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.     

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。     

Neuronal regeneration and protection by collagen-binding BDNF in the rat middle cerebral artery occlusion model

It has been well confirmed that brain-derived neurotrophic factor (BDNF) has therapeutic effects following stroke. However, it is difficult to be maintained at a sufficient concentration of BDNF in the infarcted hemisphere. We have shown in our previous work that BDNF fused with a collagen-binding domain (CBD-BDNF) could specifically bind to collagen. The ventricular ependyma of the brain is rich in collagen. Therefore, we have speculated that in the infarcted hemisphere, CBD-BDNF will bind to the collagen of the ventricular ependyma and stimulate the cell proliferation in the subventricular zone (SVZ). Using a rat middle cerebral artery occlusion model (MCAO), we injected CBD-BDNF into the lateral ventricle of MCAO rats. The results demonstrated that CBD-BDNF was retained at high levels in the infarcted hemisphere, promoted neural regeneration and angiogenesis, reduced cell loss, decreased apoptosis, and improved functional recovery. In addition, brain perfusion and metabolism, as evaluated by SPECT and PET, were improved in the CBD-BDNF treated group.     

大鼠大脑中动脉缺血后海马星形胶质细胞反应的研究

目的:观察大鼠大脑中动脉缺血后皮层损伤侧海马星形胶质细胞反应的变化。方法:采用大鼠大脑中动脉阻塞再灌流模型,应用免疫印迹和免疫组织化学方法测定脑缺血后3 d、7 d以及30 d皮层损伤侧海马胶质纤维酸性蛋白(GFAP)以及增殖细胞核抗原(PCNA)蛋白的表达,观察星形胶质细胞增殖的变化。结果: GFAP免疫组化结果显示,脑缺血后7d皮层损伤侧海马CA1、CA2区星形胶质细胞数量较假手术组增加且胞体增大;脑缺血后30 d皮层损伤侧海马CA1、CA2区呈胶质疤痕样改变。同时,免疫印迹法显示脑缺血后7 d皮层损伤侧海马GFAP表达增强;脑缺血后30 d皮层损伤侧海马GFAP表达增高更加明显。此外,免疫印迹法显示脑缺血后3 d皮层损伤侧海马PCNA蛋白表达水平升高;脑缺血后7 d PCNA蛋白表达水平达到峰值;脑缺血后30 d,PCNA蛋白表达水平降低,但仍高于假手术组。结论: 大鼠大脑中动脉缺血后可引起其皮层损伤侧海马星形胶质细胞过度反应和增殖。     

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。     

人参皂甙Rd对大鼠大脑中动脉栓塞模型NMDA受体磷酸化的影响

目的:研究人参皂甙Rd(Ginsenoside Rd,GSRd)对大鼠大脑中动脉栓塞(MCAO)模型损伤后,NMDA受体NR2B亚基的表达及其磷酸化的影响。方法:建立大鼠MCAO模型,并在术前及术后给予人参皂甙Rd,之后进行神经行为学评分,通过TTC(2,3,5-氯化三苯基四氮唑)染色观察脑梗死体积变化,采用Western blot方法检测脑缺血区不同时间点NR2B及其磷酸化的Ser1303及Tyr1472的表达情况。结果:GSRd能够改善大鼠神经行为学评分,减小模型大鼠脑梗死体积;MCAO模型大鼠脑缺血损伤处的NR2B亚基、磷酸化Ser1303及Tyr1472的表达量均较假手术组升高;在MCAO术前及术后给予GSRd,能够有效降低损伤侧NR2B亚基、磷酸化Ser1303及Tyr1472的表达量,并且GSRd对于上述各项表达量的影响呈剂量依赖性。结论:人参皂甙Rd能够有效降低MCAO模型中NR2B及磷酸化Ser1303及Tyr1472的水平,提示人参皂甙Rd可能是通过调节脑缺血再灌注损伤后NMDAR中NR2B亚基及该亚基磷酸化Ser1303、Tyr1472位点的水平,从而发挥其对脑缺血再灌注损伤的保护作用。     

Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model

Middle cerebral artery occlusion (MCAO) models have become well established as the most suitable way to simulate stroke in experimental studies. The high variability in the size of the resulting infarct due to filament composition, rodent strain and vessel anatomy makes the setup of such models very complex. Beside controllable variables of homeostasis, the choice of anesthetics and the grade of ischemia and reperfusion played a major role for extent of neurological injury. Transient MCAO was induced during either isoflurane or ketamine/xylazine (ket/xyl) anesthesia with simultaneously measurement of cerebral blood flow (CBF) in 60 male Wistar rats (380–420 g). Neurological injury was quantified after 24 h. Isoflurane compared with ket/xyl improved mortality 24 h after MCAO (10 vs. 50 %, p = 0.037) and predominantly led to striatal infarcts (78 vs. 18 %, p = 0.009) without involvement of the neocortex and medial caudoputamen. Independent of anesthesia type, cortical infarcts could be predicted with a sensitivity of 67 % and a specificity of 100 % if CBF did not exceed 35 % of the baseline value during ischemia. In all other cases, cortical infarcts developed if the reperfusion values remained below 50 %. Hyperemia during reperfusion significantly increased infarct and edema volumes. The cause of frequent striatal infarcts after isoflurane anesthesia might be attributed to an improved CBF during ischemia (46 ± 15 % vs. 35 ± 19 %, p = 0.04). S-100β release, edema volume and upregulation of IL-6 and IL-1β expression were impeded by isoflurane. Thus, anesthetic management as well as the grade of ischemia and reperfusion after transient MCAO demonstrated important effects on neurological injury.     

Hyperbaric oxygenation mitigates focal cerebral injury and reduces striatal dopamine release in a rat model of transient middle cerebral artery occlusion

The usefulness of the administration of hyperbaric oxygen (HBO) in the treatment of acute focal cerebral ischemia remains debatable. A significant association exists between focal cerebral injury and an excessive release of extracellular dopamine (DA). In vivo microdialysis was used in the present study to examine the effect of HBO on DA release in the striatum during ischemia and reperfusion in rats. The histological changes occurring were also evaluated. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCA) using a surgically placed intraluminal filament. Control rats (n=8) were subjected to 1 h of ischemia, whilst the study rats (n=8) were in addition treated with HBO (2.8 atmospheres of absolute pressure 100% O₂) during ischemia. Both groups were returned to breathing room air at normal pressure during reperfusion. Microdialysis samples were continuously collected at 15 min intervals at 2 μl·min^–1. The [mean (SE)] increase in release of striatal DA attained significance after 30 min of occlusion of MCA [170 (24)%], and continued to increase [268 (26)% at 45 min] reaching a peak level at 60 min [672 (59)%] before returning to the baseline level during the late reperfusion phase. There was no significant change in the level of DA in HBO treated rats during the period of ischemia. A significant reduction in edema and neuronal shrinkage were observed by histological examination in HBO treated rats when compared to the control rats. The results showed that HBO, when administered during ischemia, offered significant neuroprotection in our experimental model of transient focal cerebral ischemia in the rat. The mechanism seems to imply, at least in part, a reduced level of DA. Electronic Publication     

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.     

ADAMTS-9 expression is up-regulated following transient middle cerebral artery occlusion (tMCAo) in the rat

The ADAMTS enzymes (adisintegrin and metalloproteinase with thrombospondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24 h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24 h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.     

恒河猴大脑中动脉M1段栓塞模型的麻醉新方法

目的为成功构建恒河猴M1段栓塞模型提供简捷、迅速、安全的麻醉方法。方法雄性成年恒河猴25只,年龄7~9岁,体重7~11 kg,由中国人民解放军军事医学科学院实验动物中心提供。以0.1 ml/kg氯胺酮及速眠新的混合液肌肉注射,待动物入睡后气管插管(ID:4.5-5.5#),妥善固定后送入介入中心。入室后连接监护、建立静脉通路并导尿。术中用丙泊酚2~4 mg/(kg·h)持续泵入维持麻醉,呼吸机控制呼吸,并根据动物的生命体征和肢体活动情况调整麻醉深度,必要时追加上述氯胺酮与速眠新的混合液,并根据手术的需要调整心率、血压、体温等。溶栓前后行脑MRI造影,造影前适时停止麻醉药物输注,确保动物在造影室已恢复自主呼吸。术中监测实验动物的心率、体温、血压及血氧饱和度,并于股动脉穿刺及溶栓后抽取动脉血行血气分析。结果所有动物均按预计方案实施完成,术中未发生动物躁动、呼吸抑制、心律失常等严重并发症。实验结束停药后,实验动物很快清醒并送回动物实验中心,进行后续处理。其中18只实验动物存活24 h以上,7只死于溶栓后严重的脑出血和脑梗死。结论以氯胺酮及速眠新的混合液进行麻醉诱导行气管插管,丙泊酚静脉维持全身麻醉可以为此类较复杂的介入及MRI实验的顺利完成提供一种安全、实用的麻醉方法。     

Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat

Endothelin-1 (1 nmol) was applied to the exposed left middle cerebral artery (MCA) in anaesthetised adult male Sprague-Dawley rats. Local cerebral blood flow (1CBF), using [14C]iodoantipyrine and quantitative autoradiography, was measured in 27 anatomically defined structures, 10 min after topical application of endothelin-1. In those areas supplied by the MCA, 1CBF was markedly reduced beyond the threshold for ischaemic damage (e.g. dorsolateral caudate nucleus reduced from 131 +/- 3 to 29 +/- 25 ml.100 g-1.min-1, sensorimotor cortex from 109 +/- 5 to 31 +/- 21 ml.100 g-1.min-1). Distant areas were not affected.     

