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The putative role of the ventral septal area of the brain in the integration of cardiovascular regulation… McCashin, Marianne Roberta Hamilton Marshall 1993

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THE PUTATIVE ROLE OF THE VENTRAL SEPTAL AREA OF THE BRAIN IN THE INTEGRATION OF CARDIOVASCULAR REGULATION DURING ENDOTOXIN INDUCED FEVER by MARIANNE ROBERTA HAMILTON MARSHALL McCASHIN B.A., The University of Regina, 1977 B.L.T., The University of Regina, 1977 R.T. with the CSLT, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1993 © Marianne R.H. McCashin, 1993 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my wri t ten permission. Department of Phsiology The University of British Columbia Vancouver, Canada Date (Signature) o DE-6 (2/88) ABSTRACT During fever, cardiovascular changes have been seen to accompany the change in body temperature. There is evidence to implicate an area of the brain called the ventral septal area/ diagonal band of Broca (VSA/DBB) in both thermoregulation and cardiovascular control. This thesis investigates the possibility that neurons of the VSA/DBB which are known to be involved in thermoregulation during fever, may also be involved in some aspect of cardiovascular control during fever induced by endotoxin. The investigation was performed by infusing lidocaine HC1 into the VSA of freely moving conscious rats while the parameters of body temperature, heart rate and mean arterial blood pressure were monitored. Body temperature and cardiovascular parameters were measured in two separate groups of rats. The first set of experiments before brain infusion with lidocaine show that endotoxin has an effect on all of the above parameters. Body temperature showed the typical hypothermic phase followed by an increase to febrile levels in response to injection of endotoxin. Mean arterial blood pressure showed a biphasic response to injection of endotoxin as did heart rate. In the second set of experiments, the animals were injected with endotoxin while the animals received infusion of lidocaine into the VSA/DBB. The results show lidocaine infusion had no significant effect on body temperature, heart rate or mean arterial blood pressure in response to endotoxin. In conclusion the results do not support the idea that the VSA mediates an interaction between body temperature and mean arterial blood pressure, but neither does the data necessarily refute the possibility that the VSA is the site of this interaction. Hi TABLE OF CONTENTS ABSTRACT. ii TABLE OF CONTENTS iv LIST OF FIGURES .vii LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS jd DEDICATION. xii I. INTRODUCTION 1 A. Normal Thermoregulation 1 B. Fever. 2 1 .Pyrogens 4 i) Endotoxin 4 ii) Site of action of endogenous pyrogens 4 iii) Prostaglandins 5 iv) Summary. 5 2. Neural pathways involved in the production of fever 6 3. Antipyresis 7 i) Antipyretic drugs 7 ii) Endogenous antipyretic agents 8 4. Argenine vasopressin 9 i) Central neuroanatomy of AVP 9 ii) Antipyretic actions of AVP 10 iii) Role of the VSA in antipyresis 11 IV C. Central Regulation of the Cardiovascular System 11 1. Baroreceptor reflexes 12 2. Central sites of cardiovascular regulation 13 i) Nucleus tractus solitarius 14 ii) Ventrolateral medulla 15 iii) Paraventricular nucleus 16 iv) Supraoptic nucleus 17 v) Circumventricular organs 18 vi) Bed nucleus of the stria terminalis 19 vii) Locus coeruleus 20 viii) Median preoptic area 20 ix) Medial septum-diagonal band of Broca 20 D. The Interaction of Cardiovascular Function and Thermoregulation during Fever 22 1. Cardiovascular changes during fever 23 2. Central connections implicated in both cardiovascular and thermoregulatory control 25 H. OBJECTIVE 28 m METHODS 30 A. Surgical Preparation 30 1. Temperature group 30 2. Cardiovascular group 30 B. Computer Setup 32 1. Blood pressure telemetry system 32 i) Implantable transmitter. 33 ii) Telemetry receiver 35 iii) Consolidation matrix 35 v iv) Dataquest in data aquisition system 35 2. Body temperature telemetry system 35 C. Experimental Series 1 36 1. Saline injections 36 2. Endotoxin injections 36 D. Experimental Series II 37 1. Brain surgery. 37 2. Brain Cannulae Hardware 41 i) Guide cannulae template 41 ii) Guide cannula. 41 iii) Stylette plugs 41 iv) Injection stylettes 42 3. Osmotic minipumps 42 4. Protocol 44 5. Histology. 44 E. Statistical Analysis 45 IV. Results 47 A. The effects of endotoxin injection on heart rate, blood pressure, and body temperature 47 B. The effects of endotoxin injection on heart rate, blood pressure and body temperature in rats receiving saline or lidocaine infusions into the VSA. 47 Saline infused group 53 lidocaine infused group 59 C. Comparison of effects of endotoxin injection in lidocaine and saline infused animals 64 V. Discussion and Conclusion 68 VL References 77 VI LIST OF FIGURES FIGURE PAGE 1 Cardiovascular transmitter 31 2 Telemetry system 34 3. Guide cannulae 3 8 4 Guide cannulae template 39 5 Stylette plugs 40 6 Injection stylette 43 7 Brain slice at Bregma + 2.8 46 8 Change in body temperature following injection of endotoxin or saline 50 9 Change in mean arterial blood pressure following injection of endotoxin or saline 51 10 Change in heart rate following injection of endotoxin or saline....52 11 Effects of endotoxin on body temperature in saline infused animals 56 12 Effects of endotoxin on MABP in saline infused animals 57 13 Effects of endotoxin on heart rate in saline infused animals 58 14 Effects of endotoxin on body temperature in lidocaine infused animals 61 15 Effects of endotoxin on MABP in lidocaine infused animals 62 16 Effects of endotoxin on heart rate in lidocaine infused animals....63 17 Comparison of the effects of endotoxin on body temperature in saline and lidocaine infused animals 65 vii 18 Comparison of the effects of endotoxin on MABP in saline and lidocaine infused animals 66 19 Comparison of the effects of endotoxin on heart rate in saline and lidocaine infused animals 67 LIST OF ABBREVIATIONS AVP argenine vasopressin BNST bed nucleus of the stria terminalis CA anterior commissure CC corpus callosum DBB diagonal band of Broca EP endogenous pyrogen GABA gamma aminobutyric acid HC1 hydrochloride IL-1 interleukin-1 IML intermediolateral cell column LC locus coeruleus LS lateral septum LPS lipopolysaccharide MABP mean arterial blood pressure MS/DBB medial septum diagonal band of Broca mPOA medial preoptic area NTS nucleus tractus solitarius OVLT organum vasculosum of the lamina terminalis POA preoptic area PO/AH preoptic area of the anterior hypothalamus PGE prostalandin E be PVN paraventricular nucleus RES reticulo-endothelial system SON supraoptic nucleus SFO subfornical organ V ventricle VLM ventrolateral medulla VSA ventral septal area x ACKNOWLEDGEMENTS I would like to acknowledge the following: My supervisors: Dr. Norman Kasting, in whose lab the experiments took place, for his patience; Dr. Ruth Cridland, for her great tenacity in driving me onward, and her everlasting encouragement and support; and my committee: for their support, encouragement, and forthright insights: Dr. Nadine Wilson, Dr. John Ledsome, and Dr. Tony Pearson. Last but not least, I would like to acknowledge Sue Cho for being glad to see me and pointing out the difference between male and female rats. XI DEDICATION To the strongest most determined woman I have ever known, daughter of prairie pioneers, selfless and tenacious mother of four: Marion Evelyn Draper Marshall February 17, 1914 to December 14,1992 xu I. INTRODUCTION A. NORMAL THERMOREGULATION Thermoregulation is the process by which the body maintains a fairly constant temperature regardless of the ambient temperature (for review see Bligh, 1973). There is a normal daily variation or circadian rhythm in body temperature which reflects a difference between sleep and wake cycles; these daily fluctuations in body temperature function independently of ambient temperature or fever. Heat is produced by the sum of the many biochemical reactions which occur throughout the body. These reactions are essential in maintenance of ionic gradients, the assimilation of food, and in muscle movements. The effector mechanisms of thermoregulation regulate heat loss and this ensures a narrow range of body temperature allowing enzymatic activites to be maintained at optimal levels. Autonomic, somatic, endocrine and behavioral mechanisms are all involved in the process of maintaining the balance between heat production and heat loss which determines body temperature. One familiar mechanism by which the body maintains this narrow range of temperature is the involuntary action of shivering in response to cold, a mechanism which produces heat. An example of a heat conserving mechanism is vasoconstriction of the cutaneous vasculature. Heat loss mechanisms are illustrated by sweating in response to heat, or panting in the case of fur bearing mammals. Vasodilation allows heat to escape from the surface of the body. (For details on normal thermoregulation, see Gordon, 1990; Boulant, 1991.) It is reasonable to assume that the coordination of the various systems involved in thermoregulation would require one central brain area whose role would be to integrate the incoming and outgoing 1 information pertaining to body temperature. The hypothalamus performs the role of integrating incoming messages from central and peripheral temperature sensors and redirects this information via efferent pathways to effect heat loss or conservation (see review by Dinarello et al., 1988). Electrophysiological studies have shown that the preoptic area of the anterior hypothalamus (PO/AH) contains the thermosensitive neurons. Eisenman (1982) demonstrated in electrophysiological studies that 70% of neurons in the PO/AH and the ventromedial portions of the septal area respond with significant changes in their firing rates to local changes in temperature. The area under investigation in this study is the ventral septal area (VSA) located anterior to the PO/AH and ventral to the ventromedial portion of the septal area. B. FEVER Fever is defined as a controlled elevation of body temperature above the normal range resulting from a shift in the hypothalamic set-point *. Liebermeister in 1887 performed some basic investigations into the nature of fever (cited in Kluger, 1991). He noted that warming and cooling febrile patients always resulted in body temperature returning to the original febrile level. It was concluded therefore that fever was not the result of an inability to regulate body temperature but rather the regulation of body temperature at a higher level, i.e. a rise in the thermoregulatory set point. (For a detailed explanation of set-point theory, see J. Bligh, 1973 andl982.) * The concept of set-point is employed by control system engineers to achieve a variable set-point for a regulating system. It assumes there is a re-settable reference-signal generator, a sensor of the regulated variable (body temperature in this case), and a comparator of the two signals giving rise to a correction-effector activating signal. Scientists have attempted to apply this concept to the interactions of neuronal systems. 2 Set-point has been defined as the value of the controlled variable at which the control action is zero (Hensel, 1981). Snell and Atkins (1968) have provided a palpable classification of body temperature as follows: a) Normothermia occurs when the set-point and actual temperature coincide and are normal for the particular conditions. Exercise, ovulation and diurnal rhythms would be included here. b) Hypothermia occurs when the set-point is normal but temperature is reduced by disease, drugs, or cold exposure. c) Hyperthermia occurs when the set-point is normal but actual temperature is higher because of exposure to excessive heat, drugs, or diseases which are accompanied by an increase in metabolic rate. d) Fever occurs when the set-point is raised and may or may not be coincident with actual temperature. The various stages of fever are reviewed by Kluger (1991) and are briefly summarized below. During the rising phase of fever when the body is actively increasing temperature (via shivering, vasoconstriction, and behavioral effectors) to meet the elevated set-point, the febrile person or animal is actually hypothermic relative to the set-point. Physiological and behavioural effectors for conserving heat and producing heat continue until the temperature of blood bathing the hypothalamus matches the elevated hypothalamic set-point. Once body temperature has reached the set-point, the subjective feeling of cold disappears and the person or animal is normothermic at this elevated set-point. When defervescence begins, the body feels too warm as the set-point is now below body temperature. Throughout this stage the individual is hyperthermic and a variety of heat-losing physiological reflexes and behaviors now lower body temperature. 