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1 EFFECT OF CHRONIC EX ERCISE ON CENTRAL OP IOID MEDIATED RESPONSES TO HEMORRHAGE By JOSLYN K. AHLGREN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FO R THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Joslyn K. Ahlgren
3 To all the rats who gave their lives in s upport of this body of research
4 ACKNOWLEDGMENTS I would like to acknowledge Dana Townsend, my undergraduate anatomy and pysiology and cadaver dissection instructor. Without her contribution to my education, I would have neither the wonderful a natomy and physiology foundation nor the teaching skills that have resulted in my success as a graduate student and professor. Additionally, I would also like to acknowledge my undergraduate research mentor, Dr. Timothy Musch, who introduced me to the excitement and fulfillment of life in the lab as well as the intrigue of exercise physiology research. I w ill always remember and appreciate such mentorship and guidance. I would also like to recognize those individuals under whom I taught anatomy during my time at the University of Florida: Drs. Kevin Anderson and Floyd Thompson. These professors granted me a huge amount of freedom in the classroom which allowed me to come into my own as an anatomy instructor. For their trust in me as an anatomist and their willingness to allow me creative liberties in my teaching, I am ever grateful. My dissertation work would not have been possible without the supervision of my graduate committee: Drs. Roger Reep, Paul Davenport, Rob Caudle, Charles Wood, and Linda Hayward. A special and heartfelt thanks to my mentor, Linda Hayward who has guided me through my graduate s tudies and research with gentle prodding and great humor She has been a source of stability and sensibility for me over the past five years and I am comforted knowing that I can always rely on her in the future for advice, direction, and encouragement (both personal and professional) if needed. Thank you is not enough, but its all I can say.
5 As a graduate student, you work way more than you would ever want to. And sometimes the only thing that makes it tolerable is knowing that you are sharing that exp erience with other grad students doing the same. Having all my office and lab mates around for discussions, venting sessions, and straight up joking and fun has made my long hours tolerable. My sincerest thanks and gratitude to Drs. Sarah Miller, Pei Yin g Chan, Weirong Zhang Yang Ling Chou, and soonto be Drs. Kat i e Pate, Vipa Bernhardt, Ana Bassit, Karen Porter, and Carie Reynolds. I would like to acknowledge Mabelin Castellanos and Jessie Stanley for their technical assistance and enlightening and controversial political exchanges. Finally, Id like to give madd kudos to my husband, Jeremey. He has put up with my long hours away from home, too many nights without a dinner companion to count, some near nervous breakdowns brought on by experimental frus trations, lack of sleep, and a constant quest for perfection. I never had to ask for more support. I never had to ask for more encouragement. I never had to ask for forgiveness for my time spent away or for my psychotic schedule. For his patience, love, and cheerleading, I will be forever indebted.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 15 HEMORRHAGE, OPIOIDS, AND EXERCISE ............................................................... 18 Physiology of Hemorrhage ...................................................................................... 18 Phase I Hemorrhage: Compensation ............................................................... 18 Phase II Hemorrhage: Decompensation ........................................................... 21 Phase III Hemorrhage: Recompensation .......................................................... 23 Cen tral Nervous System Control of Hemorrhage ............................................. 24 Central Opioid Modulation of Hemorrhage .............................................................. 26 Opioid Peptides and Associated Receptor Function ........................................ 27 Opioidergic Modulation of Hemorrhage in the Brainstem ................................. 27 Exercise Training and the Central Nervous System ............................................... 29 Exercise Alters Central Opioids and Opioid Receptor Function ....................... 30 Dynorphins ....................................................................................................... 31 Specific Aims .......................................................................................................... 32 Specific Aim 1 ................................................................................................... 32 Specific Aim 2 ................................................................................................... 33 Spec ific Aim 3 ................................................................................................... 33 Specific Aim 4 ................................................................................................... 33 Summary ................................................................................................................ 34 HEMODYNAMIC RESPONSES AND CFOS CHANGES ASSOCIATED WITH HYPOTENSIVE HEMORRHAGE: STANDARDIZING A PROTOCOL FOR SEVERE HEMORRHAGE IN THE CONSCIOUS RAT ........................................... 41 Introduction ............................................................................................................. 41 Methods .................................................................................................................. 43 General Preparation ......................................................................................... 43 Experimental Protocol ...................................................................................... 44 Fos Immunocytochemistry ................................................................................ 46 Neuroanatomical Quantification of Fos Like Immunoreactivity ......................... 46 Cardiovascular Measureme nts ......................................................................... 47 Results .................................................................................................................... 49 Cardiovascular Response to Hypotension Versus Severe Hemorrhage .......... 49
7 Fos Like Immunoreactivity in Brainstem Nuclei Following Severe Hemorrhage .................................................................................................. 50 Discussion .............................................................................................................. 52 Methodological C onsiderations ........................................................................ 54 Cardiovascular Response to Hemorrhage ........................................................ 55 Pattern of Fos Like Immunoreactivity in the Rostral Brainstem Followin g Hemorrhage .................................................................................................. 56 Conclusions ...................................................................................................... 59 DESCENDING PROJECTIONS FROM PERIAQUEDUCTAL GRAY TO THE LATERAL PARABRACHIAL NUCLEUS ARE NOT ACTIVATED BY SEVERE HEMORRHAGE IN THE CONSCIOUS RAT .......................................................... 70 Introduction ............................................................................................................. 70 Methods .................................................................................................................. 71 Surgical Instrumentation ................................................................................... 71 Experimental Protocol ...................................................................................... 72 Immunohistochemistry and Data Collection ..................................................... 73 Data Analysis ................................................................................................... 74 Results .................................................................................................................... 75 Brain Injection Sites .......................................................................................... 75 Heart Rate and Mean Arterial Pressure in Hemorrhage Versus Control .......... 75 Neural Activation/CoLabeled Neurons ............................................................ 76 Discussion .............................................................................................................. 77 VOLUNTARY WHEEL RUNNING ALTERS THE AUTONOMIC RESPONSE TO HEMORRHAGE IN CONSCIOUS MALE RATS ..................................................... 84 Introduction ............................................................................................................. 84 Methods .................................................................................................................. 86 General Preparation ......................................................................................... 86 Experimental Prot ocol ...................................................................................... 87 Fos Immunocytochemistry ................................................................................ 88 Neuroanatomical Identification and Quantification of Fos Like Immunoreactivity ........................................................................................... 89 Cardiovascular Measurements ......................................................................... 89 Statistical Analysis ............................................................................................ 9 0 Results .................................................................................................................... 91 Confirmation of Exercise Training Effect .......................................................... 91 Effect of Exercise on the Hemodynamic Response to Hemorrhage ................. 92 Heart Rate Variability During Hemorrhage ....................................................... 94 Effect of Exercise on the Fos Like Immunoreactivity Response to Hemorrhage .................................................................................................. 95 Discussion .............................................................................................................. 97 Methodological Considerations ...................................................................... 105 Conclusions .................................................................................................... 109
8 OPIOID RECEPTOR BLOCKADE IN THE LATERAL PARABRACHIAL NUCLEUS PREVENTS EXERCISEINDUCED TOLERANCE TO HEMORRHAGE .............. 116 Introduction ........................................................................................................... 116 Methods ................................................................................................................ 118 General Preparation ....................................................................................... 118 Cranial Cannulation ........................................................................................ 119 Arterial Cannulation ........................................................................................ 120 Experimental Protocol .................................................................................... 120 Western Blot Analysis of Kappa Opioid Receptor C ontent ............................. 122 Statistical Analysis .......................................................................................... 123 Results .................................................................................................................. 124 Measures of Exer cise Training ....................................................................... 124 Injection Site Verification ................................................................................ 124 Cardiovascular Response. ............................................................................. 125 Western Analysis of Kappa Opioid Receptor Content .................................... 127 Discussion ............................................................................................................ 128 SUMMARIES AND CONCLUSIONS ........................................................................... 141 Summaries of the Study Findings ......................................................................... 141 Study #1 Summary ......................................................................................... 141 Study #2 Summary ......................................................................................... 142 Study #3 Summary ......................................................................................... 142 Study #4 Summary ......................................................................................... 143 Discussion ............................................................................................................ 144 Study Limitations and Directions for Future Studies ............................................. 148 Rate of Hemorrhage. ...................................................................................... 148 Indices of Exercise Training. .......................................................................... 149 Effect of Exercise on Plasma Arginine Vasopressin Response to Hemorrhage. ............................................................................................... 150 Effect of Exercise on Opioidergic Influence in the Lateral Parabrachial Nucleus ....................................................................................................... 151 Effect of Exercise on GammaAminobutyric Acid Influence in the Lateral Parabrachial Nucleus .................................................................................. 152 Effect of Exercise on Serotonergic Influence in the Lateral Parabrachial Nucleus ....................................................................................................... 153 Conclusions .......................................................................................................... 154 LIST OF REFERENCES ............................................................................................. 156 BIOGRAPHICAL SKETCH .......................................................................................... 178
9 LIST OF TABLES Table page 1 1 Three main classes of endogenous opioids, their precursor proteins, and the primary opioid receptor on which each ligand acts. ............................................ 40 2 1 Baseline mean arterial pressure (MAP) and hear t r ate (HR) of treatment groups ................................................................................................................ 61 2 2 Mean arterial pressure (MAP) and heart rate (HR) averaged at the time o f the offset of hemorrhage .......................................................................................... 64
10 LIST OF FIGURE S Figure page 1 1 Effect of hemorrhage on mean arterial pressure and heart rate in anesthetized (left panel) versus conscious (right panel) rats .............................. 35 1 2 Heart rate (HR) and mean arterial pressure (MAP) recording from a conscious rat during 30% estimated total blood volume hemorrhage. ................ 36 1 3 Brainstem and spinal cord pathways that subserve the baroreceptor reflex control of sympathetic output to the heart and blood vessels. ............................ 37 1 4 Effect of synaptic blockade in the ventrolateral periaqueductal gray (vlPAG) on the change in mean arter ial pre ssure ............................................................ 38 1 5 Diagram of connections between the ventrolateral periaqueductal grey (vlPAG) and lateral par abrachial nucle us ........................................................... 39 1 6 Effect of opioid receptor blockade in the caudal ventrolateral periaqueductal grey (vlPAG) on mean arterial pres sure (MAP) during hemorrhage. .................. 40 2 1 Schematic of periaqueductal grey (PAG) and lateral parabrachial nucleus (LPBN) areas imaged for quantification of Fos positive staining. ....................... 61 2 2 Mean arterial pressure (MAP; A) and heart rate (HR; B) responses to saline (SAL; n = 5) or hydralazine (HYDRAZ; n = 5) infusion. ...................................... 62 2 3 Mea n arterial pressure (MAP; A) and heart rate (HR; B) responses ................... 63 2 4 Representative images of Fos like immunoreactive neurons in the middle a nd caudal periaqueductal gray .......................................................................... 65 2 5 Average Fos positive cell co u nts ........................................................................ 66 2 6 Representative images of Fos like immunoreactive neurons in the rostral, middle, and caudal lateral parabrachial nucleus ................................................. 67 2 7 Average Fos positive cell counts from selected sub n uclei ................................. 68 2 8 Fos positive cells quantified in the Cuneiform Nucleus ...................................... 69 3 1 Periaqueductal grey (PAG) area analyzed and examples of immunohistochemical staining. ........................................................................... 80 3 4 Quantification of immunohistochemical staining in the caudal periaq ueductal grey .................................................................................................................... 83 4 1 Evidence of exercise training. ........................................................................... 110
11 4 2 Hemodynamic response to 30% total blood volume hemorrhage (HE M) in exercised (EX) vs. sedentary (SED) conscious rats. ........................................ 111 4 3 Heart rate variability (HRV) analysis of exercised (EX) and sedentary (SED) gr oup response to hemorrhage. ....................................................................... 112 4 4 Representative images of Fos like immunoreactivity throughout the rostrocaudal extent of the lateral parabrachial nucleus ............................................. 113 4 5 Average FLI and schematic represe ntations ................................................... 114 4 6 Average Fos like immunoreactivity and schematic representations for the caudal ventrolateral periaqueductal grey .......................................................... 115 5 1 Measures of exercise training. .......................................................................... 135 5 2 Injection sites.. .................................................................................................. 136 5 3 Effect of naloxone versus vehicle injection into the lateral parabrachial nucleus (LPBN) on the heart rate (HR) and mean arterial pressur e ................. 137 5 4 Effect of naloxone versus vehicle injection into the lateral parabrachial nucleus (LPBN) on the heart rate (HR) and mean arterial pressure ................. 138 5 5 Effect of naloxone injection into the lateral parabrachial nucleus (LPBN) on the heart rate (HR) and mean arterial pressure ................................................ 139 5 6 Western blot analysis of relative kappa opioid receptor (KOR) content in the rostral pons of exercised (EX) and sedentary (SED) rats. ................................ 140 6 1 Schematic of revised research hypothesis ....................................................... 155
12 LIST OF ABBREVIATIONS HEM Hemorrhage HR Heart rate AP Arterial pressure CNS Central nervous system NTS Nucleus tractus solitarius CVLM Caudal ventrolateral medulla RVLM Rostral ventrolateral medulla IML Intermediolateral cell column MAP Mean arterial pressure SAD Sinoaortic barodenervation TBV Total blood volume HIS Hemorrhageinduced sympathoinhibition AVP Vasopressin, arginine vasopressin PAG Periaqueductal gray vlPAG Ventrolateral PAG LPBN Lateral parabrachial nucleus RVMM Rostral ventromedial medulla dlPAG Dorsolateral PAG SAL Saline control HYDRAZ Hydralazine S HEM Slow rate of hemorrhage I HEM Intermediate rate of hemorrhage
13 F HEM Fast rate of hemorrhage FLI Fos like immunoreactivity GS PBSTX Goat serum PBStriton X100 solution DAB Diaminobenzidine hydrochloride SCP Superior cerebellar peduncle LC Locus coeruleus CnF Cuneif orm nucleus KF Kolliker Fuse nucleus ANOVA Analysis of variance SEM Standard error of measure dmPAG Dorsomedial PAG CON Non hemorrhaged control FGI Fluorogold immunoreactivity FG Fluorogold EX Exercise group SED Sedentary group PVN Paraventricular nucleus of the hypothalamus LF Low frequency HF High frequency CMM Caudal midline medulla CVLM Caudal ventrolateral medulla CRH Corticotropin releasing hormone WKY Wistar Kyoto rats
14 HPA Hypothalamic pituitary adrenal CeA Central nucleus of the amygdala MnPO Median preoptic nucleus KOR Kappaopioid receptor PRE Before drug injections POST After drug injections IOD Integrated optical density IC Inferior colliculus CSF Cerebral spinal fluid DOR Delta opioid receptor MOR Mu opi oid receptor GABA Gamma aminobutyric acid SHR Spontaneously hypertensive rat SON Supraoptic nucleus GAD Glutamate decarboxylase ARC Arcuate nucleus POMC Pro opiomelanocortin 5 HT Serotonin
15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF CHRONIC EX ERCISE ON CENTRAL OP IOID MEDIATED RESPONSES TO HEMORRHAGE By Joslyn K. Ahlgren December 2009 Chair: Lind a F. Hayward Major: Veterinary Medical Sciences Based on observations that central opioids are modulated by chronic exercise and the cardiovascular response to hemorrhage ( HEM ) involves opioid release in the central nervous system ( CNS ) the present studi es were undertaken to test the hypothesis that chronic exercise protects against centrally mediated hemorrhagic shock These studies are particularly important in light of the paucity of information focusing on exercise induced plasticity of CNS regions involved in cardiovascular control and health. Study #1 tested the hypothesis that the hemodynamic responses and neural activation of various brainstem nuclei would differ when blood was withdrawn at a slow, intermediate, or fast rate. Results from this s tudy support our hypothesis and identified that a rate of blood withdrawal of 1 ml/kg/min most reliably induces severe HEM in conscious rats. This rate of HEM also produced the most significant and least variable increases in neural activation of rostral brainstem sites, including the ventrolateral periaqueductal grey ( vlPAG ) and the lateral parabrachial nucleus ( LPBN ) Study #2 tested the hypothesis that LPBN projecting afferents from the vlPAG are involved in the cardiovascular response to severe HEM in the conscious rat. Results showed no difference in neural activation between LPBN projecting and nonLPBN -
16 projecting vlPAG neurons. These findings do no support our initial hypothesis that this projection is actively involved in the descending control of autonomic responses to HEM. Study #3 tested the hypothesis that exercise would modulate the cardiovascular and neural responses to severe, conscious HEM. Exercised animals displayed blunted HEM induced bradycardia and hypotension and displayed altered neural activation in brain regions involved in cardiovascular control. Heart rate variability analysis also pointed to a possible change in basal respiratory function following exercise which may have contributed to an enhanced tolerance to HEM in the trained rats. These results suggest that exercise results in an augmented tolerance to severe HEM. Study #4 tested the hypothesis that opioid receptors within the LPBN play a role in the exercise induced tolerance to HEM. While there was no difference in K OR content in the dorsolateral rostral pons between EX and SED animals, exercise induced tolerance to HEM was abolished by opioid receptor blockade in the LPBN. This study suggests that in EX animals, the inhibitory function of the LPBN may rely more heav ily upon opioidergic mechanisms, perhaps due to decreased gamma aminobutyric acid ( GABA) inputs following training. Together, these studies confirm our global hypothesis that chronic, voluntary exercise protects against the deleterious effects of acute, s evere blood loss in the conscious rat. Additionally, this body of work has identified several important brain structures which may be modulated by chronic exercise that contribute to the protective effects of exercise against HEM. These studies are parti cularly important for future
17 research evaluating specific regions of the CNS which may serve as potential targets for drug therapies and treatments for trauma victims.
18 CHAPTER 1 HEMORRHAGE, OPIOIDS, AND EXERCISE Physiology of Hemorrhage The first record ed observations regarding the arterial blood pressure response to hemorrhage (HEM) were performed by Stephen Hales in 1733 (Evans et al., 2001) Hales reported that upon initial removal of blood from a conscious horse, the blood pres sure response was disproportional to the change in blood volume. It was not until 1415 quarts of blood were removed that blood pressure significantly dropped and the animal fell into a state of shock. These foundational observations by Hales gave us a g limpse into the complex cardiovascular response to HEM. Much later, the hemodynamic response to HEM was characterized more completely by directly measuring right atrial pressure and use of the direct Fick method for calculating cardiac output (Barcroft et al., 1944; Dishman et al., 1995) In response to blood loss, two phases were identified in which the body was initially able to compensate for the blood loss with increases in heart rate (HR) and vascular resistance (phase I) followed by an inability to maintain normal blood pressure and HR (phase II), which eventually resulted in loss of consciousness. Phase I Hemorrhage: Compensation Prior to the late 1960s, the classical textbook description of the hemodynamic response to HEM was a monophasic decline in blood pressure and HR (Schadt and Ludbrook, 1991) This discrepancy from previous reports and experiments performed in conscious humans and horses was determined to be the result of the use of anesthetics in research animals subjected to HEM protocols. Figure 11 is modified from a study that evaluated the cardiovascular response to HEM in both anesthetized and conscious
19 rats (Leskinen et al., 1994) Clearly, anesthesia alters the response by eliminating the phase I response to acute blood loss. Accordingly, more recent experiments evaluating the underlying physiological mechanisms regulating the complex hemodynamic response to HEM have largely begun utilizing conscious animals. Figure 12 shows an arterial blood pressure (AP) and HR recording from a conscious rat during a 30% total blood volume (TBV) HEM. The principal mechanism regulating the initial, sympathoexcitatory, phase of hemorrhage is the withdrawal of baroreceptor input to the c entral nervous system (CNS) (Schadt and Ludbrook, 1991) Transient changes in blood pressure (resulting from blood volume loss) are sensed by these stretch activated receptors in the aortic arch and carotid sinuses. In situations of increased blood pressure, baroreceptors increase neural signals to the CNS via the glossopharyngeal (from the carotid sinuses) and vagus nerves (from the aortic arch). These afferents synapse first in the nucleus tractus solitarius (NTS) located in the caudal, dorsal brainstem. Excitat ory projections from the NTS are sent to the caudal ventrolateral medulla (CVLM) which, in turn, sends inhibitory projections to the rostral ventrolateral medulla (RVLM). The RVLM sends direct excitatory projections to the sympathetic preganglionic neurons of the intermediolateral cell column (IML) of the spinal cord (T111). Thus, decreased activation of the baroreceptors, as occurs during acute loss of blood, results in an augmented sympathetic drivevia disinhibition of the RVLM --and thus an elevated h eart rate and increased peripheral vascular resistance (Li and Dampney, 1994) Baroreceptor unloading also leads to inhibition of vagal preganglionic neurons found in the nucleus ambiguus, thus resulting in decreased cardiac vagal drive and further increases in HR (Li and Dampney, 1994; Henderson et
20 al., 2000) Because of this baroreflex mechanism early in HEM, the decline in cardiac output at the onset of HEM does not elicit a decline in mean art erial pressure (MAP) (Ludbrook and Graham, 1984; Schadt et al., 1984) Figure 13 (modified from (Dampney et al., 2002) ) shows a simplified diagram outlining this neural network. The bulk of support for barorec eptor unloading being the primary contributor to the acute response to HEM comes from a large body of literature in which animals underwent sinoaortic barodenervation (SAD) prior to being subjected to some degree of HEM (Goetz et al., 1984; Ludbrook and Graham, 1984; Schadt and Gaddis, 1986; Quail et al., 1987; Meller et al., 2003) Following SAD, animals do not display the typical compensatory hemodynamics seen in barointact animals (tachycardia and inc reased vascular resistance) (Schadt and Ludbrook, 1991) There are other afferent signals, however, that may also contribute to the sympathoexcitation elicited by the onset of HEM in some species. Morita and Vatner (1985) measured renal sympathetic nerve activity i n conscious dogs during a HEM that produced a MAP of 4050 mmHg following various levels of barodenervation. The results of this study demonstrated that both arterial baroreceptors as well as cardiac receptors contributed to the sympathoexcitation recorded at the renal sympathetic nerves during the normotensive phase of HEM, suggesting that the low pressure sensing cardiopulmonary receptors located in atrial and ventricular walls, in the coronary and pulmonary arteries, and in the inferior and superior vena cavae also play a role in the acute response to HEM (Morita and Vatner, 1985) Multiple other studies, however, argue against any major role of t he cardiopulmonary receptors in the initial response to blood loss in conscious animals (e.g., dogs and rabbits) since chemical blockade or complete denervation of the cardiac
21 nerves does not alter the blood pressure response to nonhypotensive HEM (Burke and Dorward, 1988; Ludbrook and Graham, 1984; Morita an d Vatner, 1985; Quail et al., 1987; Evans et al., 1989; Meller et al., 2003) Experiments performed on human heart transplant recipients show a role for cardiopulmonary receptors in the early rise in sympathetic nerve activity during acute HEM. Heart tr ansplant patients who lacked atrial cardiac receptor innervation but maintained ventricular cardiac receptor innervation displayed a significant attenuation in vasoconstriction (indicative of sympathoexcitation) in response to simulated moderate HEM using lower body negative pressure (Mohanty et al., 1987) Since previous studies evaluating the role of cardiac receptors have been performed in a variety of human and animal models an d have resulted in varying outcomes, the effect of species should certainly not be overlooked as a possible explanation for differing results. Phase II Hemorrhage: Decompensation When blood loss exceeds a critical value (~1525%) of total blood volume (TBV ), a decompensatory phase ensues (see Figure 1 2) (Schadt and Ludbrook, 1991) This phase is characterized by severe bradycardia and hypotension. The bradycardia is due to both a withdrawal of sympathetic nerve activity (Schadt and Ludbrook, 1991; Evans et al., 1994; Schadt and Hasser, 2004) and a rise in vagal drive (Barcroft et al., 1944; Murray and Wise, 1996; Porter et al., 2009) Decompensatory hypotension is the product of a centr ally mediated sympathoinhibition and a resultant fall in total peripheral resistance. Multiple studies in which sympathetic nerves were recorded during HEM or simulated HEM (e.g., caval occlusion) demonstrate an initial increase in nerve activity followed by a precipitous fall in activity coincident with the onset of hypotension (Morgan et al., 1988; Hasser and Schadt, 1992) Additionally, studies have shown decreased
22 vascular resistance in conscious rabbits and decreased blood flow in specific r egional vascular beds of humans that occurs with the onset of hypotension following decreases in central blood volume (Barcroft et al., 1944; Schadt et al., 2006) While arterial baroreceptors play a chief role in the sympathoexcitatory response to acute HEM, the mechanisms mediating the transition from compensation to decompensation are less clear. Multiple studies, however, support a role for the cardiac receptors in the hemodynamic response to blood loss. For example, Oberg and Thoren demonstrated increased nerve fiber activity in cardiac afferents during caval occl usion (Lund et al., 2002) and, later, Thoren reported that vagal cooling could entirely block the associated bradycardia (Thoren, 1979) Studies performed in conscious rabbits also support a role for cardiac afferents in the initiation of HEM induced sympathoinhibition (HSI). In one study, cardiac afferent nerve blockade prevented the sympathoinhibition and hypotension following blood loss (Burke and Dorward, 1988) In a separate study, rabbits were subjected to caval occlusion coupled with either saline or procaine (to induce a reversible cardiac nerve blockade) applied within the cardiac space. Results from this study showed that procaine was able to prevent the decline in vascular resistance, suggesting that stimulation of cardiac receptors precedes the sympathoinhibition associated with decreased central blood volume (Evans et al., 1989) Once HEM has advanced to this second, decompensatory phase, continued bleeding can result in massive organ failure and cardiac death. Although this suggests that the decompensatory phase of hemorrhage initiates eminent death, it may actually serve as a protective mechanism. Thoren and others have postulated that the
23 sympathoinhibit ion may act to guard against excessive heart rate during progressively declining diastolic filling and/or prevent increases in perfusion pressure from prolonging uncontrolled bleeding (Thoren, 1979; Blair and Mickelsen, 2006) slowing the flow of blood and perhaps aiding in clotting. Additionally, the severe hypotension characteristic of this phase of HEM coincides with the significant increases in the release of pressor hormones (e.g., renin, vasopressin [AVP], and catecholamines) (Korner et al., 1990; Schadt and Ludbrook, 1991; Hager et al., 2009) involved in recovery from blood loss. Although the release of all these hormones are increased during the compensatory phase of HEM due to sympathetic stimulation, they do not reach concentrations that result in a pressor response until a fall in MAP is induced (Hall and Hodge, 1971; Darlington et al., 1986; Thrasher and Keil, 1998; Haberthur et al., 2003) Phase III Hemorrhage: Recompensation If blood loss is stopped before cardiac arrest occurs, animals display a spontaneous recovery (see Figure 1 2). During this period, both MAP and HR slowly return to baseline or near baseline values (Ahlgren et al., 2007) This recovery process is the result o f acute elevations in circulating angiotensin and AVP (Fejes Toth et al., 1988; Schadt and Hasser, 1991; Ponchon and Elghozi, 1997) coupled with a r estoration of sympathetic tone to arterial (Blair and Mickelsen, 2006) and venous (Potas et al., 2003) vascular beds. Following this initial recovery process, a more gradual course of vascular refilling takes place via a shift of fluids from the interstitium into the plasma (Blair and Mickelsen, 2006) This volume restoration is initiated by a decreased capillary hydrostatic pressure drawing proteinfree fluid into the capillaries which begins at the onset of recovery from blood loss. As fluid shifts from the interstitium to the plasma, extracellular osmolality increases. H ours after fluid restoration has begun,
