Relationships between neurons expressing neuronal nitric oxide synthase, degree of microglia activation and animal survival. A study in the rat cortex after transient ischemia

The focal ischemia obtained in an animal model of middle cerebral artery occlusion (MCAo) causes the "core" of damage in the striatum and the "penumbra" of damage in the fronto-parietal cortex. The latter is mainly functionally affected and shows changes in nNOS and iNOS expression during the acute phase of ischemia. With the aim to study possible relationships between these changes and the affection entity during the animal recovery, we investigated from 24 up to 144 h after reperfusion the expression and content of these two NOS isoforms in the neurons and microglia and the degree of microglia reactivity in the fronto-parietal cortices of rats undertaken to transient MCAo. Evaluation of motor-sensory performances and survival allowed dividing the animals into two groups. Immunohistochemistry, western blot and quantitative analysis demonstrated, both in the ischemic and contralateral cortex of the rats with longer survival, wellness and significantly increased number of the nNOS-IR neurons at 24 h and moderately activated microglia up to 144 h. In the rats not recovering, injured and significantly decreased nNOS-IR neurons, intensely activated microglia and appearance of iNOS-IR were seen at all time points. In conclusion, since the recovery occurs when nNOS-IR neurons are greatly increased, we presume nNOS protect the tissue likely controlling the passage from the state of reactive to that of activated microglia. Moreover, the morphological signs of wellness and the two-fold increase in number of the nNOS-IR neurons appear to be characteristic of the "penumbra" area and could explain why this region is mainly functionally affected.     

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.     

一氧化氮合酶在脑缺血再灌注中的双重作用

目的 探讨短暂脑缺血再灌注后大鼠脑内3型一氧化氮合酶(nitric oxide synthase，NOS)的表达及作用，为脑缺血治疗提供理论依据。方法 采用免疫组织化学方法，用3型NOS的多克隆抗体检测大鼠局灶性脑缺血2h再灌注15min及22h NOS在脑内的表达情况。结果 大鼠脑缺血2h再灌注15min，在脑缺血边缘区的血管壁及神经细胞出现内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)上调表达；脑缺血2h再灌注22h，在脑梗死区内表达神经元型一氧化氮合酶(neuronal mitric oxide synthase，nNOS)的神经细胞减少，并出现表达诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的胶质细胞，同时梗死边缘区血管及神经细胞出现eNOS及iNOS的上调表达。结论 在短暂脑缺血再灌注早期，缺血区周围可能有eNOS相关的保护机制；亚急性期eNOS及iNOS的保护及损伤机制并存；因此，在短暂脑缺血早期恢复灌注后予选择性iNOS抑制剂及促进eNOS活性有可能减少迟发性神经损伤。     

慢性间断性缺氧诱导一氧化氮合酶表达的研究

目的:建立大鼠缺氧模型,检测神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(iNOS)的表达情况。方法:1.建立缺氧模型:将SD大鼠置于常压低氧舱中,充入氮气调节氧浓度至所需氧浓度。2.动物分组:(1)急性缺氧组:在低氧舱中缺氧1.5小时。(2)慢性间断性缺氧组:每日在低氧舱中6小时。每周缺氧6天,共缺氧28天。3.采用免疫组化法检测nNOS和iNOS的表达。4.统计学分析检验。结果:急性缺氧后,iNOS、nNOS阳性神经元增加;慢性缺氧后,iNOS、nNOS阳性神经元仍持续增多,慢性缺氧时增加iNOS-IR细胞远远多于nNOS-IR细胞。结论:我们的研究表明缺氧可引起iNOS、nNOS阳性神经元增加,NOS亚型表达时间的不同说明其脑损伤具有阶段性。     

Time dependent changes of striatal interneurons after focal cerebral ischemia in rats

