Experimental Physiology

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1 1272 Exp Physiol (2012) pp Research Paper Blood flow in internal carotid and vertebral arteries during orthostatic stress Kohei Sato 1, James P. Fisher 2, Thomas Seifert 3, Morten Overgaard 3,NielsH.Secher 3 and Shigehiko Ogoh 1,4 1 Research Institute of Physical Fitness, Japan Women s College of Physical Education, Tokyo, Japan 2 School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK 3 Department of Anaesthesia, The Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark 4 Department of Biomedical Engineering, Toyo University, Saitama, Japan Experimental Physiology It remains unclear whether orthostatic stress evokes regional differences in cerebral blood flow. The present study compared blood flow in the internal carotid (ICA) and vertebral arteries (VA) during orthostatic stress (60 deg head-up tilt; HUT) in six healthy young men. The ICA and VA blood flow were measured using Doppler ultrasonography. Dynamic cerebral autoregulation was also determined during supine (Supine) and HUT conditions, from the rate of regulation (RoR) in cerebrovascular conductance of the ICA and VA during acute hypotension induced by the release of bilateral thigh-cuffs. The HUT decreased ICA blood flow by 9.4 ± 1.7% (P < 0.01 versus Supine), leaving ICA conductance unchanged. In contrast, there was no significant difference in VA blood flow between Supine and HUT, and VA conductance increased (+12.9 ± 0.8%, P < 0.01). In addition, dynamic cerebral autoregulation in both the ICA and VA was attenuated during HUT, and the magnitude of the attenuation in RoR was greater in the VA [0.25 ± 0.03 s 1 Supine versus 0.16 ± 0.02 s 1 HUT ( 33.9 ± 5.8%); P < 0.05] compared with the ICA [0.23 ± 0.02 s 1 Supine versus 0.20 ± 0.03 s 1 HUT ( 10.6 ± 13.4%); P > 0.05]. These data indicate that orthostatic stress evokes regional differences in cerebral blood flow and possible differences in dynamic cerebral autoregulation between two main brain vascular areas in response to an acute change in blood pressure during orthostatic stress. (Received 24 January 2012; accepted after revision 6 June 2012; first published online 11 June 2012) Corresponding author K. Sato: Research Institute of Physical Fitness, Japan Women s College of Physical Education, Kita-Karasuyama, Setagaya-ku, Tokyo , Japan. ksato@jwcpe.ac.jp In humans, cerebral blood flow (CBF) is greater when supine compared with when seated or in an upright position (Alperin et al. 2005). Likewise, CBF velocity, measured in the middle cerebral artery (MCA) by transcranial Doppler (TCD), is reduced by 15 20% during simulation of orthostatic stress using head-up tilt (HUT) or lower body negative pressure (Levine et al. 1994; Immink et al. 2006; Zhang & Levine, 2007) and this cannot be explained by the concomitant reduction in the arterial carbon dioxide tension (P aco2 ; Immink et al. 2009). An important question regarding the reduction in CBF during orthostatic stress concerns whether it is a general phenomenon or whether it affects only the anterior part of the circle of Willis (the internal carotid system) as evaluated by mean blood flow velocity in the MCA (MCA V mean ; Madsen & Secher, 1999; Van Lieshout et al. 2003; Panerai, 2009). Sparse information is available on blood flow in the posterior part of the cerebral circulation (the vertebro-basilar system) during orthostatic stress (Haubrich et al. 2004; Sorond et al. 2005; Deegan et al. 2010). The vertebral artery (VA) and offshoots from the VA, including the anterior spinal and the posterior inferior cerebellar arteries, supply blood to the medulla oblongata, which is the location of important cardiac, vasomotor and respiratory control centres (Tatu et al. 1996). Many of the manifestations associated with development of presyncopal symptoms are likely to result from hypoperfusion in the vertebro-basilar system (Shin et al. 1999). Therefore, it is hypothesized that hypoperfusion of the medulla oblongata rather than the cerebral cortex during orthostatic stress could impair cardiac, vasomotor DOI: /expphysiol

