Suramin reduces infarct volume in a model of focal brain ischemia in rats

Extracellular adenosine 5'-triphosphate (ATP) provides excitatory transmission in the central nervous system. Stimulation by ATP of ionotropic ligand-gated ion channel purinoceptors (P_2X) leads to increased intracellular calcium levels, and activation of P_2X receptors may be involved in the process of excitotoxic neuronal injury caused by stroke. Suramin, as an agent that is known to block P_2X receptors at a specific concentration, was assessed for its neuroprotective potential in a model of experimental stroke in the rat. We propose that the effectiveness of suramin is limited to those concentrations where it is an effective P_2X receptor antagonist. Focal brain ischemia was produced by unilateral occlusion and transection of the middle cerebral artery (MCAT) and bilateral occlusion of the common carotid arteries (CCA). Thirty-four male Sprague-Dawley rats were randomly separated into five groups. Changes in regional cerebral blood flow in the ischemic region were verified by laser Doppler flowmetry. All rats received, over a period of 30 min before MCAT, a dose of suramin at 0 (saline), 25, 50, 100, or 250 mg/kg intravenously in a volume of 1 ml using an infusion pump. Six hours after ischemia onset, evaluation of the neurologic status and cerebral blood flow was followed by morphometric analysis of infarct volume. During the surgical procedure mean arterial pressure, blood gases (PaCO₂, PaO₂), and pH were monitored. A dose-dependent decrease in infarct volume (slope –0.049, SE 0.009, P<0.001) was observed in groups treated over the dose range of 0 to 100 mg/kg (r ²=0.55). Suramin at a dose of 100 mg/kg significantly decreased infarct volume (n=9, P<0.001) and edema volume (P=0.003). The neuroprotective effect of suramin at a dose of 100 mg/kg was supported by an improved neurologic score in this group (median 0) compared with a median of 3 in control animals (P=0.02). These findings indicate that suramin at 100 mg/kg is an effective pre-treatment neuroprotective agent. As the estimated brain concentration of 10 μM (from McNally et al., 2000, Life Sci 67:1847–1857) is the IC₅₀ for suramin-mediated P_2X antagonism, these results suggest that interference with the ATP excitatory system could provide neuroprotection from brain ischemia. Electronic Publication     

Use of 2,3,5-triphenyltetrazolium chloride-stained brain tissues for immunofluorescence analyses after focal cerebral ischemia in rats

The middle cerebral artery occlusion (MCAO) model in rodents has been widely used as model for studying brain ischemic stroke. TTC (2,3,5-triphenyltetrazolium chloride) staining in fresh tissues is used to evaluate the size of the infarct in MCAO model, and TTC-stained brain tissues are considered to be possible to bring a damage to the anatomical structure of neuronal cells and unsuitable for immunofluorescence analyses of cytology, and discarded after evaluation of infarct volume. Another group of models with in vivo fixation was required to the pathological or histological analyses of the infarct brains, which lead to double the numbers of animals in researches. However, some evidences indicate that if we properly optimized staining protocol, TTC-stained brain tissues might be suitable for cytological analyses. In this work, we have optimized the immunofluorescent staining methods of TTC-stained brain slices, and found that TTC-stained brain tissues are suitable for quantitative and qualitative analyses of microglia, astrocytes and neuroblasts, the morphology of theses cell were nearly identical to the in-vivo fixed models. Our optimized-protocol provide two advantages over traditional methods one of them is providing the precise the infarct region, which reduces the differences within groups, the other one is decreasing the total number of animals in research dramatically.     