24 proteins are slowly moved into the plasma, which facilitates even more fluid influx. This continues until blood volume has been reestablished, taking as much as 1824 hours (Byrnes et al., 1982; Pirkle et al., 1982; Slimmer and Blair, 1996) Central Nervous System Control of Hemorrhage Multiple lines of evidence point t o specific regions of the brain as well as specific modulatory neurotransmitter systems that may be involved in the complex cardiovascular response to hypotensive HEM. The focus of this part of the introduction and the following experiments will be on two brainstem nuclei, the periaqueductal gray (PAG) and the lateral parabrachial nucleus (LPBN). In the early 1990s, a study was performed in which mesencephalic decerebrate rabbits displayed complete abolition of decompensation to a simulated HEM (caval occlusion) (Evans et al., 1991) Authors of this study concluded that suprapontine centers provided the sympathoinhibitory input to brainstem regions regulating blood pressure (such as the RVLM) that were necessary for eliciting the decompensation following acute central hypovolemia. Later, however, it was shown that inactivation of specific opioid receptors in the midbrain PAG could also attenuate hemorrhagic decompensation (Cavun and Millington, 2001) suggesting that the midbrain may be more important in this later phase of HEM than the forebrain. Critics of the study by Evans et al (199 1) pos tulated that their results may have been due to accidental midbrain damage during the decerebration procedure (Troy et al., 2003) In an effort to clarify the roles of the forebrain and midbrain in decompensation, Troy et al (2003) evaluated the hemodynamic response to severe HEM in rats that had undergone ei ther a precollicular or pretrigeminal decerebration versus intact animals. In precollicular decerebrate rats, in which midbrainbrainstem connections were preserved, but
25 forebrain involvement was eliminated, there was an attenuated compensation and rec overy from HEM compared to controls. In contrast, pretrigeminal decerebrate animals, who lacked midbrainbrainstem integrity, displayed an attenuation of the decompensatory phase. The results of this study suggest a role for the forebrain in the compens atory and recovery phases of HEM while the midbrain seems to be more important for control of hemorrhagic decompensation. The importance of midbrain structures in the full expression of the cardiovascular response to severe blood loss is further supported by studies showing that the PAG -specifically, the caudal ventrolateral PAG (vlPAG) plays a crucial in hemorrhagic decompensation (Ward and Darlington, 1987; Cavun and Millington, 2001) As shown in Figure 14 (modified from (Cavun and Millington, 2001) ), chemical inhibition of the caud al vlPAG (with lidocaine or cobalt chloride) results in a significant attenuation in the fall of MAP following HEM in conscious rats. The vlPAG likely contributes to the onset of HSI via direct projections to other caudal brainstem sites, like the RVLM an d/or the rostral ventromedial medulla (RVMM), that have also been shown to mediate decompensation following HEM (Mason et al., 1985; Henderson et al., 1998; Henderson et al., 2000; Li et al., 2001) The vlPAG sends efferents to a second brainstem nucleus, the LPBN, a rostral pontine region that has also been shown to affect hemorrhagic decompensation (Figure 1 5, modified from (Krout et al., 1998) ). Following partial lesion of the LPBN, conscious animals showed a less severe bradycardia during HEM compared to both complete LPBN lesi oned and sham lesioned animals (Blair et al., 2001) Finally, in accordance with other reports (Krukoff et al., 1995; An selmo Franci et al., 1998; Keay et al., 2002)
26 work from our lab confirms neural activation of both the vlPAG and the LPBN, as marked by increased c Fos expression during severe conscious HEM (Ahlgren et al., 2007) Both the caudal vlPAG and the LPBN show increased Fos positive nuclei following withdrawal of 30% of estimated TBV in conscious rats compared to nonHEM controls. While the connectivity of these two nuclei, as well as their individual influences over the cardiovascular response to HEM are well established, the specific role they play as an integrated and functional network during HEM has yet to be described. Recent work has shown that the recovery phase following HEM is also dependent upon the structural integrity of the LPBN. Blair et al (2001) showed that complete bilateral LPBN lesions resulted in a deficient capacity for MAP and HR to recover following HEM. Additionally, infusion of kynurenic acid (a glutamate receptor antagonist) within the ventrolateral aspect of the LPBN immediately following hypotensive HEM in a conscious rat results in prolonged depression of HR and MAP. This would suggest that glutamate receptor activation within the LPBN is necessary for normal recovery from severe blood loss (Blair and Mickelsen, 2006) This same study concluded that the delayed recovery from HEM displayed by LPBN lesioned animals was the result of an impaired sympathetic drive to the vasculature which is not all together surprising since activation of specific subnuclei located in the ventrolateral region of the LPBN (e.g. external lateral subnucleus) can produce significant hypertension and tachycardia via activation of the RVLM (Chamberlin and Saper, 1992; Len and Chan, 1999; Blair and Mickelsen, 2006) Central Opioid Modulation of Hemorrhage In the last 20 years, opioidergic mechanisms in the CNS have been implicated in the hemodynamic response to HEM. Several lines of evidence connect opioid mechanisms
27 within the CNS to the onset of hemorrhagic decompensation and HSI. It is important to have a basic understanding of the structure and functions of opioid peptides and their receptors prior to discussing physiological experiments involving them. Opioid Peptides and Associated Receptor Function Opio id peptides were first described by Hans Kosterlitz et al. in 1975. Kosterlitz described an endogenous substance that behaved like morphine in the body (Chen et al., 1997) This substance was titled enkephalin (meaning in the head) as it was first characterized in brain and adrenal tissue (Bur ke and Dorward, 1988) Since the discovery of enkephalins, several other opioid peptides and their receptors have been described. Table 11 lists the three main classes of endogenous opioids, their precursor proteins, and the primary opioid receptor on which each ligand acts. Opioid receptors are Gi/o protein coupled receptors. Ligand binding of an opioid peptide to its respective opioid receptor leads to activation of a specific class of G protein, which can inhibit intracellular cyclic AMP production, inhibit voltage gated calcium channels, or stimulate inwardly rectifying potassium channels (Law et al., 2000) all of which result in hy perpolarization of the cellular membrane. Accordingly, opioid peptides are associated with inhibitory type functions and in the CNS they generally function to depress or inhibit neurotransmission. Opioidergic Modulation of Hemorrhage in the Brainstem End ogenous opioids were first associated with hemorrhagic hypotension in the late 1970s with the work of Faden and Holaday (1979) when they showed that the opioid receptor antagonist naloxone was able to produce increases in MAP in hemorrhaged, but not normotensive, animals (Faden and Holaday, 1979) Multiple studies since then have further described the contribution of endogenous opioid
28 peptides and opioid receptor activation in HEM. From these studies it is clear that opioid systems within the brainstem are integrally involved in the hemodynamic response to severe HEM particularly the onset of HSI and the resultant decompensation. Furthermore, multiple lines of evidence hav e isolated central opioid modulation of HEM to the rostral brainstem, including the vlPAG and the LPBN. Both the vlPAG and LPBN contain considerable amounts of opioid peptides and their associated receptors (Atweh and Kuhar, 1977; Seeger et al., 1984; Mansour et al., 1987; Kalyuzhny and Wessendorf, 1998) The PAG has been shown to express mu, delta, and kappaopioid receptors (Mansour et al., 1995) While the LPBN has an abundance of both mu and kappaopioid receptors (Unterwald et al., 1991; Yasuda et al., 1993; Mansour et al., 1995; Chamberlin et al., 1999) there is only one report suggesting the sparse presence of deltaopioid receptors within the parabrachial complex (Arvidsson et al., 1995) It is generally accepted that there is a functional lack of delta opioid receptors in the LPBN (Mansour et al., 1995; Imai et al., 1996) General l esion or blockade of either the vlPAG or the LPBN results in altered blood pressure and/or HR responses to HEM (Blair et al., 2001; Cavun and Millington, 2001) More specifically, opioid agonists injected into the vlPAG cause HEM like symptoms. Keay et al (1997) reported decreased arterial blood pressure and bradycardia following microinjection of a deltaopioid receptor agonist into the vlPAG (Keay et al., 1997) Opposite results (hypertension and tachycardia) were seen when injections of the same drug were directed toward the dorsal aspect of the PAG an area more closely associated with autonomic modulation of respiration (Hayward et al., 2004) and fight or flight responses (Dielenberg et al., 2001) In the LPBN, bilateral
29 administration of a muopioid receptor agonist results in an increas ed hypertonic saline consumption by sodium depleted as well as normohydrated conscious rats (De Oliveira et al., 2008) impli cating opioids within the LPBN in volume regulation. Opioid antagonists injected into the dorsolateral pons markedly attenuate hemorrhagic decompensation. Figure 16 demonstrates that naloxone microinjected bilaterally into the vlPAG nearly aboli shes HEM evoked hypotension (Cavu n et al., 2001) Animals from the same study that were subjected to HEM in which naloxone was microinjected into the dorsal part of the PAG did not show an attenuation of the hypotensive response to HEM. Similarly, bilateral microinjection of a deltaopioid receptor antagonist, but muor kappaspecific receptor antagonists, effectively prevented a fall in MAP, suggesting involvement of a fairly specific deltaopioid mechanism within the vlPAG. In a separate study by Iwasaki and colleagues (1993), the nonselective opioid antagonist diprenorphine was microinjected into the LPBN prior to acute hypovolemia (Iwasaki et al., 1993) This study reported that opioid antagonism in the LPBN caused an inhibition of vasopressin release that would normally occur in response to hypovolemia or HEM. Unfortunately, blood pressure and HR responses to low blood volume were not reported in this study. Exercise Training and the C entral Nervous System A large body of research has clearly shown that exercise induces overall health outcomes and enhances general brain function (Warburton et al., 2006; Engesser Cesar et al., 2007) With regard to cardiovascular health benefits, much of the basic and clinical research has focused primarily on peripheral effects of exercise and/or alluded to some modulation in CNS involvement, but failed to give indepth explanations or discussions regarding such (Meredith et al., 1989; Hambrecht et al., 2000) More
30 recently, however, research has documented the ability of exercise to alter the molecular machinery (Engesser Cesar et al., 2007) of the CNS --anatomically and functionally --in ways that promote cardiovascular health (Warburton et al., 2006) and reduce and prevent a host of cardiovascular pathologies such as hypertension (Appel et al., 2003) heart disease (Dunn et al., 1996) and type II diabetes (Knowler et al., 2002) In addition to stim ulating neurotrophic factors in the brain (Engesser Cesar et a l., 2007) exercise has been shown to modulate the synthesis, metabolism, and dynamic release of a range of neurotransmitters throughout the brain and spinal cord, many of which are involved in autonomic control and cardiovascular regulation (Brown et al., 1979; Meeusen and De Meirleir, 1995; Dunn et al., 1996; Dishman et al., 1997; Engesser Cesar et al., 2007) Exercise Alters Central Opioids and Opioid Receptor Function Acute bouts of exercise stimulate the release of endogenous opioids ( Sommers et al., 1990; Goldfarb et al., 1991; Boone et al., 1992) which can result in increased nociceptive thresholds in both humans (Janal, 1996) and rats (Smith and Yancey, 2003) The fact that this is an opioidmediated phenomenon is supported by the observation that pretreatment with naloxone is able to completely block the exercise induced change in pain threshold (Janal, 1996; Smith and Yance y, 2003) Conversely, chronic exercise, and thus continued release of these endogenous opioids, has been shown to lead to a developed tolerance or decreased sensitivity to exogenous opioid agonists (Kanarek et al., 1998; D'Anci et al., 2000) While no study to date has actually reported a decreased number of opioi d receptors in any given loci within the CNS as the result of chronic exercise training, a number of studies do support this theory. Houghten et al (1 endorphin binding sites in
31 rats given access to running wheels for as little as five weeks (Houghten et al., 1986) and proposed that the effect was modulated by a compensatory decrease in opioid receptors. Similarly, other studies have shown a downregulation of opioid receptors following chronic administration of exogenous muopioids (morphine) (Malatynska et al., 1996; Chen et al., 1997) Additionally, one study evaluated the sensitivity of exercised versus sedentary rats to a number of different analgesic drugs with varying efficacy for opioid receptors. Chroni c opioid exposure leads to greater degrees of tolerance when lower efficacy opioids are administered compared to more potent analgesics (Paronis and Holtzman, 1992; Smith et al., 1999; Walker and Young, 2001) This is due to the fact that lower efficacy opioid drugs require ac tivation of a larger number of opioid receptors and therefore have a larger receptor pool from which to subtract. Therefore, authors reasoned that if chronic exercise leads to the development of opioid tolerance similar to that resulting from chronic exog enous opioid administration, then exercised rats should display less significant changes in sensitivity to opioid drugs as they increased in magnitude of relative efficacy at the muopioid receptor. This was, in fact, the outcome of the study (Smith and Yancey, 2003) Taken together, these studies indicate that increased muop ioid receptor stimulation, whether by endogenous (chronic exercise) or exogenous (drug abuse) means, elicits a functional alteration in opioid (most likely mu opioid) receptors. Dynorphins Dynorphins, a family of opioid peptides that preferentially bind to kappa opioid receptors, have been shown to be upregulated in response to both short term and chronic exercise (Aravich et al., 1993; Persson et al., 1993) Studies showing a decreased kappaopioiddependent antinociception in rats allowed access to running
32 wheels for 56 weeks suggest that, similar to what is seen with muopioid receptors following exercise training or chronic morphine administration (Smith and Yancey, 2003) long term exercise leads to either a decreased sensitivity or a downregulation of kappa receptors (D'Anci et al., 2000) Specific Aims In summary, based on observations that central opioids are modulated by chronic exercise and the cardiovascular response to HEM involves opioid release in specific regions of the brain, the present studies were undertaken to evaluate the impact of chronic exercise on the physiological response to blood los s. These studies are particularly important in light of the paucity of information focusing on the central effects of exercise on cardiovascular health. A better understanding of the centrally mediated effects of exercise in the response to HEM is import ant for the development of individual treatment plans for trauma victims. If, indeed, trained individuals respond differently to blood loss than do sedentary individuals, a difference in effective treatment may exist. There is significant translational r elevance and potential for clinical application for the results of these studies in light of the large number of soldier deaths associated with trauma HEM (Alam et al., 2005) A more developed picture of how chronic exercise affects the CNS will enhance our ability to determine opt imal doses of drugs targeting central sites for patients in different physical conditions (e.g., trained verses sedentary, or soldier versus civilian). This is also potentially important for the prescription of certain psychoactive and analgesic drugs many of which target central opioid receptors. Specific Aim 1 Assess the neural responses to different rates of HEM in order to develop a standard protocol that best demonstrates HSI during severe HEM in the conscious rat.
33 Hypothesis Specific Aim 2 : We hypothesized that the fastest rate of HEM would induce the earliest and greatest level of decompensation and that this would be associated with increased levels of c Fos immunoreactivity in the vlPAG and LPBN. Identify whether vlPAG neurons that projec t to the LPBN are activated in response to severe HEM in the conscious rat. Hypothesis Specific Aim 3 : We hypothesized that a greater percentage of vlPAG area neurons projecting to the LPBN would be activated, as indicated by the cellular marker c Fos, compared to the dorsolateral PAG (dlPAG), in response to severe HEM. Identify the effect of chronic exercise on the hemodynamic response to HEM. Hypotheses Specific Aim 4 : Exercise trained animals will display an altered hemodynamic and neural response to hypotensive HE M compared to sedentary animals. Specifically, we hypothesized that exercise training would result in an augmented compensatory response to HEM as evidenced by either a greater increase in HR early in HEM and/or a prolonged time to decompensation. Additi onally, we anticipated a blunted decompensatory response to HEM in exercised versus sedentary rats. Determine whether the effect of chronic exercise on the modulation of the hemodynamic response to HEM is due to a modification of opioid receptors in the LPBN. Hypotheses : Chronic exercise will cause a downregulation of central kappa opioid receptors, specifically in the dorsolateral pons and blockade of opioid receptors in the LPBN will potentiate the protective effects of chronic exercise in response to HEM.
34 Summary The overall goal of these studies was to gain a more comprehensive understanding of how voluntary exercise training alters central mechanisms involved in the hemodynamic response to HEM. Based on previous work showing that opioid receptors present in specific midbrain nuclei (vlPAG and LPBN) are involved in the cardiovascular response to HEM and that chronic exercise functionally alters opioid receptor populations, we hypothesized that exercise trained animals would display an a ltered neural and cardiovascular response to HEM compared to sedentary animals. Since exercise has been shown repeatedly to be both cardioand neuroprotective, we anticipated an enhanced tolerance to severe HEM in the trained versus sedentary conditions We further hypothesized that a downregulation of opioid receptors in the dorsolateral pons may underlie such a modified response. These studies will, additionally, provide novel information regarding central neuroanatomical networks mediating the cardiovascular response to HEM that may be affected by chronic exercise.
35 Figure 11 Effect of hemorrhage on mean arterial pressure and heart rate in anesthetized (left panel) versus conscious (right panel) rats. Open circles represent group averages for rats in which 1.5 ml of blood was removed every 10 minutes (arrows; n=6). Closed circles represent group averages for control animals (n=6) that underwent the same hemorrhage protocol, except the removed blood volume was immediately replaced with donor bl ood. Modified from Leskinen et al., 1994.
36 Figure 12. Heart rate ( HR) and mean arterial pressure ( MAP ) recording from a conscious rat during 30% estimated total blood volume hemorrhage. Grey bar indicates time of blood withdrawal (20 minutes). Progr essive phases of the cardiovascular response to hemorrhage are outlined by boxes and labeled accordingly.
37 Figure 1 3. Brainstem and spinal cord pathways that subserve the baroreceptor reflex control of sympathetic output to the heart and blood vessels Open triangles indicate excitatory synapses and filled triangles indicate inhibitory synapses. CVLM = caudal ventrolateral medulla; IML = intermediolateral cell column in the spinal cord; NTS, nucleus tractus soli t ar i us. Modified from Dampney et al., 1994.
38 Figure 1 4. Effect of synaptic blockade in the ventrolateral periaqueductal gray (vlPAG) on the change in mean arterial pressure ( MAP ) from baseline during severe hemorrhage (HEM). Cobalt chloride (top panel) or lidocaine (bottom panel) was bila terally injected into the caudal vlPAG of conscious, chronically instrumented rats five minutes prior to HEM at a rate of ~1.25 ml/kg/min. Modified from Cavun and Millington, 2001.
39 Figure 1 5 Diagram of connections between the ventrolateral periaqued uctal grey ( vlPAG ) and lateral parabrachial nucleus ( LPBN ) that subserve cardiovascular regulation. SCP = superior cerebellar peduncle; NTS = nucleus tractus solitarius. Modified from Krout et al., 1998.
40 Table 11. Three main classes of endogenous opioids, their precursor proteins, and the primary opioid receptor on which each ligand acts. Opioid Peptide Precursor Protein Primary Receptor Endorphins Pro opiomelanocortin Mu, Enkephalins Pro enkephalin Dynorphins Pro dynorphin Figure 16. Effect of opioid receptor blockade in the caudal ventrolateral periaqueductal grey (vlPAG) on mean arterial pressure ( MAP ) during hemorrhage. Bilateral injections of naloxone (10nmol; 5nmol/cannula) or saline (0.5 l/cannula) were placed into the caudal vlPAG of halothaneanesthetized rats five minutes prior to hemorrhage at a rate of ~1 ml/kg/min. Reproduced with permission from Cavun et al., 2001.
41 CHAPTER 2 HEMODYNAMIC RESPONSE S AND CFOS CHANGES ASSOCIAT ED WITH HYPOTENSIVE HEMORRHA GE: S TANDARDIZING A PROTO COL FOR SEVERE HEMORRHAGE IN THE CO NSCIOUS RAT Introduction Conscious mammals subjected to severe, progressive hemorrhage ( HEM ) have been shown to pass through two hemodynamically distinct phases. Upon initial blood loss, an augmented sympathetic drive results in an elevated heart rate ( HR) and increased peripheral resistance. In this compensatory phase, the declining cardiac output is not large enough to cause a fall in mean arterial pressure ( MAP ) When blood loss approaches 2530% of total blood volume, a decompensatory phase consisting of sympathetic withdrawal, hypotension, and bradycardia ensues. Anesthesia can significantly alter the response pattern to blood loss (Evans et al., 1989; Heslop et al., 2002) Anesthetized animals, in general, show little if any compensatory response to HEM and therefore enter the decompensatory phase earlier than conscious animals following loss of much smaller volumes of blood (Heslop et al., 2002) Additionally, under anesthesia, MAP drops more quickly and is typically proportional to the amount of blood withdrawn. The duration of the decompensatory phase is often associated with increased peripheral organ damage and a reduced chance of survival (Runciman and Skowronski, 1984) Over the last ten years there has been increased interest in understanding the central neural mechanisms underlying hemorrhageinduced sympathoinhibition (HIS) with the hope of identifying therapies that might delay the onset of the decompensatory response and facilitate recovery. While the mechanisms underlying the initial, compensatory phase of hemorrhage are fairly well accepted (baroreceptor unloading)
42 (Runciman and Skowronski, 1984; Courneya et al., 1991; Evans et al., 2001) the signal that prompts the transition to a decompensatory state is less clear. There is some evidence, however, that input to the midbrain is critical for inducing HSI (Evans et al., 1991) For example, blockade of the ventrolateral periaqueductal grey ( vlPAG ) has been shown to both delay and attenuate hemorrhageevoked hypotension (Cavun and Millington, 2001) Furthermore, in a recent brain transection study, it was demonstrated that pretrigeminal decerebrate rat, lacking midbrainbrainstem communication, had a markedly attenuated HSI response to severe HEM compared to controls (Troy et al., 2003) In contrast, precollicular decerebrate animals (in which midbrainbrainstem connections were still present) displayed a prolonged decompensation compared to cont rols. These findings support the current idea that midbrain structures play a pivotal role in the onset of HSI. Interestingly, in the same study it was shown that prolongation of the decompensatory response observed following precollicular decerebration was only observed in animals that underwent a 30% total blood volume ( TBV) HE M over 20 minutes but was not present when 30% TBV HEM occurred over 40 minutes. Thus, the rate of HEM may profoundly impact brain mechanism(s) contributing to or perhaps initiating HSI. To our knowledge, no previous studies have combined a thorough evaluation of the cardiovascular outcome of constant volume HEM over different withdrawal times with the identification of the associated specific central nervous system ( CNS ) regional sites of activation. Furthermore, an indepth evaluation of the impact of r ate of HEM on the brain mechanisms involved in HSI might be considered necessary at the present time, since a review of the literature reveals a distinct lack of consistency in HEM
43 protocols between studies (Shirley et al., 1991; Buller et al., 1999; Heslop et al., 2002; Scrogin, 2003; Molina et al., 2004; Schadt and Hasser, 2004; Ditting et al., 2005) The purpose of this project was to assess the neural responses to different rates of HEM in order to develop a standard protocol that best demonstrates HSI during severe HEM in conscious rats for use in future studies. Because specific regions throughout the CNS have been shown to play integral roles in regulation of homeostasis during HEM (Buller et al., 1999; Cavun and Millington, 2001; Troy et al., 2003; Heslop et al., 2004) we fe lt that, in addition to hemodynamics, evaluating neural activity in response to hemorrhagic hypotension would offer a more complete picture of the natural consequences resulting from severe blood loss. Accordingly, this study aimed to evaluate hemodynamic responses and c Fos immunoreactivity in specific regions of the brain following different rates of severe (30% TBV) HEM in the conscious rat. Two areas of particular interest were the vlPAG and the lateral parabrachial nucleus ( LPBN ) because of the indepe ndent and integrated roles they have been shown to play in response to cardiovascular challenge (Ward, 1989; Polson et al., 1995; Krout et al., 1998; Blair et al., 2001; Cavun and Millington, 2001; Dean and Woyach, 2004) We hypothesized that the fastest rate of hemorrhage would induce the earliest and greatest level of decompensation and that this would be associated with increased levels of c Fos immunoreactivity in the vlPAG and LPBN. Methods General Preparation All experimental procedures were approved by the Animal Care and Use Committee at the University of Florida. Male SpragueDawley rat s (3576 g, Harlan Industries, Minneapolis, IN) were anesthetized with an i.p. injection of
44 ketamine/xylazine/acepromazine (80100/820/1 3 mg/kg, respectively) and then randomly placed into one of three groups: hemorrhage (HEM); saline volume control (SAL ); or hydralazine pressure control (HYDRAZ). Following assignment to a group, all rats were surgically instrumented with catheters (PE 10 connected to PE 50 tubing, Braintree Scientific, Braintree, MA) filled with heparinized saline (100 IU/ml). HEM rat s were instrumented with two femoral arterial catheters. SAL and HYDRAZ rats were instrumented with a femoral venous and a femoral arterial catheter. Catheters were then tunneled subcutaneously, exteriorized at the nape of the neck, and sealed with 23ga uge obturators until the day of the experiment. Analgesics (Rimadyl, 0.01 ml/kg; Buprenorphine, 0.01 ml/kg) were administered subcutaneously following catheterization and animals were allowed 48 hours to recover. During recovery, animals were housed sing ly under controlled illumination (12 hour cycle) with food and water ad libitum. The day following catheter placement, animals were brought to the lab to ensure catheter patency and for acclimating purposes. Animals were weighed, lightly handled, and all owed to sit quietly for 2 3 hours in the testing chamber (9x9 inch bucket) they would be placed in for the experiment. Animals were returned to their home cages following acclimation. Experimental Protocol On the day of the experiment, animals were brought to the lab, weighed, placed in the testing chamber and a single arterial catheter was connected to a calibrated pressure transducer in series with an amplifier (Stoelting, Wooddale, IL). The arterial and venous catheters were then attached to a swivel system (Instech, Plymouth Meeting, PA) so the animals could move unrestrained about the testing chamber. Both pulsatile and MAP were recorded online at 100 Hz using a Cambridge Electronics
45 Design computer interface and Spike2 data software. HR was deriv ed online from the interval between peak systolic pressure waves in the arterial pressure ( AP) trace. After 60 90 minutes of quiet rest, the experiment began. First, baseline AP, MAP, and HR were recorded for a 30minute baseline period. Next, animals underwent one of 5 experimental procedures: 1) slow hemorrhage (S HEM, 0.5 ml/kg/min, n = 7); 2) intermediate hemorrhage (I HEM, 1.0 ml/kg/min, n = 7); 3) fast hemorrhage (F HEM, 2.0 ml/kg/min, n = 6); 4) saline control (SAL n = 5); or 5) hydralaz ine control (HYDRAZ, n = 5). All hemorrhaged animals underwent a 30% estimated TBV extraction through the second arterial catheter. TBV was calculated using a previously reported equation for estimation of rat blood volume: (0.06 ml/g)*(body weight in g)+(0.77) (Lee and Blaufox, 1985) S HEM, I HEM, and F HEM were performed over 40, 20, and 10 minutes, respectively. For HYDRAZ animals, 1 ml of hydralazine (3mg/kg) was infused over 60seconds through the venous catheter in order to induce a level of hypotension similar to that induced by HEM (Graham et al., 1995; Pelaez et al., 2002) For the SAL animals, 1 ml of heparinized saline (0.9% NaCl, 2 IU/ml) was infused over 60second s through the venous catheter. Ninety minutes after the cessation of HEM or drug infusion protocols, animals were administered a lethal dose of sodium pentobarbital (100150 mg/kg) and transcardially perfused with heparinized saline followed by 4% paraformaldehyde 90minutes. Brains were removed and post fixed in 4% paraformaldehyde for 24 hours followed by 2448 hours of immersion in cryoprotectant solution (30% sucrose) prior to cryostat sectioning
46 Fos Immunocytochemistry Extracted brains were cut into 40 micrometer coronal sec tions and processed for Fos like immunoreactivity (FLI) as previously described (Hayward and Von Reitzenstein, 2002) Briefly, free floating sections were washed in sodium phosphate buffered saline (PBS, pH 7.4) followed by a second wash in a 3% goat serum PBS triton X100 solution (3% GS PBSTX) to prevent nonspecific binding. Sections were then incubated for 24 hours in rabbit anti cFos primary antibody (1:2000 dilution, sc 52r, Santa Cruz Biotechnology). Following another wash in 1% GS PBSTX, sections were incubated in goat anti rabbit biotin (Jackson ImmunoResearch Laboratories, Inc., 111065 144) for two hours and rewashed (1% GS PBSTX) prior to being placed in avidin biotin peroxidase complex (ABC Vectastain Kit, Vector, Burlingame, CA). Sections were put through a final wash (1% GS PBSTX) followed by visualization of the FLI with a chromagen solution (0.05% diaminobenzidine hydrochloride [DAB], 2.5% ammonium sulfate, 0.033% hydrogen peroxide in 0.05 M Tris HCl, Vector). Sections were then mounted onto glass slides, air dried, dehydrated in a graded alcohol and CitriSolv (Fisher Scientific) series, and covserslipped. Neuroa natomical Quantification of Fos L ike Immunoreactivity For each animal, two representative sections from each brain area of interest were imaged (Axioskop, Carl Zeiss; 5X) and analyzed for the number of FLI neurons present by a technician blinded to the experimental conditions. The software used for FLI quantification (Metamorph) allows the investigator to assign color, object size (710 m), and/or density ranges specific to Fos positive cells, as determined by the investigator. Once these ranges have been preset, the software is then able to recognize and
47 record FLI in a specified field of the image. This allows greater consistency and decreased human error in quantifying FLI between images. Figure 21 shows a schematic of the rostral, middle, and caudal coronal sections of the LPBN and PAG analyzed in this study. The criteria used for selecting specific sections of the LPBN included the shape of the superior cerebellar peduncle (SCP), the width of the LPBN from the SCP to the ventral spinocerebellar column, and the width of the ventral spinocerebellar tract. The criteria for choosing specific PAG sections were based the shape of the central aqueduct, the shape and width of the dorsal and ventrolateral columns, and the presence of the oculomotor nucl eus. Other areas imaged and quantified for FLI included the locus coeruleus (LC, interaural 0.68 to 0.80), the cuneiform nucleus (CnF, interaural 0.48 to 0.60), Kolliker Fuse (KF) and A7 (imaged in the same section and at the level of the rostral LPBN) (Paxinos and Watson, 2005) Several standardized masks were prepared for each level of each brain nucleus using counterstained brain sections as well as guidance from previously diagramed images of the brain areas of interest (Behbehani, 1995; Henderson et al., 1998; Krout et al., 1998) T hese masks were superimposed over corresponding images using Adobe Photoshop 7.0 in order to outline boundaries of selected brain areas and the different subnuclei within the PAG (Behbehani, 1995) and the LPBN (Krout et al., 1998) prior to FLI analysis. Masks were prepared in such a way as to allow fitting to individual brain dimensions as well as various angles of c ut, but to still maintain the integrity of the approximate shapes and proportions of relative subnuclei. Cardiovascular Measurements MAP and HR were averaged over fiveminute intervals for each experiment. MAP and HR values from 5 minutes prior to the ons et of HEM or drug were averaged to give
48 a single baseline value (0 min.). Following the onset of HEM or drug infusion, the first two 5 minute averages (5 min. and 10 min.) and then every other 5 minute average (20 min., 30 min. etc.) were used for calculation of group averages. Statistical Analysis. A oneway analysis of variance ( ANOVA) was used to determine if there were any significant differences in baseline MAP or HR between treatment groups. A twoway ANOVA with repeated measures was used to iden tify the effects of treatment (i.e. HEM or HYDRAZ) on MAP and HR over time (minutes 0, 5, 10, 20, 30, 40, 50 and 60). When indicated, paired or unpaired Bonferroni t tests were then used to isolate differences relative to baseline (minute 0) within treatm ent groups or between treatment groups at specific time points. The accepted P value (P<0.05) was adjusted for the number of t tests performed (n = 7, P<0.007). To determine whether MAP or HR at the offset of HEM within each treatment group were significa ntly different from the HYDRAZ treatment group at the same time point, an unpaired t test was used. Differences were considered significant when P<0.05. FLI data from all regions except the LPBN were analyzed using a oneway ANOVA comparing the effect of treatment (i.e. HEM or HYDRAZ) on FLI levels within each specific rostral caudal section chosen for analysis. In the LPBN, FLI data were analyzed using a twoway ANOVA comparing the effect of treatment and subnuclei on FLI levels within each specific rost ral caudal section. If a significant effect was indicated, unpaired Bonferroni t tests were used to reveal differences between SAL versus other treatments on FLI levels with each brain region. The accepted P value (P<0.05) was adjusted for the number of t tests performed (n = 4, P<0.012), All data are presented as mean SEM.
