normotensive andhypertensive rabbits, the slope of %HLC during hemorrhage was not significantlychanged from when the autonomic nervous system was intact. Other studies haveshown that AVP and ANG II are released early during hemorrhage when theautonomic nervous system is blocked in both normotensive (Oliver et al. 1990) andhypertensive rabbits (Courneya and Weichert, 1995). This augmented hormone92release could account for the full preservation of the hind-limb vasoconstrictiveresponse during ANSB hemorrhage. Despite preservation of hind-limbvasoconstriction MAP was not as well maintained as when the autonomic nervoussystem was intact. During ganglionic blockade, HR remained stable as blood volumewas removed. This lack of support from HR certainly contributed to the faster rate ofMAP decline during hemorrhage with the autonomic nervous system blocked. At thesame time, we cannot predict whether all vascular beds maintain the same level ofvasoconstriction with hormonal pressor stimuli alone. The ability of AVP and ANG IIto induce hind-limb vasoconstriction when the autonomic nervous system wasblocked was further exemplified by the hemorrhagic response when the actions ofthese hormones were blocked in addition to the autonomic nervous system(ANSB+HB). Under these conditions, all vasoconstrictive stimuli were removed and%HLC increased as blood volume was removed. This was believed to be a localvasodilation response typical of tissue challenged by hypoxic stimuli (Jones andBerne, 1964). The rate of MAP decline increased further during ANSB+HB, indicatingan overall loss of peripheral vascular tone as blood volume was removed.In general, the hypertensive rabbits exhibited an impaired vascular reflexresponse to hemorrhage. Under control conditions with all systems intact, the rate ofhind limb vasoconstriction as blood volume was removed was 2.9 times slower thanthat of the normotensive group. Blocking the hormones had no effect on thisresponse as discussed earlier. This impairment could have been the result of either areduction in baroreflex efferent activity, a central dampening of the reflex response, ora lack of responsiveness of the vasculature to sympathetic stimulation. In this study,93hemorrhage caused a maximum change in HLC of 33% in the hypertensive rabbits. Ithas been shown previously in conscious renal wrap hypertensive rabbits that a 90%reduction in HLC can be achieved with the administration of exogenousvasoconstrictors (Wright et al. 1987). This indicated that the impaired hind limbvasoconstrictive response in the hypertensive rabbits during hemorrhage was not afunction of a lack of vascular responsiveness. The attenuation of the hind-limbvasoconstrictor reflex response was more likely an alteration in baroreceptorsensitivity or a central dampening of the baroreflex pathway. Arterial baroreceptorsand cardiopulmonary receptors have both been shown to contribute to the control ofhind limb vasoconstriction during hemorrhage (Courneya et al. 1991). Reflexpathways from each set of these receptors have been studied in hypertension. Guoet al showed that arterial baroreflex reductions in hind limb conductance due tonitroglycerin infusion were unchanged in renal hypertensive rabbits (Guo et al. 1983).Angell-James and George (1980) reported renal hypertensive rabbits to have anaugmented hind limb vasoconstrictive response to step-wise reductions in carotidsinus pressure. However, when these data were normalized with respect to baselinedifferences in hind limb conductance, the response was similar betweennormotensive and hypertensive rabbits. In general there was no strong evidence foralterations in the arterial baroreflex control of hind limb conductance in hypertension.This was true despite evidence for hypertrophy induced alterations in arterialbaroreceptor firing (Angell-James, 1973).Past studies provide evidence for alterations in the cardiopulmonary receptorcontrol of vasoconstriction in hypertension. Renal hypertensive rabbits exhibit less of94a reduction in renal nerve activity with blood volume expansion (Thames andJohnson, 1985). Human studies have revealed that hypertensive patients with leftventricular hypertrophy had an impaired forearm vasoconstrictive response to lowerbody negative pressure (Grassi et al. 1988a; Grassi et al. 1988b). In these studies,lower body negative pressure was applied without any changes in mean arterialpressure. Grassi et al. (1988a) reported that this impairment could be markedlyimproved after one year of treatment induced regression of left ventricularhypertrophy. This suggested that left ventricular hypertrophy was perhaps alteringthe sensitivity of cardiopulmonary receptors to central blood volume changes. Thistheory has been disputed. Thames (1987) has given evidence for central alterationsof chemosensititve cardiopulmonary receptor afferents in hypertensive rabbits.Similar to the local mechanoreceptors, these chemosensitive receptors are able tomediate changes in lumbar and renal sympathetic nerve activity. The study indicatedthat left ventricular hypertrophy was not necessary for an impairment of0cardiopulmonary reflex control of sympathetic activity.In another human study, lower body negative pressure caused similarincreases in forearm conductance in both normotensive and hypertensive subjects(Trimarco et al. 1986). Propranolol, a blocker of ventricular non-medullated vagalafferents, had an inhibitory effect on cardiopulmonary reflex actions in normotensivesubjects only. This suggested that ventricular receptors were non-functional inhypertensive subjects and that other receptors compensated for this loss such thatthe overall response to lower body negative pressure was similar to normotensivesubjects. In contrast, Mark and Kerber (Mark and Kerber, 1982) have shown that95lower body negative pressure caused a slight enhancement of the forearmvasoconstrictive response in hypertensive humans. These subjects were consideredto be borderline hypertensive with no evidence of cardiac hypertrophy. It has beenspeculated that the cardiopulmonary reflex may be preserved or even enhanced inthe initial stages of hypertension, but that it undergoes progressive impairment as thestructural changes associated with hypertension develop (Grassi et al. I 988a).In the Dresent study, the rate of hind limb vasoconstriction was impaired in thehypertensive rabbits. There was no strong evidence in the literature for an alterationin arterial baroreceptor mediated peripheral vasoconstriction in hypertension. Therabbits of this study had a significantly greater ratio of left ventricular to whole bodyweight thus indicating the presence of cardiac hypertrophy. Either cardiachypertrophy or central modifications of reflex afferents may be responsible forattenuating the cardiopulmonary reflex control of hind limb conductance duringhemorrhage in the hypertensive rabbits of this study.When the autonomic nervous system was blocked, the hind-limbvasoconstrictive response to hemorrhage remained impaired in the hypertensivegroup, although the difference between groups was not statistically significant due toa large standard error. It is possible that the hypertensive rabbits had less circulatingAVP and ANG II, and, therefore, less of a vasoconstrictive response duringhemorrhage. We have previously observed that renal wrap hypertensive rabbits havean attenuated release of AVP and renin during hemorrhage with the autonomicnervous system blocked (Courneya and Weichert, 1995). The reasons for thisattenuated release are unclear. In this study, it was possible that the hypertrophic or96central alterations responsible for reducing the autonomic baroreflex response to thehind-limb vascular bed similarly attenuated the release of AVP during hemorrhage.Release of renin is reported to be independent of baroreflex mechanisms inconscious rabbits during hemorrhage (Courneya et al. 1992). In this study,hypertrophic changes to the renal vasculature may have been responsible forimpaired renin release via the renal barostat mechanism. However, there is atpresent, little evidence to suggest structural alterations to arterioles of the diameter inwhich the renal barostat mechanism is known to reside (Mulvany and Aalkjaer, 1990).Overall, an impaired release of AVP and renin was likely responsible for the observedimpairment of hormonally induced hind-limb vasoconstriction during hemorrhage withthe autonomic nervous system blocked.It has been well established that gain of the baroreflex control of heart rate isreduced in hypertension. In renal wrap hypertensive rabbits, the average gain of thepressure-heart rate curve has been reported to be 50% of the normotensive value(West and Korner, 1974). The attenuation of this MAP-HR relationship has beenattributed mainly to a reduction in the vagal component of the reflex (Korner, 1989).Similar findings have been reported in studies of one-kidney renal wrap hypertension(Fletcher, 1984; Guo et al. 1983) and in hypertensive humans (Korner et al. 1974).In this study, the rate of HR increase during hemorrhage with the autonomicnervous system intact (CT and HB) was faster in the hypertensive rabbits. However,hypertensive rabbits had a faster rate of MAP decline due, in part, to an impairment ofthe hindlimb vasoconstrictive response during hemorrhage. The increased slopevalue for HR in the hypertensive rabbits described the response as %BV was97removed and did not account for the more rapid decline in MAP during hemorrhage.If the overall change in HR (baseline-breakpoint) was instead related to the change inMAP during hemorrhage, a blunting of the baroreflex response was suggested. Onaverage, this represented a 55% reduction in the MAP-HR relationship duringhemorrhage and was similar to previous studies of the baroreflex-HR curve in renalwrap hypertensive rabbits (West and Korner, 1974).In summary, we anticipated that hypertrophic alterations in the vasculature ofthe hypertensive rabbits would lead to an augmented hind limb vasoconstrictorresponse during hemorrhage. Contrary to this hypothesis, we have found thathypertensive hind limb vasoconstriction was significantly impaired duringhemorrhage. This suggests that there was a substantial attenuation of the hind limbvasoconstrictor baroreflex response in the hypertensive rabbits. Although thehypertensive vasculature may have been hyper responsive, it was likely that thepressor stimulus was greatly reduced such that hind limb vasoconstriction could notbe maintained at the level exhibited in the normotensive rabbits. Hypertensive rabbitswere also found to have an impaired heart rate response during hemorrhage. Theattenuation in both the hind limb conductance and heart rate response contributed tothe lack of blood pressure control in the hypertensive compared to the normotensiverabbits during hemorrhage. As expected, the relative roles played by the autonomicnervous system as well as AVP and ANG Il in inducing hind limb vasoconstriction andcontrol of blood pressure during hemorrhage were similar between normotensive andhypertensive rabbits. This indicated that the basis of the baroreflex impairment of98hind limb vasoconstriction was not specific to either autonomic activation or tohormone release.a99CHAPTER 2 - THE CONTROL OF RENAL AND MESENTERIC VASCULARCONDUCTANCE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITSINTRODUCTIONThere were four aims for the experiments described in this chapter. First, wewanted to ensure that the hypertensive rabbits were hemodynamically stable for foursuccessive weeks of hemorrhage experiments. Therefore, a control study wasperformed using the protocol suggested in Chapter 1. The second aim was todetermine the roles played by a) the autonomic nervous system, as well as b) thehormones AVP and ANG II in the control of mesenteric and renal conductance(MESC and RENC) in hypertensive rabbits during hemorrhage. The third aim was tocompare the ability of normotensive and hypertensive rabbits to control MESC andRENC during hemorrhage.To date, there have been no studies performed on structural alterations toblood vessels in renal wrap hypertensive rabbits. The fourth aim of this chapter wasto perform a preliminary characterization of morphological alterations to arteries inrenal wrap hypertensive rabbits. Vessels from the vascular beds studied in thehemorrhage experiments were chosen specifically for study. These were themesenteric, hindlimb, and renal vascular beds.100HYPOTHESES1) Baseline hemodynamics and the hemodynamic response to control (no treatment)hemorrhage will be stable for four consecutive weeks of study in hypertensiverabbits.2) The relative roles of the autonomic nervous system as well as AVP and ANG II inthe control of MESC and RENC will be similar between normotensive andhypertensive rabbits.3) Hypertensive rabbits will