3 1. Pyrogens Fever is caused by substances called pyrogens. Exogenous pyrogens are substances not produced by the body; some examples include gram-negative and gram-positive bacteria, viruses, fungi, and some antigens of non-bacterial origin. Exogenous pyrogens stimulate the cells of the reticulo-endothelial system (RES) to synthesize and release endogenous pyrogen (EP). Endogenous pyrogens are heat-labile proteins secreted by a variety of white blood cells-including macrophages and lymphocytes. The term cytokine is used to describe these proteins which act as intercellular chemical messengers. Some known EPs are cytokines such as interleukins, especially IL-1, interferons, and tumor necrosis factor. Prostaglandins are also considered to be endogenous pyrogens. i) Endotoxin Endotoxin is an exogenous pyrogen which has been used extensively in experimental studies aimed at determining the mechanisms of the febrile response. Endotoxin is lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria. The LPS consists of a region responsible for the antigenicity, a core polysaccharide and a lipid 'A' region. It appears that the lipid A moiety is responsible for the pyrogenic quality of endotoxin (Reitshel et al, 1973; Luederitz et al 1973). The responses to LPS are repeatable and are not lethal. ii) Site of action of endogenous pyrogens E n d o g e n o u s pyrogens (EPs) stimulate the thermosensitive neurons of the PO/AH which in turn control the events leading to fever (Kluger, 1979; Gander, 1982). King and Wood (1958) performed experiments to elucidate the site of action of endogenous pyrogens. They infused EP into the carotid artery or into the marginal ear veins of rabbits. Infusion into the carotid artery resulted in a shorter latency of fever onset than that caused by injection 4 of EP into the ear veins. On the basis of these results, they concluded that EP acts directly on thermoregulatory centres of the brain. Cooper et al (1967) found that injection of lipopolysaccharide (LPS) into the PO/AH resulted in fever with a longer latency period compared to the fever produced by injection of EP into the same area. These results suggest that LPS stimulated the immune system to release EP. Electrophysiological studies have shown that iontophoretic applications of pyrogen activates cold-sensitive neurons while inhibiting warm-sensitive neurons in PO/AH both in vitro (Hori et al, 1984; Nakashima et al, 1985) and in vivo (Wit and Wang, 1968). iii) Prostaglandins Pyrogens increase the levels of prostaglandin E (PGE), an arachidonic acid metabolite, in the hypothalamus (Dinarello et al., 1983). It is these increases in PGE which are hypothesized to raise the set point of body temperature (Dinarello et al, 1988; Milton, 1982). Once the set-point is raised, peripheral mechanisms then act to increase the core temperature. A series of experiments using microinjection of PGE into the cerebral ventricles and PO/AH of cats and rabbits produced fever while similar injections into posterior hypothalamus and midbrain reticular formation failed to produce any change in body temperature. This was the first evidence that pyrogens induce fever through PGE (Milton and Wentlandt, 1971). iv) Summary The series of events by which pyrogens induce fever is summarized as follows: exogenous pyrogens are ingested by macrophages - this results in activated macrophages. Activated macrophages secrete endogenous pyrogens which stimulate the anterior hypothalamus to elevate synthesis of prostaglandins, especially of series E (PGE). A current theory is that PGE elevates the set-point by stimulating 5 temperature sensitive neurons of the PO/AH which in turn stimulate the vasomotor centre and peripheral efferents to initiate heat conservation and/or heat production. The result of these events is fever. 2. Neural Pathways Involved in the Production of Fever The role for PO/AH neurons in production of fever is well established (For review see Cooper, 1987; Hori, 1991; also see section A). Other regions in the CNS, such as ventromedial hypothalamus, mesencephal ic reticulospinal and rubrospinal neurons, limbic sytem, supraoptic and septal neurons have thermosensitive properties (Hori, 1991). It is therefore possible that divergent areas may also be involved in the febrile process. Recently Sagar and Price (1991) investigated the functional neuroanatomy of the rat brain during fever using the c-fos technique. This immunocytochemical technique acts as a metabolic marker for brain activity. The proto-oncogene c-fos, a nuclear protein, has been implicated in the regulation of gene expression. Elevated expression of c-fos has been observed following neuronal activation both in-vivo and in-vitro. Increased c-fos mRNA and/or protein levels are thought to result from the action of neurotransmitters on membrane receptors, and they seem to be induced by physiological stimuli (Kaczmarek and Nikolajew, 1990). LPS infused through femoral vein catheters into male Long-Evans rats induced Fos nuclear staining in the following areas: the circumventricular neuronal organs (organum vasculosum of the lamina terminalis, subfornical organ, and area postrema); the anterior hypothalamic region (AV3V); magnocellular neurons of the PVN and SON; the parvocellular neurons of the PVN, and the central nucleus of the amygdala, the arcuate nucleus; the locus coeruleus, nucleus tractus solitarious, and the Al cell group of the VLM. These experimental results 6 suggest involvement of the above areas of the brain during the production of fever (Sagar and Price, 1991). 3. Antipvresis An antipyretic is defined by the Encyclopedia of Medicine and Nursing (Miller and Keane, 1972) as "an agent that is effective against fever". Cold packs, aspirin and quinine are all considered to be antipyretics. i) Antipyretic drugs. The antipyretic drug most commonly used throughout the last century is salicylate. Salicylate was first isolated from willow bark by Piria in 1838, and synthesized by von Gerhardt in 1853 (cited in Collier, 1963; Hanzlik, 1926; Rainsford, 1984) Antipyretics reduce the increases in body temperature induced by exogenous pyrogens and endogenous pyrogens such as interleukin-1, tumor necrosis factor, and interferons (Clark, 1991). As antipyretic drugs in therapeutic doses do not affect normal body temperature, it is thought that antipyretic drugs act by interfering with the action of pyrogen upon the thermoregulatory set-point. Some medical textbooks indicate that antipyretics cause sweating or other peripheral effects, suggesting that antipyretic action is a peripheral one on sweat glands or blood vessels. However, these peripheral effects are secondary to the primary action of the antipyretic on the central thermoregulatory system. Evidence for a central site of action of antipyretics is supported by experiments where peripheral administration of antipyretic drugs antagonizes centrally administered pyrogens and central antipyretics inhibit peripherally administered pyrogen (see Clark, 1991). Furthermore, the direct application of salicylate onto thermosensitive neurons has been shown to 7 reverse pyrogen-induced firing changes in these neurons (Hori et al.,1984; Wit and Wang, 1968). The most widely accepted version of the mechanism of action of antipyretic drugs is the inhibition of cyclooxygenase, an enzyme which forms prostaglandins from arachidonic acid (Vane, 1971) . Experiments by Alexander et al (1987,1989) have shown that infusion of salicylate into the ventral septum can inhibit the pyretic action of prostaglandins. ii) Endogenous Antipyretic Agents. The presence of an endogenous antipyretic agent seems plausable when one considers that fevers rarely exceed a two degree increase in body temperature, 41°C being the highest recorded fever in most cases of infection (Dubois, 1949). It would seem logical that the body would have a built in mechanism to prevent damage to the cellular machinery which operates optimally at normal body temperature of 37°C and is disrupted at temperatures above 43°C. Scientific evidence supporting the presence of endogenous antipyretic was first provided in 1931 by Cushing who injected extract of pituitaries into the lateral ventricles of febrile patients and observed a decrease in body temperature. Kasting et al. (1978a,b) observed that newborn lambs and ewes near the end of pregnancy could not respond to endotoxin with fever, indicating an endogenously active antipyretic system. Several endogenous compounds, all peptides, have been shown to have antipyretic properties. Adrenocorticotropic hormone, corticotropin releasing factor, alpha-melanocyte stimulating hormone (alpha-MSH) and arginine vasopressin have all been implicated as antipyretic agents (Glyn and Upton, 1981). 8 While alpha-MSH, a fragment of ACTH (adrenocorticotropic hormone) has antipyretic actions at lower doses, it can also cause hypothermia in afebrile animals. (For a more detailed discussion of this peptide, see Glyn and Iipton, 1981.) The role of AVP in antipyresis will be addressed in greater detail in the following section. 4. Arginine Vasopressin i) Central Neuroanatomy of AVP Originally this peptide was identified as a peptide hormone of the posterior pituitary, produced by hypothalamic neurons and released into blood vessels of the neurohypophysis. It was noted for its antidiuretic properties and ability to contract smooth muscle. Immunohistochemical and radioimmunoassay techniques applied to the study the distribution of vasopressin have clearly shown AVP to be distributed throughout the CNS. Magnocellular AVP neurons in the SON and PVN project mainly to the posterior pituitary, whereas parvocellular AVP neurons inside and outside the hypothalamus do not project to the posterior pituitary (reviewed in Sofroniew, 1983). Changes in activity in the rat brain as a result of endotoxin induced fever have been shown to involve vasopressinergic pathways. Kasting and Martin (1983) found that endotoxin produces changes in immunoreactive vasopressin concentration in many areas such as the medial and lateral septum, the PO/AH, amygdala, NTS, and projections to the diagonal band. The largest increase in AVP-like immunoreactivity (pg/jug protein) was observed in the PO/AH, while the largest decrease was found in the medial septum during the febrile phase. A decrease in immunoreactivity could mean a depletion AVP due to increased release from nerve terminals or alternatively it could mean decreased synthesis of the neurotransmitters. 9 The findings of Sagar and Price regarding the neural pathways involved in the production of fever (see section B2) are consistent with the areas of the brain identified by AVP immunocytochemical techniques, i.e. the magnocellular neurons of the PVN and SON, BST (bed nucleus of the stria terminalis), the medial amygdaloid nucleus, and the ventral part of the vertical limb of the diagonal band of Broca (Rhodes et al.,1981; DeVries et al., 1985). These studies indicate that many of the areas and pathways involved in fever production are areas containing AVP, implicating AVP as a neurotransmitter involved in fever. Many of these areas identified as being activated during fever and identified as containing AVP are also known to be involved in cardiovascular function; for example: AVP containing neurons project to the dorsal motor nucleus and NTS from parvocellular PVN neurons. The PVN is known to send AVP projections to medial septum and amygdala (Buijs, 1978). This will be discussed in greater detail in section C. ii) Antipyretic actions of AVP AVP has been traditionally known for its peripheral effects such as water retention and vasoconstriction. Specific stimuli known to release vasopressin (hypertonic saline and hemorrhage) cause a reduction in fever, implicating AVP as an endogenous antipyretic substance (Kasting, 1986). Other evidence which implicates AVP as an endogenous antipyretic is that the levels of this peptide in push-pull perfusates from the septum are inversely proportional to the magnitude of fever (Cooper et al., 1979). In addition, perfusion of AVP into the VSA/DBB of sheep (Kasting et al., 1979), cats, rabbits (Naylor et al., 1985a,b, 1986) and rats (Ruwe et al., 1985) caused defervescence. AVP exhibited antipyretic actions in these species only when it was administered into the VSA. Antisera to AVP or a Vi-antagonist infused into the VSA prior to injections of pyrogen 10 enhanced the subsequent development of fever (Cridland and Kasting, 1992; Landgraf et al., 1990; Malkinson et al., 1987; Naylor, 1988). iii) Role of the VSA in antipvresis The VSA is situated ventral to the medial septum and rostral to the PO/AH, bounded laterally by the anterior commissure and medially by the vertical limbs of the diagonal band of Broca (Ruwe et al., 1985). As indicated above, the VSA/DBB has been implicated in thermoregulation during fever. Cooper et al, 1979, investigated the antipyretic effect of AVP administered by push-pull perfusion into various brain areas. The only region in which they found AVP-induced antipyresis was in the septal region. In a separate procedure using push-pull perfusion, they found that the amount of AVP in perfusate from the septal regions was negatively correlated with body temperature (for further support of this see Kasting et al, 1982a). AVP administered into the VSA supresses fever in a dose-dependant manner (see Veale et al, 1984). Kasting and Wilkinson (1985) have shown antagonism of the antipyretic effects of AVP with icv administration of an AVP Vi-antagonis t (d(CH2)5Tyr(Me)AVP) into the VSA. The other brain regions Sagar and Price (1991) found to be activated (as indicated by c-fos staining) during fever may be involved in the activation of the physiological effectors necessary to produce the fever, such as sending messages for vasoconstriction and/or shivering. Many of these labelled areas are known to be involved in cardiovascular regulation. (See section C for greater detail.) C. CENTRAL REGULATION OF THE CARDIOVASCULAR SYSTEM Central control of the cardiovascular system is an essential homeostatic element which compensates for varied requirements of the 11 circulation according to metabolic demands. Information regarding the metabolic demands of different parts of the body is received and integrated in brain areas concerned with cardiovascular function and the appropriate effector messages sent to the periphery. Without the central integrative input to the cardiovascular system, the sinoatrial node (which functions as the pacemaker of the heart) would maintain a steady heart rate regardless of the body's activity (for review see Green, 1987). These homeostatic mechanisms are executed by reflexes involving centres in the medulla which control heart activity, blood pressure and blood vessel diameter. 