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Object Cerebrovascular dysfunction after subarachnoid hemorrhage (SAH) may contribute to ischemia, but little is known about the contribution of intracerebral arterioles. In this study, the authors tested the hypothesis that SAH inhibits the vascular reactivity of intracerebral arterioles and documented the time course of this dysfunction. Methods Subarachnoid hemorrhage was induced using an endovascular filament model in halothane-anesthetized male Sprague-Dawley rats. Penetrating intracerebral arterioles were harvested 2, 4, 7, or 14 days postinsult, cannulated using a micropipette system that allowed luminal perfusion and control of luminal pressure, and evaluated for reactivity to vasodilator agents. Results Spontaneous tone developed in all pressurized (60 mm Hg) intracerebral arterioles harvested in this study (from 66 rats), with similar results in the sham and SAH groups. Subarachnoid hemorrhage did not affect dilation responses to acidic pH (6.8) but led to a persistent impairment of endothelium-dependent dilation responses to adenosine triphosphate (p < 0.01), as well as a transient attenuation (p < 0.05) of vascular smooth muscle–dependent dilation responses to adenosine, sodium nitroprusside, and 8-Br-cyclic guanosine monophosphate (cGMP). Impairment of NO-mediated dilation was more sustained than adenosine- and 8-Br-cGMP–induced responses (up to 7 days postinsult compared with 2 days). All smooth muscle–dependent responses returned to sham levels by 14 days after SAH. Conclusions Subarachnoid hemorrhage led to a persistent impairment of endothelium-dependent dilation and a transient attenuation of vascular smooth muscle–dependent dilation responses to adenosine. Impairment of NOmediated dilation occurred when the response to cGMP was intact, suggesting a change in cGMP levels rather than an alteration in intracellular mechanisms downstream from cGMP.

Object This study was undertaken to test the hypothesis that cerebral blood flow (CBF) and the intrinsic optical signal could be dissociated by altering adenosine receptor activity and to uncover the origin of the optic signal using a cranial window in the anesthetized rat. Methods In anesthetized, ventilated, and temperature-controlled rats with closed cranial windows, the authors evaluated simultaneously the alterations in pial arteriolar diameter, intrinsic optical signals (690 nm), and somatosensory evoked potentials during cortical activation evoked by contralateral sciatic nerve stimulation (SNS). To dissociate the vascular and intrinsic signal, they topically applied the adenosine receptors antagonists theophylline (5 μM), which affects A1 and A2A receptors, and 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 μM), which blocks the A1 receptor. The former interacts primarily with the vasculature whereas the latter influences the parenchyma exclusively. Results During 20 seconds of contralateral SNS, pial arterioles in the hindlimb somatosensory cortex dilated in a characteristic peak and shoulder pattern. As compared with mock cerebrospinal fluid alone, theophylline significantly (p < 0.05) attenuated SNS-induced vasodilation (mean ± standard deviation 8.1 ± 2.5% vs 21.7 ± 1.9%; 4 rats in each group). In contrast, CPX potentiated vasodilation significantly (p < 0.05) during SNS (54.7 ± 15.8% for the CPX group vs 20.1 ± 1.9% for the controls; 5 rats in each group). The change in optical signal persisted after cessation of SNS in all the animals. Thus, the pattern of change of the optical signal was distinctly different from the pattern of changes in arteriolar diameter (which returned rapidly to baseline). Moreover, the optical signal during SNS was increased by 50% by theophylline and by almost 5-fold by CPX (p < 0.05). The area of change of the intrinsic signal was also increased by the topical application of theophylline and CPX. The somatosensory evoked potential recordings revealed no significant changes after theophylline application, but CPX caused a small diminution of the N1 wave (p < 0.01). Conclusions The noncongruent temporal profiles of the changes in pial arteriolar diameter and optical signal, imaged at 690 nm, indicate that the optical signal at 690 nm is not related to CBF. Alteration of adenosine receptor activity independently changed cortical activity, as measured by the optical signal, and CBF, as determined by pial arteriolar diameter. Manipulation of the adenosine receptor activity during increased cortical activity confirmed the temporal dissociation of optical signal and CBF and provided further evidence for the role of adenosine in regulating CBF.

Chronic fetal alcohol exposure impairs neural and vascular development. We have previously shown that fetal alcohol exposure is associated with attenuated hypoxic cerebral vasodilation and reduced neuronal vasoactive intestinal peptide (VIP) expression in fetal sheep. In the present study, we tested the hypothesis that fetal alcohol exposure alters vascular development, leading to altered cerebral vascular reactivity to VIP in adulthood. Penetrating intracerebral arterioles were harvested from the brains of adult (10–13 months old) offspring of ewes that had received intravenous infusions of alcohol (1.5 g/kg) or same-volume saline (90 min/day, 5 days/week) during days 30–82 of gestation (full term = 145 days). The isolated arterioles were cannulated with a micropipette system that allowed luminal perfusion and control of luminal pressure and developed spontaneous tone at 40°C and 60 mm Hg luminal pressure. There was no difference in myogenic tone between arterioles exposed prenatally to alcohol (n = 18) and saline controls (n = 17). However, fetal alcohol exposure significantly (p = 0.03) enhanced the dilator responses of adult intracerebral arterioles to VIP [0.1 nM to 1 µM, logEC50: –8.6 ± 0.2 (alcohol) vs. –7.4 ± 0.8 (saline)]. In contrast, there was no difference in dilator responses to H+ (pH 6.8 buffer), to adenosine (10 nM to 0.1 mM), or to CGS21680 (an adenosine A2A receptor agonist, 0.01 nM to 10 µM). Thus, fetal alcohol exposure alters vasomotor sensitivity to VIP in adult intracerebral arterioles – perhaps a compensatory response to alcohol-induced underdevelopment of neurotransmitter pathways involved in cerebral vascular regulation.