have an impaired ability to control MESC and RENCduring hemorrhage compared to normotensive rabbits.4) Mesenteric, hindlimb, and renal arteries from the hypertensive rabbits will have anincreased vessel wall-to-lumen diameter ratio compared to normotensive rabbits.PROTOCOLHemorrhage StudiesExperiments were carried out in 6 control rabbits (hypertensive only) and 18test rabbits (9 normotensive/9 hypertensive). Three weeks after sham/wrap surgery,all rabbits underwent flow probe surgery. Control rabbits received a hindlimb flowprobe. A hindlimb flow probe was chosen so that the control data collected in thischapter could be compared to the results obtained in the control study in Chapter 1.In order to study mesenteric and renal blood flow in the test rabbits, both a renal anda mesenteric flow probe were implanted. Experiments began two weeks later (5weeks after sham/wrap surgery). Control rabbits underwent four successivehemorrhages at least one week apart and with no treatment. Test rabbits also101underwent four successive hemorrhages. Drug treatments for the test rabbits areoutlined in General Methods. Briefly, hemorrhage treatments were: 1) Control (CT),2) Hormonal blockade of vasopressin and angiotensin II (HB), 3) Autonomic nervoussystem blockade with pentolinium (ANSB), and 4) Combined autonomic andhormonal blockade (ANSB+HB).In order to further examine the role of AVP and ANG Il during hemorrhagewhen the autonomic nervous system was blocked (ANSB and ANSB+HB), data wasalso analyzed for a two minute interval Dost-hemorrhace. This interval began at thetermination of hemorrhage and ended two minutes later. Data was analyzed bydetermining the hemodynamic values at the last data point pertaining to thehemorrhage, and at two minutes later. Blood reinfusion began after this two minuteinterval. Statistical analysis of this data was performed using the tests describedearlier (see Methods, Statistical Analysis).Morphological Study of Blood VesselsAfter the test rabbits had completed their series of hemorrhage experiments,they were used for the morphological characterization of blood vessels. Rabbits werefirst anaesthetized with 120 mg of Euthanol (M.T.C.). An i.v. line was placed in aperipheral ear artery and 1000 units of heparin (Sigma) were administered, followedby a slow infusion of 0.1 mg of Procaine (Sigma). Procaine is a potassium channelblocker which was used to arrest the heart.A midline thoracic incision was made to expose the heart and the majorvessels. The descending aorta and the ascending vena cava were both cannulated.500 ml of saline were flushed through the descending aorta at 100 mmHg in order to102remove most of the blood. 500 ml of Bouin’s fixative were then flushed through thedescending aorta at 100 mmHg.Vessels were sampled from the renal, mesenteric, and hindlimb vascular beds(Figure 19). Specifically, the left renal artery was sampled, three type 3 arteries weresampled from the mesenteric bed, and the first branch of the iliac artery (left andright) was sampled. Vessels were placed in Bouin’s fixative for one hour, followed bytwo changes of 70% ethanol (one hour each). Vessels were then stored in 70%ethanol.Vessel samples were processed in a tissue processor (Fisher, model 166) andparaffin embedded with the lumenal surface down. The paraffin blocks weresectioned with a microtome at a thickness of 10 pm. This resulted in a cross-sectionof the lumenal surface. Sections were then mounted on glass slides. The glassslides were washed in xylene to remove the paraffin, stained with hemotoxylin(Sigma) and eosin (Sigma), and sealed with a cover slip.Vessel measurements employed the use of an image analyzer. Slides wereplaced on the microscope, and a video camera sent the image to a monitor. Animage analyzer program (VIDAS) captured the video image after which on-screenimages could be traced using a computer mouse attached to a drawing board. In thisway, on-screen measurements (in pm) of vessels were made. The circumferences ofthe lumen and of the outer surface of the vessel were measured. Circumference wasused to measure radius. From radius, lumen diameter and vessel wall thickness werecalculated. All vessel measurements were made blind (i.e. normotensive andhypertensive vessels were not marked).103descending aortabranchsarnpfediliac arteriesSuperior artery3 arteries sampledIMESENTERICHINDLIMBRENAL ao i-taceliac-mese ntericleft renal artery sam pledFigure 19- Sampling of blood vessels for morphological study. In the mesentericvascular bed, type 3 arteries were sampled. In the hindlimb bed, arteries for, the firstbranch of the iliac artery were sampled. In the renal bed, the left renal artery wassampled. See Protocol for a complete description of sampling procedures.104Due to the difficulty of aligning fixed vessel samples in the paraffin blocks,some vessel samples were lost. Therefore, the number of useful vessel sections pervascular bed studied varied across the different animals. The average values forlumen diameter, media thickness, and media-to-lumen ratio were analyzed betweenthe normotensive and hypertensive rabbits with a t-test.RESU LTSControl RabbitsBased on the results obtained in Chapter 1, the control study in this chapterbegan at 5 weeks after wrap surgery. Table IV shows the pre-hemorrhage baselinevalues for the four weeks of experiments. Pre-hemorrhage MAP, HR and HLC wereunchanged over the four weeks. Figure 20 depicts the slope values for MAP duringhemorrhage. The response was similar for each experiment over the four weekperiod. The same was true for the slope values corresponding to HR (Figure 21), andHLC (Figure 22) over the four weeks of experiments. These results indicate that,starting at 5 weeks post-wrap surgery, the hypertensive rabbits werehemodynamically stable for 4 weeks.Test RabbitsBaseline (pre-drug treatment) hemodynamics over timeIn order to ensure that the test rabbits were hemodynamically stable for thefour weeks of hemorrhage experiments, baseline (pre-drug treatment) hemodynamicTableIV-Hypertensivecontrolrabbits:Baselinepre-hemorrhagevaluesfor 4successivehemorrhagesHEMORRHAGEIHEMORRHAGE2HEMORRHAG..2HEMORRHAGE4MAP124.6±5.2117.9±2.8120.8±5.1125.1±4.7(mmHg)HR193.2±4.7176.3±5.8179.5±5.0180.1±5.0(beatslmin)HLC0.120±0.0200.120±0.0150.120±0.0200.133±0.025(mllminlkglmmHg)Allvaluesaremean±S.E..MAP(meanarterial pressure),HR(heartrate),andHLC(hindlimb conductance).0 011060.0--0.5- / / /SLOPEOF / / /MAP/%BV // / /—1.0 .Henil Hem2 Hem3 Hem4Figure 20 - Slope of mean arterial pressure (MAP, mmHg) as % blood volume (%BV)is removed during four successive control hemorrhages (Hem) in hypertensiverabbits. All slope values are expressed as mean ± SE. Negative slope valuesindicate a decline in MAP as %BV is removed. Hemorrhages were performed once aweek for four weeks.1074-3/2 /SLOPE OFHR/%BV /0• -Hemi Hem2 Hem3 Hem4Figure 21- Slope of heart rate (HR, beatslmin) as % blood volume (%BV) is removedduring four successive control hemorrhages (Hem) in hypertensive rabbits. All slopevalues are expressed as mean ± SE. Positive slope values indicate an increase inHR as %BV is removed. Hemorrhages were performed once a week for four weeks.1080.0 7 7 r-0.2- / / / /SLOPEOF%HLC/%BV /—0.8Hemi Hem2 Hem3 Hem4Figure 22 - Slope of % hindlimb conductance (%HLC, ml/min/kg/mmHg expressed asa % of baseline) as % blood volume (%BV) is removed during four successive controlhemorrhages (Hem) in hypertensive rabbits. HLC is expressed as a percentage ofpre-hemorrhage baseline value. All slope values are expressed as mean ± SE.Negative slope values indicate a decline in %HLC (vasoconstriction) as %BV isremoved. Hemorrhages (Hem) were performed once a week for 4 weeks.109values were analyzed (Tables V). Values were sorted according to the successiveexperiments, and analyzed for any changes from the first week of experiments. Ingeneral, pre-drug baseline values for MAP, HR, MESC, and RENC were stable for thefour weeks of experiments. This supports the data from the control rabbits, and alsoensures that there are no developmental changes to the mesenteric or renal vascularbeds in the hypertensive rabbits over the four weeks of experiments.Baseline (post-drug treatment) hemodynamicsA. Normotensive rabbitsIn the normotensive rabbits hormonal blockade of AVP and ANG II had noeffect on MAP or HR (Table VI). However, HB did cause a significant decrease inMESC indicating that, in the absence of AVP and All, there is a vasoconstriction inthe mesenteric vascular bed. In contrast, HB caused a significant increase in RENC,indicating that AVP and All contributed to baseline renal vascular tone. Blockade ofthe autonomic nervous system (ANSB) caused an increase in HR, but no change inMAP due to careful adjustment of NA infusion prior to data collection. After ANSB,MESC was significantly decreased, suggesting that, in the absence of autonomicinputs, an alternate vasoconstrictor agent was unmasked. This was not due to theactions of AVP or All since combined ANSB+HB caused a further vasoconstriction inthe mesenteric bed. The RENC was unaffected by ANSB. However, when aftercombined ANSB+HB, there was a vasodilation in the renal bed. This indicated that)when the autonomic nervous system was blocked, AVP and All may not have beencontributing to baseline renal vascular tone.TableV-Baselinepre-drughemodynamicvariablesinnormotensiveandhypertensiverabbitsduringfoursuccessiveweeksof experimentsNORMOTENSIVEHemIHem2Hem3Hem4MAP(mmHg)70.6±2.473.3±2.274.0±1.473.1±1.6HR(beats/mm)193.9±5.3196.9±5.5208.5±9•3*199.0±8.8MESC0.304±0.0180.296±0.0130.264±0.0120.254±0.012(mI/min/kg/mmHg)RENC0.165±0.0190.146±0.0150.145±0.0140.141±0.013(ml/min/kg/mmHg)HYPERTENSIVEHemIHem2Hem3Hem4MAP(mmHg)121.5±2.3121.5±3.6121.3±3.1121.9±2.1HR(beats/mm)184.0±4.9194.7±6.9176.5±5.7178.5±6.5MESC0.213±0.0070.230±0.0060.203±0.0080.210±0.013(ml/minlkg/mmHg)RENC0.057±0.0070.061±0.0060.058±0.0050.054±0.006(ml/min/kg/mmHg)Valuesareexpressedasmean±SE, n=9.VariablesrecordedareMAP(meanarterial pressure),HR(heartrate),MESC(mesentericvascular conductance),andRENC(renal vascular conductance).Predrugbaselinevaluesareshownfor four successiveweeksof hemorrhage(Hem)experiments.*representsp<0.05ascomparedtoHem1.TABLEVI-Baselinepre-hemorrhagehemodynamicvariablesinnormotensiveandhypertensiverabbitsunderfour combinationsof efferentblockadeNORMOTENSIVEANSBANSB+HBMAP(mmHg)72.1±2.170.2±1.370.9±1.575.9±1.5HR(beats/min)200.9±5.4184.1±6.3271.5±8.6*262.5±14.8*MESC0.297±0.0180.248±0.013*0.213±0.006*0.189±0.012*(mI/minlkg/mmHg)RENC0.163±0.0140.212±0.012*0.147±0.0150.186±0.014t(ml/minlkg/mmHg)HYPERTENSIVECTANSBANSB+HBMAP(mmHg)124.1±2.7127.6±3.4119.8±2.2120.9±2.3HR(beats/mm)186.3±6.9176.9±4.2231.2±6.6*229.5±5.9*MESC0.226±0.008§0.206±0.010§0.174±0.005*0.159±0.010*(mllmmnlkglmmHg)RENC0.057±0.007§0.067±0.006§0.059±0.005§0.068±0.006§(ml/minlkg/mmHg)Valuesareexpressedasmean±SE,n=9.VariablesrecordedareMAP(meanarterialpressure),HR(heartrate),MESC(mesentericvascularconductance),andREN(renal vascularconductance).TreatmentconditionsareCT(control),HB(hormonalblockade),ANSB(autonomicblockade),andANSB+HB(combinedautonomicandhormonalblockade).*representsp<0.05ascomparedtoCT; trepresentsp<0.05ascomparedtoANSB;§representsnormotensivecomparedtohypertensive.- -112B. Hypertensive rabbitsIn general, the hypertensive rabbits had a higher baseline MAP and lowerbaseline MESC and RENC (Tale VI), suggesting a contribution of the renal andmesenteric beds to the increased resistance in this model of hypertension. Hormonalblockade (HB) alone did not alter MESC or RENC, suggesting that, when theautonomic nervous system is intact, AVP and ANG II have no contribution to baselinevascular tone on these two beds. Blockade of the autonomic nervous system (ANSB)caused a significant rise in HR, but no change in MAP due to NA infusion prior to datacollection. Similar to the normotensive rabbits, ANSB resulted in a significantincrease in MESC but no change in RENC. Combined ANSB+HB caused a furthervasoconstriction in the mesenteric bed, suggesting that a vasoactive substance otherthan AVP or ANG II was contributing to mesenteric vascular tone in theseautonomically blocked hypertensive rabbits.HemorrhageFigures 23 and 24 serve as a visual representation of the response tohemorrhage in normotensive and hypertensive rabbits. The pattern of hemodynamicchange associated with hemorrhage will be described. Following this section,Figures 25-27 will outline the statistical differences between the normotensive andhypertensive response to hemorrhage, and between the four different treatmentconditions.113Figure 23 - The relationship of % blood volume removed versus mean arterialpressure (MAP, mmHg), mesenteric conductance (%MESC, ml/min/kg/mmHgexpressed as a % of baseline), and renal conductance (%RENC, ml/min/kg/mmHgexpressed as a % of baseline) in normotensive rabbits. Hemorrhage conditions were:control (CT; solid lines), hormonal blockade of AVP and All (HB; long dashed lines),autonomic nervous system blocked (ANSB; short dashed lines), and combinedautonomic and hormonal blockade (ANSB+HB; dotted lines). Lines are drawn usingthe regression equations (see Results).114NORMOTENSIVE RABBITS120100_- 80CT60 HB.ANSB+HB.ANSB400 10 20 30120,ANSB+HB:ANSB••111B.4VI.