1. Baroreceptor Reflexes Baroreceptors are stretch sensitive receptors strategically situated at the bifurcation of the common carotid and in the aortic arch. These stretch receptors monitor pressure at the entrance of blood flow into the vessels of the brain and also at the access of blood to the lower part of the body. Stimulation of the baroreceptors of the carotid sinus results in increased activity of the sinus nerve to the cardioinhibitory centre of the medulla oblongata. Afferent input from the aortic arch baroreceptors is conducted via the aortic depressor nerve to the cardioinhibitory centre. According to the Reis model (Reis et al 1984) the primary afferents from arterial baroreceptors project via the IX and Xth cranial nerve to terminate in the intermediate to caudal portions of the NTS and the dorsal motor nucleus of the vagus. The barosensitive neurons of the NTS send secondary projections to neurons containing adrenalin in the rostral VLM. The rostral VLM is also known as the tonic vasomotor centre. This is the primary source of sympathetic vasomotor discharges. Descending projections from these 12 adrenergic neurons travel to the thoracic spinal cord in the intermediolateral cell column where they directly innervate sympathetic preganglionic neurons which then send messages to the blood vessels, adrenal medulla, and heart. Electrical stimulation of baroreceptor afferent neurons results in the following: decreased sympathetic discharges to the heart and blood vessels; inhibition of renal nerve discharges, resulting in diminished renin release and therefore reduced formation of angiotensin II and aldosterone; reduced peripheral secretion of AVP; increased efferent vagal discharges. The final result of these effects are a decrease in cardiac output and a decrease in peripheral resistance, leading to the restoration of arterial pressure to a set level (reviewed in Kumada et al.,1990). 2. Central Sites of Cardiovascular Regulation Stimulation of the arterial baroreceptors has been a useful method of identifying CNS sites which are involved in control of the circulation. A multitude of these sites have been identified (Calaresu et al.,1984). Some of these areas include the nucleus tractus solitarious (NTS), the ventrolateral medulla (VLM), the paraventricular nucleus (PVN), the supra-optic nucleus (SON) and the nucleus centralis of the amydala. It is important to note with respect to the work presented in this thesis that many of these areas have been shown to have neuronal connections with the diagonal band of Broca (DBB) (Donevan and Ferguson, 1988; Randle et al., 1986b; Jhamandas and Renaud, 1987). This is relevant because the DBB is immediately adjacent to the VSA and is also considered by many investigators to play an important role in antipyresis. The roles of different brain nuclei in cardiovascular regulation will be addressed briefly in the following section. While emphasis will be 13 placed on evidence implicating AVP as a neurotransmitter in these nuclei, several other transmitters such as glutamate, noradrenalin and adrenalin also play important roles in central control of cardiovascular regulation (Kumada, 1990). i) Nucleus Tractus Solitarius The nucleus tractus solitarius (NTS) receives primary viscerosensory afferent inputs from baroreceptors, chemoreceptors, and viscera through the vagal and glossopharyngeal nerves (cranial nerve X and DC). The NTS is the site of the first synapse of incoming baroreceptor information. The NTS also receives information from other brain areas such as the PVN. The convergence of input from a variety of sources suggests that the NTS plays a role in the integration of information before the effector messages are carried out. The NTS sends projections to the septal region (Donevan and Ferguson, 1988), the dorsal motor nucleus of vagus and the nucleus ambiguus (Matsuguchi et al., 1982). The dorsal motor nucleus of vagus and the nucleus ambiguus in turn send efferents to the heart, the preganglionic vagal cardiomotor neurons. L-glutamate is thought to be the excitatory neurotransmitter of the primary afferents of the baroreceptor reflex which synapse with neurons in the NTS (Jordan and Spyer, 1986). The NTS is also densely innervated by AVP neurons; the major source of these neurons being from the PVN (Sofroniew, 1980). AVP is known to influence blood pressure through the activation of Vl-receptors in the NTS (Matsuguchi et al., 1982; King and Pang, 1987). Injection of AVP into the NTS resulted in a dose dependent increase in blood pressure. Rascher et al. (1985) have shown that the increases of blood pressure and heart rate produced by AVP are blocked by pre treatment with a VI-14 antagonist. Administration of an AVP antagonist into the NTS also abolished the cardiovascular responses illicited by electrical stimulation of the PVN (Pittman and Franklin, 1985). Peripheral administration of VI-antagonist was without effect on the baroreceptor response illicited by AVP administration into the NTS (Rascher et al., 1985; King and Pang, 1987). This evidence suggests that the NTS may be a site of the central pressor action of AVP. ii) Ventrolateral medulla The ventrolateral medulla (VLM) is considered to consist of two separate functional areas: a) The Caudal VLM mediates the baroreceptor reflex control of AVP secretions into the circulation via noradrenergic projections to the magnocellular neurons in SON and PVN (Randle et al., 1986a; Day, 1989; Sawchenko and Swanson, 1982). The Al neurons of the caudal VLM are an essential component of the baroreceptor-initiated control of vasopressin secretion; these neurons are tonically active and inhibited by GABA (Blessing and Willoughby, 1985). Electrical or chemical stimulation of the caudal VLM in rabbits and rats causes hypotension by activation of vasodepressor neurons which inhibit sympathetic vasomotor activity (Blessing and Reis, 1982). The caudal VLM has reciprocal connections with the PVN (see Kumada et al., 1990). Electrophysiological stimulation of the caudal VLM is facilitory to PVN magnocellualar neurosecretory neurons. The caudal VLM receives input from the amygdala, SON, and dorsomedial hypothalamus as well. The efferent fibres travel to the NTS, rostral VLM, SON and PVN. Unloading of peripheral baroreceptors by hemorrhage or volume loss enhances the activity of AVP neurons and seems to be preferentially channeled through the Al neurons of the caudal VLM (Day and Sibbald, 15 1990) L-glutamate injected into the Al regions selectively excites cell bodies resulting in a dose-dependent increase in plasma vasopressin. Administration of a GABA receptor agonist into the Al region abolished the reflex increase in plasma AVP following hypotension caused by acute hemorrhage. Administration of a GABA antagonist into the same region resulted in increased plasma vasopressin (Blessing and Willoughby, 1985). b) The Rostral VLM is also known as the tonic vasomotor centre and contains noradrenergic neurons. This area receives secondary projections from barosensitive neurons in the NTS. The rostral VLM is the primary source of sympathetic vasomotor discharges. These descending projections travel in thoracic spinal cord to innervate sympathetic preganglionic neurons, which in turn sends messages to the vessels, adrenal medulla, and the heart. The VLM, therefore, participates in control of sympathetic vasomotor discharges as well as secretion of catecholamines and vasopressin (Ciriello et al, 1989). iii) Paraventricular nucleus The paraventricular nucleus (PVN) receives projections from the NTS and the caudal VLM. Electrophysiological stimulation in the caudal VLM and caudal NTS has been shown to excite the paraventricular magnocellular neurosecretory neurons (Ciriello et al., 1989). The caudal VLM is thought to control the release of AVP in arterial baroreceptor reflex through noradrenergic projections to magnocellular AVP-secreting neurons (Randle et al, 1986b; Day, 1989). Sofroniew and Schrell (1981) have shown that direct projections exist from PVN to centres of blood pressure regulation in the medulla. Increases in blood pressure have been shown to inhibit the DBB neurons which project to the PVN (Donevan and Ferguson, 1988). It has 16 been suggested that the PVN neurons receiving projections from the DBB are involved with autonomic cell groups rather than the tuberinfundibular cell groups which are involved in the systemic release of AVP (circulating AVP produces vasoconstriction through activation of receptors on arterioles rather than a neuronal activation employed by autonomic cell groups). Projections from the PVN to the amydgala, locus coeruleus, dorsal raphe nucleus, parabrachial nucleus, NTS, dorsal motor nucleus of vagus (DMN), nucleus ambiguus and the intermediolateral (IML) cell column of the spinal cord have been shown to contain AVP (Weindl and Sofroniew, 1985). Pittman (1989) has suggested that the AVP released at these sites may influence cardiovascular function. Swanson and Sawchenko (1980) describe the PVN as a site for the integration of neuroendocrine and autonomic mechanisms. Lawrence et al (1984) have demonstrated vasopressinergic projections from PVN to brain stem centres involved in cardiovascular control such as the dorsal vagal nucleus. Electrical stimulation of the PVN has been found to increase the release of AVP and oxytocin from NTS/DMN area, as well as increasing blood pressure (Landgraf et al., 1990; Kannan et al., 1988). Electrical stimulation of the PVN has also been shown to excite vagal motor neurons and sympathetic preganglionic neurons of IML cell column. (Gilbey et al, 1982; Lawrence and Pittman, 1985). Thus, there is evidence to suggest a function for the PVN in cardiovascular regulation in addition to its well known role in the control of the pituitary gland. iv) Supraoptic nucleus The supraoptic nucleus (SON) receives projections from the caudal VLM which synapse with the magnocellular vasopressin secreting neurons (Randle et al, 1986a,b; Day, 1989). 17 Electrophysiological studies by Harris, (1979) have found that a rise in mean arterial blood pressure causes a decrease in the excitability of neurosecretory neurons in SON and PVN. Increased levels of plasma AVP are observed following injection of noradrenalin into the SON (Blessing and Willoughby, 1985) Day and Sibbald (1990) have shown that SON and PVN neurosecretory AVP cells are inhibited by acute activation of arterial baroreceptors. Electrical stimulation in the DBB has been found to selectively inhibit the supraoptic vasopressin secreting magnocellular neurons (Randle et al, 1986b). Administration of a GABAA antagonist onto the SON reversibly abolished the depression of AVP release induced by DBB stimulation or by activation of the baroreceptor reflex (Jhamandas and Renaud, 1987). v) Circumventricular organs These organs are brain structures accessible from the blood circulation. a) area postrema: This area has been implicated in the modulatory action of circulating AVP on baroreceptor reflex sensitivity (Unger et al., 1988) Circulating AVP produces a greater bradycardia per increase in MABP in rats whose area postrema is ablated than in those with an intact area postrema (Peuler et al., 1990). This suggests that AVP in the circulation may act through the area postrema in normal rats to selectively augment reflex control of autonomic function b) subfornical organ (SFO): This circumventricular organ sends efferent connections to the organum vasculosum of the lamina terminalis (OVLT), another circumventricular organ, as well as to the MS/DBB (Lind et al., 1982). Studies using electrical stimulation have identified a polysynaptic pathway from the SFO to the PVN which involves the MS/DBB 18 (Donevan and Ferguson, 1988). Connections to the SON have also been verified anatomically (Oldfield et al., 1985). Some of these SFO projections to the PVN and SON have direct synaptic contact with oxytocin and vasopressin secreting neurons. Lesion studies have shown that the SFO is a necessary component in angiotensin II induced release of plasma AVP (Knepel et al., 1982) and the subsequent increase of MABP (Mangiapane and Simpson, 1980). vi) Bed Nucleus of the Stria Terminalis (BNST) Electrophysiological studies of the BNST have identified vasopressinergic