大鼠局灶性脑缺血再灌注中nNOS来源的NO对细胞凋亡的影响

目的探讨大鼠局灶性脑缺血再灌注早期nNOS来源的NO对细胞凋亡的影响.方法闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用原位末端标记法及流式细胞术检测缺血2h再灌注6h细胞凋亡的变化.结果 50mg/kg、25mg/kg剂量的7硝基吲唑可使1、NO含量显著降低.2、NT阳性细胞荧光强度及阳性细胞百分比显著减少.3、TUNEL阳性细胞明显减少.4、细胞凋亡百分率降低,AP峰降低.结论 nNOS来源的NO参与介导脑缺血再灌注早期的细胞凋亡.     

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite

Background

The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia–reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury.

Results

Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon.

Conclusions

These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.     

Caffeic acid phenethyl ester ameliorates cerebral infarction in rats subjected to focal cerebral ischemia

The effects of caffeic acid phenethyl ester (CAPE), an antioxidant derived from propolis, on the infarct volume elicited by focal cerebral ischemia were studied on Long-Evans rats. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of bilateral common carotid arteries (CCA) for 60 min. The rats were sacrificed 24 h later and serial brain slices of 2 mm thickness were taken and stained for the measurement of infarct area. CAPE was administered intravenously 15 min before MCA occlusion. Pretreatment of CAPE (0.1, 1 and 10 microg/kg) significantly reduced the total infarct volume from 169.6 +/- 14.5 mm3 (control) to 61.0 +/- 24.1 mm3 (0.1 microg/kg CAPE), 47.4 +/- 9.1 mm3 (1 microg/kg CAPE), and 42.4 +/- 8.7 mm3 (10 microg/kg CAPE), respectively. Plasma nitric oxide (NO) content was significantly increased in rats subjected to focal cerebral ischemia. It is concluded that CAPE possesses neuroprotective properties in focal cerebral ischemia injury in rats possibly through its antioxidant effect and/or via the upregulation of NO production.     

Nitric Oxide Synthetase Activity in Cerebral Post-Ischemic Reperfusion and Effects of L-NG-Nitroarginine and 7-Nitroindazole on the Survival

Nitric Oxide (NO) mediates a series of physiological processes including regulation of vascular tone, macrophage-mediated cytotoxicity, platelet aggregation, learning and long-term potentiation, neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in the pathophysiology of focal cerebral ischemia. In the present study NOS activities were evaluated in cerebellum and cerebral cortex of ischemic and post-ischemic reperfused rats using an experimental model of partial cerebral ischemia; moreover, the effects of L-N^GNitroarginine (NA, nonselective NOS inhibitor) or 7-Nitroindazole (7-NI, selective neuronal NOS inhibitor) administration were assayed on percentage survival of ischemic rats. An increase of NOS activity in the cerebellum and in cerebral cortex of ischemic and post-ischemic reperfused rats was observed. NA administration failed to induce neuroprotective effects, by increasing percentage of mortality of treated ischemic rats with respect to control group. In contrast, the treatment with the selective neuronal NOS inhibitor, 7-NI, induced a significant neuroprotective effect.     

大鼠局灶性脑缺血后扩布性阻抑的发作及电针对其影响

目的：观察Wistar大鼠局灶性脑缺血后扩布性阻抑（SD）的发作情况及缺血后电针的影响。方法：线检法闭塞大鼠大脑中动脉,制备局灶性脑缺血模型。用神经电生理、神经病理等方法检测局灶性脑缺血后3h内SD发作情况及电针“合谷”穴（LI4）对SD的影响。结果：电针可减少局灶性脑缺血时SD的发作。结论：电针减少局灶性脑缺血时SD的发作,可能与电针缩小局灶性脑梗塞体积有关。     

Dragon's blood dropping pills have protective effects on focal cerebral ischemia rats model

Dragon's blood is a bright red resin obtained from Dracaena cochinchinensis (Lour.) S.C.Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has great traditional medicinal value and is used for wound healing and to stop bleeding. Its main biological activity comes from phenolic compounds. In this study, phenolic compounds were made into dropping pills and their protective effects were examined by establishing focal cerebral ischemia rats model used method of Middle Cerebral Artery Occlusion (MCAO), and by investigating indexes of neurological scores, infarct volume, cerebral index, cerebral water content and oxidation stress. Compared to model group, high, middle and low groups of Dragon's blood dropping pills could improve the neurological function significantly (p < 0.01) and reduce cerebral infarct volume of focal cerebral ischemia rats remarkably (p < 0.05–0.01). Meanwhile, each group could alleviate cerebral water content and cerebral index (p < 0.05–0.01) and regulate oxidative stress of focal cerebral ischemia rats obviously (p < 0.05–0.01). Activities of middle group corresponded with that treated with positive control drug. The results obtained here showed that Dragon's blood dropping pills had protective effects on focal cerebral ischemia rats.     