49 Results Cardiovascular R esponse t o Hypotension V ersus S evere H emorrhage Baseline MAP and HR for all groups of animals is shown in Table 21. There was no significant difference in r esting MAP and HR between groups at the start of the experiments. Figures 22 & 2 3 illustrate the average change in MAP and HR over time for all groups following treatment. For SAL animals, there was no change in MAP or HR from baseline throughout the experiment (Figure 22). HYDRAZ animals, on the other hand, showed a significant decrease in MAP from baseline starting at 5 min. following HYDRAZ administration and continued throughout the experiment (Figure 22A). Additionally, at 20 minutes and for the remainder of the measurement period, the MAP of HYDRAZ treated animals was significantly different from SAL animals. In response to the HYDRAZ induced hypotension, HR increased significantly above both baseline and the HR of SAL animals at 10 minutes po st injection and remained elevated throughout the experiment (Figure 22B). HEM also induced a persistent hypotension, but in all groups, MAP did not drop significantly below baseline until > 15% TBV had been withdrawn (Figure 23A). In the S HEM group at min. 30, when approximately 23% of the TBV had been withdrawn, MAP fell significantly below baseline (Figure 23A). In both the I HEM and F HEM groups, MAP was identified to be significantly below baseline at 20 and 10 min. post HEM onset, respectively In all three HEM groups, the lowest MAP was recorded at the offset of HEM when 30% of TBV had been withdrawnat which point MAP was not significantly different between HEM groups. Furthermore, a comparison of MAP at the time of 30% TBV withdrawal in all HEM groups versus HYDRAZ at a similar time point (Table 22) demonstrated that for both S HEM and I HEM, the decrease in MAP at the
50 time of HEM completion was similar to that induced by HYDRAZ. In contrast, at corresponding time points, the decrease i n MAP for the F HEM group was significantly different from the HYDRAZ group. HR showed an increase from baseline during the first half of the HEM protocol for all groups. However, the peak increase in HR was only significantly different from baseline (F igure 23B) in the I HEM group during this initial compensatory period. In all HEM groups the peak drop in HR occurred at the offset of HEM. However, because of a large amount of inter individual variability, the decrease in HR for the S HEM group was not significantly different from baseline at any time point following the onset of HEM. Furthermore, the decrease in HR for both the F HEM and I HEM groups was only significantly different from baseline at 20 and 30 minutes following the onset of HEM, res pectively (Figure 23B). The HR of the F HEM group was also significantly different from baseline at 30 min. following HEM onset. HR in the I HEM group was significantly different from baseline at 50 and 60 minutes following the onset of HEM. HR values recorded at the offset of the HEM when 30% of TBV had been withdrawn were not significantly different between HEM groups. However, HR values at the offset of HEM for all groups, at the time of peak hypotension, were significantly lower than HYDRAZ values at the same time points (Table 2 2). Fos Like I mmunoreactivity in B rains tem Nuclei Following S evere H emorrhage Figure 24 shows representative middle and caudal sections of the PAG for visual comparison of FLI labeling in a SAL and an I HEM rat. Region s of the PAG chosen for quantification of FLI included both the dorsomedial PAG (dmPAG) and the vlPAG, based on their known physiological contributions to sympathoexcitation and sympathoinhibition, respectively (Cavun and Millington, 2001; Hayward and
51 Castellanos, 2003) As shown in Figure 24, IHEM induced increased levels of FLI, relative to SAL, throughout the dorsal and ventral PAG. FLI labeling for the SAL group depicts basal levels of neural activation in these conscious animals, as MAP and HR were unchanged for the duration of the experiment. Accordingly, all changes in FLI following HEM or HYDRAZ were compared to FLI levels in the SA L treated animals. Figure 25 illustrates average FLI levels in the PAG for all treatment groups. In general, both HEM and HYDRAZ treatment induced large changes in FLI in the vl PAG compared to SAL treatment and there was a significant effect of treatm ent (P<0.001) in both the caudal and middle vlPAG. In the caudal vlPAG, however, only I HEM and F HEM induced a significant increase in FLI compared to SAL. In the middle vlPAG, all HEM rates and HYDRAZ induced a significant increase in FLI above SAL. I n contrast, in the caudal dmPAG there was no significant effect of treatment on FLI. In the middle dmPAG there was a treatment effect (P<0.002), but only I HEM induced a significant inc rease in FLI compared to SAL. Figure 26 shows representative rostral, middle, and caudal sections of the LPBN from a SAL treated versus an I HEM and an F HEM rat. The top three panels show increased levels of FLI in the central subnucleus of the rostral LPBN in both HEM groups compared to SAL. In the middle and caudal LPB N, increases in FLI in the HEM above SAL treated animals were primarily located in the external and dorsal subnuclei. The average increase in FLI induced within subnuclei of the rostral, middle and caudal LPBN following HEM and HYDRAZ is shown in Figure 27. In the rostral LPBN, a main effect of treatment (P<0.0004) and subnuclei (P<0.0001), as well as an interaction between factors (P<0.008), was observed. Comparisons within the
52 individual subnuclei demonstrated that within the central subnucleus of the rostral LPBN both I HEM and F HEM increased FLI significantly above SALinduced levels. In contrast, in the superior lateral subnucleus of the rostral LPBN, I HEM as well as HYDRAZ treatment induced a significant increase in FLI above SAL treatment. In the middle and caudal LPBN, there was a significant effect of subnuclei (P<0.001 for both) and treatment (middle: P<0.0001; caudal: P<0.02), but no interaction between these two factors (middle: P<0.4; caudal: P<0.8). Thus, a comparison of significant ef fects of treatment on FLI within individual subnuclei was not permitted. In the middle LPBN however, irrespective of individual subnuclei within the middle LPBN (Figure 2 7B), all HEM and HYDRAZ treatments induced increased levels of FLI relative to SAL. In the caudal LPBN, only F HEM and HYDRAZ showed significantly more FLI than SAL (combined subnuclei). Figure 28 shows the average increase in FLI across treatment groups for four other rostral brainstem regions also quantified, including the CnF, LC, K F (a subnucleus of the PBN), and A7. In all four regions, there was a significant effect of treatment on FLI (P<0.01). In both the CnF and A7 nuclei, only I HEM induced a significant increase in FLI compared to SAL. In the LC (Figure 28B), both I HEM a nd F HEM, but not HYDRAZ, induced a significant increase in FLI labeling compared to SAL. In contrast, both HYDRAZ and I HEM induced significant increases in FLI compared to SAL treatment in KF (Figures 28B & C, respectively) Discussion It is generally accepted that the amount of blood loss necessary to induce hypovolemic decompensation in a rat is between 1530% of the animals TBV (Schadt and Ludbrook, 1991) However, the rate at which this volume is lost may impact the
53 transition from compensation to decompensation and, thus, brain mechanism(s) recruited to meet the physiological challenge (Troy et al., 2003) In the present study, all rates of HEM induced clear compensatory and decompensatory stages. In all instances, during blood loss of up to 15%, there was a compensatory tachycardia, and MAP was well maintai ned. Yet, only during I HEM was the increase in HR during the compensatory phase significantly different from baseline. Following 30% TBV withdrawal, all hemorrhaged animals had MAPs that were significantly reduced from baseline. Both F HEM and I HEM grou ps showed a corresponding drop in HR that was significantly different from baseline at 10 minutes following the offset of HEM. Furthermore, in the I HEM group a significant reduction in HR was observed between 30 and 40 min. following the offset of HEM. These observations demonstrate that there are marked differences in autonomic regulation of MAP and HR when severe HEM occurs at different rates of blood loss. Examination of FLI in the rostral brainstem identified several regions that might be selectively involved in autonomic regulation during severe HEM. These regions showed significant increases in FLI following I HEM but not in response to HYDRAZ treatment; including the middle dmPAG, the caudal vlPAG, the central lateral subnucleus of the rostral LPB N, LC, A7, and the CnF. Other brain regions examined demonstrated increased levels of FLI associated with both HEM and HYDRAZ compared to SAL controls. This suggests that activation of these specific brainstem sites was more closely related to autonomic regulation in response to hypotension than the hypovolemia and HEM associated adjustments in autonomic regulation. Interestingly, the outcome of our study did not support our original hypothesis that F HEM would
54 induce the greatest change in both cardiovas cular parameters and FLI. In contrast, our results suggest that utilization of a hemorrhage at an intermediate rate of 1 ml/kg/min for 30% TBV HEM may be most useful for investigating the potential role of the rostral brainstem regions in mediating hemorr hagic decompensation in conscious rats. Methodological C onsiderations Several methodological factors must be considered when interpreting the results of the present study. First, in the present study we chose to use HYDRAZ for our nonvolume depleted hypotensive controls because of its use in previous studies (Graham et al., 1995; Pelaez et al., 2002) as well as its known and reliable hypotensive actions. However, HYDRAZ proved to be a potent and long lasting vasodilator. The induction of such a long lasting hypotension may have added additional stress to the animals. In retrospect, a shorter acting vasodilator, such as sodium nitroprusside, may have been a more suitable control and may have better mimicked the response seen with our selected method of HEM. The second factor to consider in this study is the use of FLI to identify specific regions of the brain involved in cardiovascular control when multiple stimuli and physiological changes occur over a relatively short time period. FLI is induced following neuronal excitation and depolarization and is an indicator of changes in neuronal activation associated with a stimulus. However, the resolution of these changes to specific time points is limited. In our study, all animals were sacrificed at 90 minutes following the offset of HEM. Accordingly there should have been a good correlation between FLI levels and the maximum drop in MAP (Chan and Sawchenko, 1994) However, during severe HEM many other physiological changes occur in an attempt to survive the insult ( Brown et al., 2005; Osei Owusu and Scrogin, 2006) As a result, we
55 can only correlate changes in FLI with the recorded cardiovascular changes. Further studies are needed to more definitively identify the role of each region identified in the present stu dy in mediating hemorrhagic decompensation or recovery. Cardiovascular R esponse to H emorrhage The only other study that we are aware of that has previously addressed the issue of HEM rate maintaining the volume withdrawn constant was by Troy and colleagues (Troy et al., 2003) In that study, 30% TBV HEM were performed in conscious rats over 20 or 40 minutes. Blood pressure responses were similar to those reported in the present study. That is, MAP was well maintained for the first 15% of TBV loss; but by the time 30% TBV had been withdrawn there was approxim ately a 50% drop in MAP irrespective of HEM rate. The HR responses, however, were considerably different. In their study, the faster rate of HEM, which corresponded to our intermediate rate (I HEM), produced a reflexive tachycardia that peaked at ~38% ab ove baseline following 30% hemorrhage, and HR remained elevated compared to the preHEM baseline during recovery. The slower rate of HEM (30% TBV loss over 40 minutes corresponding to our S HEM group) also produced a tachycardia in response to HEM, but it was much more variable and reached a peak of only ~10% above baseline. In contrast, we reported a transient tachycardia in both the S HEM and I HEM groups, which was only ~815% above baseline following 15% TBV loss. As blood loss continued, HR began to drop below baseline the peak drop in HR occurred at or just following 30% TBV loss and persisted throughout recovery. The discrepancy between studies might be explained by differences in protocol. For example, animals in our study were allowed 48 hours t o recover from surgery, while the rats in the previous study were hemorrhaged between 70 and 120 minutes following catheterization, in which inhalant halothane anesthesia
56 was used. Since halothane can remain in the bodys tissues for at least two hours fo llowing anesthetic levels of exposure (Divakaran et al., 1981) residual anesthesia may have modified the HR response to HRM in their study. Similarities between our study and other HEM studies with longer post surgery recovery times suggest that anesthesia, even 4 to 6 hours following withdrawal (Blair et al., 2001; Cavun and Millington, 2001; Scrogin, 2003) can markedly alter autonomic control of HR (Lee et al., 2002) and presumably brain mechanisms recruited for autonomic adjustments during HEM. Pattern of Fos Like Immunoreactivity in the Rostral Brainstem F ollowing H emorrhage Brain regions previously identified to be responsible for the switch from a compensatory response to decompensation during HEM have been isolated to the rostral brainstem (Evans et al., 1991) More specifically, both the vlPAG in the midbrain (Cavun and Millington, 2001) and the LPBN in the rostral pons (Blair et al., 2001) have been shown to play important roles in hemorrhagic decompensation. In the present study, increased levels of FLI were observed in both the vlPAG and LPBN in response to HEM compa red to control (SAL). However, only in the caudal vlPAG and the rostral LPBN were the HEM induced changes in FLI distinguished from changes in FLI induced by HYDRAZ or hypotension alone. This raises the possibility that neurons in these regions are critica l in mediating autonomic responses associated with hemorrhagic decompensation. In the present study increases in FLI were consistently observed in the vlPAG in response to all three rates of HEM. This observation supports physiological data from Cavun and Millington (Cavun and Millington, 2001) demonstrating that synaptic
57 blockade in the vlPAG markedly attenuates both the hemorrhag ic hypotension and HEM induced changes in HR. Furthermore, in a recent study by Schadt and colleagues (Schadt et al., 2006) neurons in the vlPAG wer e shown to display discharge patterns indicative of mediating HSI. Our observation that HEM induced a significant increase in FLI in the vlPAG also corroborates the results of a previous study investigating FLI following HEM (Keay et al., 2002) However, because hypotension alone can also induce FLI in the vlPAG (Li and Dampney, 1994; Murphy et al., 1995) in the present study we also evaluated the effect of hypotension (HYDRAZ) on FLI in the vlPAG. Our results identified that, similar to HEM, HYDRAZ induced a s ignificant increase in FLI in the middle vlPAG compared to control. In the caudal VvlPAG however, the response to HYDRAZ was more variable and was consequently not significant. Yet, increases in FLI in the caudal vlPAG in response to both I HEM and F HEM were significant. This raises the possibility that neurons in the middle vlPAG may be more important in regulating autonomic responses to hypotension, whereas caudal vlPAG neurons may be more involved in regulating cardiovascular function during severe HEM This observation is supported by data demonstrating that both the neuroanatomical connectivity (Behbehani, 1995) and control over different vascul ar beds of the middle vs. caudal vlPAG are distinctive (Dielenberg et al., 2001) In the present study we also observed a small but significant increase in FLI in the middle section of the dmPAG following I HEM but not HYDRAZ. Since activation of the dmPAG induces sympathoexcitation (Dielenberg et al., 2001) and th e I HEM group was the only group that showed a significant increase in HR during the compensatory phase of HEM, this raises the possibility that activation of dmPAG area neurons plays an
58 important role in maintaining MAP during the initial compensatory phase of HEM. Accordingly, an increase in FLI was also reported, but not quantified, the middle region of the dmPAG by Keay et al. in response to 15% TBV withdrawal in conscious rats (Keay et al., 2002) On the other hand, it should be noted that chemical blockade of the dorsal PAG has been reported to have no effect o n the cardiovascular response to severe HEM (Cavun and Millington, 2001) Yet, in that study severe HEM was induced 4 6 hours af ter isofluorane anesthesia and no compensatory change in HR was noted in the control conditions. Thus, it remains to be determined what role dorsal PAG neurons play in modulating cardiovascular responses to I HEM in an anesthesiafree animal. In the present study, increased levels of FLI following I HEM compared to control were observed in all three rostral caudal regions of the LPBN, including KF. Yet, only in the rostral LPBN was the increase in FLI during HEM separated from the effects of HYDRAZ or hyp otension alone. More specifically, in the central lateral subnucleus of the rostral LPBN both I HEM and F HEM selectively induced increased levels of FLI. In contrast, the effect of HYDRAZ was not significantly different from SAL controls. These resul ts comple ment recent observations by Blair et al. (Blair et al., 2 001) that smaller lesions of the LPBN, which encompassed only the dorsolateral portion, attenuated the bradycardic response to severe HEM in conscious rats. In contrast, larger lesions involving the entire LPBN (dorsolateral and ventrolateral subnuclei, possibly including KF) had little effect on the decompensatory response to severe HEM but impaired recovery. This raises the possibility that that the central lateral subnucleus of the rostral LPBN may be involved in mediating or relaying signals associated with HSI. Indeed, this region of the rostral LPBN receives a large projection from the vlPAG (Krout et al.,
59 1998) though the physiological function of this interconnection has yet to be determined. Several regions out side of the PAG and LPBN were also quantified for FLI levels in the present study. These regions included LC, the CnF nucleus, and A7. All three regions have been shown to be involved in responses to different types of stress (Korte et al., 1992; Van Bockstaele et al., 2001) and cardiovascular regulation (Murphy et al., 1994; Guyenet et al., 2001) In the present study all three regions demonstrated significant increases in FLI in response to I HEM but not HYDRAZ. Since all three regions are interconnected with the PAG (Bajic and Proudfit, 1999; Van Bockstaele et a l., 2001) all three may be well positioned to contribute to cardiovascular adjustments during the transition from compensation to decompensation during HEM. Future studies should focus on determining the role of each region in HSI. Additionally, future studies should identify regions of the CNS actively inhibited during severe conscious HEM since the use of FLI is limited to activated neurons only. Conclusions In summary, the results of the present study provide investigated the impact of different rat es of severe HEM on the cardiovascular outcome coupled with regional CNS activation. We have confirmed previous observations that suggested there are marked differences in autonomic regulation when severe HEM occurs at different rates (Troy et al., 2003) The results of our study suggest that a constant withdrawal rate of 1 ml/kg/min until 30% TBV has been removed produces the most reliable pattern of tachycardia and compensation followed by hypotension and bradycardia for the study of experimental severe HEM in conscious rats. Associated with this rate of H EM were indicators of increased levels of excitation localized to the caudal vlPAG, the middle
60 dmPAG, the rostral central lateral subnucleus of the LPBN, and LC. Other brain regions newly identified to be potentially involved in mediating HEM responses include CnF and the A7 region. Together these results provide further evidence of the potential importance of activation of the rostral brainstem in mediating the response to severe HEM.
61 Figure 2 1. Schematic of periaqueductal grey (PAG) and lateral par abrachial nucleus (LPBN) areas imaged for quantification of Fos positive staining. All figures and numbers were adapted from Paxinos and Watson, 2005. Approximate middle, and caudal PAG (A) and rostral, middle, and caudal LPBN (B) areas used. Numbers di splayed with each representative section indicate approximate c oordinates caudal to Bregma. DM PAG, dorsa l medial periaqueductal grey; VLPAG, ventrolateral periaqueductal grey; Sup, superior lateral parabrachial; Ctr, central lateral parabrachial; KF, Koll iker Fuse nucleus; SCP, superior cerebellar peduncle; Dor, dorsal lateral parabrachial; Cres, crescent lateral parabrachial; Ext, external lateral parabrachial. Table 2 1. Baseline mean arterial pressure ( MAP ) and heart rate ( HR) of treatment groups No significant different between groups (P>0.05). Treatment Group MAP (mmHg) HR (bpm) SALINE (n=5) 1094 35114 HYDRAZ (n=5) 1224 34924 S HEM (n=7) 1204 3535 I HEM (n=7) 1235 35613 F HEM (n=6) 1234 36623
62 Figure 22. Mean arterial pres sure ( MAP ; A) and heart rate ( HR; B) responses to saline (SAL; n = 5) or hydralazine (HYDRAZ; n = 5) infusion. Minute 0 represents baseline. Indicates significantly different (P<0.05) from baseline within treatment group. # Indicates significantly dif ferent (P<0.05) from HYDRAZ treated group at specific time point.
63 Figure 2 3. M ean arterial pressure ( MAP ; A) and heart rate ( HR; B) responses to slow (0.5 ml/kg/min; S HEM; n=7), intermediate (1.0 ml/kg/min; I HEM, n=7), and fast (2.0 ml/kg/min; F HE M, n=6) rates of hemorrhage (HEM). Minute 0 represents baseline. The lowest MAP for each rate of HEM corresponds with the point at which 30% of total blood volume had been removed (i.e. cessation of blood withdrawal). @ Indicates significantly different from baseline for the F HEM group. Indicates significantly different from baseline for the I HEM group. $ Indicates significantly different from baseline for the S HEM group. # Indicates F HEM value was significantly different from both I HEM and S HEM at specified time point. & Indicates S HEM value was significantly different from both I HEM and F HEM at specified time point.
64 Table 22. Mean arterial pressure (MAP) and heart rate (HR) averaged at the time of the offset of hemorrhage (HEM) co mpared to hydralazine ( HYDRAZ ) at the corresponding time point. Indicates significant difference from HYDRAZ average (P<0.01) Treatment Group Time Point (min.) MAP (mmHg) HR (bpm) S HEM (n=7) HYDRAZ (n=5) 40 766 803 29423* 48011 I HEM (n=7) HYDRAZ (n=5) 20 7315 845 27734* 48714 F HEM (n=6) HYDRAZ (n=5) 10 686* 895 30535* 48120
65 Figure 2 4. Representative images of Fos like immunoreactive neurons in the middle and caudal periaqueductal gray (PAG) of two individual animals that underwent either saline injection (SAL) or severe hemorrhage at an intermediate (I HEM) rate. Aq, central aqueduct; DMPAG, dorsal medial PAG; DLPAG, dorsal lateral PAG; LPAG, lateral PAG; VLPAG, ventrolateral PAG. Numbers in the lower left of the ventral sec tions of SAL animal indicate approximate location of representative sections relative to bregma in mm (Paxinos and Watson, 2005)
66 Figure 2 5. Average Fos positive cell counts from the middle and caudle dorsomedial periaqueductal grey ( DMPAG ; A &B) and ventrolateral periaqueductal grey ( VLPAG ; C& D) following different rates of severe hemorrhage (HEM) and hydrazaline (HYDRAZ) induced hypotension. M = middle PAG (defined as 7.5 to 7.8 mm caudal from bregma); C = caudal PAG (defined as 8.0 to 8.4 mm caudal from bregma, Paxinos and Watson, 2005) SAL = saline control (n=5); S HEM = slow rate of 30% hemorrhage (n=7); I HEM = intermediate rate of 30% hemorrhage (n=7); F HEM = fast rate of 30% hemorrhage (n=6); HYDRAZ = hydrazaline treated animals (n=5). Significantly different from SAL.
67 Figure 2 6. Representative images of Fos like immunoreactive neurons in the rostral, middle, and caudal lateral parabrachial nucleus (LPBN) of rats that underwent either saline injection (SAL) or severe hemorrhage at a fast (F HEM) or intermediate (I HEM) rate. Subnuclei of the LPBN outlined include: Sup=superior; Ctr=central; Dor=dorsal; Cres, lateral crescent; Ext, external. SCP is the superior cerebellar peduncle. Numbers in the lower left of SAL animal indic ate approximate location of representative sections relative to bregma in mm (Paxinos and Watson, 2005)
68 Figure 2 7. Average Fos positive cell counts from selected subnuclei the rostral (A) middle (B) and caudle (C) lateral parabrachial nucleus ( LPBN ) following different rates of severe hemorrhage (HEM) and hydral a z ine (HYDRDAZ) induced hypotension. Rostral LPBN was defined as 8.8 to 9.0 mm caudal from bregma. Middle LPBN was defined as 9.05 to 9.3 mm caudal to bregma. Caudal LPBN was defined as 9 .35 to 9.6 mm caudal from bregma (Paxinos and Watson, 2005) SAL = saline control (n=5); S HEM = slow rate of 30% hemorrhage (n=7); I HEM = intermediate rate of 30% hemorrhage (n=7); F HEM = fast rate of 30% hemorrhage (n=6); HYDRAZ = hydrazaline treated animals (n=5). Significantly different from SAL. Horizontal bars indicate that only the group of subnuclei, not individual subnuclei, was significantly different from SAL. Note the difference in y axis scales.
69 Figure 2 8. Fos positive cells quant ified in the Cuneiform Nucleus (A), Locus Coeruleus (B), Kolliker Fuse Nucleus (C), and A7 cell group (D) following different rates of severe hemorrhage and hydrazalineinduced hypotension. SAL = saline control (n=5); S HEM = slow rate of 30% hemorrhage ( n=7); I HEM = intermediate rate of 30% hemorrhage (n=7); F HEM = fast rate of 30% hemorrhage (n=6); HYDRAZ = hydrazaline treated animals (n=5). Indicates significantly different from SAL.
70 CHAPTER 3 DESCENDING PROJECTIO NS FROM PERIAQUEDUCT AL GRAY TO THE LATERAL PARABRACHIAL NUCLEUS ARE NOT ACTIVATED BY SEVERE HEMORRHAGE IN THE CONSCIOUS RAT Introduction The bodys compensatory response to blood loss involves a baroreflex mediated sympathoexcitation which is necessary for maintenance of arterial pressure ( AP) When blood loss is in excess of 2530% of total blood volume (TBV) however, the compensatory response of the body change and a centrally mediated sympathetic withdrawal leads to a precipitous fall in blood pressure and heart rate ( HR) This second phase of hemorrhage ( HEM ) or the hemorrhageinduced sympathoinhibitory (HIS) phase, has been attributed to the activation of cardiac vagal afferents responding to reduced blood volume (Thoren, 1979) as well as activation of specific brain nuclei (Pelaez et al., 2002; Dean and Woyach, 2004; Frithiof et al., 2007) Two brain regions within the rostral brainstem have been identified as critical contributors to HSI: the caudal ventrolateral periaqueductal grey ( vlPAG ) and the lateral parabrach ial nucleus ( LPBN ) Both of these regions have been shown to be activated in response to severe HEM in the conscious rat and blockade or lesion of either of these nuclei markedly alters HSI (Chan and Sawchenko, 1994; Blair et al., 2001; Cavun and Millington, 2001; Jaworski et al., 2002; Keay et al., 2002) While there is a well documented descending projection fr om the vlPAG to the LPBN (Krout et al., 1998) the physiological role of thi s interconnection is unknown. Previous work in our lab has shown that LPBN area neurons play a significant role in mediating descending changes in sympathetic drive originating from the PAG (Hayward et al., 2004) Because the projection of the vl PAG to the LPBN is significantly more dense compared to the projection from the dorsal PAG
71 (Krout et al., 1998) the present study was undertaken to identify whether vlPAG neurons that project to the LPBN are activated in response to severe HEM in the conscious rat. We hypothesized that a greater percentage of vlPAG area neurons projecting to the LPBN would be activated, as indicated by the cellular marker c Fos, compared to the dorsolateral PAG ( dlPAG ) in response to se vere HEM. Methods Male SpragueDawley rats (Harlan Industries, Minneapolis, IN) weighing 355 6 g were utilized for the present study. Rats were pair housed in rooms with a 12hour light dark cycle and allowed standard rat chow and water ad libitum. All animal protocols were in accordance with guidelines established by the University of Floridas Institutional Animal Care and Use Committee. Surgical Instrumentation Three days prior to experimentation, animals were deeply anesthetized with isofluorane mix ed in 100% oxygen (4% 2.5%). Each animal was instrumented with two femoral arterial catheters one in each leg that were tunneled subcutaneously and exteriorized between the scapulae (PE 10 connected to PE 50 tubing, Braintree Scientific, Braintree, MA). Catheters were filled with heparinized saline (100 IU/ml) and sealed with stainless steel obturators (Braintree Scientific, Braintree, MA). Immediately following catheter placement, rats were placed in a stereotaxic head holder (Kopf Instruments) and a small craniotomy directly overlying the left LPBN was performed with a high speed microdrill. A glass micropipette filled with 1% Fluorogold (FG) solution was then lowered into the LPBN using a high speed microdrive (Model 662, Kopf Instruments). The coordinates of the LPBN were as follows: 9.3 mm caudal to bregma, 2.1 mm lateral to midline, and 5.5 mm deep to the dorsal brain surface
7 2 (Paxinos and Watson) (Paxinos and Watson, 2005) Approximately 45 nl of Fluorogold (FG) was pneumatically injected into the left LPBN. The glass pipett e was allowed to remain in place for 25 minutes prior to removal. The skin overlying the craniotomy was sutured closed and topical antibiotics were applied to both the head and leg incisions. During recovery, animals were given subcutaneous injections o f sterile 0.9% NaCl (1 ml, for rehydration) and buprenorphine (0.01 ml/kg, for pain management). Each day following surgical instrumentation, rats were brought to the lab where the experiment would take place, weighed, catheters checked for patency, and allowed to sit quietly in order to acclimate to the testing container in which experiments would be performed. Experimental Proto col On the third day following surgery, animals were brought to the lab, weighed and placed in the testing chamber in a quiet ro om. Animals were then randomly assigned to one of two treatment groups: hemorrhage (HEM) or nonHEM control (CON). One of the arterial catheters was connected to a calibrated pressure transducer inseries with an amplifier (Stoelting, Wooddale, IL) and both pulsatile and mean arterial pressure ( MAP ) were recorded online at 100 Hz using a Cambridge Electronics Design computer interface and Spike2 data software. HR was derived online from the interval between peak systolic pressure waves in the AP trace. The second arterial catheter was connected to additional tubing for blood withdrawal or sham manipulation. Next, baseline AP, MAP, and HR were recorded for a 30minute baseline period. Next, animals assigned to the HEM group underwent a 20minute hemorr hage in which 30% of estimated TBV was removed. TBV was calculated using a previously reported equation for estimation of rat blood volume: (0.06 ml/g)*(body weight in g)+(0.77) (Lee and Blaufox, 1985) CON animals did not undergo any blood removal, but their free
73 catheter was moderately stimulated to simulate catheter movements imposed on HEM animals. Following cessation of blood withdrawal (for HEM) or ca theter stimulation (for CON), animals remained in the testing chamber for 90 min, after which animals were administered a lethal dose of sodium pentobarbital (100150 mg/kg) and transcardially perfused with heparinized saline followed by 4% paraformaldehyde. Brains were removed and post fixed in 4% paraformaldehyde for 24 hours followed by 2448 hours of immersion in cryoprotectant solution (30% sucrose) prior to cryostat sectioning. Immunohis tochemistry and Data Collection Extracted brains were cut into 3 0 micrometer coronal sections and processed for Fos like immunoreactivity ( FLI ) and FG immunoreactivity (FGI). Briefly, free floating sections were washed in sodium phosphate buffered saline (PBS, pH 7.4) followed by a second wash in a 1% goat serum PBSt riton X100 solution (1% GS PBSTX) to prevent nonspecific binding. Sections were then incubated for 24 hours in rabbit anti cFos primary antibody (1:2000 dilution, sc 52r, Santa Cruz Biotechnology) at 4C. Following another wash in 1% GS PBSTX, section s were incubated in goat anti rabbit biotin (Jackson ImmunoResearch Laboratories, Inc., 111065 144) for two hours and rewashed (1% GS PBS TX) prior to being placed in avidinbiotin peroxidase complex (ABC Vectastain Kit, Vector, Burlingame, CA). Sections were put through a final wash (1% GS PBSTX) followed by visualization of the FLI with a chromagen solution (0.05% diaminobenzidine hydrochloride [DAB], 2.5% ammonium sulfate, 0.033% hydrogen peroxide in 0.05 M Tris HCl, and nickel to produce black nuclear stain, Vector). FGI staining was performed on the same tissue slices immediately following FLI staining utilizing the same protocol with rabbit anti FG primary antibody (1:50:000) except the chromagen solution used did not contain nickel (0.05% diamino benzidine hydrochloride
74 [DAB], 2.5% ammonium sulfate, 0.033% hydrogen peroxide in 0.05 M Tris HCl, to produce brown cytosolic staining, Vector). Sections were then mounted onto glass slides, air dried, dehydrated in a graded alcohol and CitriSolv (Fisher Scientific) series, and covserslipped. FG injection sites were identified using light microscopy, imaged, and rostral caudal extent of FG spread was recorded. For each animal, two representative sections from the caudal vlPAG (ipsilateral and contralateral to the FG injection site) were imaged (Axioskop, Carl Zeiss) and analyzed for the number of FLI positive, FGIpositive, and FLI+FGI co labeled neurons present by a technician blinded to the experimental conditions (Figure 31). A FLI positive cell was identified with black nuclear staining; a FGI positive cell was identified with brown cytosolic staining with dendritic/axonal processes present; and co labeling was identified as cells containing both FLI and FGI staining (Figure 31C). Other regions anal yzed include the dorsomedial PAG ( dmPAG ) and dlPAG (Figure 31A). Because the dmPAG is a midline nucleus, it was not analyzed bilaterally (ipsi vs. contralateral). Data Analysis MAP and HR were averaged over oneminute intervals, at times 5, 0, 5, 10, 1 5, 20, 25, 30 and 60 minutes relative to the onset of the treatment (HEM or CON). A twoway analysis of variance ( ANOVA) with repeated measures was used to identify main effects of treatment (i.e. HEM vs. C ON) on MAP and HR across time. Immunohistochemic al data were analyzed using a oneway ANOVA comparing the effect of treatment (i.e. HEM or CON) on cellular staining levels within the caudal vlPAG, dmPAG, and dlPAG. When indicated, Student Newman Keuls multiple
75 comparison analyses were used to isolate differences within or between treatment groups. All data are presented as mean SEM. Results Brain Injection Sites FG injections were centered in what we have previously defined as the middle LPBN (Ahlgren et al., 2007) (Bregma 9.16 mm) for all animals with the exception of one HEM animal whose injection site was centered more in the caudal LPBN (Bregma 9.30 mm; Fig 1C). Figure 32A shows a typical rostrocaudal dis tribution of injected FG. Reconstruction of the injection site identified that a 45 nl bolus of FG spread less than 0.5 mm rostral and .75 mm caudal from the injection site center for all brains. Heart R ate and Mean Arterial P ressure in H emorrhage Versus C ontrol Baseline HR and MAP were not different between treatments (HEM HR: 379 9 bpm; CON HR: 383 15 bpm; HEM MAP: 120 4 mmHg; CON MAP: 115 4 mmHg). CON animals did not display any deviation of HR or MAP from baseline at any time during the experi ment. Animals that underwent HEM displayed a significant rise in both MAP and HR at 5 minutes following the onset of HEM compared to CON. At 10 min, however, there was no significant difference between groups. Following 10 min. of HEM or blood loss gr eater than 15% both HR and MAP dropped significantly below both baseline and CON values (Figure 33). HR reached its lowest point (261 9 bpm) at minute 20 of HEM the very end of blood withdrawal. HR in the HEM group remained significantly lower than CO N animals and baseline measures for the remainder of the experiment. MAP reached its lowest (62 6 mmHg) in the HEM group at minute 15 when approximately 23% of TBV had been removed and was significantly different from CON at minutes 15 and 20. Following the offset of HEM, MAP in the HEM group
76 remained significantly lower than the CON animals through minute 25 (5 minutes post HEM offset) and was statistically less than HEM baseline for the remainder of the experiment. Neural Activation/CoLabeled Neurons In both HEM and CON animals, a greater abundance of LPBN projecting neurons in the left vlPAG (ipsilateral to the FG injection) relative to the right vlPAG were identified (Figure 34). Thus, while the vlPAG does project bilaterally to the LPBN, there is a much denser ipsilateral projection (approximately 72% greater). This observation is in agreement with previous work (Krout et al., 1998) Similar to reports from our lab and others (Ahlgren et al., 2007; Vagg et al., 2008) HEM animals displayed a significantly greater amount of Fos positive neurons within the vlPAG compared to CON animals. This relat ionship was true both ipsi and contralateral to the FG injection site (Figure 34). However, only a small number of FLI+FGI colabeled neurons were identified in the vlPAG (either in the ipsi or contralateral vlPAG) following HEM (Figure 34A). For co mparison, the dorsomedial and dorsolateral subnuclei of the caudal PAG were also evaluated for FLI, FGI, and FLI+FGI colabeled neurons (Figures 34B & C). Similar to the vlPAG, the dorsal PAG showed heavier ipsilateral versus contralateral projections to the LPBN; however, both subnuclei of the dorsal PAG showed noticeably fewer neurons with projections to the LPBN compared to the vlPAG (roughly 50% less). Similar patterns were seen for FLI immunoreactivity and FLI+FGI colabeling: no significant difference in the amount of FLI seen between ipsi and contralateral dlPAG; substantially less FOS positive neurons in both the dm and dlPAG compared to vlPAG; and little to no colabeled neurons in either dorsal PAG subnucleus.