projections to the VSA (Disturnal et al, 1985). Vasopressinergic fibres from the BNST project to the lateral septum, anterior amygdala, lateral habenular nucleus, periventricular grey and locus coeruleus and the diagonal band of Broca (according to lesion and dye injection studies of DeVries and Buijs,1983). The BNST is considered to be the main source of AVP released into the VSA during fever. The presence of AVP in the VSA causes antipyresis. The BNST also appears to be involved in cardiovascular regulation. Jhamandas and Renaud (1989) proposed that the established baroreceptor-induced inhibition of hypothalamic vasopressinergic neurons is mediated through DBB neurons. Electrical stimulation of the DBB depresses the excitability of VP-secreting neurons in the SON. These DBB neurons were recorded during baroreceptor activation and were found to have an increase in firing rate coincident with the rise in arterial pressure. Retrograde labeling studies indicate that the neural lobe of the pituitary receives fibres not only from the vasopressin and oxytocin containing neurones of the PVN and SON, but also from the vasopressin 19 and oxytocin containing neurones of the BNST (Weindl and Sofroniew, 1985). vii) Locus coeruleus The locus coeruleus (LC) is known to receive vasopressinergic fibres from hypothalamic vasopressin synthesizing nuclei ( Unger et al., 1988; Sofroniew, 1980) Microinjections of AVP into the LC resulted in dose dependent increases of heart rate and blood pressure (Berecek et a l , 1984). Alpha and beta noradrenergic blockade prevented these effects, suggesting that AVP acts via the LC to stimulate sympathetic outflow. viii) Median preoptic area The median preoptic area (mPOA) is believed to be an integral part of the neural loop by which AVP facilitates baroreflex-mediated bradycardia. The mPOA receives extensive inputs from PVN, SFO, NTS, parabrachial nucleus, LC, VLM and sends projections to the dorsal motor nucleus of vagus which is the primary source of vagal efferents to affect heart rate. Patel and Schmid, 1988 have shown that administration of Udocaine into the mPOA attenuated the decrease in heart rate mediated by AVP. These investigators conclude that local administration of lidocaine blocks neural activity in the mPOA and therefore that this nucleus plays an important role in the action of AVP in baroreflex-mediated bradycardia. ix) Medial septum-diagonal band of Broca Barorecep to r activation has been shown to increase the activity of neurons in the diagonal band of Broca (DBB) which project to the SON. These neurons are thought to mediate the decreased activity of the AVP neurons in the SON (Jhamandas and Renaud, 1986a,b;1987; Renaud et al., 1988a). 20 Jhamandas and Renaud (1986a) have proposed that DBB neurons relay a GABAergic baroreceptor input to vasopressin-secreting neurons in the SON. This proposal is based on the observation that a GABA antagonist reversibly blocks the DBB and baroreceptor induced depression of vasopressin secretion by SON neurons. The MS-DBB receives input from the SFO (Lind et al , 1982) and from vasopressinergic neurons in the BNST and medial amygdala (Oldfield et al.,1985). The septal region may receive baroreceptor input from NTS directly or polysynaptically (Donevan and Ferguson, 1988). Septal efferents synapse directly on vasopressinergic dendrites in the PVN (Oldfield et al., 1985). Large numbers of parvocellular vasopressin neurons are found in the septal region/DBB. The connections with the PVN have been verified electrophysiologically. Following an increase in blood pressure, there is a decreased activity of the DBB neurons which project to the PVN (Donevan and Ferguson, 1988). Baroreceptor reflex activation results in increased activity of DBB neurons which project to the SON (Jhamandas, 1986). The studies of Donevan and Ferguson (1988) have shown that MS-DBB neurons receive afferent input from the SFO and the cardiovascular system. This information is relayed to the neurons in the PVN. Thus, neurons in the MS-DBB are thought to activate neurons involved in the control of a variety of autonomic functions. In summary, a brief drug induced elevation in mean arterial blood pressure which is sufficient to activate peripheral baroreceptors causes a transient depression in the activity of vasopressin secreting neurons. It is thought that this depression is due to the GABA input to the baroreceptor reflex from the DBB. 21 D. THE INTERACTION OF CARDIOVASCULAR FUNCTION AND THERMOREGULATION DURING FEVER The basic premise of a relationship between cardiovascular function and fever seems obvious when one considers the mechanisms by which body temperature is maintained. It is the cardiovascular system which circulates the blood to the thermoregulatory centres of the hypothalamus, and via a precise orchestration of homeostatic elements, constricts the surface vessels to prevent heat loss and thus cause an increase in core temperature. The heart rate, blood pressure and cardiac output are all adjusted to implement the elevation of body tempera ture . Thermoregulation, therefore, cannot occur separately from the actions of other homeostatic systems, such as regulation of cardiovascular function. In section A, normal thermoregulation was discussed and the role of vasoconstriction and vasodilation in maintaining body temperature was explained. In section B, the mechanisms by which body temperature is elevated to a febrile level were discussed. When the normal firing rates of mammalian thermoregulatory neurons are shifted as a consequence of the action of EPs, the sympathetic nervous system is activated to cutaneous vessels, as well as to other organs (Greisman, 1991). The neural pathways involved in the production of fever were also discussed in section B. In section C, many neural pathways involved in cardiovascular function were outlined. There is some obvious physiological and anatomical redundancy in that some of these areas serve more than one function, i.e. thermoregulation as well as cardiovascular regulation. The neurotransmitter AVP has not only been implicated as an endogenous antipyretic, but is also seen to have a role in cardiovascular 22 control both centrally and peripherally. Centrally, AVP has been shown to play an integral part in cardiovascular control centers. AVP also enters the peripheral circulation from the posterior pituitary with the effect of constricting peripheral vessels and causing renal sympathetic stimulation. 1. Cardiovascular changes during fever Malkinson et al. (1988) noted that fever was accompanied by shivering and increases in heart rate and arterial blood pressure, during the rising phase of the fever in the rat. Vasodilation of the paws occurred during defeveresence. In human studies, during the chill phase of fever, there is an increase in cardiac rate and contractility, as well as an increase in total peripheral resistance and systemic venoconstriction (reviewed in Greisman, 1991). The net cardiac output appears to be variable, rising in some subjects, and decreasing in others. The appearance of a rise in arterial blood pressure at the onset of the chill phase was documented by Chasis et al (1942) when humans were injected intravenously with endotoxin. This rise in blood pressure was absent when the pyrogenic response was blocked with an antipyretic. Rises in blood pressure have also been observed during the chill phase of malaria (Perera, 1941). It is important to note that while MABP may not change appreciably during the chill phase, systolic and diastolic pressures may change considerably in humans (Greisman, 1991). Mean arterial blood pressure may remain unchanged in subjects because total peripheral resistance can rise in compensation for any decrease in cardiac output. As core temperature rises during fever, the discharge rate of the thermoregulatory neurones returns to prefebrile levels and the stimulus for the elevated sympathetic nervous system activity to the cutaneous vascular beds (responsible for restriction of heat loss) is withdrawn. The cutaneous blood flow returns to near prefebrile levels in man during the 23 steady phase of naturally occuring fever (Weinberg et al., 1989). The temperature of the skin is also elevated because the blood is now circulating at an elevated temperature, During the steady phase of fever, total peripheral resistence is reduced. A subsequent decline in MABP may not be significant because of compensatory increases in cardiac output. MABP decreased an average of 18 mm Hg in 12 human volunteers, at the peak (102°F) of endotoxin induced fever (Suffredini et al., 1989). In other studies, where a maximum mean core temperature of 101°F was reached, the results varied from no significant change in MABP to minimal decreases of MABP from 2 mm Hg to 7 mm Hg (Greisman, 1991). Heart rate in humans during the steady phase of fever was found to be elevated by 10 beats/min for each degree Fahrenheit rise in core temperature. Most studies have noted a significant difference in the response observed between individuals. The initial rise in heart rate during the chill phase is considered to be due to increased sympathetic nervous system activity and the withdrawal of vagal tone. However, during the steady febrile phase, increased heart rate is thought to result from the direct effect of elevated blood temperature on the sinoatrial node (Greisman, 1991). The appUcation of a heat stimulus to the terminal part of the superior vena cava causes an acceleration in heart rate, whereas similar heat applied to the ventricular part of the heart is without effect (McWilliam, 1888). The isolated heart of the dog and cat have also been reported to be extremely sensitive to changes in temperature (Knowlton and Starling, 1912). It has also been noted that even though mammals may have different resting heart rates in relation to their size, the incremental change in heart rate in proportion to the increase in core temperature is fairly consistant (Tanner, 1951). Thus, the increase in 24 heart rate during fever is probably due to a direct effect of the temperature of blood on the sinoatrial node, whereas the alterations in total peripheral resistance, cardiac output, and mean arterial blood pressure appear to be the result of different mechanisms (Greisman, 1991). There is a marked dilation of the surface vessels d u r i n g defeveresence. It is estimated that 50-70% of the cardiac output is delivered to effect heat loss as body temperature returns to normal (Rowell, 1983). If cardiac output fails to increase during defeveresence, then MABP may decline during this stage because of the marked reduction in cutaneous vascular resistance. In humans, given an iv bolus of endotoxin, a 24% decline in MABP occurred when defeveresence was most pronounced (Suffredini et al., 1989). In summary, changes in cutaneous circulation are critical for the production of the rising, steady and defervescent phases of fever. The action of endogenous pyrogens on the thermoregulatory system evokes these changes in sympathetic cutaneous system. Circulatory changes in other vascular areas during fever are complex and may result from pathogenesis of the febrile state (for further information, see Greisman, 1991). 2. Central connections implicated in both cardiovascular and thermoregulatory control There are many areas of the brain which have been shown to respond to more than one type of stimuli. The neurons in these areas have been called "multimodal" because of their ability to respond to divergent stimuli (Hori, 1991). For example, Miyazawa et al. (1988) found neurons in the septum which respond to activation of baroreceptors and 25 chemoreceptors. Conversely, chemical microstimulation of the septal area lowers blood pressure in the rat (Gelsema, 1987). The PO/AH is an established centre in thermoregulation, and appears to contain multimodal neurons, in that they respond to temperature and cardiovascular changes as well as other stimuli. Hori et al. (1988) showed that thermosensitive neurons in this region exhibited responsiveness to blood pressure changes of less than 15 mm Hg. Bilateral section of the glossopharyngeal, and vagus nerves abolished the PO/AH neuronal responses to changes in blood pressure. This suggests that these PO/AH thermosensitive neurons respond to input from baro/volume receptors. It has been estimated that up to 75% of PO/AH thermosensitive neurons in the rat receive afferent input from peripheral baro/volume receptors (Koga et al., 1987a,b). The PO/AH not only responds to blood pressure changes, but electrical stimulation of this area causes changes in blood pressure as well (Faiers et al., 1976). The thermosensitive neurons which Koga et al.