Selective attenuation by perivascular blood of prostanoid-dependent cerebrovascular dilation in piglets

Cerebral hemorrhagic insults are common in neonates. However, the consequences of intracranial blood on cerebral hemodynamics are poorly understood. We examined the effects of perivascular blood on cerebrovascular dilator responses in 29 piglets. Fresh, autologous blood (n = 15) or cerebrospinal fluid (n = 14) was placed under the dura mater over the parietal cortex, and the piglets were allowed to recover from anesthesia. One to four days later, a closed cranial window was placed over the parietal cortex and pial arteriolar responses to arterial hypercapnia (PaCO2 greater than 55 mm Hg), hemorrhagic hypotension (mean arterial blood pressure less than 35 mm Hg), or topical application of 10(-6) and 10(-4) M isoproterenol were determined. Pial arterioles in the cerebrospinal fluid group dilated 27 +/- 4% (mean +/- SEM) (n = 11) in response to hypercapnia, 26 +/- 5% (n = 9) in response to hypotension, and 26 +/- 3% in response to 10(-6) M and 40 +/- 4% in response to 10(-4) M isoproterenol (n = 11). In the group in which blood was placed on the parietal cortex, pial arterioles did not dilate significantly in response to hypercapnia (8 +/- 3%, n = 11) or hypotension (2 +/- 5%, n = 13) but dilated normally in response to isoproterenol (25 +/- 5% in response to 10(-6) M and 36 +/- 7% in response to 10(-4) M, n = 13). We conclude that prolonged contact of pial arterioles with extravascular blood selectively attenuates cerebrovascular dilation in piglets.     

Segmental vascular resistance after mild controlled cortical impact injury in the rat.

In an effort to localize the site at which increased resistance occurs after brain trauma, pial arteriole diameter and pressure were assessed after mild controlled cortical impact (CCI) injury in rats using an open cranial window technique. The authors tested the hypothesis that an increase in resistance accompanied by vasoconstriction occurs at the level of the pial arterioles within the injured cortex of the brain. At 1 hour after mild CCI injury, ipsilateral cerebral blood flow was significantly reduced by 42% compared with sham injury (n = 4; < 0.05). Pial arteriole diameter and pressure remained unchanged. Resistance in the larger arteries (proximal resistance), however, was significantly greater after CCI injury (1.87 +/- 0.26 mm Hg/[mL. 100 g. min]) compared with sham injury (0.91 +/- 0.21 mm Hg/[mL. 100 g. min]; < 0.0001). Resistance in small vessels, arterioles, and venules (distal resistance) was also significantly greater after CCI injury (1.13 +/- 0.05 mm Hg/[mL. 100 g. min]) compared with sham injury (0.74 +/- 0.13 mm Hg/[mL. 100 g. min]; = 0.0001). The authors conclude that, at 1 hour after mild CCI injury, changes in vascular resistance comprise a 53% increase in the resistance distal to the area of injury and, surprisingly, a 105% increase in resistance in the arteries proximal to the injury site.     

Cerebral vasoconstriction in response to hypocapnia is maintained after ischemia/reperfusion injury in newborn pigs.

BACKGROUND AND PURPOSE: Hypocapnic cerebral vasoconstriction is used therapeutically to reduce elevated intracranial pressure caused by cerebral edema. Because cerebral ischemia/reperfusion injury causes a selective loss of prostanoid-dependent responses, including vasodilation to hypercapnia, we designed these experiments to examine the effect of ischemia/reperfusion on hypocapnic cerebral vasoconstriction. METHODS: Microvascular responses were studied in 10 newborn pigs (closed cranial window) in response to hyperventilation-induced hypocapnia (PaCO2, 22 +/- 2 mm Hg) both before and 45 minutes after 20 minutes of global cerebral ischemia. Responses to hypercapnia (PaCO2, 63 +/- 3 mm Hg), topical isoproterenol (10(-7) M), and norepinephrine (10(-4) M) were also studied before and after ischemia in the same animals for comparison. RESULTS: Before ischemia/reperfusion, pial arterioles vasoconstricted to hypocapnia (-17 +/- 2%) and norepinephrine (-35 +/- 4%) and vasodilated to CO2 (37 +/- 7%) and isoproterenol (25 +/- 2%). After ischemia/reperfusion, the constriction of pial arterioles to hypocapnia (-19 +/- 2%) was similar to that before ischemia. This is in contrast to the loss of dilation to hypercapnia. Dilation to isoproterenol and constriction to norepinephrine were not affected by ischemia. CONCLUSIONS: Hypocapnic cerebral vasoconstriction is maintained after ischemia/reperfusion. Since prostanoid-dependent responses, such as hypercapnic dilation, are lost following cerebral ischemia, these data suggest that hypocapnic constriction is not dependent on an intact prostanoid system and that cerebral vascular responses to CO2 involve multiple mechanisms, depending on whether CO2 is increasing or decreasing from baseline.     