77 Discussion Anatomically, the LPBN i s ideally positioned to act as a relay center for descending projections from the vlPAG to the hindbrain (Blair et al., 2001) T his study investigated potential physiological stimuli that might activate descending projections from the vlPAG to the LPBN. In accordance with other neuroanatomical studies (Krout et al., 1998) we identified a large number of retrogradely labeled neurons throughout the PAG following FG microinjections into the LPBN. Furthermore, we confirmed previous findings from both our laboratory (Ahlgren et al., 2007) as well as others (Vagg et al., 2008) that severe HEM induces a significant increase in FLI label ed vlPAG neurons compared to control animals. Quantification of FLI FGI colabeled neurons in vlPAG however identified that relatively few FGI labeled neurons also stained for FLI following severe HEM. Moreover, there were equal amounts of both FLI FGI l abeled neurons in the vlPAG of HEM and CON animals. Taken together, these data do not support the proposed hypothesis that vlPAG activation of the LPBN is important in relay to the hindbrain mediating autonomic or other responses during severe conscious h emorrhage in the rat. Consequently, the stimulus responsible for activating this relatively dense projection, between these two important autonomic nuclei, remains to be identified. It is well documented that the vlPAG plays a significant role in blood pressure regulation during hemorrhage (Ward and Darlington, 1987; Evans et al., 1991; Cavun and Millington, 2001; Tr oy et al., 2003) At present, the cardiovascular and behavioral adjustments associated with vlPAG excitation have been shown to be mediated, in part, through descending activation of neurons in the RVMM (Lovick, 1993; Schenberg and Lovick, 1995; Hermann et al., 1997; Odeh and Antal, 2001) Furthermore, the release
78 of serotonin from this region of the hindbrain has been shown to participate in the blood pressure and heart rate responses to HEM (Scrogin et al., 1998; Henderson et al., 2000; Scrogin et al., 2000; Dean and Bago, 2002; Pelaez et al., 2002; Scrogin, 2003) The LPBN is situated dorsolaterally in the caudal mesencephalon/rostral pons, immediately lateral to the brachium conjunctivum (Paxinos and Watson, 2005) In addition to being reciprocally connected to both the dorsal and vlPAG, the LPBN has been shown to be interconnected with a number of other central nuclei involved in mediating autonomic control including the nucleus tractus solitarius ( NTS ) the amygdala, the hypothalamus, the rostral ventrolateral medulla ( RVLM ) and the rostral ventromedial medulla ( RVMM; Fulwiler and Saper, 1984; Herbert et al., 1990) Furthermore, LPBN connections with the RVMM have been established (Henderson et al., 1998; Heslop et al., 200 4; Vagg et al., 2008) Based on these studies and others showing the importance of the LPBN in modulating descending sympathetic drive to the heart from the dlPAG (Hayward et al., 2004) we hypothesized that vlPAG activation of lower brainstem nuclei may be mediated through a polysynaptic pathway involving the LPBN. The results of the present study howev er support more recent findings by Vagg et. al. (Vagg et al., 2008) that vlPAG activation of ventral medullary regions arise from a direct descending projection from the vlPAG to the RVMM. In the present study we also quantified the response of descending projections from the dorsal PAG to the LPBN. Activation of the dorsal PAG induces an increase in MAP, HR and respiratory rate and these changes are known to mediated, in part, through act ivation of LPBN area neurons (Hayward et al., 2004) However, to date there is evidence that dorsal PA G neurons do not respond to changes in venous return
79 during simulated HEM in conscious rabbits (Schadt et al., 2006) Furthermore, chemical blockade of the dorsal PAG does not alter the time course of HSI (Cavun and Millington, 2001) In a previous study however we did identif y a small but significant increase in FLI labeling in the dorsal PAG in response to severe HEM (Ahlgren et al., 2007) Similarly, in the present study, we also identified a significant increase in the number of FLI positive cells in the dlPAG following hemorrhage compared to CON. Yet, we did not identify a significant number of FLI labeled neurons with projections to the LPBN. This observation supports previous work sug gesting the dorsal PAG neurons probably play little role in mediating cardiovascular responses to HEM. In summary, the present study was undertaken to test the hypothesis that descending projections from the vlPAG to the LPBN are activated in response t o severe HEM and potentially play a role in mediating HSI. The results of this study confirmed previous observations that a large number of vlPAG neurons send projections to the LPBN. However, our results suggest that this pathway is not activated in res ponse to severe hemorrhage and therefore must be involved in mediating other types of responses associated with activation of the vlPAG (Wang and Lovick, 1993; Snowball et al., 2000; Lumb, 2004)
80 Figure 31. Periaqueductal grey ( PAG ) area analyzed and examples of immunohistochemi cal staining. (A) Adapted image of the specific midbrain level selected for Fos like and Fluorogold immunoreactivity ( FLI FGI, respectively) quantification within the caudal PAG. Number in the bottom right hand corner represents the distance in mm caudal to Bregma. Image adjusted from Paxinos and Watson, 2005. (B) Light microscope image (1.25x) of the PAG. (C) Light microscope image (20x) of FLI positive (black arrows), FGI positive (white arrows), and FLI FGI colabeled (red arrows) neurons in the cau dal vlPAG. DMPAG = dorsomedial PAG, DLPAG = dorsolateral PAG, VLPAG = ventrolateral PAG, Aq = cerebral aqueduct.
81 Figure 32. Fluorogold (FG) injection sites. (A) Reconstruction of the rostrocaudal spread of a typical 45 nl FG injection. (B) Reconst ruction of locations of focal point of all injection sites (red dots). For illustration purposes, injection sites recovered from control ( CON) animals are shown on the left and hemorrhage ( HEM ) animals on the right. (C) Light microscope image of an actual injection site. Numbers beside reconstructed brain sections indicate distance in mm caudal to Bregma. Images adjusted from Paxinos and Watson 2005)
82 Figure 33. Heart rate (HR; A) and mean arterial pressure (MAP; B) responses to 30% total blood volume removal over 20 minutes (HEM) or no treatment (CON). indicates significant difference from CON at indicated time point (P < 0.05). # indicates significant difference from baseline within group (P < 0.05).
83 Figure 34. Quantification of immunohi stochemical staining in the caudal periaqueductal grey ( PAG ) Fluorogold immunoreactivity ( FGI ) positive, Fos like immunoreativity ( FLI ) positive, and FLI FGI colabeled neurons were counted in the ventrolateral PAG ( vlPAG ; A) and dorsolateral PAG ( dlPAG ; B) ipsilateral (IPSI) and contralateral (CONTRA) to the lateral parabrachial nucleus ( LPBN ) injection site. The dorsomedial PAG ( dmPAG ; C) was counted as a single nucleus because it is a midline structure. indicates significant difference from control ( CON; P < 0.05).
84 CHAPTER 4 VOLUNTARY WHEEL RUNNING ALTERS THE AUTONOMIC RESPONSE TO HEMORRHAGE IN CONSCI OUS MALE RATS Introduction The hemodynamic response to hemorrhage ( HEM ) follows a tri phasic pattern. At the onset of HEM, the initial loss of blood v olume, and the associated deviation in arterial pressure ( AP) is sensed by the arterial baroreceptors which stimulate a reflex increase in heart rate ( HR) and sympathetic drive to the vasculature to maintain a normotensive state. This first compensatory phase of HEM is typically sustained until blood loss reaches ~1530% of TBV (Schadt and Ludbrook, 1991) When blood loss exceeds this critical value, the bodys initial compensatory response quickly transitions into a decompensatory or sympathoinhibitory phase w hich triggers a sudden decline in both HR and AP (Schadt and Ludbrook, 1991; Hasser and Schadt, 1992; Evans et al., 2001) These changes in autonomic drive trigger a HEM induced hypotension that is paralleled by increasing plasma levels of renin, vasopressin, and epinephrine (Schadt and Ludbrook, 1991) These neurohumoral factors aid in the third phase of HEM or the recovery phase, which follows the offset of blood loss. During the recovery phase, sympathetic tone is restored to the vasculature (Scrogin, 2003) The longer the body is exposed to reduced perfusion pressures during the sympathoinhibitory phase, the chance of recov ery is decreased (Kauvar et al., 2006) Thus, interventions that can modulate central or peripheral mechanisms activated during HEM to delay the onset and limit the magnitude of the sympathoinhibitory phase and/or facilitate the recovery phase would be beneficial to survival outcomes. Exercise has been shown to ameliorate a host of cardiovascular pathologies including hypertension (Grassi et al., 1992; Sutoo and Akiyama, 2003) heart failure
85 (Coats et al., 1992; Bensimhon et al., 2007) and a number of other diseases in which there is marked autonomic dysregulation (Warburton et al., 2006; Souza et al., 2007; Felber Diet rich et al., 2008) While the peripheral effects of exercise are well documented (Blomqvist, 1983; Long et al., 2004; Bensimhon et al., 2007) and constitute a large portion of ex ercise research, recent work has begun identifying centrally mediated adaptations that also contribute to enhanced health outcomes associated with chronic exercise (Zhu et al., 2004; Nelson et al., 2005; Mueller and Hasser, 2006; Bakos et al., 2007; Kleiber et al., 2008) In particular, recent data suggest that some of the benefits of exercise may be mediated through increased inhibition of central sympathoexcitatory circuits (Mueller, 2007) Current evidence suggests that chronic exercise can affect many areas of the brain involved in autonomic control of AP and HR (Zhu et al., 2004; Nelson et al., 2005; Mueller and Hasser, 2006; Bakos et al., 2007; Mueller, 2007; Kleiber et al., 2008) Interestingly, some of these same central sites have been identified to be involved in mediating autonomic changes during the different phases of severe HEM (Ward and Darlington, 1987; Krukoff et al., 1995; Krukoff et al., 1997; Chan and Sawchenko, 1998; Jhamandas et al., 1998; Buller et al., 1999; Kakiya et al., 2000; Pelaez et al., 2002) This raises the possibility that exercise training may impact the bodys ability to withstand severe hemorrhage via a change in neuronal responsiveness of specific brain nuclei known to play a role in HEM. The present study was undertaken to test the hypothesis that wheel exercised rats would display altered autonomic responses to severe HEM and they would be better able to tolerate a significant loss of blood compared to sedentary rats. Specifically, we hypothesized that chronic voluntary
86 exercise would result in an enhanced compensatory response (Brum et al., 2000) and/or an attenuated decompensatory phase in response to severe HEM Accordingly, we also hypothesized that voluntary exercise t raining would alter the pattern of neuronal activation (as marked by c Fos immunoreactivity) in rostral brainstem regions identified to be involved in autonomic control during HEM including the Locus Coeruleus ( LC ; compensation), ventrolateral periaqueduct al grey ( vlPAG ; d ecompensation) and the lateral parabrachial nucleus ( LPBN ; recovery) Methods General Preparation All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Male SpragueDawl ey rats (175200 g, Harlan Industries, Minneapolis, IN) were randomly placed into one of two groups: exercise (EX) or sedentary (SED). Following a preliminary study on singlehoused EX rats, all subsequent animals were pair housed for six weeks in cages t hat did (EX) or did not (SED) contain a running wheel. Animals were lightly handled and weighed weekly and were maintained in a 12 hour lights on: 12 hour lights off, temperature controlled environment with food and water ad libitum. Following six weeks of training, each animal was surgically instrumented with bilateral femoral arterial catheters (PE 10 connected to PE 50 tubing, Braintree Scientific, Braintree, MA) under isofluorane anesthesia (4% 2.5%). Catheters were subcutaneously routed and exteriorized between the scapulae, filled with heparinized saline (100 IU/ml), and plugged with stainless steel obturators (23gauge, Braintree Scientific, Braintree, MA). Analgesics (Rimadyl, 0.01 ml/kg; Buprenorphine, 0.01 ml/kg) were administered subc utaneously following catheterization and animals were allowed
87 48 hours to recover. During recovery, animals were housed singly. The day following catheter placement, animals were brought to the lab to ensure catheter patency and for acclimation to the testing chamber. Animals were weighed, lightly handled, and allowed to sit quietly for 2 3 hours in the testing chamber (9x9 inch bucket) to be used on the day of the experiment. Animals were then returned to their home cages for another 24 hours of recovery. Experimental Protocol On the day of the experiment, animals were brought to the lab, weighed, and both arterial catheters were connected to additional heparinized salinefilled tubing (1050 IU/ml; PE 50). The animal was then placed in the testing chamber and the catheters were routed through a hole in the lid of the testing chamber in such a way that the animal could move freely within the testing chamber but not excessively twist the catheters. One of the arterial catheters was connected to a calibr ated pressure transducer inseries with an amplifier (Stoelting, Wooddale, IL). Both pulsatile and mean arterial pressure ( MAP ) were recorded online at 100 Hz using a Cambridge Electronics Design computer interface and Spike2 data software. HR was deriv ed online from the interval between peak systolic pressure waves in the AP trace. AP, MAP, and HR were collected for 3060 minutes during which the animal was undisturbed in order to ensure a stable baseline measurement. Animals were randomly assigned to one of two groups: hemorrhage (HEM) or no treatment (time control; CON). Animals assigned to the HEM group were then subjected to a 30% TBV HEM over 15 minutes (EX HEM, SED HEM, n=8 per group) followed by 90 minutes of recovery. Animals assigned to the CON group were allowed to sit quietly for 105 minutes (EX CON, n=5; SED CON, n=4). For the HEM groups TBV was estimated using a
88 previously reported equation: (0.06 ml/g)*(body weight in g)+(0.77) (Lee and Blaufox, 1985) Ninety minutes following HEM offset or after 105 minutes of quiet sitting in the CON groups, animals were administered an overdose of sodium pentobarbital (100150 mg/kg) and transcardially perfused with heparinized saline followed by 4% paraformaldehyde. Brains were removed and post fixed in 4% paraformaldehyde for 24 hours followed by 2448 hours of immersion in cryoprotectant solution (30% sucrose) prior to cryostat sectioning. Fos Immunocytochemistry Extracted brains were cut into 30micrometer coronal sections and processed for Fos like immunoreactiviyt ( FLI ) as previously described (Hayward and Von Reitzenstein, 2002) Briefly, free floating sections were washed in sodium phosphate buffered saline (PBS, pH 7.4) followed by a second wash in a 3% goat serum PBS triton X100 solution (3% GS PBSTX) to prevent nonspecific binding. Sections were then incubated for 24 hours in rabbit anti cFos primary antibody (1:2000 dilution, sc 52r, Santa Cruz Biotechnology) at 4C. Following another wash in 1% G S PBSTX, sections were incubated in goat anti rabbit biotin (Jackson ImmunoResearch Laboratories, Inc., 111 065144) for two hours and rewashed (1% GS PBSTX) prior to being placed in avidinbiotin peroxidase complex (ABC Vectastain Kit, Vector, Burlinga me, CA). Sections were put through a final 1% GS PBSTX wash followed by visualization of the FLI with a chromagen solution (0.05% diaminobenzidine hydrochloride [DAB], 2.5% ammonium sulfate, 0.033% hydrogen peroxide in 0.05 M Tris HCl, Vector). Sections were then mounted onto glass slides, air dried, dehydrated in a graded alcohol and CitriSolv (Fisher Scientific) series, and coverslipped.
89 Neuroanatomical Identific ation and Quantification of Fos Like I mmunoreactivity For each animal, two representative s ections from each brain area of interest were imaged (Axioskop, Carl Zeiss; 540X) and analyzed for the number of FLI neurons present by a technician blinded to experimental conditions. A stereotaxic rat brain atlas was referenced for identification of al l areas imaged and quantified (Paxinos and Watson, 2005) Rostral, middle, and caudal aspects of the LPBN were imaged at approximately 9mm, 9.16mm, and 9.3mm caudal to bregma, respectively. Criteria used for selecting specific sections of the LPBN included the shape of the superior cerebel lar peduncle ( SCP ) the width of the LPBN from the SCP to the ventral spinocerebellar column, and the width of the ventral spinocerebellar tract. Caudal vlPAG sections were imaged at approximately 8mm caudal to bregma. Other areas imaged and quantified f or FLI included the Kolliker Fuse ( KF ; imaged in the same section and at the level of the rostral LPBN), LC (rostral: 9.39.68mm caudal to bregma; caudal: 9.8mm caudal to bregma), and the main body of the paraventricular nucleus of the hypothalamus (PVN; 1 .8mm caudal to bregma). Cardiovascular Measurements MAP and HR values averaged over 60second intervals beginning one minute prior to, and ending 30 minutes following the onset of HEM, as well as at 60 minutes following the onset of HEM were used for deter mining group averages. For nonHEM control experiments, MAP and HR values averaged over 60second intervals for a time period that corresponded to the minute prior to blood withdrawal (baseline), 15 minutes of blood withdrawal, and 90 minutes of recovery in HEM experiments were used for determining group averages.
90 Changes in the autonomic control of HR during HEM was also evaluated using heart rate variability (HRV) analysis (Porter et al., 2009) HRV was analyzed using 45 minute time segments taken from three different time points during HEM. The three segments chosen for HRV analys is included: baseline, (within 10 minutes prior to the onset of HEM); peak (the 45 minute interval just preceding the drop in AP and HR associated with hemorrhagic sympathoinhibition); and nadir (the last 45 minutes immediately preceding the offset of HE M). Within each segment, HR was converted into a tachogram --a record of the time interval between heart beats (RRI). Next, the filtered tachogram was analyzed in the frequency domain using HRV software (Biosignal Analysis Group; University of Kuopio, Finland) (Niskanen et al., 2004) In t he software used, the tachogram was interpolated at 10 Hz and detrended via the smoothness priors formulation (alpha=1000) (Tarvainen et al., 2002; Niskanen et al., 2004) The autoregressive model was set to the 40th order. The Welchs Periodogram window width was designated to 512 points with an overlap of 256 points in the Hanning window. In the rat, the frequency components of HRV are designated by the following frequency ranges: 0.16 0.6 Hz (Low Frequency, LF), and 0.63. 0 Hz (High Frequency, HF) (Japundzic et al., 1990) Frequency domain characteristics analyzed included the power of the LF and HF components, the ratio of LF/HF power, and the freq uency associated with the HF peak. Statistical Analysis Within treatment groups, data were averaged and reported as the mean SEM. A oneway analysis of variance ( ANOVA) was used to determine if there were any significant differences in baseline MAP or H R between treatment groups. A twoway ANOVA with repeated measures was used to identify the effects of experimental
91 treatment (HEM) and group (EX vs. SED) on MAP and HR across time (every minute: 1 through 30, and 60). When indicated, Student NewmanKue ls post hoc analysis or multiple paired t tests with a Bonferonni adjustment for number of comparisons were used to isolate differences relative to baseline (minute 1) within treatment groups or between treatment groups at specific time points. HRV data were also analyzed by a two way ANOVA with repeated measures comparing treatment groups against the designated time points. When indicated, a oneway ANOVA with a Scheffe Post Hoc or multiple paired t tests with a Bonferonni adjustment for the number of co mparisons (time points within groups) was utilized to determine significance. FLI data from all brain regions evaluated were analyzed using a oneway ANOVA to compare differences between experimental groups (EX vs. SED) within and between treatments (HEM vs. CON). Differences were considered significant when P<0.05 for all statistical analyses performed. Results Exercise Training Effect A preliminary study was performed in a small group (n=4) of singlehoused animals to compare average daily running distances and weight gain of singleversus pair housed (n=13) animals (Figure 41). Two way ANOVA with repeated measures comparison of average daily distances run by singlehoused rats versus estimated daily distances run by pair housed rats (Figure 41A) show ed neither a treatment effect (P=0.99) nor an interaction (P=0.12). Furthermore, the weight gained over six weeks of voluntary running was also not significantly different between pair and singlehoused EX animals (P=0.5; see Figure 41B). Based on the similarities between groups, pair housing was chosen as the preferred method of housing for all experiments described
92 below to eliminate the potential confounding stress of solitary housing in these social animals. Figure 41B shows the average weight gain over six weeks for all pair housed animals. Twoway ANOVA with repeated measures demonstrated no significant difference in body weight between EX versus SED animals before wheel running began (1937g versus 189.14g, respectively; P=0.43). From week t hree through the end of the training period, however, the EX rats weighed significantly less compared to their SED counterparts. At week six, EX animals displayed ~60% increase in body weight compared to ~88% gain in SED rats. In addition to weighing les s, there was subjective evidence of less body fat in EX compared to SED rats (observation made by the technician during surgical instrumentation). Effect of E xercise on the Hemodynamic R esponse to H emorrhage Despite significant body weight differences, there was no evidence of a training induced bradycardia at rest in the EX animals compared to the SED group (Figure 42A). After the onset of HEM, HR increased in both EX and SED groups. In the SED animals, between minutes seven through ten during HEM, the i ncrease in HR was significantly different from baseline. In contrast, the increase in HR in the EX group was only significantly different from baseline at minutes eight and nine during HEM. In both groups, a noticeable decline in HR began around minute eleven and within a minute of HEM offset (minute 15), HR reached its lowest value in both groups. The absolute drop in HR from baseline at the offset of HEM was greatest in the SED group. The mean HR at minute 15 in the SED group was statistically lower than baseline and remained statistically below baseline through minute 19. Conversely, while EX animals did display a drop in HR during the latter half of blood withdrawal, at no time point during or
93 following the offset of HEM did HR fall statistically below baseline. At minute 16 (one minute following the offset of HEM), the average HR in EX animals was 50 bpm higher than that observed in the SED animals (32526 vs. 27518 bpm, respectively; P=0.04). SED animals displayed a significantly lower HR compared to EX animals from the offset of HEM through minute 30. At 60 minutes following the onset of HEM, HR for SED animals (3275 bpm) was not significantly different from baseline (3305 bpm). In contrast, at minute 60, HR in the EX group (368bpm) was elev ated compared to baseline (3399 bpm). This slight tachycardia failed to reach statistical significance following the Bonferonni correction (P=0.045). At this time point, however, HR in the EX animals (368bpm) was significantly greater than HR in the S ED animals (3275 bpm, P=0.02). Figure 42B shows the corresponding changes in MAP response to HEM in EX and SED animals. Similar to the HR results, prior to the onset of HEM, MAP was not significantly different between groups (SED: 1234, EX: 1203; P=0. 38). Moreover, from the beginning of blood withdrawal, both EX and SED groups maintained MAP through minute nine, after which both groups displayed a drop in MAP until the offset of HEM. MAP for SED animals fell significantly below baseline starting at minute 13 and this HEM induced hypotension lasted through minute 30 and at minute 60, MAP (1052 mmHg) remained significantly below baseline (P=0.003). For EX animals, however, MAP was not significantly different from baseline at any time point and at mi nute 60, values for MAP in the EX group (1104 mmHg) were not different from baseline (1203mmHg, P=0.14). Both groups displayed the lowest MAP values immediately at the offset of HEM (minute 15). At this time point, MAP in EX animals was significantly
94 g reater than MAP in the SED rats (93.76 mmHg compared to 58.55 mmHg, respectively; P=0.0002). Overall, EX animals displayed an attenuated drop in MAP in response to HEM compared to SED animals and MAP in the EX group was significantly higher than the SED group from minutes 14 to 25. Heart Rate V ariability D uring H emorrhage Figure 43A illustrates the typical HRV frequency profile of an EX rat at rest. In both groups of animals, the area under the curve or power of the HF component was greater than the pow er of the LF curve at baseline. As a consequence, prior to the onset of HEM, the LF/HF ratio was less than 1.0 for all animals (Figure 43B, Base). During the baseline period, there was no significant difference between the EX and SED animals in the LF/HF ratio (Figure 43B), the HF power (Figure 43C), or peak frequency of the HF curve (Figure 43D). Analysis of HRV during the 45 minute period prior to the onse t of the drop in MAP during HEM indicate d a significant increase in the LF/HF ratio (Figure 43 B, Peak) relative to baseline in both EX and SED animals. There was no significant change in HF in either group at the same time point. Analysis of HRV during the 45 minute period just prior to the offset of HEM (Figure 4 3B, Nadir) demonstrated that the LF/HF ratio remained elevated relative to baseline when both EX and SED groups were combined (P<0.003). HF power at the nadir was also elevated relative to baseline (Figure 43C) but was significantly different from baseline only when both groups (EX and SED) were combined (P<0.016). Finally, analysis of the frequency of the HF peak --which can be indicative of respiratory rate (Yang and Kuo, 1999) --identified a significant shift to a lower peak frequency during the nadir time point compared to baseline when the SED and EX groups were combined
95 (P=0.01; Figure 43D). Additionally, the HF peak in the SED group was identified to be located at a significantly (P<.0.006) higher frequency (1.40.08 Hz) compared to the EX group (1.160.06 Hz) when all time points were combined. Effect o f E xercise on the Fos Like I mmunoreactivity R esponse to H emorrhage To further evaluate the impact of exercise on autonomic control during HEM, three regions of the rostral brainstem and the PVN wer e evaluated for changes in FLI following HEM in EX versus SED groups. Two groups of control animals were added for this analysis: SED CON (n=4) and EX CON (n=5). These animals underwent the same treatments as their corresponding experimental groups but did not undergo the HEM protocol. At no point during the control experiments did HR and MAP values deviate from baseline for either SED CON (3523 bpm and 1275 mmHg) or EX CON (35511 bpm and 1325 mmHg) animals. Figure 44 illustrates the typical FLI pattern observed throughout the LPBN from a SED and an EX animal following HEM. In general, HEM induced a greater amount of FLI in the SED versus EX animals. Based on previous work in our lab on FLI in the LPBN following HEM (Ahlgren et al., 2007) the LPBN was divided into three rostrocaudal divisions (rostral, middle, and caudal). Figure 45 shows the average FLI for the subnuclei of the LPBN evaluated. In the rostral division (Figure 45A), four subnuclei were evaluated for changes in FLI: superior, central, external and KF. Following HEM, a difference in FLI compared to CON within groups (SED and EX) was identified in two of the four subnuclei. In the central subnucleus, there was a significant increase in FLI in the EX HEM compared to EX CON. There was also a trend for FLI to increase in the SED HEM animals versus SED CON animals in the central subnucleus, but this comparison failed to reach statistical significance following the Bonferonni adjustment
96 (P=0.05). In the external subnucleus, SED HEM, but not EX HEM, animals displayed a significant increase in FLI compared to SED CON. Comparisons of SED HEM versus EX HEM and SED CON versus EX CON did not reveal signi ficant differences in the subnuclei of the rostral LPBN. In the middle LPBN, four separate subnuclei were evaluated: dorsal, central, external, and crescent subnuclei (see Figure 45B). In both the dorsal and external subnuclei, SED HEM animals had a si gnificantly greater number of FLI positive cells compared to SED CON. EX HEM animals also had greater FLI compared to EX CON in both of these subnuclei; however, statistical comparisons failed to reach significance following Bonferonni correction (P=0.06 and P=0.02, respectively). FLI in the external subnucleus of the SED HEM was significantly greater than FLI in the external subnucleus of the EX HEM animals. There were no significant differences in FLI between groups in either the central and crescent s ubnuclei. The same four subnuclei evaluated in the middle LPBN were evaluated in the caudal LPBN. Figure 45C shows that --similar to what was seen in the middle LPBN -only SED HEM animals had significantly greater FLI compared to controls in the dor sal and external subnuclei alone. Additionally, EX HEM animals had significantly less FLI compared to SED HEM animals in the external subnucleus. The average FLI counted in the caudal VLPAG, LC, and the PVN is shown in Figure 46. In the caudal vlPAG (Figure 4 6A) both SED HEM and EX HEM animals showed significant increases in FLI compared to controls, but, there was no difference between FLI in SED HEM versus EX HEM animals (P=0.64).
97 The LC was divided into rostral and caudal regions based on previous work showing functional differences along the rostrocaudal extent of this nucleus (Figure 46B) (Bajic et al., 2000) In the rostral LC, only SED animals displayed increased FLI following HEM compared to controls; however, there was not a difference in the amount of FLI present in SED HEM versus EX HEM animals (P=0.15). In the caudal LC, both SED HEM and EX HEM animals displayed significantly greater FLI compared to controls. A comparison between FLI in SED HEM and EX HEM animals, however, failed to reveal any significant differences (P=0.22). Finally, the PVN was evaluated in three parts: the magnocellular, parvocellular, and dorsal cap regions. Shown in Figure 46C, both SED HEM and EX HEM animals displayed a significant increase in FLI in all PVN subnuclei assessed compared to controls. In the dorsal cap region alone, however, there was a significant difference bet ween SED HEM and EX HEM animals with EX HEM animals displaying an attenuated increase in FLI compared to SED HEM animals (P=0.01). Discussion This study evaluate d the effect of voluntary exercise training on the cardiovascular and neural responses to 30% T BV HEM in conscious rats. The EX animals in this study did not train at a high enough intensity to induce a resting bradycardiaan effect typically seen in more rigorously trained individuals. Nonetheless, following six weeks of voluntary wheel running, the EX rats displayed an attenuated cardiovascular decline during severe blood loss. These results suggest that even modest daily activity may help attenuate cardiovascular consequences associated with hypovolemic shock and thus potentially improve surv ival outcome in trauma situations.
98 During HEM, the initial response or compensatory phase is primarily mediated by baroreflex adjustments in sympathetic and parasympathetic drive, and there is evidence that exercise training can enhance baroreflex functi on (Brum et al., 2000) Based on these observations, we initially hypothesized that EX animals would display an enhanced compensatory response to blood loss compared to SED animals. This, however, was only partially confirmed. During the initial period of blood withdrawal, both EX and SED rats displayed the typical compensatory increase in HR and maintenance of MAP. Yet, although the EX animals did produce a slightly greater tachycardic compensatory response compared to SED animals, these differences were not statistically significant. HRV analysis, however, did identify a significant increase the LF/HF ratio at the peak time point in the EX group, potentially indicative of elevated sympathetic activity. The SED animals also showed an increase in LF/HF ratio at the peak time point, but the increase was not significantly different from baseline. Conversely, the increase in HR in EX animals appeared to reach a peak value earl ier (at minute 8) and then began declining slightly sooner compared to SED animals (albeit at a slower rate). At the time of transition from compensation to a decompensatory or sympathoinhibitory response, MAP was not significantly different between the t wo groups. These results suggest that modest exercise training may augment the peak compensatory response to hemorrhage but the ability to sustain a peak response may be blunted compared to SED animals. Baroreflex mediated compensatory responses to HEM involve a decrease in baroreceptor afferent input into the brain. The primary central termination site of baroreceptor afferents is in the dorsal medulla, in the NTS. Baroreceptor related
99 information integrated within the NTS is then relayed to other ce ntral nervous system ( CNS ) nuclei which either directly or indirectly activate sympathetic preganglionic neurons. Within the brainstem, the pontine noradrenergic cell group A6, LC, has been shown to play a pressor role in response to blood withdrawal (AnselmoFranci et al., 1998) AnselmoFranci et. al. (1998) showed that lesioning the caudal, but not the rostral, component of the LC, resulted in a greater drop in MAP in response to 20% TBV HEM compared to sham lesioned animals. The same study also showed an enhanced cFos expression following HEM in the caudal versus rostral LC (Anselmo Franci et al., 1998) In the present study, c Fos expression in the LC was also evaluated. Consistent with other investigators (Anselmo Franci et al., 1998) we did observe a greater increase in c Fos in the caudal versus rostral LC following HEM compared to CON groups. However, there was no difference in FLI following HEM between EX and SED animals. Thus, the modest effect we observed in the compensatory response following exercise training (as evidenced by the increase in the LF/HF ratio and the slightly higher HR) was not sufficient to be detected by evaluating c Fo s expression in the LC. Since a change in the reflex response of HR and MAP were seen with HEM in EX vs. SED animals, we can conclude that some central neural system was modulated by the voluntary exercise. Quantification of HEM activated neurons (FLI) did not, however, identify this region as one modified by exercise. Furthermore, peripheral changes in hormone release, potentially from the adrenal gland, which in turn may augment compensatory responses (Erdem et al., 2002; Fediuc et al., 2006) should be evaluated in future studies as possibly contributing to the protective effect of exercise training in situations of hypovolemia.