Summary. The cellular damage over time and the alterations of neuronal subtypes was characterized in the striatum after 90-min middle cerebral artery occlusion and reperfusion in rats. We investigated the immunohistochemical alterations of choline acetyltransferase (ChAT)-positive (cholinergic-positive), γ-aminobutyric acid (GABA)ergic parvalbumin (PV)-positive, GABAergic nNOS (neuronal nitric oxide synthase)-positive interneurons, neuronal nuclei (NeuN)-positive spiny projection neurons, glial fibrillary acidic protein (GFAP)-positive strocytes and microglial response factor-1 (MRF-1)-positive microglia in the striatum after focal cerebral ischemia in rats. In the present study, transient focal cerebral ischemia in rats caused severe damage against interneurons as well as spiny projection neurons in the striatum. In contrast, a significant increase in the number of GFAP-immunopositive astrocytes was observed in the ipsilateral striatum 15 days after focal cerebral ischemia. Furthermore, a significant increase of MRF-1 immunoreactivity was observed in microglia of the ipsilateral striatum 7 days and 15 days after focal cerebral ischemia. Among three types of cholinergic interneurons, GABAergic PV-positive interneurons and GABAergic nNOS-positive interneurons, the severe damage of cholinergic and GABAergic PV-positive interneurons was more pronounced than that of GABAergic nNOS-positive interneurons after transient focal cerebral ischemia in rats. Furthermore, the present results suggest that GABAergic nNOS-positive interneurons in the striatum after focal cerebral ischemia undergo cellular death in a delayed manner. Correspondence: Tsutomu Araki, Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan     

脑室内注射乙酰胆碱受体抗体IgG对大鼠神经元凋亡及一氧化氮合酶表达的影响

目的研究乙酰胆碱受体抗体(AchRab)对大鼠脑内神经元的损害及一氧化氮合酶(NOS)在损害中所起的作用，探讨重症肌无力(MG)中枢神经系统损害的机制。方法将AchRab IgG或健康人的IgG注入大鼠侧脑室。HE染色、TUNEL法检测细胞凋亡；免疫组化方法观察大鼠皮质、海马及杏仁核神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化。结果2周后实验组皮质、海马及杏仁核凋亡细胞明显增多，对照组仅见少量凋亡。实验组皮质、海马及杏仁核nNOS神经元数目明显减少。实验组及对照组脑内细胞均来见iNOS表达。结论AchRab脑内注射可诱导神经元凋亡；损伤皮质。海马及杏仁核nNOS神经元；但未能诱导脑内细胞iNOS表达。神经元凋亡损害参与了AchRab对中枢神经损害的机制；nNOS神经元的减少，可能与MG认知功能障碍有密切关系；而神经元的损伤可能与NO的毒性作用无关。     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P < 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P < 0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time. Key Words: focal cerebral ischemia; hyperlipidemia; ischemia/reperfusion injury; neuronal nitric oxide synthase     

Quantitative analysis of neuronal nitric oxide synthase-immunoreactive neurons in the mouse hippocampus with optical disector.

A detailed quantitative analysis of immunocytochemically identified nonprincipal neurons containing neuronal nitric oxide synthase (nNOS) was performed on the mouse hippocampus, with particular reference to the dorsoventral gradient. The present study applied two variations of a stereologic technique, the optical disector--one that used confocal laser-scanning microscope optical sections to examine colocalization of nNOS and glutamic acid decarboxylase 67 (GAD67), and the other that used conventional thick sections to examine numerical densities (NDs) and cell sizes of nNOS-immunoreactive (IR) neurons. Colocalization analysis indicated that practically all nNOS-IR neurons (97.6%) were GAD67-IR, whereas a part of the GAD67-IR neurons (about 30%) were nNOS-IR in the whole hippocampus at both dorsal and ventral levels. The percentages of GAD67-IR neurons containing nNOS were higher in the dentate gyrus (DG, about 50%), and lower in the Ammon's horn (about 20%). Laminar analysis revealed that the majority of GAD67-IR neurons contained nNOS in the stratum lacunosum-moleculare of the CA3 region (about 60%) and in the molecular layer of the DG (about 80%). The NDs of nNOS-IR neurons in the whole hippocampus showed a dorsoventral gradient, which increased from dorsal (1.6 x 10(3)/mm3) to ventral (2.2 x 10(3)/mm3) levels. The NDs were relatively higher in the principal cell layers, where about 40% of nNOS-IR neurons were situated both in the Ammon's horn and DG. The mean cell sizes of nNOS-IR neurons showed no remarkable laminar differences or dorsoventral gradient in the Ammon's horn, but they were extensively larger in the hilus of the DG than in other layers. These results indicate that nNOS-IR neurons in the mouse hippocampus represent a subpopulation of gamma-aminobutyric acid (GABA)ergic neurons and suggest that the laminar distributions of nNOS-IR neurons related to possible functional heterogeneity of GABAergic neurons in each hippocampal layer.     

