Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.     

Relative changes in cerebral blood flow and neuronal activity in local microdomains during generalized seizures.

There is broad agreement that generalized tonic-clonic seizures (GTCS) and normal somatosensory stimulation are associated with increases in regional CBF. However, the data regarding CBF changes during absence seizures are controversial. Electrophysiologic studies in WAG/Rij rats, an established animal model of absence seizures, have shown spike-wave discharges (SWD) that are largest in the perioral somatosensory cortex while sparing the visual cortex. Recent functional magnetic resonance imaging (fMRI) studies in the same model have also shown localized increases in fMRI signals in the perioral somatosensory cortex during SWD. Because fMRI signals are only indirectly related to neuronal activity, the authors directly measured CBF and neuronal activity from specific microdomains of the WAG/Rij cortex using a specially designed probe combining laser-Doppler flowmetry and extra-cellular microelectrode recordings under fentanyl/haloperidol anesthesia. Using this approach, parallel increases in neuronal activity and CBF were observed during SWD in the whisker somatosensory (barrel) cortex, whereas the visual cortex showed no significant changes. For comparison, these measurements were repeated during somatosensory (whisker) stimulation, and bicuculline-induced GTCS in the same animals. Interestingly, whisker stimulation increased neuronal activity and CBF in the barrel cortex more than during SWD. During GTCS, much larger increases that included both the somatosensory and visual cortex were observed. Thus, SWD in this model produce parallel localized increases in neuronal activity and CBF with similar distribution to somatosensory stimulation, whereas GTCS produce larger and more widespread changes. The normal response to somatosensory stimulation appears to be poised between two abnormal responses produced by two physiologically different types of seizures.     

Dynamic cerebral microcirculatory changes in transient forebrain ischemia in rats: involvement of type I nitric oxide synthase.

The diameter of surface microvessels and the erythrocyte velocity and flux through intraparenchymal capillaries in the parietal cortex were measured during transient global cerebral ischemia and reperfusion using laser-scanning confocal fluorescence microscopy in anesthetized rats. The role of nitric oxide (NO) from neurons in the microcirculatory changes was also investigated using 7-nitro-indazole (7-NI, 25 mg/kg, i.p.). Wistar rats (4 per group) equipped with a closed cranial window were given fluorescein isothiocyanate (FITC)-Dextran and FITC-labeled erythrocytes intravenously to respectively visualize the microvessels and the erythrocytes in the capillaries. Experiments were videorecorded on-line. Forebrains were made ischemic for 15 minutes and then reperfused for 120 minutes under the microscope. Ischemia was associated with a flattened EEG, a low persistent blood flow, and a transient leakage of fluorescein across the arteriole wall. Unclamping the carotid arteries led to immediate high blood flow in the arterioles, but it was not until 5 minutes later that the arterioles dilated significantly (181% +/- 27%) and erythrocyte velocity in the capillaries increased significantly (460% +/- 263%). Neither nonperfused capillaries nor erythrocyte capillary recruitment occurred. 7-Nitro-indazole significantly reduced the arteriole dilatation and prevented the increase in erythrocyte velocity and flux through capillaries in early reperfusion. 7-Nitroindazole had no influence on the fluorescein leakage. The current study suggests a partial role for NO released from neurons in the postischemic microcirculatory changes and provides new findings on the timing of arteriole dilatation and blood-brain barrier opening, and on erythrocyte capillary circulation in global ischemia.     

Changes of cerebral blood flow in rats with acute cerebral ischemia and the effect of nitric oxide donor S-nitroso-N-acetyl-penicillamine

INTRODUCTION In cerebral ischemia, the production of nitric oxide increases, which leads to the increase of cyclic guanosine monophosphate (cGMP) and thus the dilation of the cerebral vessles. cGMP is an important regulatory factor mediating a vasodilatoy…     

Cerebral blood flow and neuronal damage during progressive cerebral ischemia in gerbils

A combined autoradiographic and immunohistochemical method was used to correlate the extent of focal cerebral ischemia and morphologic ischemic damage following unilateral carotid occlusion in 16 gerbils for 5-30 minutes. Immunohistochemical lesions detectable by the reaction for microtubule-associated proteins 1 and 2 were visible in the subiculum-CA1 and CA2 regions of the hippocampus and layer III/IV of the cerebral cortex after 5 minutes of ischemia (n = 4). Local blood flow was promptly reduced but still heterogeneous after 10 minutes of ischemia (n = 4); local blood flow in immunohistochemical lesions was less than 5 ml/100 g/min except in highly vulnerable regions, where flow values of 5-15 ml/100 g/min were observed. After 15 minutes of ischemia (n = 4) local blood flow in less vulnerable regions including the thalamus and caudoputamen also declined to less than 5 ml/100 g/min, and immunohistochemical lesions became visible in those regions after 30 minutes of ischemia (n = 4). On the other hand, many brain regions tolerated local blood flow of less than 5 ml/100 g/min without ischemic damage. The present study demonstrates that selective tissue vulnerability during progressive cerebral ischemia depends on the degree of hypoperfusion and on factors inherent to neurons in various brain regions.     