2 Exp Physiol (2012) pp Cerebral blood flow during orthostatic stress 1273 and respiratory control, possibly causing many of the haemodynamic changes that take place preceding syncope. Thus, blood flow regulation in the vertebro-basilar system is likely to be particularly important for orthostatic tolerance. Although the relative contribution of the internal carotid artery (ICA) and VA to global CBF at rest is believed to be balanced in resting humans ( 75% in ICA and 25% in VA, respectively), Sato & Sadamoto (2010) and Sato et al. (2011) reported that dynamic exercise evoked different blood flow responses in the internal carotid and vertebro-basilar systems. However, the effect of orthostatic stress on the distribution of blood flow in the ICA and VA remains unclear. There are anatomical and physiological differences between the internal carotid and vertebro-basilar systems, including regional heterogeneity in the sympathetic innervation of intracranial arterioles (Edvinsson et al. 1976; Hamel et al. 1988) and cerebral CO 2 reactivity (Sorond et al. 2005; Reinhard et al. 2008), which may lead to differences in the cerebrovascular responses to an orthostatic challenge. Studies have compared dynamic cerebral autoregulation (CA) during orthostatic stress between the internal carotid and vertebro-basilar system (Haubrich et al. 2004; Deeganet al. 2010). Haubrich et al. (2004) reported that dynamic CA evaluated using transfer function analysis (TFA) was less effective in the posterior cerebral artery (PCA) than in the MCA at rest and during HUT. In contrast, there appears to be no significant difference in dynamic CA between the MCA and VA during combined HUT and lower body negative pressure to presyncope in healthy subjects (Deegan et al. 2010). However, these previous studies used CBF velocity as an index of CBF using TCD measurement. Thus, the actual and global CBF response to orthostatic stress remains unknown but can be evaluated by determination of blood flow in the ICA and VA. Figure 1. Schematic illustration of experimental protocol After baseline measurements of cerebrovascular and cardiorespiratory variables during supine (Supine) and 60 deg head-up tilt (HUT) conditions, subjects underwent a thigh-cuff test in order to assess dynamic cerebral autoregulation (CA). Abbreviations: ICA, internal carotid artery; and VA, vertebral artery. The purpose of the present study was to identify blood flow in the ICA and VA using Doppler ultrasonography and to determine the potential differences in the response of the CBF between the ICA and VA to orthostatic stress in healthy young subjects. As orthostatic syncope is related to a sudden decrease of blood flow and is generated within the vertebro-basilar system (Shin et al. 1999; Haubrich et al. 2004), we hypothesized that the VA would demonstrate a more pronounced reduction in blood flow during orthostatic stress and a lower dynamic CA than the ICA. Methods Six young men (mean ± SD: 26 ± 3 years, 177 ± 7cm and 74 ± 8 kg) participated in this study. The subjects were non-obese, normotensive and free from overt cardiovascular, pulmonary, metabolic or neurological diseases. Subjects were moderately active, non-smokers, who were not taking any medications. Written informed consent was obtained according to the regional Ethics Committee (H-A ), and the study was conducted in accordance with the principles of the Declaration of Helsinki. Subjects were requested to abstain from caffeinated beverages for 12 h and from strenuous physical activity and alcohol for at least 24 h before experimental sessions. On the experimental day, the subjects arrived at the laboratory a minimum of 2 h following a light meal. All subjects were familiarized with the equipment and procedures before any experimental sessions. Experimental protocol Subjects were supine throughout the instrumentation and stabilization period ( 45 min). After supine rest, 2 min of baseline data were recorded (Fig. 1). Subsequently, bilateral thigh-cuffs were inflated to 250 mmhg, maintained at that pressure for 3 min and then rapidly deflated, causing a transient drop in arterial pressure. After thigh-cuff deflation, measurements were continued for 2 min to assess dynamic CA (Aaslid et al. 1989; Ogoh et al. 2008), and the protocol was repeated at least twice in order to measure both ICA and VA blood flow (defined as ICA or VA trials). After recovery to re-establish resting values, the subjects were passively tilted to 60 deg headup tilt (HUT), followed by 2 min of data recording. Then, the thigh-cuff test was repeated. Thigh-cuff tests that were performed during supine rest (Supine) and HUT that resulted in a 15 mmhg or greater decrease in mean arterial pressure (MAP) were used for analysis for dynamic CA. In all experimental protocols, the subjects were asked to maintain their eyes open, and the room was illuminated.

