The Role of the Sympathetic Nervous System on Cerebral Blood Flow Regulation

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1 University of North Texas Health Science Center UNTHSC Scholarly Repository Theses and Dissertations The Role of the Sympathetic Nervous System on Cerebral Blood Flow Regulation Sushmita Purkayastha University of North Texas Health Science Center at Fort Worth, Follow this and additional works at: Recommended Citation Purkayastha, S., "The Role of the Sympathetic Nervous System on Cerebral Blood Flow Regulation" Fort Worth, Tx: University of North Texas Health Science Center; (2010). This Dissertation is brought to you for free and open access by UNTHSC Scholarly Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UNTHSC Scholarly Repository. For more information, please contact

2 Purkayastha, Sushmita, The Role of the Sympathetic Nervous System on Cerebral Blood Flow Regulation. Doctor of Philosophy (Biomedical Science), November, 2010\ 99 pp; 2 tables; 15 figures; bibliography Despite evidence of an abundance of sympathetic and parasympathetic nerve fibers innervations of the α-adrenergic receptors in animals, the contribution of the sympathetic nervous system in the regulation of the cerebral vasculature in humans remains controversial. Previous investigators state that catecholamines do not penetrate the blood brain barrier (BBB), however, the tight junctions of the endothelium of the brain s arterioles are leaky and subject to modulation. An investigation of healthy human subjects and neurologically impaired patients using the norepinephrine spillover technique identified the presence of functional α adrenergic receptors within the cerebral vasculature. Therefore, we propose that the more permeable BBB surrounding the arterioles of the cerebral circulation allows the α-adrenergic receptors access to circulating as well as locally released norepinephrine within the brain, especially during exercise. Furthermore, arterial baroreceptor control of the cerebral vasculature has not been confirmed. However, our laboratory has demonstrated that α-1 adrenergic receptor blockade during acute hypotension influences dynamic cerebral autoregulation (dca) and baroreflex mediated changes in cerebral blood flow (CBF). Therefore, the purpose of the investigations in this dissertation was to: i) determine if pharmacologic activation and blockade of α-1 adrenergic receptors resulted in

3 cerebral vasoconstriction and vasodilatation, respectively, during rest and dynamic exercise; and ii) investigate the role of arterial baroreflex control of SNA on CBF regulation during rest and dynamic exercise. Pulsatile stimulation of the carotid baroreflex (CBR) with neck pressure (NP) and neck suction (NS) was used to entrain the variability of the reflex changes of the cerebral hemodynamic. In the first investigation we demonstrated an increase in cerebrovascular tone with activation of α-1 adrenergic receptor agonist with phenylephrine and with dynamic exercise. However, blockade of α-1 adrenergic receptors abolished the increase in vascular tone and caused impairment in dynamic CA. In the second investigation we demonstrated an increase in variability of the CBF and cerebral tissue oxygenation with pulsatile NP and this variability was abolished with α-1 adrenergic receptor blockade with prazosin. These findings identify the functional role of the α-1 adrenergic receptors in establishing cerebral vascular tone and their effects on dynamic CA and beat-to-beat dynamic CBF regulation.

4 THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM ON CEREBRAL BLOOD FLOW REGULATION DISSERTATION Presented to the Graduate Council of the Graduate School of Biomedical Sciences University of North Texas Health Science Center at Fort Worth In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSPHY By SUSHMITA PURKAYASTHA Fort Worth, Texas, U.S.A. November 2010

5 ACKNOWLEDGEMENTS I thank everyone who has provided me with their technical, intellectual, and emotional support and assistance throughout the duration of the completion of these projects. I especially thank my mentor, Dr. Peter B. Raven who has provided me with continued support, guidance and encouragement throughout from the conception to the completion of these projects, as well as with the development of myself as a graduate student and researcher. He has continually encouraged and guided me throughout my time here and has certainly made me a better scientist and person for it. He has demonstrated nothing less than excellence as a mentor. Special thanks go out to each member of my committee for their assistance and guidance, Dr. James Caffrey, Dr. Steve Mifflin, Dr. Michael Smith and Dr. Shaohua Yang. I would also like to recognize the valuable inputs and guidance provided by Dr. Jeff Potts during my Research Proposal Defense. In addition, I would like to recognize and thank all of my former and current laboratory partners, Dr. Robert Brothers, Dr. Megan Murphy, Quinton Barnes and Dr. Ashwini Saxena, who were instrumental in each phase of my project. Special thanks go out to Dr. Shigehiko Ogoh, Dr. Wendy L. Eubank and Dr. Christina F. Kneip for their help and troubleshooting during my experiments. Thanks to Darice Yoshishige for her support in the technical aspect of catecholamine measures. I certainly thank my family for their support of me as long as I remember. I also thank my husband Biswadip for showing patience and being supportive of me throughout and my sons Sujoy and Sujosh for their love and support and I appreciate it all. Thank You. The project was supported by Department of Integrative Physiology and the Cardiovascular Research Institute at UNTHSC. iii

6 My education and research training was supported by the Department of Integrative Physiology at UNTHSC and the NIH RO-1 # HL45547 awarded to Dr. Raven. The following are original articles published and presentations as a result of this support. Original Articles Purkayastha, S., Saxena, A., Barnes, Q., Eubank, W.L., Hoxha, B., Raven, P.B. Alpha- 1adrenergic receptor control of the cerebral vasculature in humans during rest and exercise. In submission to The Journal of Physiology. Purkayastha, S., Barnes, Q., Saxena, A., Eubank, W.L., Hoxha, B., Raven, P.B Influence of the Carotid Baroreflex on the cerebral vasculature in humans during rest and exercise. In submission to The Journal of Physiology. Hawkins MN, Barnes Q, Purkayastha S, Eubank W, Ogoh S, Raven PB. The effects of aerobic fitness and beta1-adrenergic receptor blockade on cardiac work during dynamic exercise. J Appl Physiol Feb; 106 (2): Cramer JT, Beck TW, Housh TJ, Massey LL, Marek SM, Danglemeier S, Purkayastha S, Culbertson JY, Fitz KA, Egan AD. Acute effects of static stretching on characteristics of the isokinetic angle - torque relationship, surface electromyography, and mechanomyography. J Sports Sci Apr 25(6): Ogoh S, Fisher JP, Purkayastha S, Dawson EA, Fadel PJ, White MJ, Zhang R, Secher NH, Raven PB. Regulation of middle cerebral artery blood velocity during recovery from dynamic exercise in humans. J Appl Physiol Feb; 102(2): Purkayastha S, Cramer JT, Trowbridge CA, Fincher AL, Marek SM. Surface electromyographic amplitude-to-work ratios during isokinetic and isotonic muscle actions. J Athl Train Jul-Sep; 41(3): Ogoh S, Brothers RM, Barnes Q, Eubank WL, Hawkins MN, Purkayastha S, O-Yurvati A, Raven PB. Effects of changes in central blood volume on carotid-vasomotor baroreflex sensitivity at rest and during exercise. J Appl Physiol Jul; 101(1): Ogoh S, Brothers RM, Barnes Q, Eubank WL, Hawkins MN, Purkayastha S, O-Yurvati A, Raven PB. The effect of changes in cardiac output on middle cerebral artery mean blood velocity at rest and during exercise. J Physiol Dec 1; 569(Pt 2): iv

7 S, Brothers RM, Barnes Q, Eubank WL, Hawkins MN, Purkayastha S, O-Yurvati A, Raven PB. Cardiopulmonary baroreflex is reset during dynamic exercise. Ogoh J Appl Physiol Jan; 100(1):51-9. Marek SM, Cramer JT, Fincher AL, Massey LL, Dangelmaier SM, Purkayastha S, Fitz KA, Culbertson JY. Acute Effects of Static and Proprioceptive Neuromuscular Facilitation Stretching on Muscle Strength and Power Output. J Athl Train Jun; 40(2): Presentations: The Influence of Autonomic Neural Control on Human Cerebral Vessels in vivo. Purkayastha, S, Zhang, R, Levine, B, Raven, PB. Will be presented at 21st International Symposium on the Autonomic Nervous System. Effect of angiotensin (AT1) receptor blockade on cerebral autoregulation in healthy human subjects at rest. Sushmita Purkayastha, R. Mathew Brothers, Peter B. Raven FACSM and Shigehiko Ogoh.Department of Integrative Physiology, University of North Texas, Fort Worth, TX, 76107, USA. Presented at Texas chapter of American College of Sports Medicine (TACSM), Modulation of vascular impedance in the femoral artery of humans with neck pressure and neck suction with KATP channel blockade. Sushmita Purkayastha, Shigehiko Ogoh, David M. Keller, and Peter B. Raven, FACSM. Presented at Texas chapter of American College of Sports Medicine (TACSM), Electromyographic amplitude-to-work (EMG:WK) ratios during isotonic and isokinetic leg extension exercises. Sushmita Purkayastha, Joel T. Cramer, PhD. Department of Kinesiology, University of Texas at Arlington. Presented at Texas chapter of American College of Sports Medicine (TACSM), Abstracts: Ogoh, S, Brothers, R.M., Barnes, Q., Eubank, W.L., Hawkins, M.N., Purkayastha, S., Albert O-Yurvati and P.B. Raven. The Cardiopulmonary baroreflex is reset during dynamic exercise. 2 nd Joint Meeting of the EFAS and AAS, 2005 (Los Cabos, Mexico). v

8 Raven, P.B., Brothers, R.M., Barnes, Q., Eubank, W.L., Hawkins, M.N., Purkayastha, S., Albert O-Yurvati, and Ogoh, S. Changes in cardiac output effect the middle cerebral artery blood velocity at rest and during exercise. 2 nd Joint Meeting of the EFAS and AAS, 2005 (Los Cabos, Mexico). Brothers, R.M., Purkayastha, S., Ogoh, S., O-Yurvati, A., Caffrey, J., and Raven, P.B. Angiotensin II release during maximal and sub-maximal exercise. MSSE, Ogoh, S., Brothers, R.M., Barnes, Q., Eubank, W.L., Hawkins, M.N., Purkayastha, S., Albert O-Yurvati, Raven, P.B. The effects of changes in central blood volume on carotid baroreflex sensitivity at rest and exercise , FASEB, vi

9 TABLE OF CONTENTS ACKNOWLEDGEMENTS... iii LIST OF TABLES... ix LIST OF FIGURES... x LIST OF ABBREVIATIONS... xii INTRODUCTION... 1 REVIEW OF RELATED LITERATURE... 1 SPECIFIC AIMS... 8 EXPERIMENTAL DESIGN:... 9 METHODS: REFERENCES: CHAPTER II Alpha-1 adrenergic receptor control of the cerebral vasculature in humans during rest and exercise ABSTRACT: INTRODUCTION: METHODS: RESULTS: DISCUSSION: ACKNOWLEDGEMENTS: REFERENCES: FIGURE LEGENDS: CHAPTER III Influence of the Carotid Baroreflex on the Cerebral Vasculature in Humans during Rest and Exercise ABSTRACT: INTRODUCTION: METHODS: RESULTS: vii

10 DISCUSSION: ACKNOWLEDGEMENTS: REFERENCES: FIGURE LEGENDS FIGURES CHAPTER IV CONCLUSIONS CHAPTER V SUGGESTIONS FOR FUTURE RESEARCH REFERENCES: viii

11 Chapter I LIST OF TABLES Chapter II Table 1: Hemodynamic variables at rest and during low exercise intensity at heart rate 88 (Ex88) and moderate exercise intensity (Ex130) during control, with phenylephrine (PE) and 2 hours following prazosin ingestion Chapter III Table 2: Hemodynamic variables at rest and during low exercise workload at heart rate 84 bpm (Ex84) and moderate exercise workloaaad (Ex130) during control and 2 hours following prazosin ingestion ix

12 LIST OF FIGURES Chapter I Chapter II Figure 1. Protocol outline for the controland drug (phenylephrine and prazosin) conditions Average mean arterial pressure and middle cerebral artery blood velocity at rest, Ex88 and Ex130 during control, PE and with prazosin Average norepinephrine concentrations at rest, Ex88 and Ex130 during control PE and with prazosin Average low frequency gain at rest, Ex88 and Ex130 during control, PE and prazosin Representative critical closing pressure calculation graph Average critical closing pressure at rest, Ex88 and Ex130 during control, PE and prazosin Average cerebrovascular conductance index at rest, Ex88 and Ex130 during control. PE and prazosin Chapter III Figure 1. Protocol outline for the control and prazosin conditions Group power spectral density of MAP Group power spectral density of MCA V Group power spectral density of ScO Peak power spectral density of MAP at 0.1 Hz Peak power spectral density of MCA V at 0.1 Hz x

13 7. Peak spectral density of ScO 2 at 0.1 Hz Group averaged percentage change in power spectral density from baseline xi

14 LIST OF ABBREVIATIONS ABP BBB CA CBF CCP CVCi E ECG h HF HR IL-6 LF MAP MCA V mmhg NE NP NIRS NS PE PSD rpm arterial blood pressure blood brain barrier cerebral autoregulation cerebral blood flow critical closing pressure cerebrovascular conductance index epinephrine Electrocardiogram hour high frequency heart rate Interleukin-6 low frequency mean arterial pressure middle cerebral artery blood velocity millimeters of mercury norepinephrine neck pressure near infrared spectroscopy neck suction phenylephrine power spectral density revolutions per minute xii

15 ScO 2 SE TCD VLF W WL cerebral tissue oxygenation standard error transcranial Doppler very low frequency watt workload α-1 alpha-1 adrenergic receptor β-1 beta-1 adrenergic receptor xiii