(1987a,b) found to be responsive to blood pressure changes were located in the mPOA, BNST, anterior hypothalamus and paraventricular nuclei. The BNST is of interest in this study because it is the source of the AVP synthesizing neurons which release AVP into the VSA. An anatomical study using HRP (Mathieson et al.,1989) has shown that reciprocal connections exist between the ventral septal area (VSA) and the BNST, lateral septum and medial preoptic area of the forebrain. Electrophysiological evidence suggests that the PVN and BNST provides the source of AVP to the VSA (Disturnal et al., 1985). Autoradiographic evidence (Swanson and Cowan, 1979) shows projections from the VSA to the preoptic area. This was confirmed by Anderson and Shen (1980) who 26 found medial preoptic axons which projected to the diagonal band of Broca, and septum, specific to the area investigated in this thesis. Thermosensitive and thermoregulatory stuctures also appear to exist in the midbrain, pons, medulla, and spinal cord; areas which are known to be involved in cardiovascular control. Simon et al. (1986) has suggested that a hierarchy of neural integration and control exists in the thermoregulatory response. Neurons responsive to several types of input may cause the selective production of specific effects. Changes in internal and external conditions cause a variety of autonomic and behavioral thermoregulatory responses. Thermoregulation does not occur in isolation, but is intricately interelated with the action of other regulatory systems (Nadel, 1983; Hori et al., 1988). Renaud et al. (1988b) found that electrical stimulation of neurons in the DBB selectively depress AVP secreting neurons in the SON. These AVP secreting neurons cease firing while the DBB neurons are activated in response to hypertension. This would suggest that neurons of the DBB receive cardiovascular input and in turn cause inhibition of vasopressin secreting neurons in response to an increase in blood pressure. The DBB neurons investigated by Renaud et al. are anatomically in the same experimental area of the VSA/DBB investigated for the research of this thesis. This thesis investigates the possiblity that thermosensitive neurons of the VSA/DBB which are involved in thermoregulation during fever, may also be involved in some aspect of cardiovascular control during fever induced by endotoxin. 27 H. OBJECTIVE The ventral septal area (VSA) has been implicated as an important centre for thermoregulation during fever (see reviews by Kasting, 1989; Cooper, 1987). Arginine vasopressin immunoreactive nerve terminals and receptors have been identified in the VSA (DeVries et al., 1983, 1985; Sofroniew, 1980, 1983; Sofroniewand Schrell, 1981; Poulin et al., 1988), and abundant evidence suggest that AVP in this region has important antipyretic or fever reducing effects. The role of circulating AVP, from the pituitary, has well documented effects in cardiovascular control. In addition, AVP has been implicated as a neurotransmitter in the central control of cardiovascular function (see section C). During fever, cardiovascular changes accompany the change in temperature (see section D). Electrophysiological studies, physiological evidence, and anatomical tracing techniques suggest that there are important connections between central cardiovascular control centres and thermoregulatory centres. The VSA is one of the areas which may be involved in both cardiovascular control and thermoregulation during fever. The VSA/Diagonal band of Broca (DBB) has reciprocal connections with the bed nucleus of the stria terminalis, amygdala, lateral septum, medial preoptic area of the forebrain, the lateral habenular nucleus, many nuclei of the hypothalamus, and midbrain serotonergic cell groups which innervate the brain stem. Some of these areas have been implicated in cardiovascular function, and some in thermoregulation. Neurons in the area of the VSA/DBB have been shown to exhibit changes in their firing rate in response to stimulation of barorecepter reflexes (Jhamandas and Renaud, 1986a). These neurons thus appear to have physiological connections to the cardiovascular control centres of the 28 brain stem suggesting that they may function in cardiovascular control in addition to their well established role in antipyresis. This thesis explores the possibility that, during fever, not only is the VSA involved in thermoregulation, but that it may also control the accompanying cardiovascular changes seen during a febrile episode. Specifically, this study examines the effect of interfering with neural transmission in the VSA during fever by infusing lidocaine HCl into the area. It was expected that the lidocaine would prevent the release of AVP resulting in a higher and more prolonged fever. The effect of a lack of AVP in the VSA on cardiovascular parameters could then be examined 29 III. METHODS A. SURGICAL PREPARATION Two separate experimental groups of male Sprague Dawley rats (325-375g) were utilized. Body temperature was monitored in one group, and cardiovascular variables in the other. All rats were housed at a 12:12 light-dark cycle with food and water freely available. 1. Temperature Group Rats in which body temperature was monitored were anesthetized with chloral hydrate (300 mg/Kg). The rats were placed on a heating pad to maintain body temperature during surgery. The abdomen was then shaved and swabbed with 70% alcohol (isopropyl) and a longitudinal incision, approximately 3-4 cm long, was made in the skin below the sternum to expose the underlying muscle. A pair of rat-tooth forceps was used to hold the abdominal wall, while a midline incision was made to allow insertion of a radio-telemetric transmitter (Minimitter Inc.) into the peritoneal cavity. This transmitter allowed constant monitoring of body temperature. After suturing and stapling of the wound, the rat remained under a heat lamp until body temperature returned to normal. 2. Cardiovascular Group Brietal (methohexital sodium, 50 mg/Kg, i.p.) was administered to anesthetize rats in preparation for implantation of transmitters which measured cardiovascular variables. The abdomen was shaved, swabbed with 70% isopropyl alcohol, and an incision made from sternum posteriorly 30 for 3 to 4 cm. Sterile cotton gauze was inserted into the cavity to push the intestines aside and expose the dorsal aorta from the bifurcation of the femoral arteries to approximately 0.5 cm above the iliolumbar veins. Fat and connective tissue were removed from the area of the dorsal aorta by gently scraping with cotton swabs. A pair of curved forceps was inserted between the dorsal aorta and the inferior vena cava to separate the connective tissue between the two vessels. Curved vascular clamps placed at the rostral end of the exposed aorta temporarily interrupted the flow of blood from the heart. Curved forceps placed between the vessels below the clamp were used to lift the aorta into an accessible position. Releasing the pressure on the forceps caused them to expand, thus stretching the aorta and preventing any back flow of blood into the working area. A 21 gauge needle bent at an angle of 90° was used to puncture the dorsal aorta as close to the bifurcation of the femoral arteries as possible. The flexible plastic end of a cardiovascular transmitter was then inserted into the dorsal aorta to a distance of about 1.5 cm (see arrow in Figure 1). 4-FIGURE 1 : Cardiovascular transmitter (DataSciences Inc.). The transmitter is 12 mm by 20 mm with 90mm of flexible tubing attached. The antithrombogenic end of the tubing is inserted into the dorsal aorta to a distance of approximately 15 mm. (not to scale). 31 Tissue adhesive (Vetbond) was used to glue the transmitter into the aorta and to prevent leakage of blood around the tubing. After a few seconds to allow hardening of the glue, the clamp and the forceps were removed. It was necessary to complete these manipulations within 3 minutes to avoid tissue ischemia. Proper cannulation was verified by placing the animal on a receiver board to monitor the telemetry signal by observing the blood pressure wave form on the computer screen. After the gauze was removed and the intestines put back into place, the transmitter was stitched by its net covering to the abdominal wall during the suturing of the incision. A recovery period of approximately 1 week ensured the reinstatement of circadian rhythms before experiments were begun. Brietal was chosen as an anesthetic because it is not hypotensive. The success rate of implantation of the cardiovascular devices was much higher using Brietal anesthetic. In initial experiments, chloral hydrate anesthetized animals were found to recover easily from temperature transmitter implantation and brain surgery, but these animals exhibited a fall in blood pressure severe enough to cause clotting at the site of insertion of the cannula into the dorsal aorta leading to ischemia and paralysis of the lower Hmbs. Thus, the success rate of implantation of the cardiovascular devices was much higher using Brietal because this anesthetic allowed the blood to continue to flow past the point of cannulation with normal pressure immediately after the surgery. B. COMPUTER SETUP 1. Blood Pressure Telemetry System This system consists of 5 major components: 32 - implantable transmitter -the dual ambient pressure monitor -the telemetry receiver -the consolidation matrix -the data acquisition system. Together these components function to provide a system for measurement, collection, storage, and retrieval of blood pressure variables from animals. (see Figure 2) i) Implantable transmitter This device (Figure 1) contains a catheter which is inserted into a blood vessel. The device consists of a magnetically activated on-off mechanism, a pressure sensor, electronics which convert the signal from the pressure sensor, and a radio-frequency transmitter which sends the pressure signal to the telemetry receiver. The catheter tip is specially constructed to provide for a high level of blood compatibility. The distal 1.5 cm of the tip is a very thin membrane coated with an antithrombogenic film. The distal end of this thin-walled section is filled with a gel. This implantable transmitter measures absolute pressure. Barometric pressure is subtracted from the telemetered pressure measurement automatically by the Dataquest III System and all pressure values recorded on the Dataquest III System are in mm Hg relative to atmospheric pressure. The transmitters were received factory calibrated from Datasciences Inc. Three calibration values for each transmitter were entered into the software program before monitoring the signal. Before the transmitters were used a second time, the calibration was checked by placing into a pressure chamber and monitoring the signal at each of the calibration 33 fr rat with transmitter receiver boards pressure reference consolidation matrix Dataquest IGM-PC FIGURE 2 34 pressures. Any corrections were then entered for each individual transmitter into the software program. ii) Telemetry receiver The signal from the transmitter easily passes through the walls of the plastic cage in which the animals are housed. The receiver board detects and amplifies the radio signal, converting it to a series of digital pulses which can be decoded by the computer. In addition, the telemetry receiver provides a digital signal to the Dataquest III System for recording of heart rate. The receiver plugs into the consolidation matrix via a modular cable. iii) Consolidation matrix The consolidation matrix provides a link between the receiver and the dataquest III system and allows the signal inputs from each of the receivers to be reconfigureable via the Dataquest III software menu, iv) Dataquest III data aquisition system A plug-in card and software for an IBM PC was provided by DataSciences Inc., Minneapolis, MN, USA. This system collected the data at preset timed intervals, stored the information and allowed retrieval for later analysis. 2. BODY TEMPERATURE TELEMETRY SYSTEM Basic operation of this system was identical to the above description. No pressure reference is necessary, and a simple adjustment to the software program Dataquest III allowed for the collection of body temperature data. Both body temperature and mean arterial blood pressure experiments were run simultaneously on separate channels by simple software manipulations. Small transmitters powered by hearing aid batteries were encased in plastic covers which were sealed with wax to prevent the entrance of liquid. 35 Body temperature transmitters were calibrated at 35°C and 39°C. The transmitter was immersed in a water bath at each body temperature while the signal was monitored by the computer. These values were then entered into the software program. By entering the value for the signal at 35°C and 39°C, the software program was then able to interpolate the body temperature of the animal from the signal it received. C. EXPERIMENTAL SERIES I Body temperature and the blood pressure groups of rats were treated similarly. After a recovery period of approximately 1 week when the circadian rhythms were seen to have returned to normal, the preliminary set of experiments was begun. All injections were given intraperitoneally (i.p.) after the 0950 a.m. reading collected by the computer. Readings of blood pressure and heart rate or body temperature were recorded by the computer every 10 minutes via the software program Dataquest IE for the duration of the experiment. 1. Saline Injections All animals, after recovery from surgery, were injected with 0.5 cc of physiological saline (0.9%) intraperitoneally. The heart rate, mean arterial blood pressure or body temperature were monitored for l h previous to the injection and for six hours post-injection. 2. Endotoxin Injections All rats were then injected with endotoxin (Lipopolysaccharide E.Coli serotype 026:B6; 50 pg/rat in 0.5 cc of physiological saline) on day 2. Again, all variables were recorded 1 hr previous to and 6.6 hours post injection. 