Protective effect of pitavastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, on ischemia-induced neuronal damage

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg(-1) did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

Protective effect of pitavastatin,a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor,on ischemia-induced neuronal damage

Abstract

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg^-1 did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia

Adult neurogenesis is modulated by growth factors, physical conditions, and other alterations in the physical microenvironment. We studied the effects of focal ischemia on neurogenesis in the subventricular zone (SVZ), olfactory bulb (OB), and hippocampal dentate gyrus (DG) (known to be persistent neurogenic regions) in the adult non-human primate, the cynomolgus monkey. Three monkeys underwent middle cerebral artery occlusion-induced focal ischemia and were given multiple BrdU injections during the first 2 weeks after ischemia. Twenty-eight days later, the animals were perfused. The number of new neurons (3182 +/- 408/mm3) in the ipsilateral DG of ischemic monkeys was 4.7-fold that in the DG of non-operated monkeys. The number of new neurons (9176 +/- 2295/mm3) in the ipsilateral olfactory bulb of ischemic monkeys was 18.0-fold that in normal olfactory bulb. These observations suggest an increase in the number of new OB neurons, as well as new DG neurons, after focal ischemia in a primate. This substantial increase in new neurons after focal ischemia could result from the enhancement of cell proliferation rather than a change in the rate of cell commitment. Of the three monkeys subjected to ischemia, only one animal possessed a unique progenitor cell type at the most anterior aspect of the ipsilateral SVZ. Within this region, a short migration (approximately 500 microm) of doublecortin-expressing immature neuronal progenitor cells was observed.     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P< 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P<0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.     

Nitric oxide synthase immunoreactivity in the rat hippocampus after status epilepticus induced by perforant pathway stimulation

Nitric oxide has recently been implicated in mediation of neuronal excitotoxicity and damage. This study aimed at elucidating the changes in the expression of neuronal isoform of nitric oxide synthase (nNOS) in the hippocampus after status epilepticus induced by perforant pathway stimulation. nNOS-immunoreactivity (nNOS-ir) and neuronal damage, assessed by silver staining, were evaluated separately in different hippocampal subfields 2 weeks after induction of status epilepticus. Perforant pathway stimulation resulted in an increase in the number of nNOS-immunoreactive neurons in the stratum radiatum of the CA1 and CA3 subfields of the hippocampus proper, and the hilus of the dentate gyrus. The morphology and distribution of the nNOS-ir neurons resembled that of interneurons. No correlation of the number of nNOS-ir neurons to the neuronal damage score was observed. Our results suggest that status epilepticus provokes a de novo expression of nNOS protein, and the nNOS expressing neurons may be selectively resistant to epileptic brain injury.     

Prenatal stress causes gender-dependent neuronal loss and oxidative stress in rat hippocampus

Our purpose was to investigate the effects of prenatal stress on neuronal changes in the hippocampus and the possible involvement of oxidative stress in female and male rats. Female and male offspring (1-month-old), whose dams were restrained in middle or later pregnant stage (MS or LS), were studied to observe changes in the number of hippocampal neurons and the expression of neuronal nitric oxide synthase (nNOS) in the hippocampus. Both MS and LS induced an increase in the number of nNOS-positive expression in female and male offspring in the hippocampus; however, both MS and LS caused a significant decrease in the number of hippocampal neurons in the female, but not in the male offspring. In addition, significant increases in calcium content and oxidant generation were induced by LS in the hippocampal CA3 region in female rats. These data suggest that prenatal stress can cause oxidative stress and consequent damage to neurons, leading to neuronal loss in the brain of offspring during development.     

