Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brains angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.     

盐酸法舒地尔对慢性脑低灌注损伤大鼠海马细胞的保护作用

目的 观察Rho激酶抑制剂盐酸法舒地尔(hydroxy fasudil, HF)对慢性低灌注脑缺血所致大鼠海马神经细胞损伤的保护作用。方法 采用永久性结扎大鼠双侧颈总动脉(permanent occlusion of the bilateral CCA, 2VO)制备大鼠慢性不完全性全脑缺血模型,将SD大鼠随机分为假手术组、脑缺血模型组和HF治疗组,运用Morris 水迷宫行为学方法检测大鼠空间学习记忆能力; 用HE染色观察海马组织形态学改变。结果 Morris 水迷宫检测发现模型组大鼠学习记忆能力受损,与假手术组比较逃避潜伏期延长、空间辨别能力下降; 组织学观察模型组大鼠海马CA1细胞发生丢失,组织结构异常。连续给予盐酸法舒地尔30 d能改善大鼠学习记忆功能,减少脑缺血所致的大鼠海马神经细胞丢失。结论 盐酸法舒地尔可减少慢性脑缺血所致的大鼠海马神经元的丢失,改善学习记忆功能。     

Dextromethorphan attenuates post-ischemic hypoperfusion following incomplete global ischemia in the anesthetized rat

The effects of dextromethorphan (DM) were tested in an in vivo model of incomplete global cerebral ischemia. Anesthetized rats were divided into 4 groups: Group 1 (saline); Group 2 (DM pre-treatment, 20 mg/kg i.v. bolus followed by 10 mg/kg/h DM infusion); Group 3 (DM post-treatment, 2 mg/kg i.v. bolus followed by 10 mg/kg/h DM infusion at the onset of post-ischemic hypoperfusion); and Group 4 (sham-operated, drug-treated). Groups 1–3 underwent 15 min of 4-vessel occlusion followed by 3 h of reperfusion. Administration of DM in sham-operated animals (Group 4) had no effect on cerebral blood flow or electroencephalographic (EEG) activity. In contrast, when compared to the Group 1 saline controls, significant attenuation of post-ischemic hypoperfusion and EEG dysfunction was demonstrated in ischemic rats treated with DM (both pre- and post-treatment), suggesting an ability of DM to improve cerebral blood flow (CBF) and brain function in cerebral ischemia.     

Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion

White matter lesions are thought to result from chronic cerebral ischemia and constitute a core pathology of subcortical vascular dementia. This rarefaction has been known to be associated with microglial activation. We investigated whether minocycline, a microglial inhibitor, attenuates the white matter damage induced by chronic cerebral hypoperfusion that is used as a model of vascular dementia. Male Wistar rats were subjected to bilateral, permanent occlusion of the common carotid arteries (BCCAO) to induce chronic cerebral hypoperfusion. Minocycline or saline was injected daily for 2 weeks after BCCAO. In the corpus callosum and the optic tract, white matter damage observed with Klüver-Barrera staining was significantly attenuated in the minocycline-treated group compared to saline-treated controls. In control rats, immunoreactivities of major basic protein (MBP), Ox-42 as a microglial marker, and matrix metalloproteinase (MMP)-2 were increased in the corpus callosum. Minocycline significantly reduced these changes. Co-expression of Ox-42 and MMP-2 was confirmed by double immunofluorescence histochemistry. Our results suggest that chronic treatment with minocycline could be protective against at least some ischemic white matter damage, and its mechanism may be related to suppressing microglial activation.     