We tested the hypothesis that adenosine (Ado) mediates glutamate-induced vasodilation in the cerebral cortex by monitoring pial arteriole diameter in chloralose-anesthetized rats equipped with closed cranial windows. Topical application of 100 μM glutamate and 100 μM N-methyl-d-aspartate (NMDA) dilated pial arterioles (baseline diameter 25 ± 2 μm) by 17 ± 1% and 18 ± 4%, respectively. Coapplication of the nonselective Ado receptor antagonist theophylline (Theo; 10 μM) significantly reduced glutamate- and NMDA-induced vasodilation to 4 ± 2% ( P < 0.01) and 6 ± 2% ( P < 0.05), whereas the Ado A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 μM) had no effect. Moreover, application of the Ado A2A receptor-selective antagonist 4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol (ZM-241385), either by superfusion (0.1 μM, 1 μM) or intravenously (1 mg/kg), significantly inhibited the pial arteriole dilation response to glutamate. Neither Theo nor ZM-241385 affected vascular reactivity to mild hypercapnia induced by 5% CO2 inhalation. These results suggest that Ado contributes to the dilation of rat cerebral arterioles induced by exogenous glutamate, and that the Ado A2A receptor subtype may be involved in this dilation response.

Adenosine (ADO) is a potent cerebral vasodilator and has been proposed as a metabolic regulator of cerebral blood flow. However, the signal transduction pathway by which ADO causes vasodilation in cerebral microvessels is currently unknown. The current study was designed to investigate the role of cyclic nucleotides and cyclic nucleotide-dependent protein kinases in ADO-induced dilation of resistance-sized rat cerebral arterioles that develop spontaneous tone. Arterioles were cannulated and perfused intraluminally at constant flow (2 microl/min) and pressure (60 mm Hg). ADO (29.7 +/- 2.0%; 1 microM), CGS-21680 (16 +/- 4%, 1 microM), 8-bromo-cyclic guanosine monophosphate (8 Br-cGMP; 29.9 +/- 3.9%; 100 microM), sodium nitroprusside (SNP; 30.6 +/- 3.3%, 1 microM), cyclic guanine monophosphate-dependent protein kinase activator (Sp-8-pCPT-cGMPS, 25.9 +/- 4.2%; 10 microM), forskolin (30.5 +/- 5.9%; 0.1 microM), and pH 6.8 all produced large dilations. The selective cGMP-dependent protein kinase inhibitor, Rp-8-pCPT-cGMPS (10 microM), had no effect on resting diameter or reactivity to acidic pH, but significantly ( < 0.05) attenuated arteriolar dilations to ADO (59%, n = 8), CGS-21680 (60%, n = 4), SNP (62%, n = 3), 8 Br-cGMP (88%, n = 3), and Sp-8-pCPT-cGMPS (98%, n = 3). H8, the less-selective cyclic nucleotide-dependent protein kinase inhibitor, had similar effects as Rp-8-pCPT-cGMPS. Additionally, the inhibitor of the soluble guanylate cyclase, 1H-[1,24]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), blocked the response to SNP (70% inhibition) and significantly inhibited the ADO response (43% inhibition). In contrast, inhibition of the cyclic ADO monophosphate (cAMP)-dependent protein kinase Rp-8-CPT-cAMPS had no effect on the ADO, SNP, or pH responses, but significantly blocked forskolin-induced vasodilation (53%). It is concluded that ADO-induced vasodilation in cerebral microvessels, at least in part, involves cGMP and cGMP-dependent protein kinase, but not cAMP or cAMP-dependent kinase. Our data therefore provides a new insight into mechanisms by which ADO invokes vasodilation in cerebral microvascular arterioles.

Vascular control mechanisms have been studied extensively in mice. However, an in vitro characterization of penetrating intracerebral arterioles has not been reported. We describe methods for isolation and cannulation for mouse intracerebral arterioles. This technique allows analysis of mouse cerebral arteriolar physiology and pharmacology without the confounding influences of the surrounding brain elements. Penetrating intracerebral arterioles from adult C57/BL6 wild-type (WT) mice were isolated at 4 degrees C, transferred to an inverted microscope and cannulated at both ends using a dual glass micropipette system, wherein intraluminal flow (0.2 microl/min) and pressure (60 mmHg) were maintained. The arterioles developed spontaneous tone when the chamber was warmed to 37 degrees C, with the resulting diameter reaching 68.4+/-0.9% of passive diameter (29.8+/-1.1 microm). After the development of spontaneous tone, incremental changes in luminal pressure from 20 to 140 mmHg induced myogenic responses. Acidosis (pH 6.8) and alkalosis (pH 7.6) caused dilation (20.0+/-1.4%) and constriction (17.2+/-1.4%), respectively. Extraluminal adenosine (ADO (10 microM); 24.3+/-3.6%) and sodium nitroprusside (SNP (10 microM); 28.6+/-4.1%) and intraluminal adenosine 5'-triphosphate (ATP (10 microM); 20.0+/-3.9%) resulted in vasodilation similar in magnitude to that observed in rat arterioles. This information provides a foundation for elucidating cerebral vascular control mechanisms in genetically engineered mice.