The effect of desflurane on ameliorating cerebral infarction in rats subjected to focal cerebral ischemia-reperfusion injury

To obtain more information on the cerebral ischemia and reperfusion injury under desflurane anesthesia, we compared the infarct volume and lactate dehydrogenase (LDH) activity in rats subjected to focal cerebral ischemia during different concentration of desflurane anesthesia. Male Long-Evans rats weighing 270-350 g were anesthetized with desflurane in air at 1.0, 1.25 or 1.5 MAC whereas rats in the control group received intraperitoneal chloral hydrate (400 mg/kg) anesthesia. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of the bilateral common carotid arteries (CCA) for 60 minutes. The rats were sacrificed 24 hours later, serial brain slices of 2mm thickness were taken and stained for the measurement of the infarct area. Cellular damage was evaluated by measuring the LDH level in the plasma. Desflurane (1.0, 1.25 or 1.5 MAC by inhalation) and chloral hydrate (400 mg/kg; ip.) did not produce any changes in pH, blood gases, heart rate or mean arterial blood pressure. In the rats subjected to focal cerebral ischemia, the volume of infarction was significantly less in the desflurane groups in all three different concentrations than in the chloral hydrate group. The changes of LDH activity in plasma also correlated with the result of the infarct volume. Our study suggests that desflurane may offer a neuroprotective effect such as decreased infarct volume after focal cerebral ischemia.     

Intervention effect of total flavonoids of ilex pubesceus on tolerant rat models under cerebral anoxia

Objective: To observe the intervention effect of total flavonoid of ilex pubesceus on animal models of cerebral ischemic tolerance. Methods: A rat model of global-focal cerebral ischemic tolerance was established by blocking bilateral common carotid artery blood flow and occluding left middle cerebral artery using thread-occlusion method. After the first operation, the Ginaton group and large-dosage, medium-dosage and small-dosage groups of total flavonoid of ilex pubesceus were given intragastric administration of corresponding drugs. The sham-operated group, pretreatment model group and ischemia-reperfusion group were given intragastric administration of the same volume of normal saline, 1 time a day, and administrated for 4d. At 24 h after the second operation, the neurological deficit was assessed, the whole blood viscosity, plasma viscosity, iNOS activity as well as NO level, IL-1β content and TNF-α content in the brain tissue of the rats were determined, and the morphological changes of brain tissue of the rats were observed by HE staining. Results: All the rat models of cerebral ischemic tolerance were established successfully. The total flavonoid of ilex pubesceus can obviously or significantly reduce the neurological deficit score, whole blood viscosity and plasma viscosity, obviously or significantly increase the NO level in the brain tissue of the rats, and significantly reduce the pathological damage of brain tissue of the rats. But compared with the ischemia-reperfusion group, the total flavonoid of ilex pubesceus can significantly or obviously increase the iNOS activity, IL-1β content and TNF-α content in the brain tissue of the rats.     

丹参酮ⅡA与丹皮酚配伍对大鼠脑缺血再灌注损伤保护作用研究

目的：研究丹参酮ⅡA与丹皮酚配伍（简称双丹配伍）对大鼠局灶性脑缺血再灌注损伤的脑梗死体积、自由基变化的影响,探讨双丹配伍对脑缺血损伤的保护作用。方法：复制大鼠中动脉缺血再灌注模型,分别给予双丹配伍干预,观察和评价受试动物行为学、脑梗死率、脑指数、脑含水量、SOD、MAD等指标的变化。结果：①双丹配伍的各试药组均具有明显改善局灶性脑缺血再灌注损伤大鼠的神经行为学评分,降低脑梗死率、脑指数、脑含水量、提高脑组织SOD活性、降低MDA含量;②双丹配伍各组中1：3配伍组的总体药效作用优于1：2和1：4组,但数据未见统计学差异;⑧双丹配伍尾静脉注射的药效作用明显优于预先灌胃组。结论：双丹配伍脂质体给大鼠口服和注射对脑缺血再灌注损伤具有显著保护作用。     