大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化

目的研究大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化。方法用线栓法制作SD大鼠大脑中动脉阻塞的脑缺血/再灌注动物模型,用免疫细胞化学法结合图像分析技术,检测缺血脑区缓激肽样免疫反应阳性物的平均光密度(A)值作为缓激肽的相对含量,比较局灶性脑缺血3h组、假手术对照组、正常对照组和再灌注30min、2h、4h、16h组缓激肽的相对含量。结果缺血脑区缓激肽样免疫反应阳性物的A值于脑缺血3h/再灌注2h后明显增高(P<0.05),随后下降至增高前水平。结论大鼠局灶性脑缺血/再灌注后,缺血脑区缓激肽含量于再灌注2h后明显增高,其可能在脑缺血后脑水肿的发生中起着重要作用。     

Thalidomide protects against ischemic neuronal damage induced by focal cerebral ischemia in mice

We aimed to examine whether thalidomide might inhibit the neuronal damage resulting from focal cerebral ischemia, and if so to explore the neuroprotective mechanism. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) in mice, and thalidomide was intraperitoneally administered a total of three times (at 10 min before, just before, and 1 h after MCAO). Thalidomide significantly reduced (a) the infarct area and volume at 24 and 72 h after MCAO and (b) the neurological score at 72 h after MCAO. Brains were also histochemically assessed for apoptosis and lipid peroxidation using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and an antibody recognizing 8-hydroxy-2′-deoxyguanosine (8-OHdG), respectively. Thalidomide reduced both the number of TUNEL-positive cells and the oxidative damage. However, post-treatment of thalidomide [20 mg/kg, three times (at just after, 1 h after, 3 h after MCAO)] did not reduce the infarct volume. In an in vitro study, we examined the effects of thalidomide on lipid peroxidation in mouse brain homogenates and on the production of various radical species. Thalidomide inhibited both the lipid peroxidation and the production of H₂O₂ and O₂ · ⁻ (but not HO⁻) radicals. We also measured the brain concentration of TNF-α by ELISA. The TNF-α level in the brain was significantly increased at 9–24 h after MCAO. However, thalidomide did not reduce the elevated TNF-α level at either 12 or 24 h after MCAO. These findings indicate that thalidomide has neuroprotective effects against ischemic neuronal damage in mice, and that an inhibitory action of thalidomide against oxidative stress may be partly responsible for these neuroprotective effects.     

Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain

Expression of cyclooxygenase-2 (cox-2) mRNA and inducible heat-shock protein-70 (hsp-70) mRNA was studied with in situ hybridization techniques at 30 min and 4 h following 1 h transient middle cerebral artery (MCA) occlusion in the rat brain. In addition, immunoreactivity for cox-2 was studied after 8 h of reperfusion. Induction of hsp-70 and cox-2 mRNA was found in the brain side ipsilateral to MCA occlusion. Hsp-70 mRNA was induced in the parietal cortex and striatum within the territory of the occluded MCA. Induction of cox-2 mRNA was particularly seen in cortical layer II in the brain side ipsilateral to MCA occlusion. At 30 min of reperfusion, areas showing cox-2 mRNA induction included the cingulate and frontal cortices located perifocally to the areas showing hsp-70 mRNA induction, and the piriform cortex. At 4 h of reperfusion, induction of cox-2 mRNA was seen within the parietal cortex. At 8 h of reperfusion, immunoreactivity for cox-2 was mainly seen in the ipsilateral cortex. These results demonstrate that transient focal ischemia induces the expression of cox-2 mRNA and protein in discrete areas of the rat brain during reperfusion, which might lead to local increases of arachidonic acid metabolism     

Preconditioning with +Gz acceleration (head-to-foot inertial load) produces neuroprotection against transient focal cerebral ischemia in rats

Ischemic preconditioning is considered to be the most robust endogenous neuroprotectant. However, the conventional ischemic preconditioning protocol is both invasive and impractical to apply. The aim of the present study was to evaluate whether preconditioning with +Gz centrifuge acceleration (head-to-foot inertial load) which could induce brief episodes of sublethal ischemia in brain had neuroprotection against focal cerebral ischemic injury. A total of 85 male Sprague-Dawley rats were randomly assigned to five groups (n = 17 in each). The 2 Gz, 4 Gz, 6 Gz and 8 Gz groups were subjected to 3 min exposures at +2 Gz, +4 Gz, +6 Gz and +8 Gz, respectively for consecutive three times in animal centrifuge, with a 30-min rest period between each centrifuge run. The control group had no exposure to +Gz acceleration. Twenty-four hours after the last pretreatment, 12 rats in each groups were subjected to focal cerebral ischemia for 120 min and the other five rats in each group were sacrificed to measure the expression of heat shock protein 70(HSP70) in hippocampus by Western blot analysis. The results indicated that the 6 Gz and 8 Gz groups showed smaller infarct volume and lower neurologic deficit scores than the control group. The expression of HSP70 was significantly increased in 6 Gz and 8 Gz groups than those in the control group. Therefore, preconditioning with +Gz acceleration produced delayed neuroprotection against focal cerebral ischemia and that the neuroprotection may be related to the induction of HSP70.     