Increased cerebral lactate output to cerebral venous blood after forebrain ischemia in rats

Increased cerebral lactate levels are a well-known aspect of the sequelae of the metabolic derangements that follow cerebral ischemia. A new technique has recently become available to sample cerebral venous blood from the superior sagittal sinus on a long-term basis in conscious rats. We report the applicability of this method to assess serial biochemical responses to brain injury. Serum samples were obtained from the superior sagittal sinus, the common carotid artery, and the external jugular vein of nine anesthetized rats before and up to 7 days after 10 minutes of forebrain ischemia was produced by carotid occlusion and hypovolemic hypotension (mean arterial blood pressure 50 +/- 4 mm Hg). The cerebral venous-arterial difference in serum lactate concentration was increased for up to 3 hours after ischemia, while there was no significant change in the difference in serum lactate concentrations in the common carotid artery and the external jugular vein. This indicates an elevated output of lactate from brain tissue to blood, detectable only in the superior sagittal sinus, which underlines the usefulness of the technique. We observed a persistent elevation in brain lactate production after virtually complete recovery from the acute insult.     

Role of endothelial nitric oxide synthase in cerebral blood flow changes during kainate seizures: a genetic approach using knockout mice

The role of endothelial nitric oxide (NO) in the cerebrovascular response to partial seizures was investigated in mice deleted for the endothelial NO synthase gene (eNOS-/-) and in their paired wild-type (WT) congeners. Local cerebral blood flow (LCBF, quantitative [14C]iodoantipyrine method) was measured 3-6 h after unilateral kainate (KA) injection in the dorsal hippocampus; controls received saline. In WT mice, KA seizures induced a 22 to 50% LCBF increase restricted to the ipsilateral hippocampus, while significant LCBF decreases (15-33%) were noticed in 22% of the contralateral areas, i.e., the parietal cortex, amygdala and three basal ganglia areas, compared to saline-injected WT mice. In eNOS-/- mice, no LCBF increases were recorded within the epileptic focus and generalized contralateral LCBF decreases (22-46%) were noticed in 2/3 of the brain areas, compared to saline-injected eNOS-/- mice. Thus, endothelial NO is the mediator of the cerebrovascular response within the epileptic focus and participates in the maintenance of LCBF in distant areas.     

Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia

Progressive brain damage after transient cerebral ischemia may be related to changes in postischemic cerebral blood flow and metabolism. Regional cerebral blood flow (rCBF) and cerebral glucose utilization (rCGU) were measured in adult rats prior to, during (only rCBF), and serially after transient forebrain ischemia. Animals were subjected to 30 minutes of forebrain ischemia by occluding both common carotid arteries 24 hours after cauterizing the vertebral arteries. Regional CBF was measured by the indicator-fractionation technique using 4-iodo-[¹⁴C]-antipyrine. Regional CGU was measured by the 2-[¹⁴C]deoxyglucose method. The results were correlated with the distribution and progression of ischemic neuronal damage in animals subjected to an identical ischemic insult. Cerebral blood flow to forebrain after 30 minutes of moderate to severe ischemia ( < 10% control CBF) was characterized by 5 to 15 minutes of hyperemia; rCBF then fell below normal and remained low for as long as 24 hours. Postischemic glucose utilization in the forebrain, except in the hippocampus, was depressed below control values at 1 hour and either remained low (neocortex, striatum) or gradually rose to normal (white matter) by 48 hours. In the hippocampus, glucose utilization equaled the control value at 1 hour and fell below control between 24 and 48 hours. The appearance of moderate to severe morphological damage in striatum and hippocampus coincided with a late rise of rCBF above normal and with a fall of rCGU; the late depression of rCGU was usually preceded by a period during which metabolism was increased relative to adjacent tissue. Further refinement of these studies may help identify salvageable brain after ischemia and define ways to manipulate CBF and metabolism in the treatment of stroke.     