100 At the point during HEM corresponding to approximately 20% TBV loss, both EX and SED animals began a sympathoinhibitory or decompensatory response, including bradycardia and declining MAP. For SED animals, MAP dropped below baseline levels at minute 13 and both MAP and HR reached levels that were significantly lower than baseline at the time of 30% TBV withdrawal. In contrast, although MAP began to decline at the same time point, in the EX group MAP and HR did not significantly deviate from basal values at any time point during or after the onset of decompensatory phase (minute 10). Moreover, from minute 1415 dur ing HEM and for 15 minutes following the offset of HEM, MAP and HR were elevated above the SED animals. Multiple studies have implicated the vlPAG in the sympathoinhibitory phase that occurs following severe blood loss (Cavun and Millington, 2001; Cavun et al., 2004; Dea n, 2004; Schadt et al., 2006) Cavun and Millington (2001) showed a significant attenuation of HEM induced hypotension by blocking neural activity within the caudal vlPAG and it has been suggested that the descending innervation of brainstem depressor regions, such as the caudal midline medulla (CMM) and caudal ventrolateral medulla (CVLM), by the vlPAG may mediate the sympathoinhibitory phase of HEM (Cavun and Millington, 2001) In the present study, EX animals displayed an attenuated drop in MAP when equal amounts of blood were removed compared to SED animals. Based on this result, one might expect to see less neural activity ( i.e., less FLI) within the vlPAG of EX versus SED rats following HEM (Vagg et al., 2008) Interestingly, this result was not observed. HEM induced a significant increase in FLI within the caudal vlPAG that was essentially the same between EX and SED animals. This lack of tr eatment effect within the vlPAG could mean that the attenuated depressor response
101 seen in EX compared to SED animals during HEM was not the result of differences in ac tivation of the vlPAG, but may have been downstream. Alternatively, Mueller (2007) recently showed that exercise training leads to an increase in baseline inhibition of sympathetic activity initiated by activation of the RVLM, an area that projects direct ly to sympathetic preganglionic motor neurons in the IML of the spinal cord and has been shown to cause sympathoexcitation (Dampney et al., 2003) This work supports the idea that the rostral ventrolateral medulla ( RVLM ) is less sensitive to activation by excitatory inputs in exercise trained subjects. The same may be true for depressor regions such as the caudal midl ine medulla ( CMM) and/or caudal ventrolateral medulla ( CVLM ) a hypothesis yet to be investigated. Thus, it may be the case that HEM elicits equal levels of output from the vlPAG to downstream depressor regions in both EX and SED populations, but the overall excitability of these depressor regions may be blunted following exercise training. In the present study, HRV analysis identified that the drop in HR during the sympathoinhibitory phase was mediated in part by an increase in vagal drive, as indicated by a significant increase in HF power during the last five minutes of HEM. Although there was no significant difference in total power between groups, the average HF power in the EX group at the nadir time point was ~50% less than that observed in the SED group. This raises the possibility that, in addition to modulating central sympathoinhibitory circuits, exercise training may also modulate central circuits involved in parasympathetic control. Indeed, analysis of HF peak location demonstrated that the p eak of the EX group was located at a lower frequency compared to the SED group at all time points. Since the location of the HF peak is reported to reflect respiratory rate
102 (Baekey et al., 2008) this observation raises the possibility that some of the difference between EX and SED groups in response to hemorrhage may have also resulted from a training induced change in respiratory control. Although the respiratory response to severe hemorrhage has not been extensively studied in conscious rats, there is recent evidence from Strittmatter and Schadt (2007) that respi ratory rate is unchanged during the initial phase of HEM followed by an increase in rate during the sympathoinhibitory phase of HEM in conscious male in rabbits (Strittmatter and Schadt, 2007) Our results indirectly confirm that respiratory rate may not have changed during the initial compensatory phase (i.e., no change in HF peak frequency location). During the sympathoinhibitory phase of HEM however, there was a decrease in the HF peak frequency, suggesting a drop in the respiratory rate. This raises the possibility that the respiratory response to hemorrhage may be species specific and in rodents may involve a decrease in respiratory rate possibly coupled with an increase in tidal volume. Since respiratory pattern (changes in tidal volume versus frequency) can influence venous return, and there was a trend for the HF peak of the EX HEM animals to be located at a lower frequency, one of the central adaptations to exercise training may have been an overall increase in tidal volume in response to HEM which facilitated venous return. This observation supports our hypothesis that EX animals would better tolerate the decompensatory phase of blood loss and raises the possibility that some of the exercise training effect may be mediated by alterations in central respiratory control (Pellegrino et al., 1999; Eastwood et al., 2001) At the offset of HEM (minute 15), both EX and SED animals showed immediate signs of recovery with spontaneous increases in HR and MAP. At 30 minutes following
103 the onset of HEM, SED rats were still significantly hypotensive compared to baseline and MAP was significantly lower compared to the EX group at all time points. In contrast, the EX group recovered quickly and appeared to return to baseline levels within seven minutes. One brain region known to be important for recovery from HEM is the LPBN (Ward, 1989) --more specifically, the ventrolateral region of the LPBN, including the external subnucleus has been implicated in HEM recovery. For example, Blair et al (2002) showed that lesions of the ventrolateral LPBN prevented the norm al recovery response following HEM (Blair et al., 2001) In anot her study, these investigators showed that activation of glutamate receptors within the LPBN contributes to the recovery phase following HEM (Blair and Mickelsen, 2006) In the present study, FLI was significantly increased compared to nonHEM contro ls in the external subnucleus of the LPBN for SED but not EX animals throughout the rostrocaudal extent of the LPBN. Furthermore, FLI following HEM was significantly less in EX versus SED rats in the middle and caudal regions of the LPBN. These FLI data support previous findings that correlate neural activation within the external LPBN with spontaneous recovery following HEM. Greater activation of this subnucleus fits with our cardiovascular measures demonstrating that SED animals displayed a greater hyp otension and therefore required greater activation of this group of neurons for recovery. Alternatively, only one subnucleus of the LPBN showed a trend for greater increase in FLI compared to controls in the EX HEM versus SED HEM animals. This nucleus, t he central lateral nucleus of the rostral LPBN has been previously identified by our lab to be activated by central sympathoexcitatory pathways (Hayward and Castellanos, 2003) raising the possibility that EX training mediates some of its effects
104 through modulation of cardiorespiratory control circuits in the rostral central subnucleus of the LPBN (Baekey et al., 2008) Finally, the hypothalamic PVN is another particularly interesting neural site that mediates cardiovascular responses to changes in blood volume. In the present study, all subn uclei of the PVN displayed an increased FLI following HEM in EX animals compared to controls; however, there was no difference seen in HEM induced FLI within the parvocellular PVN between EX and SED groups. In addition to producing corticotropinreleasing hormone (CRH), this group of neurons produces arginine vasopressin ( AVP) that acts as a neurotransmitter within the CNS (Swanson and Sawchenko 1983; Badoer, 2001) Central blockade of AVP V1 receptors during HEM results in complete abolition of bradycardia and an attenuated hypotension in Wistar Kyoto (WKY) rats (Budzikowski et al., 1996) Other studies also support a role for central release of AVP in HEM induced bradycardia and hypotensionpart of the decompensatory phase (Johnson et al., 1988; Evans et al., 1991; Shoji et al., 1993; Imai et al., 1996) Since EX animals displayed an attenuation of both the bradycardia and hypotension during HEM, it might be expected that these animals would express less FLI in the parvocellular PVN compared to SED animals. This, however, was not observed. Similarly, the FLI quantified in the magnocellular PVN which contains neurons that produce peripherally acting AVP did not show any differences between the EX and SED animals in response to HEM. Again, one might have anticipated a greater neural activation in this region in SED animals since they produced a greater degree of hypotension comp ared to trained animals. These data suggest that six weeks of voluntary exercise training may not impact the role of either the centrally or
105 peripherally projecting AVP neurons within the PVN during HEM. The dorsal cap region of the PVN, however, did sh ow significantly less FLI in EX versus SED animals following HEM. This response was unexpected as this region of the PVN projects directly to the intermediolateral cell column ( IML ) and RVLM. Thus a decreased activation of the dorsal PVN would presumably translate into a decreased sympathoexcitatory response to HEM in the EX animals. Such a finding is intriguing and paradoxical as EX animals displayed higher mean values for HR and MAP compared to SED animals in the current set of experiments. It is well documented that exercise training leads to a downregulation of sympathetic activity in pathological states such as congestive heart failure (Zucker et al., 2004) and in healthy animals as well (Mueller, 2007) The present results may simply reflect this training induced decrease in sympathetic drive. Alternately, a decreased activation of neurons in this region of the PVN may reflect the fact that th e stimulus triggering activation of this area was less intense for EX versus SED rats. Indeed, EX rats displayed a blunted hypotension, which may have resulted in a weaker signal to the dorsal PVN to activate sympathetic preganglionic motor neurons in the IML or sympathetic premotor neurons in the RVLM. Methodological Considerations There are four main methodological constraints to consider in the context of the present study. The first methodological consideration in this study is that we chose to utiliz e a voluntary wheel based model of exercise training rather t han forced exercise training. Forced exercise regimens typically result in training adaptations indicative of high intensity and/or high endurance exercise rather than the more moderate exercise routines prescribed to cardiovascular patients and the general population. In addition to the clinical limitations of forced exercise studies, forced treadmill and swimming has
106 been shown to cause alterations in the hypothalamic pituitary adrenal (HPA) axis that mimic chronic stress (Noble et al., 1999; Moraska et al., 2000) Such an impact on the HPA axis may further limit the clinical applicability of studies utilizing forced models of exercise training. While voluntary exercise (e.g., wheel running) has not been shown to induce the level or degree of training adaptations seen in forced exercisers, it does lead to both peripheral (Sexton, 1995) and central (van Praag et al., 1999; Mabandla et al., 2004) adaptations that benefit the animal Furthermore, voluntary wheel running does not appear to cause deviations in the HPA axis that would imply any stress was induced in the animal (Dishman et al., 1995; Droste et al., 2003) This is important for the present study because differences in basal HPA axis functionas is the case with stressed animals --significantly alters the response to HEM (Darlington et al., 1989; Graessler et al., 1989) A second methodological consideration in the present study is that rats were pair housed in order to eliminate the stress of social isolation shown to occur in naturally social creatures, like rats (Gavrilovic et al., 2008) A preliminary evaluation of singly housed wheel exercised rats proved extremely difficult. Individually housed runners were exceptionally anxious, appeared to be more defensive to experimenter handling, and baseline HR for t hese rats was higher and less stable (data not utilized in the present study) compared to the pair housed rats used in this study. It has been reported that exposure to repeated physical exercise can offset the stress of isolated living conditions in rats (Filipovic et al., 2007) A majority of stu dies, however, show contrary results that social isolation blunts some of the positive effects of chronic exercise (Stranahan et al., 2006; Leasure and Decker, 2009) Although the exact
107 distances run by each animal could not be discriminated in the present study, other studies have used similar methods for estimating individual running distances in grouphoused rats (Stranahan et al., 2006) and the estimated distances run that we have reported for our pair housed runners coincide very well with data collected from other investigators and from the singly housed runners used in our preliminary study (Figure 1A). Finally, the average weight gain in the pair housed and singlehoused EX rats were not different (Figure 1B) and both were less than SED animals at weeks three through six, showing that running, and ther efore at least a moderate training effect, did still occur. A third consideration is the use of c Fos to quantify the exercise training effects on central neural circuitry involved the autonomic response to HEM. It is generally accepted that c Fos immunohistochemistry is an accurate indication of neuronal activation in response to sustained stimuli. In the present study, animals were sacrificed 90 minutes after MAP had reached its lowest value (minute 15, Figure 4 2) coincident with the cessation of blood withdrawal (or an equivalent time period in CON animals). Because FLI typically shows maximal expression 90120 minutes following a stimulus (Chan and Sawchenko, 1994) the quantification of Fos positive neurons in the present study may not accurately reflect changes in neural responsiveness to one spec ific phase of the HEM. Rather, the data presented here are intended to represent an overall look at the altered neuronal activation in response to the complete experience of 30% TBV HEM. Another limitation associated with the use of changes in FLI to id entify central circuits involved in mediating a response is the inability of this technique to identify those neurons inhibited by a stimulus. Since changes in sympathoinhibition
108 have been proposed to mediate the effect of exercise training on cardiovascu lar control, these circuits would not necessarily be isolated by this technique. Finally, the peripheral effects of chronic exercise, although not evaluated in the current study, cannot be excluded from the possible explanations of the enhanced tolerance t o HEM seen in EX rats. Of particular relevance to the present study, is the effect of chronic exercise on blood plasma volume. Hypervolemia is a well documented outcome of long term exercise training. If, indeed, EX animals in the present study developed an increased blood plasma volume as a result of six weeks of voluntary wheel running, the use of the same equation for TBV estimation based on body weight would be inappropriate. While blood volume was not directly measured in the present study, blood v olume was not likely changed significantly by the modest amount of exercise performed. Other studies using a similar wheel based model of exercise have reported no difference in plasma protein concentration and, presumably, therefore, no functional change in plasma volume between exercised and sedentary rats (Stranahan et al., 2006) Additionally, had blood volume been greater in the EX animals in this study, a delay in the onset of the decompensatory stage of HEM mi ght have been expected. That is, had EX animals started off with more blood, the average HR for that group should have began declining later compared to the SED group. Yet, as Figure 4 2A shows, the peak HR for the EX group occurred slightly prior to and began decreasing slightly sooner compared to the SED group. Taken together, these findings contest the likelihood that the attenuated cardiovascular response to HEM in EX animals was the result of a greater initial blood volume.
109 Conclusions The results o f this study demonstrate that six weeks of voluntary exercise leads to an enhanced ability to tolerate severe blood loss in conscious male rats. While this study does not pinpoint an exact mechanism or mechanisms of action directly resulting in this protection against hypovolemic shock, this study e valuated specific brain regions involved in modulating the hemodynamic response to HEM that may be altered by chronic voluntary exercise. While alterations within such brain regions, including the dorsal cap region of the hypothalamic PVN, the external subnucleus, or central lateral subnucleus of the LPBN, may contribute to the enhanced compensation, attenuated decompensation, and faster rate of recovery following HEM, it is unclear whether these changes are specific to the response to hypovolemic hemorrhage or if there are neurochemical changes induced by exercise that have resulted in adjusted function of these regions. Further studies are needed to fully elucidate the specific role of each of these central nuclei in the exercise induced prevention of hypovolemic shock reported in this set of experiments. Additionally, more studies are needed to further identify any peripheral adaptations that may also be critical for the altered response to HEM seen in trained subjects. Finally, more investigation into the respiratory response to HEM in EX versus SED animals is required to further elucidate whether such a change is significantly contributing to a training benefit during blood loss and whether or not such a ch ange is central or peripheral in nature.
110 Figure 41. Evidence of exercise training. (A) Average distance run per day over six weeks of wheel access in pair and singlehoused rats. Data points for the pair housed rats represent estimated distance run per day for individual animals (see results section for further explanation). (B) Average body weights over six weeks for pair housed sedentary ( SED ) and exercised ( EX ) rats as well as singlehoused EX rats. Significant difference from Pair EX rats.
111 Figure 42. Hemodynamic response to 30% total blood volume hemorrhage (HEM) in exercised ( EX ) vs. sedentary ( SED ) conscious rats. Heart rate ( HR; A) and mean arterial pressure ( MAP ; B) response prior to (minute 1), during (minutes 015), and after (minutes 16 30) HEM. Grey box indicates time of blood withdrawal. Significant difference from SED. # Significant difference from baseline (minute 1).
112 Figure 43. Heart rate variability ( HRV ) analysis of exercised ( EX ) and sedentary ( SED ) group response to hemorrhage (HEM). (A) Typical frequency spectrum of a heart rate (HR) interval in a conscious SpragueDawley EX rat at rest prior to HEM (Base). Low frequency (LF) and high frequency (HF) peak components are shown in (B). Ratio of power in the LF and HF ranges before HEM (Base), during the peak increase in HR (Peak), and just prior to the offset of HEM (Nadir). (C) HF power before and during different phases of HEM (D) Frequency of the peak power in the HF range. Significant difference from pr e values for EX HEM group. # Significant difference from pre values for EX and SED groups across all time points combined. P<0.016.
113 Figure 4 4. Representative images of Fos like immunoreactivi ty throughout the rostrocaudal extent of the lateral parabrachial nucleus ( LPBN ) in a sedentary ( SED ) and exercised ( EX ) animal following 30% total blood volume hemorrhage SCP = superior cerebellar peduncle; CTR = central subnucleus; SUP = superior subnucleus; EXT = external subnucleus.
114 Figure 4 5. Average FLI and schematic representations for the rostral (A), middle (B) and caudal (C) lateral parabrachial nucleus ( LPBN ) in exercised ( EX) versus sedentary ( SED ) rats following either 30% total blood volume hemorrhage ( EX HEM and SED HEM) or quiet rest (EX CON and SEDCON). Numbers accompanying the schematics represent distance in millimeters caudal to bregma (adapted from Paxinos and Watson, 2005). Ctr = central subnucleus; Sup = superior subnucleus ; Ext = external subnucleus; KF = Kolliker Fuse nucleus; SCP = superior cerebellar peduncle. # Significant difference from control. Significant difference from SED HEM.
115 Figure 4 6. Average F os like immunoreactivity and schematic representations for t he caudal ventrolateral periaqueductal grey (vl PAG ; A), Locus Coeruleus ( LC ; B), and hypothalamic paraventricular nucleus (PVN; C) in exercised ( EX ) versus sedentary ( SED ) rats following either 30% total blood volume hemorrhage (EX HEM and SED HEM) or quie t rest (EX CON and SED CON). Numbers accompanying the schematics represent distance in millimeters caudal to bregma (adapted from Paxinos and Watson, 2005). Magno = magnocellular; Parvo = parvocellular; Dorsal = dorsal cap. # Significant difference from control. Significant difference from SED HEM.
116 C HAPTER 5 OPIOID RECEPTOR BLOC KADE IN THE LATERAL PARABRACHIAL NUCLEUS PREVENTS EXERCISEINDUCED TOLERANCE TO HEMORRHAGE Introduction At the onset of blood loss, slight decreases in baroreceptor activation trigger an increase in sympathetic drive, which acts to speed heart rate ( HR) and maintain mean arterial pressure ( MAP ) If bleeding continues, the body changes from a strategy involving the recruitment of compensatory mechanisms to maintain a sympathoexcitatory state to a strategy of sympathetic withdrawal. The sympathoinhibitory phase in response to severe blood loss leads to a drop in both HR and MAP. Upon cessation of blood loss, prior to cardiac arrest, HR and MAP spontaneously recover to baseline or near baseline values. Multiple sites within the central nervous system ( CNS ) have been implicated in the modulation of each of these phases of hemorrhage ( HEM ) Of particular interest to our laboratory is the lateral parabrachial nucleus ( LPBN ) l ocated dorsolateral to the superior cerebellar peduncles ( SCP ) in the rostral pons (Paxinos and Watson, 2005) This nucleus is interconnected with forebrain regions that regulate volume control and electrolyte balance (e.g., hypothalamic paraventricular nucleus [ PVN ] central nucleus of the amygdala [CeA], and median preoptic nucleus [MnPO] (Ciriello et al., 19 84; Krukoff et al., 1993; Bianchi et al., 1998; Krout et al., 1998) ) as well as hindbrain regions that receive and relay baroreceptor and blood volume information (e.g., nucleus tractus solitaries [ NTS ] (Herbert et al., 1990) ). Thus, the LPBN is ideally situated in the CNS to act as an integration site for monitoring and influencing blood volume regulation. De Oliveira and colleagues (2008) recently reported that opioid activation within the LPBN induces salt and water intake in both sodium depleted and normohydrated
117 rats, demonstrating an opioidmediated mechanism of osmoor volume regulation at the LPBN. This effect was blocked by co application of naloxone (De Oliveira et al., 2008) a predominantly muopioid receptor antagonist that is also able to antagonize deltaand kappaopioid receptors when applied at higher concentrations. Furthermore, Blair and Mickelson (2006) showed that activation of the LPBN via application of excitatory amino acid resulted in augmented recovery following HE M an effect blocked by chemical lesions of the same region (Blair and Mickelsen, 2006) Combined with the observation that the LPBN displays significantly increased FLI positive staining following hypotensive HEM (Ahlgren et al., 2007) it is clear that the LPBN plays some role in the complex cardiovascular response to perturbations in blood volume, such as occurs during HEM. Since the LPBN contains large amounts of opioid receptors (Unterwald et al., 1991; Mansour et al., 1995) and manipulation of these receptors with agonists or antagonists can effect volume regulation (De Oliveira et al., 2008) it seems likely that th e role of the LPBN in HEM may also involve an opioid mediated component. Physical exercise is beneficial for both healthy and unhealthy individuals and has been shown to positively impact both the systemic and central nervous systems (Ma, 2008; Harrington et al., 2009) During an acute bout of exercise, opioid peptides are released both peripherally and centrally (Shyu et al., 1982; Carmody and Cooper, 1987; Tierney et al., 1991) Chronic exercise, and coinciding chronic opioid release, has repeatedly been shown to mimic the response of opioid abuse, resulting in increased tolerance to opioid drugs (Kanarek et al., 1998; Mathes and Kanarek, 2001) and a probable downregulation of opioid receptors (Houghten et al., 1986) Previous work from our lab has demonstrated that as little as six weeks of running wheel activity can
118 protect against HEM induced hypotension and bradycardia (see Chapter 4), suggesting that physi cal activity confers an increased ability to buffer the cardiovascular decline associated with severe perturbations in blood volume. Several studies have shown beneficial effects (increased MAP and survival) of kappaopioid antagonists delivered centrall y or peripherally following HEM (Ang et al., 1999; Henderson et al., 2002; Liu et al., 2005) The dorsal lateral subnucleus of the LPBN has been shown to play a role in the complex response to hypovolemic stimuli (Iwasaki et al., 1993) This area contains the vast majority of dynorphinergic neurons and kappaopioid receptors (KORs) in the LPBN and neurons in this area send afferents directly to rostral brain nuclei involved in homeostatic responses to changes in blood volum e (Moga et al., 1990; Wolinsky et al., 1996; Hermanson et al., 1998) The present study was performed to test the hypothesis that exercise induced tolerance to severe HEM involves an opioidergic m echanism within the LPBN possibly resulting from an exercise induced decrease in KORs in the LPBN. Accordingly, we hypothesized that application of an opioid receptor antagonist into the LPBN of sedentary rats would result in an increased tolerance to HEM as is observed with voluntary exercise trained rats and that Western blot analysis of rostral pontine brain tissue from exercise trained rats would display less KOR protein content compared to sedentary controls. Methods General Preparation All experime ntal procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Male SpragueDawley rats (Harlan Industries, Minneapolis, IN) were randomly placed into one of two groups: exercise (EX,
119 n=17) or sedentary (SED n=17). All animals were pair housed in cages that did (EX) or did not (SED) contain a single running wheel. EX animals were introduced to the running wheel at seven to eight weeks of age and allowed six weeks of access to the running wheel. Because voluntary exercise on a running wheel slows weight gain over a six week period compared to nonexercised controls (see Chapter 4), SED animals were agematched rather than weight matched to EX animals. EX animals were lightly handled and weighed weekly and all animals were maintained in a 12 hour lights on: 12 hour lights off, temperature controlled environment with food and water ad libitum. Cranial Cannulation Twelve animals from each group were instrumented with bilateral brain cannulas to allow injectio n of drug or vehicle into the LPBN. Seven days before the day of the experiment, animals were deeply anesthetized with isofluorane gas in pure oxygen (4 2 2.5%) and placed into a stereotaxic head holder (Kopf Instruments, Tejunja, CA, USA). A mid sagitta l incision was then made in the skin overlying the skull and connective tissue was carefully scraped away from the surface of the skull using a scalpel blade. The edges of the incision were held out of the surgical field with 4O monofilament suture weighted with a surgical hemostat instrument. Excess bleeding on the skull surface was controlled with a sterile cauterizing device. The skull was leveled between bregma and lambda. A bilateral craniotomy directly overlying the right and left LPBN was perfor med with a highspeed microdrill. Following the craniotomy procedure, dura mater was carefully removed from the surface of the brain. Stainless steel, 23gauge guide cannulas (Plastics One, Roanoke, VA, USA) were bilaterally implanted into the brain usi ng the following coordinates: 9.3mm caudal to bregma, 2.1mm lateral to midline. Guide cannulas had a 5.5mm projection such that the tip of
120 the cannula was positioned approximately 1.5mm dorsal to the LPBN. Cannulas were fixed to the cranium using dental acrylic resin and a single watch screw that was drilled into a region of the skull rostral to cannula placement. Stainless steel, 30gauge dummy cannulas were inserted into and screwed onto the guide cannulas (Plastics One). The skin incision was closed using 4O monofilament suture and a topical antibiotic was applied. Following removal from the head holder, animals received subcutaneous injections of sterile saline (1ml 0.09% NaCl, for rehydration) and analgesics (Buprenorphine, 0.1ml/kg; Rimadyl, 0.1m l/kg). Following cranial cannulation, animals were singly housed and allowed five to seven days to recover. EX animals were allowed access to a running wheel during this recovery time. Arterial Cannulation One to two days prior to the experiment, animals were re anesthetized with isofluorane (4 2 2.5%) and surgically instrumented with bilateral femoral arterial catheters (PE 10 connected to PE 50 tubing, Braintree Scientific, Braintree, MA). Catheters were subcutaneously routed and exteriorized between t he scapulae, filled with heparinized saline (100 IU/ml), and plugged with stainless steel obturators (23gauge, Braintree Scientific, Braintree, MA). Analgesics (Rimadyl, 0.01 ml/kg; Buprenorphine, 0.01 ml/kg) were administered subcutaneously following ca theterization. Following arterial cannulation, EX animals were housed in cages without a running wheel in order to ensure than on the day of the experiment any effects of an acute bout of exercise did not confound the results of the study. Experimental Pr otocol On the day of the experiment, animals were brought to the lab, weighed, and both arterial catheters were connected to additional heparinized salinefilled tubing (1050
121 IU/ml; PE 50). Dummy cannulas were carefully unscrewed and removed from t he imp lanted guide cannulas. Each animal was then placed in the testing chamber and the catheters were routed through a hole in the lid of the testing chamber in such a way that the animal could move freely within the testing chamber but not excessively twist t he catheters. One of the arterial catheters was connected to a calibrated pressure transducer inseries with an amplifier (Stoelting, Wooddale, IL). Both pulsatile and MAP were recorded online at 100 Hz using a Cambridge Electronics Design computer inte rface and Spike2 data software. HR was derived online from the interval between peak systolic pressure waves in the AP trace. Baseline recordings of AP, MAP, and HR were collected for 3060 minutes during which the animal was undisturbed in order to ens ure a stable baseline measurement. Next, bilateral injections into the LPBN were made using a 10l Hamilton syringe connected to internal cannulas (1.5mm longer than the guide cannulas) with polyethylene tubing (50PE 10PE). The internal cannula was fir st carefully advanced into the right guide cannula. Approximately 60 seconds later, a bolus (200500 nL) of either naloxone (5 or 20M in 0.09% NaCl) or vehicle (0.09% NaCl) was administered. Another 60 seconds was allowed before removing the cannula and placing it into the contralateral guide, afterwhich a 60second time period was permitted prior to injecting the same type and volume of drug. Following another 60 seconds, the internal cannula was removed. The animal was then allowed 10 minutes without handling. Ten minutes after the final removal of the internal cannula, animals underwent a 30% estimated TBV HEM over 15 minutes followed by 45 minutes of recovery. AP, MAP and HR were recording continuously during the hemorrhage and recovery periods.
122 T BV was estimated using a previously reported equation: (0.06ml/gram)*(body weight in grams)+(0.77) (Lee and Blaufox, 1985) Animals were then administered a lethal dose of sodium pentobarbital (100150ml/kg). The right soleus muscle was immediately dissected and removed from the hindlimb and weighed. The same internal cannula used to administer drugs during the experiment was used to inject a fluorescent marker (Fluorogold) into the brain for verification of cannula placement. Brains were then removed and allowed to sit in 4% paraformaldehyde for at least 24 hours prior to being sectioned with a cryostat, slidemounted, and evaluated for accuracy of injecti ons. Western Blot Analysis of Kappa Opioid R eceptor C ontent Western blot analysis was performed on rostral pontine brain tissue from EX and SED groups of animals (n=5 per groups) for relative KOR protein density as previously described (Tanaka et al., 2005) An imals were brought to the lab and allowed to sit quietly for 23 hours followed by brief isofluorane anesthesia (4%) and decapitation. Brains were immediately harvested, snapfrozen in methyl butane at 40C, and stored at 80C. A 200m section of brain tissue from the rostral pons of each animal (to include the LPBN) was collected on a freezer microtome. Each sec tion was divided into quadrants The right dorsal quadrant from each animal was pooled for each group and homogenized in lysis buffer (20mM T ris HCl [pH 7.4], 5mM EDTA [pH 8.0], 10mM EGTA [ph 7.0], 2mM DTT, 1mM Na3VO4, 0.1mg/mL PMSF, 0.01mg/mL leupeptin, 1% triton x glycerophosphate). Extracted protein samples were boiled for 5 minutes after being diluted 1:2 (v/v) with sampleloading buffer (ph 6.8, 62.5 mM Tris HCl, 20% glycerol, 4% SDS, 0.2% bromphenol blue). Equal amounts of protein (25 g/35L) per sample were separated by SDS PAGE (NuPAGE Novex 4 12% BisTris Gel) at 200 volts for 60 mi nutes followed
123 by Electrophoretic transfer to a nitrocellulose membrane (Invitrogen, LC 2001, 0.45m pores) at 200 volts for 60 minutes. A biotinylated protein latter detection kit (Cell Signaling, 7727) was utilized to identify molecular weights of the t argeted protein (KOR, ~46 kDa). All samples were run in duplicate. Immunoblots were blocked with 5% nonfat milk in Tris buffered saline with 0.05% Tween 20 (TBS T) for 60 minutes at room temperature followed by overnight incubation with a rabbit polyclonal antibody raised against the internal region of the rat KOR (diluted 1:1000 with 5% nonfat milk in TBS T, Biosource, 44302G). Immunoblots were then incubated for 1 hour at room temperature with secondary antibody (goat anti rabbit HRP, diluted 1:500 wi th 5% nonfat milk in TBS T, BioRad, 1706515). Targeted proteins were visualized with enhanced chemiluminescence (ECL, Pierce) and band intensities were quantified using Quantity One software (BioRad). Relative protein abundance is expressed as integrated optical density (IOD) of KOR factored for Ponceau red stain (total protein loaded). Whole brain lysate was used for control comparison. Statistical Analysis For animals that underwent the HEM protocol, paired t tests were employed to evaluate difference s in average body weight at week six and soleus/body weight ratios between EX and SED rats. Oneminute averages of HR and MAP values were calculated prior to the first brain injection (PRE), within one minute following the second brain injection (POST), and every five minutes from 60 seconds prior to the onset of HEM (minute 0) though the end of recovery (minute 60). Within treatment groups, HR and MAP data were averaged and reported as mean SEM. A twoway analysis of variance ( ANOVA) with repeated meas ures was used to compare HR and MAP between treatment groups across time. When significant main effects and/or
124 interactions were present, Student NewmanKeuls post hoc analyses were performed to isolate differences between groups at certain time points, w ithin groups across time, and/or across time irrespective of treatment or group. For animals used in Western blot analysis of pontine KOR density, a paired t test was used to compare KOR integrated optical density (IOD) between groups. Significance was determined as P<0.05 for all statistical analyses. Results Measures of Exercise Training Upon initial exposure to running wheels, EX rats weighed 192 4 grams. Following six weeks of running wheel access, EX animals weighed 313 5 grams -significantly le ss compared to agematched SED rats (346 6 grams; P<0.001). EX animals were pair housed rather than singly housed, therefore daily running distances were collected per cage and divided by two. These data are displayed in Figure 51A and show an increas e in average distance run per animal per day from week one to week five, after which daily running distances reached a plateau. By week six, the estimated average distance run per day per rat was 6.2 0.5 km. Figure 51B shows no difference in soleus muscle/body weight ratio (g/kg) for EX versus SED animals (P=0.2837). Injection Site Verification Figure 52 shows photomicrograph images of brain slices taken at the level of injection sites. The cardiovascular response to HEM was not different between bil ateral (Figure 5 2A) and unilateral (Figure 52B) injection of either vehicle or naloxone; therefore, all brains in which at least one injection was correctly placed into the LPBN were included for statistical analyses. The primary reason for unilateral i njections was
125 incorrect placement of one of the two implanted guide cannulas. Usually, one of the guide cannulas was not implanted deeply enough such that injections were placed into fiber tracts or nuclei located dorsal to the LPBN (see Figure 52B). In a couple of cases, however, one of the guide cannulas became clogged during the post surgical recovery period, preventing advancement of the internal injection cannula during the experiment. For the SED vehicle group, two animals in which injections were placed into the inferior colliculus (IC) were included in the statistical analyses since cardiovascular responses to HEM were not different from those in which vehicle injections were correctly placed within the LPBN (see top panel of Figure 52D). Addit ionally, the cardiovascular response to HEM was not different between vehicleinjected rats and rats in which both injections of naloxone were placed outside the LPBN (n=1 per group; see Figure 52C & D); therefore, animals in which both injections were mi splaced (not in the LPBN and not in the cerebral spinal fluid [CSF]) were included with the vehicletreated group for statistical comparisons. Cardiovascular Response. Figure 53 shows the hemodynamic response to vehicle or naloxone injections prior to s evere HEM in SED rats. For HR, a repeated measures ANOVA identified a main effect of time (P<0.0001), but no main effect of drug treatment (P=0.9145) and no interaction (0.6020) between factors. I njection of either drug resulted in a nonsignificant inc rease in HR that fell slightly prior to the onset of HEM. A typical response to HEM was recorded for vehicleand naloxoneinjected rats: HR rose initially, followed by a precipitous fall by minute 15, which remained significantly lower than minute 0 (just prior to HEM onset, i.e., baseline) though minute 20, irrespective of the drug injected. HR was also found to be significantly lower than baseline towards the end of recording
126 at minutes 55 and 60. Similar to what was recorded for HR, analysis of MAP data with a repeated measures ANOVA identified a main effect only for time (P<0.0001). There was not a main effect of drug treatment (P=0.8101) or an interaction between factors (P=0.1184). Irrespective of drug, there was a nonsignificant rise in MAP im mediately following injection, which dropped slightly by minute 0. Once HEM was initiated, MAP was not different from baseline for either group until minute 15. When data for both groups were combined, MAP was significantly lower than baseline from minut e 15 through the end of recording, minute 60. Naloxoneinjected rats appear to have a lower value for both HR and MAP at minute ten compared to vehicleinjected rats, however, these values are not statistically different. Figure 54 shows the hemodynami c response to vehicle or naloxone injections prior to severe HEM in the EX rats. For HR, a repeated measures ANOVA identified a main effect across time (P<0.0001) but not treatment group (P=0.0611). There was however a significant interaction between the se factors (P=0.0289). Similar to what was seen for SED rats, HR increased slightly (nonsignificant) following injection of either vehicle or naloxone and then fell slightly by minute 0. HR rose during the early part of HEM, and fell to below baseline v alues during minutes 1520 for the naloxonetreated rats only. Vehicleinjected rats also displayed a decline in HR by minute 15, but this value was not different from baseline. Furthermore, at no point during the experiment were HR values for vehiclein jected EX rats different from baseline. Naloxonetreated rats experienced a delayed recovery from HEM compared to vehicle rats as these animals had significantly lower HR from minute 1530 relative to vehicle controls. For MAP data, a repeated measures A NOVA identified a main effect of time (P<0.0001) but
127 not treatment group (P=0.1581) and an interaction was identified between factors (P=0.0254). In naloxonetreated EX rats, MAP was not lower than baseline until minute 15 of HEM and this hypotension last ed through minute 30. MAP fell below baseline for vehicle treated rats as well, but only for minute 15. By minute 20, vehicle controls had already recovered to near baseline values while naloxone rats remained hypotensive. Although vehicletreated rats displayed significant hypotension by minute 15, MAP values were significantly greater in vehicletreated rats compared to naloxonetreated rats at both minute 15 and minute 20. Figure 55 shows the hemodynamic response to naloxone injections prior to sev ere HEM in SED versus EX rats. Statistically and functionally speaking, these groups were not different in their cardiovascular response to HEM. For HR data, a repeated measures ANOVA isolated a main effect for time only (P<0.0001). There was no main ef fect of exercise training (P=0.4048) and no interaction between time and training factors (P=0.8336). Thus, when the two groups were combined, HR was maintained during HEM through minute 10, afterwhich it fell significantly from baseline through minute 20. Similarly, MAP data subjected to a repeated measures ANOVA revealed only a main effect of time (P<0.0001), but not for exercise training (P=0.4827) and no interaction was present (P=0.8921). Irrespective of whether rats were sedentary or trained, MAP f ell significantly lower than base line at minute 10 and this hypotension lasted through the end of recovery (minute 60). Western Analysis of Kappa Opioid Receptor C ontent Figure 56B shows a chemiluminescent image of an immunoblot of pooled brain tissue fr om the rostral pons of EX and SED rats along with the relative KOR abundance.