36 D. EXPERIMENTAL SERIES II 1. Brain Surgery Following completion of preliminary experiments and allowing a one week recovery period after the endotoxin induced fever, brain surgery was performed to allow infusion into the VSA. All rats received the same anesthetic for the brain surgery (chloral hydrate; 300mg/Kg, i.p.). and were operated on using identical protocols. A heating pad placed over the base of the stereotaxic device kept the rat warm during brain surgery. The rat was placed securely into the ear bars of the stereotaxic device with the nose bar set at 5 mm above horizontal. An incision of approximately 2 cm long in the skin over the saggital suture was made. The fascia was scraped away to expose Bregma and the surrounding working area. The guide cannula (Figure 3) was placed over the guide cannulae template (Figure 4). The non-beveled ends of the template were firmly secured into the movable arm of the stereotaxic device. A point was marked on the skull on the saggital suture at a distance of 2.8 mm anterior to Bregma where a hole was drilled. This hole extended 1 mm on either side of the sagittal suture. Forceps were used to carefully remove bone chips from the drilled hole, and the area was then washed with sterile physiological saline. Four screw holes were drilled around this opening. Stainless steel jeweler's screws were then set into the screw holes. The guide cannula was positioned with each side of the tubing placed 1 mm on either side of the superior sagittal venous sinus. A sterile needle was used to puncture the dura, and the cannula lowered to 3.0 mm below the dura and cemented into place with a mixture of dental acrylic. The dental acrylic was applied over the hole and around the screws. Once dry, the cannula were separated from the cannula template while the arm of the stereotaxic device was raised. Sterile stylette plugs (Figure 5) were 37 GUIDE CANNULA Wf 1 20g 15 mm to centre of bevel u FIGURE 3 38 GUIDE CANNULAE TEMPLATE 20 g tubing 2mm apart Two 23g needles total length does not exceedl5mm FIGURE 4 39 STYLETTE PLUG v | 5mm 20g | | 18mmof25p, 30g 25 mm FIGURE 5 40 inserted into the guide cannulae to prevent infection. (These plugs were not removed until the osmotic minipumps were attached.) After the rat was removed from the stereotaxic device and body temperature returned to normal, it was then placed into a plastic cage on the specific receiver board programmed for the specific transmitter implanted into each rat. The computer continued to collect data on the variables measured by Dataquest III during the recovery from surgery until it was established by normal circadian rhythms that the rat had recovered adequately for the last set of experiments to take place. 2, Hardware i) Guide Cannulae Template Thin-walled 20 gauge stainless steel tubing was soldered exactly 2mm apart (see Figure 4). Two 23g needles were cut from their hubs and inserted into the 20g tubing to extend no more than 15 mm. The 20g tubing was then crimped to hold the needles in place. ii) Guide Cannula The guide cannula were built from 20 gauge stainless steel tubing. Lengths of 16 mm were cut and bevelled until they were 15 mm long in the center of the bevel. Two of these lengths placed over the guide cannula template were soldered together. The two lengths of tubing were then 2 mm apart (see Figure 3). iii) Stvlette Plugs A length of 30 gauge stainless steel tubing was placed inside an 18 mm length of 23 gauge tubing. A 5 mm cap of 20 gauge stainless steel tubing placed over the other two pieces was crimped into place. Two plugs were used for each rat; one for each side of the guide cannula (see Figure 5). 41 iv) Injection Stvlettes A 30 mm piece of 30g stainless steel tubing was beveled on one end. A piece of 23g HTX was placed over the 30g piece, leaving 6 mm from the beveled end (see Figure 6). A 5 mm cut of 20 g tubing was then placed over the 23g, flush with the end farthest from the bevel. These 3 pieces were then soldered together. Two injection stylettes were prepared for each rat. 3. Osmotic Minipumps After recovery from brain surgery, the animals were prepared for the second stage of the experiment. Osmotic minipumps (Model 1003D , Alzet Co, Palo Alto, CA, USA) were filled with vehicle or treatment (5% lidocaine hydrochoride, Sel-Win Chemicals, Vancouver, Canada). The minipumps were incubated at 37° for 4 hours to ensure the pumps were primed at the time of implantation and to prevent any premature release of contents (Theewes and Yum, 1976) The pumps dispensed their contents at a rate of 0.5 juL/h over the course of the next 72 hours. Pumps were attached via polyethylene tubing to injection stylettes (Figure 6). The PE tubing was filled with either vehicle or treatment solution (to match the contents of the pump which was being attached to it) to ensure that the infusion into the brain began immediately. The rats were anesthetized with chloral hydrate, stylette plugs removed, and the injection stylettes inserted into the guide cannula. The length and construction of the injection stylettes ensured that the tips were situated bilaterally 7.5 mm below dura to the level of the ventral septal area (VSA). The pumps and tubing were inserted subcutaneously at the back of the neck. 42 INJECTION STYLETTE Z7 2 0 g 23gHTX 5mm 18mm 3Qg 30mm FIGURE 6 43 4. Protocol Any rat whose baseline heart rate and blood pressure appeared hypertensive, had not regained their presurgical weight, had above normal temperatures, or appeared unhealthy in any way were deleted from the experiments. A mash of chocolate chip cookies with evaporated milk aided their recovery to pre-surgical weight. Following implantation of osmotic mini-pumps, the rats were then replaced into their plastic cages on the receiver boards and the computer continued to collect data. This was considered day zero. The following day (day 1), the animals were monitored throughout the course of the day, which provided the baseline values. On the morning of day 2, immediately after the 9:50 a.m. reading, all animals were injected with endotoxin. The effects of endotoxin injection were monitored throughout the day, 400 minutes past the injection time was considered to be the end of the experiment. 5. Histology At the termination of the experiment, the animals were perfused with a 10% solution of buffered neutral formalin (BDH). The brains were then preserved in a solution of 10% buffered formalin with 20% sucrose and kept refrigerated until slicing. A Microtome Cryostat (IEC Minotome) was used to cut 20^m thick sections. The sections were "melted" onto agar-alum coated slides and dried at room temperature overnight. The slides were then stained in a 0.1% solution of cresyl violet, cleared in xylene substitute from BDH ( a biodegradable form containing terpene hydrocarbons and an oxidation inhibitor), and cover-slipped using "Permamount". 44 The injection sites were confirmed by light microscopy to be within the area defined as the VSA. Rats with bilateral injection sites falling outside this area were discarded from data analysis. Figure 7 shows a brain slice at the coordinate +2.8 anterior to Bregma. The circles represent the injection sites in this experiment that were deemed to be in an acceptable location for the definition of the VSA. E. STATISTICAL ANALYSIS All points graphed were expressed as the mean. Error bars represent plus or minus standard error of the mean. Unpaired t-tests were used, according to Neter et al., 1990. When the animals acted as their own controls, then a paired t-test was used where indicated. Occasionally when the animals were used as their own controls, a rat would die before receiving vehicle or treatment, or a faulty transmitter would result in an unequal number of animals in a group. When this occurred it was necessary to use unpaired t-tests. Significance as denoted by asterisks represents p<0.05. Mean baseline values were calculated by taking the average of the readings recorded 90 minutes prior to injection. The change scores were then the recorded temperature minus the baseline value. Each point on the graphs represents the average change score, thus error bars are standard error of the mean. 45 FIGURE 7 The cannulae tips were 2 mm apart to allow bilateral infusions into the VSA. The circles represent a few of the sites which were considered to be acceptable locations defined as the VSA. 46 IV RESULTS A. THE EFFECTS OF ENDOTOXIN INJECTION ON HEART RATE, BLOOD PRESSURE, AND BODY TEMPERATURE Heart rate, blood pressure and body temperature changes were monitored in rats following injections with either saline or endotoxin. These rats had no brain surgery, only the transmitters implanted. The purpose of this set of experiments was to investigate the effects of endotoxin injection alone, before any brain infusions, and to be assured that any changes from baseline values were indeed due to the endotoxin and not an injection artifact. The purpose of the saline injection was to eliminate the possibility of injection artifact. Figure 8 The effects of injection of endotoxin (50jL7g/rat in 0.5 cc physiological saline; n=17) on changes in body temperature over the course of 400 minutes were examined and compared to the control group which received injection of physiological saline (n=13). Unpaired t-tests were done to determine statistical significance (p<0.05) of differences. Data collected from transmitters with technical difficulties or unhealthy rats were deleted. Injected of saline showed saline failed to alter body temperature throughout the experimental period. The endotoxin injected animals showed a decrease in body temperature beginning at 40 minutes and extending to 100 minutes. A subsequent increase in body temperature occurred following 100 minutes with the peak change in body temperature (0.84°C) occurring at 200 minutes. Here the body temperature stabilized at this elevated level (0.7 to 0.8°C above baseline) before a gradual decline in the last 40 minutes of the experiment. 47 The mean baseline value for body temperature in saline injected rats was determined to be 36.8°C ± 0.1°C (± SEM)and in the endotoxin injected group, 36.8°C ± 0.1°C. Statistically significant changes in body temperature exist between the saline and endotoxin injected groups at 60, 80, and 100 minutes post-injection as well as 160 to 400 minutes inclusive. Figure 9 The effects of injection of endotoxin (n=9) on mean arterial blood pressure over 400 minutes were compared to the effects of injection of saline (n=8). The Dataquest system determines mean arterial blood pressure by taking 1/3 of the pulse pressure and adding diastolic pressure. Pulse pressure is equal to the systolic minus the diastolic pressure. Significant differences (p<0.05) were determined by unpaired t-tests due to the unequal number of rats in each group. The average baseline mean arterial blood pressure in saline and endotoxin injected rats was determined to be 98 ± 3 mm Hg and 92 ± 5 mm Hg respectively. The saline injected animals showed a relatively constant mean arterial blood pressure throughout the experimental period. The endotoxin injected animals showed a biphasic response. An increase in blood pressure occurred immediately; peaked at 20 minutes and gradually declined to 6 mm Hg above baseline at 80 minutes after injection. This marked the end of the first phase. At the beginning of the second phase, there was an upward climb immediately following 80 minutes to reach a peak (albeit a lower peak than the first phase: 12 mm Hg above baseline) at 120 minutes. After 120 minutes blood pressure declined for the remainder of the experimental period. Statistically significant differences between the animals injected with saline and animals injected with endotoxin (p<0.05) are seen at 20 to 60 minutes and 100 to 180 minutes post-injection. 48 Figure 10 The change in heart rate following injection with saline (n=8) or endotoxin (n=8) are represented in figure 10. The baseline heart rate in saline and endotoxin injected rats was determined to be 326 ± 4 bpm and 338 ± 5 bpm respectively. Heart rate proved to be relatively variable. The animals injected with saline showed a variation of -14 to +13 bpm from baseline throughout the experiment. The rats injected with endotoxin showed an immediate increase in heart rate at 20 minutes to + 47 bpm above baseline with a subsequent drop at 40 minutes to 20 bpm above baseline. This was followed immediately by another increase in heart rate to a second peak at 80 minutes to + 45 bpm above baseline. Following the 80 minute reading, heart rate dropped to a low at 220 minutes of 2 bpm above baseline. At 240 minutes post injection to the termination of the experiment heart rate maintained itself at a moderately increased level of an average of 20 bpm above baseline. There appeared to be a trend to increased heart rate in animals injected with endotoxin; there were three points at which significant differences (p<0.05) existed when compared to the saline group (at 60, 280, and 320 minutes post injection). 49 u 0 LU CL I -< LU CL z LLI > O O CO LU O z < - i — • — • — i — i — i — i — • — i — i — i — • — r 60 120 180 240 300 360 420 TIME (MIN) FIGURE 8: This graph represents mean (± SEM) changes in body temperature following administration of either saline (open circles; n=13) or endotoxin (50 ^yg/rat in 0.5 cc saline, i.p.) represented by closed circles (n=17). All injections were given at time zero. Asterisks represent significant differences as determined by unpaired t-tests (p<0.05). 