Al C. Ngai

Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide : With detailed description of the closed cranial window technique in rats

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 (<20 μm). There was limited vasoreac-tivity of pial venules during CO2 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.

Modulation of Cerebral Arteriolar Diameter by Intraluminal Flow and Pressure

We determined whether cerebral arterioles in vitro adjust their diameters in response to changes in intraluminal flow rate and pressure. Intracerebral arterioles (38- to 55-microns diameter) were isolated from Sprague-Dawley rats and cannulated with a perfusion system that permitted separate control of intraluminal pressure and flow rates. Increasing pressure at 0 flow, in 20 mm Hg steps from 20 to 100 mm Hg, resulted in myogenic constriction, which was greatest at 60 mm Hg (approximately 20%). Increasing flow rate at a constant pressure of 60 mm Hg elicited a biphasic response. At flow rates of up to 10 microL/min, the arterioles dilated by up to 14.5 +/- 2.2% of their control diameter. At higher (> 10 microL/min) flow rates, however, a progressive restoration of resting diameter was observed. Application of the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA, 0.1 mmol/L) caused a 15.4 +/- 1.7% decrease in control diameter (at 60 mm Hg, zero flow). Although L-NMMA did not affect the responses to increases in pressure or to vasodilators (adenosine and pH 6.8 buffer), it abolished the dilator responses to flow rate increases and to acetylcholine. In contrast, inhibition of prostaglandin synthesis by indomethacin (10 mumol/L) had no effect on flow-induced dilation. These results show that changes in intraluminal flow rates and pressure can independently influence cerebral arteriolar tone and suggest that the flow-induced dilator responses of cerebral arterioles are mediated by an arginine metabolite, such as nitric oxide.

Frequency-dependent changes in cerebral blood flow and evoked potentials during somatosensory stimulation in the rat.

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<0.001) with LDF responses. Thus, during somatosensory stimulation at various frequencies, cerebral blood flow is coupled to integrated neuronal activity but not to averaged evoked potentials.

Simultaneous Measurements of Pial Arteriolar Diameter and Laser-Doppler Flow during Somatosensory Stimulation

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.

Differential effects of alcohols on intracerebral arterioles. Ethanol alone causes vasoconstriction

We compared the effect of the acute application of ethanol, methanol, 1-propanol, 1-butanol, urea, and mannitol (1–100 mM) on the basal tone of isolated–cannulated rat intracerebral arterioles to determine if the response of these arterioles to ethanol could be attributed to alteration of membrane fluidity or changes in osmolality. These arterioles spontaneously developed tone to 62.0 ± 8.4% of passive diameter (44.2 ±11.9 vs. 70.9 ± 14.7 μm). Ethanol caused a dose-dependent reduction in arteriolar diameter starting at 3 mM (p = 0.03), reaching a diameter of 81.4 ± 3.0% of basal tone at 100 mM. In comparison, all other agents tested caused the arterioles to dilate, with the exception of 1-propanol, which produced inconsistent vessel responses. At 100 mM concentration, methanol, 1-butanol, urea, and mannitol dilated intracerebral arterioles by 116.1 ± 12.7, 151.5 ± 12.4, 131.1 ± 17.0, and 149.8 ± 6.6%, respectively. Thus, in a concentration range associated with acute intoxication, ethanol causes constriction of isolated intracerebral arterioles. The mechanism of action of ethanol cannot be accounted for solely based upon its physicochemical characteristics of osmolality or lipid solubility, but rather may reflect a more specific action on one or more cellular mechanisms responsible for determining basal intracerebral arteriolar tone. The characterization of the response of intracerebral arterioles to ethanol is important in view of epidemiologic links between ethanol consumption and cerebrovascular disease.

Effects of Topical Adenosine Analogs and Forskolin on Rat Pial Arterioles in vivo

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.

Effect of Caffeine on Cerebral Blood Flow Response to Somatosensory Stimulation

Despite caffeines 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<0.05; n=6) attenuated both resting diameter and cerebral blood flow (CBF) responses to somatosensory stimulation. Intravenous administration of theophylline (10, 20, and 40 mg/kg), another adenosine receptor antagonist, also significantly reduced SNS-induced vasodilatation in a dose-dependent manner. Hypercarbic vasodilatation was unaffected by either caffeine or theophylline. The results of the present study show that caffeine significantly reduces cerebrovascular responses to both adenosine and to somatosensory stimulation and supports a role of adenosine in the regulation of CBF during functional neuronal activity.

Changes in Pial Arteriolar Diameter and CSF Adenosine Concentrations during Hypoxia

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 μM, 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.

Postischemic Augmentation of Conducted Dilation in Cerebral Arterioles

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.

Adenosine Release and Changes in Pial Arteriolar Diameter during Transient Cerebral Ischemia and Reperfusion

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 μM, while inosine and hypoxanthine were 0.35 ± 0.17 and 1.23 ± 0.47 μM, 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 values 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 μm and was not significantly changed after 20 min of ischemia. However, during the first 5 min of reperfusion, arteriolar diameter increased significantly (p < 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.