Role of neuronal and inducible nitric oxide synthases in the guinea pig ileum myenteric plexus during in vitro ischemia and reperfusion

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western‐immunoblotting and real time RT‐PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS‐expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS‐immunoreactive neurons, protein and mRNA levels were up‐regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by N^ω‐propyl‐l ‐arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R.     

局灶性鼠脑缺血时一氧化氮及一氧化氮合酶抑制剂的作用机制

目的研究一氧化氮在鼠脑局灶性脑缺血再灌注损伤中的作用。方法用线栓法建立大鼠大脑中动脉区缺血再灌注模型，分别用选择性和非选择性诱导型一氧化氮合酶抑制剂对鼠脑局灶性缺血再灌注过程中脑组织一氧化氮的变化规律及可能作用进行探讨。结果非选择性一氧化氮合酶抑制剂（Ｌ－ＮＡＭＥ）可加重局灶性脑缺血性损害，而选择性诱导型一氧化氮合酶抑制剂（ａｍｉｎｏｇｕａｎｉｄｉｎｅ，ＡＧ）具有明确的脑保护作用。结论不同类型的一氧化氮合酶所产生的一氧化氮在脑局灶性缺血性损害中具有不同的作用。     

Role of nitric oxide in cerebral blood flow changes during kainate seizures in mice: genetic and pharmacological approaches

The role of neuronal nitric oxide (NO) in the cerebrovascular response to partial seizures induced by intrahippocampal injection of kainate (KA) was investigated in mice deleted for the neuronal NO synthase gene (nNOS-/-) and in wild-type controls (WT). A second group of WT mice received the nNOS inhibitor, 7-nitroindazole (WT-7NI). Local cerebral blood flow (LCBF) was measured using the quantitative (14)C-iodoantipyrine method. Within the epileptic focus, all three groups of seizing mice (WT, WT-7NI, and nNOS-/-) showed significant 26-88% LCBF increases in ipsilateral hippocampus, compared to saline-injected mice. Contralaterally to the epileptic focus, KA seizures induced a 21-47% LCBF decreases in hippocampus and limbic cortex of WT mice and in most contralateral brain structures of nNOS-/- mice, while WT-7NI mice showed no contralateral CBF change. Neuronal NO appears to be not involved in the cerebrovascular response within the epileptic focus, but may rather have a role in the maintenance of distant LCBF regulation during seizures.     

Decreased neuronal nitric oxide synthase expression and cell migration in the peri-infarction after focal cerebral ischemia in rats

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in the normal developing brain, but the role of nNOS in neurogenesis of the adult ischemic brain remains unclear. The aim of this study was to investigate the temporal and spatial relationship between cell migration from the ependymal/subventricular zone (SVZ) to peri-infarction and nNOS expression in the rat. Ependymal/subventricular zone cells were prelabeled with fluorescence dye DiI. Focal cerebral ischemia was induced by occlusion of the left middle cerebral artery. At 1, 3, 7, 14 and 21 days after ischemia, the rats were killed in order to determine the number of migrating cells, the colocalization of DiI and nNOS as well as nNOS quantity in specific regions. Compared to non-ischemic control and 1 day post-ischemia, the number of DiI-labeled cells in the selected regions increased at 3 days and peaked 14 days following ischemia. During 3–7 days post-ischemia, none of the migrating cells expressed nNOS and decreased nNOS expression was observed in the regions where migrating cells passed through. These results suggest the possible association between ependymal/SVZ cell migration and decreased nNOS expression within the areas including the migrating routes towards the peri-infarction.     

Effect of ibuprofen on regional eicosanoid production and neuronal injury after forebrain ischemia in rats