16 CHAPTER I INTRODUCTION REVIEW OF RELATED LITERATURE Functional role of the sympathetic nervous system on the cerebral vasculature: Perivascular nerves have been identified in close proximity to smooth muscles in cerebral vessel (Willis, 1664; Benedikt, 1874; Aronson, 1890) and the density of innervations of cerebral resistance vessels have been reported to be similar to the mesenteric and/or the femoral arterial beds (Rosenblum & Chen, 1976; Rosenblum, 1976). Animal studies have also identified that the cerebral arteries are richly innervated with sympathetic nerve fibers (Nielsen & Owman, 1967; Nelson & Rennels, 1970; Edvinsson, 1975). However, the role of autonomic neural control of the cerebral blood flow (CBF) regulation remains controversial (Ogoh et al., 2008; Zhang et al., 2009; Hamner et al., 2010). The traditional thinking is that in the presence of normocapnia and normotension changes in sympathetic tone appear to have limited effects on CBF (Fitch et al., 1975; Heistad et al., 1978). However, activation of the sympathetic nervous system on cerebral vessels is effective during hypertension, hypoxia, hypercapnia, and hemorrhagic states (Busija & Heistad, 1984). Furthermore, several investigators have identified a direct effect of sympathoexcitation on CBF in pathophysiology (Pearce & D'Alecy, 1980; Jordan et al., 1998). In addition, the blood flow of primates in to the brain via internal carotid and vertebral arteries was reduced by direct stimulation of the cervical and stellate ganglia (Meyer et al., 1967). In humans, a decrease in middle cerebral artery blood velocity (MCA V) was reported during unilateral trigeminal ganglion stimulation (Visocchi et al., 1994). There is also strong evidence 1

17 that increases in sympathetically mediated vasoconstriction protects cerebral vessels from overperfusion during hypertension. In an animal model it has been demonstrated that hypertension breaksdown the blood brain barrier (BBB) in the cerebrum and this disruption was prevented by electrically induced sympathetic stimulation (Bill & Linder, 1976). Together these findings suggest a functional role of sympathetic nervous system in CBF regulation. Indeed, the extension of the high pressure value (> 160 mmhg arterial pressure) at which cerebral autoregulation fails in hyperadrenergic diseases has been proposed to be linked to the degree of sympathoexcitation (Immink et al., 2004; Low et al., 2009). In addition, the exponential increase in sympathetic activity that occurs during increasing work load exercise to maximum in healthy subjects where systolic blood pressure exceeds (200 mmhg in dynamic exercise, more than 400 mmhg in weight lifting) the autoregulatory range of 160 mmhg will increase cerebrovascular tone, and will buffer the pulsatile increases in cerebral perfusion and pressure (Ogoh et al., 2005). The role of α-1 adrenergic receptors on the cerebral vasculature: Similar to the systemic vasculature, cerebral vessels are richly innervated with sympathetic nerve fibers connected to α-1 adrenergic receptors that appear to be located on the cerebral arterioles (Edvinsson, 1982).Stimulation of stellate and superior cervical ganglia results in cerebral vasoconstriction (Meyer et al., 1967). In the vascular smooth cells α-1 adrenergic receptors bind to norepinephrine, or other sympathomimetic drugs, and via receptor coupling with its Gq-Protein stimulates Phospholipase C (PLC) activity which then promotes hydrolysis of phosphatidylinositol bisphosphate producing inositol triphosphate and diacylglycerol. These molecules act as second messengers mediating intracellular Ca 2+ release from non-mitochondrial pools and activate protein kinase C, which then leads to activation of contractile proteins and 2

18 vasoconstriction (Guimaraes & Moura, 2001). Strandgaard et al. (Strandgaard & Sigurdsson, 2008) have stated that vasoactive amines like catecholamines do not penetrate the BBB and only influence cerebral resistance vessels from outside the BBB. Consequently they conclude that the sympathetic nerves do not effectively control the inner vascular smooth muscle of the cerebral vessels. Sandor (Sandor, 1999) refuted the impermeability of the BBB and reported that the peripheral adrenergic neurons come in close contact with the smooth muscle layer of the cerebral vessels. Although, there are reports of impermeable tight junctions on the BBB on the capillary endothelium, the tight junctions of the BBB in brain arterioles and venules of the cerebral microvasculature are found to be leaky and subject to modulation (Abbott et al., 2006). Furthermore, there are reductions in the tightness of the BBB by increases in arterial pressure (Bill & Linder, 1976), free radicals and IL-6 (Abbott, 2005) which are abundant during hyperadrenergic states and dynamic exercise (Pedersen & Febbraio, 2008). Vasoactive substances, such as, bradykinin and nitric oxide, also appear to increase permeability of the BBB via activation of second messenger pathways (Mayhan, 2001). The resultant increase in permeability of the BBB makes the α-1 adrenergic receptors accessible to the circulating norepinephrine along with local release of NE from nerve endings within the brain. Mitchell et al. have (Mitchell et al., 2009) demonstrated norepinephrine (NE) spillover from the cerebral vasculature into the internal jugular vein and identified that NE spillover originates primarily from the cerebral vasculature outside the BBB, or more specifically the accessible arteriolar α-1 adrenergic receptors, and that the lipophilic metabolite spillover originates from both sides of the BBB. In Mitchell et al. s (Mitchell et al., 2009) findings it was noted that pure autonomic failure patients had 77 % lower brain NE spillover than found in the healthy subjects indicating that the sympathetic degeneration associated with pure autonomic failure limited the NE spillover. 3

19 Moreover, evidence of functional α-1 adrenoreceptors was demonstrated by an increase in cerebral vascular conductance (CVCi) following an experimentally induced acute hypotension with thigh cuff occlusion/release technique designed to stimulate the sympathetic nerve activity (Ogoh et al., 2008). The increase in CVCi was attenuated by selective α-1 adrenergic blockade (prazosin) reflecting a sympathetically mediated dynamic control of the cerebral vasculature. In addition, attenuation of sympathetically mediated vascular tone induced by spinal cord stimulation (SCS) resulted in increases in CBF due to withdrawal of sympathetic activation of the α-1 adrenergic receptors (Visocchi et al., 1994; Patel et al., 2003). In healthy subjects Brassard et al. (Brassard et al., 2009) demonstrated a reduction in cerebral tissue oxygenation after infusion of NE. These findings identify the presence of functional α-1 adrenoreceptors in the cerebral vasculature. Zhang et al. (Zhang et al., 2009) demonstrated a progressive increase in cerebrovascular resistance (CVRi) with incremental dosage of phenylephrine (PE) in healthy subjects at rest and concluded that cerebral autoregulatory changes in myogenic tone were primarily responsible for the increase in CVRi. However, when the data was presented as changes in CVCi, where the PE induced increases in blood pressure was mathematically accounted for (O'Leary, 1991), a α-1 adrenergic mediated vasoconstriction was identified. Recently, Brassard et al. (Brassard et al., 2010) reported a decrease in cerebral tissue oxygenation at rest with bolus PE infusion which was attenuated during light intensity cycling exercise and abolished with high intensity exercise mediated increase in cerebral metabolism indicating the presence of functional sympatholysis (Remensnyder et al., 1962) as evidenced in the peripheral vasculature (Keller et al., 2004). Ogoh et al. (Ogoh et al., 2010) have also demonstrated increases in critical closing pressure (CCP), an index of cerebral vascular tone, in 4

20 relation to increases in exercise intensity. These data strongly indicate a functional role of the α-1 adrenergic receptor mediated sympathetic nerve activity in the cerebral vasculature. Measurement of static versus dynamic changes in cerebral blood flow: Static cerebral autoregulation (sca) refers to the relative steady-state relationship between CBF and arterial blood pressure (ABP), characterized by a positive slope of 0.8% (often referred to as a plateau) increase in CBF/mmHg between the autoregulatory range of mmhg ABP (Heisted & Kontos, 1983; Lucas et al., 2010). Dynamic cerebral autoregulation (dca) corresponds to the transient response of CBF to beat-to-beat fluctuations in ABP and acute changes in arterial pressure (Tiecks et al., 1995; Panerai, 2008, Aaslid et al. 1989). Hence, dynamic dca is important for rapidly adjusting the CBF to its steady-state flow, when challenged by acute changes in perfusion pressure (Ogoh & Ainslie, 2009). The importance of dca on sca and its effect on oxygen delivery to the brain is at the forefront of current research (Ogoh & Ainslie, 2009). Historically the predominant measurement technique for assessing sca was the Kety-Schmidt technique using 133 Xenon which required one to establish steady-state conditions for the measure to be valid. This process resulted in elimination of dynamic assessment of changes in CBF resulting from transient changes in ABP. Therefore, the dynamic changes in CBF were undetected resulting in a reported constant CBF. Consequently the cerebral metabolic rate for oxygen during exercise has been reported as unchanged (Madsen et al., 1993). Furthermore, the jugular venous sampling required for the Kety-Schmidt method was compromised, because it is now known that the internal jugular vein is collapsed in the upright position (Dawson et al., 2004; Gisolf et al., 2004). In the upright posture, venous drainage from the brain is dependent on the spinal veins of the vertebral plexus (Valdueza et al., 2000; Cirovic et al., 2003; Alperin et al., 2005). In addition, it is known that cerebral activation was directly 5

21 associated with increases in regional CBF and metabolism, as detected by positron emission tomography (PET) (Secher et al., 2008).With the advent of dynamic measurement techniques, such as transcranial Doppler (TCD) of middle cerebral blood velocity (MCA V) changes and near infrared spectroscopic (NIRS) analysis of cerebral tissue oxygenation (ScO 2 ), the dynamic regulation of CBF by dca, when ABP is rapidly changing, can be accomplished. Because of these techniques, there is a growing body of evidence identifying that increases in sympathetic neural activity result in local release of NE within the brain and circulating NE which can bind to the accessible α-1 adrenergic receptors on the smooth muscle of the cerebral arterioles and influence dca and sca (Ogoh et al., 2008; Brassard et al., 2009). The MCA V obtained from TCD does not take into account the vessel diameter which could possibly be increased due to sympathetically mediated decrease in vessel diameter. However, during exercise the MCAV increases in parallel with the internal carotid artery blood flow (Huang et al., 1992; Hellstrom et al., 1996). This suggests that sympathetic modulation does not significantly alter the diameter of the MCA and that sympathetic regulation of CBF occurs mainly in the cerebral resistance arterioles rather than in the large cerebral conductance arteries. It is reasonable to propose that if the diameter of the MCA decreases in response to sympathoexcitation the MCA V would increase, in fact the opposite occurs and conductance is decreased, indicating that sympathetic vasoconstriction of the cerebral vessels occurs at the small resistance arterioles. Arterial baroreflex interaction with the cerebral vasculature: Studies in the past were unable to identify any relationship between the cerebral vasculature and its response to carotid baroreceptor stimulation (Rapela et al., 1967; Heistad et al., 1980) despite cerebral arteries being richly innervated with sympathetic nerve fibers (Nelson & Rennels, 1970; Edvinsson, 1982). However, a reduction in CBF was observed with sino-aortic 6

22 de-afferentation, a procedure that produces marked elevation of peripheral sympathetic tone and extreme hypertension (20). Stimulation of the carotid baroreceptors of baboons decreased their CBF, while at the same time maintaining the cerebral inflow pressure (Ponte & Purves, 1974). Similar results were obtained in a human study where middle cerebral artery blood velocity (MCA V) decreased during unilateral trigeminal ganglion stimulation (Visocchi et al., 1994). Zhang et al. (Zhang et al., 2002) have demonstrated that dca is altered with ganglion blockade using Trimethaphan and thus speculated a tonic control of the autonomic nervous system in beatto-beat CBF regulation. Patients with idiopathic orthostatic intolerance exhibit an excessive decline in CBF upon orthostasis despite sustained systemic blood pressure. However, this decline in CBF was abolished with Phentolamine blockade of the α-adrenoreceptors during head up tilt (Jordan et al., 1998). β-1 adrenergic blockade during dynamic cycling exercise in healthy humans diminished cardiac output as well as MCA V. In addition, because stellate ganglion blockade eliminated the β-1 blockade induced decreases in MCA V, it was concluded that the exercise pressor reflex induced sympathoexcitation resulting from underperfused exercising muscle was the cause of the reduction in MCA V (Ide et al., 2000). The decrease in MCA V is indicative of an augmentation of sympathetic mediated vasoconstriction in the cerebral vessels (Ide & Secher, 2000). During recovery from acute hypotension induced by an ischemic thigh cuff occlusion/release protocol (Aaslid et al., 1989) sympathoexcitation was evident from the recovery in cerebral vascular conductance (CVCi) identified by the rate of return (RoR). The RoR was attenuated by α-1 adrenoreceptor blockade (Ogoh et al., 2008) indicating arterial baroreflex control of the cerebral circulation via the sympathetic nervous system. Therefore, we conclude that the arterial baroreflex mediated alterations of sympathetic nervous system are crucial in the beat-to-beat regulation of CBF. 7

23 SPECIFIC AIMS: The findings summarized in the above introduction clearly indicate the presence of a dynamic sympathetically mediated control of the cerebral vasculature. This conclusion is in marked contrast to the generally accepted suggestion that sympathetic control of the cerebral vasculature is minimal (Heistad et al., 1983; Edvinsson & Hamel, 2002; Strandgaard & Sigurdsson, 2008). Therefore, we hypothesize that functional sympathetic neural activity is present in the cerebral vasculature and operates independent and in consort with arterial carbon dioxide tension and cerebral autoregulation in cerebral blood flow regulation. Activation of the sympathetic nervous system causes α-1 adrenergic receptor mediated modulation of vascular smooth muscle in cerebral vessels. To test this global hypothesis investigation into the following specific aims will be accomplished I. To test the hypothesis that pharmacologic activation (phenylephrine) and blockade (prazosin) of the α-1adrenergic receptors, respectively, during rest and steady state dynamic exercise will result in i.) cerebral vasoconstriction and increased cerebral vascular tone; and ii.) cerebral vasodilatation and decreased cerebral vascular tone, respectively. II. To test the hypothesis that dynamic arterial baroreflex control of sympathetic neural activity reflexly regulates cerebral blood flow at rest and during steady state dynamic exercise. 8