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.

We have previously shown that topically applied N G -nitro- l -arginine ( l -NNA), a nitric oxide synthase (NOS) inhibitor, suppressed both somatosensory evoked potentials (SEPs) and vascular responses during sciatic nerve stimulation in rats. Due to the normal tight coupling between cerebral blood flow and neuronal activity, we surmise that the vascular response attenuation may be secondary to the SEP decrease. However, a recent study, in which SEPs were recorded with a `non-contact' electrode placed longitudinally across the cranial window without touching the cortex, did not find a SEP decrease following NOS inhibition. In the present study, we compared SEPs recorded with `contact' and `non-contact' electrodes. Regardless of stimulation methods (sciatic nerve or hindpaw), an electrode in contact with the pial surface overlying the hindlimb somatosensory cortex recorded a steady SEP decline during l -NNA application. In contrast, a `non-contact' electrode did not detect a significant SEP change in the presence of l -NNA. The present results thus confirm the attenuation of SEPs by NOS inhibition.

We tested the hypothesis that a shear stress-dependent mechanism is involved in the dilation of pial arterioles during somatosensory stimulation. In alpha-chloralose-anesthetized rats implanted with cranial windows, we simultaneously measured the diameter and flow velocity of pial arterioles with video and dual-slit methods. Stimulation (0.2-0.3 V, 5 Hz, 0.5 ms pulses for 20 s) of the contralateral sciatic nerve evoked consistent dilator responses in pial arterioles (36 +/- 1 micron diam) without affecting blood pressure. The dilator responses consisted of an initial transient peak dilation of 30 +/- 3%, followed by a sustained dilation of 13 +/- 1% (n = 11). Mean velocity increased by 16.4 +/- 5.7% at 5 s after stimulus onset. Wall shear rate and volume flow were calculated from diameter and velocity data by assuming a parabolic flow profile. There was no significant change in wall shear rate, whereas flow rate increased significantly during sciatic nerve stimulation. The present findings suggest that a flow (shear stress)-mediated mechanism does not play an important role in the dilator response of pial arterioles to sciatic nerve stimulation.

We studied the local cerebrovascular response and somatosensory-evoked potentials (SEPs) to stimulation of the sciatic nerve during both short- (< 30 min) and long-term (90-150 min) exposure to topically applied NG-nitro-L-arginine (L-NNA). The pial circulation was visualized through a cranial window in alpha-chloralose-anesthetized rats. The diameter of pial arterioles (25-45 microns) and laser-Doppler flow (LDF) in the hindlimb sensory cortex were simultaneously measured during sciatic nerve stimulation. Short-term (< 30 min) treatment with L-NNA (1 mM) abolished the dilation of pial arterioles induced by acetylcholine, whereas the response to sciatic nerve stimulation was not affected. When applied for > 30 min, L-NNA induced severe vasomotion and attenuated the vascular responses to sciatic nerve stimulation. Long-term exposure to topically (1 mM) or systemically (10 mg/kg i.v.) applied L-NNA also attenuated cortical SEPs to sciatic nerve stimulation. Thus L-NNA-induced inhibition of vascular responses may be secondary to suppression of neuronal activity and an L-arginine metabolite, such as nitric oxide, may be involved in neurotransmission in the cerebral cortex during somatosensory activity.

We utilized the closed cranial window technique in the anesthetized rat to determine changes in CSF concentrations of adenosine, inosine, and hypoxanthine and pial arteriolar diameter during transient (20 min) forebrain ischemia and reperfusion. After mock CSF under the cranial window was allowed to equilibrate with cerebral interstitial fluid, endogenous adenosine concentration was found to be 0.16 +/- 0.05 microM, while inosine and hypoxanthine were 0.35 +/- 0.17 and 1.23 +/- 0.47 microM, respectively. The concentration of adenosine in CSF increased 4.2-fold during ischemia and 13.8-fold during the first 5 min of reperfusion. Inosine and hypoxanthine concentrations were also significantly increased during ischemia and reperfusion. After 1 h of reperfusion, CSF adenosine and inosine levels had decreased from peak value but remained significantly above preischemic values. In contrast, hypoxanthine remained at peak concentrations even after 60 min of reperfusion. Preischemic arteriolar diameter was 42.6 +/- 11.3 microns and was not significantly changed after 20 min of ischemia. However, during the first 5 min of reperfusion, arteriolar diameter increased significantly (p less than 0.05), coincident with peak adenosine concentrations. By 60 min of reperfusion, arteriolar diameter had returned to baseline. These results indicate that during the postischemic period, adenine nucleosides and hypoxanthine in CSF are elevated and could affect reperfusion.

We studied the influences of sympathetic and cholinergic mechanisms on pial arteriolar responses during cortical activation in the rat. Adult male Sprague-Dawley rats were anesthetized with α-chloralose and urethane and mechanically ventilated. Pial arterioles on the somatosensory cortex were visualized on a video monitor through a closed cranial window. Changes in arteriolar diameter induced by sciatic nerve stimulation (0.2 V, 5 Hz, 5 ms, for 20 s) were measured before and after (a) ipsilateral superior cervical ganglionectomy (n = 5), (b) intravenous (0.5 mg/kg) administration and topical (10−5 M) application of atropine ( n = 5), and (c) lesion of the nucleus basalis magnocellularis (the major source of intracerebral acetylcholine neurons, n = 7). Unilateral nucleus basalis magnocellularis lesions were performed stereotactically by injection of ibotenic acid (25 nmol/μl). Sensory cortex cholinergic denervation was confirmed histologically. These treatments had no significant effect on arteriolar responses to sciatic nerve stimulation. Thus, the present results suggest that neither sympathetic nor cholinergic mechanisms play a significant role in somatosensory evoked cerebral vasodilation.