128 Data are displayed as averages for each group of the duplicate samples. There was no difference seen in KOR IOD between EX and SED rats (P=0.57). Discussion There are three mai n findings from the present set of experiments. First, exercise induced tolerance to severe conscious HEM is mediated centrally, since the effect can be completely blocked by central administration of an opioid receptor blockade in the LPBN. Second, six weeks of voluntary wheel running results in central alterations that has a greater inhibitory effect on opioidergic mechanisms within the LPBN. This is evidenced by the fact that opioid receptor blockade within the LPBN caused a significant change in the hemodynamic response to severe conscious HEM in EX rats and no change in SED rats. Finally, six weeks of voluntary wheel running does not result in a decreased KOR content in the brain region including the LPBN, suggesting that the exercise induced tolerance to HEM is not likely the result of a downregulation of KORs in this brain region. Previous literature has shown that opioids in the CNS are involved in the onset of HEM induced decompensation. In the periaqueductal grey, deltaopioid receptor (D OR) antagonism prevents the decompensatory phase of HEM in conscious rabbits (Ludbrook and Ventura, 1994) Adminis tration of mu (Evans et al., 1989) and kappa(Evans et al., 1989) opioid receptor antagonists into the 4th ve ntricle also prevent the onset of decompensated hypotension in conscious rabbits, suggesting the possibility that more than a single type of opioid receptor is involved in mediating the reflex bradycardia and hypotension during severe HEM. In rats, blockade of both DORs and mu opioid receptors (MORs) within the brainstem have been shown to modulate the decompensatory response to HEM (Ang et al., 1999)
129 Previous work from our lab demonstrates that six weeks of voluntary wheel exercise is able to protect conscious rats from the decompensation that typically occurs with 30% TBV loss (see Chapter 4). Results from the present study confirm a voluntary EX induced tolerance to HEM. At the end of blood withdrawal (minute 15), when hypotension and bradycardia were maximal, vehicleinjected EX rats displayed a 12% drop in HR while vehicleinjected SED animals dropped 27% from baseline (P= 0.04). In the sam e vehicletreated group of animals, MAP in EX animals dropped 41% from baseline, while SED animals dropped 54% (P= 0.002). Chronically exercised individuals exhibit decreased sensitivity to opioids (Smith and Yancey, 2003) perhaps due to an overall downregulation of central opioid receptors. Thus, it is possible that exercise induced tolerance to severe HEM could, in part, be explained by decreased opioidtriggered decompensation at one or multiple central loci. The fact that naloxone injections into the LPBN were able to completely block this effect of exercise argues that th is is, indeed, a central ly regulated phenomenon. In addition to the well established role of the LPBN in the relay of cardiovascular afferent information to more rostral brain sites (Fulwiler and Saper, 1984; Herbert et al., 1990; Krukoff et al., 1993; Jhamandas et al., 1996) and its ability to alter autonomic tone via anatomical connections with both forebrain and brainstem nuclei (Saper and Loewy, 198 0; Hubbard et al., 1987; Saleh et al., 1997) an opioidergic mechanism specifically within the LPBN has been identified to have a modulatory function in the response to perturbations in blood volume. Iwasaki et al (2001) showed that bilateral injection o f naloxone into the LPBN prior to simulated HEM prevented hypovolemiainduced release of AVP (Iwasaki et al., 1993) suggesting that opioid receptor activation within the LPBN
130 is critical for the humoral response to hypovolemia, as oc curs with severe HEM. Since opioid receptor activation in the LPBN (as well as other CNS sites) acts to inhibit neuronal activity (Milner et al., 1984; Xia and Haddad, 1991) it seems likely that opioid receptor activation within the LPBN leads to arginine vasopressin ( AVP ) release f rom forebrain regions such as the magnocellular paraventricular nucleus ( PVN ) via inhibition of tonically inhibitory neurons involved in relaying osmoor volume information. De Oliveira et al (2008) showed that opioid receptor blockade in the LPBN of conscious rats with naloxone resulted in significantly increased sodium intake in both normohydrated and salt deprived groups of animals (De Oliveira et al., 2008) which could functionally translate to an enhanced hypotensive signal. This study also proposed a disinhibitory mechanism of opioid receptor activation within the LP BN. We originally hypothesized that naloxone would hav e a greater effect in SED compared to EX rats based on previous literature supporting a chronic opioid exposureinduced alteration in central opioid receptor systems in EX rats (Smith and Yancey, 2003) By blocking opioid receptors, we expected naloxone in the LPBN to prevent the severe fall in HR and MAP normally seen during s evere HEM in the SED but not EX rats. However SED rats displayed no differences in HR or MAP response to HEM following unilateral or bilateral injection of naloxone or salinevehicle. On the other hand, the training induced tolerance to HEM was completely blocked by naloxone injected either unilaterally or bilaterally into the LPBN of EX rats. In fact, naloxoneinjected EX rats actually had a greater fall in HR (though not significantly different) compared to naloxonetreated SED rats. This is likely d ue to the high concentration of naloxone used. It should be noted that this effect of naloxone could not be the result of
131 drug leakage into the 4th ventricle as naloxone injections into the cerebrospinal fluid (CSF) have been shown to postpone decompensat ory phase of HEM in both conscious sheep (Frithiof and Rundgren, 2006; Frithiof et al., 2007) and rabbits (Evans et al., 1989) Additionally, in the conscious rat, systemic administration of naloxone is able to reverse HEM induced hypotension (Faden and Holaday, 1979) and intrathecal administration of naloxone prior to the onset of HEM can totally block HEM induced decompensation (Ang et al., 1999) Indeed, when naloxone injections were misplaced into the 4th ventricle in the present study (n=2, data not shown or used in group averages for statistical analysis), there was no deviation in HR or MAP from baseline values during HEM. A large amount and high concentration of naloxone was intentionally utilized in these experiments in order to identify a clear effect, if any. While injections in the present study were placed into the LPBN (see Figure 5 1), the impact of naloxone at other, nearby, locations cannot be excluded from consideration as alternative sites of action. The fact that naloxone impacted the EX rats but had no effect at all on SED rats contradicts our hypothesis that a decr eased opioid tone within the LPBN may underlie the tolerance to HEM seen following exercise training or at a minimum, is an over simplified view of how EX induced alterations in central opioid systems may impact the cardiovascular response to severe blood loss. An alternate explanation for the present results may be a decreased gammaaminobutyric acid (GABA) tone in EX versus SED rats rather than a downregulation of opioid receptors. Although the reported effects of exercise on central GABA are somewhat mixed, there is general agreement that exercise can decrease GABAergic inhibition in the CNS. It is well documented that
132 overactivity of the GABAergic system within the NTS occurs with the development of hypertension (Catelli an d Sved, 1988; Tsukamoto and Sved, 1993; Vitela and Mifflin, 2001) and it is postulated that a decreased GABA release within the NTS is involved in the post exercise hypotension consistently seen in hypertensive patients and less consistently seen in normotensive individuals (Chen et al., 2009) A study performed in spontaneously hypertensive rats (SHRs) demonstrated decreased GABA mediated inhibitory post synaptic potentials in NTS neurons immediately following an acute bout of exercise and concluded that dynamic exercise caused a decreased GABAergic synaptic input to the NTS (Chen et al., 2009) Dishman (1997) reported an elevated GABA concentration in rats given eight weeks of free access to running wheels relative to sedentary controls, results from the same study also showed a concomitant decrease in GABAA receptor density (as measured by 3H bicuculine binding) in EX versus SED rats (Dishman et al., 1997) Finally, Mueller and Hasser (2006) showed that exercise trai ning blunts the bradycardic effect of bilateral microinjection of bicuculline, a GABA A receptor antagonist, into the NTS, suggesting that exercise training decreases tonic GABAA receptor mediated inhibition of NTS neurons involved in modulating HR (Mueller and Hasser, 2006) While these studies do not give conclusive evidence that exercise training results in a suppression of GABAergic mechanisms within the CNS, they do point to the plasticity of the GABA inhibitory system as a function of exercise training. Activation of either GABA A or opioid receptors within the LPBN results in a reduced overall inhibition of this nucleus (Kobashi and Bradley, 1998; De Oliveira et al., 2008) indicating that these neurotransmitters act on inhibitory neurons. While thes e
133 neurotransmitter systems work via different cellular mechanisms, they may represent individual, parallel systems which function as an inhibitory influence over other inhibitory neurons important for the relay of volume information or salt appetite (De Oliveira et al., 2008) If GABAergic tone is dampened as a result of EX training, the effect of opioid receptor blockade in th e LPBN of trained rats could represent a near complete loss of inhibitory influence within this nucleus and a significant alteration in the relay of osmoor volume information. In SED rats, however, the effect of opioid blockade would be less significant because of the remaining inhibitory presence of GABAergic mechanisms. This may explain the severe decompensation seen in the present study in EX but not SED rats following naloxone injection into the LPBN prior to HEM. In summary, the results of this stu dy reveal a centrally mediated exercise induced tolerance to severe blood loss in the conscious rat that involves an alteration of an opioidergic mechanism within the LPBN. The present study was unable to identify a difference in KOR protein content between EX and SED rats. Combined with the generally accepted lack of DORs present in this particular brain locus, these data point to an exercise induced alteration of MORs in the LPBN. Naloxone was used in the present study to antagonize opioid receptors wi thin the LPBN. This drug preferentially binds to MORs; but, when used at higher concentrations such as was utilized in the present study, naloxone will also block KORs and DORs. Thus, further studies are needed to fully elucidate the exact group (or groups) of opioid receptors that may be altered following chronic exercise. Furthermore, results from this study were contrary to our original hypothesis that opioid receptor blockade within the LPBN would replicate the exercise induced tolerance to HEM in SE D rats. Because the opposite was
134 observed, it seems implausible to attribute the observed exercise induced tolerance to blood loss to a downregulation of central opioid receptors, at least within the LPBN. In light of these unexpected results, more studies are warranted to elucidate the exact central mechanisms underlying the protection against hypovolemic shock afforded by modest amounts of physical exercise. Finally, the trained rats in the present study did not display a resting bradycardia or a diff erence in soleus muscle/body weight ratio compared with sedentary rats. These data lend support to the notion that even modest physical activity can lead changes within the brain that may benefit the overall health of the individual.
135 Figure 51. Measures of exercise training. A) Estimated average distance run per animal per day (see results section for detailed description). B) Soleus/body weight ratio (g/kg) for exercised ( EX ) and sedentary ( SED ) rats. EX rats did not display an increased soleus/bo dy weight ratio relative to SED rats following six weeks of voluntary wheel running (P=0.2837).
136 Figure 52. Injection sites. Photomicrograph images (1.25X) of brain slices showing typical patterns and locations of internal cannula placement. A) Bilater al placement of tip of injection cannula into the lateral parabrachial nucleus ( LPBN ) B) Unilateral placement of tip of injection cannula into the LPBN. Tip of the contralateral injection cannula was located dorsal to the LPBN. C) Bilateral misplacemen t of internal cannulas. D). Schematic composite of injection sites used in group analyses (modified from Paxinos and Watson). Injections for exercised ( EX ) animals are displayed on the left and injections for sedentary ( SED ) animals are displayed on the right. For simplification, only one injection site is displayed for animals that received bilateral injections. In two SED animals, vehicle was bilaterally injected into the inferior colliculus (IC), represented by red squares labeled V. Data from the se animals was included in the group averages for the vehicletreated SED group. Red boxes labeled N represent bilateral injections of naloxone outside the LPBN. Data from these animals was included in the group averages for vehicletreated rats within their respective groups. SCP = superior cerebellar peduncle.
137 Figure 5 3. Effect of naloxone versus vehicle injection into the lateral parabrachial nucleus ( LPBN ) on the heart rate (HR) and mean arterial pressure (MAP) responses to severe conscious hemo rrhage ( HEM ) in sedentary ( SED ) rats. Grey box indicates time of blood withdrawal (minute 015). # indicates significant difference from preHEM baseline (minute 0) irrespective of drug treatment.
138 Figure 5 4. Effect of naloxone versus vehicle injection into the lateral parabrachial nucleus ( LPBN ) on the heart rate (HR) and mean arterial pressure (MAP) responses to severe conscious hemorrhage ( HEM ) in exercise ( EX ) rats. Grey box indicates time of blood withdrawal (minute 015). $ indicates significan t difference from preHEM baseline (minute 0) within treatment group. indicates significant difference from vehicle at the indicated time point.
139 Figure 5 5. Effect of naloxone injection into the lateral parabrachial nucleus ( LPBN ) on the heart rate ( HR) and mean arterial pressure (MAP) responses to severe conscious hemorrhage ( HEM ) in exercised ( EX ) versus sedentary ( SED ) rats. Grey box indicates time of blood withdrawal (minute 015). # indicates significant differences from preHEM baseline (minut e 0) irrespective of treatment group.
140 Figure 56. Western blot analysis of relative kappa opioid receptor ( KOR ) content in the rostral pons of exercised ( EX ) and sedentary ( SED ) rats. A) Schematic demonstrating the way in which brain slices from the rostral pons were separated into quadrants for Western blot analysis of the dorsolateral region only. Dashed lines indicate where each section of brain tissue was sectioned. B) Chemiluminescent photograph of an immunoblot showing KOR protein expression bet ween 40 and 50 kDa for EX and SED animals and averages of the quantified KOR integrated optical density (IOD) for both groups. There was not a statistical difference in KOR IOD between groups (P=0.57).
141 CHAPTER 6 SUMMARIES AND CONCLUSIONS Summaries of the Study Findings Study #1 Summary This study evaluated the hemodynamic responses and neural activation of various rostral brainstem nuclei (as marked by c Fos immunoreactivity) during three different rates of hemorrhage ( HEM ) in conscious rats. This study was important for identifying a reliable HEM protocol that would best simulate the bi phasic hemodynamic response to severe blood loss and induce distinct changes in neural activation within nuclei previously shown to be involved in regulating the cardiovascular and autonomic responses to blood loss. Previous studies have utilized a wide range of HEM protocols which makes interpreting results across studies somewhat difficult. The results of study #1 demonstrated that while the fastest rate of HEM (2.0 ml /kg/min) produced a significant bradycardia and hypotension at the end of HEM, there was not a clear compensatory tachycardia at the onset of blood withdrawal. Animals that underwent the slowest rate of HEM (0.5 ml/kg/min) showed neither a compensatory tachycardia nor a decompensatory bradycardia or hypotension. Only animals from the intermediate rate of blood withdrawal (1.0 ml/kg/min) displayed clear compensatory and decompensatory phases that resulted in deviations in heart rate and mean arterial press ure that were significantly different from preHEM baseline values. Additionally, the intermediate rate of HEM produced the most significant and least variable increases in neural activation of rostral brainstem sites (including, but not limited to, the c audal ventrolateral periaqueductal grey [ vlPAG ] and the central subnucleus of the rostral lateral parabrachial nucleus [ LPBN ] ) when compared to hypotension alone. Results from this
142 study further substantiate the integral role of the rostral brainstem in t he autonomic response to severe HEM and provide a dependable protocol for future HEM experiments in this lab as w ell as for other investigators. Study #2 Summary This study evaluated whether or not LPBN projecting afferents from the vlPAG are involved in s ome component of the cardiovascular response to severe HEM in the conscious in the rat. Results from this study corroborated previous work which showed a dense axonal projection from the vlPAG to the LPBN. However, these data do not support our initial hypothesis that this projection is actively involved in the descending control of autonomic responses to HEM. This was evidenced by a lack of difference in Fos Fluorogold (FG) co labeling within the vlPAG neurons of HEM verses nonHEM animals which had rec eived a unilateral FG injection into the LPBN. Since the neuroanatomical networks underlying the transition from hemorrhagic compensation to decompensation are not well understood, the results of this study will allow investigators interested in elucidati ng these neural pathways to redirect and focus their attention and resources on other anatomic al connections involved in hemorrhage induced sympathoinhibition (HIS) Study #3 Summary This study evaluated the effect of chronic, voluntary exercise on the hem odynamic and neural responses to severe HEM in conscious rats. This study identified an exercise induced tolerance to severe HEM. Rats that were allowed access to running wheels for six weeks displayed a significantly blunted bradycardia and hypotension associated with the decompensatory phase of HEM compared to sedentary rats. While exercised rats displayed a fall in heart rate ( HR ) and mean arterial pressure ( MAP ) that
143 corresponded with the time point and therefore the same percentage of blood volume r emoved--at which decompensation occurred in sedentary animals, at no point did the fall in HR or MAP for exercised animals differ from preHEM baseline values. Additionally, exercised animals displayed significantly less neural activation (as marked by Fo slike immunoreactivity [ FLI ] ) in subregions of the LPBN and hypothalamic paraventricular nucleus ( PVN ) suggesting that exercise induced tolerance to HEM may result from the suppression of central circuits involved in autonomic control during blood loss. Heart rate variability ( HRV ) analysis of data collected before, during, and after HEM demonstrated a difference in heart rate regulation between exercised and sedentary rats which may correspond to differences in basal respiratory function that may have contributed to an enhanced ability to cope with the internal stress of severe blood loss in the exercised group. Finally, it is noteworthy that exercised (EX) animals in this study did not demonstrate a lower resting HR compared to sedentary (SED) rats, w hich is commonly used to indicate a training effect. Therefore, even moderate physical activity seems to be sufficient to induce central changes that may contribute to an exercise induced protection against the cardiovascular decline associated with sever e HEM. Study #4 Summary This study evaluated whether opioid receptors in the LPBN play a role in the exercise induced tolerance to HEM. One part of this study compared the protein expression of kappa opioid receptors ( KORs ) within the dorsolateral rostral pons of EX and SED rats. Although we expected to see significantly less KOR protein expression in this region of the brain in EX versus SED rats, no difference was seen between groups. Since the LPBN contains mainly KORs and mu opioid receptors ( MORs ) these
144 data point to a possible MOR mediated mechanism. Accordingly, either saline or a MOR antagonist (naloxone) was administered bilaterally to the LPBN of EX and SED conscious rats prior to HEM. Interestingly, the cardiovascular response to HEM was not different in SED rats given saline or naloxone. On the other hand, while saline had no effect on the HR and MAP of exercised rats in response to HEM, naloxone in the LPBN completely blocked the exercise induced tolerance to HEM. In fact, there was no di fference in the cardiovascular response of naloxonetreated EX and SED rats following HEM. The combined results of this study indicate that although there is an opioidergic mechanism within the LPBN that is involved in the exercise induced protection agai nst hypovolemic decompensation, it is not likely due to a downregulation of opioid receptors. Rather, chronic exercise may decrease other inhibitory mechanisms within the LPBN (such as gamma aminobutyric acid [GABA] ) that leads to an increased influence of opioidergic inhibition within the LPBN. Discussion The peripheral effects of exercise have been studied more thoroughly than those in the central nervous system ( CNS ) A number of studies, however, have identified both anatomical and neurochemical changes in the brain and spinal cord that occur following chronic exercise (Nelson et al., 2005; Vaynman and Gomez Pinilla, 2005) While the effect of exercise training on brain opioidergic systems has been studied to some extent, there is still little known about the exact locations in the brain at which exercise affects opioids and opioid receptors particularly those that contribute to cardiovascular control. Some investigators have postulated that chronic exercise, like chronic morphine administration, leads to the downregulation of opioid receptors in the brain (Smith and Yancey, 2003) Partly based on these studies, we also initially
145 hypothesized that exercised animals would display less opioid receptors in the LPBN and that there would be less of an o pioid mediated influence on the response to HEM in exercised versus sedentary rats. Surprisingly, the results from the present studies indicate something quite different. No difference in KOR protein expression was identified between groups. Additionall y, exercise trained animals responded in such a way as to indicate that this group of animals possessed a greater opioidergic influence within the LPBN compared to their sedentary counterparts. In addition to its influence on autonomic reflexes, such as the arterial baroreflex (Hayward and Felder, 1998) the LPBN also plays a si gnificant role in salt appetite (Johnson and Thunhorst, 1997) which implicates this group of neurons in volume or osmo regulation, as ingestion of salt and water are stimulated by circumstances such as cellular dehydration or low plasma volumes ( De Castro e Silva et al., 2006) It is possible that such stimuli are interpreted by the brain in a similar way as hypotensive signals as salt induced water retention and, later, increases in plasma volume may act long term to rectify decreases in mean arterial pressure. A number of forebrain regions (like the PVN, median preoptic nucleus supraoptic nucleus, and the central nucleus of the amygdala) (Ciriello et al., 1984; Krukoff et al., 1993; Bianchi et al., 1998; Krout et al., 1998) act to stimulate salt ingestion. The LPBN is interconnected with all of these forebrain regions and its overall influence in salt appetite is to inhibit the aforementioned forebrain regions (Johnson and Thunhorst, 1997) thereby dampening afferent signals which would drive salt appetite ( see Figure 61). Activation of a number of neurotransmitters, such as serotonin and corticotrophin releasing hormone ( CRH) within the LPBN results in decreased sodium ingestion in rats exposed to blood volume
146 expansion or peripheral angiotensin II, both of which produce an elevation in blood pressure (De Castro e Silva et al., 2006; Margatho et al., 2008) On the other hand, activation of GABA A and opioid receptors within the LPBN result in inhibition of neurons within this nucleus and withdrawal of inhibitory drive to forebrain regions, which leads to increased sodium and water intake (De Oliveira et al., 2008) suggesting that this may be more closely associated with hypotensivelike input to the brain. As mentioned in the discussion of study #4 (chapter 5), a possible mechanism of enhanced opioidergic tone within the LPBN following exercise training may result from a decreased GABAergic tone within the LPBN. While some studies have identified increases in GABA levels following wheel exercise in spontaneously hypertensive rats ( SHRs ) (Kramer et al., 2000) others have shown decreases in GABAergic synaptic input and GABA A receptors following similar exercise regimens in SHRs (Chen et al., 2009) Furthermore, in normotensive rats, as little as 28 days of activity wheel exposure has been shown to decr ease the gene expression of both GABA A receptor and glutamate decarboxylase (GAD) in the brain (Molteni et al., 2002) Thus, exercise may result in decreased inhibitory GABAergic tone in the LPBN placing a greater proportion of inhibitory influence on opioid receptors in this region (irrespective of any possible change in opioid receptor density or function induced by the exercise training). In Figure 61, this is depicted by the tipped scale balancing GAB Aergic and opioidergic inputs to the LPBN It should be noted, however, that this figure is not intended to imply any change (increase or decrease) in the density of opioidergic input to the LPBN following exercise training. Alternatively, based on a greater abundance of evidence that GABA mediated inhibitory systems display some degree of neural plasticity as a
147 function of exercise training, we have focused our revised model of the influence of exercise training on neurotransmission in the LPBN to reflec t decreased GABAergic control during HEM. The HRV analysis from study #3, which evaluated alterations in HRV before, during, and after HEM in EX versus SED rats, did not show any difference in the way these two groups responded to severe blood loss. Howev er, the location of the high frequency ( HF ) peak of EX rats was significantly lower across all time points compared to SED rats. Since this HF peak supposedly reflects respiratory rate (Baekey et al., 2008) these data suggest an exercise induced plasticity within the respiratory control system. If exercise result s in an altered respiratory pattern that facilitates increased venous return (increased tidal volume), this may contribute to the enhanced tolerance of these animals to severe HEM. A number of investigators have evaluated the role of the LPBN in respiratory control and have concluded that activation of LPBN neurons results in an increased respiratory rate (Miura and Takayama, 1991; Chamberlin and Saper, 1994; Lara et al., 1994) Recently, Hayward and colleagues (2004) reported a significant change in baseline respiratory pattern following bilateral blockade of LPBN neurons with muscimol (a GABA A receptor agonist), clearly demonstrating an inhibitory role of GABA on LPBN neurons which project to and activate respiratory control centers (Hayward and Castellanos, 2004) Thus, a decreased GABAergic tone within the LPBN may feasibly contribute to an exercise induced alteration in the central control and modulation of respiratory timing which may, in turn, underlie a training induced tolerance to HEM.
148 Study Limitations and Directions for Future Studies Whil e the present set of experiments have contributed meaningful information to what is currently known about the central effects of exercise and the central control of HEM, there are limitations to these studies which make the reported results less comprehens ive. Accordingly, the following is a brief discussion of methodological considerations which may have enhanced the present studies along with some proposals for experiments that could potentially broaden this body of work. Rate of Hemorrhage Multiple s tudies have evaluated the cardiovascular responses to HEM. These previous studies have utilized a number of animal models, including sheep (Frithiof and Rundgren, 2006) dogs (Thrasher and Keil, 1998) rats (Ahlgren et al., 2007) pigs (Salerno et al., 1981) mice (Liaudet et al., 2000) cats (Hall and Hodge, 1971) and rabbits (Schadt and Ludbrook, 1991) It is beneficial to investigators to compare results from experiments performed on such a wide range of species because it allows inter species differences to be teased out and permits investigators to select a species which they feel may be best suited for their own experiments in terms of time management, cost, translation, etc. What is less beneficial about the current large body of literature regarding HEM is the variety of protocols used. For example, much of the earlier HEM experiments were performed on anesthetized animals, which (as mentioned earlier in the introduction) has been shown to significantly alter the phasic response to severe blood loss. Accordingly, mor e recent studies have implemented a conscious model of HEM. Even still, the actual method of blood removal remains inconsistent between investigators, making results difficult to translate and repeat. Since the focus of this series of studies was to eval uate the complex hemodynamic and neural responses to
149 HEM, it seemed warranted to determine the rate of blood removal that produced the most reliable, phasic, hemodynamic and neural responses to blood loss. It was determined from study #1 that removal of blood at a rate of 1 ml/kg/min over 20 minutes was ideal for inducing a clear compensatory tachycardia, decompensatory bradycardia and hypotension, and a recompensatory recovery to baseline or near baseline values. Although this specific protocol was found to be reliable and repeatable, blood removal over a 20 minute time period was sometimes infeasible. In our hands, several experiments had to be stopped because blood clots developed at the tip of the catheter a problem that was not encountered when blood was removed faster (2.0 ml/kg/min over 10 minutes). When blood was removed too quickly, however, a clear compensatory phase was not present. Consequently, hemorrhages performed in studies #2, #3, and #4 utilized a blood withdrawal rate of approximately 1.2 1.3 ml/kg/min over 15 minutes. This rate was sufficient to produce all three phases of HEM as well as neural activation similar to that seen at the slightly slower rate. Indices of Exercise Training The indices used in the present studies to docume nt an exercise training effect, though commonly used, are relatively primitive and somewhat inconclusive. Evidence of a resting bradycardia and blunted weight gain over time are not consistently seen following running wheel activity under 812 weeks durat ion and often times require a much greater training stimulus, such as forced treadmill exercise. Additionally, soleus muscle/body weight ratios were not different between exercised and sedentary rats in study #4. Evaluation of soleus and/or cardiac muscl e citrate synthase activity would have greatly enhanced our conclusions about the level of exercise performed on the rats utilized in these experiments, especially considering the fact that the rats were
150 paired rather than individually housed with running wheels. We observed a significant difference in the HR and MAP response to HEM seen in exercised versus sedentary rats, thus a more critical means of determining the level of exercise training was not all together necessary. However, future studies comparing the effect of voluntary versus forced exercise would need to implement such measures. It would also be of interest to evaluate the time course over which moderate levels of activity confer HEM tolerance to conscious animals. In other words, how long after one starts/stops exercising will a benefit be present in the face of blood loss? Effect of Exercise on Plasma Arginine Vasopressin Response to Hemorrhage The role of AVP in hypotensive hemorrhage has been well established. Release of this hormon e corresponds with the decompensatory drop in mean arterial pressure observed late in HEM (Schadt and Hasser, 1991) and is important for blood pressure recovery following HEM (Korner et al., 1990) Additionally, there is evidence for a central effect of endogenous opioids on AVP release during hypotensive HEM (Korner et al., 1990) Naloxone pretreated rabbits display significantly greater plasma AVP content following conscious HEM compared to saline pretreated co ntrols an occurrence which could not be explained simply a pressor affect of naloxone (Schadt and Hasser, 1991) Authors of this study concluded that naloxone was augmenting the AVP response to HEM through blockade of the opioidmediated suppression of AVP release, which supported previous work showing that KOR agonists inhibit AVP release in situations of hypovolemia (Oiso et al., 1988) Comparison of plasma AVP concentrations in exercised versus sedentary rats during and following HEM would have added an interesting c omponent to the present studies and certainly would have been manageable in terms of blood collection during and following HEM experiments since
151 animals were chronically instrumented with vascular catheters. Additionally, evaluation of brain tissue co lab eled for AVP and c FOS following HEM might have further elucidated differences in central control mechanisms regulating the humoral response to HEM in EX versus SED rats which may involve a central opioidergic component. Effect of Exercise on Opioidergic I nfluence in the Lateral Parabrachial Nucleus Although study #4 evaluated the KOR content within the dorsolateral quadrant of the rostral pons, more molecular studies are needed to better and more accurately quantify any changes in opioid receptors within t he LPBN. Mastery of the micropunch technique specific to the LPBN would have greatly enhanced the specificity of the Western analysis of KOR protein content in study #4. Additionally, future studies are warranted to evaluate whether exercise training alt ers MOR protein expression and/or opioid peptides release within the LPBN. When opioid ligands bind their receptor, the receptor is phosphorylated, resulting in internalization of the receptor (Gaudriault et al., 1997) Literature supports the idea that opioid phosphorylation reflects level of opioid peptide release (Wang et al., 1996) as well as opioid receptor desensitization (Pan, 2003) Throughout the body, the opioidergic system is very plastic, showing signs of receptor upand downregulation and even functional shifts from overall inhibitory to excitatory influences in response to varying degrees of opioid peptide exposure (Wang et al., 1996) Thus, measures of opioid phosphorylation within the LPBN would seemingly be an appropriate means to evaluate the effect of exercise on opioidergic control and modulation within this (or any) brain locus.