Real-time measurement of glutamate release from the ischemic penumbra of the rat cerebral cortex using a focal middle cerebral artery occlusion model

Following permanent middle cerebral artery occlusion, extracellular penumbral glutamate levels, measured by a real-time glutamate electrode, increased in two different patterns. In 7/11 rats, glutamate increased from baseline levels of 19+/-4 (mean+/-SEM) to 208+/-29 microM and then declined towards baseline levels. Blood flow in the penumbral area declined to 30% of pre-ischemic levels with recovery to 60 and 70% of baseline values by 3 and 6 h, respectively. Four of 11 rats in the study also exhibited late peaks of glutamate release (120+/-40 microM ) 2 h after the onset of ischemia. There were no changes in the EEG recordings or cerebral blood flow during these late glutamate peaks.     

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.     

急性缺血性脑梗死大鼠皮层神经蛋白聚糖的表达

目的： 研究神经蛋白聚糖（neurocan）在急性缺血性脑梗死大鼠皮层内的表达。方法: 用电凝法制成大鼠右侧大脑中动脉梗死模型，在1周、2周和4周不同时期，采用免疫组化、实时荧光定量RT-PCR和Western blotting技术检测梗死灶同侧和对侧的neurocan表达。结果: Neurocan和GFAP的双标结果提示neurocan阳性区域要大于GFAP阳性区域，neurocan阳性染色主要分布于胶质细胞及细胞间隙，4周组较1周组与2周组neurocan mRNA表达显著降低，差异显著（P<0.01），neurocan表达显著降低，差异显著（P<0.05），各组的neurocan C-末端糖蛋白无显著差异（P>0.05）。结论: Neurocan主要分布于脑梗死周围皮质的细胞间隙。脑梗死后neurocan表达升高，第1-2周是高峰期，4周下降至低水平，提示neurocan参与脑梗死后病理生理过程。     

Spermine reduces infarction and neurological deficit following a rat model of middle cerebral artery occlusion: a magnetic resonance imaging study

The role of nitric oxide (NO) in post-ischemic cerebral infarction has been extensively examined, but few studies have investigated its role on the neurological deficit. In the present study, we investigated the effect of spermine on the temporal evolution of infarct volume, NO production and neurological deficit using magnetic resonance imaging in a model of permanent focal cerebral ischemia in rats. Spermine given at 10 mg/kg 2 h after ischemia reduced the infarct volume by 40% and abolished brain NO production and improved the neurological score 24 h, 48 h and 72 h after ischemia. Spermine also reduced the neurological deficit as evaluated by rotamex, grip strength and neurological severity score tests.     

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.     

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.     

Xenon preconditioning confers neuroprotection regardless of gender in a mouse model of transient middle cerebral artery occlusion

Xenon preconditioning induces tolerance to the consequences of an injurious stimulus such as cerebral ischaemia. There have been surprisingly few studies investigating gender difference in the efficacy of pharmacological preconditioning, despite the known ability of oestradiol to exert neuroprotectant activity. We explored this paradigm using a mouse model of transient middle cerebral artery occlusion. C57BL/6 mice both male and female received either 2 h of 70% xenon (preconditioning) or 70% nitrogen (control) balanced with oxygen. Twenty-four hours later animals underwent 1 h of middle cerebral artery occlusion and then allowed to recover. After a further 24 h, functional neurological outcome and cerebral infarct size were evaluated. Western blotting was used to detect activity of signalling pathways involving hypoxia-inducible factor (HIF)-1α and phospho-Akt for the preconditioning effect. Both xenon preconditioned male and females showed improved functional outcome on focal deficit scales (P<0.05). Cerebral infarct volumes were significantly reduced in both xenon treated male and females (P<0.01). There was no significant difference between the male and female cohorts. HIF-1α and phospho-Akt were quantitatively upregulated in both sexes. Our data suggested that xenon preconditioning improved histological and neurological functional outcome in both gender in a stroke model of mice.