We utilized the closed window technique to study the in vivo responses of rat pial arterioles to superfused adenosine agonists. Adenosine and its analogs dilated pial arterioles and exhibited the following order of potency: 5′ N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine (2-CADO) > adenosine = R-N6-phenylisopropyladenosine ( R-PIA) = S-PIA > N6-cyclohexyladenosine (CHA). This potency profile suggests that cerebral vasodilation is mediated through the A2 receptor. Forskolin (10−9 M) potentiated the vasodilation caused by 10−6 M NECA, thus implicating adenylate cyclase activation during NECA-induced vasodilation and providing further support for involvement of the A2 receptor.

This study was undertaken to test the hypothesis that cerebral blood flow (CBF) and the intrinsic optical signal could be dissociated by altering adenosine receptor activity and to uncover the origin of the optic signal using a cranial window in the anesthetized rat.In anesthetized, ventilated, and temperature-controlled rats with closed cranial windows, the authors evaluated simultaneously the alterations in pial arteriolar diameter, intrinsic optical signals (690 nm), and somatosensory evoked potentials during cortical activation evoked by contralateral sciatic nerve stimulation (SNS). To dissociate the vascular and intrinsic signal, they topically applied the adenosine receptors antagonists theophylline (5 microM), which affects A1 and A2A receptors, and 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 microM), which blocks the A(1) receptor. The former interacts primarily with the vasculature whereas the latter influences the parenchyma exclusively.During 20 seconds of contralateral SNS, pial arterioles in the hindlimb somatosensory cortex dilated in a characteristic peak and shoulder pattern. As compared with mock cerebrospinal fluid alone, theophylline significantly (p0.05) attenuated SNS-induced vasodilation (mean+/-standard deviation 8.1+/-2.5% vs 21.7+/-1.9%; 4 rats in each group). In contrast, CPX potentiated vasodilation significantly (p0.05) during SNS (54.7+/-15.8% for the CPX group vs 20.1+/-1.9% for the controls; 5 rats in each group). The change in optical signal persisted after cessation of SNS in all the animals. Thus, the pattern of change of the optical signal was distinctly different from the pattern of changes in arteriolar diameter (which returned rapidly to baseline). Moreover, the optical signal during SNS was increased by 50% by theophylline and by almost 5-fold by CPX (p0.05). The area of change of the intrinsic signal was also increased by the topical application of theophylline and CPX. The somatosensory evoked potential recordings revealed no significant changes after theophylline application, but CPX caused a small diminution of the N1 wave (p0.01).The noncongruent temporal profiles of the changes in pial arteriolar diameter and optical signal, imaged at 690 nm, indicate that the optical signal at 690 nm is not related to CBF. Alteration of adenosine receptor activity independently changed cortical activity, as measured by the optical signal, and CBF, as determined by pial arteriolar diameter. Manipulation of the adenosine receptor activity during increased cortical activity confirmed the temporal dissociation of optical signal and CBF and provided further evidence for the role of adenosine in regulating CBF.

Fetal alcohol syndrome is a leading cause of mental retardation, but mechanisms of alcohol-associated brain damage remain elusive. Chronic alcohol exposure attenuates fetal and neonatal hypoxic cerebral vasodilation in sheep. We therefore hypothesized that alcohol could alter development of cerebrovascular responses to adenosine, a putative mediator of hypoxic cerebral vasodilation. The objective of this study was to examine the effect of earlier fetal alcohol exposure on later reactivity to adenosine in fetal sheep cerebral arterioles. Penetrating intracerebral arterioles were harvested from the brains of third trimester fetal sheep previously exposed in the second trimester to maternal alcohol “binges” (1.5 g/kg IV over 90 min, 5 days/week for 4 weeks) or same-volume saline infusions. Arterioles were cannulated with a micropipette system and luminally pressurized. Fetal alcohol exposure did not affect spontaneous myogenic tone, but enhanced the dilator response of penetrating arterioles to extraluminal acidosis (pH 6.8). Alcohol exposure also resulted in an increase in maximal vessel response to CGS-21680, an adenosine A 2A receptor agonist, but did not alter the concentration-dependent response curves to adenosine. Our results suggest that earlier alcohol exposure does not impair the subsequent responsiveness of fetal cerebral arterioles to vasodilator agents. Thus, alteration in cerebral vascular response to hypoxia in fetal sheep may not be attributed to changes in vascular reactivity to adenosine.