Neurotrophin-4/5 treatment reduces infarct size in rats with middle cerebral artery occlusion

The aim of this study was to determine whether neurotrophin-4/5 (NT-4/5) treatment alters infarction volume following permanent focal cerebral ischemia in the rat. Permanent focal cerebral ischemia was produced in adult male rats by intraluminal occlusion of the right middle cerebral artery. NT-4/5 was administered intraventricularly one day before and immediately following occlusion. Rats were sacrificed at 1, 4 and 7 days after occlusion. NT-4/5 treatment reduced infarction volume by 34% when compared to control rats 1 day after occlusion. Infarction volume was unaltered by treatment 4 to 7 days after occlusion. Middle cerebral artery occlusion led to a significant reduction in levels of mRNAs coding for catalytic and truncated TrkB receptors. This expression was unaffected by NT-4/5 treatment. Special issue dedicated to Dr. Hans Thoenen.     

NG-硝基-L-精氨酸对大鼠局灶性缺血脑区线粒体损伤的影响

目的:观察非选择性一氧化氮合酶抑制剂NG-硝基-L-精氨酸(NG-nitro-L-arginine,L-NA)对局灶性脑缺血大鼠脑线粒体的损伤作用,以探讨其改善缺血性脑损伤的作用机制。方法:将大鼠随机分为假手术组、缺血对照组、L-NA治疗组,采用线栓法阻断大鼠大脑中动脉(MCAO)复制局灶性脑缺血模型,分别于缺血后2h、6h、12h给药治疗3d,迅速断头取脑,差速离心法提取缺血侧脑组织线粒休,迅速测定线粒体膜肿胀度及线粒体活力,测定线粒体总ATP酶、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性,以及线粒休一氧化氮(NO)、丙二醛(MDA)含量:电镜观察缺血后皮层神经元超微结构的改变及L-NA对其影响。结果:在大鼠MCAO后线粒体膜肿胀度增加,线粒体活力下降,线粒体NO、MDA含量明显增加,线粒体总ATP酶、SOD、GSH-Px活性均明显下降:缺血后2h、6h、12h给予L-NA治疗3d与缺血对照组相比NO含量明显下降,缺血后12h治疗组线粒体膜肿胀度、线粒体活力、总ATP酶、SOD、GSH-Px活性均显著升高、MDA含量下降。电镜结果显示脑缺血后皮层神经元水肿,线粒体肿胀、嵴断裂、溶解、消失,且随缺血时间延长损伤加重;缺血后12h给予L-NA治疗能明显改善脑缺血引起的神经元水肿、线粒体肿胀和空泡化。结论:L-NA能明显抑制脑缺血后线粒体NO生成,在缺血早期给予L-NA对缺血性脑损伤无改善作用:缺血后期给予L-NA,能明显降低线粒体膜肿胀程度,改善线粒体能量供应,增强线粒体抗氧化作用及其活力,从而减轻脑缺血损伤。     

Angiogenic protection from focal ischemia with angiotensin II type 1 receptor blockade in the rat

Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.     

Ucf-101对大鼠局灶性脑缺血再灌注后Caspase-3蛋白表达的影响

目的观察Ucf—101对大鼠脑缺血再灌注后神经元caspase-3蛋白表达及细胞凋亡的影响,研究其对缺血性脑损伤是否具有保护作用。方法将36只雄性WiStar大鼠随机分为3组：假手术组、缺血组及Ucf—101组,采用线栓法建立大鼠右侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）2h再灌注模型,于再灌注后6h和24h断头取脑,采用TTC法测梗死体积,TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化,免疫组化法观察脑皮质神经元caspase-3的表达。结果脑缺血再灌注后不同时间点（6h、24h）,Ucf-101组与缺血组相比梗死体积明显缩小,有显著性差异（P〈0．05）;假手术组未见梗死现象。缺血组TUNEL阳性细胞数较假手术组明显增多（P〈0．05）,脑皮质caspase-3的表达较假手术组亦显著增强（P〈0．05）,给予Ucf-101处理后,TUNEL阳性细胞数较缺血组明显减少（P〈0．05）,caspase-3的表达较缺血组亦明显减弱（P〈0．05）。结论Ucf-101能有效地抑制脑缺血再灌注损伤,下调脑皮质神经元Caspase-3蛋白的表达,抑制神经元的凋亡,发挥神经保护作用。     

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity.