—100S800 10 20 30130120,ANSB+HBIpANSB110%%%.— •4100900 10 20 30% BLOOD VOLUME REMOVED115Normotensive Response to HemorrhageThe average linear regression curves of the normotensive response tohemorrhage under the four conditions of efferent blockade are shown in Figure 23.The HR response to hemorrhage was not significantly different from the hemorrhageresults in Chapter 1 and, therefore, is not shown here. As blood volume was removed(%BV) under control (CT) conditions, MAP decreased slowly while %MESC and%RENC declined indicating a vasoconstriction response in the mesenteric and renalvascular beds. Hormonal blockade did not change the pattern of MAP decline, butdid cause a %MESC and %RENC to increase during hemorrhage, indicating avasodilation response. Although this suggests a contribution by AVP and ANG II inthe control of mesenteric and renal conductance during hemorrhage, the slope of%MESC and %RENC during hemorrhage was not statistically different from control(CT) hemorrhage. The statistical analysis of these slope values will be outlined in afollowing section.Blockade of the autonomic nervous system (ANSB) caused MAP to declinerapidly as %BV was removed. %MESC and %RENC both increased rapidly duringANSB hemorrhage, indicating a rapid vasodilation response. Combined blockade ofthe autonomic nervous system as well as the hormones AVP and ANG II (ANSB+HB)caused no further change in the pattern of MAP decline or %MESC and %RENCincrease. This suggested that, in this study, there was no evidence for a contributionby AVP and ANG II in the control of MAP or mesenteric and renal conductance duringhemorrhage with the autonomic nervous system blocked.116Figure 24 - The relationship of % blood volumeo removed versus mean arterialpressure (MAP, mmHg), mesenteric conductance (%MESC, ml/min/kg/mmHgexpressed as a % of baseline), and renal conductance (%RENC, ml/min/kg/mmHgexpressed as a % of baseline) in hypertensive rabbits. Hemorrhage conditions were:control (CT; solid lines), hormonal blockade of AVP and All (HB; long dashed lines),autonomic nervous system blocked (ANSB; short dashed lines), and combinedautonomic and hormonal blockade (ANSB+HB; dotted lines). Lines are drawn usingthe regression equations (see Results).117HYPERTENSIVE RABBITS120 CTblp.4100‘..ANSB+HB80 ANSB60400 10 20 30bE120..ANSB+HB...100RBCT80o io 20 30.—‘ 130bEpANSB120 /.ANSB+HBS..110 ‘/I’!‘/.‘/S 100 ./— — — — —— - RBCT0 10 20 30Z BLOOD VOLUME REMOVED!118Hypertensive Response to HemorrhageThe average linear regression curves corresponding to the hypertensiveresponse to hemorrhage are shown in Figure 24. There were some specificdifferences between the normotensive and the hypertensive response to hemorrhageunder the four different treatment conditions (compare Figures 23 & 24).Hypertensive rabbits had a faster rate of MAP decline under all four conditions ofhemorrhage compared to the normotensive rabbits. Similar to the normotensiveresponse, MAP decline did not change between control (CT) and hormonal blockade(HB) conditions. Also, MAP decline did not differ between conditions of autonomicblockade (ANSB) and combined hormonal and autonomic blockade (ANSB+HB).During CT hemorrhage, %MESC and %RENC declined in a manner similar to thenormotensive response. During HB hemorrhage, %MESC and %RENC declined(vasoconstriction response) similar to the CT hemorrhage. This was different fromthe normotensive rabbits where %MESC and %RENC increased (vasodilationresponse) during HB hemorrhage. This difference between the normotensive andhypertensive rabbits was only significant for %MESC, suggesting that AVP and ANGII contributed to control of mesenteric conductance during hemorrhage with theautonomic nervous system intact in the normotensive rabbits only.During hemorrhage with the autonomic nervous system blocked (ANSB), therewas an increase in %MESC and %RENC, indicating a vasodilation response. The%RENC response was similar to the normotensive rabbits. However, the rate of0increase in %MESC during hemorrhage was significantly slower than in thenormotensive rabbits. The hypertensive rabbits were, therefore, exhibiting less119vasodilation in the mesenteric bed compared to the normotensive rabbits at the samepercent loss of blood volume. After combined autonomic and hormonal blockade(ANSB+HB), the pattern of %MESC and %RENC increase during hemorrhage did notchange significantly from ANSB hemorrhage. This indicated that, similar to thenormotensive rabbits, there was no role played by AVP and ANG II in the control ofmesenteric or renal vascular conductance when the autonomic nervous system wasblocked.StatisticalAnalysis of Hemodynamic Slope Values During HemorrhageStatistical comparisons were obtained from an evaluation of the slope valuesof the hemodynamic variables measured during the hemorrhage period (Figures 23and 24). Comparisons were made across the treatment conditions, and between thenormotensive and hypertensive rabbits. The following results specifically addressthese slope value comparisons.Mean Arterial Pressure (MAP)Figure 25 emphasizes the slope values for MAP during hemorrhage under thefour treatment conditions. A more negative slope value indicated a more rapiddecline in MAP as % blood volume (BV) is removed. With regard to the normotensiverabbits (open bars), when the autonomic nervous system was intact, blockade of AVPand ANG II (HB) did not significantly alter the rate of MAP decline. Blockade of theautonomic nervous system (ANSB) resulted in rapid rate of MAP decline. Curiously,combined ANSB+HB caused no further change in the slope of MAP decline duringhemorrhage. This was in contrast to the results obtained in Chapter 1 which120SLOPE OF /MAP/%BVCT HB ANSB ANSB+HBI I NORMOTENSIVEVVJI HYPERTENSIVEFigure 25 - Slope of the regression line representing mean arterial pressure (MAP,mmHg) as % blood volume (%BV) was removed in normotensive and hypertensiverabbits during hemorrhage under four conditions of efferent blockade (Chapter 2). Allslope values are expressed as mean ± SE. Treatment conditions were control (CT),hormonal blockade (HB), autonomic nervous system blockade (ANSB) and combinedautonomic and hormonal blockade (ANSB+HB). Negative slope values indicateddeclining MAP as % blood volume was removed. * p<0.05 for normotensive versushypertensive; p<0.05 for CT versus ANSB.121indicated that AVP and All did contribute to the control of MAP during ANSBhemorrhage.With respect to the hypertensive rabbits (Figure 25, hatched bars), under alltreatment condition, the slope of MAP during hemorrhage was significantly morenegative compared to the normotensive rabbits, indicating a faster rate of MAPdecline. This was consistent with the data from Chapter 1. Similar to thenormotensive rabbits in this study, hormonal blockade with the autonomic nervoussystem intact (HB) had no effect on the slope of MAP. Blockade of the autonomicnervous system (ANSB) caused a profound increase in the rate of MAP decline.However, as was observed in the normotensive rabbits, combined ANSB+HB causedno further increase in the MAP/%BV relationship. Thus, in the hypertensive rabbits,the hormones AVP and ANG II did not contribute to control of MAP duringhemorrhage with the autonomic nervous system blocked.Mesenteric Conductance (MESC)Figure 26 depicts the slope values for %MESC during hemorrhage under the 4treatment conditions. A negative slope value indicated a vasoconstriction response,while a positive slope value indicated a vasodilation response. In the normotensiverabbits (open bars), the slope was negative during control (CT) hemorrhage,indicating a vasoconstriction response. When the autonomic nervous system wasintact and the hormones AVP and ANG II were blocked (HB) there was a positiveslope value for %MESC during hemorrhage, indicating a vasodilation response.Although this was suggestive of a contribution by AVP and ANG II in the control of1223.2TSLOPE OF%MESC/%BV1•CT HB ANSB ANSB+HBI I NORMOTENSIVEIYJI HYPERTENSIVEFigure 26 - Slope of the regression line representing mesenteric conductance(%MESC; ml/min/mmHg expressed as a % of baseline) as % blood volume (%BV)was removed in normotensive and hypertensive rabbits during hemorrhage under fourconditions of efferent blockade. All slope values are expressed as mean ± SE.Treatment conditions were control (CT), hormonal blockade (H B), autonomic nervoussystem blockade (ANSB) and combined autonomic and hormonal blockade(ANSB+HB). Negative slope values indicated a decline in %MESC as % bloodvolume was removed (vasoconstriction) while positive slope values represented anincrease in %MESC as % blood volume was removed (vasodilation). * p<O.05 fornormotensive versus hypertensive; @ p<O.05 for CT versus ANSB.123mesenteric conductance during hemorrhage, it should be noted that the slope valuefor %MESC/%BV was not statistically different between CT and HB hemorrhage.After the autonomic nervous system was blocked (ANSB), the slope value for%MESC/%BV was strongly positive in the normotensive rabbits, indicating avasodilation in the mesenteric bed. This was significantly different from the slopevalue for CT hemorrhage. Combined ANSB+HB did not alter the %MESC slope valuefrom hemorrhage during ANSB, indicating that AVP and ANG II were not contributingto control of mesenteric vascular tone when the autonomic nervous system wasblocked. This was in contrast to what was found in the hindlimb bed in Chapter 1.In the hypertensive rabbits (hatched bars), the slope of %MESC/%BV wasnegative during CT hemorrhage, indicating a vasoconstriction response. Themagnitude of the %MESC/%BV slope was not different from the normotensive rabbits,indicating a similar vasoconstriction response in the mesenteric bed. In contrast tothe normotensive rabbits, HB did not alter the rate of %MESC decline duringhemorrhage in the hypertensive rabbits, indicating that AVP and ANG II were notcontributing to the control of mesenteric conductance when the autonomic nervoussystem was intact. Thus, during HB hemorrhage, the slope values for %MESC/%BVwere significantly different between normotensive and hypertensive rabbits.After autonomic blockade, the slope of %MESC/%BV during hemorrhage waspositive in the hypertensive rabbits, indicating a vasodilation response. CombinedANSB+HB caused no further change in the slope of %MESC during hemorrhage inthe hypertensive rabbits. Similar to the normotensive rabbits, this indicated that AVPand ANG II were not contributing to mesenteric tone during hemorrhage when the124autonomic nervous system was blocked. This was contrary to the results obtained inChapter 1 for the hindlimb bed, where AVP and ANG II were shown to contribute tomesenteric tone during hemorrhage with the autonomic nervous system blocked. It isimportant to note that the magnitude of the vasodilatory response (as indicated by apositive slope value) in the hypertensive mesenteric bed was significantly attenuatedcompared to the normotensive rabbits when the autonomic nervous system wasblocked. This suggests an impairment in the mesenteric vasodilation response in theautonomically blocked hypertensive rabbits.Renal Conductance (RENC)The slope values for %RENC/%BV during the four hemorrhage treatmentconditions are shown in Figure 27. As for Figure 26, a positive slope value indicateda vasoconstriction response while a negative slope value indicated a vasodilationresponse. In the normotensive rabbits (open bars), %RENC declined during control(CT) hemorrhage, indicating a vasoconstriction response. Blockade of AVP and ANGII (HB) caused a slight vasodilation in the renal bed. However, the slope value for%RENC/%BV was not significantly different from CT hemorrhage. Therefore, AVPand ANG II did not contribute significantly to renal vascular tone during hemorrhagewith the autonomic nervous system intact. Similar to the mesenteric bed, autonomicblockade (ANSB) caused a profound vasodilation in the renal bed during hemorrhagein the normotensive rabbits. Combined ANSB+HB had no further effect on the slopevalue for %RENC/%BV, suggesting that AVP and ANG II played no role in the controlof renal conductance when the autonomic nervous system was blocked.1253. 