3 1274 K. Sato and others Exp Physiol (2012) pp Cerebral blood flow The right-sided ICA and VA blood flow were measured by colour-coded ultrasonography (Vivid-e; GE healthcare, Tokyo, Japan), equipped with a 10 MHz linear transducer. The ICA blood flow measurements were made cm distal to the carotid bifurcation, and VA blood flow was measured between the transverse processes of C3 and the subclavian artery (Fig. 2). Blood flow measurement in the VA is commonly targeted between C3 and C6. The VA runs parallel with the vertebral vein; therefore, it is difficult to measure between C3 and C6 in some subjects. It is also difficult to visualize the entire VA on an ultrasound screen due to acoustic shadow caused by the transverse process of the cervical vertebra. In such cases, it is easier to visualize the blood vessel by measuring between C3 and the subclavian artery. We identified the most clearly visualized blood vessel area between C3 and the subclavian artery for blood flow measurement in each subject. In addition, the right MCA mean blood flow velocity (MCA V mean ) was measured by TCD (Multidop X; DWL, Sipplingen, Germany). A 2 MHz Doppler probe was adjusted over the temporal ultrasound window of the MCA until an acoustical signal was identified. The baseline Supine and HUT ICA and VA blood flow were averaged over 2 min. For blood flow measurements, we used the brightness mode (B mode) to measure the mean diameter of each vessel in a longitudinal section, and the Doppler velocity spectrum was subsequently identified by pulsed wave mode (PW mode), and these data were stored on the hard disk of the machine. To calculate average CBF, we analysed mean vessel diameter and mean blood flow velocity a minimum of three times over 2 min during Supine and HUT. Specifically, the systolic and diastolic diameters were measured and then the mean diameter (D mean ; in centimetres) was taken as D mean = [(systolic diameter 1 / 3 )] + [(diastolic diameter 2 / 3 )]. Moreover, the time-averaged mean flow velocity obtained using the PW mode was defined as the mean blood flow velocity (V mean ; in centimetres per second). The recordings of the V mean were taken from the average of 15 cardiac cycles in order to eliminate effects caused by the breathing cycle. Care was taken to ensure that the probe position was stable, that the insonation angle did not vary, and that the sample volume was positioned in the centre of the vessel and adjusted to cover the width of the vessel diameter. Finally, baseline blood flow in Supine and HUT were calculated by multiplying the cross-sectional area [π (D mean /2) 2 ] by V mean ; blood flow = V mean area 60 (in millilitres per minute). The coefficient of variation in test retest measurements of ICA and VA blood flow was 4.3 ± 2.2 and 3.6 ± 2.2% during Supine, and 4.2 ± 1.8 and 3.9 ± 1.9% during HUT, respectively. For assessment of dynamic CA in ICA and VA, beat-by-beat D mean and V mean in each cardiac cycle were measured using the same Doppler ultrasonography procedures as described above, and beatby-beat ICA and VA blood flows were calculated by multiplying the cross-sectional area by V mean during the thigh-cuff test. Cardiovascular measurements Heart rate (HR) was monitored using a lead II electrocardiogram (ECG; Dialogue-2000; IBC-Danica Electronic, Copenhagen, Denmark). Beat-to-beat arterial blood pressure was measured from a catheter in the left brachial artery and a transducer (Edwards Life Science, Irvine, CA, USA) placed at heart level. Beat-to-beat stroke volume (SV) was estimated from the arterial pressure wave according to the Modelflow method (Beat Scope 1.1; Finapres Medical Systems BV, Amsterdam, The Netherlands), and cardiac output (CO) was SV HR. All cardiovascular data were sampled at 1 khz using an analog-to-digital converter interfaced with a computer (Powerlab; AD Instruments, Colorado Springs, CO, USA). An arterial blood