The present study describes modifications to the endovascular filament model of subarachnoid hemorrhage (SAH) in rats. Specifically, we sought to improve the percentage yield of SAH, reduce mortality rates and better simulate human cerebral aneurysmal rupture. Instead of using a 4-0 prolene suture to induce SAH in the existing endovascular filament model, a hollow and flexible polyetrafluoroethylene (PTFE) tube was maneuvered into the proximal anterior cerebral artery (ACA) to ensure that advancement occurred without producing trauma to the vessels. SAH was induced by advancing a tungsten wire through this tube, perforating the ACA at the desired location. These modifications produced significant improvements over the endovascular filament model. Mortality rate declined from 46 to 19%, and SAH was produced more frequently. With the prolene suture, only 48% of our attempts produced a SAH, and unsuccessful attempts typically resulted in an acute subdural hematoma (ASDH). In contrast, the wire/tubing technique was 90% successful at inducing SAH, and led to a significant reduction of ASDH incidence from 44 to 6%. Additionally, the modified technique produced vasospasm in basilar and middle cerebral arteries post-SAH as well as pseudoaneurysms in the proximal ACA which indicated the location of vessel perforation.

Background and Purpose— Disturbances in cerebral arteriolar function, in addition to large vessel vasospasm, may be responsible for ischemia after subarachnoid hemorrhage. The purpose of this study was to test the hypothesis that subarachnoid hemorrhage alters cerebral microvascular reactivity. Methods— An endovascular filament model was used to induce subarachnoid hemorrhage in halothane-anesthetized male Sprague-Dawley rats. We evaluated pial arteriolar responses to sciatic nerve stimulation, topically applied vasoactive agents (adenosine or sodium nitroprusside), and CO 2 inhalation in rats subjected to subarachnoid hemorrhage at 1 to 5 days after insult. Results— In sham-operated rats, sciatic nerve stimulation evoked a 23.5±1.8% increase in arteriolar diameter, which was significantly attenuated to 13.7±0.9%, 12.8±2.5%, and 18.8±2.9% at 24, 48, and 72 hours after subarachnoid hemorrhage, respectively ( P P 2 reactivity was unaffected by subarachnoid hemorrhage. Conclusions— Pial arteriolar responses to cortical activation may be decreased in the initial 2 to 3 days after experimental subarachnoid hemorrhage.

Background and Purpose— Conducted vasomotor responses likely play an important role in cerebrovascular regulation, but it is unclear how these responses may be affected by ischemia. The purpose of this study was to evaluate the hypothesis that cerebral ischemia and reperfusion (I/R) alters vascular conduction in cerebral arterioles. Methods— Middle cerebral artery occlusion (MCAO) was induced by an intraluminal filament technique in 4 groups of rats: (A) 2-hour MCAO/24-hour reperfusion (n=14); (B) 2-hour MCAO/1-hour reperfusion (n=7); (C) 1-hour MCAO/24-hour reperfusion (n=6); and (D) 1-hour MCAO/1-hour reperfusion (n=5). Neurological status and infarction (2,3,5-triphenyltetrazolium chloride staining) were evaluated after I/R. Conducted vasomotor responses were assessed in intracerebral branches of the MCA, by following the longitudinal spread of vasodilation or vasoconstriction to localized microapplication of ATP or adenosine. Results— Local microapplication of ATP evoked a biphasic constriction (17±3%) and dilation (7±2%) response, whereas adenosine elicited only dilation (11±2%). These local responses spread longitudinally along sham-control arterioles (1 mm conduction distance) with rapid spatial decay. Ischemia followed by 24-hour reperfusion (groups A and C) led to a marked potentiation of conducted dilation responses: dilation to ATP conducted with virtually no decay in I/R arterioles. Augmentation of conductivity was not observed in the 1-hour reperfusion groups (B and D). Moreover, I/R did not alter conducted constriction. Conclusions— Ischemia-reperfusion led to a specific augmentation of conducted vasodilation in cerebral arterioles. Presumably, enhanced conductivity may improve cerebral perfusion after ischemia.

Despite caffeine's wide consumption and well-documented psychoactive effects, little is known regarding the effects of caffeine on neurovascular coupling. In the present study, we evaluated the effects of caffeine, an adenosine receptor antagonist, on intracerebral arterioles in vitro and subsequently, on the pial circulation in vivo during cortical activation induced by contralateral sciatic nerve stimulation (SNS). In our in vitro studies, we utilized isolated intracerebral arterioles to determine the effects of caffeine (10 or 50 μmol/L) on adenosine-induced vasodilatation. At the lower concentration, caffeine was without effect, but at the higher concentration, caffeine produced significant attenuation. In our in vivo studies, we determined the cerebrospinal fluid (CSF) caffeine concentrations at 15, 30, and 60 mins after intravenous administration of 5, 10 and 40 mg/kg. At the latter two concentrations, CSF levels exceeded 10 μmol/L. We then evaluated the pial arteriolar response during cortical activation caused by contralateral SNS after administering caffeine intravenously (0, 5, 10, 20 30, and 40 mg/kg). The pial circulation was observed through a closed cranial window in chloralose-anesthetized Sprague—Dawley rats. The contralateral sciatic nerve was isolated, positioned on silver electrodes and stimulated for 20 secs (0.20 V, 0.5 ms, and 5 Hz). Arteriolar diameter was quantified using an automated video dimension analyzer. Contralateral SNS resulted in a 23.8%±3.9% increase in pial arteriolar diameter in the hindlimb sensory cortex under control conditions. Intravenous administration of caffeine at the lowest dose studied (5 mg/kg) had no effect on either resting arteriolar diameter or SNS-induced vasodilatation. However, at higher doses (10, 20, 30, and 40 mg/kg, intravenously), caffeine significantly ( P