Post-ischemic metabolism of arachidonic acid by cyclooxygenase results in the elaboration of numerous eicosanoids and in the generation of free radicals. Accordingly, the effect of cyclooxygenase inhibition by ibuprofen on post-ischemic eicosanoid production and delayed neuronal death was evaluated in Wistar-Kyoto rats subjected to incomplete forebrain ischemia. In control (C) and ibuprofen-treated groups (n = 5 each), pre- and post-ischemic eicosanoid production in the caudate nucleus (CN) and dorsal hippocampus (HPC) were evaluated by microdialysis. The ibuprofen-treated animals were given ibuprofen, 15 mg/kg i.v., prior to insertion of microdialysis probes. Forebrain ischemia was induced by bilateral carotid artery occlusion (BCAO) for 10 min with simultaneous hypotension to 35 Torr. The concentrations of thromboxane B₂ (TxB₂), 6-keto-PGF_1α and PGF_2α in the microdialysate were measured by radioimmunoassay. In two additional concurrent groups of rats (>n = 10 each), neuronal injury in the HPC, CN and cortex (parietal, temporal and entorhinal regions) was evaluated histologically three days after 10 min of forebrain ischemia with and without pre-ischemic ibuprofen administration. In the control microdialysis group, levels of TxB₂, 6-keto-PGF_1α and PGF_2α increased in both CN and HPC after probe insertion. These probe related increases were substantially reduced in the ibuprofen group. After ischemia and reperfusion in the control group, the levels of TxB₂ and PGF_2α increased in both CN and HPC. Levels of 6-keto-PGF_1α increased in the CN but not in the HPC. The administration of ibuprofen substantially reduced post-ischemic TxB₂ and PGF_2α levels in both CN and HPC and decreased 6-keto-PGF_1α levels in the CN. The results of these initial microdialysis studies left the possibility that, in the ibuprofen group, the reduction in eicosanoid levels after probe penetration might have influenced the subsequent post-ischemic eicosanoid production. Therefore, in an additional group of animals (n = 5), ibuprofen was administered after probe insertion. Only PGF_2α levels were measured in this group. Increased levels of PGF_2α comparable to the original control group were detected after probe penetration. Nonetheless, after ibuprofen administration, the pre- and post-ischemic levels of PGF_2α were again significantly reduced. In the histologic evaluation groups, overall neuronal injury was significantly less in the ibuprofen treated animals. This protective effect of ibuprofen was most clearly evident in the CA3 sector of the HPC. The data suggest that metabolism of arachidonic acid by cylooxygenase may contribute to post-ischemic neuronal injury, though the relative contributions of eicosanoids per se and of free radicals remains undefined.     

Effects of dextromethorphan on regional cerebral blood flow in focal cerebral ischemia

Dextromethorphan (DM), a noncompetitive NMDA antagonist, has been demonstrated to reduce ischemic neuronal damage and edema, but DM's influence on cerebral blood flow has not been extensively studied. In this investigation, it is shown that DM has significant effects on regional cerebral blood flow (rCBF) patterns in a rabbit model of focal cerebral ischemia. rCBF was measured using radioactive microspheres following a 1 h permanent occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries in rabbits. Somatosensory evoked potentials (SEPs) were used to assess the degree of ischemia; only animals where SEPs were completely abolished were used for a frequency distribution analysis of rCBF. It was found that there were significantly more regions with lower flows in animals treated with normal saline (NS) (n = 7) compared to animals treated with DM (n = 7) (p less than 0.05, ipsilateral left side; p less than 0.001, contralateral right side). The frequency distribution medians were 27.5 ml 100 g-1 min-1 (left) and 70.0 ml 100 g-1 min-1 (right) in the NS group vs. 34.5 ml 100 g-1 min-1 (left) and 80.5 ml 100 g-1 min-1 (right) in the DM group. The left and right hemispheric regional means were 29.4 +/- 20 and 74.3 +/- 23 ml 100 g-1 min-1, respectively, in the NS group vs. 34.4 +/- 16 and 91.0 +/- 28 ml 100 g-1 min-1, respectively, in the DM group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential cerebral blood flow changes in short-term cerebral ischemia in gerbils

Using quantitative autoradiography, we studied sequential changes in regional cerebral blood flow during and after 2 minutes of bilateral common carotid artery occlusions in 18 gerbils. Occlusion (n = 4) led to severe ischemia in the forebrain (regional cerebral blood flow less than 5% of control [n = 4]) and midbrain (regional cerebral blood flow less than 10% of control), but was morphologically nonlethal. Reperfusion of the brain was complete, and regional cerebral blood flow was not different from control 1 minute after ischemia (n = 4), but hypoperfusion (regional cerebral blood flow 30-50% of control) occurred at 5 minutes (n = 3) and was pronounced at 1 hour (n = 4); at this stage blood flow was inhomogeneous. Hypoperfusion had disappeared at 4 hours (n = 3). Our results indicate that the well-documented sequence of cerebral blood flow changes (i.e., ischemia, initial recovery of blood flow, and delayed hypoperfusion) takes place even after nonlethal cerebral ischemia.     

黄芪对大鼠脑缺血血脑屏障及脑血流的影响

利用大鼠局灶性脑缺血再灌流和全脑缺血再灌流损伤两种动物模型，观察黄芪注射液对脑缺血后再灌注期间血脑屏蔽及脑血流的保护作用。结果显示，与相庆对照组比较，不论是全脑缺血还是局灶性脑缺血１ｈ后再灌流３ｄ，应用黄芪的各组动物脑水肿明显减轻，血脑屏障通透性改善，大脑局部血流量显著增加。