24 The experimental techniques and methodology designed to explore specific aims I, and II are specifically explained in chapters II and III. However, we propose below a general description of the experimental design, experimental protocols and methods used to address the specific aims. EXPERIMENTAL DESIGN: Middle cerebral artery blood velocity (MCA V) measurements allowed for assessment of cerebral blood flow (CBF) at rest and during low and moderate workload intensity on an upright cycle ergometer. Beat-to-beat arterial blood pressure was monitored using finger photoplethysmographic arterial blood pressure cuff and subsequently the relationship between mean arterial pressure (MAP) and MCA V were evaluated using transfer function analysis. In studies addressed in chapter II, low frequency (LF) transfer function gain was utilized to identify dynamic cerebral autoregulation (dca). A decrease in gain is interpreted as an increase in effective CA and an increase in gain is interpreted as a reduction in the effectiveness of dca. The calculation of changes in critical closing pressure (CCP) was used to identify the changes in the vascular tone of the cerebral circulation (Panerai et al., 1995; Aaslid et al., 2003, Ogoh et al., 2010). The CCP was estimated using systolic and diastolic arterial blood pressure values and their consecutive systolic and diastolic velocities of MCA V from twenty cardiac cycles during each condition. An increase in CCP is reflective of an increase in vascular tone and decrease in CCP indicates a decrease in vascular tone. Changes in LF transfer function gain and CCP were obtained during rest, low and moderate exercise workloads with a α-1 adrenergic agonist, phenylephrine, and an antagonist, prazosin, to examine dca and estimate cerebrovascular tone with and without activation of α-1 adrenergic receptors. 9

25 In studies addressed in chapter III, carotid baroreflex (CBR) entrainment with pulsatile neck pressure (NP at + 40 mmhg) and pulsatile neck suction (NS 40 mmhg) stimulus was performed at a predetermined frequency of 0.1 Hz during rest, low and moderate workloads. This dynamic input of the CBR at a constant frequency was presumed to be transduced to all end organs influenced by the CBR. Therefore, we evaluated measures of variability in the frequency domain of 0.1 Hz by analysis of power spectral density of MCA V and cerebral tissue oxygenation SCO 2 values obtained from trans cranial Doppler and near infrared spectroscopy (NIRS), respectively. Pharmacologic activation and blockade of α-1 adrenergic receptors: The functional role of sympathetic nervous system was examined by α-1 adrenergic receptors agonist phenylephrine (PE) and antagonist prazosin. In Specific aim I, a bolus injection (1.0 µg / kg body weight) of PE was administered during steady-state rest, low and moderate workload. An oral dose of prazosin (1 mg / 20 kg body weight) was given to assess the effect of antagonist blockade of the α-1 adrenergic receptors during steady-state rest, low and moderate workloads. A bolus of PE was infused prior to 2 h after prazosin ingestion to estimate the percentage of blockade of blood pressure response achieved by the drug. Dynamic CA was estimated by LF transfer function gain and estimation of cerebral vascular tone was obtained from CCP analysis. In specific aim II, the entrainment of CBR via NP induced sympathoexcitation and NS induced sympathetic withdrawal were examined in the control conditions and following α-1 adrenergic receptor blockade with prazosin during steady state rest, low and moderate workloads. 10

26 METHODS: A detailed description of the methods used to assess LF transfer function gain and CCP is provided in chapter II of the dissertation and CBR entrainment via NP and NS and power spectral analysis used to quantify the variability of CBR entrainment transduced to MCA V and ScO 2 are discussed in chapter III of the dissertation. A brief description of the methods used to measure beat-to-beat arterial blood pressure; MCA V and ScO 2 are provided in this chapter. Beat-to-beat arterial blood pressure recordings were obtained for each subject using finger photoplethysmographic arterial blood pressure cuff (Finometer). In six subjects beat-tobeat blood pressure was simultaneously acquired using a catheter inserted into the radial artery. The overall correlation between mean arterial pressure obtained by Finometer (y-axis) and direct radial arterial line (x-axis) was 0.88 and the regression equation y = 1.0x -3. MCA V was measured by transcranial Doppler (TCD) ultrasonography to assess CBF. In humans the MCA diameter remains relatively constant under a variety of conditions described previously (Schreiber et al., 2000; Serrador et al., 2000) and the diameter of large cerebral arteries do not change significantly during exercise and regulation of CBF takes place in smaller arteries and arterioles (Giller et al., 1993). Frontal lobe oxygenation was measured with near infrared spectroscopy (NIRS) and the near-infrared HbO 2 signal (arbitrary units, a.u.) was used as an index of cerebral tissue oxygenation (ScO 2 ) and provides indirect indication of vascular responses in the microcirculation (Fadel et al., 2004). The first four subjects tested were also instrumented with a sensor attached to a clip on the earlobe with a drop of electrolyte solution for detection of transcutaneous PCO 2. The PCO 2 measured by this technology reflects arterial PCO 2 and was found to remain constant across all the conditions of the experiment. 11

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33 ZHANG, R., BEHBEHANI, K. & LEVINE, B. D. (2009). Dynamic pressure-flow relationship of the cerebral circulation during acute increase in arterial pressure. J Physiol 587, ZHANG, R., ZUCKERMAN, J. H., IWASAKI, K., WILSON, T. E., CRANDALL, C. G. & LEVINE, B. D. (2002). Autonomic neural control of dynamic cerebral autoregulation in humans. Circulation 106,

34 CHAPTER II Alpha-1 adrenergic receptor control of the cerebral vasculature in humans during rest and exercise. Sushmita Purkayastha 1, Ashwini Saxena 1, Quinton Barnes 1, Wendy L. Eubank 1, Besim Hoxha 1, Peter B. Raven 1. In submission to The Journal of Physiology 19

35 ABSTRACT: Despite cerebral vessels being richly innervated with sympathetic nerve fibers connected to α-1 adrenergic receptors, the influence of the sympathetic nervous system on the cerebral vasculature remains equivocal. Blockade of the α-1 -adrenoreceptors led to impairment of cerebral autoregulation following acute hypotension in healthy humans which demonstrated a sympathetically mediated influence on cerebral autoregulation. Therefore, we tested the hypothesis that pharmacologic activation and blockade of the α-1 adrenoreceptors, respectively, during rest and steady-state dynamic exercise will result in cerebral vasoconstriction and increased cerebral vascular tone; and cerebral vasodilatation and decreased cerebral vascular tone, respectively. In ten subjects (7 men and 3 women) beat-to-beat arterial blood pressure (ABP) and middle cerebral artery blood velocity (MCA V) were determined during rest, and two workload (WL) intensities (low WL at 10W and moderate WL achieving a heart rate of 130 bpm) on an upright bicycle ergometer. The measurements were during control (no drug), with bolus phenylephrine (PE) injection and 2 h following prazosin ingestion. A decrease in transfer function gain in the low frequency (LF) range between mean arterial blood pressure and MCA V was observed at Ex130 from rest indicating a strengthening of cerebral autoregulation (CA) in the control condition. Prazosin caused an increase in LF gain at rest, Ex88 and Ex130 identifying impairment in CA. A stepwise increase in critical closing pressure (CCP) was observed from rest to Ex88 to Ex130 (from 18 ± 3 to 23 ± 4 to 31 ±4 mmhg) identifying an increase in cerebral vascular tone with exercise mediated augmentation of sympathetic activity. However, withdrawal of functional α 1 -adrenoreceptors with prazosin abolished the increases in CCP during Ex88 and Ex130 (from 21 ±4 to 22 ±4 to 24 ± 4 mmhg) despite the exercise induced activation of the sympathetic nervous system. These findings identify the functional role of the cerebral vascular α-1 adrenergic receptors in establishing cerebral vascular tone in conjunction with the 20

36 myogenic properties of the cerebral vasculature in modulating CA during rest and dynamic exercise in humans. Key Words: Critical Closing Pressure; Cerebral Vascular Tone; Cerebral Autoregulation; Transfer Function Gain. 21

37 INTRODUCTION: Despite the abundance of sympathetic nerve fibres emanating from the sympathetic ganglia that are connected to α-adrenergic receptors and innervate the cerebral arteries (Nielsen & Owman, 1967; Nelson & Rennels, 1970; Edvinsson, 1975; Heistad et al., 1978; Edvinsson, 1982; Hamel, 2006), the role of the sympathetic nerve activity in the regulation of the cerebral vasculature in humans remains equivocal. Previously, many authors have relied on the classic work of Olesen (Olesen, 1972) as documenting the inability of phenylephrine (PE) to cross the blood brain barrier (BBB). Olesen referred to the work of Weil-Malherbe et al (Weil-Malherbe et al., 1961) in which they radioactively tagged NE infused into brain circulation of cats at rest and identified regional areas of radioactivity not thought to require passage across the BBB. It is not clear whether the data obtained in these studies was confounded by species differences in the integrity of the BBB of cats compared to humans (Sandor, 1999). However, the actual experimental data of Olesen reported on the intra-arterial infusion of 133 Xenon for the steady-state measures of cerebral blood flow (CBF) following 2 min intra-carotid artery infusions of NE, epinephrine (E) and angiotensin (AII). However, the data did not provide evidence of vasoconstriction. Importantly, the investigator goes on to say that the results obtained in the present study do not speak against the vasoconstrictor effect of the sympathetic nerves on cerebral vasculature, as local release of transmitters from nerve endings might produce much higher concentrations locally on the receptor site that could possibly be achieved by diffusion from blood (Olesen, 1972). Furthermore, 133 Xenon technique of measuring CBF would be confounded by requiring a new steady state and would not identify dynamic changes in CBF. Similar to the systemic vasculature, the α-1 adrenoreceptors of the cerebral vessels appear to be located on the cerebral arterioles (Edvinsson, 1982). Strandgaard et al. (Strandgaard & Sigurdsson, 2008) have stated that vasoactive amines like catecholamines do not penetrate the 22

38 BBB and only influence cerebral resistance vessels from outside the BBB. Consequently they conclude that the sympathetic nerves do not effectively control the inner vascular smooth muscle of the cerebral vessels. However, Sandor (Sandor, 1999) refuted the impermeability of the BBB and reported that the peripheral adrenergic neurones come in close contact with the smooth muscle layer of the cerebral vessels. Although the BBB on the capillary endothelium have been found to have impermeable tight junctions, the BBB in the endothelium of brain arterioles and venules of the cerebral microvasculature are found to be leaky and subject to greater modulation (Abbott et al., 2006). Furthermore, there are data indicating reductions in the tightness of the BBB by increases in arterial pressure (Bill & Linder, 1976) and free radicals and IL-6, (Abbott, 2005), which are abundant during hyperadrenergic states and dynamic exercise (Pedersen & Febbraio, 2008). Vasoactive substances, such as, Bradykinin and Nitric Oxide, also appear to increase permeability of BBB via activation of second messenger pathways (Mayhan, 2001). We propose that the resultant increase in permeability of the BBB of the arterioles and venules would make the α-1 adrenoreceptors accessible to circulating norepinephrine (NE) and exogenous vasoactive drugs, in addition to the local release of norepinephrine (NE) from nerve fibers within the brain. This proposal is supported by the findings of Mitchell et al. (Mitchell et al., 2009) who utilized NE spill-over technology and analyzed brain radioactive NE and lipophilic brain NE metabolite spillover sampled at the jugular vein and identified a discordance between the NE and its metabolites in a sub-sample of healthy subjects with: ganglionic blockade; central sympathetic inhibition; neuronal NE blockade; and in pure autonomic failure (PAF) patients. They identified that NE spillover originates primarily from the cerebral vasculature outside the BBB, or more specifically the accessible arteriolar α-1 adrenoreceptors, 23

39 and that the lipophilic metabolite spillover originates from both sides of the BBB. The PAF patients had 77% lower brain NE spillover than found in the healthy subjects indicating that the sympathetic nerve degeneration associated with PAF had spread to the cerebral circulation. Further evidence for the functional role of the α-1 adrenoreceptors was demonstrated by an increase in cerebral vascular conductance (CVCi), as measured by the rate of regulation (RoR), following an experimentally induced acute hypotension designed to stimulate sympathetic nerve activity (Ogoh et al.2008). The increase in CVCi and RoR was attenuated by α-1 adrenoreceptor blockade (prazosin) indicating a sympathetically mediated influence on the cerebral autoregulation (CA) control of the cerebral vasculature. Ide et al. (Ide et al., 2000) identified sympathetically mediated cerebral vasoconstriction in exercising humans with exercise pressor reflex activated sympathoexcitation, which was eliminated by stellate ganglion blockade. In a clinically relevant investigation Brassard et al. (Brassard et al., 2009) reported a decline in frontal lobe tissue oxygenation, internal jugular vein oxygen saturation and a decline in middle cerebral artery blood velocity (MCA V) with incremental dosages of NE infusion. These findings clearly indicate the presence of a dynamic sympathetically mediated control of the cerebral vasculature. Zhang et al. (Zhang et al., 2009) demonstrated a progressive increase in cerebrovascular resistance with incremental dosages of phenylephrine (PE) at rest and concluded that cerebral autoregulatory changes in myogenic tone were primarily involved in the increase in the cerebral vascular resistance. However, when presented as changes in the cerebral vascular conductance index (CVCi), where the PE induced increases in blood pressure were mathematically accounted for (O'Leary, 1991), we identified an α-1 adrenergic receptor mediated vasoconstriction. Spinal cord stimulation (SCS) induced decreases in sympathetically mediated vascular tone resulted in 24

