构建大脑中动脉阻塞脑缺血模型大鼠的类型分析

背景：线栓法造成短暂性大脑中动脉阻塞是研究大鼠局灶性脑缺血普遍使用的模型制作方法。但制作大鼠脑缺血模型的类型存在一定差异,可能导致实验结果的偏差。 目的：分析大脑中动脉阻塞线栓法制作大鼠脑缺血模型的类型及其影响因素。 方法：雄性SD大鼠166只,参照Longa线栓法造模,术后24 h行MRI扫描,根据扫描结果将大鼠分成皮质梗死组、皮质下梗死组及无梗死组,分析造模时线栓插入的深度。 结果与结论：皮质梗死组、皮质下梗死组和无梗死组大鼠的线栓插入深度分别为(19.9±0.9),(19.0±1.1)和(17.7±1.3) mm,皮质梗死组大鼠的线栓插入最深,而无梗死组的线栓插入最浅(P < 0.01)。提示插入深度不同导致的大鼠脑梗死的类型也不同,线栓插入越深,皮质梗死的概率可能越大。     

大鼠脑缺血后IFN-γ mRNA的表达

目的：检测SD大鼠脑缺血后缺血脑组织和外周免疫淋巴细胞干扰素-γ（IFN-γ）mRNA的表达，探讨IFN-γ在脑缺血中的作用。方法：用线拴封闭大脑中动脉的方法制作脑缺血动物模型，采用原位杂交的方法检测缺血脑组织及外周淋巴组织中IFN-γ mRNA动态的变化情况。采用免疫组织化学方法检测缺血脑组织中浸润的T淋巴细胞数量。结合免疫组化和寡核苷酸探针杂交方法，检测T细胞表达IFN-γ。结果：①缺血脑半球IFN-γ mRNA含量较假手术组明显增高（P〈0．001），且随着脑缺血时间延长其表达量增加；②IFN-γ mRNA表达数量随损伤面积扩大而增加（R=0．9780，P〈0．001）；③缺血组外周血单核细胞IFN-γ mRNA水平较假手术组明显增高，12小时达高峰（P〈0．001），而脾淋巴细胞、淋巴结细胞IFN-γ mRNA水平也均比假手术组明显增高，且随着脑缺血时间延长表达量增加（P〈0．05、P〈0．01、P〈0．001）；④随着脑缺血时间的延长，脑内浸润的T细胞数量显著增加（P〈0．05、P〈0．01、P〈0．001）。结论：IFN-γ mRNA表达主要参与大脑损伤后期反应过程，可能加重脑缺血后期的炎症反应。     

Expression of heparanase in nestin-positive reactive astrocytes in ischemic lesions of rat brain after transient middle cerebral artery occlusion

Heparanase is an enzyme that cleaves heparan sulfate proteoglycans, an important component of the extracellular matrix to generate heparan sulfate fragments, leading to the remodeling of the extracellular matrix and the basement membrane particularly during cancer metastasis. A growing body of evidence suggests that heparanase serves multiple functions in normal tissues including the central nervous system. In this study, we showed that heparanase is expressed in reactive astrocytes in the peri-infarct lesion of a rat brain whose middle cerebral artery was transiently occluded for 90 min. RT-PCR and Western blot analyses revealed that heparanase expression was markedly upregulated during the subacute phase of ischemia (from 3 to 7 days post-reperfusion (dpr)). As revealed by immunohistochemical study, heparanase was localized in astrocytes located in the peri-infarct region. Heparanase+ astrocytes expressed nestin that is known as a marker of reactive astrocytes. Infiltrated neutrophils were weakly heparanase+. After 7 dpr, the expression level of heparanase+ astrocytes considerably decreased. Therefore, the maximum expression of heparanase by astrocytes may correlate with the time of migration of reactive astrocytes toward the ischemic core, which may result in astrogliosis. These findings suggest a novel role of heparanase in the pathophysiology of brain ischemia.     

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.     