Leukocyte adherence contributes to disruption of the blood-brain barrier during activation of mast cells

The goal of this study was to examine the role of leukocytes in disruption of the blood-brain barrier during activation of mast cells using compound 48/80. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood-brain barrier (clearance of fluorescent-labeled dextran; molecular weight 10000 daltons; FITC-dextran-10 K) was determined while suffusing with vehicle or compound 48/80 (10 or 25 microg/ml). During superfusion with vehicle (saline), clearance of FITC-dextran-10 K from pial vessels was modest and remained relatively constant during the experimental period (0.52+/-0.05 ml/sx10(-6) at 80 min). In addition, diameter of pial arterioles remained constant (32+/-5 microm) while suffusing with vehicle. In contrast, topical application of compound 48/80 produced marked disruption of the blood-brain barrier to FITC-dextran-10 K. For example, suffusion with compound 48/80 (25 microg/ml) increased clearance of FITC-dextran-10 K about 4-fold to 2.26+/-0.25 ml/sx10(-6) at 80 min. In addition, superfusion with compound 48/80 (25 microg/ml) constricted pial arterioles by 26+/-9% at 80 min. To determine a potential role for leukocyte adhesion to endothelium in disruption of the blood-brain barrier during suffusion with compound 48/80, we examined permeability during suffusion with compound 48/80 (25 microg/ml) in the presence of WT.3 (2 mg/kg i.v.), a monoclonal antibody directed against the functional epitope of the leukocyte adhesive glycoprotein (CD18; LFA-1beta). We found that infusion of WT.3 markedly attenuated disruption of the blood-brain barrier to FITC-dextran-10 K in response to compound 48/80. The clearance of FITC-dextran-10 K during superfusion with compound 48/80 in the presence of WT.3 was 1.29+/-0.14 ml/sx10(-6) at 80 min (P<0.05). Thus, the findings of the present study suggest that application of compound 48/80, to degranulate mast cells, activates the adhesion of leukocytes to cerebral venular endothelium which contributes to disruption of the blood-brain barrier.     

Stimulus duration modulates the interaction between opioids and nitric oxide in hypoxic pial artery dilation

Since recent studies show that pial artery dilation during a 20 or 40 min hypoxic exposure was less than that observed during a 5 or 10 min exposure, stimulus duration determines the nature of the vascular response to hypoxia. Decremented hypoxic pial dilation during longer exposure periods results, at least in part, from decreased release of methionine enkephalin (Met), an opioid known to contribute to dilation during hypoxia. Nitric oxide and cGMP contribute to both release and the vascular response to this opioid. The present study was designed to determine if the stimulus duration modulates the interaction between opioids and NO in hypoxic pial dilation using newborn pigs equipped with a closed cranial window. Elevation of CSF cGMP during hypoxia (Po2 approximately 35 mmHg) was dependent on stimulus duration (435+/-31, 934+/-46, 747+/-25, and 623+/-17 fmol/ml cGMP during normoxia and after 10, 20, and 40 min of hypoxia). Met-induced pial dilation during hypoxia was also stimulus duration dependent (7+/-1, 10+/-1, and 15+/-1, vs. 4+/-1, 6+/-1, and 8+/-2 vs. 2+/-1, 3+/-1, and 5+/-1% for 10(-10), 10(-8), 10(-6) M Met during normox and after 20, and 40 min of hypoxia). Additionally, the release of cGMP by Met during hypoxia was also stimulus duration dependent (1.8+/-0.1 vs. 1.6+/-0.1 vs. 1.3+/-0.1 fold change in CSF cGMP for 10(-8) M Met during normoxia and after 20 and 40 min of hypoxia). These data indicate that the diminished role of Met in pial dilation during longer hypoxic exposure periods results from a diminished capacity of this opioid to elicit dilation. Such impaired dilation is correlated with diminished stimulated cGMP release. These data also suggest that diminished CSF cGMP release during prolonged hypoxia contributes to decreased release of Met during longer hypoxic periods. Therefore, stimulus duration modulates the interaction between opioids and NO in hypoxic pial artery dilation.     