40 increases in cerebral blood flow (CBF). The SCS mediated decreases in cerebral vascular tone involves withdrawal of sympathetic activation of the α-1 adrenergic receptors (Patel et al., 2003; Visocchi, 2008). Recently Brassard et al. (Brassard et al., 2010) reported a decrease in cerebral tissue oxygenation at rest with bolus PE infusion which was abolished with high intensity exercise mediated increase in cerebral metabolism indicating the presence of functional sympatholysis (Remensnyder et al., 1962) in the brain. These reports are in marked contrast to the generally accepted didactic notion that sympathetic control of the cerebral vasculature is minimal (Edvinsson, 1982; Heisted & Kontos, 1983; Strandgaard & Sigurdsson, 2008) and suggests that agonist activation of the α-1 adrenergic receptors on the cerebral vasculature increases cerebral vascular tone and improves the myogenic mechanisms of CA. In support of this suggestion is the identification of a relationship between circulating NE and exercise intensity related increases in critical closing pressure (CCP), an index of cerebral vascular tone (Ogoh et al., 2010). Therefore, we hypothesized that pharmacologic activation (phenylephrine) and blockade (prazosin) of the α-1 adrenoreceptor, respectively, during rest and steady-state dynamic exercise would result in i.) cerebral vasoconstriction and increased cerebral vascular tone; and ii.) cerebral vasodilatation and decreased cerebral vascular tone, respectively. METHODS: Subjects: Seven men and three women (age, 27 ± 1 years, height, 176 ± 4 cm, weight, 76± 4 kg; mean ± SE), volunteered to participate in the present investigation. All subjects were healthy, free of known cardiovascular and respiratory disease, and were not using any prescription or over-the counter medication at the time of participation in the study. The subjects were also 25

41 asked to abstain from drinking alcohol and caffeine and to not exercise for 24 h period prior to any scheduled experiments. The protocol required two days of participation. On Day 1, subjects were informed of the study protocol, signed a written informed consent, completed a health history questionnaire, and were familiarized with all the testing protocols, underwent seated and standing 12-lead electrocardiogram (ECG) and performed a cycling exercise stress test for detection of arrhythmias or orthostatic intolerance. On Day 2, the experimental protocol was performed. All experimental procedures were approved by the Institutional Review Board at the University of North Texas Health Science Center (IRB # ) and were in accordance with the guidelines of the Declaration of Helsinki. Experimental protocol: On Day 1, subjects were instrumented with 12-lead ECG and finger photoplethysmographic arterial blood pressure cuff (Finometer Pro, Finapres Medical Systems, Amsterdam, Netherlands) and performed a progressive exercise work load stress test on a stationary electrically braked upright cycle ergometer (Scifit) to volitional exhaustion. The subjects were asked to maintain a cadence of ~60 rpm throughout the stress test. On Day 2, the subjects exercised at two workload intensities and the heart rate (HR) data recorded on Day 1 was used to identify each subject s workload that would be required to achieve a steady state HR of 130 bpm and identified as a moderate workload (Ex130). Because of the variability in the individual resting HRs each subject pedaled at a 10W workload for the light workload regardless of their baseline HR. The experimental Day2 was separated from Day 1 by at least 2 days after their individual exercise stress test. The protocol for Day 2 is outlined in Figure 1. All subjects arrived in the laboratory in the morning approximately two hours after having a light breakfast. After catheterization and instrumentation, the subjects were seated on an upright cycle ergometer for 26

42 fifteen minutes while their resting data was acquired. Following the rest protocol, each subject began pedaling at 60 rpm at a low workload of 10W for ten minutes. The group average HR achieved for the 10W workload was 88 bpm (Ex88). Without pause the workload was increased to achieve each subject s respective moderate workload at a steady state target HR of 130 bpm (Ex130) for ten minutes. After the control condition, which incorporated the rest and the two exercise protocols, the subjects recovered seated at rest in an armchair for minutes to enable recovery of HR and MAP from the preceding exercise trials. The rest and the two exercise protocols were repeated with intravenous bolus phenylephrine (PE) injection (1.0 µg/kg body weight) at steady state for each exercise workload. The subjects were allowed to rest seated in an armchair for 2 hours and at the beginning of the recovery period, the subjects ingested an oral dose of prazosin (1 mg/ 20 kg of body weight). The rest and the two exercise protocols were repeated after the 2 h recovery period. After the subject reached steady state for each condition (control, PE and prazosin), 5 min of data were collected for spectral and transfer function analysis. Hemodynamic variables were obtained by averaging the data segment over 3 min for each rest and exercise trial. Blood samples were obtained at the end of each rest and exercise trial. There were a total of nine blood samples taken throughout the experiment. The control condition was always first followed by PE condition after minute rest. A bolus dose of PE (1µg/kg body weight) was injected during steady state at rest and two exercise conditions. Each subject ingested their individual dose of prazosin (1 mg/kg of body weight) at the end of the cool down period after the PE condition and had a 2 h seated recovery period. The same dose of PE was injected after the 2 h seated recovery to identify the percentage block of blood pressure response with α-1 adrenoreceptor blockade was achieved. The difference between the pressor response 27

43 obtained during the rest trial with PE and that following the 2 h recovery period was used to determine the percent blockade achieved. Rest and the two exercise trials for the prazosin condition were resumed after the 2 h. recovery period. Immediately after completion of the prazosin condition another bolus dose of PE was injected to determine whether the effectiveness of the α-1 adrenoreceptor blockade was maintained Measurements: Beat-to-beat blood pressure recordings were also obtained for each subject using finger photoplethysmographic arterial blood pressure cuff (Finometer). In six subjects beat-to-beat blood pressure was simultaneously acquired using a catheter (2.54 cm, 22 gauge catheter, Terumo Corporation, Tokyo, Japan) placed in the radial artery of the non-dominant arm using sterile techniques under local anesthesia with 1ml of Lidocaine and aseptic conditions. The catheter was connected to a pressure transducer (Argon Medical Devices, Inc., Tx, USA) positioned at the level of the right atrium in the midaxillary line. The overall correlation between mean arterial pressures obtained by Finometer (y-axis) and direct radial arterial line (x-axis) was 0.88 and the regression equation between the two values was y= 1.0x- 3. In addition, a venous catheter was inserted into the median antecubital vein enabling the bolus PE injections and the obtaining of blood samples. The HR was monitored using a three lead ECG (model 78342A, Hewlett Packard) for continuous recording throughout the rest and cycling exercise trials. Middle cerebral artery blood velocity (MCA V) was measured by transcranial Doppler (TCD) ultrasonography (Multidop X, DWL; Sipplingen, Germany). A 2-MHz Doppler probe was placed over the temporal window and fixed with an adjustable head band and adhesive ultrasonic gel (Tensive, Parker Laboratories, Orange, NJ, USA). The first four subjects tested were also instrumented with a sensor attached to a clip on the earlobe with a drop of electrolyte solution for detection of transcutaneous PCO 2 (TOSCA 28

44 500, Radiometer, Copenhagen, Denmark). The PCO 2 measured by this technology reflects arterial PCO 2 and was found to remain constant across all the conditions of the experiment. Drugs: i.) Phenylephrine Hydrochloride (PE) (Baxter Healthcare Corp., Deerfield, IL, USA): PE was used as a selective α-1 adrenoreceptor agonist in the protocol. A bolus injection (1.0 µg/kg body weight) of the drug was administered at steady-state during rest and the two exercise conditions. PE was also used prior to and 2 h following prazosin ingestion to determine the percentage of α-1 adrenoreceptor blockade achieved. Another PE bolus dose was infused at the end of the experiment to confirm that the same degree of blockade was maintained throughout the experiment. ii.) Prazosin (Mylan Pharmaceuticals, Morgantown, WV, USA): The subjects ingested an oral dose of α-1 adrenoreceptor antagonist prazosin after completion of the control and PE rest and exercise conditions. The dose of prazosin ingested was equal to 1 mg/20 kg of body weight. Blood Sampling: A 3 ml blood sample was drawn from the antecubital venous catheter at the end of rest and the two exercise trials for control, PE and prazosin conditions. The blood samples were transferred to tubes pre-treated with heparin and glutathione. A total of nine blood samples were obtained throughout the experiment. Plasma Catecholamines: The blood samples were centrifuged and the plasma samples were stored at -70ºC for future analysis. Samples were then thawed and plasma concentrations of norepinephrine and epinephrine were separated and analyzed by high-performance liquid chromatography (HPLC) as described previously (Napier et al., 1998). 29

45 Data analysis: All data were sampled at 1 KHz using an analogue-to-digital convertor system interfaced with a personal computer (Dell Laptop). Beat-to-beat mean arterial pressure (MAP) and middle cerebral artery blood velocity (MCA V) were obtained from each waveform. MAP and mean MCA V were calculated using one minute steady-state data at the end of each condition. Transfer function analysis: The relationship between MAP and MCA V were evaluated using transfer function analysis (DADisp 4.1) and has been described previously (Zhang et al., 1998; Ogoh et al., 2005) Briefly, the spectral power of MAP and MCA V and transfer function phase, gain and coherence were calculated in the very low frequency (VLF; Hz), low frequency (LF; Hz), and high frequency (HF; Hz). These frequency ranges reflect patterns of the dynamic pressure-flow relationships, as identified by transfer function analysis (Zhang et al., 1998; Ogoh et al., 2005). The blood pressure fluctuations in the HF range are induced primarily by respiration, whereas those in the LF range are independent of respiratory frequency and are dampened by cerebral autoregulation (CA). The VLF range of both pressure and flow variability appears to reflect multiple physiological mechanisms that confound interpretations (Ogoh et al., 2005). Thus the LF range for the pressure and flow variables was used to calculate power spectral density and identify dynamic CA. The transfer function gain reflects the relative amplitude between the changes in perfusion pressure and blood flow over the specified frequency range. Effective CA decreases the transmission effect of pressure on flow. Therefore, an increase in transfer function gain can be interpreted as an increase in transmission, suggesting impairment in CA. Critical Closing Pressure (CCP) of the cerebral circulation was estimated in the present study as an index of cerebral vascular tone (Panerai, 2003; Panerai et al., 2005). Changes in CCP that 30

46 occur in response to dynamic exercise and the vasoactive drugs (PE and prazosin) reflect changes in vasomotor tone. The CCP was calculated from twenty pairs of systolic and diastolic arterial blood pressures and were associated with the systolic and diastolic velocities of their consecutive MCA V waveforms during steady-state rest and exercise trials across control, PE and prazosin conditions. Linear regression between consecutive pairs of systolic and diastolic values of ABP and MCA V waveforms from the twenty cardiac cycles were determined and the ABP axis (abscissa) intercept of the MCA V/ABP regression line determines the CCP (Panerai, 2003; Ogoh et al., 2010), see figure 5. Cerebrovascular conductance index (CVCi) was calculated by dividing beat-to-beat MCA V by MAP for each condition and an average was reported. The CVCi was used as an estimate of changes in cerebrovascular conductance. Conductance changes normalize the change in flow velocity to a 1.0 mmhg change in arterial pressure. Hence changes in CVCi provide evidence of vasoconstriction or vasodilatation resulting from changes in blood vessel diameter (O'Leary, 1991) Statistics: Subject comparison was made across three exercise conditions (rest, Ex88, Ex130) and across three drug conditions (control, phenylephrine and prazosin). Two (3x3) factor ANOVA with repeated measures was across each factor was used to assess the differences in hemodynamic variables, transfer function phase, gain and coherence in each frequency (SigmaStat, Jandel Scientific Software, SPSS, Chicago, IL). Significant main effects were analyzed using a student-newman-keuls post hoc test. Statistical significance was set at P < All data is expressed as means ± SE. 31

47 RESULTS: The mean values for cardiovascular and hemodynamic variables obtained at rest and the two exercise workloads during control, PE injection and with prazosin ingestion are presented in Table 1. Moderate exercise intensity at HR of 130bpm (Ex130) resulted in significant increases in MAP compared to low intensity exercise at HR of 88bpm (Ex88) and rest during control, PE injection and following prazosin ingestion. Compared to control condition, PE injection increased MAP at rest, Ex88, and Ex130 (P<0.001). However, prazosin ingestion caused a decrease in MAP at rest, Ex88 and Ex 130 (P<0.001), see Table 1 and Figure 2. During control rest, a bolus dose of PE yielded 12 ± 1 mmhg increases in MAP. However, two hours postprazosin ingestion the same dose of PE challenge yielded an increase of only 3 ± 1 mmhg increase in MAP indicating a 74 ± 2% blockade of blood pressure response after 2 h of prazosin and 71 ± 4 % at the end of the experimental protocol. MCA V was unchanged from rest to the two exercise workloads during control and with PE injection. However, following prazosin ingestion, a significant decline in MCA V was observed from control (P=0.009) and PE (P= 0.032), see Figure 2. In addition, MCA V was maintained at its rest value with prazosin during the two exercise workloads. This lack of response in MCA V to exercise induced sympathetic stimulation could be attributed to the prazosin induced decrease in perfusion pressure, as well as to impairment in CA to maintain the flow velocity within the cerebral autoregulatory range, indicating an attenuation of sympathetic activity in establishing the cerebral vascular tone. The average HR achieved during low exercise intensity was 88 ± 4 bpm (Ex88) and for moderate WL was 126 ± 1 bpm (Ex130). Slight decreases in HR were observed with PE injection at rest and the two exercise workloads during control conditions (Table 1). However, 32