152 Effect of Exercise on Gamma Aminobutyric A cid Influence in t he Lateral Parabrachial N ucleus As suggested earlier, the GABAergic inhibitory influence within the LPBN may potentially be blunted following chronic exercise. Although examination of this hypothesis was not within the realm of the current set of studies, it is, nonetheless an interesting and testable hypothesis. Presumably, application of a GABA A agonist, such as muscimol, within the LPBN, would prevent the naloxoneinduced augmentation of the hemorrhagic decompensation observed in exercised rats (study #4). Another brain region of interest regarding GABAergic input to the LPBN and GABA/opioid interaction within the CNS is the arcuate nucleus of the hypothalamus (ARC). This nucleus, situated next to the third ventricle and the median eminence [Paxinos] is one of only two groups of neurons in the brain containing proopioimelanocortin (POMC) neurons (which produce melanocortin and endorphins) (Palkovits et al., 1987) and is integrally involved in the regulation of energy balance and appetite (Minor et al., 2009) The ARC provides both GABAergic and melanocortin (via POMC neurons) input to the LPBN (Cone, 2005) Since POMC neurons produce both melanocortin and endorphins, there is a possibility that endorphins acting in the LPBN may also, at least in part, originate from the ARC. This notion is supported by the results from study #2 which demonstrated that another possible source of opioid release in the LPBN during HEM, the vlPAG, is not activated. In addition to their inhibitory role within the LPBN, ARC originatin g GABAergic neurons inhibit local POMC neurons with efferent connections to regions of the brain involved in fluid and electrolyte balance, such as the PVN (Dietrich and Horvath, 2009) GABAA receptor blockade within the LPBN and decreased GABA mediated inhi bition
153 of POMC neurons within the ARC both, independently, suppress the drive to eat (Cone, 2005; Wu et al., 2009) indicating that both GABAergic and POMC projections from this region significantly alter afferent visceral signals (such as hunger) via connections with the LPBN and other hypothalamic nuclei. Combined with the fact that lidocaine inactivation of ARC neurons is able to dramatically attenuate the fall in arterial pressure during severe HEM (Goktalay et al., 2006) future studies should focus on this nucleus as an interesting modulator of the autonomic response to HEM via its projections to the LPBN. Effect of Exercise on Serotonergic Influence in the L ateral Parabrachial N ucleus Finally, serotonin (5HT) is another neurotransmitter system that has bee n identified to play an important role both in the sensory processing within the LPBN and modulation of the cardiorespiratory response to HEM. Specifically within the LPBN, 5HT has been shown to modulate sodium and water ingestion (Menani et al., 2000) via projections from areas such as the NTS and dorsal raphe nucleus (Margatho et al., 2008) In addition, activation of 5HT 1A receptor s in the hindbrain has been shown to reverse the hypotensive and sympathoinhibitory responses to severe blood loss in conscious rats (Scrogin, 2003; Osei Owusu and Scrogin, 2004) Moreover, some of the influence of 5HT 1A receptor activation on modulating the response to HEM was shown to be mediated by a change in respiration. This was identified by a significant increase in the HF or respiratory related component of spectral analysis of renal sympathetic nerve activity (Osei Owusu and Scrogin, 2004) Chronic exercise has been shown to increase central 5HT synthesis and release (Hong et al., 2003; Min et al., 2003) and decrease the sensitivity of 5HT 1A autoreceptors (Dwyer and Browning, 2000) which act to slow 5HT release.
154 Additionally, as little as 3 days of activity wheel running results in increased serotonergic n euron fiber length (Engesser Cesar et al., 2007) fu rther supporting the possibility of an exercise induced plasticity of the serotonergic systems of the CNS. Although such exercise induced changes in 5HT mechanisms within the CNS have been proposed to underlie mood enhancement in depressed patients (Chaouloff, 1997) whether these changes can or do affect modulation of the autonomic responses to changes in blood volume remains to be seen. Future studies should evaluate whether or not exercise induces changes in the way 5 HT acts within the LPBN to alter the cardiovascular or respiratory resp onses to HEM. Conclusions Results from all four studies presented support previous work showing that the LPBN is an integral site for the complex cardiovascular and respiratory response to severe HEM in the conscious rat. Additionally, this body of work supports the idea that even modest amounts of physical exercise can induce changes within the central nervous system that are beneficial for autonomic regulation and general health. Specifically, chronic exercise may induce changes within the LPBN which result in greater dependence on opioidergic mechanisms supporting the overall inhibitory role of this nucleus in body fluid regulation.
155 Figure 61. Schematic of revised res earch hypothesis regarding the impact of chronic exercise on the balance of gamma aminobutyric acid (GABA) and opioid contributions to the overall inhibitory influence of the lateral parabrachial nucleus ( LPBN ) on salt appetite. Exercise training may incr ease the opioidergic component of the inhibitory mechanisms acting within the LPBN.
156 LIST OF REFERENCES Ahlgren, J., Porter, K. Hayward, L. F., 2007. Hemodynamic responses and c Fos changes associated with hypotensive hemorrhage: standardizing a protocol for severe hemorrhage in conscious rats. Am J Physiol Regul Integr Comp Physiol. 292, R186271. Alam, H. B., Burris, D., DaCorta, J. A. Rhee, P., 2005. Hemorrhage control in the battlefield: role of new hemostatic agents. Mil Med. 170, 63 9. Ang, K. K., McRitchie, R. J., Minson, J. B., Llewellyn Smith, I. J., Pilowsky, P. M., Chalmers, J. P. Arnolda, L. F., 1999. Activation of spinal opioid receptors contributes to hypotension after hemorrhage in conscious rats. American Journal of Physio logy (Heart Circ Physiol). 276, H1552H1558. AnselmoFranci, J. A., Peres Polon, V. L., da RochaBarros, V. M., Moreira, E. R., Franci, C. R. Rocha, M. J., 1998. C fos expression and electrolytic lesions studies reveal activation of the posterior region of locus coeruleus during hemorrhage induced hypotension. Brain Res. 799, 27884. Appel, L. J., Champagne, C. M., Harsha, D. W., Cooper, L. S., Obarzanek, E., Elmer, P. J., Stevens, V. J., Vollmer, W. M., Lin, P. H., Svetkey, L. P., Stedman, S. W. Young, D. R., 2003. Effects of comprehensive lifestyle modification on blood pressure control: main results of the PREMIER clinical trial. Jama. 289, 208393. Aravich, P. F., Rieg, T. S., Lauterio, T. J. Doerries, L. E., 1993. Beta endorphin and dynorphin abnormalit ies in rats subjected to exercise and restricted feeding: relationship to anorexia nervosa? Brain Res. 622, 18. Arvidsson, U., Riedl, M., Chakrabarti, S., Vulchanova, L., Lee, J. H., Nakano, A. H., Lin, X., Loh, H. H., Law, P. Y., Wessendorf, M. W. et al. 1995. The kappa opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids. Proc Natl Acad Sci U S A. 92, 50626. Atweh, S. F. Kuhar, M. J., 1977. Autoradiographic localization of opiate receptors in rat brain. II. The brain stem. Brain Res. 129, 112. Badoer, E., 2001. Hypothalamic paraventricular nucleus and cardiovascular regulation. Clin Exp Pharmacol Physiol. 28, 959. Baekey, D. M., Dick, T. E. Paton, J. F., 2008. Pontomedullary transe ction attenuates central respiratory modulation of sympathetic discharge, heart rate and the baroreceptor reflex in the in situ rat preparation. Exp Physiol. 93, 803 16. Bajic, D. Proudfit, H. K., 1999. Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception. J Comp Neurol. 405, 35979.
157 Bajic, D., Proudfit, H. K. Van Bockstaele, E. J., 2000. Periaqueductal gray neurons monosynaptically innervate extranuclear noradrenergic dendrites in the rat pericoerulear region. J Comp Neurol. 427, 64962. Bakos, J., Hlavacova, N., Makatsori, A., Tybitanclova, K., Zorad, S., Hinghofer Szalkay, H., Johansson, B. B. Jezova, D., 2007. Oxytocin levels in the posterior pituitary and in the heart are modified by voluntary wheel running. Regul Pept. 139, 96101. Barcroft, H., McMichael, J., Edholm, O. Sharpey Shafer, E., 1944. Posthaemrrhagic fainting: study by cardiac output and forearm flow Lancet. 48991. Behbehani, M. M., 1995. Functional ch aracteristics of the midbrain periaqueductal gray. Prog Neurobiol. 46, 575605. Bensimhon, D. R., Adams, G. L., Whellan, D. J., Pagnanelli, R. A., Trimble, M., Lee, B. A., Lee, K. L., Ellis, S. J., Kraus, W. E., Rendall, D. S., Iskandrian, A. E., O'Connor, C. M. Borges Neto, S., 2007. Effect of exercise training on ventricular function, dyssynchrony, resting myocardial perfusion, and clinical outcomes in patients with heart failure: a nuclear ancillary study of Heart Failure and A Controlled Trial Investiga ting Outcomes of Exercise TraiNing (HF ACTION); design and rationale. Am Heart J. 154, 46 53. Bianchi, R., Corsetti, G., Rodella, L., Tredici, G. Gioia, M., 1998. Supraspinal connections and termination patterns of the parabrachial complex determined by th e biocytin anterograde tract tracing technique in the rat. J Anat. 193 ( Pt 3), 41730. Blair, M. L., Jaworski, R. L., Want, A. Piekut, D. T., 2001. Parabrachial nucleus modulates cardiovascular responses to blood loss. American Journal of Physiology (Integrative Comp Physiol). 280, R1141R1148. Blair, M. L. Mickelsen, D., 2006. Activation of lateral parabrachial nucleus neurons restores blood pressure and sympathetic vasomotor drive after hypotensive hemorrhage. Am J Physiol Regul Integr Comp Physiol. Blo mqvist, C. G., 1983. Role of exercise training in secondary prevention of ischemic heart disease. Prev Med. 12, 22832. Boone, J. B., Jr., Sherraden, T., Pierzchala, K., Berger, R. Van Loon, G. R., 1992. Plasma Met enkephalin and catecholamine responses to intense exercise in humans. J Appl Physiol. 73, 38892. Brown, B. S., Payne, T., Kim, C., Moore, G., Krebs, P. Martin, W., 1979. Chronic response of rat brain norepinephrine and serotonin levels to endurance training. J Appl Physiol. 46, 1923. Brown, J. W., Whitehurst, M. E., Gordon, C. J. Carroll, R. G., 2005. Thermoregulatory set point decreases after hemorrhage in rats. Shock. 23, 23942.
158 Brum, P. C., Da Silva, G. J., Moreira, E. D., Ida, F., Negrao, C. E. Krieger, E. M., 2000. Exercise training increases baroreceptor gain sensitivity in normal and hypertensive rats. Hypertension. 36, 101822. Budzikowski, A. S., Paczwa, P. Szczepanska Sadowska, E., 1996. Central V1 AVP receptors are involved in cardiovascular adaptation to hypovolemia in WKY but not in SHR. Am J Physiol. 271, H105764. Buller, K. M., Smith, D. W. Day, T. A., 1999. NTS catecholamine cell recruitment by hemorrhage and hypoxia. Neuroendocrinology (NeuroReport). 10, 38533856. Burke, S. L. Dorward, P. K., 1988. Influence of endogenous opiat es and cardiac afferents on renal nerve activity during haemorrhage in conscious rabbits. J Physiol. 402, 927. Byrnes, G. J., Engeland, W. C. Gann, D. S., 1982. Restitution of blood volume after hemorrhage: inhibition by somatostatin. Endocrinology. 110, 19459. Carmody, J. Cooper, K., 1987. Swim stress reduces chronic pain in mice through an opioid mechanism. Neurosci Lett. 74, 35863. Catelli, J. M. Sved, A. F., 1988. Enhanced pressor response to GABA in the nucleus tractus solitarii of the spontaneously hypertensive rat. Eur J Pharmacol. 151, 2438. Cavun, S., Goktalay, G. Millington, W. R., 2004. The hypotension evoked by visceral nociception is mediated by delta opioid receptors in the periaqueductal gray. Brain Research. 1019, 237245. Cavun, S. Millington, W. R., 2001. Evidence that hemorrhagic hypotension is mediated by the ventrolateral periaqueductal gray region. American Journal of Physiology (Integrative Comp Physiol). 281, R747R752. Cavun, S., Resch, G. E., Evec, A. D., Rapacon Baker, M. M. Mil lington, W. R., 2001. Blockade of delta opioid receptors in the ventrolateral periaquaductal gray region inhibits fall in arterial pressure evoked by hemorrhage. Journal of Pharmacology and Experimental Therapeutics. 297, 612619. Chamberlin, N. Saper, C. B., 1994. Topographic organizatin of respiratory responses to glutamate microstimulation of the parabrachial nucleus in the rat. The Journal of Neuroscience. 14, 65006510. Chamberlin, N. L., Mansour, A., Watson, S. J. Saper, C. B., 1999. Localization of m u opioid receptors on amygdaloid projection neurons in the parabrachial nucleus of the rat. Brain Res. 827, 198204.
159 Chamberlin, N. L. Saper, C. B., 1992. Topographic organization of cardiovascular responses to electrical and glutamate microstimulation of the parabrachial nucleus in the rat. J Comp Neurol. 326, 24562. Chan, R. K. Sawchenko, P. E., 1994. Spatially and temporally differentiated patterns of cfos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J Comp Neurol. 348, 43360. Chan, R. K. W. Sawchenko, P. E., 1998. Differential timeand dose related effects of haemorrhage on tyrosine hydroxilase and neuropeptide Y mRNA expression in medullary catecholamine neurons. European Journal of Neuroscience. 10, 37473758. Chaouloff, F., 1997. Effects of acute physical exercise on central serotonergic systems. Med Sci Sports Exerc. 29, 5862. Chen, C. Y., Bechtold, A. G., Tabor, J. Bonham, A. C., 2009. Exercise reduces GABA synaptic input onto nucleus tractus solitarii baroreceptor secondorder neurons via NK1 receptor internalization in spontaneously hypertensive rats. J Neurosci. 29, 275461. Chen, J. J., Dymshitz, J. Vasko, M. R., 1997. Regulation of opioid receptors in rat sensory neurons in culture. M ol Pharmacol. 51, 66673. Ciriello, J., Lawrence, D. Pittman, Q. J., 1984. Electrophysiological identification of neurons in the parabrachial nucleus projecting directly to the hypothalamus in the rat. Brain Res. 322, 38892. Coats, A. J., Adamopoulos, S., Radaelli, A., McCance, A., Meyer, T. E., Bernardi, L., Solda, P. L., Davey, P., Ormerod, O., Forfar, C. et al., 1992. Controlled trial of physical training in chronic heart failure. Exercise performance, hemodynamics, ventilation, and autonomic function. Circulation. 85, 211931. Cone, R. D., 2005. Anatomy and regulation of the central melanocortin system. Nat Neurosci. 8, 5718. Courneya, C. A., Korner, P. I., Oliver, J. R. Woods, R. L., 1991. Afferent vascular resistance control during hemorrhage in norm al and autonomically blocked rabbits. Am J Physiol. 261, H38091. D'Anci, K. E., Gerstein, A. V. Kanarek, R. B., 2000. Long term voluntary access to running wheels decreases kappaopioid antinociception. Pharmacol Biochem Behav. 66, 3436. Dampney, R. A., Coleman, M. J., Fontes, M. A., Hirooka, Y., Horiuchi, J., Li, Y. W., Polson, J. W., Potts, P. D. Tagawa, T., 2002. Central mechanisms underlying short and long term regulation of the cardiovascular system. Clin Exp Pharmacol Physiol. 29, 2618.
160 Dampney, R A., Horiuchi, J., Tagawa, T., Fontes, M. A., Potts, P. D. Polson, J. W., 2003. Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone. Acta Physiol Scand. 177, 20918. Darlington, D. N., Keil, L. C. Dallman, M. F., 1989. Potentiati on of hormonal responses to hemorrhage and fasting, but not hypoglycemia in conscious adrenalectomized rats. Endocrinology. 125, 1398406. Darlington, D. N., Shinsako, J. Dallman, M. F., 1986. Responses of ACTH, epinephrine, norepinephrine, and cardiovascu lar system to hemorrhage. Am J Physiol. 251, H6128. De Castro e Silva, E., Fregoneze, J. B. Johnson, A. K., 2006. Corticotropinreleasing hormone in the lateral parabrachial nucleus inhibits sodium appetite in rats. Am J Physiol Regul Integr Comp Physiol. 290, R1136 41. De Oliveira, L. B., De Luca, L. A., Jr. Menani, J. V., 2008. Opioid activation in the lateral parabrachial nucleus induces hypertonic sodium intake. Neuroscience. 155, 3508. Dean, C., 2004. Hemorrhagic sympathoinhibition mediated through t he periaqueductal gray in the rat. Neurosci Lett. 354, 7983. Dean, C. Bago, M., 2002. Renal sympathoinhibition mediated by 5HT(1A) receptors in the RVLM during severe hemorrhage in rats. Am J Physiol Regul Integr Comp Physiol. 282, R12230. Dean, C. Woyach, V., 2004. Serotonergic neurons of the caudal raphe nuclei activated in response to hemorrhage in the rat. Brain Research. 1025, 159168. Dielenberg, R. A., Carrive, P. McGregor, I. S., 2001. The cardiovascular and behavioral response to cat odor in rat s: unconditioned and conditioned effects. Brain Research. 897, 228237. Dietrich, M. O. Horvath, T. L., 2009. GABA keeps up an appetite for life. Cell. 137, 11779. Dishman, R., Renner, K., Youngstedt, S., Reigle, T., Bunnell, B., Burke, K., Yoo, H., Mougey, E. Meyerhoff, J., 1997. Activity wheel running reduces escape latency and alters brain monoamine levels after footshock. Brain Research Bulletin. 42, 399406. Dishman, R. K., Warren, J. M., Youngstedt, S. D., Yoo, H., Bunnell, B. N., Mougey, E. H., Meye rhoff, J. L., Jaso Friedmann, L. Evans, D. L., 1995. Activity wheel running attenuates suppression of natural killer cell activity after footshock. J Appl Physiol. 78, 154754.
161 Ditting, T., Hilgers, K. F., Scrogin, K. E., Stetter, A., Linz, P. Veelken, R., 2005. Mechanosensitive cardiac C fiber response to changes in left ventricular filling, coronary perfusion pressure, hemorrhage, and volume expansion in rats. Am J Physiol Heart Circ Physiol. 288, H54152. Divakaran, P., Rigor, B. M. Wiggins, R. C., 1981. Halothane accumulation in rat brain and liver. Neurochem Res. 6, 7785. Droste, S., Gesing, A., Ulbricht, S., Muller, M., Linthorst, A. Reul, J., 2003. Effects of long term voluntary exercise on the mouse hypothalamic pituitary adrenocortical axis. Endocr inology. 144, 30123023. Dunn, A. L., Reigle, T. G., Youngstedt, S. D., Armstrong, R. B. Dishman, R. K., 1996. Brain norepinephrine and metabolites after treadmill training and wheel running in rats. Med Sci Sports Exerc. 28, 2049. Dwyer, D. Browning, J., 2000. Endurance training in Wistar rats decreases receptor sensitivity to a serotonin agonist. Acta Physiol Scand. 170, 2116. Eastwood, P. R., Hillman, D. R. Finucane, K. E., 2001. Inspiratory muscle performance in endurance athletes and sedentary subjec ts. Respirology. 6, 95104. Engesser Cesar, C., Ichiyama, R. M., Nefas, A. L., Hill, M. A., Edgerton, V. R., Cotman, C. W. Anderson, A. J., 2007. Wheel running following spinal cord injury improves locomotor recovery and stimulates serotonergic fiber growt h. Eur J Neurosci. 25, 19319. Erdem, S. R., Demirel, H. A., Broxson, C. S., Nankova, B. B., Sabban, E. L. Tumer, N., 2002. Effect of exercise on mRNA expression of select adrenal medullary catecholamine biosynthetic enzymes. J Appl Physiol. 93, 4638. Eva ns, R. G., Ludbrook, J. Potocnik, S. J., 1989. Intracisternal naloxone and cardiac nerve blockade prevent vasodilatation during simulated haemorrhage in awake rabbits. J Physiol. 409, 114. Evans, R. G., Ludbrook, J. S, V., 1994. Role of vagal afferents in the haemodynamic response to acute central hypovolaemia in unanaesthetized rabbits. Journal of Autonomic Nervous System. 46, 251260. Evans, R. G., Ludbrook, J. Van Leeuwen, A. F., 1989. Role of central opiate receptor subtypes in the circulatory response s of awake rabbits to graded caval occlusions. J Physiol. 419, 1531. Evans, R. G., Ludbrook, J., Woods, R. L. Casley, D., 1991. Influence of higher brain centres and vasopressin on the haemodynamic response to acute central hypovolaemia in rabbits. Journal of the Autonomic Nervous System. 35, 114.
162 Evans, R. G., Ventura, S., Dampney, R. A. Ludbrook, J., 2001. Neural mechanisms in the cardiovascular responses to acute central hypovolaemia. Clin Exp Pharmacol Physiol. 28, 47987. Faden, A. I. Holaday, J. W., 1979. Opiate antagonists: a role in the treatment of hypovolemic shock. Science. 205, 3178. Fediuc, S., Campbell, J. E. Riddell, M. C., 2006. Effect of voluntary wheel running on circadian corticosterone release and on HPA axis responsiveness to restraint stress in SpragueDawley rats. J Appl Physiol. 100, 186775. Fejes Toth, G., Brinck Johnsen, T. Naray Fejes Toth, A., 1988. Cardiovascular and hormonal response to hemorrhage in conscious rats. Am J Physiol. 254, H94753. Felber Dietrich, D., AckermannL iebrich, U., Schindler, C., Barthelemy, J. C., Brandli, O., Gold, D. R., Knopfli, B., Probst Hensch, N. M., Roche, F., Tschopp, J. M., von Eckardstein, A. Gaspoz, J. M., 2008. Effect of physical activity on heart rate variability in normal weight, overweig ht and obese subjects: results from the SAPALDIA study. Eur J Appl Physiol. 104, 55765. Filipovic, D., Gavrilovic, L., Dronjak, S. Radojcic, M. B., 2007. The effect of repeated physical exercise on hippocampus and brain cortex in stressed rats. Ann N Y Ac ad Sci. 1096, 20719. Frithiof, R., Eriksson, S. Rundgren, M., 2007. Central inhibition of opioid receptor subtypes and its effect on haemorrhagic hypotension in conscious sheep. Acta Physiol (Oxf). 191, 2534. Frithiof, R. Rundgren, M., 2006. Activation o f central opioid receptors determines the timing of hypotension during acute hemorrhageinduced hypovolemia in conscious sheep. Am J Physiol Regul Integr Comp Physiol. 291, R98796. Fulwiler, C. Saper, C., 1984. Subnuclear organization of the efferent conn ections of the parabrachial nucleus in the rat. Brain Research Reviews. 7, 229259. Gaudriault, G., Nouel, D., Dal Farra, C., Beaudet, A. Vincent, J. P., 1997. Receptor induced internalization of selective peptidic mu and delta opioid ligands. J Biol Chem. 272, 28808. Gavrilovic, L., Spasojevic, N., Tanic, N. Dronjak, S., 2008. Chronic isolation of adult rats decreases gene expression of catecholamine biosynthetic enzymes in adrenal medulla. Neuro Endocrinol Lett. 29, 101520. Goetz, K. L., Wang, B. C. Sun det, W. D., 1984. Comparative effects of cardiac receptors and sinoaortic baroreceptors on elevations of plasma vasopressin and renin activity elicited by haemorrhage. J Physiol (Paris). 79, 4405.
163 Goktalay, G., Cavun, S., Levendusky, M. C., Resch, G. E., Veno, P. A. Millington, W. R., 2006. Hemorrhage activates proopiomelanocortin neurons in the rat hypothalamus. Brain Res. 1070, 4555. Goldfarb, A. H., Hatfield, B. D., Potts, J. Armstrong, D., 1991. Beta endorphin time course response to intensity of exer cise: effect of training status. Int J Sports Med. 12, 2648. Graessler, J., Kvetnansky, R., Jezova, D., Dobrakovova, M. Van Loon, G. R., 1989. Prior immobilization stress alters adrenal hormone responses to hemorrhage in rats. Am J Physiol. 257, R6617. G raham, J. C., Hoffman, G. E. Sved, A. F., 1995. c Fos expression in brain in response to hypotension and hypertension in conscious rats. J Auton Nerv Syst. 55, 92104. Grassi, G., Seravalle, G., Calhoun, D., Bolla, G. B. Mancia, G., 1992. Physical exercise in essential hypertension. Chest. 101, 312S 314S. Guyenet, P. G., Schreihofer, A. M. Stornetta, R. L., 2001. Regulation of sympathetic tone and arterial pressure by the rostral ventrolateral medulla after depletion of C1 cells in rats. Ann N Y Acad Sci. 940, 259 69. Haberthur, C., Schachinger, H., Seeberger, M. Gysi, C. S., 2003. Effect of non hypotensive haemorrhage on plasma catecholamine levels and cardiovascular variability in man. Clin Physiol Funct Imaging. 23, 15965. Hager, P., Permert, J. Strommer L., 2009. An experimental model of intestinal resection and compensated nonhypotensive blood loss. J Surg Res. 154, 18. Hall, R. C. Hodge, R. L., 1971. Changes in catecholamine and angiotensin levels in the cat and dog during hemorrhage. Am J Physiol. 221, 13059. Hambrecht, R., Wolf, A., Gielen, S., Linke, A., Hofer, J., Erbs, S., Schoene, N. Schuler, G., 2000. Effect of exercise on coronary endothelial function in patients with coronary artery disease. N Engl J Med. 342, 45460. Harrington, M., Gibson S. Cottrell, R. C., 2009. A review and meta analysis of the effect of weight loss on all cause mortality risk. Nutr Res Rev. 22, 93108. Hasser, E. M. Schadt, J. C., 1992. Sympathoinhibition and its reversal by naloxone during hemorrhage. Am J Physiol. 262, R444 451. Hayward, L. F. Castellanos, M., 2003. Increased c Fos expression in select lateral parabrachial subnuclei following chemical versus electrical stimulation of the dorsal periaqueductal gray in rats. Brain Res. 974, 15366.
164 Hayward, L. F. Castellanos, M., 2004. Activation of the dorsal periaqueductal gray in the rat induces Fos like immunoreactivity in select noncholinergic mesopontine neurons. Neurosci Lett. 360, 58. Hayward, L. F., Castellanos, M. Davenport, P. W., 2004. Parabrachial neurons mediate dorsal periaqueductal gray evoked respiratory responses in the rat. Journal of Applied Physiology. 96, 11461154. Hayward, L. F. Felder, R. B., 1998. Lateral parabrachial nucleus modulates baroreflex regulation of sympathetic nerve activity. Ameri can Journal of Physiology (Integrative Comp Physiol). 43, R12741282. Hayward, L. F. Von Reitzenstein, M., 2002. c Fos expression in the midbrain periaqueductal gray after chemoreceptor and baroreceptor activation. Am J Physiol Heart Circ Physiol. 283, H19 7584. Hayward, L. F. Von Reitzenstein, M., 2002. c Fos expression in the midbrain periaqueductal gray after chemoreceptor and baroreceptor activation. American Journal of Physiology (Heart Circ Physiol). 283, H1975H1984. Henderson, L. A., Keay, K. A. Bandler, R., 1998. The Ventrolateral Periaqueductal Gray Projects to Caudal Brainstem Depressor Regions: A Functional Anatomical and Physiological Study. Neuroscience. 82, 201221. Henderson, L. A., Keay, K. A. Bandler, R., 2000. Caudal midline medulla mediat es behaviourally coupled but not baroreceptor mediated vasodepression. Neuroscience. 98, 779 92. Henderson, L. A., Keay, K. A. Bandler, R., 2002. Deltaand kappa opioid receptors in the caudal midline medulla mediate haemorrhageevoked hypotension. Neuror eport. 13, 72933. Herbert, H., Moga, M. M. Saper, C. B., 1990. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J Comp Neurol. 293, 54080. Hermann, D. M., Luppi, P. H., Peyron, C., Hinckel, P. Jouvet, M., 1997. Afferent projections to the rat nuclei raphe magnus, raphe pallidus and reticularis gigantocellularis pars alpha demonstrated by iontophoretic application of choleratoxin (subunit b). J Chem Neuroanat. 13, 121. Hermans on, O., Telkov, M., Geijer, T., Hallbeck, M. Blomqvist, A., 1998. Preprodynorphin mRNA expressing neurones in the rat parabrachial nucleus: subnuclear localization, hypothalamic projections and colocalization with noxious evoked fos like immunoreactivity. Eur J Neurosci. 10, 35867.