102SLOPE OF%RENC/%BV/I. /a. ///T /OyTflT—1CT HB ANSB ANSB+HBI I NORMOTENSIVEFYA HYPERTENSIVEFigure 27 - A. Slope of the regression line representing renal conductance (%RENC;mllmin/mmHg expressed as a % of baseline) as % blood volume (%BV) was removedin normotensive and hypertensive rabbits during hemorrhage under four conditions ofefferent blockade. All slope values are expressed as mean ± SE. Treatmentconditions were control (CT), hormonal blockade (H B), autonomic nervous systemblockade (ANSB) and combined autonomic and hormonal blockade (ANSB+HB).Negative slope values indicated a decline in %RENC as % blood volume wasremoved (vasoconstriction) while positive slope values represented an increase in%RENC as % blood volume was removed (vasodilation). c p<O.05 for CT versusANSB.126In general, the hypertensive slope values (hatched bars) for %RENC/%BVwere similar to the normotensive values for all conditions of hemorrhage. Also, theDattern of renal conductance changes during the different hemorrhage conditions wassimilar to what was observed in the mesenteric vascular bed. However, there is oneimportant observation to note. In the hypertensive rabbits, there was no evidence foran impaired renal vasodilation response during hemorrhage with the autonomicnervous system blocked. In contrast, there was evidence for an impaired mesentericvasodilation response during hemorrhage in the hypertensive rabbits.Two Minute Interval Post-HemorrhageWe examined MAP, MESC and RENC data during the post-hemorrhage periodwhen the autonomic nervous system was blocked. This interval was a two minutepause at the end of hemorrhage, prior to the reinfusion of blood. Interval data wasanalyzed because we could find no role for AVP and ANG II in the control of MAP,MESC or RENC durin hemorrhae with the autonomic nervous system blocked.This was contrary to the results obtained in Chapter 1 which showed that, duringhemorrhage with the autonomic nervous system blocked, AVP and ANG II contributedto control of MAP and hindlimb vascular tone. If release of AVP and ANG II wassomehow delayed in this study, we reasoned that we might observe a contribution ofthese hormones to the control of MAP, MESC, and RENC during the interval periodpost-hemorrhage. The results obtained are presented in Figures 28-30. The changein MAP, MESC and RENC from the last data point during hemorrhage, to two minuteslater was calculated. For MAP, this change was expressed as an absolute change.127zE INORMOTENSIVEK/A HYPERTENSIVEI IJ-*15Lii50—5151050ANSB ANSB+HB©©—5ANSB ANSB+HBFigure 28 - Change in mean arterial pressure (MAP, mmHg) during a two minuteinterval post-hemorrhage. Open bars normotensive rabbits, hatched bars indicatehypertensive rabbits. Treatment conditions were autonomic nervous system blockade(ANSB) and combined autonomic and hormonal blockade (ANSB+HB). * p<O.05 forANSB versus ANSB+HB; p<O.05 for normotensive compared to hypertensiveresponse.128p4C,,zIIKI I NORMOTENSIVEk/A HYPERTENSIVEANSB ANSB +HB10______0—10—20—30100—10—20—30ANSB ANSB+HBFigure 29 - Percent change in mesenteric conductance (MESC) during a two minuteinterval post-hemorrhage. MESC expressed as a % change from the end ofhemorrhage to the end of the interval. Decrease in %MESC represents avasoconstriction, an increase in %MESC represents a vasodilation. Open barsindicate normotensive rabbits, hatched bars indicate hypertensive rabbits. Treatmentconditions were autonomic nervous system blockade (ANSB) and combinedautonomic and hormonal blockade (ANSB+HB). * p<O.05 for ANSB versusANSB+HB.129zKANSB ANSB+HBI I NORMOTENSIVEF-VA HYPERTENSIVE10- *0—1010-0—10ANSB ANSB+HBFigure 30 - Percent change in renal conductance (RENC) during a two minute intervalpost-hemorrhage. RENC expressed as a % change from the end of hemorrhage tothe end of the interval. Decrease in %RENC represents a vasoconstriction, anincrease in %RENC represents a vasodilation. Open bars indicate normotensiverabbits, hatched bars indicate hypertensive rabbits. Treatment conditions wereautonomic nervous system blockade (ANSB) and combined autonomic and hormonalblockade (ANSB+HB). * p<0.05 for ANSB versus ANSB+HB.130For MESC and RENC, this change was expressed as a percent from the end ofhemorrhage to the end of the two minute interval.When the autonomic nervous system was blocked (ANSB), MAP recovered by—7 mmHg in the normotensive rabbits during the post-hemorrhage interval (Figure28). When the actions of AVP and ANG II were also blocked (ANSB+HB), MAP failedto recover in the normotensive rabbits, and in fact declined by —3 mmHg. In thehypertensive rabbits, MAP recovered by —13 mmHg during the post-hemorrhageinterval under ANSB. This represented a greater recovery in MAP compared to thenormotensive response. ANSB+HB treatment resulted in a reduced (but nonsignificant) recovery of MAP in the hypertensive rabbits. Thus, although the actionsof AVP and ANG II do appear to contribute to recovery of MAP during the post-hemorrhage interval in the normotensive rabbits, the same may not be true in thehypertensive rabbits.Figures 29 and 30 show that, during hemorrhage with the autonomic nervoussystem blocked (ANSB), there was a vasoconstriction (negative % change) in themesenteric and renal beds during the interval period. This could have contributed tothe recovery of MAP seen in Figure 28. AVP and ANG II contributed to the observedvasoconstriction since blocking the hormones (ANSB+HB) caused a vasodilation(positive % change) in the mesenteric and renal beds during the post-hemorrhageinterval. There was no difference in the normotensive and hypertensive rabbits withrespect to the observed changes in mesenteric and renal conductance during thepost-hemorrhage interval.131To summarize, AVP and ANG II played a significant role in mesenteric andrenal vasoconstriction during the post-hemorrhage interval in both the normotensiveand the hypertensive rabbits. In the normotensive rabbits, this vasoconstriction couldhave contributed to the observed recovery of MAP during the interval. In thehypertensive rabbits, despite a significant contribution of AVP and ANG II tomesenteric and renal vasoconstriction during the post-hemorrhage interval with theautonomic nervous system blocked, the recovery of MAP did not seem to bedependent on the presence of these hormones.Morphological Study of Blood VesselsLumen diameter and media (vessel wall) thickness for the mesenteric,hindlimb, and renal vessels are shown in Table VII. The final number of rabbitsrepresented in each group is indicated. The mesenteric vessels were the smallestvessel sampled in the study. The hypertensive mesenteric vessels had a significantlyincreased media thickness compared to the normotensive rabbits with no change inlumen diameter. The hindlimb vessels were of a slightly larger diameter compared tothe mesenteric vessels. There was no change in lumen diameter or media thicknesswhen comparing the normotensive to the hypertensive hindlimb ve&sels. The renalvessels had the largest diameter of the three vessel types. Hypertensive renalvessels had a significantly increased media thickness with no change in lumendiameter.Figure 31 shows the wall-to-lumen ratio of the three vessel types sampled.The hypertensive rabbits had a significant increase in the wall-to-lumen ratio in therenal and mesenteric vessels compared to the normotensive rabbits. The wall-to-ñ132Table VII - Morphological characteristics of blood vessels in normotensive rabbits andin renal wrap hypertensive rabbitsMESENTERIC Normotensive (n=7) Hypertensive (n7)Lumen diameter (pm) 358 ±39 386±23Media thickness (pm) 27.8 ± 1.3 44.1 ± 4.5 *HINDUMB Normotensive (n=7) Hypertensive (n=8)Lumen diameter (pm) 701 ± 105 534 ±54Media thickness (pm) 76.6 ± 10.4 80.2 ± 6.9RENAL Normotensive (n=7) Hypertensive (n8)Lumen diameter (pm) 1,189 ± 91 1,029 ± 83Media thickness (pm) 101.6 ±7.7 149.5 ± 18.0 *All measurements are mean ± S.E.. *p<0O5compared to normotensive133100MEDIA—TO—LUMENRATIO (%)806040200I I NORMOTENSIVEKNN HYPERTENSIVEFigure 31 - Wall-to-lumen ratio (%) in mesenteric, hindlimb, and renal vessels fromnormotensive and hypertensive rabbits. * p<O.05 as compared to normotensivevalue.SMESENTERIC HINDLIMB RENAL134lumen ratio was also higher in the hypertensive hindlimb vessels, but this increasewas not significantly different from the normotensive value.DISCUSSIONA. Control RabbitsThe control study was performed in order to ensure that the hyiertensiverabbits were hemodynamically stable and gave a consistent response to hemorrhageover a four week period. The control experiments began at five weeks after wrapsurgery. Over the four week experimental period, the hypertensive control rabbitshad stable pre-hemorrhage baseline values for MAP, HR and HLC, and the slopevalues of these variables during hemorrhage were unchanged. These results aredifferent from those obtained in Chapter 1, where the control study began at fourweeks after renal wrap surgery. In Chapter 1, hypertensive baseline values for HRand HLC, and the slope values for MAP and HLC were not stable over three weeks ofexperiments. The results of the present study confirm that the protocol of waiting fiveweeks after renal wrap surgery before commencing experiments on the hypertensiverabbits is appropriate in order to ensure the hemodynamic stability of thehypertensive rabbits. Although a control study on the normotensive rabbits using thismodified protocol (i.e. a five week wait after sham surgery, and four weeks ofsuccessive hemorrhage experiments) was not performed, it can be assumed that thenormotensive rabbits are hemodynamically stable. This assumption is based on thestability of the normotensive hemodynamics in Chapter 1 (i.e. a four week wait aftersham wrap surgery, and three weeks of successive hemorrhage experiments).135The experiments performed on the test rabbits in this chapter are based on theprotocol tested in this control study on hypertensive rabbits. This protocol eliminatedthe experimental error of developmental hemodynamic alterations in the hypertensiverabbits during successive hemorrhage experiments, as seen in Chapter 1.B. Test RabbitsBaseline values (Effects of Time)In order to confirm that the test rabbits were hemodynamically stable for thefour weeks of hemorrhage experiments, baseline (pre-drug treatment) values wereanalyzed according to the sequential weeks of experimentation. Since the controlstudies focused only on the hindlimb vascular bed, this analysis was performed inorder to ensure that the renal and mesenteric vascular beds were similarlyhemodynamically stable. We have shown that, over the four week experimentalperiod, baseline (pre-drug treatment) values were unchanged. Thus, we could beconfident that hypertensive developmental changes to the renal and mesentericvascular beds had stabilized by the beginning of the experimental period. Theseresults confirmed the appropriateness of the five week wait after renal wrap surgerybefore the beginning of experiments.Baseline Values (Effects of Drug Treatments)I. Control (no drug treatment)Under control conditions, MAP was elevated 52 mmHg, mesentericconductance was decreased by 23% and renal conductance was decreased by 65%136in the hypertensive as compared to the normotensive rabbits. These results are inagreement with those of Bolt and Saxena (Bolt and Saxena, 1983) who showed that,in established renal wrap hypertension in rabbits, vascular conductance is decreasedin the kidneys and in the gastrointestinal tract.ii. Hormonal Blockade of A VP and ANG IIBasal endogenous levels of AVP and ANG Il had significant but oppositeeffects on the mesenteric and renal vascular beds in the normotensive rabbits.Hormonal blockade caused vasoconstriction in the mesenteric bed and vasodilationin the renal bed. A similar, but not statistically significant, pattern of mesenteric andrenal vascular change was seen in the hypertensive rabbits. This lack of significanteffect in the hypertensive rabbits could not have been due to lower basal levels ofcirculating ANG II and AVP since resting plasma renin and AVP levels are notsignificantly different between normotensive and hypertensive rabbits (Courneya andWeichert, 1995). Previous studies in conscious normotensive rabbits have describedno change in renal or mesenteric conductance after blockade of AVP and ANG II(Courneya and Korner, 1991; Korner et al. 1990).In evaluating the hemodynamic alterations associated with pharmacologicalblockade of the actions of AVP and ANG II, it is impossible to separate the relativecontribution of each hormone. What is known of the effects of blocking eachhormone individually will be discussed. The effect of angiotensin converting enzymeinhibition (ACE-I) on renal vascular conductance has been previously studied. ACE-Ihas been shown to cause renal vasodilation without alterations in MAP in rabbits (Hofet al. 1987), dogs (Gavras et al. 1978), rats (Arendshorst and Finn, 1977), pigs137(Bulkley et al. 1985), and humans (Hollenberg et al. 1981). This renal vasodilationresponse was more pronounced when the renin-angiotensin II system was