Cumulative white matter changes in the gerbil brain under chronic cerebral hypoperfusion

Summary An animal model of chronic brain hypoperfusion has been developed by applying coiled clips to the bilateral carotid artery of Mongorian gerbils. The brain tissue damage was neuropathologically studied after 1, 4, 8, and 12 weeks of hypoperfusion. The hippocampus, basal ganglia, and cerebral cortex of the chronically hypoperfused gerbil showed lesions with various severity which are probably due to ischemic episodes. In the cerebral white matter, however, two types of lesions were observed; one similar to those in the gray matter, and the other observed only in the white matter after more than an 8-week duration of brain hypoperfusion. The lesion specific to the white matter showed rarefaction and gliosis without locally associated ischemic changes. This type of the white matter lesion was never found in the gerbil brain before 8 weeks and, significantly, increased in number and size by 12 weeks post operation. The accumulation of the white matter lesions is characteristic in the gerbil with chronic hypoperfusion. The observed white matter-specific lesion resembles the histological changes in aged brain with cerebrovascular diseases.     

Secondary hypotensive insults in a rat forebrain ischemia model

Previous studies have shown that the recently injured brain has an increased sensitivity to subsequent brief episodes of severe ischemia. This investigation was designed to assess whether less severe secondary insults, which alone would be incapable of producing injury, exacerbate brain damage resulting from a primary episode of global ischemia. Rats were subjected to either 10 min of 2-vessel forebrain ischemia (primary insult alone), 20 min of hypotension (mean arterial pressure, MAP = either 40 or 25 mmHg) without vessel occlusion (secondary insult alone), or 10 min ischemia followed 1 h later by the hypotensive challenge (primary + secondary insult). Seven days later, the animals were neurologically evaluated and the brains then prepared for histologic analysis. Neither magnitude of secondary insult alone was found to produce injury. In contrast, the primary insult alone caused moderate damage in the hippocampus, caudoputamen and neocortex. With the exception of increased neuronal necrosis in the hippocampal CA1 sector in rats receiving the primary + secondary insult (MAP = 25 mmHg), no worsening of outcome could be attributed to the secondary insults. These results indicate that the recovering brain may not be as sensitive to hypoperfusion as has previously been suggested.     

YM796, a novel muscarinic agonist, improves the impairment of learning behavior in a rat model of chronic focal cerebral ischemia

We studied effects of YM796, a novel muscarinic agonist, on behavioral, histological and regional cerebral blood flow changes in the chronic phase after focal cerebral ischemia in rats. YM796 (0.03, 0.1, 0.3 and 1 mg/kg) was administered orally once a day from the 7th to the 13th day after the permanent occlusion of left middle cerebral artery. On the 7th day, rats were trained in one-trial step-through passive avoidance task 45 min after drug administration. Test trials were carried out on the 8th and 14th days. Neurological deficits, including hemiplegia and abnormal posture, were observed on the 7th and 14th days. After the completion of behavioral studies, the rats were decapitated and cerebral infarction was measured. Regional cerebral blood flow was also measured by the hydrogen clearance technique 7 days after MCA occlusion. YM796 (0.1 – 1 mg/kg) significantly (P < 0.05) attenuated the impairment of learning behavior in a dose-dependent manner without affecting spontaneous locomotor activity. The ameliorating effect of YM796 (0.3 mg/kg) on the impaired learning behavior was significantly (P < 0.05) suppressed by intracerebroventricular injection of pirenzepine (10 μg/rat), an M₁ antagonist. No significant difference in either neurological deficits or cerebral infarction was found between the vehicle- and YM796-treated groups. Further, YM796 (0.3 mg/kg) had little effect on the reduced blood flow in the ipsilateral frontal cortex 7 days after occlusion. These results suggest that YM796 improves the impaired learning behavior probably by activating central M₁ receptors in a rat model of chronic focal cerebral ischemia.     

Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia

The influence of the interval of the repeated reversible transient cerebral ischemia on the neuronal damage in the hippocampal CA1 sector was investigated in the rats using a 4-vessel occlusion (4-VO) model. A single 3-min 4-VO did not produce any significant neuronal damage in the hippocampal CA1 sector, whereas the rats subjected to three 3-min 4-VO at 1-h intervals revealed a very severe neuronal damage which was much more severe than that in the rats subjected to a single 9-min 4-VO. In contrast, the rats subjected to three 3-min 4-VO at 6-h intervals revealed only a mild neuronal damage. The degree of the neuronal damage in the rats subjected to three 3-min 4-VO at 5-min intervals was similar to that in the rats subjected to a single 9-min 4-VO. The present study indicates that even such a brief, non-lethal ischemia as 3-min 4-VO can produce a severe neuronal damage if it occurs repeatedly at 1-h intervals.     