Effects of cocaine on pial arterioles in cats

We used the closed cranial window technique to observe the responses of pial arterioles to topical application of cocaine in 29 anesthetized cats. Alterations in arteriolar diameter were dependent on the concentration of cocaine applied. Cocaine dissolved in artificial cerebrospinal fluid at concentrations of 10(-8) or 10(-7) M was without effect. Concentrations of 10(-6) and 10(-5) M produced dilation (4.9 +/- 1.5% [mean +/- SEM] and 5.9 +/- 2.0%, respectively) in large arterioles (greater than 100 microns) but no significant change in the diameter of small arterioles (less than 100 microns). A concentration of 10(-4) M dilated both large and small arterioles (20.3 +/- 3.1% and 12.0 +/- 7.1%, respectively). Pretreatment with 1 mg/kg i.v. propranolol blocked the increase in pial arteriolar diameter after application of 10(-4) M cocaine and produced significant vasoconstriction in small arterioles (-8.3 +/- 3.1%). Cocaine produces vasodilation of cat cerebral arterioles. This effect appears to be mediated, at least in part, by mechanisms that depend on stimulation of beta-adrenergic receptors.     

Administration of selective endothelin receptor type A antagonist Ro 61-1790 does not improve outcome in focal cerebral ischemia in cat.

The authors examined the effect of selective endothelin (ET) receptor type A (ET(A)) antagonism on histological and functional recovery in cat at 24 hours after reversible middle cerebral artery occlusion (MCAO). A novel and specific ET(A) antagonist, Ro 61-1790 [5-methylpyridine-2-sulfonic acid-6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(2-1H-tetrazol-5-y l-pyridin-4-yl)-pyrimidin-4-ylamide sodium salt (1:2)] (Roche, Basel, Switzerland), was used at doses that produced steady-state plasma concentrations and abolished ET-induced pial arteriolar vasoconstriction. In a cranial window preparation, 8 nmol/L ET constricted pial arterioles by 33 +/- 18% (mean +/- SD), but this response was ablated by intravenous Ro 61-1790 treatment (10-mg/kg bolus, 4-mg/kg/h infusion). In additional animal cohorts, halothane-anesthetized cats were treated with 90 minutes of MCAO and 24 hours of reperfusion. Animals received Ro 61-1790 infusion beginning at the onset of reperfusion and continuing for 6 or 24 hours (n = 41). Control cats were treated with 0.9% saline by intravenous infusion throughout reperfusion. There was no difference in injury volume or neurologic evaluation score in saline-treated cats (n = 11; caudate 24 +/- 28%, cortical injury 7.5 +/- 5% of ipsilateral structure; score 52 +/- 8) versus the results in cats treated with Ro 61-1790 for either 24 hours (n = 6; caudate 22 +/- 23%, cortex 6 +/- 5%, injury volume of ipsilateral structure; score 55 +/- 3) or 6 hours (n = 11; caudate 33 +/- 30%, cortex 12 +/- 14%, injury volume of ipsilateral structure; score 50 +/- 10). Mortality was greatest in the 24-hour drug treatment group. These data suggest that blockade of ET(A) receptor activity is not beneficial to tissue or functional outcomes from experimental stroke in cat.     