previouslyactivated with a sodium restricted diet (Gavras et al. 1978; Fisher et al. 1994). ACE-Iis also known to inhibit the degradation of kinins. Therefore, when assessing theability of ACE-I to induce renal vasodilation, one must also consider the vasodilatoractions of bradykinin buildup. However, Hollenberg et al (1981) measured bradykininlevels in response to ACE-I in humans and found them to be unchanged. Fisher et al(Fisher et al. 1994) confirmed that the renal vasodilatory effects of ACE-I are directlydue to a direct loss of ANG Il production by showing that a similar amount of renalvasodilation occurs with an ANG II receptor antagonist. It should be emphasized thatthe ability of ACE-I to cause renal vasodilation does not allow for a determination ofthe relative contribution of locally formed ANG II as compared to circulating ANG II(Navar and Rosivall, 1984).ACE-I has been shown to cause renal vasodilation and a decrease in MAP inspontaneously hypertensive rats (Takishita et al. 1994), in rats with renovascularhypertension (Wang et al. 1992), and in human essential hypertension (Hollenberget al. 1981). Therefore, in these hypertensive states, ANG II appears to contribute todecreased renal vascular conductance and to increased blood pressure. It wouldappear that, in our model of hypertension, ANG II does not play a role in maintainingrenal tone since hormonal blockade resulted in a significant renal vasodilation in thenormotensive rabbits only.The mesenteric response to ACE-I is less clear. ACE-I has been shown tocause mesenteric vasodilation in cats (McNeill et al. 1977) and in sodium deplete138humans (Stadeager et al. 1989). Others studies have described a transientmesenteric vasoconstriction after ACE-I in dogs (Gavras et at. 1978). ACE-I causedno change in mesenteric blood flow in rats (Pang, 1983) and in rabbits (Hof et al.1987).Studies using vasopressin Vi -antagonists (AVPA) have not generallydescribed any effects on resting renal vascular tone. Although AVPA has beenshown to increase vasa recta blood flow (Zimmerheckl et at. 1985), other studieshave shown no overall change in total renal vascular conductance following AVPAinfusion (Jover et al. 1987). AVPA infusion in rats causes a decrease in blood flow tothe intestine (Pang, 1983). Although the reason for this AVPA induced mesentericvasoconstriction is unknown, this effect may have been responsible for the decreasein mesenteric conductance seen after AVPA in the normotensive rabbits of this study.Overall, the results of the present study showed that combined treatment withAVPA and ACE-I caused significant renal vasodilation and mesentericvasoconstriction in the normotensive rabbits. Based on the results of previousstudies described above, it is possible that ACE-I was responsible for the renalvasodilation, and that AVPA was responsible for the mesenteric vasoconstriction.iii. Blockade of the Autonomic Neivous SystemCompared to control conditions, autonomic blockade caused avasoconstriction in the mesenteric vascular bed, and no change in renal vasculartone in both normotensive and hypertensive rabbits. Others have described nochange in mesenteric vascular conductance following denervation of the gut (McNeillet al. 1977; Bulkley et at. 1985). Alternatively, infusion of a ganglionic blocker has139been shown to cause a vasoconstriction in the mesenteric bed in conscious rabbits(Korner et al. 1990). Previous studies in conscious rabbits have reported renalvasodilation following ganglionic blockade (Courneya and Korner, 1991). Othershave described that renal vascular tone is relatively independent of renal nerveactivity, and that denervation has little effect on renal hemodynamics (Lemley andKriz, 1994). The finding that mesenteric and renal conductance remained decreasedin the hypertensive rabbits after autonomic blockade indicates the contribution ofstructural and other non-autonomic factors to the reduced conductance. Thisstatement can be made with confidence since, as outlined by West et al (West et al.1975), the comparison of vascular tone before and after autonomic blockade wasmade at the same level of resting MAP.iv. Combined Autonomic and Hormonal BlockadeCombined autonomic and hormonal blockade caused renal vasodilation in thenormotensive rabbits compared to autonomic blockade alone. This suggested thatAVP and ANG II may have been contributing to renal vascular tone when theautonomic nervous system was blocked. However, AVP and ANG II have previouslybeen shown not to contribute to renal vascular tone when the autonomic nervoussystem is blocked in conscious normotensive rabbits (Courneya and Korner, 1991).HemorrhageI. Autonomic Neivous System IntactUnder control conditions, hemorrhage in both normotensive and hypertensiverabbits was characterized by mesenteric and renal vasoconstriction, and well140maintained MAP up to the breakpoint (removal of — 30% blood volume). The level ofmesenteric and renal vasoconstriction was similar to what has previously beenreported in conscious normotensive rabbits during hemorrhage (Coumeya andKorner, 1991; Korner et al. 1990). Despite the similarity of the vasoconstrictiveresponse in the normotensive and hypertensive rabbits, the hypertensive rabbits stillhad an impaired ability to control MAP during hemorrhage. This impaired ability tocontrol MAP in the hypertensive rabbits was not due to a lack of support by thehormones AVP and ANG II since blocking these hormones had no effect on control ofMAP during hemorrhage. The findings from Chapter 1 serve to explain part of thisimpaired control of MAP in the hypertensive rabbits. In Chapter 1, it was shown thatthe hypertensive rabbits had an impaired ability to control heart rate, and an impairedvasoconstriction of the hindlimb bed during hemorrhage. Both of these findings likelycontributed to the impaired control of MAP in the hypertensive rabbits. In the Dresentstudy, renal and mesenteric vasoconstriction was not impaired in the hypertensiverabbits during hemorrhage. If anything, the normotensive rabbits exhibited less of anautonomically mediated, mesenteric vasoconstriction response. This was apparentfrom the slight mesenteric vasodilation seen in the normotensive rabbits duringhemorrhage with the autonomic nervous system intact and the actions of AVP andANG II blocked. Therefore, the impaired ability of the hypertensive rabbits to controlMAP during hemorrhage was not due to an impaired ability to control renal andmesenteric vascular tone.Several studies of autonomic reflex control of the renal and mesentericvascular beds in hypertension have been performed. However, most of these141experiments have focused on sympathetic nerve activity to these vascular beds.Studies in spontaneously hypertensive rats and in high-sodium borderlinehypertensive rats have shown that arterial baroreflex control of renal sympatheticnerve activity is unchanged (Lundin et al. 1984; DiBona and Jones, 1992). In thesestudies, pressor drugs were administered to elicit arterial baroreflex inhibition of renalsympathetic nerve activity. Using similar techniques, others have reported animpairment in the arterial baroreflex inhibition of renal sympathetic nerve activity inspontaneously hypertensive rats (Coote and Sato, 1977; Judy and Farrell, 1979) andin anaesthetized renal wrap hypertensive rabbits (Thames et al. 1984). In their studyon renal wrap hypertensive rabbits, Thames et at. (1984) attributed this impairment toa central abnormality since pressor-induced changes in aortic nerve traffic weresimilar between normotensive and hypertensive rabbits.Other studies have been aimed at selectively stimulating the cardiopulmonaryreceptors. Ricksten et at. (1979) showed that a smaller volume expansion wasrequired in spontaneously hypertensive rats to cause a similar degree of reflexinhibition of renal sympathetic nerve activity compared to normotensive control rats.This increased sensitivity was attributed to a decreased atrial distensibility in the faceof increased blood volume in spontaneously hypertensive rats compared tonormotensive control rats. This observation that a similar volume load causes agreater increase in left atrial pressure has been confirmed in renal wrap hypertensiverabbits (Thames and Johnson, 1985). However, renal wrap hypertensive rabbits havebeen shown to have an impaired cardiopulmonary reflex control of renal sympatheticnerve activity in response to a volume expansion (Thames and Johnson, 1985). This142impairment has been attributed to a central abnormality of the cardiopulmonary reflexarc (Thames, 1987). In summary, spontaneously hypertensive rats have beendescribed as having an increased, decreased, or unchanged baroreflex control ofrenal sympathetic nerve activity. However, in the renal wrap hypertensive rabbit,studies have consistently shown an impaired baroreflex control of renal sympatheticnerve activity. The results from the present study show no impairment of baroreflexcontrol of renal vascular conductance in the hypertensive rabbits. This does notnecessarily contradict pervious findings of impaired baroreflex control of renalsymapthetic nerve activity since local vascualr responsiveness must also beconsidered. This point will be discussed further at the end of this section.Fewer studies have been performed to characterize the reflex control ofmesenteric sympathetic nerve activity in hypertension. Arterial baroreflex control ofsplanchnic sympathetic nerve activity in response to pressor drugs has been shownto be decreased in spontaneously hypertensive rats (Judy et al. 1976). Others haveshown no change in this reflex in spontaneously hypertensive rats (Brown andThames, 1982). Luft eta!. (1986) studied reflex changes in splanchnic nerve activityin response to both pressor and depressor drugs in stroke-prone spontaneouslyhypertensive rats. Their results showed an increased gain in the hypertensive rats.Studies of the cardiopulmonary reflex control of splanchnic sympathetic nerve activityshow similar contradictions. Volume expansion in Dahl-sensitive hypertensive ratscaused less reflex inhibition of splanchnic sympathetic nerve activity compared tonormotensive control rats (Ferrari et al. 1984). However, volume expansion inspontaneously hypertensive rats resulted in an augmented reflex inhibition of143splanchnic sympathetic nerve activity compared to controls (Ricksten and Thoren,1980). The authors of this study suggested that decreased distensibility of thecapacitance vessels in spontaneously hypertensive rats, as described by Ricksten etal. (1979), could cause this augmented reflex control of sympathetic splanchnic nerveactivity.It is clearly difficult, given the conflicting evidence in the literature, to findconsistent support for the results presented in the present study. In evaluating theautonomic reflex control of vascular conductance and blood pressure duringhemorrhage, it is important to consider inputs from both the arterial and thecardiopulmonary baroreceptors. Courneya et al. (1991) have shown that, duringhemorrhage in conscious normotensive rabbits, the relative contribution of the arterialversus the cardiopulmonary receptors is —3:1. In general, arterial andcardiopulmonary reflex control of renal and mesenteric sympathetic nerve activity isunchanged or even augmented in the spontaneously hypertensive rat (Brown andThames, 1982; Lundin et al. 1984; DiBona and Jones, 1992; Luft et al. 1986; Rickstenet al. 1979; Ricksten and Thoren, 1980). However, studies performed in renal wraphypertensive rabbits provide evidence for impaired arterial and cardiopulmonaryreflex control of renal sympathetic nerve activity (Thames et al. 1984; Thames, 1987;Thames and Johnson, 1985). To date, there have been no studies of reflex control ofsplanchnic nerve activity performed in renal wrap hypertensive rabbits.The results presented in the present study are unique in that renal andmesenteric vascular conductance (as opposed to sympathetic nerve activity) weremeasured during reflex control of blood pressure in a hypertensive model. Our144finding that there was no impaired autonomic control of mesenteric and renalconductance during hemorrhage in renal wrap hypertensive rabbits has twoimplications. First, it is possible that sympathetic discharge to these vascular bedswas not impaired in the hypertensive rabbits. This implies that, given a similarfrequency of sympathetic discharge, renal and mesenteric vessels respond with thesame degree of vasoconstriction in normotensive and hypertensive rabbits. However,Wright et al. (1987) have shown that structural changes to the hypertensive hindlimbvasculature in renal wrap hypertensive rabbits leads to a non-specific hyperresponsiveness to pressor stimuli. Therefore, given a similar frequency ofsympathetic discharge, the renal and mesenteric hypertensive