Expression of CD200 by macrophage-like cells in ischemic core of rat brain after transient middle cerebral artery occlusion

Brain ischemia causes the death of neurons and glial cells. Such brain cells are believed to inevitably undergo degeneration in the core of ischemic lesions, whereas neurons and glial cells may survive in the region surrounding the core that is often referred to as the ischemic penumbra. However, many cells, particularly immune cells infiltrate and survive in the core. In this study, we characterized macrophage-like cells that accumulated in the ischemic core of a rat brain whose right middle cerebral artery was transiently occluded for 90 min. At 7 days post-reperfusion, we observed macrophage-like cells expressing CD200, a cell surface glycoprotein belonging to an immunoglobulin superfamily and that elicits suppressive effects on myeloid cells including microglia by interacting with the CD200 receptor (CD200R). RT-PCR and immunoblot analyses revealed the presence of CD200-mRNA and protein in the ischemic core as well as in the contralateral region. As revealed by immunohistochemistry, CD200 is located on the cell membrane of spherical Iba1(+) cells with many cytoplasmic granules. CD200(-)/Iba1(+) macrophage-like cells were also present, which have a more irregular shape than CD200(+)/Iba1(+) cells. CD200 was detected in isolated spherical Iba1(+) macrophage-like cells. Thus, CD200 is expressed in some populations of macrophage-like cells that may be responsible for the suppression of CD200R(+) myeloid cell functions in the ischemic core.     

Diameter and length changes in cerebral collaterals after middle cerebral artery occlusion in the young rat

Rapid occlusion of the middle cerebral artery (MCA) in the 36-day-old normal Wistar rat results in change of the dorsal collaterals joining branches of the anterior and middle cerebral arteries. As compared to similarly positioned vessels on the opposite hemisphere and arterioles in unoperated rats of 56 days, of age, there were significantly (P < 0.001) more large (60–120 μm)diameter collaterals on the occluded side 20 days after MCA occlusion. There were fewer small (0-59μm) diameter collaterals on the occluded side as compared to unoperated rats. The data suggest small diameter arterioles existing at occlusion became large diameter collaterals. The mean number of collaterals per hemisphere was not significantly different (P > 0.05) between occluded and unoperated rats. There was no evidence that new vessels were added during the 20-day ligation period. The mean collateral tortuosity value was significantly (P < 0.01) greater for large diameter vessels on the right occluded side as compared to vessels on the left hemisphere or vessels in unoperated age-matched control rats. The greater tortuosity values of vessels on the occluded side were evidence that collateral vessel length was increased by 24–29% after MCA occlusion. Speculation was made about possible mechanisms responsible for these vascular changes.     

Temporal and spatial changes of free cholesterol and neutral lipids in rat brain after transient middle cerebral artery occlusion

In order to examine lipid metabolism in relation to neural process following brain ischemia, we investigated temporal and spatial changes of free cholesterol (FC) and neutral lipids (NLs) after 90 min of transient middle cerebral artery occlusion (MCAO). Filipin and Nile Red stainings were performed to detect mainly FC and NLs, respectively. Double stainings for Nile Red plus ED1, MAP2, or GFAP were performed in order to identify cell type of positive stainings. Filipin stanining decreased during 1-7 day and lost at 21 day after transient MCAO in the ischemic core, but did not change in the penumbra. Nile Red positive droplets reached the maximum at 7 day after transient MCAO and gradually decreased in the core, while the peak time delayed in the penumbra. MAP2 immunoreactivity lost at 7 day in the core, and increased in the penumbra during 7-56 day. Most Nile Red positive droplets were double positive for ED1 in the core, and were localized within GFAP positive cells in the penumbra. These results suggest that changes of FC and NLs are different temporally and spatially between the core and penumbra in relation to degenerative and regenerative neural processes following brain ischemia.     