✓ Anesthetic agents are often administered in the presence of ethyl alcohol, both in research and in the clinical setting. The authors tested the hypothesis that anesthetic agents may affect cerebrovascular responses to ethanol. A closed cranial window preparation in the rat was used to compare the response of pial arterioles to topically applied ethanol (0.01% to 1% vol/vol) in the presence of α-chloralose/urethane (50 and 600 mg/kg, respectively) or halothane (0.5% to 1%) anesthesia. Heart rate, mean arterial blood pressure, and blood gas levels were maintained stable and within the physiological range throughout each experiment. Ethanol induced significant vasoconstriction in α-chloralose/urethane-anesthetized animals (multivariate analysis of variance (MANOVA), p = 0.039); conversely, ethanol induced significant vasodilation of the pial arterioles in halothane-anesthetized animals (MANOVA, p = 0.017). These responses were significantly different from one another (MANOVA, p = 0.001). Thus, the choice of anesthetic agent alters the cerebrovascular response to ethanol, and care should be taken to ascertain the influence of anesthesia in both research and clinical settings.

The purpose of this study was to investigate the receptor subtypes that mediate the dilation of rat intracerebral arterioles elicited by adenosine. Penetrating arterioles were isolated from the rat brain, cannulated with the use of a micropipette system, and luminally pressurized to 60 mmHg. Both adenosine and the A2A receptor-selective agonist CGS-21680 induced dose-dependent vasodilation (−logEC50: 6.5 ± 0.2 and 8.6 ± 0.3, respectively). However, adenosine, which is capable of activating both A2A and A2B receptors, caused a greater maximal dilation than CGS-21680. The A2Areceptor-selective antagonist ZM-241385 (0.1 μM) only partially inhibited the dilation induced by adenosine but almost completely blocked CGS-21680-induced dilation. Neither 8-cyclopentyl-1,3-dipropylxanthine (0.1 μM), an A1receptor-selective antagonist, nor MRS-1191 (0.1 μM), an A3 receptor-selective antagonist, attenuated adenosine dose responses. Moreover, ZM-241385 had no effect on the dilation induced by ATP (10 μM) or acidic (pH 6.8) buffer. We concluded that the A2A receptor subtype mediates adenosine-induced dilation of intracerebral arterioles in the rat brain. Furthermore, our results suggest that A2B receptors may also participate in the dilation response to adenosine.

Contrary to the concept of neuronal-vascular coupling, cortical evoked potentials do not always correlate with blood flow responses during somatosensory stimulation at changing stimulus rates. The goal of this study is to clarify the effects of stimulus frequency on the relationship between somatosensory evoked potentials (SEPs) and cerebral blood flow. In rats anesthetized with alpha-chloralose, we measured SEPs by signal-averaging field potentials recorded with an electrode placed on dura overlying the hindlimb somatosensory cortex. Regional blood flow was simultaneously assessed in the same region with a laser-Doppler flow (LDF) probe. The contralateral sciatic nerve was stimulated with 0.1 A pulses at the frequencies of 1, 2, 5, 10 and 20 Hz. SEPs (both P1 and N1 components) declined with increasing frequency regardless whether stimulus duration (20 s) or number (100) were kept constant, suggesting that frequency is an important determinant of neuronal activity. In contrast, LDF responses increased to a maximum at 5 Hz, and do not correlate with SEPs. Because CBF should reflect integrated neuronal activity, we computed the sum of SEPS (summation operatorSEP = SEP x stimulus frequency) as an index of total neuronal activity at each frequency. Summation operatorSEP indeed correlates positively (P

We simultaneously measured pial arteriolar diameter and changes in cortical blood flow during activation of the somatosensory cortex by sciatic nerve stimulation. The pial vasculature was visualized with a closed-cranial window technique in chloralose-anesthetized rats ( n = 13). Local blood flow was monitored with laser-Doppler flowmetry. During stimulation of the sciatic nerve (0.2 V, 5 Hz, 20 s), vascular diameter and laser-Doppler flow consistently displayed similar response profiles. With 0.5-ms stimulation pulses, the responses showed an initial peak followed by a smaller but sustained plateau dilation. In contrast, 5-ms pulses evoked a monotonically rising response. Our results support the concept that pial arteriolar diameter changes reflect cortical blood flow responses during somatosensory stimulation.

We measured the changes in pial arteriolar diameter and CSF concentrations of adenosine, inosine, and hypoxanthine during hypoxia in the absence and presence of topically applied dipyridamole (10−6 M) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 10−5 M). Closed cranial windows were implanted in halothane-anesthetized adult male Sprague–Dawley rats for the observation of the pial circulation and collection of CSF. The mean resting arteriolar diameter in mock CSF was 31.2 ± 5.9 μm. Topically applied dipyridamole and EHNA, in combination, caused a slight but significant ( p < 0.05) increase in resting arteriolar diameter (33.8 ± 4.3 μm). With mock CSF, moderate hypoxia caused a 22.1 ± 9.7% increase in pial vessel diameter. Topically applied dipyridamole and EHNA significantly ( p < 0.01) potentiated pial arteriolar vasodilation in response to hypoxia. Moreover, the potentiating effects of dipyridamole and EHNA during hypoxia were completely abolished by theophylline (0.20 μmol/g, i.p.; p < 0.05), an adenosine receptor antagonist. Resting concentrations of adenosine, inosine, and hypoxanthine in the subwindow CSF were 0.18 ± 0.09, 0.35 ± 0.21, and 0.62 ± 0.12 μ M, respectively. In the absence of dipyridamole and EHNA, these levels were not affected by sustained moderate hypoxia (Pao2 = 36 ± 6 mm Hg). However, in the presence of dipyridamole and EHNA, the concentration of adenosine in the CSF during hypoxia was significantly ( p < 0.05) increased. Our data indicate that dipyridamole and EHNA potentiate hypoxic vasodilation of pial arterioles while simultaneously increasing extracellular adenosine levels, thus supporting the hypothesis that adenosine is involved in the regulation of cerebral blood flow.