vascular beds wouldbe expected to have a greater vasoconstriction response. It is more likely that renaland mesenteric sympathetic discharge was indeed impaired, as suggested by otherstudies. Overall, the integration of an impaired sympathetic nerve discharge and anincreased vascular responsiveness to this discharge could have resulted in a renaland mesenteric vasoconstriction response which was not different betweennormotensive and hypertensive rabbits. This is a possible explanation for thesimilarity between the normotensive and hypertensive rabbits with respect toautonomic control of renal and mesenteric conductance during hemorrhage.ii. Autonomic Neivous System BlockedWhen the autonomic nervous system was blocked, blood pressure declined ata much faster rate during hemorrhage compared to when the autonomic nervoussystem was intact. Part of this augmented rate of MAP decline was due to a loss ofheart rate support during hemorrhage under the conditions of ganglionic blockade.145Under these hemorrhage conditions, the hypertensive rabbits had an impaired abilityto control MAP compared to the normotensive rabbits. The mechanisms serving tocontrol MAP in normotensive and hypertensive rabbits during hemorrhage with theautonomic nervous system blocked will be discussed. It has been shown previouslythat, under conditions of autonomic blockade, AVP and ANG II can contributesignificantly to hindlimb, mesenteric and renal vascular tone in consciousnormotensive rabbits during hemorrhage (Korner et al. 1990; Courneya and Korner,1991; Courneya et al. 1991). In Chapter 1, it was suggested that the hypertensiverabbits had an impaired ability to control hindlimb vascular conductance duringhemorrhage with the autonomic nervous system blocked, but with AVP and ANG IIintact. This is supported by our recent findings that the release of AVP and plasmarenin are attenuated in hypertensive rabbits during hemorrhage with the autonomicnervous system blocked (Courneya and Weichert, 1995). The impairedvasoconstriction observed in the hindlimb bed supports the finding that thehypertensive rabbits were less able to control MAP during hemorrhage with theautonomic nervous system blocked.In the present study, the experiments were designed to determine whether therenal and mesenteric vascular response to hemorrhage after autonomic blockade wassimilarly impaired. If this were true, we would have expected less vascular control ofthe renal and mesenteric vascular beds during the hemorrhage in the hypertensiverabbits. What was found was a vasodilation response in the renal and mesentericbeds during hemorrhage in both the normotensive and hypertensive rabbits. Thisvasodilation response is supported by a previous study of mesenteric conductance146during hemorrhage in conscious normotensive rabbits during hemorrhage (Korner eta!. 1990). However, Courneya and Korner (1991) found that renal vasoconstrictionwas similar in the presence and in the absence of an intact autonomic nervoussystem in normotensive rabbits during hemorrhage.What was unexpected about the results of this chapter was the finding that therenal and mesenteric response to hemorrhage under the conditions of autonomicblockade was not impaired in the hypertensive rabbits. The rate of renal vasodilationwas similar between normotensive and hypertensive rabbits, but the hypertensiverabbits had j of a mesenteric vasodilation response during hemorrhage comparedto the normotensive rabbits. It appears that, in the absence of autonomic inputsduring hemorrhage, the mesenteric vascular bed of hypertensive rabbits is less proneto vasodilation in the hypertensive rabbits. If anything, these results contradict thefinding that blood pressure declines more rapidly in the hypertensive rabbits duringhemorrhage with the autonomic nervous system blocked. Studies in otherhypertensive models have described an impairment in endothelium-dependentvasodilation (Dohi et al. 1991; Diederich et al. 1990). It is possible that a similarimpairment in the hypertensive mesenteric bed could be responsible for theattenuated vasodilation during hemorrhage observed in this study.When AVP and ANG II were blocked in addition to the autonomic nervoussystem, we expected a more profound vasodilation in the renal and mesenteric beds,and a further decrease in the slope value for MAP. What we found was that therewas no further change in the slope values for MAP or renal and mesentericconductance during hemorrhage. This indicated that there was no contribution by147AVP and ANG II in the control of these parameters during hemorrhage with theautonomic nervous system blocked. These results are contrary to those found inChapter 1 which showed that, after blocking AVP and ANG II in addition to theautonomic nervous system, MAP declined even faster and the hindlimb bed lost theability to vasoconstrict. Therefore, in Chapter 1, AVP and ANG II were contributing tohindlimb vasoconstriction and to control of MAP when the rabbits were autonomicallyblocked. Other studies have shown that, in the absence of autonomic inputs, AVPand ANG II are released in vasoactive levels early during hemorrhage innormotensive (Oliver et a!. 1990) and hypertensive rabbits (Courneya and Weichert,1995). This early increase in AVP and ANG II has been shown to contribute to renaland mesenteric vascular tone in normotensive rabbits during hemorrhage when theautonomic nervous system is blocked (Courneya and Korner, 1991; Korner et al.1990). The results of the present study suggest that AVP and ANG II had notachieved vasoconstrictive levels during the hemorrhage period. As a result, therewas no evidence for the expected vasoconstrictive contribution of AVP and ANG II inthe control of renal and mesenteric vascular tone and in the control of MAP duringhemorrhage with the autonomic nervous system blocked. Therefore, the resultsobtained in this study cannot provide us with any additional insight on the impairedability of hypertensive rabbits to control MAP during hemorrhage with early release ofAVP and ANG II (after autonomic blockade).One alteration to the protocol used in this study was the use of pentolinium asthe ganglionic blocker as opposed to mecamylamine, which was used in the studydescribed in Chapter 1. It is possible that pentolinium was 1) interfering with the148reflex release of AVP or ANG II during hemorrhage, or 2) interfering with thevasoconstrictive actions of AVP and ANG II. There is however, no evidence in theliterature to support either claim. Furthermore, we have compared the release of AVPand plasma renin during hemorrhage in conscious rabbits treated with eithermecamylamine or pentolinium. In both normotensive and hypertensive rabbits,release of AVP and renin was similar during hemorrhage with either ganglionicblocker (C.A. Courneya and G.Weichert, unpublished observations).The lack of evidence for AVP and ANG II mediated vasoconstriction duringhemorrhage with the autonomic nervous system blocked was unexpected consideringOliver et al’s (1990) observation that AVP and ANG II are released in vasoactivelevels early during hemorrhage with the autonomic nerovus system blocked. In orderto examine this anomaly in our results further, we chose to analyze MAP as well asrenal and mesenteric conductance during the two minute period of data collectionwhich began immediately after hemorrhage. During the interval period with theautonomic nervous system blocked but AVP and ANG II intact, MAP recoveredsomewhat and there was a vasoconstriction response in the renal and mesentericbeds. This response was similar between normotensive and hypertensive rabbitswith the exception of MAP which increased —8 mmHg more in the hypertensiverabbits. In the interval period following hemorrhage with the additional blockade ofAVP and ANG II, MAP declined and there was a vasodilation in both the renal andmesenteric vascular beds in the normotensive rabbits. The hypertensive responsewas similar to the normotensive response with the exception of MAP, which stillshowed a slight recovery during the interval with combined blockade treatment. In149general, blocking the actions of AVP and ANG Il led to a loss of renal and mesentericvasoconstriction and to a loss of MAP control. This was evidence for the presence ofvasoconstrictive levels of AVP and ANG II during the interval period post-hemorrhage. It is possible that, in this study, AVP and ANG II only achievedvasoconstrictive levels during the interval period after hemorrhage had terminated.The reasons for this delayed release are unclear.The similarity between the normotensive and the hypertensive vasoconstrictiveresponse in the renal and mesenteric vascular beds during the interval could beinterpreted in two ways. First, it could suggest that AVP and ANG II were released insimilar levels in both groups of rabbits, and that the normotensive and hypertensivevasculature responded similarly to the resultant vasoconstrictive effects of these twohormones. However, we have shown that, durinQ hemorrhae with the autonomicnervous system blocked, there is impaired release of AVP and ANG ii in hypertensiverabbits (Courneya and Weichert, 1995). There is also evidence that the vasculatureof renal wrap hypertensive rabbits is hyper-responsive to the actions of pressoragents due to structural alteration related to the hypertension (Wright et a!. 1987).Therefore, we suggest that release of AVP and ANG II was impaired even afterhemorrhage (during the interval period), and that structural alterations in thehypertensive renal and mesenteric vascular beds compensated for the reduced levelsof circulating AVP and ANG II such that the normotensive and hypertensivevasoconstriction was similar. This suggestion does not adequately explain theenhanced recovery of MAP in the hypertensive rabbits with the hormones active. It is150possible that other vascular beds were responding with an augmentedvasoconstriction in the hypertensive rabbits.C. Morphological Study of Blood VesselsIt was hypothesized that vessels from renal wrap hypertensive rabbits wouldhave an increased wall-to-lumen ratio. The results of this study showed that renaland mesenteric arteries in the hypertensive rabbits had an increased wall-to-lumenratio compared to the normotensive rabbits. The hypertensive hindlimb vessels alsohad an increased wall-to-lumen ratio, however this was not statistically different fromthe normotensive values. An increased wall-to-lumen ratio in hypertension is anindex of non-specific vascular hyper-responsiveness (Schiffrin, 1992). Theimplications of the increased wall-to-lumen ratio found in the hypertensive vessels ofthis study must be discussed with respect to the caliber of the vessels measured. Allvessels in this study had a lumen diameter of greater than 300 pm. The vesselscontributing most importantly to vascular resistance are the small arteries with alumen diameter of 150-300 pm, and arterioles with a lumen diameter of 50-150 pm(Bohlen, 1986). The vessels studied in this chapter were not true resistance vesselsand, therefore, they played a limited role in the overall contribution of the vasculatureto total peripheral resistance. The presence of an increased wall-to-lumen ratio oflarger non-resistance arteries in the hypertensive samples, coupled with the previousfinding that renal wrap hypertensive rabbits exhibit non-specific amplification ofpressor stimuli (Wright et al. 1987), indicated the likely presence of an increased wallto-lumen ratio in smaller resistance vessels.151The finding that the vessels of this study had an increased wall-to-lumen ratiocould represent different types of structural alterations. An increased wall-to-lumenratio can occur as a result of vessel wall growth. However, an increased wall-to-lumen ratio can also occur in the absence of growth. This phenomenon, known asvascular remodeling (Baumbach and Heistad, 1989), occurs when vessel wallmaterial is rearranged such that lumen diameter is decreased, but cross-sectionalarea of the vessel wall is unchanged. Due to the small number of rabbits included inthis preliminary study, the source of the increased wall-to-lumen ratio was notdetermined. Other studies have shown great variability in the nature of the structuralchanges contributing to an increased wall-to-lumen ratio in hypertension. Studies ofaorta samples from spontaneously hypertensive rats and renal hypertensive rats haveshown evidence for vessel wall growth (Wolinsky et al. 1970; Owens, 1987; Dilley etal. 1994). Small mesenteric arteries in spontaneously hypertensive rats also exhibitincreased wall growth (Mulvany et al. 1985). However, small mesenteric arteries fromtwo-kidney one-clip renal hypertensive rats and from human hypertensive patientshave shown vessel remodeling in the absence of growth (Schiffrin et at. 1993; Dengand Schiffrin, 