sample was obtained during the 2 min baseline period before thigh-cuff test and immediately analysed for P aco2 using an ABL725 (Radiometer, Copenhagen, Denmark). Data analysis The ratio of ICA and VA blood flow to MAP at each vessel level (flow/map) was used to calculate local cerebrovascular conductance. Estimated MAP at the ICA or VA was calculated by measuring the vertical distance from the heart to the Doppler probe in centimetres and subtracting mmhg cm 1 from MAP at heart level. For evaluation of dynamic CA, beat-by-beat ICA and VA blood flow data were obtained, and their conductance Figure 2. Internal carotid artery (ICA) and vertebral artery (VA) imaging and blood flow velocities measured by Doppler ultrasonograpy

4 Exp Physiol (2012) pp Cerebral blood flow during orthostatic stress 1275 Table 1. Baseline haemodynamic values in each trial in the supine position (Supine) and during 60 deg head-up tilt (HUT) Supine HUT Parameter ICA trial VA trial ICA trial VA trial Cardiorespiratory P aco2 (mmhg) 40.1 ± ± ± ± 0.8 MAP at heart level (mmhg) 88 ± 2 87 ± 3 82 ± 3 81 ± 3 Estimated MAP at ICA level (mmhg) 74 ± 3 Estimated MAP at VA level (mmhg) 75 ± 3 HR (beats min 1 ) 63 ± 2 64 ± 3 78 ± 5 79 ± 5 Cardiac output (l 1 min 1 ) 6.1 ± ± ± ± 0.3 Cerebral blood flow ICA blood flow (ml 1 min 1 ) 346 ± ± 16 Mean blood flow velocity (cm s 1 ) 30.4 ± ± 2.0 Mean diameter (cm) 0.49 ± ± 0.01 VA blood flow (ml 1 min 1 ) 115 ± ± 19 Mean blood flow velocity (cm s 1 ) 18.4 ± ± 2.2 Mean diameter (cm) 0.37 ± ± 0.04 MCA V mean (cm s 1 ) 56.3 ± ± 2.3 Cerebrovascular conductance ICA conductance (ml 1 min 1 mmhg 1 ) 3.95 ± ± 0.24 VA conductance (ml 1 min 1 mmhg 1 ) 1.30 ± ± 0.21 Data are means ± SEM. Abbreviations: HR, heart rate; ICA, internal carotid artery; MAP, mean arterial pressure; MCA V mean, mean blood flow velocity in middle cerebral artery; P aco2, arterial partial pressure of CO 2 ; and VA, vertebral artery. Differences between Supine and HUT are indicated as follows: P < 0.05 and P < was calculated as the ratio between blood flow and MAP. Control values for MAP, ICA and VA blood flow and conductance were defined as their means during the 4 s immediately before the thigh-cuff release. The changes in these variables during recovery from thigh-cuff release were determined relative to their concomitant control values. Although at s following thigh-cuff release the rate of change in vascular conductance expresses its dynamic CA and was used in the original study (Aaslid et al. 1989), we extended this time window to calculate the index of dynamic CA to ensure beat-by-beat blood flow data for at least three cardiac cycles. In detail, the rate of regulation (RoR) was calculated as an index of dynamic CA from the slope of the regression line between ICA or VA conductance and time (t) attime1.0to 4.0 s from thigh-cuff release, normalized by the cuff-release-induced hypotension, as follows: RoR = ( ICA or VA conductance/ t)/ MAP where ICA or VA conductance/ t is the slope of the linear regression between ICA or VA conductance and t, and MAP, the magnitude of the step, was calculated by subtracting control MAP from MAP averaged during the interval from 1.0 to 4.0 s. Consistent with previous studies (Ogoh et al. 2008, 2010; Tzeng et al. 2010), the RoR was calculated using conductance (flow/pressure). Statistics Values are expressed as means ± SEM. Repeated-measures one-way ANOVAs with Bonferoni post hoc tests were used to compare the changes in haemodynamic and dynamic CA variables between Supine and HUT, for the ICA and VA trials. All statistical analyses were conducted using SPSS (19.0, IBM SPSS Statistics, Tokyo, Japan), and P < 0.05 was considered to be a significant difference. Results Cardiorespiratory and cerebrovascular responses to head-up tilt Baseline cardiorespiratory and cerebrovascular data in ICA and VA trials obtained during Supine and HUT are presented in Table 