绞股蓝总皂甙对慢性脑缺血性白质氧化性损伤的保护作用

目的 探讨绞股蓝总皂甙对慢性脑缺血大鼠脑白质氧化性损伤的保护作用以及对脑缺血大鼠认知功能的影响.方法 将57只成年雄性SD大鼠按随机数字表法分成假手术组(n=12)、模型组(n=15)、绞股蓝总皂甙200 mg组(n=15)、绞股蓝总皂甙400 mg组(n=15),后3组采用双侧颈总动脉结扎法制备慢性脑缺血模型,且在造模后3h分别将等量生理盐水,200 mg/kg、400mg/kg绞股蓝总皂甙溶液灌胃,1次/d,持续33 d.应用Morris水迷宫实验测试各组大鼠空间学习与记忆能力的改变,酶联免疫吸附法(ELISA)测定大鼠胼胝体、视束内超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,免疫组化染色用8-羟基脱氧鸟苷(8-OHdG)抗体测定中枢神经细胞的氧化损伤水平.结果 与模型组相比,绞股蓝总皂甙400 mg组大鼠的逃避潜伏期明显缩短及在原平台象限的游泳时间明显延长,差异有统计学意义(P＜0.05).与假手术组比较,模型组大鼠胼胝体及视束内MDA含量明显增加,SOD活性明显降低,差异有统计学意义(P＜0.05).与模型组相比,绞股蓝总皂甙400 mg组MDA含量明显降低,SOD活性明显升高,8-OHdG阳性细胞数明显减少,差异有统计学意义(P＜0.05).与模型组相比,绞股蓝总皂甙200 mg组SOD活性、MDA含量及8-OHdG阳性细胞数差异无统计学意义(P＞0.05).结论 绞股蓝总皂甙能有效改善慢性脑缺血大鼠脑白质氧化性损伤,提示其可能是一种有效的抗痴呆药物,但其作用的确切机制还有待进一步研究.     

氧化应激在慢性缺血性脑白质损伤中的作用

目的 阐明氧化应激是否参与大鼠慢性脑缺血所致的脑白质损伤.方法 健康雄性Wistar大鼠按照完全随机数字表法分为假手术组,持久性双侧颈总动脉结扎3 d组、7 d组、3周组及6周组,每组6只.应用大鼠双侧颈总动脉结扎制备慢性脑缺血模型,检测大鼠脑白质内超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷胱甘肽(GSH)含量以及脂质过氧化产物丙二醛(MDA)和4-羟基壬烯醛(4-HNE)加合物的变化.结果 与假手术组比较,慢性脑缺血大鼠脑白质内MDA含量在手术后3周明显增加,手术后6周进一步增高,差异有统计学意义(P<0.05).手术后3d至6周,慢性脑缺血大鼠脑白质内4-HNE蛋白加合物逐渐增高,与假手术组比较有差异有统计学意K(P<0.05).SOD活性在手术后3周和6周才明显降低,与假手术组比较差异有统计学意义(P<0.05).此外,慢性脑缺血大鼠脑白质内GSH含量在手术后7d即开始降低,而在手术后3周及6周则进一步下降,与假手术组比较差异有统计学意义(P<0.05).结论 慢性脑缺血导致大鼠脑白质氧化性损伤增加,抗氧化防御能力降低:氧化性损伤的增加和抗氧化防御能力的降低与慢性脑缺血所致的脑白质损伤密切相关.     

Inhibition of Na/Ca exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury

Reversal of the Na⁺/Ca²⁺ exchanger (NCX) occurs during ischemia–reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 μM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 μM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na⁺/Ca²⁺ exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na⁺/Ca²⁺ exchange inhibitors in ischemia–reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.     