1991). Alternatively, small mesenteric arteries from one-kidney one-cliphypertensive rats, and cerebral arterioles from spontaneously hypertensive ratsexhibit both remodeling and growth (Deng and Schiffrin, 1991; Baumbach andHeistad, 1989). Therefore, the nature of the hypertension, the vascular bed studied,and the size of the vessel itself may each have independent characteristics withrespect to the structural alterations exhibited in hypertension.152Certain studies have made attempts to identify the mechanisms inducingstructural alterations in hypertension. This is a very important area of study sincecurrent hypertensive therapies can effectively reduce blood pressure, but have notbeen shown to cause complete regression of structural changes to the vasculature(Mulvany and Aalkjaer, 1990). Possible stimuli for vessel wall growth in hypertensioninclude the intrinsic response to elevated blood pressure, the actions of circulatingsubstances, or genetic factors. Vasoactive peptides such as ANG II, AVP, NA, andendothelin as well as certain growth factors have been implicated in vessel wallgrowth in hypertension (Schiffrin, 1992; Mulvany and Aalkjaer, 1990). Inspontaneously hypertensive rats, there is evidence for a genetic predisposition tovessel wall growth which, in some studies, has been shown to precede the elevationin blood pressure (Heagerty et al. 1993). Growth of a vessel wall can occur as aresult of increased cell volume (hypertrophy) or increased call number (hyperplasia).In small arteries of spontaneously hypertensive rats, vessel wall growth occurs as aresult of hyperplasia (Mulvany et al. 1985). However, in small arteries ofexperimentally induced hypertensive models, there is evidence for cellularhypertrophy (Schiffrin, 1992). It has been suggested that cellular hypertrophy in smallarteries is an adaptive response to elevated blood pressure, while hyperplasia has agenetic component (Heagerty et al. 1993). The mechanisms governing vascularremodeling in hypertension are not understood. Studies in human essentialhypertension suggest that vascular remodeling of small arteries is a major componentof the structural alterations found (Schiffrin et al. 1993).153In this preliminary study, we have made vessel measurements usinghistological sections of fixed tissue. The is a relatively simple technique which has itsdisadvantages. The fixation of tissue samples and the subsequent staining processis known to cause shrinkage. The difficulty in handling samples of very small vesselslimits the size of the vessels studied. Recent morphological studies have employedthe use of the wire myograph which allows for the study of blood vessels under highlycontrolled isometric conditions (Schiffrin, 1992). The more recent development ofvideo dimension analysis allows pressurized vessels to be studied in vivo, therebyproviding a more physiological environment for the study of vessel characteristics(Schiffrin, 1992). Video dimension analysis and the wire myograph technique bothallow for the structural and functional study of very small vessels (small arteries andarterioles). A more thorough investigation of structural alterations to renal wraphypertensive resistance vessels would require the use of these techniques.In conclusion we have shown that artery samples from the mesenteric andrenal vascular beds of renal wrap hypertensive rabbits have an increased wall-to-lumen ratio. A similar trend was suggested in the hypertensive hindlimb vessels.The vessels sampled were considered to be large arteries and therefore, did notcontribute substantially to vascular resistance. However, the presence of thesestructural alterations might be considered to be good evidence for similar structuralalteration in smaller resistance vessels of renal wrap hypertensive rabbits.SummaryIn summary, it was anticipated that reflex control of renal and mesentericvascular conductance would be impaired in the renal wrap hypertensive rabbits, and154that this would contribute to the impaired ability of hypertensive rabbits to controlMAP during hemorrhage. The results of this study showed that the autonomic reflexcontrol of renal and mesenteric conductance was unchanged in the hypertensiverabbits. This conclusion does not contradict previous findings of impaired control ofsympathetic nerve activity in renal wrap hypertensive rabbits, but suggests thatstructural changes to the vasculature could compensate for such an impairment.Therefore, the impaired ability of the hypertensive rabbits to control MAP duringhemorrhage was not due to an impaired ability to control renal and mesentericvascular tone.Under conditions of autonomic blockade, it was expected that augmentedrelease of AVP and ANG II during hemorrhage would contribute to renal andmesenteric vascular tone and to the support of MAP. An impairment in thehormonally mediated, hypertensive control of the renal and mesenteric vascular bedwas expected. In contrast to the findings in Chapter 1, there was no evidence forhormonally mediated control of the renal and mesenteric vascular bed, or to thecontrol of MAP with the autonomic nervous system blocked. The results suggestedthat AVP and ANG II did not achieve vasoconstrictive levels during the hemorrhageperiod. Due to the absence of a role for AVP and ANG II during hemorrhage with theautonomic nervous system blocked, it was not possible to determine whether thehypertensive rabbits had an impairment in the hormonally mediated control of renaland mesenteric vascular tone during hemorrhage.The role of AVP and ANG II in the absence of an intact autonomic nervoussystem was further examined in the interval period immediately after hemorrhage was155terminated. It was expected that the vasoconstrictive actions of these hormoneswould be apparent, and that the hypertensive rabbits would have an impaired renaland mesenteric vasoconstriction and support of MAP. Evidence for AVP and ANG IImediated renal and mesenteric vasoconstriction and control of MAP was found.However, the normotensive and hypertensive response was similar. We speculatethat renal and mesenteric vascular hyper-responsiveness was compensating forattenuated AVP and ANG II levels during the interval period post-hemorrhage.It was also hypothesized that the relative contribution of the autonomic nervoussystem as well as AVP and ANG II in the control of renal and mesentericvasoconstriction would be similar between normotensive and hypertensive rabbits.This was verified with the exception of the suggested contribution of AVP and ANG IIin the control of mesenteric conductance in the normotensive rabbits only, duringhemorrhage with the autonomic nervous system intact.The morphological study of hypertensive blood vessels indicated the presenceof an increased wall-to-lumen ratio in the mesenteric and renal arteries. A similarincrease was suggested in the hypertensive hindlimb arteries. Although an increasedwall-to-lumen ratio is indicative of increased structural reactivity, the vessels studiedwere not true resistance vessels. However, it might be assumed that a similarincrease in wall-to-lumen ratio also characterized the smaller resistance vessels ofthe renal wrap hypertensive rabbits. This finding supports to contention that therewas increased vascular reactivity in the hypertensive rabbits. This will be discussedfurther in the following general discussion.156GENERAL DISCUSSION - CONTROL OF BLOOD PRESSURE AND VASCULARCONDUCTANCE DURING HEMORRHAGE IN CONSCIOUS RENAL WRAPHYPERTENSIVE RABBITSThe experiments described in Chapters 1 and 2 examined the control ofhindlimb, renal and mesenteric conductance as well as the control of blood pressureduring hemorrhage in renal wrap hypertensive rabbits. The roles of the autonomicnervous system as well as the hormones AVP and ANG II were determined. Also, inChapter 2, morphological alterations to hindlimb, mesenteric and renal vessels inrenal wrap hypertensive rabbits were characterized. This discussion will compile thefindings from the three chapters with respect to the reflex cardiovascular controlmechanisms during hemorrhage in conscious renal wrap hypertensive rabbits.RENAL WRAP HYPERTENSIVE RABBITSThis study is the first to characterize directly peripheral blood flow in consciousrenal wrap hypertensive rabbits. We have demonstrated a decrease in baselinevascular conductance in the hindlimb, renal and mesenteric vascular beds. Bolt andSaxena (1983) previously studied this model of hypertension using microspheres tostudy indirectly peripheral blood flow. Their study showed that organs could beranked according to the degree of decrease in vascular conductance(kidneys>skeletal muscle>gastrointestinal tract). The results from our direct bloodflow measurements confirm this observation. When comparing the degree ofdecrease in vascular conductance in the hypertensive rabbits (compared to thenormotensive rabbits), we found that the renal bed had the greatest decrease in157vascular conductance, followed by the hindlimb bed, and then the mesenteric bed.These findings further support the conclusion that renal wrap hypertensive rabbitsexhibit a resistance mediated form of hypertension.HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM INTACTWhen the autonomic nervous system was intact, there was little evidence forhormonally mediated (AVP and ANG II) vasoconstrictive inputs during hemorrhage ineither the normotensive or hypertensive rabbits. This is consistent with previousstudies which have demonstrated that there is minimal release of AVP and ANG II inconscious rabbits during hemorrhage with the autonomic nervous system intact(Oliver et al. 1990). In the present studies, it was shown that the hypertensive rabbitshad an impaired ability to control blood pressure during hemorrhage (i.e. bloodpressure declined faster for the same volume of blood removed compared to thenormotensive rabbits). One contribution to this impaired control of blood pressurewas the impaired control of hea!t rate during hemorrhage in the hypertensive rabbits.It was demonstrated in Chapter 1 that, by the end of the hemorrhage period, thenormotensive rabbits had a greater increase in heart rate per mmHg decrease inblood pressure compared to the hypertensive rabbits. This finding was consistentwith previous studies which have shown impaired baroreflex control of heart rate inrenal wrap hypertensive rabbits (West and Korner, 1974; Fletcher, 1984). Our studywas the first to characterize this impairment in conscious hypertensive rabbits duringa hemorrhagic stimulus.The control of peripheral vascular conductance is another important parameterto consider in the control of blood pressure during hemorrhage. The hindlimb158(Chapter 1), renal and mesenteric (Chapter 2) vascular beds were studied duringhemorrhage. In the hypertensive rabbits, there was an impaired control of hindlimbvascular conductance, but no change in the profile of renal and mesentericvasoconstriction during hemorrhage compared to the normotensive rabbits.Therefore, the finding that hypertensive rabbits had an impaired ability to controlblood pressure during hemorrhage is explained (in part) by the observation ofimpaired control of hindlimb conductance, but not by the observation that control ofrenal and mesenteric conductance was unchanged.The differential response seen in the hindlimb bed versus the renal andmesenteric bed in the hypertensive rabbits during hemorrhage warrants a discussion.There are two factors to consider in the autonomic reflex control of peripheralvascular conductance. One is the frequency of efferent sympathetic nerve activity. Itis possible that, in the hypertensive rabbits, reflex sympathetic nerve activity wasimpaired to a greater extent in the hindlimb bed compared to the renal and mesentericbeds during hemorrhage. Such a statement implies that reflex control mechanisms indifferent vascular beds are non-uniform. Baroreflex control of different vascular bedsis influenced by the relative contribution of the arterial baroreceptors and thecardiopulmonary baroreceptors (Brown and Thames, 1982). During hemorrhage innormotensive rabbits, there is little contribution by the cardiopulmonary receptors inneural control of renal vascular tone (Courneya and Korner, 1991). However, thecardiopulmonary receptors are responsible for about one quarter of the neural controlof hindlimb vascular tone during hemorrhage in conscious normotensive rabbits(Courneya at el. 1991). The relative contribution of the arterial and cardiopulmonary159baroreceptors in control of mesenteric vascular tone during hemorrhage is unknown.If arterial and cardiopulmonary baroreceptor control mechanisms are altered indifferent proportions in hypertension, this will have a direct relationship on the degreeto which reflex control of blood flow is altered in the various vascular beds. Ingeneral, there