1. When compared with Supine, the P aco2, MAP and CO were reduced (P < 0.05 for P aco2 and CO; and P < 0.01 for MAP at heart, ICA and VA levels) during HUT in both trials, while HR increased (P < 0.05). The ICA blood flow decreased during HUT by 9.4 ± 1.7% (P < 0.01), with no change in conductance (Table 1 and Fig. 3A). The decrease in ICA blood flow (Supine 346 ± 16 versus HUT 314 ± 16 ml min 1 ; P < 0.01) was caused by a decrease in both ICA diameter (Supine 0.49 ± 0.01 versus HUT 0.47 ± 0.01 cm; P < 0.05) and mean blood flow velocity (Supine 30.4 ± 1.3 versus HUT 28.6 ± 2.0 cm s 1 ; P < 0.05; Table 1). Likewise, MCA V mean decreased by 8.8 ± 2.1% (Supine 56.3 ± 3.0 versus HUT 51.2 ± 2.3 cm s 1 ; P < 0.05). In contrast, there was no significant difference in VA blood flow between Supine and HUT, and VA conductance increased (Supine 1.30 ± 0.19 versus HUT 1.46 ± 0.21 ml min 1 mmhg 1 ; P < 0.01; Table 1 and Fig. 3B).

5 1276 K. Sato and others Exp Physiol (2012) pp Thigh-cuff release In both Supine and HUT conditions, thigh-cuff release elicited a decrease in blood pressure (Fig. 4A). Changes in MAP from baseline to nadir in HUT were larger than those in Supine (P < 0.01), yet there were no significant differences between ICA and VA trials (Table 2). The degree of the decrease in ICA blood flow and VA blood flow responses to cuff release was not different during Supine, while during HUT changes in VA blood flow to thigh-cuff release were larger than those in ICA blood flow (VA 32.2 ± 1.7 versus ICA 26.1 ± 1.8%; P < 0.05; Table 2 and Fig. 4B). Immediately after thigh-cuff release, the increase in VA conductance (slope) was attenuated in HUT (Supine ± versus HUT ± 0.004; P < 0.05; Table 2), and RoR in VA was lower (Supine 0.25 ± 0.03 versus HUT 0.16 ± 0.02 s 1 ; P < 0.05; Table 2 and Fig. 5B). In contrast, dynamic CA in the ICA was not significantly attenuated during HUT (Supine 0.23 ± 0.02 versus HUT 0.20 ± 0.03 s 1 ;Table2andFig.5A). Discussion The major novel findings of the present investigation are that there is a differential blood flow response in the ICA and VA in response to orthostatic stress. While blood flow in the ICA and MCA V mean were reduced during HUT, VA blood flow was well maintained. This preservation of VA blood flow may be a beneficial response for regulating the systemic circulation during orthostatic stress. Moreover, the attenuation in RoR during HUT in the VA suggests that there may be differences in dynamic CA between two major brain vascular areas in response to an acute change in perfusion pressure during orthostatic stress. Deegan et al. (2010) attempted to demonstrate the responses of CBF for the different vessels to orthostatic stress in healthy adults. They reported that the decline in blood flow velocity during orthostatic stress was similar in the MCA and VA. Moreover, they did not find any differences in dynamic CA between MCA and VA. This discrepancy may relate to differences in the period of examination and/or the degree of the orthostatic stress, because Deegan et al. (2010) used severe orthostatic stress, which combined HUT at 70 deg with lower body negative pressure to presyncope. Furthermore, in the present study, we evaluated the blood flow extracranially, using the portion of the VA in the cervical region, while Deegan et al. (2010) used TCD measurements to assess intracranial blood flow velocity (from the transforaminal window) in the VA. Given the possibility for differences in regulatory mechanisms controlling intracranial and extracranial blood flow, this may also contribute to inconsistencies between the present study and the work of Deegan et al. (2010). Importantly, Deegan et al. (2010) Figure 3. Change in ICA and VA blood flow from Supine to HUT (A and B) and change (%) in ICA and VA blood flow from Supine to HUT (C) Difference between Supine and HUT (P < 0.01). Difference between VA and ICA (P < 0.05).