We have previously demonstrated that rat pial arterioles located on the somatosensory cortex dilated in response to contralateral sciatic nerve stimulation (SNS). We hypothesized that the vasodilation was mediated by adenosine, released as a result of somatosensory cortex activation. To test this hypothesis, we examined the effects of SNS (0.15-0.2 V, 5 ms, 5 Hz for 20 s) on pial arterioles under conditions of altered adenosine availability. Cerebrospinal fluid (CSF) adenosine was altered by perfusing mock CSF, under a cranial window in anesthetized rats, containing either an adenosine uptake competitor (dipyridamole or inosine) or an adenosine receptor blocker (theophylline). With CSF only, SNS caused pial arterioles (resting diam, 29 +/- 1 micron) to dilate by 38 +/- 10% (peak magnitude) for 32 +/- 2 s. Dipyridamole (10(-6) M) significantly (P less than 0.02) enhanced both the magnitude (to 62 +/- 12%) and duration (to 68 +/- 10 s) of the response. Similarly, inosine (10(-3) M) significantly (P less than 0.02) potentiated the vasodilative response from resting values of 27 +/- 5% and 34.8 +/- 4.1 s to 37 +/- 6% and 89.6 +/- 14.1 s. In contrast, theophylline (5 x 10(-5) M) significantly (P less than 0.001) attenuated arteriolar vasodilation from resting values of 38 +/- 5% and 29.3 +/- 1.2 s to 18 +/- 3% and 22.0 +/- 0.9 s. Neither dipyridamole nor theophylline had a significant effect on neuronal response (sensory-evoked response) recorded from the somatosensory cortex. These results suggest that adenosine is involved in the regulation of pial vasodilation during cerebral cortical activation.

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to electrical stimulation of the contralateral sciatic nerve. The mechanism of this response is not well understood. One possibility involves the cell-to-cell conduction of dilator signals via gap junctions from parenchymal to pial vessels. To test the hypothesis that sciatic nerve stimulation (SNS)-induced dilation of pial arterioles involves gap junctional communication, we determined if pharmacological gap junction blockers may prevent the dilation response. We performed a craniotomy over the somatosensory cortex of adult chloralose-anesthetized rat to create an open cranial window. A surface electrode was placed in the same area for somatosensory evoked potential (SEP) recording. Through the craniotomy, a segment of a pial arteriole overlying the hindlimb somatosensory cortex was chosen for study (Figure 1). Microscopic streams of the gap junction blockers octanol (4 mM) and carbenoxolone (75 microM) were applied continuously onto a segment of the chosen pial arteriole by pressure-ejection using micropipettes, while vessel diameter was monitored at sites upstream and downstream from the site of application relative to the direction of blood flow (Fig. 1). Warmed mock cerebrospinal fluid was continuously superfused to disperse drugs applied by micropipette. At the upstream site, octanol (n=6) and carbenoxolone (n=6) reversibly attenuated SNS-induced dilation to 35% and 30% of the baseline response, respectively. The gap junction blockers did not alter the dilation response at the downstream site. Moreover, the blockade was not due to nonspecific impairment of the response by octanol and carbenoxolone, because 1) dilation to hypercapnia remained intact at both upsteam and downstream sites, and 2) SEP did not change. In addition, we evaluated the effect of connexin mimetic peptides homologous to extracellular loop motifs of vascular connexins, on SNS-induced pial dilation. The gap junction peptides, Gap27(37,43) [SRPTEKTIFII, 250 microM, n=5] and Gap26(37,40) [VCYDQAFPISHIR, 250microM, n=5], were superfused over the pial surface for 120 minutes while the dilation response to SNS was observed at 15-minute intervals. Both peptides significantly reduced SNS-induced dilation (by 70% and 50%, respectively), without affecting SEP. These results suggest that the dilation response of pial arterioles to somatosensory stimulation involves the conduction of dilator signals via gap junctions along the vascular wall.

Despite its wide consumption and well-documented psychoactive effects, little is known regarding the effects of caffeine, an adenosine receptor antagonist, on neurovascular coupling. In the present study, we evaluated the effects of caffeine on intracerebral arterioles in vitro and on the pial circulation in vivo during cortical activation induced by contralateral sciatic nerve stimulation (SNS). In our in vitro studies, we utilized isolated intracerebral arterioles to determine the effects of caffeine (10 or 50uM) on vasodilatation induced by extraluminal application of adenosine. Adenosine caused dose-dependent dilations. Pretreatment with caffeine alone, at either of the concentrations tested, had no effect on resting diameter. The higher concentration (50uM) of caffeine resulted in significant attenuation of adenosine-induced vasodilatation (p

This study describes a closed cranial window technique that allows the observation and measurement of rat pial arterioles and venules in situ. The resolving power of this system is 1–2 μm. Using this sensitive technique, we characterized the responses to 7% carbon dioxide inhalation and adenosine in arterioles (10–70 μm) and venules (15–100 μm). During carbon dioxide inhalation, larger arterioles (>40μm) dilated more than smaller arterioles (2 inhalation. Dilation of arterioles was initially observed with an adenosine concentration of 10−8 M. Almost a twofold increase in diameter was noted at 10−3 M. In contrast to the effect of CO2 inhalation, the degree of dilation with topical application of adenosine was not size dependent. Pial venules did not respond to adenosine. The technique for observation of pial vessels using the closed cranial window and for measurement of vessel diameter by video camera system microscopy is a powerful tool for studying in vivo the cerebral circulation in the rat.