are many studies which have demonstrated that cardiopulmonarycontrol of skeletal muscle blood flow is impaired in hypertension (see Chapter 1).However, there is also evidence for impaired control of both arterial andcardiopulmonary control of renal and mesenteric blood flow (see Chapter 2). It ispossible that the cardiopulmonary reflex is more profoundly impaired than the arterialbaroreflex in hypertension. Studies aimed at characterizing this effect have beencomplicated by the difficulties in measuring afferent and central components ofbaroreflex mechanisms and by comparing the results from different species. Atpresent, there is controversy regarding which of the two baroreflex mechanisms areimpaired (and to what extent) in hypertension. If the cardiopulmonary reflex is moreimpaired than the arterial baroreflex in our hypertensive rabbits, this would have agreater effect on those vascular beds which are more influenced by thecardiopulmonary receptors (such as the skeletal muscle bed). Such an effect couldbe responsible for the observation from our studies that reflex control of hindlimbconductance is impaired in hypertensive rabbits but that there was a lack ofimpairment in reflex control of renal and mesenteric conductance. This statementassumes that the relative influence of the arterial and cardiopulmonary receptors issimilar between the renal and mesenteric vascular beds.160The other component to consider in the reflex control of vascular conductanceis the degree of vascular responsiveness to a given level of sympathetic nerveactivity. In Chapter 2, we provided evidence for structural alterations to the hindlimb,renal and mesenteric vascular beds in renal wrap hypertensive rabbits. An increasedwall-to-lumen ratio was found in all three vascular beds. This parameter is anindication of increased vascular responsiveness to pressor agents (Heagery et at.1993). Although true resistance vessels (diameter <300pm) were not specificallycharacterized, we speculated that similar structural alterations were present in thesmall arteries and large arterioles of the vascular beds studied. This is supported bythe observation that renal wrap hypertensive rabbits exhibit a non-specific hindlimbvascular hyper-responsiveness to pressor agents (Wright et al. 1987). A differentialdegree of vascular hyper-responsiveness in the resistance vessels of the hindlimb,renal and mesenteric vasculature could have accounted for the differences seen withrespect to baroreflex control of these beds during hemorrhage. The renal andmesenteric beds may have had an enhanced degree of hyper-responsiveness, thusnormalizing an impaired level of sympathetic nerve activity. To date, there have beenno studies aimed at specifically characterizing the relative hyper-responsiveness ofdifferent hypertensive vascular beds in vivo or in vitro.HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM BLOCKEDDuring hemorrhage with the autonomic nervous system blocked, thehypertensive rabbits still exhibited an impaired control of blood pressure. In otherwords, blood pressure declined at a faster rate for the same volume of blood removedcompared to the normotensive rabbits. One contribution to this impairment was the161impaired control of hindlimb conductance during hemorrhage (shown in Chapter 1)which was associated with the vasoactive effects of AVP and ANG II. After autonomicblockade, AVP and ANG II are known to be released in vasoactive quantities earlyduring hemorrhage in normotensive rabbits (Oliver et at. 1990). We have shownpreviously that the release of AVP and ANG II under these conditions is impaired inhypertensive rabbits (Courneya and Weichert, 1995). However, it has also beenshown that the hindlimb vasculature of renal wrap hypertensive rabbits is hyperresponsive to pressor agents (Wright et al. 1987). We have confirmed the structuralalterations implicated in this vascular hyper-responsiveness in the morphologicalstudy presented in Chapter 2. However, in the face of attenuated levels of AVP andANG II, this hyper-responsiveness in the hindlimb bed could not achieve the samedegree of vasoconstriction as was observed in the normotensive rabbits.In Chapter 2, the renal and mesenteric beds were studied in order to determinetheir contribution to impaired hormonally-mediated control of blood pressure in thehypertensive rabbits during hemorrhage with the autonomic nervous system blocked.Unexpectedly, the results did not show a role for AVP and ANG II in contributing torenal or mesenteric vascular tone or to the control of blood pressure. With theexception of changing the protocol to use pentolinium as a ganglionic blocker, theprotocol was similar to what we (Chapter 1) and others have used previously (Oliveret al. 1990; Korner et al. 1990; Courneya and Korner, 1991; Courneya et at. 1991).Given the lack of hormonally mediated control of renal and mesenteric vascular tone,we could conclude that the impaired hormonally-mediated control of blood pressure in162the hypertensive rabbits was not related to an impaired control of the renal andmesenteric beds during hemorrhage.We chose to examine the effects of AVP and ANG II on the renal andmesenteric vascular beds during the two minute interval period after hemorrhage.During this period, there was AVP and ANG II mediated vasoconstriction of the renaland mesenteric vascular beds and recovery of blood pressure. Overall, this responsewas similar between the normotensive and hypertensive rabbits. Based on ourobservations that AVP and ANG II release during hemorrhage is impaired inhypertensive rabbits, it might be assumed that release of AVP and ANG II wassimilarly impaired during the interval period. A hyper-responsiveness in thehypertensive renal and mesenteric mesenteric beds could have compensated forattenuated levels of AVP and ANG II during the interval.HEMORRHAGE WITH THE AUTONOMIC NERVOUS SYSTEM AS WELL AS AVPAND ANG II BLOCKEDWhen both the autonomic nervous system as well as AVP and ANG II wereblocked, the rate of blood pressure decline remained faster in the hypertensiverabbits. Under these conditions of hemorrhage, it is the actions of local vasodilatorswhich are responsible for vascular regulation. It was expected that, given a fasterrate of blood pressure decline, the hypertensive rabbits would also exhibit anenhanced vasodilation in the vascular beds studied. However, the renal and hindlimbvasodilation observed during hemorrhage with combined blockade was similar to thenormotensive rabbits. In the mesenteric bed, there was less of a vasodilation in thehypertensive compared to the normotensive rabbits. If anything, these findings163contradict the observation of a faster loss of blood pressure during hemorrhage thehypertensive rabbits. During such a rapid loss of blood pressure, local vasodilationwould be mediated by such factors as hypoxia, acidosis, adenosine, and increasedosmolality (Qlsson, 1981; Granger and Kvietys, 1981). Some of these factors exerttheir effects via the production of nitric oxide from the endothelium. In hypertension,alterations in endothelium dependent vasodilation have been described. Suchalterations include attenuated release of nitric oxide (Lockette et al. 1986), theenhanced local production of vasoconstricting factors such as endothelin andprostaglandin-H2(Fu-Xiang et al. 1992). If anything, it might have been expected thatall hypertensive vascular beds studied in our experiments would have exhibited lessvasodilation in the absence of pressor inputs compared to the normotensive rabbits.This was only observed in the mesenteric vascular bed. In general, an impairedendothelium-mediated vasodilation does not contribute to our understanding of whythe hypertensive rabbits still exhibited a faster rate of blood pressure decline duringhemorrhage in the absence of pressor inputs. It is possible that other vascular bedsstudied (such as the skin, upper gut, pulmonary, cerebral, coronary) werevasodilating at a faster rate, thereby contributing to the faster rate of blood pressuredecline. However, the combination of the renal, mesenteric and hindlimb vascularbeds constitute a large portion of the cardiovascular system.The finding that there was a differential degree of local vasodilation in thethree hypertensive vascular beds studied has important implications on thediscussion of reflex control of vascular conductance during hemorrhage. Pressorinputs by the sympathetic nervous system or AVP/ANG II are superimposed upon the164local vasodilatory response during hemorrhage (Korner et at, 1990). In the hindlimbbed, the hypertensive rabbits exhibited a faster (but not significant) rate ofvasodilation compared to the normotensive rabbits. This might be suggestive of atrue difference between the normotensive and hypertensive rabbits with respect to thelocal response to hypoxia (Jones, 1964). Such an effect could have contributed tothe impaired hindlimb vasoconstriction observed in the hypertensive rabbits duringhemorrhage with pressor inputs intact. In the renal and mesenteric beds, thehypertensive rabbits exhibited a slower rate of vasodilation during hemorrhage withall exogenous pressor inputs blocked. This difference was only significant for themesenteric bed. If we were to assume that reflex autonomic and hormonalvasoconstriction of the renal and mesenteric beds was impaired in the hypertensiverabbits, then such an impairment could be partially off-set by a blunted localvasodilation response to hemorrhage compared to the normotensive rabbits. Such aneffect could have been responsible for the similarity between the normotensive andhypertensive rabbits with respect to renal and mesenteric vasoconstriction whenpressor inputs were intact.SUMMARY AND FUTURE DIRECTIONSThe research presented in this thesis has demonstrated that reflex control ofblood pressure in renal wrap hypertensive rabbits is impaired during hemorrhage.Our results have, for the first time, directly characterized control of peripheral bloodflow in three vascular beds during reflex control of blood pressure in a conscioushypertensive model. Our major findings are summarized:1651) When the autonomic nervous system is intact, impaired control of blood pressurein the hypertensive rabbits was associated with impaired control of heart rate andhindlimb conductance.2) When the autonomic nervous system is blocked, impaired control of bloodpressure in the hypertensive rabbits was associated with a loss of hormonallymediated (AVP and ANG II) control of hindlimb vascular tone.3) In the absence of all pressor inputs, (autonomic nervous system as well as AVPand ANG II) hypertensive rabbits still had a faster rate of blood pressure decline forthe same loss of blood volume compared to normotensive rabbits.4) Renal wrap hypertensive rabbits have structural alterations to hindlimb, mesentericand renal vessels which are indicative of non-specific vascular hyper-responsiveness.The hemorrhage studies performed were comprehensive in that both arterialand cardiopulmonary baroreceptors were involved, and in that the reflex vascularresponse was an integration of neural inputs and local responsiveness of the bloodvessels. These findings raise some important questions. It would be of interest toidentify the relative contribution of the arterial and cardiopulmonary receptors in thecontrol of the hindlimb, renal and mesenteric beds in the hypertension rabbits duringhemorrhage. It might also be interesting to determine whether the hypertensivevascular beds studied had a differential degree of non-specific hyper-responsiveness.In order to characterize fully blood pressure control mechanisms in hypertension, itwould be beneficial to study further the control of blood pressure immediately after ahemorrhage is terminated, and during a reinfusion of shed blood. These results alsosuggest that the local vascular vasodilatory response was not similar in all vascular166beds. Recent studies have only just begun to characterize hypertension inducedalterations to the vascular endothelium which may have been responsible for thiseffect. Furthermore, we have characterized structural alterations to larger arteries ofrenal wrap hypertensive rabbits. These findings warrant a more comprehensive studyof resistance vessels using well controlled techniques such as the wire myograph orvideo dimension analysis.Understanding the mechanisms governing control of blood pressure duringhemorrhage in a hypertensive model may thus have some significant clinicalapplications. Characterizing alterations in control of blood pressure allows us tounderstand further how hypertension might induce such alterations, It also allows usto understand how hypertensive individuals must regulate blood pressure on a dailybasis (i.e. in response to orthostatic changes). 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