Evidence against parenchymal metabolites directly promoting pial arteriolar dilation during cortical spreading depression in rabbits

The role of parenchymal metabolic factors in directly promoting pial arteriolar dilation during cortical spreading depression (CSD) in anesthetized rabbits was examined by direct measurement of periarachnoid cerebrospinal fluid (CSF) levels of a representative metabolite (i.e., K+) or superfusion of the cerebral cortical surface with artificial CSF. CSD was induced by KCl microinjection or tissue puncture and its movement was monitored electrophysiologically. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. CSD propagated across the cortex under the window with a velocity of 2.9 +/- 0.2 mm/min, and caused pial arteriolar diameter to increase from 87 +/- 9 microns to 133 +/- 11 microns (53%, n = 23) for 1.6 +/- 0.1 min. At the same time, K+ concentration increased from 3.0 +/- 0.2 mM to a maximum of 4.6 +/- 0.3 mM. Topical application of 6 mM K+ increased pial arteriolar diameter by only 8%. Continuous superfusion of the cortical surface with aCSF at a rate of 3.0-4.5 ml/min (window volume = 0.5 ml) did not affect pial arteriolar dilation during CSD, but virtually abolished pial arteriolar dilation during inhalation of 10.2% CO2. These results suggest that pial arterioles dilate via a mechanism which does not involve diffusion of vasoactive metabolites released from the parenchyma during CSD.     

Superoxide generation links nociceptin/orphanin FQ (NOC/oFQ) release to impaired N-methyl-D-aspartate cerebrovasodilation after brain injury

BACKGROUND AND PURPOSE: Although activation of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to altered cerebrovascular regulation after traumatic brain injury, the effects of such injury on the vascular response to NMDA itself has been less well appreciated. The newly described opioid nociceptin/orphanin FQ (NOC/oFQ) elicits pial artery dilation, at least in part, in a prostaglandin-dependent manner and is released into cerebrospinal fluid after fluid percussion brain injury (FPI). Generation of superoxide anion (O(2)(-)) occurs after FPI, and a byproduct of cyclooxygenase metabolism is the generation of O(2)(-). This study was designed to determine whether NOC/oFQ generates O(2)(-), which in turn could link NOC/oFQ release to impaired NMDA-induced pial artery dilation after FPI. METHODS: Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O(2)(-) generation. RESULTS: Under non-brain injury conditions, topical NOC/oFQ (10(-)(10) mol/L, the concentration present in cerebrospinal fluid after FPI) increased superoxide dismutase-inhibitable NBT reduction from 1+/-1 to 20+/-3 pmol/mm(2) but had no effect itself on pial artery diameter. Indomethacin (5 mg/kg IV) blunted such NBT reduction (1+/-1 to 6+/-2 pmol/mm(2)), whereas the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH(2) (10(-)(6) mol/L) blocked NBT reduction. [F/G] NOC/oFQ (1-13) NH(2) and indomethacin also blunted the NBT reduction observed after FPI (1+/-1 to 15+/-1 versus 1+/-1 to 4+/-1 versus 1+/-1 to 4+/-1 pmol/mm(2) for sham, NOC/oFQ antagonist, and indomethacin-treated animals, respectively). NMDA (10(-)(8) and 10(-)(6) mol/L)-induced pial artery dilation was reversed to vasoconstriction after FPI, and [F/G] NOC/oFQ (1-13) NH(2) attenuated such vasoconstriction (sham 9+/-1% and 16+/-1% versus FPI -7+/-1% and -12+/-1% versus FPI-[F/G] NOC/oFQ (1-13) NH(2)-pretreated animals -2+/-1% and -3+/-1%). Indomethacin and the free radical scavengers polyethylene glycol superoxide dismutase and catalase also partially restored NMDA-induced vasodilation. CONCLUSIONS: These data show that NOC/oFQ, in concentrations present in cerebrospinal fluid after FPI, increased O(2)(-) production in a cyclooxygenase-dependent manner and contributes to such production after FPI. These data show that NOC/oFQ contributes to impaired NMDA-induced pial artery dilation after FPI. Therefore, these data suggest that cyclooxygenase-dependent O(2)(-) generation links NOC/oFQ release to impaired NMDA-induced cerebrovasodilation after brain injury.     