Glial and neuronal localization of neuronal nitric oxide synthase immunoreactivity in the median eminence of female rats

Localization of neuronal nitric oxide synthase-immunoreactivity (nNOS-IR) in the median eminence of female rats (n=4) was examined by electron microscopy to explore the possibility that nitric oxide is involved in the terminal regulation of neurosecretory peptides such as GnRH. Under light microscopy, a dense distribution of nNOS-IR was observed in this region. Electronmicroscopically, nNOS-IR was found in glial elements and nerve terminals containing dense-core vesicles. We also found a few nNOS-immunopositive synapses, in which intense immunoreactivity was found on the postsynaptic density and mitochondrial membrane. The localization of nNOS-IR in nerve terminals and glial elements in the median eminence might indicate that nNOS plays a role in regulating the release of neurosecretory peptide.     

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.     

Nitric oxide synthase in cerebral ischemia

The results of our continuing studies on the role of nitric oxide (NO) in cellular mechanisms of ischemic brain damage as well as related reports from other laboratories are summarized in this paper. Repetitive ip administration ofN ^G-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor, protected against neuronal necrosis in the gerbil hippocampal CA1 field after transient forebrain ischemia with a bell-shaped response curve, the optimal dose being 3 mg/kg. Repeated ip administration of L-NNA also mitigated rat brain edema or infarction following permanent and transient middle cerebral artery (MCA) occlusion with a U-shaped response. The significantly ameliorative dose-range and optimal dose were 0.01–1 mg/kg and 0.03 mg/kg, respectively. Studies using a NO-sensitive microelectrode revealed that NO concentration in the affected hemisphere was remarkably increased by 15–45 min and subsequently by 1.5–4 h after MCA occlusion. Restoration of blood flow after 2 h-MCA occlusion resulted in enhanced NO production by 1–2 h after reperfusion. Administration of L-NNA (1 mg/kg, ip) diminished the increments in NO production during ischemia and reperfusion, leading to a remarkable reduction in infarct volume. In brain microvessels obtained from the affected hemisphere, Ca²⁺-dependent constitutive NOS (cNOS) was activated significantly at 15 min, and Ca²⁺-independent inducible NOS (iNOS) was activated invariably at 4 h and 24 h after MCA occlusion. Two hour reperfusion following 2 h-MCA occlusion caused more than fivefold increases in cNOS activity with no apparent alterations in iNOS activity. Thus, we report here based on available evidence that there is good reason to think that NOS activation in brain microvessels may play a role in the cellular mechanisms underlying ischemic brain injury.     

依达拉奉预处理对小鼠脑缺血再灌注损伤后皮质一氧化氮合酶表达的影响

目的探讨依达拉奉预处理对小鼠脑缺血再灌注(IR)损伤后皮质一氧化氮合酶(NOS)表达的影响。方法 48只健康ICR小鼠被分为假手术组、对照组和依达拉奉组。依达拉奉组和对照组分别给予依达拉奉3 mg/(kg.d)和同等体积的生理盐水腹腔注射共7 d,然后建立小鼠IR模型;缺血1 h、再灌注24 h时应用2,3,5-氯化三苯基四氮唑(TTC)染色法测量各组脑梗死体积,应用免疫组化法检测各组小鼠皮质神经元型、、诱导型和内皮型NOS(nNOS、iNOS、eNOS)阳性细胞数。结果与假手术组比较,对照组小鼠皮质nNOS、iNOS和eNOS阳性细胞数明显增多(均P<0.05);与对照组比较,依达拉奉组脑梗死体积明显缩小,皮质nNOS和iNOS阳性细胞数明显减少,eNOS阳性细胞数明显增多(均P<0.05)。结论依达拉奉预处理可以影响IR小鼠皮质nNOS、iNOS和eNOS的表达,发挥神经保护作用。