50 20-1 JZ E £ a. CD < Z LU o < u 120 180 240 300 360 420 TIME (MIN) FIGURE 9: This graph represents mean (± SEM) changes in mean arterial blood pressure following either injection with saline (open circles; n=8) or injection with endotoxin (closed circles; n=9). Significant differences between saline and endotoxin injected animals are represented by asterisks (unpaired t-tests, p<0.05). For further details see Fig. 8. 51 100-80-4 0 H — i — i — i — i — i — i — i — • — i — i — i — i — • — • — i — " — i — i — i — i — i — > — i 0 60 120 180 240 300 360 420 TIME (MIN) FIGURE 10: This graph represents mean (± SEM) changes in heart rate following injection of saline (open circles; n=8) or endotoxin (closed circles; n=8). Statistically significant differences (p<0.05) as determined by unpaired t-tests are represented by asterisks. For further details, see Figure 8. 52 B. THE EFFECTS OF ENDOTOXIN INJECTION ON HEART RATE, BLOOD PRESSURE AND BODY TEMPERATURE IN RATS RECEIVING SALINE OR LIDOCAINE INFUSIONS INTO THE VSA 1. Saline Infused Group Blood pressure, heart rate and body temperature were monitored over the course of a complete day during which osmotic minipumps infused physiological saline into the VSA. The parameters thus measured constituted the control day of this set of experiments. On the second day, each animal was given an injection of endotoxin (50 jvg dissolved in 0.5 cc of physiological saline) Figures 11, 12, and 13 compare the control day (saline infusion alone-day one) to the endotoxin day (saline infusion with endotoxin injection-day two). The purpose of this set of experiments was to investigate the effects of endotoxin induced changes in each of the parameters measured while receiving saline infusions into the VSA. Figure 11 The effects of endotoxin injection on body temperature in saline infused animals were compared to the control day (day 1). Unpaired t-tests were used to determine statistical significance (p<0.05). Mean baseline values for body temperature on day 1 and day 2 are 37.5 °C ± 0.1 °C and 37.3 °C ± 0.1 °C respectively. In saline infused animals, injection of endotoxin significantly increased body temperature from 20 to 380 minutes post-injection compared to the respective values on the control day. Infusion of saline alone into the VSA was without effect on body temperature. Figure 12 The effects of endotoxin injection on mean arterial blood pressure in saline infused animals are represented in Figure 12. Each animal acted as its own control, and since there were equal number of 53 animals in control and endotoxin injected groups, paired t-tests were done to determine statistical significance (p<0.05). The baseline value for the animals receiving saline infusions alone was 104 mm Hg ± 2.9 mm Hg and 104 mm Hg ± 2.7 on the second day. The animals receiving saline infusions alone (control day; n=7) showed changes in MABP ranging from -7 to +3 mm Hg from baseline. The endotoxin injected animals (n=7) showed an increase in MABP immediately following the 20 minute reading to a peak of 12 mm Hg above the baseline. At 80 minutes post-injection, MABP dropped slightly with a second peak occurring at 100 minutes. Following the second peak, MABP returned to baseline. MABP then appears to vary with three smaller peaks, the last of which is significantiy different from the control day due to the control animals dropping below baseline. Statistically significant differences between the saline infused endotoxin injected animals and the saline infused alone were seen at 40, 60, 100, 340, and 360 minutes following endotoxin injection. Figure 13 The effects of endotoxin injection on heart rate in animals receiving saline infusion into the VSA are shown in Figure 13. On day 1 (saline infusion alone) the mean baseline value for heart rate was determined to be 353 ± 18 bpm. On day 2 the mean baseline value was determined to be 330 ± 6.7 bpm. The animals infused with saline alone (control group; n=6) showed changes in heart rate from 10 bpm to -20 bpm around the baseline for the duration of the experiment. Injection with endotoxin (50/vg/rat) affected on heart rate. There was an initial rise in heart rate over the course of 80 minutes post-injection to 85 bpm above baseline. After the 80 minute reading, heart rate then dropped to 5 bpm above baseline at 180 minutes post-injection. 54 For the rest of the experimental period, there were some minor increases, with two of the following three smaller peaks showing some significant differences from the control day. Statistically significant differences in heart rate between the saline infused endotoxin injected group and the control group existed at 20 to 80 minutes (inclusive) post-injection as well as at 200, 340, and 380 minutes post-injection. Unpaired t-tests determined statistical significance (p<0.05). 55 u 0 w I e s IS o « w u * * * * * * T * T * * T * • J I T T — • — • — r 60 120 180 240 300 360 420 TIME (MIN) FIGURE 11: This graph illustrates the mean (± SEM) changes in body temperature following injection of endotoxin in animals receiving saline infusion into the VSA (0.05jL/L/h). Open squares represent saline infusion without endotoxin (control day; n=12). Closed squares represent saline infused on day 2 after injection with endotoxin (treatment day; n=ll) . Asterisks represent significance as detennined by unpaired t-tests (p<0.05). For further details, see text. 56 -1 2 H — i — i — i — • — • — i — ' — ' — i — • — • — i — • — • — i — • — • — i — • — ' — r -0 60 120 180 240 300 360 420 TIME (MIN) FIGURE 12: This graph represents the mean changes (± SEM) of MABP in animals receiving saline infusions into the VSA. The control animals (saline infusion alone) are represented by open triangles (n=7). Animals injected with endotoxin are represented by closed squares (n=7). Asterisks represent significance as determined by paired t-tests (p<0.05). For further details, see Figure 11. 57 * z Q. CQ LU I— < < LU LU CD Z < X u 100-80-60-40-60 120 180 240 300 360 420 TIME (MIN) FIGURE 13: This graph illustrates the effects of endotoxin injection on mean (± SEM) heart rate in animals receiving saline infusions into the VSA. Animals infused with saline alone (control day) are represented by open squares (n=6). Closed squares represent animals injected with endotoxin (n=6). Asterisks mark statistically significant points as determined by unpaired t-tests (p<0.05). For further details see Figure 11. 58 2. Lidocaine Infused Group The treatment group received infusions into the VSA of 5% lidocaine HC1 (in physiological saline). Figure 14 Changes in body temperature in animals receiving only lidocaine infusion (control day- day one) were compared with those receiving endotoxin injection at time zero (day two). Paired t-tests were used to determine statistically significant differences (p<0.05) between the two days. The mean baseline value for the animals receiving lidocaine infusion alone was 37.7 ± 0.2 °C. The endotoxin injected animals (day 2) have a mean baseline temperature of 37.7 ± 0.2 °C. The effect of endotoxin injection on temperature in lidocaine infused animals appeared to be a biphasic response. There was an initial small increase in body temperature of 0.3°C followed by a larger more significant peak of 0.6°C above baseline. The second increase in body temperature remained significantly elevated until 240 minutes. The points which are significantly different from the control day occurred at 20, 40, 120,140, and 180 to 220 inclusive. Figure 15 The effects of endotoxin injection on mean arterial blood pressure in lidocaine infused animals. Each animal acted as its own control and paired t-tests were done to determine statistical significance (p<0.05). The mean absolute values for baseline in these animals was 117 ± 9.5 mm Hg and 124 ± 9.1 mm Hg for the control day and the day of injection with endotoxin, respectively. Lidocaine alone appeared to be without effect (control day). On day two following endotoxin injection, MABP increased to 12 mm Hg above baseline at 60 minutes, and remains elevated until suddenly at 260 59 minutes, MABP dropped over the next 20 minutes to baseline where it remained for the duration of the experimental period. Statistically significant differences between the two days were seen at 20, 220, and 260 minutes. Figure 16 The effects of endotoxin injection on changes in heart rate in animals receiving infusion of lidocaine into the VSA are presented in Figure 16. On day two, all animals received injections of endotoxin at time zero. Unpaired t-tests were used to calculate significant differences (p<0.05) between the two days. The mean baseline values for heart rate in these animals was 385 ± 10 bpm and 367 ± 7 bpm for the control day and the day of injection with endotoxin, respectively. Statistically significant differences in heart rate were seen at 60, 80, 220, 240, 340 and 360 minutes post-injection. Endotoxin appears to produce a biphasic effect on heart rate. 60 120 180 240 300 360 420 TIME (MIN) FIGURE 14: This graph depicts the effects of endotoxin injection on mean (± SEM) body temperature in animals receiving lidocaine infusions (5% Lidocaine HC1 in physiological saline) into the VSA (0.5 /jL/h.). Squares represent endotoxin injected animals (n=7) and triangles represent the animals receiving lidocaine infusion but no endotoxin (control day; n=7). Asterisks mark significant differences as determined by paired t-tests (p<0.05). For further details, see text. 61 20-i 16-12 -•12 0 ~l • ' 1 r-60 120 180 240 300 360 420 TIME (MIN) FIGURE 15: This graph illustrates the effects of endotoxin injection on mean arterial blood pressure in animals receiving lidocaine infusions (0.05 j;L/h) to the VSA. Animals infused with lidocaine (no endotoxin) are represented by triangles (control day; n=l l ) . Lidocaine infused animals injected with endotoxin are represented by squares (n=l l ) . Significant differences are represented by asterisks as determined by paired t-tests. (p<0.05). For further details, see Fig. 14. 62 100-— I — ' — ' — I — • — • — I — ' — ' — 1 — I — ' — I — ' — ' — I — • — ' — I — 60 120 180 240 300 360 420 TIME (MIN) FIGURE 16: This graph illustrates the effects of endotoxin injection on mean (± SEM) changes in heart rate in animals receiving lidocaine infusions (0.05 fjL/h) into the VSA. Animals receiving lidocaine infusions and no endotoxin (control day; n=9) are represented by triangles. Squares represent infused animals injected with endotoxin (n=9). Significant differences are represented by asterisks as determined by unpaired t-tests (p<0.05). For further details, see figure 14. 63 C. COMPARISON OF EFFECTS OF ENDOTOXIN INJECTION IN LIDOCAINE AND SALINE INFUSED ANIMALS This section compares the effects of injection of endotoxin in two groups of animals. Mean arterial blood pressure, heart rate and temperature parameters are compared in the lidocaine and saline infuse animals. Statistical significance was determined by unpaired t-test (p<0.05). Each point is the mean of all animals measured at that time. The baseline values are as per previous graphs of endotoxin effects. Figure 17 The effects of injection with endotoxin (i.p.) on body temperature in animals receiving infusion of saline are compared to the effects of infusion of 5% lidocaine into the VSA. There was a trend for the fever in the lidocaine treated group to be somewhat lower. When compared to the saline infused group, however, there was only one statistically significant difference at 160 minutes. Figure 18 The effects of injection with endotoxin on MABP in animals receiving either saline or lidocaine infusion into the VSA. are compare in Figure 18. Although there appeared to be an increase of MABP in the lidocaine treated group relative to the saline treated group, there were no statistically significant differences between the them. Figure 19 Comparison of the effects of endotoxin injection on heart rate in animals receiving saline or lidocaine infusion into the VSA are shown in Figure 19. The biphasic response appeared to vary between the two groups. In the first phase, the saline infused animals had a larger response; whereas in the second phase, the lidocaine animals had a larger response. There were however, no statistically significant differences between the two groups. 64 1 1 1 1— 300 360 TIME (MINI) FIGURE 17: This graph compares the effects of injection with endotoxin (i.p.) on mean (± SEM) body temperature in animals receiving infusions of saline (open squares; n=ll) or 5% lidocaine (closed squares; n=7) into the VSA (0.05 pL/h). Statistical significance (p<0.05) as determined by unpaired t-tests is marked by asterisks. Error bars represent standard error of the mean. For further details, see text. 65 TIME (MIN) FIGURE 18: This graph compares the changes in mean arterial blood pressure after injection of endotoxin into two groups of animals. Animals receiving lidocaine infusion are represented by solid squares (n=12) and animals receiving saline infusion are represented by open squares (n=7). For details, see Fig. 17. 