Endothelium-derived relaxing factor modulates noradrenergic constriction of cerebral arterioles in rabbits.

BACKGROUND AND PURPOSE: Cerebral arterioles are relatively unresponsive to norepinephrine. We tested the hypothesis that release of endothelium-derived relaxing factor is stimulated by norepinephrine and attenuates adrenergic constriction of pial arterioles. METHODS: In seven anesthetized New Zealand White rabbits, diameter of pial arterioles was measured through a cranial window. Responses to topical application of norepinephrine and arginine vasopressin were examined before and during application of NG-nitro-L-arginine, which inhibits synthesis of endothelium-derived relaxing factor. RESULTS: Norepinephrine (10(-6) M) had no effect (0 +/- 3%, mean +/- SE) on arteriolar diameter under basal conditions. Norepinephrine decreased arteriolar diameter by 15 +/- 4% during application of nitro-L-arginine (10(-4) M) (p less than 0.05 versus basal response). L-arginine inhibited the effect of nitro-L-arginine on responses of pial arterioles to norepinephrine. In contrast to norepinephrine, constrictor responses of pial arterioles to vasopressin were not potentiated by nitro-L-arginine. CONCLUSIONS: Norepinephrine, but not arginine vasopressin, releases endothelium-derived relaxing factor, which inhibits constrictor responses of cerebral arterioles in rabbits. This mechanism contributes to the finding that cerebral vessels in rabbits are relatively unresponsive to noradrenergic stimuli.     

Nitric oxide donor-induced increase in permeability of the blood-brain barrier

The goal of the present study was to determine the effect of nitric oxide (NO) donors on the permeability of the blood-brain barrier in vivo. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood-brain barrier was quantitated by calculating the clearance of fluorescent-labeled dextran (M(w)=10000 Da; FITC-dextran-10K) during suffusion with vehicle, S-nitroso-N-acetylpenicillamine (SNAP; 100 microM) and 3-morpholinosydnonimin (SIN-1; 100 microM). In addition, we examined changes in arteriolar diameter during suffusion with vehicle, SNAP and SIN-1. During suffusion with vehicle, clearance of FITC-dextran-10K from pial vessels and diameter of pial arterioles remained relatively constant during the experimental period. In contrast, suffusion with SNAP or SIN-1 markedly increased clearance of FITC-dextran-10K from the cerebral microcirculation and produced a rapid, sustained dilatation of pial arterioles. Thus, NO donors increase the permeability of the blood-brain barrier and produce pronounced dilatation of cerebral arterioles. In light of evidence suggesting that NO donors may produce their effect by the simultaneous release of NO and superoxide anion to form peroxynitrite, we elected to examine the role of superoxide anion in increases in permeability of the blood-brain barrier in response to SNAP and SIN-1. We found that suffusion with tiron (1 mM) did not alter basal permeability of the blood-brain barrier, but significantly inhibited increases in permeability of the blood-brain barrier in response to SNAP and SIN-1. In addition, tiron did not alter baseline diameter of cerebral arterioles, or SNAP- and SIN-1-induced cerebrovasodilatation. The findings of the present study suggest that NO donors produce an increase in permeability of the blood-brain barrier which appears to be related to the presence of NO and superoxide anion, to presumably form peroxynitrite. We suggest that increases in NO formation observed during brain trauma may contribute to disruption of the blood-brain barrier.     

Exogenous norepinephrine constricts cerebral arterioles via alpha 2-adrenoceptors in newborn pigs