48 the ingestion of prazosin caused an increase in HR during rest (from 79 ± 3 to 90 ± 3 bpm), Ex88 (from 88 ± 4 to 102 ± 3 bpm) and Ex130 (from 126 ± 1 to 148 ± 3 bpm) compared to the control condition. Norepinephrine (NE) concentrations increased significantly during Ex130 compared to rest (P<0.001) and Ex88 (P<0.001) in the control condition (Figure 3). Prazosin ingestion resulted in further increases in NE at rest and the two exercise workloads compared to control and PE conditions. A decrease in low frequency (LF) ( Hz) transfer function gain was observed at Ex130 in the control condition (P < 0.05); see Figure 4, indicating an increase in CA. In contrast prazosin ingestion caused a significant increase in LF gain from control (P < 0.004) and PE (P < 0.001) conditions at rest, identifying impairment in CA. Prazosin ingestion resulted in a significant increase in LF gain at Ex88 and for the Ex130 condition between control and PE injections. These findings confirm that α-1 adrenergic receptor blockade resulted in the impairment of CA. The coherence between MAP and MCA V for all the exercise and drug conditions remained above 0.5 and was accompanied by a decrease in phase shift in the prazosin condition. Compared to rest, both exercise workloads (Ex88 and Ex130) resulted in significant increases in critical closing pressure (CCP) in the control condition, see Figure 6. The Ex130 condition increased CCP above rest during control and PE injection (P < 0.05). These increases in CCP indicate significant increases in cerebral vascular tone as a result of α-1 adrenergic receptor activation. This finding was confirmed by the fact that the CVCi, see Figure 7 (an index of cerebral blood flow) was decreased from control indicating cerebral vasoconstriction during 33

49 rest and the two exercise trials with the PE activation of the α-1 adrenergic receptors. Prazosin ingestion resulted in a significant decline in CCP compared to the PE conditions regardless of exercise. In addition, prazosin significantly decreased CCP during the Ex130 condition compared to the control Ex130 conditions only. DISCUSSION: There are several primary findings in the present investigation and these are identified as follows: i.) The significant increase in low frequency (LF) transfer function gain following prazosin ingestion during steady-state rest and the two exercise workloads indicates impairment of cerebral autoregulation (CA) following blockade of the accessible α-1 adrenoreceptors on the cerebral vasculature (Ogoh et al., 2008; Mitchell et al., 2009); ii.) The increase in CCP from rest to Ex130 in the control condition indicates an exercise induced increase in vascular tone. However, this exercise induced increase in CCP was attenuated after prazosin ingestion indicating the involvement of activated α-1 adrenoreceptor in the development of vascular tone; iii.) The bolus injection of PE at rest and during exercise resulted in reductions in CVCi, i.e. identifying a reduction in CBF per unit change in arterial pressure, or cerebral vasoconstriction resulting from α-1 adrenergic receptor activation. These findings highlight the prominent role of functional α-1 adrenergic receptors in establishing cerebral vascular tone, which when activated in conjunction with the myogenic properties of the smooth muscle enhances dynamic cerebral autoregulation (Zhang et al., 2009). Perivascular nerves have been identified in close proximity to smooth muscles in cerebral vessels (Willis, 1664; Benedikt, 1874; Aronson, 1890) and the density and innervations of cerebral resistance vessels have been reported to be extensive and similar to the mesenteric and/or the femoral arterial beds (Rosenblum & Chen, 1976; Rosenblum, 1976). However, despite our 34

50 acceptance of the anatomical existence of a sympathetic neural network associated with the cerebral vessels the role of sympathetic nervous system in the regulation of cerebral blood flow has remained elusive (Ogoh et al., 2008; Zhang et al., 2009; Hamner et al., 2010). In animal models sympathetically mediated vasoconstriction during severe hypertension prevented the rupture of the BBB (Bill & Linder, 1976). Recently, Ogoh and Ainslie (Ogoh & Ainslie, 2009) have highlighted the role of the sympathetic nervous system as a potential candidate influencing dynamic cerebral blood flow regulation. Previously many authors have relied on the work of Olesen in 1972 (Olesen, 1972) as documenting the inability of PE to cross the BBB. However, the work by Olesen referred to previous work using radioactively tagged NE in animal models (Weil-Malherbe et al., 1961), which may be confounded by species differences (Sandor, 1999). Unfortunately, the experiments performed by Olesen used CBF measurement techniques that were unable to distinguish the dynamic effects of sympathetic stimulation. However, more recently Mitchell et al (Mitchell et al., 2009) have documented that intra-arterial infusions of tritiated NE crossed the BBB and identified NE spillover from the α-1 adrenergic receptors of the smooth muscle of the cerebral blood vessels. Furthermore, Brassard et al (Brassard et al., 2010) have reported, in humans at rest that PE stimulation of α-1 adrenoreceptor decreased cerebral tissue oxygenation, an indirect measurement of the decrease in cerebral blood flow, due to activated α-1 adrenoceptor vasoconstrictor mechanisms. However, the increased cerebral metabolism associated with high intensity exercise eliminated the effect suggesting a balance between cerebral metabolism and a functional lysis of sympathetic control of blood flow in the brain at higher exercise intensities (Secher et al., 2008). Similar observations were made in animal studies, where exercise increased blood flow in regions of the brain associated with motor 35

51 control. However, pharmacologically induced hypocapnia related vasoconstriction was overridden by exercise induced increases in cerebral metabolism (Gross et al., 1980). The CBF is strongly regulated by CA to having a positive slope of 0.8% increase in CBF/mmHg between arterial pressures of approximately 60 mmhg to approximately 150 mmhg, whether determined by static blood flow (Heisted & Kontos, 1983) or dynamic blood velocity (Lucas et al., 2010) measurements in humans. The regulation of CBF by CA is in contrast to the pressure passive changes in flow that occur outside the CA range. However, historically the predominant measurement techniques for assessing CA were only able to identify static CA, in that it required one to establish steady-state conditions for the measure to be valid. These conditions identified that the myogenic properties of the smooth muscle was fundamental to CA and that the arterial carbon dioxide pressures (PaCO 2 ) effects on the myogenic properties of cerebral vascular tone modulated CA. With the advent of dynamic measurement techniques, such as transcranial Doppler (TCD) of cerebral blood velocity (MCA V) changes; near-infrared spectroscopic (NIRS) analysis of cerebral tissue oxygenation (ScO 2 ); and linear dynamic analysis techniques of beat-to-beat changes in ABP and MCA V, the dynamic regulation of CBF by CA, when arterial blood pressure is rapidly changing, can be accomplished. Because of these techniques, there is a growing body of evidence identifying that increases in sympathetic nerve activity and its activation of the accessible α-1 adrenergic receptors on the smooth muscle of the cerebral arterioles has greater influence on CA than previously thought (Heisted & Kontos, 1983). This increased functional sympathetic influence on cerebral vascular function appears related to its effect on cerebral vascular tone (D'Alecy et al., 1979; Ogoh et al., 2008), not unlike that associated with changes in PaCO 2 (Heisted & Kontos, 1983). 36

52 The critical closing pressure (CCP) of the cerebral circulation indicates the value of ABP at which CBF approaches zero. The calculation of apparent CCP by extrapolation of the regression line of MCA V to the ABP axis intercept is a useful measure of the dynamics of cerebral circulation (Richards et al., 1999) and a relevant index of cerebrovascular tone (Panerai et al., 1995; Panerai et al., 1999). In the present study dynamic exercise induced an increase in CCP and is interpreted to reflect the increase in sympathetically mediated increases in cerebral vascular tone, which would serve to protect the BBB from exercise induced hypertension (Ogoh et al., 2010), especially the augmented transient peak pressure associated with systole. Increases in ABP produces increases in active wall tension leading to increases in CCP. In addition, the increases in intracranial pressure associated with exercise also results in an increased CCP (Panerai, 2003). In our present investigation there was a stepwise increase in CCP from rest to Ex88 and Ex130 suggesting an increase in cerebral vascular tone with increases in the sympathetic activity associated with exercise. The PE injections further increased the CCP at rest and the two exercise workloads. However, with prazosin the increase in CCP at Ex130 was attenuated from the control condition. It is well established that increases in exercise workloads results in sympathoexcitation (Kotchen et al., 1971; Hartley et al., 1972). In the present study the prazosin blockade of the cerebral arteriolar adrenoreceptors appear to have abolished the increase in vascular tone associated with the exercise induced increase in sympathetic activity. Therefore, we suggest that along with the myogenic tone induced stretch-tension mechanism, increases in sympathetic activity augments the tension developed in vascular tone (Davis & Hill, 1999) as initially proposed by Panerai et al.(panerai et al., 2005). This increase in cerebrovascular tone associated with α-1 adrenergic receptor activation results in cerebral vasoconstriction. The presence of 37

53 cerebral vasoconstriction is confirmed by the concomitant decrease in CVCi at Ex130 in the control condition and that PE injection further decreased CVCi at rest and during the two exercise intensities. Recently, Zhang et al. (Zhang et al., 2009) demonstrated modulations in oscillations of cerebral blood flow velocity following incremental doses of constant PE infusion at rest as a result of progressive increases in cerebrovascular resistance index (CVRi) suggesting a dominant myogenic control of cerebral vessels at rest. Without accounting for pressure changes the estimation of CVRi may undermine the role of other regulatory pathways associated with increases in cerebral vascular tone and may distort the interpretation of pressure volume relationships (Panerai et al., 2005). In the present study the concurrent PE induced increases in CCP and reductions in CVCi (pressure changes accounted for) indicate α-1 adrenergic receptor activation induced cerebral vasoconstriction. The decrease in CCP during Ex88 and Ex130 following prazosin ingestion identified a drop in cerebral vascular tone and confirmed that α-1 adrenoreceptor activation is involved in establishing cerebral vascular tone. Recently, a cholinergically mediated vasodilatation in the cerebral vasculature has been identified during exercise induced increases in CBF (Seifert et al., 2010). Future studies need to further investigate this mechanism and examine if a balance between the sympathetic and parasympathetic nervous system is involved in establishing cerebral vascular tone. Experimental Limitations: In our study CBF was estimated from MCA V using transcranial Doppler ultrasonography which is a potential limitation, as vasoconstriction of the insonated vessel would increase MCA V at any given volume of flow. However, in humans, MCA diameter appears to remain relatively 38

54 constant under a variety of conditions (Schreiber et al., 2000; Serrador et al., 2000) and the diameter of large cerebral arteries do not change significantly during exercise. Similar to the systemic circulation regulation of CBF takes place in smaller arteries and arterioles (Giller et al., 1993). Furthermore, the finding that changes in MCA V increased similarly to the inflow of the internal carotid artery (Hellstrom et al., 1996) and the initial slope index of the 133 Xenon clearance determined cerebral blood flow (Jorgensen et al., 1992) supports our use of transcranial Doppler measurements for identifying changes in cerebral blood flow. It is possible that dynamic exercise may cause signal noise in the data acquisition of MCA V, but that did not appear to be the case in the present study from visual inspection of the MCA V waveforms. In addition, the coherence between MCA V and MAP remained above 0.5 in all conditions suggesting that there was little effect of signal noise on the validity of transfer function analysis during exercise. The CCP was calculated by linear regression between twenty waveforms of consecutive pairs of systolic and diastolic values of ABP and MCA V during steady state rest and exercise conditions to minimize the short-term variability in CCP with beat-to-beat values from a cardiac cycle (Ogoh et al., 2010). Previously, we have documented that there was no difference in the calculation of CCP using only the systolic and diastolic values versus the beatto- beat data from cardiac cycles (Ogoh et al., 2010). In the present study, we achieved an average of 74% decrease in blood pressure response by blockade of α-1 adrenoreceptors with oral prazosin ingestion. However, if we had established a complete blockade the identified impairment of dynamic CA would be exacerbated and the presence of sympathetic activation of cerebral vessel α-1 -adrenoreceptors in the establishment of cerebral vascular tone would be emphasized. 39

55 Perspective: Early in 1970 Olesen (Olesen, 1972) sought to address the question whether circulating catecholamines played an important role in the pathogenesis of cerebral vasospasm associated with subarachnoid hemorrhage and the early stages of a migraine attack. In addition, he went on to postulate that as vasoconstrictor drugs were often used to support blood pressure during shock or anesthetic hypotension that any cerebral vasoconstriction resulting from drug therapy would be dangerous to the patient. In addition, to the conclusions of many previous studies (Ide et al., 2000; Ogoh et al., 2008; Brassard et al., 2009), the findings of the current investigation provides evidence of the dynamic functional role of the sympathetic neural control of the cerebral circulation. It appears that increases in sympathetic activity increases cerebral vascular tone and by interaction with the myogenic properties of the smooth muscle of the cerebral vessels results in cerebral vasoconstriction and enhanced CA. Hence, the use of vasoconstrictor drugs in the support of a patient s arterial blood pressure during triage, surgery and/or critical care requires further evaluation. 40

56 ACKNOWLEDGEMENTS: The study was performed at the University of North Texas Health Science Center (UNTHSC) with the financial support from Department of Integrative Physiology and the Cardiovascular Research Institute at UNTHSC. The authors would also like to thank Dr. James Caffrey, Ph D, and Darice Yoshishige for their expertise and assistance in the analysis of plasma catecholamines. 41