6 Exp Physiol (2012) pp Cerebral blood flow during orthostatic stress 1277 identified only CBF velocity during HUT, rather than CBF, because of the limitation of TCD measurement. Indeed, the TCD measurement is useful for identifing changes in CBF only when the arterial diameter is unchanged. Notably, in the present study a change in blood vessel diameter was one of the major causes of the difference between ICA and VA in terms of the blood flow response to HUT (Table 1). These findings raise the possibility that sole examination of CBF velocity is insufficient to characterize CBF regulation fully during orthostatic stress. Thus, the differences in the CBF responses in the ICA and VA that Figure 4. Mean response of mean arterial pressure (A), cerebral blood flow (B) and cerebrovascular conductance (C) to thigh-cuff release during Supine and HUT we report in response to HUT provide new information regarding CBF regulation during orthostatic stress. In this study, the reduction in ICA diameter was probably related to the hydrostatic pressure difference between Supine and HUT. The effect of HUT on carotid artery diameter has been examined (Steinback et al. 2005); however, data concerning the VA are lacking. The absence of a change in VA diameter during HUT may be explained by a difference in mechanical properties of the vessel for a change in hydrostatic pressure between ICA and VA. In addition, the different blood flow responses might be explained by differential autonomic innervations of the ICA and VA, or regional heterogeneity in ion channels and/or reactive oxygen species (Edvinsson et al. 1976; Andresen et al. 2006; Faraci, 2006). The posterior cerebral circulation may have less sympathetic innervation than the anterior cerebral portion (Edvinsson et al. 1976; Hamel et al. 1988). Another possible explanation for the difference between the ICA and VA in terms of blood flow responses to the HUT may be a difference in CO 2 reactivity, although posture might affect the cerebral CO 2 reactivity (Mayberg et al. 1996). Accordingly, these factors may be linked to the lower vascular tone in the VA vascular bed during orthostatic stress, although orthostatic stressinduced reductions in CBF velocity cannot be explained only by sympathetic nerve activity (Zhang & Levine, 2007) or lower P aco2 (Immink et al. 2009). An alternative explanation may be that blood flow and vascular conductance during orthostatic stress are different in the ICA and VA because they are operating in different metabolic states. As neural activity and metabolism are closely related to regional CBF, termed neurovascular coupling, the vascular beds supplying more metabolically active brain regions are likely to be dilated (Nakagawa et al. 2009). Such observations are relevant to the present study, where the territories supplied by the vertebro-basilar system (i.e. medulla oblongata, visual cortex, cerebellum and vestibular regions) are robustly and constantly activated during HUT due to sympathoexcitation, visual stimulation, postural control and gravitational stress. This would place the vertebrobasilar territories in a state of continuous vasodilatation compared with the internal carotid territories (Haubrich et al. 2004; Nakagawa et al. 2009). However, our study did not incorporate direct imaging techniques to assess regional brain metabolic rate for oxygen. Thus, we could not show any direct evidence that the orthostatic condition resulted in regional increases in blood flow within the vertebro-basilar system. The vasodilated vascular beds (increase in VA conductance) in the vertebro-basilar circulation may have induced an impaired dynamic CA response during orthostatic stress. Indeed, the decrease in RoR during orthostatic stress was more pronounced in the VA than that in the ICA (Fig. 5C). Previous study showed that dilated