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N(omega)-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.     

孕酮对缺血／再灌注大鼠脑皮层水肿的影响

目的探讨孕酮(progesterone,PROG)对脑水肿的影响.方法48只大鼠随机分为6组即缺血/再灌(I/R)组,二甲基亚砜(DMSO)组,预防(pretreatment)组,防治(pre+posttreatment)组,治疗(posttreatment)组,地塞米松(DEXA)组.采用大鼠局灶性脑缺血/再灌注(I/R)模型,测定大脑中动脉阻塞(MCAO)24h后脑皮层水、钠、钾、钙含量.结果与DMSO组相比,应用PROG预防及防治组均能明显降低缺血皮层的H2O(P＜0.01)、Na+(P＜0.01)、Ca2+(P＜0.01)含量,升高K+(P＜0.01)含量,而治疗组虽能明显降低H2O(P＜0.05)、Na+(P＜0.01),但降低Ca2+(P>0.05)和升高K+(P＞0.05)的效果不显著.DEXA组的结果与PROG预防或防治组类似.结论用PROG预防或防治能显著减轻I/R引起的脑水肿.     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

Substrain Differences, Gender, and Age of Spontaneously Hypertensive Rats Critically Determine Infarct Size Produced by Distal Middle Cerebral Artery Occlusion

1. The present work discussed the effects of substrain or genetic differences, gender, and age of the rat on infarct size produced by distal middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats (SHR). 2. In SHR/Kyushu, infarct volume was significantly larger than that of SHR/Izm, while blood pressure levels were essentially the same between the two substrains. Although SHR-SP/Izm had a higher blood pressure than SHR/Kyushu, infarct volumes were the same between SHR/Kyushu and SHR-SP/Izm. These results suggest the presence of blood pressure-independent factors which affect the infarct size after MCAO. 3. Estrogen accounted the large part of greater tolerability against focal brain ischemic injury in female compared with male SHR. 4. We found age-related vulnerability to focal cerebral ischemia in female SHR. This age-related vulnerability in aged female SHR was unrelated to the blood levels of sex hormones such as estrogens and progesterone. 5. Finally, we emphasized the importance of reproducible and least invasive focal ischemia models in stroke research.     

Nicotine-Induced Neuroprotection Against Ischemic Injury Involves Activation of Endocannabinoid System in Rats

Nicotine has been reported to exert certain protective effect in the Parkinson’s and Alzheimer’s diseases. Whether it has a similar action in focal cerebral ischemia was unclear. In the present study, rats received either an injection of (?)-nicotine hydrogen tartrate salt (1.2 mg/kg, i.p.) or the vehicle 2 h before the 120 min middle cerebral artery occlusion. Neurological deficits and histological injury were assessed at 24 h after reperfusion. The content of endocannabinoids and the expression of cannabinoid receptor CB1 in brain tissues were determined at different time points after nicotine administration. Results showed that nicotine administration ameliorated neurological deficits and reduced infarct volume induced by cerebral ischemia in the rats. The neuroprotective effect was partially reversed by CB1 blockage. The content of the endocannabinoids N-arachidonylethanolamine and 2-arachidonoylglycerol, as well as the expression of cannabinoid receptor CB1 were up-regulated in brain tissues after nicotine delivery. These results suggest that endogenous cannabinoid system is involved in the nicotine-induced neuroprotection against transient focal cerebral ischemia.     

Is neuroprotective efficacy of nNOS inhibitor 7-NI dependent on ischemic intracellular pH?

The purpose of this study was to test the hypothesis that the efficacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 h of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 min before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Confirmation of the effects of these serum glucose manipulations on brain intracellular pH (pH(i)) was confirmed in a group of acute experiments utilizing umbelliferone fluorescence. The animals were euthanized at 72 h for histology. 7-NI significantly (P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach significance because moderate hypoglycemia in itself dramatically reduced infarction volume. We hypothesize that a mechanism to explain the published discrepancies on the effects of neuronal NOS inhibitors in vivo may be due to the effects by differences in ischemic brain acidosis on the production of NO.