The purpose of this study was to determine whether exogenous norepinephrine mediates cerebrovascular constriction via alpha 1- or alpha 2-adrenoceptors in anesthetized neonatal pigs. Diameters of pial arterioles in anesthetized piglets, 1--6 days old, were investigated using a "closed" cranial window. We examined constrictor effects of norepinephrine on pial arterioles in the absence and presence of relatively selective alpha 1-(prazosin) and alpha 2-(yohimbine) adrenoceptor antagonists (1 mg/kg i.v.). Yohimbine and prazosin inhibited pial arteriolar constriction induced by topical application of clonidine and phenylephrine (10(-6) and 10(-4) M, respectively), and yohimbine did not affect the response to topical phenylephrine. In one group diameter was 188 +/- 13 (mean +/- SEM) micron during control and 146 +/- 12 micron during 10(-5) M norepinephrine (22 +/- 5% constriction). Following yohimbine the same vessels did not constrict significantly. In another group 10(-5) M norepinephrine constricted arterioles by 22 +/- 5%, and this response was unaffected by prazosin (24 +/- 5% constriction). We conclude that pial arterioles are responsive to both alpha 1- and alpha 2-adrenoceptor agonists, that intravenous administration of prazosin and yohimbine results in these drugs crossing the blood-brain barrier and inhibiting constrictor effects of agonists, and that norepinephrine constricts pial arterioles predominantly via alpha 2-adrenoceptors.     

Perivascular blood attenuates noradrenergic but not cholinergic effects on piglet pial arterioles

We examined the chronic and acute effects of perivascular blood on cerebrovascular responses to norepinephrine and acetylcholine in 35 piglets. In the chronic experiment, fresh autologous blood (n = 15) or cerebrospinal fluid (n = 14, control) was placed under the dura mater over the parietal cortex, and the piglets were allowed to recover from anesthesia. One to 4 days later, a closed cranial window was placed over the parietal cortex and baseline pial arteriolar responses and responses to topical application of the neurotransmitters norepinephrine (10(-6) and 10(-4) M) and acetylcholine (10(-4) M) were determined. We also sampled cerebrospinal fluid from under the window during baseline conditions and during application of the neurotransmitters, and we measured the concentrations of prostanoids (6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin F2 alpha, and prostaglandin E2) via radioimmunoassay. Pial arterioles in the chronic control group (n = 13) constricted by 20 +/- 2% (mean +/- SEM) in response to 10(-4) M norepinephrine and by 28 +/- 2% in response to 10(-4) M acetylcholine. In the chronic blood group (n = 14), pial arterioles did not constrict significantly in response to 10(-4) M norepinephrine but constricted normally (23 +/- 4%) in response to 10(-4) M acetylcholine. In the acute experiment, six other piglets had blood placed on the brain surface for 30 minutes and then removed; pial arterioles constricted by 21 +/- 1% in response to 10(-4) M norepinephrine (n = 5) and by 28 +/- 4% in response to 10(-4) M acetylcholine (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of impaired endothelium-dependent cerebral vasodilatation in response to bradykinin in hypertensive rats

Bradykinin produces less dilatation of pial arterioles in stroke-prone spontaneously hypertensive rats than in normotensive Wistar-Kyoto rats. The goals of this study were to determine the mediator of bradykinin-induced dilatation in cerebral arterioles of rats and to determine whether responses to this mediator are altered in hypertensive rats. Diameter of pial arterioles (20-65 microns) was measured using intravital microscopy in 18 normotensive and 17 hypertensive rats. Superfusion of 3 x 10(-7) M bradykinin dilated pial arterioles by 53 +/- 4% (mean +/- SEM) in normotensive rats but only 33 +/- 6% in hypertensive rats (p less than 0.05 versus normotensive rats). Vasodilatation in response to bradykinin was almost completely inhibited by 280 units/ml catalase in both normotensive and hypertensive rats (n = 7 and n = 7, respectively) whereas 150 units/ml superoxide dismutase (n = 6 and n = 5, respectively) and 1 mM deferoxamine (n = 5 and n = 5, respectively) did not attenuate bradykinin-induced vasodilatation. These findings suggest that hydrogen peroxide is the mediator of bradykinin-induced dilatation in cerebral arterioles of rats. We also examined responses of cerebral arterioles to hydrogen peroxide in five normotensive and six hypertensive rats. Dilator responses of cerebral arterioles to 3.2 x 10(-5) M to 1.6 x 10(-4) M hydrogen peroxide did not differ in normotensive and hypertensive rats, which suggests that impaired dilatation of cerebral arterioles in response to bradykinin is not related to altered responsiveness of smooth muscle to an endothelium-derived relaxing factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic--but not nicotinic--acetylcholine receptors mediate a nitric oxide-dependent dilation in brain cortical arterioles: a possible role for the M5 receptor subtype.