The pretectal necleus of the optic tract modulates the direction selectivity of accessory optic neurons in rats

Direction-selective singel units within the medical terminal nucleus of the rat were shown to prefer motion in upward-temporal or downward-nasal directions. Lesions of the optic tract nucleus practically abolished responses to the nasal direction of motion and increased the directional bias toward the temporal direction. Such results suggest a significant role of that pretectal nucleus in the determination of direction selectivity of accessory optic neurons.     

亚低温联合黄芪对大鼠局灶脑缺血再灌注损伤的神经保护作用

目的探讨亚低温(32±1℃)联合黄芪对大鼠局灶脑缺血再灌注损伤的协同保护作用。方法采用改良线栓法制备大鼠大脑中动脉闭塞再灌注模型,将24只SD大鼠随机分为四组(每组6只)常温组;亚低温组,再灌注后立即诱导亚低温并持续12小时;黄芪组,脑缺血后立即按1g/kg剂量腹腔注射黄芪;黄芪加亚低温组,脑缺血后立即按1g/kg剂量腹腔注射黄芪,再灌注后立即诱导亚低温并持续12小时。每组均脑缺血6小时后再灌注。观察脑梗死体积、神经功能缺损评分和病理改变。结果黄芪加亚低温组脑梗死体积为112.41±1664mm3,亚低温组为14668±14.88mm3,经两因素方差分析有显著性意义(P＜0.05)。结论亚低温联合黄芪对脑缺血再灌注损伤具有协同保护作用。     

Endogenous proteolytic activity in a rat model of spontaneous cerebral stroke

We evaluated the expression of two extra-cellular protease systems in a model of spontaneous cerebrovascular pathology: spontaneously hypertensive stroke-prone rats (SHRSP). The appearance of brain damage in individual animals was imaged and followed by means of magnetic resonance imaging (MRI). In situ zymography of brain slices obtained 3 days after the appearance of brain damage showed an increase in plasminogen activator (PA)/plasmin activity that co-localised with the cerebral damage detected by MRI; there was also concomitant accumulation/activation of inflammatory cells in the damaged area. Proteolytic activity was inhibited by the urokinase-specific inhibitor amiloride but not by an antibody against tissue-type plasminogen activator (t-PA). SDS-PAGE zymography of brain extracts revealed the presence of 58 kDa plasminogen-dependent lysis areas in the ischemic and non-ischemic tissues, and a 33 kDa lysis area in ischemic tissue only. An antibody against t-PA inhibited the former, whereas the latter was inhibited by amiloride. The specific induction of urokinase-type plasminogen activator (u-PA) in the damaged tissue was further confirmed by the fact that both u-PA protein mass and mRNA were markedly increased in the damaged cerebral areas. Concomitant metalloproteinase-2 (MMP-2) activation was only observed in the damaged area. These data suggest that u-PA is expressed and selectively catalyses proteolysis in the injured area of spontaneous brain damage in SHRSP.     

Blood-brain barrier disruption in white matter lesions in a rat model of chronic cerebral hypoperfusion.

Blood-brain barrier damage has been implicated in the pathogenesis of cerebrovascular white matter lesions. This type of lesion is responsible for cognitive impairment in the elderly and can be induced by permanent ligation of the bilateral common carotid arteries in the rat. Because it is unclear whether the blood-brain barrier is impaired, we examined whether vascular permeability to horseradish peroxidase is altered using this model. According to light microscopic results, the reaction product of horseradish peroxidase was most intensely localized to the paramedian part of the corpus callosum in the brain, occurring to a small degree at 3 hours, day 1, markedly on day 3, but reduced on days 7 and 14. By electron microscopic study of the same area, the reaction product of horseradish peroxidase was localized to the plasmalemmal vesicles in the endothelial cells 3 hours after ligation, but appeared in the cytoplasm on days 1 and 3, suggesting a diffuse leakage of horseradish peroxidase. In addition, the reaction product was dispersed into the cytoplasm of glial cells in the perivascular regions on day 3. The luminal surface of the endothelial cell cytoplasm appeared irregular on day 7, suggesting a conformational change of the endothelial cells. Collagen fibrils proliferated in the thickened basal lamina and mitochondria degenerated in the pericyte on days 7 and 14. Perivascular glial endfeet were swollen throughout the survival period. In sham-operated rats, the reaction product of horseradish peroxidase was not observed at any time interval, except in vesicular structures. These findings indicate that chronic cerebral hypoperfusion induces blood-brain barrier damage with subsequent morphologic changes of the vascular structures in the corpus callosum. An extravasation of macromolecules, such as proteases and immunoglobulins, may contribute to the pathogenesis of white matter lesions.     