大鼠脑缺血预适应对脑线粒体功能的影响

观察缺血预适应对大鼠大脑中动脉梗塞的保护作用 ,以及对大鼠脑线粒体功能的影响。观察大鼠双侧颈总动脉缺血预适应后 ,短暂缺血作用对脑梗塞面积、神经症状评分、倾斜板停留时间的影响 ,并测定线粒体复合酶活性、膜肿胀、膜电位、膜流动性的变化。与大脑中动脉梗塞 (MCAO)组比较 ,预适应可以显著降低大脑梗塞面积 (P <0 0 0 1) ;明显改善神经症状评分 ;延长在倾斜板上停留时间 (P <0 0 0 1) ;并可增强线粒体复合酶Ⅰ、Ⅲ、Ⅳ活性 ;线粒体膜肿胀降低 (P <0 0 0 1) ,流动性好于MCAO组 (P <0 0 1) ;膜电位无显著性变化。结果表明 ,缺血预适应对脑有保护作用 ;线粒体的膜受到了保护     

大鼠短暂性局灶性脑缺血早期HMGB1在半暗带区的表达

目的:探讨调节炎症反应的细胞因子HMGB1在脑缺血/再灌注损伤中的时程变化特点。方法:采用大鼠MCAO模型,利用ELISA及免疫组织化学染色方法观察缺血/再灌注后不同时间点脑内HMGB1蛋白的表达变化。结果:ELISA检测提示再灌注后大鼠缺血侧大脑HMGB1表达显著增高,在10 h和70 h形成两次高峰(P<0.05);免疫组织化学染色提示再灌注1 h后部分大鼠(1/5)和再灌注后4,10,22和70 h后全部大鼠梗死区周边半暗带区的细胞外间隙有HMGB1阳性染色颗粒,后期并在部分细胞内出现HMGB1高表达。结论:结果提示,HMGB1不仅与脑缺血/再灌注的晚期损伤有关,亦可能参与了早期损伤过程。     

G-CSF enhances stem cell proliferation in rat hippocampus after transient middle cerebral artery occlusion

Granulocyte colony-stimulating factor (G-CSF) enhances the survival and stimulates the proliferation of neutrophil progenitors. Recently, the neurogenerative effect of G-CSF has been intensely investigated. In this study, we explored the possibility that G-CSF enhanced the cell proliferation in the rat dentate gyrus (DG) after focal cerebral ischemia, using a rat transient middle cerebral artery occlusion (tMCAO) model. At 7 days after tMCAO, the number of 5-bromodeoxyuridine (BrdU)-positive cells in the G-CSF-treated group was significantly increased compared with that in the vehicle-treated group in the ipsilateral SGZ (16.6 ± 5.5/mm² in the vehicle-treated group versus 33.0 ± 7.2/mm² in the G-CSF-treated group, **p < 0.01) and in the ipsilateral GCL (14.2 ± 2.8/mm² in the vehicle-treated group versus 21.0 ± 3.8/mm² in the G-CSF-treated group, *p < 0.05). This result showed the possibility of a neurogenerative role of G-CSF after tMCAO in rats.     

c-Jun N-terminal kinase (JNK) and JNK interacting protein response in rat brain after transient middle cerebral artery occlusion

c-Jun response is involved in the development of ischemic brain injury, which is activated by c-Jun N-terminal kinase-1 (JNK-1). The activity of JNK-1 is strictly regulated, and only the phosphorylated form of JNK (phospho-JNK) which is translocated to the nucleus has an ability to activate c-Jun response. There is a protein which inhibits JNK-1 activation, and known as JNK interacting protein-1 (JIP-1). In this study, we investigated change in JNK-1, phospho-JNK, and JIP-1 immunoreactivity in rat brain after transient middle cerebral artery (MCA) occlusion. Immunoreactive JNK-1 was scant in the sham-control brain, but it was induced at 1 h after reperfusion, which was slightly increased at 3 h of reperfusion. By contrast, phospho-JNK remained negative till 3 h. At 8 h, JNK-1 and phospho-JNK became distinctly positive, and nuclei as well as cytoplasm were stained. Thereafter, immunoreactivity for JNK-1 and phospho-JNK became furthermore dense, and most neurons revealed positively stained nuclei. Immunoreactivity for JIP-1 remained negative till 8 h of reperfusion, but at 24 and 72 h, cytoplasm of cortical neurons at the MCA boundary area was positively stained. This JIP-1 induction got behind the JNK-1 activation, and therefore, may be a vain effort for neurons to survive. Inhibition of JNK-1 activation might become an innovative means of therapy for stroke treatment in the future.     