66 100-80-60-40-20 0 -20--40 - i 1 1 r - - i — . — i — i — i — r 60 120 180 240 300 360 420 TIME (MINI) FIGURE 19: This graph compares the effects of injection of endotoxin on mean (± SEM) heart rate changes in lidocaine (closed squares; n=9) and saline (open squares; n=6) infused animals. For details, see figure 17. V. DISCUSSION AND CONCLUSIONS The purpose of these experiments was to examine the possibility that the VSA of the brain is involved in cardiovascular regulation during endotoxin induced fever in the freely moving rat. This investigation was accomplished by examining the effects of injection of endotoxin on heart rate, blood pressure and body temperature, then examining these same parameters while the VSA received infusion of saline or lidocaine to block local neuronal activity. In the first set of experiments, alterations in body temperature, mean arterial blood pressure and heart rate were observed following i.p. injection of endotoxin (see Figure 8 - 10; Section IV-A). The changes in body temperature following injection of endotoxin confirm the observations of Kasting et al, 1985, and Kasting, 1986B. As shown in Figure 8, there was an initial drop in body temperature followed by an increase to febrile levels which was sustained throughout the experimental period of 6 hours. The animals injected with saline showed a minor artifactual change in body temperature. This change was probably due to the stress of handling and injection (see Dilsaver and Majchrzak, 1990). However, the significant differences between the two groups confirm that the effects of ip injection of endotoxin on body temperature are not artifactual. Mean arterial blood pressure was elevated immediately following injection of endotoxin. This effect lasted 180 minutes (Figure 9). This 180 minute mark corresponds to the same amount of time that was required to attain the peak elevated temperature in Figure 8. The increased MABP may result from changes in circulation such as peripheral vasoconstriction elicited to raise body temperature to febrile levels. Alternately, it may be due to a direct effect of endotoxin on the cardiovascular system. By 3 68 hours other mechanisms may have compensated for the increased peripheral resistance, thereby causing an decrease in MABP. The saline injected animals show an initial brief increase in MABP of 5 mm Hg, probably due to the stress of handling and injection. This effect stabiUzed within an hour, contrasting with the prolonged changes in MABP observed following endotoxin administration. These results confirm that the observed changes in the animals injected with endotoxin are due to the effects of endotoxin on the animal. The first peak of MABP (which may be artificially augmented by the stress of injection) occurs at 20 minutes. The second peak in MABP occurs at 120 minutes. The trough between the peaks occurs at 80 minutes; interestingly, this corresponds to the lowest point of body temperature in Figure 8. This may suggest that the mechanisms which caused the body temperature to drop similarly elicited the drop in MABP. At 120 minutes after administration of endotoxin, both MABP and temperature increase to above baseline values (Kasting et al., 1985). These results have been confirmed in a subsequent study by Kasting and Cridland (personal communication, 1993) where MABP showed a biphasic response. The trough at 80 minutes also corresponds to the lowest point in body temperature and the peak of body temperature occurs at the point where the MABP has finished its second peak. In this study body temperature and MABP measured simultaneously in the same animal showed a statistically significant correlation between MABP and body temperature for the first two hours following injection of endotoxin. It is not possible to directly examine the relationship between body temperature and MABP in the present study because these parameters were measured in separate groups of animals. 69 Administration of endotoxin to urethane anesthetized rabbits show a drop in blood pressure to 15 ± 4 mm Hg below baseline at 60 minutes post-injection (Saigusa, 1989). This difference could be attributed to the species differences or the urethane anesthesia which results in the animal being unable to maintain a normal body temperature, thus affecting other physiological parameters (see Malkinson et al., 1988). An anesthetized animal does not represent an accurate picture of normal physiological responses (see Brockway, 1991). The observation that hemorrhage results in antipyresis suggests that blood pressure can affect regulation of body temperature (see Kasting, 1990). The blood pressure sensitive neurons in the VSA have yet to be tested for sensitivity to thermal or pyretic stimuli (Jhamadas and Renaud, 1986a). A relationship between temperature and blood pressure also has been seen in non-febrile spontaneously hypertensive rats. These rats have a higher core temperature than normotensive rats and body temperature and MABP rose together when the animals were exposed to stressful situations (Morley et al, 1990) Heart rate was seen to be elevated during the course of these experiments, however, only a few points showed statistically significant differences from the control day (figure 10). As indicated by the larger error bars, heart rate was more variable in the endotoxin treated animals: it is, therefore difficult to interpret these results. However, there is a trend toward a biphasic response in heart rate to the effects of injection of endotoxin. The first peak is seen to occur in the hypothermic phase of fever, and the second in the plateau phase. These results have been confirmed by subsequent studies by Kasting and Cridland (personal communication, 1993). 70 Thus, endotoxin causes changes in body temperature, mean arterial blood pressure, and heart rate. Fever is the result of the changes in body temperature in response to endotoxin (see section I B). Mean arterial blood pressure responds to endotoxin by producing a biphasic response (see Figure 9) which appears to be synchronous with the changes in body temperature. Heart rate also responds to endotoxin injection with a biphasic response. A relationship among these parameters is expected, in that stimulation of temperature sensitive neurons is thought to cause an elevation of the set-point and is also thought to stimulate the vasomotor center and peripheral efferents to initiate heat conservation and/or heat production (see Section B-2). In the second set of experiments, the animals were given time to recover from brain surgery before the mini-pumps were implanted. The response of animals to the injection of endotoxin and mini-pump implantation to permit the infusion of saline (Figures 11-13) or lidocaine Figures 14-16) into the VSA (section IV -B). Lidocaine hydrochloride monohydrate (C14H23CIN2OH2O) was used in these experiments because it is a local anesthetic which is very soluble in physiological saline. Lidocaine specifically blocks activity-dependent sodium channels. These voltage-dependent Na+ channels mediate depolarization during the rising phase of the action potential. This action of lidocaine therefore prevents action potentials in fibres of passage (Bertil Hille, Ionic Channels of Excitable Membranes, 1984, Courtney, 1975). Lidocaine (2% solution-200nL/h) infused into the MnPO has been shown to have no effect on baseline blood pressure and heart rate in chloralose anesthetized rabbits and attenuated AVP-mediated bradycardia (Patel and Schmid, 1988). 71 It was expected that infusion of lidocaine would prevent the release of AVP into the VSA thus causing a higher and more prolonged fever (see section I B - antipyretic actions of AVP). The effects of lidocaine on cardiovascular parameters could then be examined. However, the predicted higher and more prolonged fever was not seen (see Figure 17). In fact, although there is only one statistically significant point which differs between the saline and lidocaine infused animals at 160 minutes, it appears that the lidocaine infused animals have an attenuated fever response when compared to the saline infused animals. Studies in rats which have used a specific AVP-V1 receptor antagonist have shown elevated and prolonged fevers (Kasting and Wilkinson, 1985a; Cridland and Kasting, 1992). Lidocaine is quite non-specific in its action and would interfere not only with the release and uptake of AVP (by preventing the action potential), but also the other neurotransmitters released into the area and fibres of passage as well (see Cunningham, 1993). Figures 11-13 show that the saline infusion failed to prevent the expected physiological changes in body temperature, MABP, or heart rate in response to injection of endotoxin. Saline infusion alone (no endotoxin) was without effect on any of the measured parameters, suggesting that osmotic minipumps are a suitable technique for the infusion of experimental agents into the VSA (see Cridland and Kasting, 1992). The use of the minipump prevents artifactual changes in the parameters measured due to handling stress or restraint required to administer drugs by other methods. The area of spread of the infused agent is concentrated at the tip of the cannula and decreases significantly with the distance from the tip. This has been verified by studies infusing dye or radioactive compounds (see Theeuwes and Yum, 1976, Sendelbeck and Urquhart, 72 1985, Kasamatsu et al. 1981, Mangano and Schwarcz, 1983). In a study by by Kroin and Penn (1982) using minipumps and stainless steel 23 gauge tubing, the concentration of the drug they administered was found to extend only 1 mm from the tip of the tubing. All placement of cannulae used in these experiments were verified histologically. All infusion sites were within the area defined as the ventral septal area. This site also includes that described in several papers as the diagonal band of Broca (Jhamandas and Renaud, 1986). Any animals with infusion sites outside of the defined area were excluded from the data. In the saline infused animals, there was an attenuation of the response of MABP to the endotoxin injection when compared to the rats which had no brain surgery which may suggest that saline caused some minor dilution of the neurotransmitters or have caused some non-specific damage resulting in minor changes affecting the magnitude of the blood pressure response. Theoretically this could verify that synapses in the VSA are involved in the control of blood pressure. Another possibility is that the animal has become accustomed to handling and thus the actifactual reaction to the stress has been attenuated. Comparing the changes in MABP in saline and lidocaine infused rats following injection with endotoxin showed that while the saline infused group had a drop in blood pressure to baseline at 160 minutes, the lidocaine infused group continued to have elevated blood pressure until 280 minutes. This would indicate that lidocaine was interfering with normal transmission in the area of the VSA which had an effect on the blood pressure response to endotoxin. The biphasic heart rate response to endotoxin injection was seen in both the saline and lidocaine infused animals. The lidocaine infused 73 animals had a lower first phase response and a higher second phase response. There were however, no significant differences between the two groups. Perhaps a larger number of animals would have narrowed the error bars and shown some significance as there appears to be a trend for the differences noted above. Chronic measurements in freely moving rats of arterial blood pressure with the telemetry system used in this thesis has been validated by comparision with direct arterial catheterization in rats (Guiol et al., 1992), marmosets (Schnell and Wood, 1993), and rhesus monkeys (Sadoff et al., 1992). It has been found that heart rate was often higher in catheterized animals due to the stress of restraint. Implantable transmitters provide an accurate view of the normal physiological state by avoiding artifact due to restraint (see Adams et al., 1988), the unpredictability of anesthesia (Brockway, 1991) or the problem of maintaining patency in direct arterial measurements. Telemetry used for the measurement of temperature also avoids hyperthermia caused by handling (Dilsaver et al., 1992). The results observed did not meet those expected. Body temperature in the lidocaine infused rats did not respond with the expected higher and more prolonged fever in response to endotoxin. Cardiovascular parameters were not significantly altered by lidocaine infusion. However, in view of the fact that temperature was not affected in the expected way, it is difficult to interpret cardiovascular results. Heart rate showed great variability thus limiting the validity of interpretation. The techniques used in this study were valid state of the art methods on attaining an accurate picture of the physiological changes of temperature, heart rate and mean arterial blood pressure. The choice of 74 drug infused however, may have limited the value of the comparison results. The body temperature and MABP had to be obtained in separate rats so they could not be directly compared with a linear regression analysis. Furthermore, the lack of any very clear changes induced by lidocaine made interpretation of the results more difficult. 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