165 Heslop, D. J., Bandler, R. Keay, K. A., 2004. Haemorrhageevoked decompensation and recompensation mediated by distinct projections from rostral and caudal midline medulla in the rat. Eur J Neurosci. 20, 2096110. Heslop, D. J., Keay, K. A. Bandler, R., 2002. Haemorrhageevoked compensation and decompensation are mediated by distinct caudal midline medullary regions in the urethaneanaesthetised rat. Neuroscience. 113, 555567. Hong, J. A., Chung, S. H., Lee, J. S., Kim, S. S., S hin, H. D., Kim, H., Jang, M. H., Lee, T. H., Lim, B. V., Kim, Y. P. Kim, C. J., 2003. Effects of Paeonia radix on 5 hydroxytryptamine synthesis and tryptophan hydroxylase expression in the dorsal raphe of exercised rats. Biol Pharm Bull. 26, 1669. Hought en, R. A., Pratt, S. M., Young, E. A., Brown, H. Spann, D. R., 1986. Effect of chronic exercise on betaendorphin receptor levels in rats. NIDA Res Monogr. 75, 5058. Hubbard, J. W., Buchholz, R. A., Keeton, T. K. Nathan, M. A., 1987. Parabrachial lesions increase plasma norepinephrine concentration, plasma renin activity and enhance baroreflex sensitivity in the conscious rat. Brain Res. 421, 22634. Imai, Y., Kim, C. Y., Hashimoto, J., Minami, N., Munakata, M. Abe, K., 1996. Role of vasopressin in neurocardiogenic responses to hemorrhage in conscious rats. Hypertension. 27, 13643. Iwasaki, Y., Gaskill, M. B., Fu, R., Saper, C. B. Robertson, G. L., 1993. Opioid antagonist diprenorphine microinjected into parabrachial nucleus selectively inhibits vasopressi n response to hypovolemic stimuli in the rat. J Clin Invest. 92, 22309. Janal, M. N., 1996. Concerning the homology of painful experiences and pain descriptors: a multidimensional scaling analysis. Pain. 64, 3738. Japundzic, N., Grichois, M. L., Zitoun, P., Laude, D. Elghozi, J. L., 1990. Spectral analysis of blood pressure and heart rate in conscious rats: effects of autonomic blockers. J Auton Nerv Syst. 30, 91100. Jaworski, R. L., Piekut, D. Blair, M. L., 2002. Pregnancy alters lateral parabrachial nucleus but not hypothalamic fos expression following hypotensive hemorrhage. Brain Research Bulletin. 57, 595602. Jhamandas, J. H., Harris, K. H., Petrov, T., Yang, H. Y. Jhamandas, K. H., 1998. Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal. J Comp Neurol. 402, 21021.
166 Jhamandas, J. H., Petrov, T., Harris, K. H., Vu, T. Krukoff, T. L., 1996. Parabrachial nucleus projection to the amygdala in the rat: electrophysiological and anatomical observations. Brain Res Bull. 39, 11526. Johnson, A. K. Thunhorst, R. L., 1997. The neuroendocrinology of thirst and salt appetite: visceral sensory signals and mechanisms of central integration. Front Neuroendocrinol. 18, 292353. Jo hnson, J. V., Bennett, G. Hatton, R., 1988. Central and Systemic Effects of a Vasopressin V1 Antagonist on MAP Recovery After Hemorrhage in Rats. Journal of Cardiovascular Pharmacology. 12, 40512. Kakiya, S., Arima, H., Yokoi, H., Murase, T., Yambe, Y. Oi so, Y., 2000. Effects of acute hypotensive stimuli on arginine vasopressin gene transcription in the rat hypothalamus. American Journal of Physiology (Endocrinol Metab). 279, E886E892. Kalyuzhny, A. E. Wessendorf, M. W., 1998. Relationship of muand delt a opioid receptors to GABAergic neurons in the central nervous system, including antinociceptive brainstem circuits. J Comp Neurol. 392, 52847. Kanarek, R. B., Gerstein, A. V., Wildman, R. P., Mathes, W. F. D'Anci, K. E., 1998. Chronic running wheel activ ity decreases sensitivity to morphineinduced analgesia in male and female rats. Pharmacol Biochem Behav. 61, 1927. Kauvar, D. S., Lefering, R. Wade, C. E., 2006. Impact of hemorrhage on trauma outcome: an overview of epidemiology, clinical presentations, and therapeutic considerations. J Trauma. 60, S311. Keay, K. A., Clement, C. I., Matar, W. M., Heslop, D. J., Henderson, L. A. Bandler, R., 2002. Noxious activation of spinal or vagal afferents evokes distinct patterns of fos like immunoreactivity in the ventrolateral periaqueductal gray of unanaesthetised rats. Brain Res. 948, 12230. Keay, K. A., Crowfoot, L. J., Floyd, N. S., Henderson, L. A., Christie, M. J. Bandler, R., 1997. Cardiovascular effects of microinjections of opioid agonists into the 'Depr essor Region' of the ventrolateral periaqueductal gray region. Brain Res. 762, 6171. Kleiber, A. C., Zheng, H., Schultz, H. D., Peuler, J. D. Patel, K. P., 2008. Exercise training normalizes enhanced glutamatemediated sympathetic activation from the PVN in heart failure. Am J Physiol Regul Integr Comp Physiol. 294, R186372. Knowler, W. C., Barrett Connor, E., Fowler, S. E., Hamman, R. F., Lachin, J. M., Walker, E. A. Nathan, D. M., 2002. Reduction in the incidence of type 2 diabetes with lifestyle interv ention or metformin. N Engl J Med. 346, 393403.
167 Kobashi, M. Bradley, R. M., 1998. Effects of GABA on neurons of the gustatory and visceral zones of the parabrachial nucleus in rats. Brain Res. 799, 3238. Korner, P. I., Oliver, J. R., Zhu, J. L., Gipps, J Hanneman, F., 1990. Autonomic, hormonal, and local circulatory effects of hemorrhage in conscious rabbits. Am J Physiol. 258, H22939. Korte, S. M., Jaarsma, D., Luiten, P. G. Bohus, B., 1992. Mesencephalic cuneiform nucleus and its ascending and descending projections serve stress related cardiovascular responses in the rat. J Auton Nerv Syst. 41, 15776. Kramer, J. M., Plowey, E. D., Beatty, J. A., Little, H. R. Waldrop, T. G., 2000. Hypothalamus, hypertension, and exercise. Brain Res Bull. 53, 7785. K rout, K. E., Jansen, A. S. P. Loewy, A. D., 1998. Periaqueductal gray matter projection to the parabrachial nucleus in rat. The Journal of Comparative Neurology. 401, 437454. Krukoff, T. L., Harris, K. H. Jhamandas, J. H., 1993. Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer Phaseolus vulgaris leucoagglutinin. Brain Res Bull. 30, 16372. Krukoff, T. L., MacTavish, D., Harris, K. H. Jhamandas, J. H., 1995. Changes in blood volume and pressure induce c Fos express ion in brainstem neurons that project to the paraventricular nucleus of the hypothalamus. Molecular Brain Research. 34, 99108. Krukoff, T. L., MacTavish, D. Jhamandas, J. H., 1997. Activation by hypotension in neurons in the hypothalamic paraventricular n ucleus that project to the brainstem. The Journal of Comparative Neurology. 385, 285296. Lara, J. P., Parkes, M. J., Silva Carvhalo, L., Izzo, P., DawidMilner, M. S. Spyer, K. M., 1994. Cardiovascular and respiratory effects of stimulation of cell bodies of the parabrachial nuclei in the anaesthetized rat. J Physiol. 477 ( Pt 2), 3219. Law, P. Y., Wong, Y. H. Loh, H. H., 2000. Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol. 40, 389430. Leasure, J. L. Decker, L., 2009. Social isolation prevents exercise induced proliferation of hippocampal progenitor cells in female rats. Hippocampus. Lee, H. B. Blaufox, M. D., 1985. Blood volume in the rat. J Nucl Med. 26, 726. Lee, J. S., Morrow, D., Andresen, M. C. Chang, K. S., 2002. Isoflurane depresses baroreflex control of heart rate in decerebrate rats. Anesthesiology. 96, 121422. Len, W. B. Chan, J. Y., 1999. Glutamatergic projection to RVLM mediates suppression of reflex bradycardia by parabrachial nucleus. Am J Physiol. 276, H148292.
168 Leskinen, H., Ruskoaho, H., Huttunen, P., Leppaluoto, J. Vuolteenaho, O., 1994. Hemorrhage effects on plasma ANP, NH2terminal proANP, and pressor hormones in anesthetized and conscious rats. Am J Physiol. 266, R193343. Li, P., Tjen A. L. S. Longhurst, J. C., 2001. Rostral ventrolateral medullary opioid receptor subtypes in the inhibitory effect of electroacupuncture on reflex autonomic response in cats. Auton Neurosci. 89, 3847. Li, Y. W. Dampney, R. A., 1994. Expression of Fos like protein in brain following sustained hypertension and hypotension in conscious rabbits. Neuroscience. 61, 61334. Liaudet, L., Soriano, F. G., Szabo, E., Virag, L., Mabley, J. G., Salzman, A. L. Szabo, C., 2000. Protection against hemorrhagic shock in m ice genetically deficient in poly(ADP ribose)polymerase. Proc Natl Acad Sci U S A. 97, 10203 8. Liu, L. M., Hu, D. Y., Pan, X. K., Lu, R. Q. Dan, F. J., 2005. Subclass opioid receptors associated with the cardiovascular depression after traumatic shock and the antishock effects of its specific receptor antagonists. Shock. 24, 4705. Long, Y. C., Widegren, U. Zierath, J. R., 2004. Exercise induced mitogenactivated protein kinase signalling in skeletal muscle. Proc Nutr Soc. 63, 22732. Lovick, T. A., 1993. The periaqueductal gray rostral medulla connection in the defence reaction: efferent pathways and descending control mechanisms. Behav Brain Res. 58, 19 25. Ludbrook, J. Graham, W. F., 1984. The role of cardiac receptor and arterial baroreceptor reflexes i n control of the circulation during acute change of blood volume in the conscious rabbit. Circ Res. 54, 42435. Ludbrook, J. Ventura, S., 1994. The decompensatory phase of acute hypovolaemia in rabbits involves a central delta 1opioid receptor. Eur J Phar macol. 252, 1136. Lumb, B. M., 2004. Hypothalamic and midbrain circuitry that distinguishes between escapable and inescapable pain. News Physiol Sci. 19, 226. Lund, T. D., Yu, L. C., Uvnas Moberg, K., Wang, J., Yu, C., Kurosawa, M., Agren, G., Rosen, A. Lekman, M., 2002. Repeated massagelike stimulation induces long term effects on nociception: contribution of oxytocinergic mechanisms. European Journal of Neuroscience. 16, 330338. Ma, Q., 2008. Beneficial effects of moderate voluntary physical exercise and its biological mechanisms on brain health. Neurosci Bull. 24, 26570. Mabandla, M., Kellaway, L., St Clair Gibson, A. Russell, V. A., 2004. Voluntary running provides neuroprotection in rats after 6hydroxydopamine injection into the medial forebrain bundle. Metab Brain Dis. 19, 4350.
169 Malatynska, E., Wang, Y., Knapp, R. J., Waite, S., Calderon, S., Rice, K., Hruby, V. J., Yamamura, H. I. Roeske, W. R., 1996. Human delta opioid receptor: functional studies on stably transfected Chinese hamster ovary cel ls after acute and chronic treatment with the selective nonpeptidic agonist SNC 80. J Pharmacol Exp Ther. 278, 10839. Mansour, A., Fox, C. A., Akil, H. Watson, S. J., 1995. Opioid receptor mRNA expression in the rat CNS: anatomical and functional implicat ions. Trends Neurosci. 18, 229. Mansour, A., Fox, C. A., Burke, S., Akil, H. Watson, S. J., 1995. Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS. J Chem Neuroanat. 8, 283305. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H. Watson, S. J., 1987. Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci. 7, 244564. Margatho, L. O., Godino, A., Oliveira, F. R., Vivas, L. Antunes Rodrigues, J., 2008. Lateral parabrachial afferent areas and serotonin mechanisms activated by volume expansion. J Neurosci Res. 86, 361321. Mason, P., Strassman, A. Maciewicz, R., 1985. Pontomedullary raphe neurons: monosynaptic excitation from midbrain sites that suppress the jaw opening reflex. Brain Res. 329, 3849. Mathes, W. F. Kanarek, R. B., 2001. Wheel running attenuates the antinociceptive properties of morphine and its metabolite, morphine6 glucuronide, in rats. Physiol Behav. 74, 24551. Meeusen, R. De Meirleir, K., 1995. Exercise and brain neurotransmission. Sports Med. 20, 16088. Meller, E., Shen, C., Nikolao, T. A., Jensen, C., Tsimberg, Y., Chen, J. Gruen, R. J., 2003. Regionspecific effects of acute and repeated restraint stress on the phosphorylation of mitogenact ivted protein kinases. Brain Research. 979, 5764. Menani, J. V., De Luca, L. A., Jr., Thunhorst, R. L. Johnson, A. K., 2000. Hindbrain serotonin and the rapid induction of sodium appetite. Am J Physiol Regul Integr Comp Physiol. 279, R12631. Meredith, C. N., Frontera, W. R., Fisher, E. C., Hughes, V. A., Herland, J. C., Edwards, J. Evans, W. J., 1989. Peripheral effects of endurance training in young and old subjects. J Appl Physiol. 66, 28449. Milner, T. A., Joh, T. H., Miller, R. J. Pickel, V. M., 1984 Substance P, neurotensin, enkephalin, and catecholaminesynthesizing enzymes: light microscopic localizations compared with autoradiographic label in solitary efferents to the rat parabrachial region. J Comp Neurol. 226, 43447.
170 Min, Y. K., Chung, S. H., Lee, J. S., Kim, S. S., Shin, H. D., Lim, B. V., Shin, M. C., Jang, M. H., Kim, E. H. Kim, C. J., 2003. Red ginseng inhibits exercise induced increase in 5hydroxytryptamine synthesis and tryptophan hydroxylase expression in dorsal raphe of rats. J Pharmacol Sci. 93, 21821. Minor, R. K., Chang, J. W. de Cabo, R., 2009. Hungry for life: How the arcuate nucleus and neuropeptide Y may play a critical role in mediating the benefits of calorie restriction. Mol Cell Endocrinol. 299, 7988. Miura, M. Takayama, K ., 1991. Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat. J Auton Nerv Syst. 32, 12133. Moga, M. M., Herbert, H., Hurley, K. M., Yasui, Y., Gray, T. S. Saper, C. B., 1990. Organization of corti cal, basal forebrain, and hypothalamic afferents to the parabrachial nucleus in the rat. J Comp Neurol. 295, 62461. Mohanty, P. K., Thames, M. D., Arrowood, J. A., Sowers, J. R., McNamara, C. Szentpetery, S., 1987. Impairment of cardiopulmonary baroreflex after cardiac transplantation in humans. Circulation. 75, 91421. Molina, P., Zambell, K., Zhang, P., Vande Stouwe, C. Carnal, J., 2004. Hemodynamic and immune consequences of opiate analgesia after trauma/hemorrhage. Shock. 21, 526534. Molteni, R., Ying Z. Gomez Pinilla, F., 2002. Differential effects of acute and chronic exercise on plasticity related genes in the rat hippocampus revealed by microarray. Eur J Neurosci. 16, 110716. Moraska, A., Deak, T., Spencer, R. L., Roth, D. Fleshner, M., 2000. Treadmill running produces both positive and negative physiological adaptations in SpragueDawley rats. Am J Physiol Regul Integr Comp Physiol. 279, R13219. Morgan, D. A., Thoren, P., Wilczynski, E. A., Victor, R. G. Mark, A. L., 1988. Serotonergic mechanism s mediate renal sympathoinhibition during severe hemorrhage in rats. Am J Physiol. 255, H496502. Morita, H. Vatner, S. F., 1985. Effects of hemorrhage on renal nerve activity in conscious dogs. Circ Res. 57, 78893. Mueller, P. J., 2007. Exercise training attenuates increases in lumbar sympathetic nerve activity produced by stimulation of the rostral ventrolateral medulla. J Appl Physiol. 102, 80313. Mueller, P. J. Hasser, E. M., 2006. Putative role of the NTS in alterations in neural control of the circu lation following exercise training in rats. Am J Physiol Regul Integr Comp Physiol. 290, R38392.
171 Murphy, A. Z., Ennis, M., Rizvi, T. A., Behbehani, M. M. Shipley, M. T., 1995. Fos expression induced by changes in arterial pressure is localized in distinct longitudinally organized columns of neurons in the rat midbrain periaqueductal gray. J Comp Neurol. 360, 286300. Murphy, A. Z., Ennis, M., Shipley, M. T. Behbehani, M. M., 1994. Directionally specific changes in arterial pressure induce differential pat terns of fos expression in discrete areas of the rat brainstem: a doublelabeling study for Fos and catecholamines. J Comp Neurol. 349, 3650. Murray, E. A. Wise, S. P., 1996. Role of the hippocampus plus subjacent cortex but not amygdala in visuomotor conditional learning in rhesus monkeys. Behav Neurosci. 110, 12611270. Nelson, A. J., Juraska, J. M., Musch, T. I. Iwamoto, G. A., 2005. Neuroplastic adaptations to exercise: neuronal remodeling in cardiorespiratory and locomotor areas. J Appl Physiol. 99, 2312 22. Niskanen, J. P., Tarvainen, M. P., RantaAho, P. O. Karjalainen, P. A., 2004. Software for advanced HRV analysis. Comput Methods Programs Biomed. 76, 7381. Noble, E., Moraska, A., Mazzeo, R., Roth, D., Olsson, M., Moore, R. Fleshner, M., 1999. Dif ferential expression of stress proteins in rat myocardium after free wheel or treadmill run training. J Appl Physiol. 85, 16961701. Odeh, F. Antal, M., 2001. The projections of the midbrain periaqueductal grey to the pons and medulla oblongata in rats. Eur J Neurosci. 14, 127586. Oiso, Y., Iwasaki, Y., Kondo, K., Takatsuki, K. Tomita, A., 1988. Effect of the opioid kappareceptor agonist U50488H on the secretion of arginine vasopressin. Study on the mechanism of U50488H induced diuresis. Neuroendocrinology. 48, 65862. Osei Owusu, P. Scrogin, K., 2006. Role of the arterial baroreflex in 5 HT1A receptor agonist mediated sympathoexcitation following hypotensive hemorrhage. Am J Physiol Regul Integr Comp Physiol. 290, R133744. Osei Owusu, P. Scrogin, K. E., 2004. Buspirone raises blood pressure through activation of sympathetic nervous system and by direct activation of alpha1adrenergic receptors after severe hemorrhage. J Pharmacol Exp Ther. 309, 113240. Palkovits, M., Mezey, E. Eskay, R. L., 1987. Pro opi omelanocortinderived peptides (ACTH/beta endorphin/alphaMSH) in brainstem baroreceptor areas of the rat. Brain Res. 436, 32338. Pan, Z., 2003, Opioid Research Methods and Protocols, Humana Press.
172 Paronis, C. A. Holtzman, S. G., 1992. Development of tolerance to the analgesic activity of mu agonists after continuous infusion of morphine, meperidine or fentanyl in rats. J Pharmacol Exp Ther. 262, 19. Paxinos, G. Watson, C., 2005, The Rat Brain in Stereotaxic Coordinates, Academic. Pelaez, N. M., Schreihof er, A. M. Guyenet, P. G., 2002. Decompensated hemorrhage activates serotonergic neurons in the subependymal parapyramidal region of the rat medulla. Am J Physiol Regul Integr Comp Physiol. 283, R68897. Pelaez, N. M., Schreihoffer, A. M. Guyenet, P. G., 20 02. Decompensated hemorrhage activates serotonergic neurons in the subependymal parapyramidal region of the rat medulla. American Journal of Physiology (Integrative Comp Physiol). 283, R688R697. Pellegrino, R., Villosio, C., Milanese, U., Garelli, G., Rod arte, J. R. Brusasco, V., 1999. Breathing during exercise in subjects with mildto moderate airflow obstruction: effects of physical training. J Appl Physiol. 87, 1697704. Persson, S., Jonsdottir, I., Thoren, P., Post, C., Nyberg, F. Hoffmann, P., 1993. C erebrospinal fluid dynorphinconverting enzyme activity is increased by voluntary exercise in the spontaneously hypertensive rat. Life Sci. 53, 64352. Pirkle, J. C., Jr., Gann, D. S. Allen Rowlands, C. F., 1982. Role of the pituitary in restitution of blood volume after hemorrhage. Endocrinology. 110, 7 12. Polson, J. W., Potts, P. D., Li, Y. W. Dampney, R. A., 1995. Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla after sustained hypertension in conscious rab bits. Neuroscience. 67, 10723. Ponchon, P. Elghozi, J. L., 1997. Contribution of humoral systems to the recovery of blood pressure following severe haemorrhage. J Auton Pharmacol. 17, 31929. Porter, K., Ahlgren, J., Stanley, J. Hayward, L. F., 2009. Modu lation of heart rate variability during severe hemorrhage at different rates in conscious rats. Autonomic Neurosci. in press, Potas, J. R., Keay, K. A., Henderson, L. A. Bandler, R., 2003. Somatic and Visceral Afferents to the 'Vasodepressor Region' of th e Caudal Midline Medulla in the Rat. European Journal of Neuroscience. 17, 11351149. Quail, A. W., Woods, R. L. Korner, P. I., 1987. Cardiac and arterial baroreceptor influences in release of vasopressin and renin during hemorrhage. Am J Physiol. 252, H1120 6. Runciman, W. B. Skowronski, G. A., 1984. Pathophysiology of haemorrhagic shock. Anaesth Intensive Care. 12, 193205.
173 Saleh, T., Bauce, L. Pittman, Q., 1997. Glutamate release in parabrachial nucleus and baroreflex alterations after vagal afferent act ivation. Am J Physiol. 272, 16311640. Salerno, T. A., Milne, B. Jhamandas, K. H., 1981. Hemodynamic effects of naloxone in hemorrhagic shock in pigs. Surg Gynecol Obstet. 152, 7736. Saper, C. B. Loewy, A. D., 1980. Efferent connections of the parabrachial nucleus in the rat. Brain Res. 197, 291317. Schadt, J. C. Gaddis, R. R., 1986. Cardiovascular responses to hemorrhage and naloxone in conscious barodenervated rabbits. Am J Physiol. 251, R90915. Schadt, J. C. Hasser, E. M., 1991. Interaction of vasopressin and opioids during rapid hemorrhage in conscious rabbits. Am J Physiol. 260, R37381. Schadt, J. C. Hasser, E. M., 2004. Hemodynamic effects of blood loss during a passive response to a stressor in the conscious rabbit. American Journal of Physiology (Integrative Comp Physiol). 286, R373R380. Schadt, J. C. Ludbrook, J., 1991. Hemodynamic and neurohumoral responses to acute hypovolemia in conscious animals. Am J Physiol. 260, H305H318. Schadt, J. C., McKown, M. D., McKown, D. P. Franklin, D., 1984. He modynamic effects of hemorrhage and subsequent naloxone treatment in conscious rabbits. Am J Physiol. 247, R497505. Schadt, J. C., Shafford, H. L. McKown, M. D., 2006. Neuronal activity within the ventrolateral periaqueductal gray during simulated hemorrh age in conscious rabbits. Am J Physiol Regul Integr Comp Physiol. 290, R71525. Schenberg, L. C. Lovick, T. A., 1995. Attenuation of the midbrainevoked defense reaction by selective stimulation of medullary raphe neurons in rats. Am J Physiol. 269, R137889. Scrogin, K. E., 2003. 5 HT1A Receptor Agonist 8OH DPAT acts in the Hindbrain to Reverse the Sympatholytic Response to Severe Hemorrhage. American Journal of Physiology (Regul Integr Comp). 284, R782R791. Scrogin, K. E., Johnson, A. K. Brooks, V. L., 2000. Methylsergide delays the decompensatory responses to severe hemorrhage by activating 5HT1A receptors. American Journal of Physiology (Integrative Comp Physiol). 279, R1776R1786. Scrogin, K. E., Veelken, R. Johnson, A. K., 1998. Central methysergide prevents renal sympathoinhibition and bradycardia during hypotensive hemorrhage. Am J Physiol. 274, H4351.
174 Seeger, T. F., Sforzo, G. A., Pert, C. B. Pert, A., 1984. In vivo autoradiography: visualization of stress induced changes in opiate receptor occupancy in the rat brain. Brain Res. 305, 30311. Sexton, W., 1995. Vascular adaptations in rat hindlimb skeletal muscle after voluntary running wheel exercise. J Appl Physiol. 79, 287296. Shirley, D., MacRae, K. Walker, J., 1991. The renal vascular response to mild and severe haemorrhage in the anaesthetized rat. J Physiol. 433, 373382. Shoji, M., Kimura, T., Kawarabayasi, Y., Ota, K., Inoue, M., Yamamoto, T., Sato, K., Ohta, M., Funyu, T., Yamamoto, T. et al., 1993. Effects of acute hypotensive hemorrhage on arginine vasopressin gene transcription in the rat brain. Neuroendocrinology. 58, 6306. Shyu, B. C., Andersson, S. A. Thoren, P., 1982. Endorphin mediated increase in pain threshold induced by long lasting exercise in rats. Life Sci. 30, 83340. Slimme r, L. M. Blair, M. L., 1996. Female reproductive cycle influences plasma volume and protein restitution after hemorrhage in the conscious rat. Am J Physiol. 271, R62633. Smith, M. A., Barrett, A. C. Picker, M. J., 1999. Antinociceptive effects of opioids following acute and chronic administration of butorphanol: influence of stimulus intensity and relative efficacy at the mu receptor. Psychopharmacology (Berl). 143, 2619. Smith, M. A. Yancey, D. L., 2003. Sensitivity to the effects of opioids in rats with free access to exercise wheels: muopioid tolerance and physical dependence. Psychopharmacology (Berl). 168, 42634. Snowball, R. K., Semenenko, F. M. Lumb, B. M., 2000. Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study. Neuroscience. 99, 35161. Sommers, D. K., Loots, J. M., Simpson, S. F., Meyer, E. C., Dettweiler, A. Human, J. R., 1990. Circulating met enkephalin in trained athletes duri ng rest, exhaustive treadmill exercise and marathon running. Eur J Clin Pharmacol. 38, 3912. Souza, S. B., Flues, K., Paulini, J., Mostarda, C., Rodrigues, B., Souza, L. E., Irigoyen, M. C. De Angelis, K., 2007. Role of exercise training in cardiovascular autonomic dysfunction and mortality in diabetic ovariectomized rats. Hypertension. 50, 78691. Stranahan, A. M., Khalil, D. Gould, E., 2006. Social isolation delays the positive effects of running on adult neurogenesis. Nat Neurosci. 9, 52633.
175 Strittmatter, R. R. Schadt, J. C., 2007. Sex differences in the respiratory response to hemorrhage in the conscious, New Zealand white rabbit. Am J Physiol Regul Integr Comp Physiol. 292, R19639. Sutoo, D. Akiyama, K., 2003. Regulation of brain function by exercise Neurobiol Dis. 13, 114. Swanson, L. W. Sawchenko, P. E., 1983. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu Rev Neurosci. 6, 269324. Tanaka, S., Fan, L. W., Tien, L. T., Park, Y., Liu Chen, L. Y., Rockhold, R. W. Ho, I. K., 2005. Butorphanol dependence increases hippocampal kappaopioid receptor gene expression. J Neurosci Res. 82, 25563. Tarvainen, M. P., RantaAho, P. O. Karjalainen, P. A., 2002. An advanced detrending method with application to HRV analys is. IEEE Trans Biomed Eng. 49, 1725. Thoren, P., 1979. Role of cardiac vagal C fibers in cardiovascular control. Rev Physiol Biochem Pharmacol. 86, 194. Thrasher, T. N. Keil, L. C., 1998. Arterial baroreceptors control blood pressure and vasopressin resp onses to hemorrhage in conscious dogs. Am J Physiol. 275, R184357. Tierney, G., Carmody, J. Jamieson, D., 1991. Stress analgesia: the opioid analgesia of long swims suppresses the nonopioid analgesia induced by short swims in mice. Pain. 46, 8995. Troy, B. P., Heslop, D. J., Bandler, R. Keay, K. A., 2003. Haemodynamic response to haemorrhage: distinct contributions of midbrain and forebrain structures. Autonomic Neuroscience: Basic and Clinical. 108, 111. Tsukamoto, K. Sved, A. F., 1993. Enhanced gammaaminobutyric acidmediated responses in nucleus tractus solitarius of hypertensive rats. Hypertension. 22, 81925. Unterwald, E. M., Knapp, C. Zukin, R. S., 1991. Neuroanatomical localization of kappa 1 and kappa 2 opioid receptors in rat and guinea pig br ain. Brain Res. 562, 5765. Vagg, D. J., Bandler, R. Keay, K. A., 2008. Hypovolemic shock: critical involvement of a projection from the ventrolateral periaqueductal gray to the caudal midline medulla. Neuroscience. 152, 1099109. Van Bockstaele, E. J., Bajic, D., Proudfit, H. Valentino, R. J., 2001. Topographic architecture of stress related pathways targeting the noradrenergic locus coeruleus. Physiol Behav. 73, 27383.
176 van Praag, H., Kempermann, G. Gage, F. H., 1999. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci. 2, 26670. Vaynman, S. Gomez Pinilla, F., 2005. License to run: exercise impacts functional plasticity in the intact and injured central nervous system by using neurotrophins. Neurorehabil Neural Repair. 19, 28395. Vitela, M. Mifflin, S. W., 2001. gamma Aminobutyric acid(B) receptor mediated responses in the nucleus tractus solitarius are altered in acute and chronic hypertension. Hypertension. 37, 61922. Walker, E. A. Young, A. M., 2001. Di fferential tolerance to antinociceptive effects of mu opioids during repeated treatment with etonitazene, morphine, or buprenorphine in rats. Psychopharmacology (Berl). 154, 13142. Wang, L., Medina, V. M., Rivera, M. Gintzler, A. R., 1996. Relevance of ph osphorylation state to opioid responsiveness in opiate naive and tolerant/dependent tissue. Brain Res. 723, 619. Wang, W. H. Lovick, T. A., 1993. The inhibitory effect of the ventrolateral periaqueductal grey matter on neurones in the rostral ventrolateral medulla involves a relay in the medullary raphe nuclei. Exp Brain Res. 94, 295300. Warburton, D. E., Nicol, C. W. Bredin, S. S., 2006. Health benefits of physical activity: the evidence. Cmaj. 174, 8019. Ward, D. G., 1989. Neurons in the parabrachial nuclei respond to hemorrhage. Brain Research. 491, 8092. Ward, D. G. Darlington, D. N., 1987. A blood pressure lowering effect of lesions of the caudal periaqueductal gray: relationship to basal pressure. Brain Res. 423, 3737. Ward, D. G. Darlington, D. N ., 1987. Lesions of the caudal periaqueductal gray prevent compensation of arterial pressure during hemorrhage. Brain Res. 407, 36975. Wolinsky, T. D., Carr, K. D., Hiller, J. M. Simon, E. J., 1996. Chronic food restriction alters mu and kappa opioid rece ptor binding in the parabrachial nucleus of the rat: a quantitative autoradiographic study. Brain Res. 706, 3336. Wu, Q., Boyle, M. P. Palmiter, R. D., 2009. Loss of GABAergic signaling by AgRP neurons to the parabrachial nucleus leads to starvation. Cell 137, 122534. Xia, Y. Haddad, G. G., 1991. Ontogeny and distribution of opioid receptors in the rat brainstem. Brain Res. 549, 18193. Yang, C. C. Kuo, T. B., 1999. Assessment of cardiac sympathetic regulation by respiratory related arterial pressure variability in the rat. J Physiol. 515 ( Pt 3), 88796.
177 Yasuda, K., Raynor, K., Kong, H., Breder, C. D., Takeda, J., Reisine, T. Bell, G. I., 1993. Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. Proc Natl Acad Sci U S A. 90, 673640. Zhu, G., Gao, L., Patel, K., Zucker, I. Wang, W., 2004. ANG II in the paraventricular nucleus potentiates the cardiac sym pathetic afferent reflex in rats with heart failure. J Appl Physiol. 97, 17461754. Zucker, I. H., Patel, K. P., Schultz, H. D., Li, Y. F., Wang, W. Pliquett, R. U., 2004. Exercise training and sympathetic regulation in experimental heart failure. Exerc Sp ort Sci Rev. 32, 107 11.
178 BIOGRAPHICAL SKETCH Joslyn K. Ahlgren was born and raised in Wichita, Kansas After graduating Suma Cum Laude with a Bachelor of Science degree in k inesiology from Kansas State University in May 2002, Joslyn began pursuing a gr aduate education in the field of autonomic n europhysiology. Joslyn joined the laboratory of Dr. Linda Hayward in June of 2004 and has spent the last five years studying the central effects of exercise training. During her graduate studies, Joslyn was awarded a Predoctoral Fellowship from the Florida/Puerto Rico affiliate of the American Heart Association. She received her Doctor of Philosophy degree in v eterinary m edical sciences from the Department of Physiological Sciences in the College of Veterinary Medicine in December 2009 and immediately began a teaching career in the Department of Applied Physiology and Kinesiology in the University of Floridas College of Health and Human Performance.