Upregulation of glutamate receptors in rat cerebral cortex with neuronal migration disorders

Neuronal migration disorders (NMDs) constitute the main pathologic substrate of medically intractable epilepsy in human. This study is designed to investigate the changes in expression of glutamate receptor subtypes on radiation-induced NMD in rats. The lesion was produced by intrauterine irradiation (240 cGy) on E17 rats, and then 10 weeks old rats were used for the study. The pathologic and immuno-histochemical findings for glutamate receptor subunit proteins on NMD cortex were correlated with development of behavioral seizures and EEG abnormality. Spontaneous seizures uncommonly occurred in NMD rats (5%); however, clinical stages of seizures were significantly increased in NMD rats by an administration of kainic acid. Brains taken from irradiated rats revealed gross and histopathologic features of NMD. Focal cortical dysplasia was identified by histopathology and immunohistochemistry with neurofilament protein (NF-M/H). Significantly strong NR1 and NR2A/B immunoreactivities were demonstrated in cytomegalic and heterotopic neurons of NMD rats. The results of the present study indicate that epileptogenesis of NMD might be caused by upregulation of glutamate receptor expression in dysplastic neurons of the rat cerebral cortex with NMDs.     

Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat.

Brains from eight rats subjected to transient focal ischemia (2 hours) and reperfusion (22 hours) revealed an ultrastructural appearance of apoptosis and light microscopic features of cells exhibiting DNA fragmentation. The number of cells exhibiting DNA fragmentation was significantly higher (P < 0.01) in the ipsilateral hemisphere (216 +/- 43 per section) than in the contralateral hemisphere and in normal (n = 4) and sham operated (n = 4) rats (0 to 3 positive cells per section in controls). Cells exhibiting DNA fragmentation were primarily located in the inner boundary zone to the infarct. Cells exhibiting DNA fragmentation provided morphological information of apoptosis. Apoptosis may contribute to the development of infarct after transient focal cerebral ischemia.     

大脑中动脉阻塞后脑组织病变过程及MDA含量变化

在大鼠中用热凝造成大脑中动脉阻塞而致脑局灶性缺血。电镜观察发现,在缺血2min后,神经细胞粗面内质网与线粒体即出现改变。光镜检查,缺氧10min后才出现神经细胞缺血改变;梗死灶周边微血管体积密度在缺血24h后即明显升高。肉眼检查,于缺血3h后可见梗死灶。在缺血2min后,脑组织内丙二醛(MOA)即升高,说明在缺血缺氧的早期,脂质过氧化作用加强。表明自由基在缺血性脑损伤早期即起重要作用。     

Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat

Summary It has become increasingly clear that a stroke lesion usually consists of a densely ischemic focus and of perifocal areas with better upheld flow rates. At least in rats and cats, some of these perifocal (penumbral) areas subsequently become recruited in the infarction process. The mechanisms may involve an aberrant cellular calcium metabolism and enhanced production of free radicals. In general, though, the metabolic perturbation in the penumbra requires better characterization. The objective of this article was to define flow distribution in a rat model of reversible middle cerebral artery (MCA) occlusion, so as to allow delineation of the metabolic aberrations responsible for the subsequent infarction. We modified the intraluminal filament occlusion model recently developed by Koizumi et al. (1986), and described in more detail by Nagasawa and Kogure (1989), adopting it for use in both spontaneously breathing and artificially ventilated rats. Successful occlusion of the MCA (achieved in about 9/10 rats) was judged by unilateral EEG depression in ventilated rats, and neurological deficits, such as circling, in spontaneously breathing ones. CBF in the ipsilateral hemisphere was reduced to nearly constant values after 20, 60, and 120 min of occlusion, flow rates in the focus being about 10% and in the perifocal ipsilateral areas about 15–20% of control (contralateral side). When the filament was left in place (permanent occlusion) 2,3,5-triphenyl tetrazolium chloride (TTC) staining and histopathology after 24 h showed a massive infarct on the occluded side, extending from caudoputamen and overlaying cortex to the occipital striate cortex. Animals recirculated after 60 min of MCA occlusion, and allowed to survive 7 days for histopathology, showed infarction of the caudoputamen (lateral part or whole nucleus) in 5/6 animals and selective neuronal necrosis in one animal. The neocortex showed either infarcts, selective neuronal necrosis, or no damage. There was some overlap between neocortical areas which were infarcted and those which were salvaged by reperfusion. In general, though, both the CBF data and the recovery studies with a histopathological endpoint define large parts of the neocortex as perifocal (penumbral) areas which lend themselves to studies of metabolic events leading to infarction.     

The effects of TRH analogues on cerebral ischaemia produced by middle cerebral artery occlusion in the rat

Summary This study examined the effects of two stabilised analogues of TRH, RX 77368 and CG 3509, in a rat cerebral ischaemia model produced by unilateral occlusion of the middle cerebral artery. The analogues were given intraventricularly after artery occlusion. The extent of the cortical ischaemia was evaluated after 10 days by somatosensory evoked potential (SEP) recording, followed by tetrazolium staining of brain slices for NADH-diaphorase activity. RX 77368 (2×10 g; 15 min, 24 h) significantly improved the survival rate, protected the SEP and reduced the area of infarct. In contrast, neither a smaller dose of RX 77368 (2×3 g) nor a 4 h delay in the treatment had any significant beneficial effects. Although CG 3509 (2×10 g) resulted in an apparent improvement in survival, its overall effects were not statistically significant. The findings indicate that stabilised TRH analogues may have beneficial effects when given to animals with focal cerebral ischaemia.     

Minimosymptomatic occlusion of the middle cerebral artery

Minimosymptomatic occlusion of the MCA before the origin of the perforating branches is an exceedingly rare occurrence. We report two cases of MCA occlusion at its origin, the second case proven by the CT scan in vivo. Published work rules out the possibility of a functionally effective deep collateral circulation in the distribution of the capsular rami of the MCA. In view of this we argue that there may be cases — admittedly very rare — in which, given the individual variability of the vascular supply, the MCA may be of negligïble functional importance to the circulation of the internal capsule. In such cases occlusion of the MCA would be relatively well tolerated.     

Long-term functional outcome following transient middle cerebral artery occlusion in the rat: correlation between brain damage and behavioral impairment

The assessment of both histological and functional long-term outcomes after cerebral ischemia is increasingly recommended for preclinical studies. Whereas correlations between behavioral impairments and primary ischemic lesion are documented, little is known about their relationships with remote nonischemic regions that undergo secondary degeneration, such as the thalamus. Anesthetized rats were subjected to mild (30 min) or severe (60 min) occlusion of the middle cerebral artery. Two months after ischemia, sensorimotor behavior was assessed according to the neurological score, limb-placing, adhesive-removal, and staircase tests; the final histological lesion was measured after this assessment. Cortical damage was correlated to all transient and long-lasting sensorimotor deficits, whereas striatal lesion was more consistently reflected by the forelimb-placing reflexes and adhesive-removal motor deficits. By contrast, the thalamic atrophy was not correlated to early neurological impairment, but rather to the late sensory deficit at the adhesive-removal test and to the skilled forepaw reaching alteration at the staircase test. This suggests that thalamus contributes, albeit moderately, to the ischemia-induced long-lasting sensorimotor deficits, some of which represent relevant targets for therapeutic interventions.