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61 SANDOR, P. (1999). Nervous control of the cerebrovascular system: doubts and facts. Neurochem Int 35, SCHREIBER, S. J., GOTTSCHALK, S., WEIH, M., VILLRINGER, A. & VALDUEZA, J. M. (2000). Assessment of blood flow velocity and diameter of the middle cerebral artery during the acetazolamide provocation test by use of transcranial Doppler sonography and MR imaging. AJNR Am J Neuroradiol 21, SECHER, N. H., SEIFERT, T. & VAN LIESHOUT, J. J. (2008). Cerebral blood flow and metabolism during exercise: implications for fatigue. J Appl Physiol 104, SEIFERT, T., FISHER, J. P., YOUNG, C. N., HARTWICH, D., OGOH, S., RAVEN, P. B., FADEL, P. J. & SECHER, N. H. (2010). Glycopyrrolate abolishes the exercise-induced increase in cerebral perfusion in humans. Exp Physiol 95, SERRADOR, J. M., PICOT, P. A., RUTT, B. K., SHOEMAKER, J. K. & BONDAR, R. L. (2000). MRI measures of middle cerebral artery diameter in conscious humans during simulated orthostasis. Stroke 31, STRANDGAARD, S. & SIGURDSSON, S. T. (2008). Point:Counterpoint: Sympathetic activity does/does not influence cerebral blood flow. Counterpoint: Sympathetic nerve activity does not influence cerebral blood flow. J Appl Physiol 105, ; discussion VISOCCHI, M. (2008). Comments on Point:Counterpoint: Sympathetic activity does/does not influence cerebral blood flow. Sympathetic activity does influence cerebral blood flow. J Appl Physiol 105, WEIL-MALHERBE, H., WHITBY, L. G. & AXELROD, J. (1961). The uptake of circulating [3H]norepinephrine by the pituitary gland and various areas of the brain. J Neurochem 8, WILLIS, T. (1664). Cerebri anatone, cui accesit nervorum, descriptio etusus. Flesher J., London. ZHANG, R., BEHBEHANI, K. & LEVINE, B. D. (2009). Dynamic pressure-flow relationship of the cerebral circulation during acute increase in arterial pressure. J Physiol 587, ZHANG, R., ZUCKERMAN, J. H., GILLER, C. A. & LEVINE, B. D. (1998). Transfer function analysis of dynamic cerebral autoregulation in humans. Am J Physiol 274, H

62 Table 1: Hemodynamic variables at rest and during low exercise intensity at heart rate 88 (Ex88) and moderate exercise workload (Ex130) during control, with phenylephrine (PE) and 2 hours following prazosin ingestion. Values are means ± S.E Abbreviations: MAP, mean arterial pressure, MCA V, middle cerebral artery velocity, HR, heart rate, SV, stroke volume, NE, norepinephrine. 47

63 FIGURE LEGENDS: Figure 1. Protocol outline for the control and drug (phenylephrine and prazosin) conditions: There were a total of three conditions: control, phenylephrine (PE) and prazosin. Each condition had three trials: i.) rest (R ) ; ii.) exercise at a very low workload (10 W), which resulted in an average HR of 88 bpm (Ex88); and iii.) exercise at a moderate workload to achieve a steady state HR of 130 bpm (Ex130). Each trial was for 10 minutes duration followed by a blood draw (bl). Figure 2: Average mean arterial pressure and middle cerebral artery blood velocity at rest, Ex88 and Ex130 during control, PE and with prazosin. A. MAP at Ex130 was significantly higher than rest and Ex88 throughout all conditions. Prazosin decreased MAP throughout all exercise workloads when comapred to control (P = 0.003) and PE injection (P < 0.001). *, when significantly different from rest within the group. #, $, when group is significantly different from control condition. B. Middle cerebral artery blood velocity (MCA V) decreased significantly following prazosin ingestion from control (P=0.009) and PE injection (P= 0.032). #, group was significantly different from control and PE conditions. Figure 3: Average Norepinephrine concentrations at rest, Ex88 and Ex130 during control, PE and prazosin. NE concentrations increased significantly during Ex130 from rest (P<0.001) and Ex88 (P<0.001) regardless of the drug condition. Prazosin led to significant increases in NE concentration compared to control (P = 0.002) and PE (P = 0.001) conditions. *, significantly different from rest and Ex88 within each group. #, group was significantly different from rest and PE conditions. Figure 4: Average low frequency gain at rest, Ex88 and Ex130 during control, PE and prazosin. LF gain at Ex130 was significantly lower than rest (P =0.05 ) within the control condition. Prazosin significantly increased LF gain at each exercise conditions compared to its similar trial within control and PE conditions. *, significantly different from rest within group. #, significantly different from rest in 48

64 control and PE conditions. $, significantly different from Ex88 in control and PE conditions., significantly different from Ex130 in control and PE conditions. Figure 5: Representative Critical Closing Pressure calculation graph. Pressure-velocity relationships for twenty waveforms during steady-state segments at rest during control, with PE injection and 2 h following prazosin ingestion: Representative data for calculation of critical closing pressure (CCP) from systolic and diastolic values of arterial blood pressure (BP) and middle cerebral artery blood velocity (MCA V) at rest during control ([ ], regression equation; y= 0.693x-15.82, r = 0.98, CCP = 22), with PE ([ ], regression equation; y= 0.71x , r = 0.98, CCP = 33) and two hours post prazosin ingestion ([x], regression equation; y= 1.1x- 27, r = 0.99, CCP = 25). Figure 6: Average Critical Closing Pressure at rest, Ex88 and Ex130 during control, PE and prazosin. There was a significant increase in CCP at Ex130 compared to rest (P < 0.001) and Ex88 (P < 0.001) in the control condition. Regardless of the exercise conditions PE had a significant increase in CCP from control (P < 0.001) and prazosin (P < 0.001) conditions. Ex130 with PE had a significant increase in CCP and Ex130 following prazosin had a significant decrease in CCP compared to the similar exercise condition in the control condition. *, significantly different from control within the group., significantly different from Ex130 in the control condition. #, group significantly different from control and prazosin conditions. Figure 7: Average Cerebrovascular conductance index at rest, Ex88 and Ex130 during control, PE and prazosin. There was a significant decrease in CVCi in the PE condition compared to control (P =0.023) and prazosin (P = 0.029) conditions regardless of the exercise conditions in the experiment. #, group significantly different from control and prazosin conditions. 49

65 FIGURES FIGURE 1: 50

66 FIGURE 2A 2B. 51

67 FIGURE 3 52

68 FIGURE 4 53

69 FIGURE 5 54

70 FIGURE 6 55

71 FIGURE 7 56

72 CHAPTER III Influence of the Carotid Baroreflex on the Cerebral Vasculature in Humans during Rest and Exercise. Sushmita Purkayastha 1, Quinton Barnes 1, Ashwini Saxena 1, Wendy L. Eubank 1, Besim Hoxha 1, Peter B. Raven 1. Ready for submission to The Journal of Physiology 57

73 ABSTRACT: Recently, a number of studies have indicated that sympathetic activity influences cerebral vascular tone during exercise and hypotension. Therefore, we tested the hypothesis that arterial baroreflex control of sympathetic activity reflexly regulates the cerebral vasculature. In seven subjects (5 men and 2 women) pulsatile stimulation of the carotid barorecptors was performed using neck pressure (NP) at + 40 mmhg and neck suction (NS) stimuli at 40 mmhg at a predetermined frequency of 0.1 Hz to entrain the responses of the mean arterial blood pressure (MAP), middle cerebral artery blood velocity (MCA V) and cerebral tissue oxygenation (ScO 2 ). The pulsatile NP/NS stimuli were performed during rest and two workload (WL) intensities; low WL at 10W and moderate WL achieving heart rates of 84 bpm (Ex84) and 130 bpm (Ex130), respectively on an upright bicycle ergometer without and with prazosin (72% decline in blood pressure response with blockade of the α-1 adrenoreceptors achieved). Measurements of beat-tobeat ABP, MCA V and ScO 2 were obtained during rest and each exercise workload. The NP entrainment augmented the power spectral density (PSD) of the MAP (P = 0.048), MCAV (P = 0.02) and ScO 2 (P = 0.006) at rest compared to no neck collar stimulation. The PSD for MCA V (P = 0.006) and ScO 2 (P = 0.018) increased with the NP stimulation of the exercise mediated increase in sympathetic activity at Ex130. However, prazosin blockade decreased the PSD of the NP stimuli of MAP (P = 0.028), MCA V (P = 0.011) and abolished the increases in ScO 2 at Ex130. The NS stimulus significantly decreased PSD of MAP (P = 0.007), MCA V (P = 0.004) at Ex 130. Discordance in percentage change of the PSD from the respective baseline measurement was observed for the MCA V and ScO 2 when compared with the PSD of the MAP. These data strongly suggest that in addition to the myogenic responses to the pulsatile changes in perfusion pressure, the NP/NS induced alterations in sympathetic neural activity are involved in the beat-to-beat regulation of the cerebral vasculature. 58

74 Key Words: Arterial Blood Pressure, Middle Cerebral Artery Blood Velocity, Cerebral Tissue Oxygenation, Power Spectral Density 59

75 INTRODUCTION: During dynamic exercise arterial baroreflexes in conjunction with central command and the exercise pressor reflex mediate the cardiovascular and hemodynamic alterations necessary to meet the metabolic demands of the body (Fadel et al., 2004b; Raven et al., 2006). In humans at rest the peripheral vasculature is controlled by the autonomic nervous system and the carotid baroreflex plays an important role in the regulation of beat-to-beat blood flow and arterial blood pressure (Wray et al., 2004) However, the role of the autonomic nervous system in the control of the cerebral blood flow (CBF) remains controversial. Despite the cerebral arteries being richly innervated with sympathetic nerve fibers (Nelson & Rennels, 1970; Edvinsson, 1982) and the fact that superior cervical ganglia innervate the cerebral vessels (Iwayama et al., 1970) in animals, the effect of carotid baroreceptor stimulation appears to be absent when measures of CBF are obtained using steady-state measurement techniques (Rapela et al., 1967; Heistad et al., 1980). In contrast, stimulation of the carotid baroreceptors of baboons decreased CBF, while at the same time the cerebral inflow pressure was unchanged suggesting the independence of CBF from perfusion pressure (Ponte & Purves, 1974). Similar results were obtained in a human study where the middle cerebral artery blood velocity (MCA V) measured by transcranial Doppler decreased during unilateral trigeminal ganglion stimulation (Visocchi et al., 1994). However, it has been established that both metabolic and myogenic mechanisms are major factors in the cerebral autoregulatory response to changes in arterial pressure, whereas, increases in sympathetic nerve activity appear to have a negligible effect on CBF during normocapnia and normotension (Fitch et al., 1975; Heistad et al., 1978). In addition, the reflex activation of the sympathetic nervous system has been reported to affect cerebral vessels during hypertension, hypoxia, hypercapnia, and hemorrhage (Busija & Heistad, 1984). In contrast to the data which 60

76 indicates that there is little or no influence of sympathetic control on CBF, dynamic cerebral autoregulation (dca) was attenuated following ganglion blockade using trimethaphan (Zhang et al., 2002). Furthermore, patients with idiopathic orthostatic intolerance exhibit an excessive decrease in CBF upon standing despite a sustained systemic blood pressure. However, this decline in CBF was abolished with Phentolamine induced α-adrenoreceptor blockade during head up tilt (Jordan et al., 1998). During recovery from acute hypotension, induced by an ischemic thigh cuff occlusion/release protocol (Aaslid et al., 1989) sympathoexcitation was evident from the increase in cerebral vascular conductance (CVCi) during rate of regulation (RoR) associated with dca and arterial baroreflex mediated changes in arterial pressure and CVCi. These responses were attenuated by α-1 adrenoreceptor blockade (Ogoh et al., 2008) suggesting that the baroreflex, in addition to dca, provides additional regulation of CBF via baroreflex mediated changes in sympathetic activity. Recently, it has been found using the norepinephrine (NE) spillover technique across the brain of humans that functional α-1 adrenoreceptors are accessible to circulating catecholamines and sympathetic neural innervations (Mitchell et al., 2009). In an investigation using selective β-1 blockade to diminish cardiac output during dynamic cycling exercise in healthy subjects, the MCA V was reduced. In addition, because stellate ganglion blockade eliminated the β-1 blockade induced decrease in MCA V, it was concluded that the exercise pressor reflex induced sympathoexcitation resulting from the underperfused exercising muscle was the cause of the reduction in MCA V (Ide et al., 2000). During progressive increases in cycling exercise intensities, achieving heart rates of 90, 120 and 150 beats/min, the transfer function gain of beat-to-beat changes in systolic arterial blood pressure and the systolic MCA V decreased (P = 0.08), thereby, indicating an increase in dca during the 61

77 systolic phase (Ogoh et al., 2005). Because the increases in dca occurred at the same time as the known exercise induced increases in sympathetic activity (Hartley et al., 1972), the investigators suggested that the increased dca was related to the increased sympathetic activity. Recently, decreases in cerebral tissue oxygenation at rest were found following bolus phenylephrine injections, which were attenuated during light intensity cycling exercise followed by complete abolition during high intensity exercise (Brassard et al., 2010). These findings indicate the presence of a functional sympatholysis related to increases in brain metabolism and which is similar to that identified in the peripheral vasculature of animals and humans during exercise (Remensnyder et al., 1962; Fadel et al., 2001; Keller et al., 2003; Keller et al., 2004; Wray et al., 2004) Recently, a modest cerebrovascular vasoconstriction was elicited by a cold pressor test performed at rest and during hand grip exercise (Hartwich et al., 2010). In summary, collectively these findings result in our hypothesizing that the arterial baroreflex mediated increases in sympathetic activity affect dca. A number of investigations have identified the role of the carotid baroreflex (CBR) regulation of heart rate (HR), mean arterial blood pressure muscle (MAP) and sympathetic nerve activity (MSNA) during rest and exercise (O'Leary & Seamans, 1993; Potts et al., 1993; Fadel et al., 2001; Raven et al., 2006). Additional investigations identified that the primary mechanism by which the CBR regulates the arterial blood pressure during exercise is by changes in systemic vascular conductance in response to CBR induced changes in systemic vascular tone (Ogoh et al., 2002; Ogoh et al., 2003). Subsequently, we developed a technique using pulsatile NP stimuli at a pre- determined frequency of 0.1 Hz which provided dynamic entrainment of the systemic vasculature by the CBR (Wray et al., 2004). This dynamic input to the systemic vasculature from the CBR at a constant period was presumed to transduce to all end organs influenced by the CBR. It has been proposed that the dynamic beat- 62