Increases in cortical cerebral blood flow are induced by stimulation of basal forebrain cholinergic neurons. This response is mediated in part by nitric oxide (NO) and reportedly involves both nicotinic and muscarinic receptors, some of which are possibly located in the vessel wall. In the present study, the vasomotor response(s) elicited by acetylcholine (ACh) on isolated and pressurized bovine and/or human intracortical penetrating arterioles were investigated, and pharmacological characterization of the receptor involved in this response was carried out. Acetylcholine (10(-11) to 10(-4) mol/L) dose dependently dilated bovine and human intracortical arterioles at spontaneous tone (respective pD2 values of 6.4+/-0.3 and 7.2+/-0.3 and E(Amax) of 65.0+/-26.8 and 43.2+/-30.1% of the maximal dilation obtained with papaverine) and bovine arterioles after preconstriction with serotonin (pD2 = 6.3+/-0.1, E(Amax) = 80.0+/-17.9% of induced tone). In contrast, nicotine (10(-8) to 10(-4) mol/L) failed to induce any vasomotor response in bovine vessels whether at spontaneous or at pharmacologically induced tone. Application of the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-5) mol/L) elicited a gradual constriction (approximately 20%) of the arterioles, indicating the presence of constitutive NO release in these vessels. Nomega-Nitro-L-argigine (10(-5) to 10(-4) mol/L) also significantly blocked the dilation induced by ACh. The muscarinic ACh receptor (mAChR) antagonists pirenzepine, 4-DAMP, and AF-DX 384 dose dependently inhibited the dilatation induced by ACh (10(-5) mol/L) with the following rank order of potency: 4-DAMP (pIC50 = 9.2+/-0.3) > pirenzepine (pIC50 = 6.7+/-0.4) > AF-DX 384 (pIC50 = 5.9+/-0.2). These results suggest that ACh can induce a potent, dose-dependent, and NO-mediated dilation of bovine and/or human intracortical arterioles via interaction with an mAChR that best corresponds to the M5 subtype.     

Nitric oxide donor-induced increase in permeability of the blood–brain barrier

The goal of the present study was to determine the effect of nitric oxide (NO) donors on the permeability of the blood–brain barrier in vivo. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood–brain barrier was quantitated by calculating the clearance of fluorescent-labeled dextran (M_w=10 000 Da; FITC–dextran-10K) during suffusion with vehicle, S-nitroso-N-acetylpenicillamine (SNAP; 100 μM) and 3-morpholinosydnonimin (SIN-1; 100 μM). In addition, we examined changes in arteriolar diameter during suffusion with vehicle, SNAP and SIN-1. During suffusion with vehicle, clearance of FITC–dextran-10K from pial vessels and diameter of pial arterioles remained relatively constant during the experimental period. In contrast, suffusion with SNAP or SIN-1 markedly increased clearance of FITC–dextran-10K from the cerebral microcirculation and produced a rapid, sustained dilatation of pial arterioles. Thus, NO donors increase the permeability of the blood–brain barrier and produce pronounced dilatation of cerebral arterioles. In light of evidence suggesting that NO donors may produce their effect by the simultaneous release of NO and superoxide anion to form peroxynitrite, we elected to examine the role of superoxide anion in increases in permeability of the blood–brain barrier in response to SNAP and SIN-1. We found that suffusion with tiron (1 mM) did not alter basal permeability of the blood–brain barrier, but significantly inhibited increases in permeability of the blood–brain barrier in response to SNAP and SIN-1. In addition, tiron did not alter baseline diameter of cerebral arterioles, or SNAP- and SIN-1-induced cerebrovasodilatation. The findings of the present study suggest that NO donors produce an increase in permeability of the blood–brain barrier which appears to be related to the presence of NO and superoxide anion, to presumably form peroxynitrite. We suggest that increases in NO formation observed during brain trauma may contribute to disruption of the blood–brain barrier.     