7 1278 K. Sato and others Exp Physiol (2012) pp Table 2. Haemodynamic response to thigh-cuff test and variables of dynamic cerebral autoregulation in each trial in Supine and HUT conditions Supine HUT Parameter ICA trial VA trial ICA trial VA trial Responses to thigh-cuff release MAP response to cuff release (%) 25.3 ± ± ± ± 3.7 ICA blood flow response to cuff release (%) 16.5 ± ± 1.8 VA blood flow response to cuff release (%) 15.6 ± ± 1.7 Dynamic CA variables Slope of ICA conductance ± ± Slope of VA conductance ± ± MAP (%) 15.1 ± ± ± ± 3.3 ICA RoR (s 1 ) 0.23 ± ± 0.03 VA RoR (s 1 ) 0.25 ± ± 0.02 Data are means ± SEM. Abbreviations: CA, cerebral autoregulation; RoR, rate of regulation; other abbreviations are as for Table 1. Differences between Supine and HUT are indicated as follows: P < 0.05 and P < Differences between ICA trial and VA trial during HUT are indicated as follows: P < 0.05 and P < vascular beds have attenuated dynamic CA compared with constricted beds. Aaslid et al. (1989) reported that the rate of dynamic CA is dependent on basal vascular tone and that the autoregulatory response rate in MCA V mean was slower when cerebral vessels were dilated when subjects breathed a hypercapnic gas mixture. Likewise, Serrador et al. (2005) showed that transfer function gain was higher in subjects with lower cerebrovascular resistance, indicating that dynamic CA is impaired when cerebral vessels are dilated. Thus, we speculate that cerebral vasodilatation in the vascular bed of the VA during orthostatic stress contributed to the slower autoregulatory response. Previous studies have reported that dynamic CA is similar in the MCA and vertebro-basilar circulation during supine rest (Park et al. 2003) and orthostatic stress (Deegan et al. 2010). In contrast, Haubrich et al. (2004) reported that dynamic CA evaluated using TFA was less effective in the PCA than in the MCA during supine rest and orthostatic conditions. Moreover, dynamic CA in the Figure 5. Rate of regulation (RoR) in ICA and VA during Supine and HUT conditions (A and B) and change (%) in RoR from Supine to HUT (C) Difference between Supine and HUT (P < 0.05). Difference between VA and ICA (P < 0.05).

8 Exp Physiol (2012) pp Cerebral blood flow during orthostatic stress 1279 PCA is dependent on the metabolic demand of the visual cortex,andwhensubjectsclosedtheireyestherewasno difference in autoregulation between the PCA and MCA (Nakagawa et al. 2009). This further supports the concept that dynamic CA is altered by metabolic demand in the territories perfused by the vessel under examination. Based on previous studies, a possible reason why dynamic CA is different in the ICA and VA territories may be because they are operating in different metabolic states during HUT. The change in CBF responses observed during acute hypotension is consistent with some previous reports (Aaslid et al. 1989; Ogoh et al. 2008; Tzeng et al. 2010). The decrease in blood flow immediately after cuff release during HUT was more marked in the VA compared with the ICA (Table 1 and Fig. 4B). An attenuated dynamic CA in the VA may mean that flow in this vessel is more sensitive to changes in perfusion pressure. However, VA blood flow regulation in the acute hypotension phase may be different from that over a longer time course. Interestingly, following about the first five heart beats after thigh-cuff release, both ICA and VA blood flow returned to baseline values, probably due to an arterial baroreflexmediated recovery of perfusion pressure (Aaslid et al. 1989; Ogoh et al. 2008, 2010; Tzeng et al. 2010). We observed no significant difference in the increase of blood flow and cerebrovascular conductance between the ICA and VA at this time during HUT. Indeed, a recent study indicated that there is an inverse relationship between dynamic CA and arterial baroreflex sensitivity (Tzeng et al. 2010), but the baroreflex control of cerebral vascular tone in humans and the effect of the baroreflex on dynamic CA remain unknown (Ogoh et al. 2008, 2010; Tzeng et al. 2010). Potential limitations of the present study should be considered. First, the validity and reliability of CBF measurement by ultrasonography are important for our study. Blood flow evaluation by ultrasonography has the advantage of allowing simultaneous evaluation of the vessel diameter and the blood flow velocity. This information