长期饮酒大鼠缺血脑组织Fas-L抗原表达的动态研究

目的 研究长期饮酒大鼠局灶性脑缺血后Fas—L抗原在各时间段的表达与正常大鼠脑缺血同一时间段有何不同。方法 7．2％酒精代水自由饮用12周的大鼠用线栓法制备局灶性脑缺血模型，用免疫组织化学法测定Fas—L抗原表达阳性细胞。结果 长期饮酒组大鼠缺血1h Fas—L抗原呈阳性，3h达高峰，6h减少，而正常组大鼠Fas—L阳性表达随缺血时间增加而出现增多的趋势。结论 长期饮酒大鼠在脑缺血后Fas—L的表达比正常大鼠脑缺血明显，且促使凋亡细胞迅速坏死。     

Effects of Fujian tablet on Nogo-A mRNA expression and plasticity of the corticospinal tract in a rat model of focal cerebral ischemia

The present study investigated the effects of Fujian tablet, a Chinese medicine compound that can nourish liver and kidney, on corticospinal tract plasticity and cervical cord microenvironment in rats with focal cerebral ischemia. Results showed that motor function of rats with right proximal middle cerebral artery occlusion was significantly improved following treatment with Fujian tablet, 9 g crude drug/kg. Anterograde tracing revealed significantly increased biotinylated dextran amine expression in the denervated (left) side of the cervical cord (C4-6) following Fujian tablet treatment, and significantly decreased Nogo-A mRNA expression was detected in the denervated side of the cervical cord (C4-6) using in situ hybridization. Pearson’s correlation analysis showed a negative correlation between biotinylated dextran amine and Nogo-A mRNA expression (r = -0.943, P < 0.01). Results demonstrated that Fujian tablet can promote corticospinal tract plasticity possibly through the inhibitory effect on Nogo-A mRNA expression in the cervical spinal cord, thereby improving motor dysfunction.     

Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia

Activation of group I metabotropic glutamate receptors (mGluR) has been implicated in the pathophysiology of acute central nervous system injury. However, the relative roles of the two group I subtypes, mGluR1 or mGluR5, in such injury has not been well examined. We compared the effects of treatment with the newly developed, selective mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) and the selective mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) in a rat intraluminal filament model of temporary middle cerebral artery occlusion (MCAo). Rats were administered MPEP or CHPG i.c.v. beginning 15 or 135 min after induction of ischemia for 2 h. Infarct size was measured after either 22 or 70 h of reperfusion, and neurological function was quantified at 2, 24, 48 and 72 h. Treatment with MPEP or CHPG at 15 min reduced 24 h infarct volume by 61 and 44%, respectively. The neuroprotective effects were dose dependent. Delaying MPEP treatment until 135 min eliminated the neuroprotective effects. In other studies, using early MPEP treatment (15 min) at optimal doses, infarct volume was reduced by 44% at 72 h and this was correlated with significant neurological recovery. These data suggest that both MPEP and CHPG are neuroprotective when administered after focal cerebral ischemia. In separate, recent studies we found that although MPEP does act as an mGluR5 antagonist and blocks agonist induced phosphoinositide hydrolysis, it also serves as a non-competitive NMDA antagonist; in contrast, other results indicate that CHPG mediated neuroprotection may reflect anti-apoptotic activity. Therefore, both types of compounds may prove to have therapeutic potential for the treatment of stroke.