Nociceptin/orphanin FQ contributes to hypoxic/ischemic impairment of hypercapnic cerebrovasodilation

Previous studies in piglets show that hypercapnic pial artery dilation was blunted following cerebral ischemia. Unrelated studies show that the newly described opioid nociceptin orphanin FQ (NOC/oFQ) is released into cerebrospinal fluid and contributes to altered cerebral hemodynamics following hypoxia/ischemia. This study was designed to determine the contribution of NOC/oFQ to hypoxic/ischemic impairment of hypercapnic pial dilation in piglets equipped with a closed cranial window. Global cerebral ischemia was produced via elevated intracranial pressure. Hypoxia decreased P(O2) to 34 +/- 3 mmHg. Topical NOC/oFQ (10(-10) M), the CSF concentration following hypoxia/ischemia, had no effect on pial artery diameter by itself but attenuated hypercapnia P(CO2) of (73 +/- 2 mmHg)-induced pial artery dilation (28 +/- 2 vs. 19 +/- 2%). Hypercapnia pial artery dilation was blunted by hypoxia/ischemia but such dilation was partially protected by pretreatment with the putative NOC/oFQ receptor antagonist, [F/G] NOC/oFQ (1-13) NH(2) (10(-6) M), (25 +/- 1, sham control; 4 +/- 1, hypoxia/ischemia; and 12 +/- 3%, hypoxia/ischemia + [F/G] NOC/oFQ (1-13) NH(2), respectively). These data suggest that NOC/oFQ release contributes to impaired hypercapnia-induced cerebrovasodilation following hypoxia/ischemia.     

Role of nitric oxide, adenosine,N-methyl-d-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation ofN-methyl-d-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles (∼ 40 μm) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-l-arginine methylester (LNAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO₂ ≈ 46 mmHg)then severe (PaO₂ ≈ 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)(and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO, ≈ 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 μM), MK-801 (10 μM), TTX (1 μM), or 8-SPT + MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT + MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO₂ = 70–85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited ≈ 20% and 35–40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45–50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT + MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.     

Role of nitric oxide, adenosine,N-methyl-d-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation ofN-methyl-d-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles ( 40 μm) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-l-arginine methylester (LNAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO₂ ≈ 46 mmHg)then severe (PaO₂ ≈ 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)(and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO, ≈ 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 μM), MK-801 (10 μM), TTX (1 μM), or 8-SPT + MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT + MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO₂ = 70–85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited ≈ 20% and 35–40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45–50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT + MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.     

Adrenomedullin reduces gender-dependent loss of hypotensive cerebrovasodilation after newborn brain injury through activation of ATP-dependent K channels.

Cerebrovascular dysregulation during hypotension occurs after fluid percussion brain injury (FPI) in the newborn pig owing to impaired K channel function. This study was designed to (1) determine the role of gender and K channel activation in adrenomedullin (ADM) cerebrovasodilation, (2) characterize the role of gender in the loss of hypotensive cerebrovasodilation after FPI, and (3) determine the role of gender in the ability of exogenous ADM to modulate hypotensive dysregulation after FPI. Lateral FPI (2 atm) was induced in newborn male and female newborn pigs (1 to 5 days old) equipped with a closed cranial window, n=6 for each protocol. Adrenomedullin-induced pial artery dilation was significantly greater in female than male piglets and blocked by the K(ATP) channel antagonist glibenclamide, but not by the K(ca) channel antagonist iberiotoxin. Cerebrospinal fluid ADM was increased from 3.8+/-0.7 to 14.6+/-3.0 fmol/mL after FPI in female but was unchanged in male piglets. Hypotensive pial artery dilation was blunted to a significantly greater degree in male versus female piglets after FPI. Topical pretreatment with a subthreshold vascular concentration of ADM (10(-10) mol/L) before FPI reduced the loss of hypotensive pial artery dilation in both genders, but protection was significantly greater in male versus female piglets. These data show that hypotensive pial artery dilation is impaired after FPI in a gender-dependent manner. By unmasking a gender-dependent endogenous protectant, these data suggest novel gender-dependent approaches for clinical intervention in the treatment of perinatal traumatic brain injury.