We examined the cerebral vasoactivity of inosine and its effect on pial arteriolar vasodilation induced by adenosine. Pial circulation was observed through cranial windows implanted in rats anesthetized with halothane. No significant change in venous or arteriolar diameter was apparent when inosine (10(-6) to 10(-3) M) was superfused. In contrast, adenosine in concentrations of 10(-7) and 10(-6) M dilated pial arterioles by 9.0 +/- 1.2 and 17.7 +/- 1.7%, respectively. Addition of 10(-5) M inosine had no effect, whereas 10(-4) M inosine enhanced the vasodilation induced by 10(-7) M adenosine to 19.4 +/- 1.7% and that by 10(-6) M adenosine to 23.3 +/- 2.3%. When theophylline (5 X 10(-5) M) was perfused together with 10(-7) M adenosine and 10(-4) M inosine, the vasodilation was almost completely abolished. The present study indicates that inosine alone does not affect pial vessel diameter but potentiates the response of pial arterioles to exogenous adenosine. This potentiating action of inosine may be attributed to an increase in perivascular adenosine and may be explained by the action of inosine as an adenosine uptake inhibitor.

We analyzed the retroglenoid venous outflow (VOF) technique in the rat to document the validity of this method of measuring cerebral blood flow (CBF). Stereotypic changes in CBF were obtained with VOF during hypercarbia and hypotension. O2 content of retroglenoid venous blood did not differ significantly from O2 content of blood obtained from the sagittal sinus, suggesting minimal extracerebral contamination of the retroglenoid venous blood. This lack of extracerebral contamination was further analyzed using a double tracer technique (125I-labeled serum albumin, 22Na) that quantitated minimal extracerebral contamination in the retroglenoid vein. CBF measurements were made simultaneously using microsphere and VOF methods, and excellent correlation was found between the two techniques over a wide range of CBF during normoxia, hypoxia, and normoxic hypocarbia and hypercarbia. However, a decrease in the ratio of VOF to microsphere CBF was observed during severe normoxic hypotension (mean arterial pressure = 41 +/- 4 mmHg). VOF represented 18% of total CBF as measured by microsphere method. This study indicates that the retroglenoid outflow technique in rats is a valid method of measuring CBF.

The present study documents the microvascular response of the pial circulation in sensory hindlimb cortex to sciatic nerve stimulation. Rats, anesthetized with alpha-chloralose and urethan, were equipped with closed cranial windows, and pial arteriolar diameter was measured during stimulation of the contralateral sciatic nerve. The effects of varying stimulus frequency, intensity, and duration were examined. Optimal stimulus frequency was 5 Hz, but response diminished significantly beyond 10 Hz. Optimal stimulus intensity was 0.2 V. At higher stimulus strength, arteriolar dilation was reduced, but systemic blood pressure rose significantly. At low stimulus frequency and intensities, pial arterioles responded to stimulation with a consistent pattern: initial delay of 1.4 s followed by abrupt dilation to a peak magnitude, subsequent decline to a lesser but still dilated state, and recovery to a resting diameter after the cessation of stimulation. No consistent response profile was discernible at high stimulus intensity and/or frequency. This vasodilatory response was discretely restricted to a limited number of arterioles, confined to the hindlimb somatosensory cortex as confirmed by sensory evoked response. The response of the pial circulation provides a well-characterized model for analysis of brain microcirculation, which presumably is linked to cerebral metabolism.

Hypocarbia results in an increase in brain adenosine concentrations, presumably because of brain hypoxia associated with hypocarbic vasoconstriction. It was hypothesized that adenosine limits the degree of hypocarbic vasoconstriction. To test this hypothesis, the effects of dipyridamole and theophylline on CO2 reactivity during hypocarbia were investigated in anesthetized rats. Dipyridamole should reduce the vasoconstriction by potentiating adenosine action, whereas theophylline should increase the vasoconstriction by blocking adenosine receptors. Cortical pial arterioles of mechanically ventilated and anesthetized rats were displayed on a video monitor system through a closed cranial window. Arterial blood pressure and oxygen tension were stable. CO2 reactivity, formulated as 100 x [Δ diameter (μm)/resting diameter (μm)]/Δ PaCO2 (mmHg), in the hypocarbic phase was calculated before and after topical superfusion of dipyridamole (10−6 M; n = 7) and theophylline (5 × 10−5 M; n = 6). CO2 reactivity was significantly decreased after superfusion of dipyridamole (0.57 ± 0.08; mean ± SEM) as compared with mock cerebrospinal fluid (CSF) (0.97 ± 0.17, p < 0.05, n = 7). On the other hand, CO2 reactivity after superfusion of theophylline was increased (1.63 ± 0.28) as compared with mock CSF (1.00 ± 0.20, p < 0.05, n = 6), indicating that adenosine is involved in hypocarbic vasoconstriction.