78 to-beat control of CA is mediated by the CBR reflex control of the sympathetic nervous system in conjunction with the myogenic properties of the vessel (Busija et al., 1980; Ogoh et al., 2008; Zhang et al., 2009). Therefore, in the present study we sought to test our hypothesis by examining the dynamic CBR entrainment of the cerebral vasculature at rest and during steady state dynamic exercise. METHODS: Subjects: Five men and two women (age, 26 ± 1 years, height, 176 ± 4 cm, weight, 73± 4 kg; mean ± SE), volunteered to participate in the present investigation. All subjects were healthy, free of known cardiovascular and respiratory disease and were not using any prescription or over-the counter medication at the time of participation in the study. The subjects were asked to abstain from drinking alcohol and caffeine and to not exercise for 24 h period prior to any scheduled experiments. The protocol required two days of participation. On Day 1, subjects were informed of the study protocol, signed a written informed consent, completed a health history questionnaire, were familiarized with all the testing protocols, underwent seated and standing 12-lead electrocardiogram (ECG) and performed a cycling exercise stress test for detection of arrhythmias or orthostatic intolerance. In addition, the subjects were evaluated for carotid baroreflex (CBR) responsiveness to ensure that the variable pressure obtained with the neck collar could adequately alter carotid sinus transmural pressure (Querry et al., 2001). On Day 2, the experimental protocol was performed. All experimental procedures were approved by the Institutional Review Board at the University of North Texas Health Science Center (IRB # ) and were in accordance with the guidelines of the Declaration of Helsinki. Experimental protocol: On Day 1, subjects were instrumented with 12-lead ECG and finger photoplethysmographic arterial blood pressure cuff (Finometer Pro, Finapres Medical Systems, 63

79 Amsterdam, Netherlands) and performed a progressive exercise work load stress test on a stationary electrically braked upright cycle ergometer (Scifit) to volitional exhaustion. The subjects were asked to maintain a cadence of ~60 rpm throughout the stress test. On Day 2, the subjects exercised at two workloads (WL) and the heart rate (HR) data recorded on Day 1 was used to identify each subject s workload that would be required to achieve a steady state HR of 130 bpm, and was defined as a moderate workload (Ex130). Because of the variability in the individual resting HRs each subject pedaled at a 10W workload for the light workload regardless of their baseline HR. The experimental Day 2 was separated from Day 1 by at least 2 days after their individual exercise stress test. The protocol for Day 2 is outlined in Figure 1. All subjects arrived in the laboratory in the morning approximately two hours after having a light breakfast. After catheterization and instrumentation, the subjects were seated on an upright cycle ergometer for fifteen minutes while their resting data was acquired. Following the rest protocol, each subject began pedaling at 60 rpm at a low workload of 10W for fifteen minutes. The group average HR achieved for the 10W workload (WL) was 84 bpm (Ex84). Without pause the WL was increased to achieve each subject s respective moderate workload at a steady state target HR of 130 bpm (Ex130) for another fifteen minutes. After the control condition, which incorporated the rest and the two exercise protocols, the subjects recovered seated at rest in an armchair for minutes to enable recovery of HR and MAP from the preceding exercise trials. The rest and the two exercise protocols were repeated with 3 min of 5 s pulses of neck pressure (NP) at +40 mmhg followed by 3 min of 5 s neck suction (NS) at - 40 mmhg in a pulsatile manner (0.1 Hz, i.e. 5 s on, 5 s off for 3 min) at steady state during rest and at each exercise WL. The subjects were allowed to recover at rest seated in an armchair for 2 hours and at the beginning of the recovery 64

80 period, the subjects ingested an oral dose of prazosin (1 mg/ 20 kg of body weight). The rest and the two exercise protocols were repeated with pulsatile NP and NS stimuli at 0.1 Hz after the 2 h recovery period. After the subject reached steady state for each condition (control with no neck collar stimulation, control with neck collar stimulation and prazosin with neck collar stimulation), 3 min of data were collected for power spectral analysis. Hemodynamic variables were obtained by averaging a steady-state data segment of three minutes for each rest and exercise trial. The control condition with no neck collar stimulation preceded control with neck collar stimulation followed by prazosin condition with neck collar stimulation for all subjects. A bolus dose of PE (1µg/kg body weight) was injected during steady state at rest in the control condition. Each subject ingested their individual dose of prazosin (1 mg/ 20 kg body weight) at the end of the cool down period after the control condition with neck collar stimulation and had a 2 h seated recovery period. The same dose of PE was injected after the 2 h seated recovery to identify the percentage degree of α-1 adrenoreceptor blockade achieved. The difference between the pressor response obtained during the rest trial in control condition and that following the 2 h recovery period was used to determine the percent blockade achieved. Rest and the two exercise workloads for the prazosin condition with pulsatile NP/ NS stimuli during each trial was resumed after the 2 h recovery period. Immediately after completion of the prazosin condition another bolus dose of PE was injected to determine whether the effectiveness of the α-1 adrenoreceptor blockade was maintained. Measurements: 5 s pulses of neck pressure (NP) at + 40 mmhg followed by 0 mmhg for 5 s (0.1 Hz) for 3 min was followed by 5 s pulses of neck suction (NS) at -40 mmhg followed by 0 mmhg for 5 s (0.1 Hz) for 3 min produced consequent sympathoexcitatory (with NP) or sympathoinhibitory (with NS) stimuli and entrained the mean arterial blood pressure (MAP), 65

81 cerebral blood velocity (MCA V) and cerebral tissue oxygenation (ScO 2 ) responses at 0.1 Hz. Beat-to-beat blood pressure recordings were also obtained for each subject using finger photoplethysmographic arterial blood pressure cuff (Finometer Pro, Finapres Medical Systems, Amsterdam, Netherlands). In a previous experiment conducted in our laboratory, we determined the correlation between MAP values obtained by Finometer (y-axis) and direct arterial line (xaxis) and it was 0.88 and the regression equation between the two values was y= 1.0x 3. The HR was monitored using a three lead ECG (model 78342A, Hewlett Packard) for continuous recording throughout the rest and cycling exercise trials. Middle cerebral artery blood velocity (MCA V) was measured by transcranial Doppler (TCD) ultrasonography (Multidop X, DWL; Sipplingen, Germany). A 2-MHz Doppler probe was placed over the temporal window and fixed with an adjustable head band and adhesive ultrasonic gel (Tensive, Parker Laboratories, Orange, NJ, USA). Frontal lobe tissue oxygenation was measured with near infrared spectroscopy (NIRS) (NIRO 300, Hamamatsu Photonics, Hamamatsu City, Japan) and the near-infrared HbO 2 signal (arbitrary units, a.u.) was used as an index of cerebral tissue oxygenation. In addition, these measurements provide an indirect indication of vascular responses in the microcirculation (Fadel et al., 2004a). Two fiber optic bundles consisting of a transmitter and a receptor with an optode separation of 4 cm was placed on the forehead over the left eye at the approximate location of the cortical externalization of the anterior cerebral artery. The near-infrared signals at four different wavelengths were sampled concurrently at a rate of 1 Hz. The first four subjects tested were also instrumented with a sensor attached to a clip on the earlobe with a drop of electrolyte solution for detection of transcutaneous PCO 2 (TOSCA 500, Radiometer, Copenhagen, Denmark). The PCO 2 measured by this technology reflects arterial 66

82 PCO 2 (Fuke et al., 2009) and was found to remain constant across all the conditions of the experiment. Drugs: i.) Phenylephrine Hydrochloride (PE) (Baxter Healthcare Corp., Deerfield, IL, USA): PE was used to determine the percentage of α-1 adrenoreceptor blockade achieved with prazosin ingestion. A bolus injection (1.0 µg/kg body weight) of the drug was administered at steady-state rest during control and prazosin conditions. Another PE bolus dose was infused at the end of the experiment to confirm that the same degree of blockade was maintained throughout the experiment. ii.) Prazosin (Mylan Pharmaceuticals, Morgantown, WV, USA): The subjects ingested an oral dose of α-1 adrenoreceptor antagonist prazosin after completion of the control and PE rest and exercise conditions. The dose of prazosin ingested was equal to 1 mg/20 kg body weight. Pulsatile Carotid Baroreflex (CBR): CBR entrainment of the reflex responses of MAP, MCA V and ScO 2 was achieved by altering the carotid sinus transmural pressure using a neck collar device at a predetermined frequency of 0.1 Hz (i.e. 5 s on, 5 s off) for 3 min. NP at + 40 mmhg increases the pressure within the neck collar leading to compression of carotid baroreceptors causing a decrease in carotid sinus transmural pressure, thereby, simulating a hypotensive stimuli. NS at 40 mmhg, on the other hand stretches the carotid baroreceptors causing an increase in carotid sinus transmural pressure and thus simulating a hypertensive stimuli (Fadel et al., 2004b). Power Spectral analysis: Dynamic CA was analyzed by examining the dynamic CBR entrainment with NP/NS stimulus at a predetermined frequency of 0.1 Hz and the variability is presumed to transduce to all end organs influenced by the CBR including the cerebral 67

83 vasculature. The CBR entrainment on the cerebral vessels was thus quantified by power spectral density as a measure of variability in the signal due to CBR entrainment. The measure of variability is observed in the frequency at which CBR entrainment was made. In our study frequency domain at 0.1 Hz reflect a discrete spectral peak at the frequency with which the sinusoidal NP/NS was applied (Wray et al., 2004). Data analysis: All data were sampled at 1 KHz using an analogue-to-digital convertor system interfaced with a personal computer (Dell Laptop). Beat-to-beat mean arterial pressure (MAP) and middle cerebral artery blood velocity (MCA V) and cerebral tissue oxygenation (ScO 2 ) were obtained from each waveform. Frequency domain spectral analysis (DADisp 4.1 software) was performed to identify changes in variability of MAP, MCA V and ScO 2 signal at low frequency (LF) of 0.1 Hz during rest and the two exercise workloads in the control and prazosin conditions. Statistics: Subject comparison was made across three exercise conditions (rest, Ex84, Ex130) and across two drug conditions (control, and prazosin). Two (3x2) factor ANOVA with repeated measures across each factor was used to assess the differences in power spectral densities (PSD) separately between NP and NS stimulus (SigmaStat, Jandel Scientific Software, SPSS, Chicago, IL). Student s paired t test was performed to test for a significant difference in baseline with no neck collar stimulation to rest with oscillatory NP/NS stimulus without and with prazosin for HR, MAP and ScO 2 hemodynamic values. Significant main effects were analyzed using a student-newman-keuls post hoc test. Statistical significance was set at P < All data are expressed as means ± SE. 68

84 RESULTS: During control rest, a bolus dose of PE injection yielded 12 ± 1 mmhg increases in MAP. However, 2 h post-prazosin ingestion the same dose of PE yielded an increase of only 3 ± 1 mmhg increase in MAP indicating a 72 ± 2% decline in blood pressure response following blockade of the functional α-1 adrenergic receptors and 72 ± 4 % at the end of the experimental protocol. Table 1 summarizes the changes in the hemodynamic variables observed with CBR entrainment at rest and during dynamic exercise. During resting condition without prazosin, pulsatile NP stimuli significantly increased HR (from 73 ± 2 to 83 ± 3 beats min -1 ), MAP (from 82 ± 2 to 87 ± 3 mmhg), and decreased MCA V (from 54.6 ± 3 to 47.1 ± 4); whereas, the pulsatile NS stimuli produced no significant differences in the measured hemodynamic variables. On the other hand during rest with prazosin, the pulsatile NP significantly decreased MAP (from 82 ± 2 to 79 ± 3 mmhg), MCAV (from 54.6 ± 3 to 44.8 ± 5), and increased HR (73 ± 2 to 94 ± 3 beats min -1 ); and with prazosin the pulsatile NS stimuli increased HR (73 ± 2 to 85 ± 3 beats min -1 ). During Ex84 and Ex130 the pulsatile NP stimuli increased MAP significantly from rest (P= and 0.001, respectively) without and with prazosin, increased MCA V at Ex130 (P= 0.002) from rest without and with prazosin. A significant increase in HR (P < 0.001) was observed during NP stimuli with prazosin regardless of the exercise condition and a further increase in HR from rest was observed at Ex84 (P =0.010) and Ex130 (P < 0.001) with prazosin. MAP with pulsatile NS stimuli was only significantly increased at Ex130 (P = 0.003) from the rest condition. MCA V was significantly increased at Ex130 compared to rest (P = 0.005) and Ex84 (P = 0.011) in the exercise condition regardless of prazosin. During pulsatile NS stimuli HR was significantly increased with prazosin regardless of exercise (P <0.001). HR at 69

85 Ex84 and Ex130 were significantly increased from rest without (P < and = respectively) and with prazosin (P = and <0.001 respectively). Power spectral analysis was performed to examine the changes in variability identified as peak power spectral density (PSD) of each physiological measurement. The degree of variability is indicative of the extent of CBR entrainment on MAP, MCA V and ScO 2 (Figure 2, 3, 4). The PSD increased significantly during control rest with the pulsatile NP stimuli at + 40 mmhg from baseline with no neck collar stimulation for MAP (P = 0.048), MCA V (P = 0.020) and ScO 2 (P= 0.006). However, the magnitude of the PSD of the MCA V response to the NP stimuli was approximately six times greater than the MAP response across all experimental conditions. The PSD of the MAP response to the pulsatile NP stimuli declined (P = 0.028) in the prazosin conditions compared to the control regardless of the exercise conditions However, there were no significant differences in PSD of the MAP response to the pulsatile NS stimuli between the control and prazosin conditions. Moreover, the pulsatile NS stimuli significantly decreased the PSD at Ex130 condition from rest (P = 0.007) and Ex84 (P = 0.024) without or with prazosin (Figure 5). The PSD for MCA V at Ex84 (P = 0.013) and Ex130 (P = 0.006) were significantly augmented from the rest condition with the pulsatile NP stimuli in control condition. On the other hand ingestion of prazosin resulted in a significantly lower PSD of the MCA V (P =0.011) with the pulsatile NP stimuli throughout all exercise workloads. In addition, the NS stimuli significantly decreased the PSD at Ex130 from control (P = 0.004) and Ex84 (P = 0.004) (Figure 6). 70