cannot be obtained by using TCD. However, measurement using a hand-held transducer without a holder, such as a head band, is expected to be a less reproducible measure because it is difficult to fix the measurement position for several minutes by using this method. However, the coefficient of variation of CBF in test retest measurements was 5% or less, both in the Supine and the HUT positions, and the values recorded were within the range of reported values at rest and during physiological stress (Schöning & Scheel, 1996; Sato & Sadamoto, 2010; Ogoh et al. 2011; Sato et al. 2011). This demonstrates a good reproducibility of the CBF measurement by ultrasonography in our study. Second, calculation of the RoR by using the cuff release method may be considered a less reproducible method for evaluating the dynamic CA (Panerai, 2009). To address this weakness in the evaluation of dynamic CA using the thigh-cuff release test, previous investigations have performed repeated trials to obtain an average RoR value (Aaslid et al. 1989; Panerai, 2009) or used the test in combination with the autoregulatory index (Tiecks et al. 1995) and TFA (Tzeng et al. 2010). The addition of autoregulatory index and/or TFA calculations would have provided support for our results. Unfortunately, this study only assessed dynamic CA using the rate of regulation. However, Haubrich et al. (2004) conducted TFA during orthostatic stress and showed that the vertebro-basilar circulation had a higher gain in blood flow in response to blood pressure variations and a weaker dynamic CA function than the internal carotid circulation. In addition, the autoregulatory index should be reflected by RoR data, and the RoR values are consistent with beat-to-beat changes in CBF within about five beats after cuff release (in the VA, lower dynamic CA probably causes a larger drop in CBF during HUT; Fig. 4B). These findings support the resultsofdynamiccainthepresentstudy. The physiological mechanism for these differential ICA and VA blood flow responses to orthostatic stress is unclear. However, our findings suggest that blood flow regulation in the vertebro-basilar system rather than in the carotid circulation is likely to be more important physiologically for maintenance of systemic circulatory control during orthostatic stress. The vertebrobasilar system provides the blood supply to the medulla oblongata, although there is a possible interaction with the internal carotid circulation. Thus, the blood supply from the basilar artery or the VA could be particularly important for cardiorespiratory regulation. We have also usedvabloodflowasanindexofbasilararterybloodflow, and the basilar artery receive their blood supply from the two VAs. Thus, VA blood flow provides more circulatory information compared with basilar artery blood flow. In summary, VA blood flow regulation during orthostatic stress was not the same as that in ICA. The ICA blood flow was reduced during HUT. In contrast, VA blood flow was preserved during orthostatic stress. This preservation of VA blood flow should be a beneficial response for regulating the systemic circulation in response to orthostatic stress. Thus, even during orthostatic stress, VA blood flow is unchanged by dilatation of territories of the vertebro-basilar system. Furthermore, the attenuation in RoR in the VA suggests possible differences in dynamic CA and CBF regulation between two main brain vascular areas in response to an acute change in perfusion pressure during orthostatic stress. References Aaslid R, Lindegaard KF, Sorteberg W & Nornes H (1989). 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Autonomic ganglionic blockade does not prevent reduction in cerebral blood flow velocity during orthostasis in humans. Stroke 38, Acknowledgements We appreciate the time and effort spent by our volunteer subjects in the present study. This study was supported by a research grant from Mr Jakob Ehrenreich and Grethe Ehrenreichs Fund (to N.H.S.); the Japanese Ministry of Education, Science, Sports and Culture (grant # to K.S.); and J.P.F. was supported by the Royal Society.