86 A significant augmentation in PSD for ScO 2 was observed at Ex130 (P =0.018) compared to rest in the control condition with the pulsatile NP stimuli. However, prazosin abolished the increase in PSD of ScO 2 at Ex130 with NP stimuli compared to that of the control condition (P= 0.003). Sympathetic withdrawal evoked by pulsatile NS stimuli with prazosin abolished any increases in PSD throughout all rest and exercise conditions (Figure 7). During the pulsatile NP and NS stimuli coherence between compared measurements was > 0.50 within each condition. DISCUSSION: There are several primary findings in the present investigation and these are summarized below: i.) The pulsatile NP and NS stimuli of + 40 mmhg and 40 mmhg of the carotid baroreceptors (CB) at a predetermined frequency of 0.1 Hz resulted in a dynamic entrainment of MAP, MCA V and ScO 2 signals during rest and the two exercise workloads via sympathetic activation and withdrawal respectively; ii.) Pulsatile NP augmented the variability in signals of MCA V and ScO 2 from rest to Ex130; whereas iii.) Pulsatile NS stimuli attenuated the variability in signals by means of sympathetic withdrawal; and iv.) prazosin dampened the fluctuations in PSD in MAP, MCA V and ScO 2 associated with the NP stimuli s induced sympathoexcitation. These findings confirm the presence of a dynamic CBR regulation of the cerebral vasculature and cerebral blood flow (CBF) in humans at rest and during dynamic exercise. In addition, the suppression of the responses of MAP, MCA V and ScO 2 to NP with prazosin ingestion confirms the involvement of the sympathetic activation of the functional α-1 adrenergic receptors (Mitchell et al., 2009) in dca and baroreflex control of the cerebral vasculature. Historically, many previous studies in humans were unable to confirm any relationship between carotid baroreceptor stimulation and their effect on the cerebral vasculature (Rapela et al., 1967; Heistad et al., 1980). However, since 1983 there has been a growing body of evidence 71

87 to indicate that there exist a functional sympathetic control of the cerebral vasculature involving dca and the beat-to=beat regulation of cerebral blood flow (Jordan et al., 1998; Ide et al., 2000; van Lieshout et al., 2001; Ogoh et al., 2008). Much of the discrepancy in the findings is probably related to the differences in the steady-state CBF measurement techniques used in humans and the surgically implanted electro-magnetic flow probes in animals used to obtain dynamic changes in CBF (Yoshida et al., 1966; Vatner et al., 1970). With the advent of the use of the transcranial Doppler measurement of cerebral blood velocity and its validation as a reliable index of beat-to-beat changes in CBF or CVCi (Bishop et al., 1986; Secher et al., 2008), the dca was reported to be altered with ganglion blockade in healthy humans postulating autonomic neural control of beat-to-beat CBF in healthy humans (Zhang et al., 2002). In the present study, we found a progressive increase in PSD, or variability, of MCA V and SCO 2 from rest to exercise with pulsatile NP stimuli at 0.1 Hz indicative of CBR mediated control of the sympathetic influence on the cerebral vasculature. These responses to NP stimuli were abolished by pulsatile NS at 0.1 Hz, indicating a CBR mediated sympathetic withdrawal. Furthermore, prazosin, a α-1 adrenergic receptor antagonist attenuated the increased variability in response to the NP stimuli confirming that the CBR control of the cerebral vasculature operates via the sympathetic nervous system. Moreover, the increased sympathetic activation during exercise and the CBR mediated sympathoexcitation resulting from the pulsatile NP stimuli resulted in an increased pulsatality of the MCA V and ScO 2 from rest to exercise. It is established that as the dynamic exercise intensity increases to 60% of maximal oxygen uptake (60 % VO 2max ), there is a proportionally linear increase in CBF from increased neuronal activity and cerebral metabolism. However, after 60 %VO 2max CBF is decreased by hyperventilation induced hypocapnia and its resultant cerebral vasoconstriction (Secher et al., 2008). Because the 72

88 exercise workloads in the present study were of light to moderate intensity it appears that the corresponding sympathetic activation resulting from the NP stimuli were transmitted to the cerebral microcirculation and not buffered or overridden by local metabolic factors (Gross et al., 1979; Brassard et al., 2010). The effect of pulsatile NP stimuli mediated sympathoexcitation was identified by the quantifiable difference between the PSD of MAP and augmentation in PSD of the MCA V and SCO 2 signals from rest to exercise. With the pulsatile NS stimuli there was disengagement of the carotid baroreceptors mediated sympathoexcitation, which resulted in significant decreases in the variability of MAP, MCA V and ScO 2. In addition, prazosin abolished the increases in PSD due to CBR responsive entrainment by blocking sympathetic stimulation of α-1 adrenergic receptors, thereby decreasing the variability in PSD of signals evoked by the pulsatile NP stimuli. The findings confirm the sympathetic nervous system s influence on the cerebral vasculature. It was previously suggested, that along with the vascular myogenic tone induced stretch-tension mechanism, that increases in sympathetic activity would augment the tension developed in cerebral vascular tone (Panerai et al., 2005), We suggest that activation of the α-1 adrenergic receptors activated the Gq pathway leading to activation of contractile properties of the smooth muscle cells increasing vascular tone (Davis & Hill, 1999; Guimaraes & Moura, 2001) and augmenting the vasoconstriction of the myogenic response to increases in vessel wall tension similar to peripheral vascular smooth muscle cells. In the present study the prazosin blockade of the cerebral arteriolar adrenoreceptors appear to have abolished the increases in vascular tone associated with the exercise and arterial baroreflex induced increases in sympathetic activity. The data indicate vasoconstriction above that which would be seen if cerebral autoregulation were the only factor in maintenance of CBF. This vasoconstriction is mediated by factors other 73

89 than perfusion pressure as evidenced by the discordance in the magnitude of variability between MAP with MCA V and ScO 2 and the significant augmentation of PSD in MCA V and ScO 2. If these responses were the same for MAP and MCA V little would be suspected about the contribution of sympathetic nervous system in the maintenance of CBF. However, even though the cerebral vascular bed is smaller compared to the sum of vascular beds involved in maintaining the MAP, the difference in PSD between MAP and MCA V as well as MAP and ScO 2 was magnified supporting the fact that in addition to perfusion pressure another unique factor plays an important role in CBF regulation. We suggest that because of the CBR mediated activation of sympathetic activity by NP and sympathetic withdrawal by NS (Fadel et al., 2001) the findings confirm the presence of a functional sympathetic neural control, see Figure 8. Experimental Limitations: CBF was measured as blood velocity rather than flow within the middle cerebral artery (MCA) using transcranial Doppler. Changes in velocity can reflect changes in flow only if the diameter of the insonated vessel remains constant. In humans, the MCA diameter remains the same under a variety of conditions (Schreiber et al., 2000; Serrador et al., 2000) indicating that velocity changes in the MCA do reflect changes in CBF. Similar to the systemic circulation regulation of CBF takes place in smaller resistance arteries and arterioles (Giller et al., 1993). Furthermore, the finding that changes in MCA V increased similarly to the inflow of the internal carotid artery (Hellstrom et al., 1996) and the initial slope index of the 133 Xenon clearance determined CBF (Jorgensen et al., 1992) supports our use of transcranial Doppler measurements for identifying changes in CBF. Another potential limitation of the present study is the use near-infrared spectroscopy (NIRS) as an index of cerebral microcirculatory blood flow, but instead provides a qualitative index of cerebral tissue oxygenation. However, several studies have demonstrated a close correlation between peripheral 74

90 blood flow values measured by plethysmography, the Fick method and NIRS (Edwards et al., 1993; Van Beekvelt et al., 2001). The confounding factor of including extracerebral tissue oxygenation within the NIRS measurement was resolved by placing the transmitter and the receptor at a distance of 4 cm (Murkin & Arango, 2009). Ambient laboratory conditions were 25 degrees Celsius and 50% relative humidity with minimal air flow. Hence, it is unlikely that the 15 minute workloads at low (Ex84) and moderate (Ex130) intensities would result in spurious NIRS signals due to increase in skin blood flow to whole body heating (Davis et al., 2006). In the present study, we achieved an average of 72 % of blockade of α-1 adrenergic receptors with oral prazosin ingestion. However, if we had established a complete blockade, the identified impairment of dca would be exacerbated and the presence of sympathetic activation of cerebral vessel α-1 adrenergic receptors in the establishment of cerebral vascular tone would be emphasized. 75

91 ACKNOWLEDGEMENTS: The study was performed at the University of North Texas Health Science Center (UNTHSC) with the financial support from Department of Integrative Physiology and the Cardiovascular Research Institute at UNTHSC. 76

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98 Table 1: Hemodynamic variables at rest and during low exercise workload at heart rate 84 bpm (Ex84) and moderate exercise workload (Ex130) during control and 2 hours following prazosin ingestion. Values are means ± S.E., Baseline refers to rest condition with no neck collar stimulation. Pulsatile neck pressure at + 40 mmhg and pulsatile neck suction at 40 mmhg for 3 minutes at a frequency of 0.1 Hz (i.e. 5s on, 5 s off) was applied during steady state at rest, Ex84 and Ex130. Abbreviations: HR, heart rate, MAP, mean arterial pressure and MCA V middle cerebral artery blood velocity. *, when there is significant difference from baseline with no neck collar stimulation. 83

99 FIGURE LEGENDS: Figure 1. Protocol outline for the control and prazosin conditions: There were a total of three conditions: Baseline, Control and prazosin. Baseline condition had no neck collar stimulation whereas control and prazosin condition included neck pressure (NP and neck suction (NS) stimuli for each trial. Each condition had three trials: i.) rest; ii.) exercise at a very low workload (10 W), which resulted in an average HR of 84 bpm (Ex84); and iii.) exercise at a moderate workload to achieve a steady state HR of 130 bpm (Ex130). Each trial was for 15 minutes duration. Three minutes each of NP at + 40 mmhg and NS at 40 mmhg was given during steady-state for each condition at a predetermined frequency of 0.1 Hz (i.e. 5s on, 5s off). Figure 2: Group power spectral density of MAP: The group average power spectral density (PSD) of mean arterial pressure (MAP) at all frequency range (0 0.4 Hz) at rest, Ex84 and Ex130 during control and prazosin conditions with pulsatile neck pressure (NP) at + 40 mmhg and neck suction (NS) stimulus at - 40 mmhg at 0.1 Hz frequency for 3 minutes. Figure 3: Group power spectral density of MCA V: The group average power spectral density (PSD) of middle cerebral artery blood velocity (MCA V) at all frequency range (0 0.4 Hz) at rest, Ex84 and Ex130 during control and prazosin conditions with pulsatile neck pressure (NP) at + 40 mmhg and neck suction (NS) stimulus at -40 mmhg at 0.1 Hz frequency for 3 minutes. Figure 4: Group power spectral density of ScO 2 : The group average power spectral density (PSD) of cerebral tissue oxygenation (ScO 2 ) at all frequency range (0 0.4 Hz) at rest, Ex84 and Ex130 during control and prazosin conditions with pulsatile neck pressure (NP) at +40 mmhg and neck suction (NS) stimulus at - 40 mmhg at 0.1 Hz frequency for 3 minutes. 84

100 Figure 5: Peak power spectral density of MAP at 0.1 Hz: Group average peak power spectral density (PSD) of MAP at 0.1 Hz at baseline with no neck collar stimulation, rest, Ex84, and Ex130 during control and prazosin conditions with pulsatile neck pressure and neck suction stimuli. A significant decrease (P = 0.028) in PSD with prazosin was observed throughout all conditions regardless of exercise during NP stimulation. Whereas, prazosin did not affect any condition during pulsatile NS stimuli. Pulsatile neck pressure stimulation at + 40 mmhg at 0.1 Hz. NS, pulsatile neck suction stimulation at -40 mmhg at 0.1 Hz Figure 6: Peak power spectral density of MCA V at 0.1 Hz: Group average power spectral density (PSD) data of MCA V at 0.1 Hz at baseline with no stimulation of the carotid baroreceptors, rest, Ex84, and Ex130 during control and prazosin conditions. A progressive increase in PSD occurred from rest to Ex84 (P = 0.024) to Ex130 (P < 0.001) in the control condition. Significant decline in PSD (P = 0.011) was observed with prazosin regardless of the exercise conditions. Ex130 had significant augmentation of PSD (P < 0.001) compared to rest in the control condition. NP, pulsatile neck pressure stimulation at + 40 mmhg at 0.1 Hz. NS, pulsatile neck suction stimulation at - 40 mmhg at 0.1 Hz. Figure 7: Power spectral density of ScO 2 at 0.1 Hz: Group average power spectral density (PSD) data of ScO 2 at 0.1 Hz at baseline with no stimulation of the carotid baroreceptors, rest, Ex84, and Ex130 during control and prazosin conditions. A significant augmentation in PSD at Ex130 (P = 0.018) was observed compared to rest at control condition. Prazosin significantly decreased the PSD (P = 0.003) at Ex130 with prazosin compared to control condition. NP, pulsatile neck pressure stimulation at + 40 mmhg at 0.1 Hz. NS, pulsatile neck suction stimulation at - 40 mmhg at 0.1 Hz. 85

101 Figure 8: Group averaged percentage change in power spectral density from baseline: Group comparison of percentage change in PSD from baseline values with no neck collar stimulation of MAP and MCAV and of MAP and ScO 2 during rest and at Ex130 for control and prazosin condition with pulsatile NP stimuli at + 40 mmhg. 86

102 FIGURES: Figure 1 87