Dynamic changes in cortical NADH fluorescence in rat focal ischemia: evaluation of the effects of hypothermia on propagation of peri-infarct depolarization by temporal and spatial analysis

Suppression of peri-infarct depolarizations (PIDs) is one of the major mechanisms of hypothermic protection against transient focal cerebral ischemia. Previous studies have shown the lack of hypothermic protection against permanent focal ischemia. We hypothesized the lack of hypothermic protection was due to the poor efficacy in suppression of PIDs. To examine the hypothesis, we elucidated the effects of hypothermia on the manner of propagation of PIDs with temporal and spatial resolutions using NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by illuminating the parietal-temporal cortex with ultraviolet light. Spontaneously hypertensive rats (n=14) were subjected to permanent focal ischemia by occlusion of the middle cerebral and left common carotid arteries. 2-h hypothermia (30 degrees C) was initiated before ischemia. Although hypothermia delayed the appearance of PIDs, it did not suppress their appearance. Furthermore, 54% of the PIDs enlarged the high-intensity area of NADH fluorescence in the hypothermia group, similar to the normothermia group (53%). The high-intensity area of NADH fluorescence widened by each PID was larger in the hypothermia group than in the normothermia group. These findings suggest that PIDs even in hypothermia are one of the major factors causing growth of infarction, emphasizing the importance of therapy that targets suppression of PIDs even during hypothermia.     

Monitoring of neurological deficit and disturbances in higher nervous activity in rats with focal cerebral ischemia

Neurological, locomotor, and behavioral changes in 20 Wistar rats with permanent proximal occlusion of the middle cerebral artery and 18 control sham-operated animals were monitored for 2 months at 10-day intervals. Neurological deficit was maximum immediately after occlusion (3.0±0.6 points), then progressively decreased, but did not completely disappear (0.70±0.06 points on day 60). In control animals neurological status returned to normal on days 10-15. The degree of ketamine-induced rotational asymmetry was 4.0±0.7 rpm over 40 days after surgery and decreased to 2.5±0.6 rpm on day 60. In control rats this parameter only transiently increased to 1.00±0.03 rpm (t=2.5, p<0.05). The time of stay on a rotarod and the latency of passive avoidance in rats with focal cerebral ischemia were lower than in control animals throughout the experiment. The results of complex tests can be used in the experimental search for new drugs for the therapy and rehabilitation of stroke patients.     

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction,cerebral edema and increased antioxidation in rat models of focal cerebral ischemia

Thrombolytic agent is increasingly being used in treating acute ischemic stroke. A novel protease with strong thrombolytic activity, Neanthes japonica (Iznka) fibrinolytic enzyme (NJF) discovered in our laboratory has been reported with characteristics of direct hydrolyzing fibrin and fibrinogen. The neuroprotective effect of NJF and urokinase (UK) was tested in rat models of middle cerebral artery occlusion (MCAO). The model was successfully produced by introducing an intraluminal suture into the left middle cerebral artery (MCA). NJF (0.25, 0.5, 1 mg/kg) was injected intravenously 1 h after the onset of reperfusion. Compared with vehicle group, MCAO animals treated with NJF showed dose dependent reduction in cerebral infarction with improved neurological outcome. Meanwhile, ischemia induced cerebral edema was reduced in a dose dependent manner. Treatment with NJF at 0.5 mg/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema. Biomedical assay showed that NJF treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activities in brain tissue. These results suggest that NJF posses neuroprotective potential in rat MCAO and reperfusion model. Neuroprotection shown by NJF may be attributed to inhibition of lipid peroxidation, increase in endogenous antioxidant defense enzymes.     

虫草素对大脑中动脉局灶性脑缺血模型大鼠氧化应激和脑损伤的影响

目的　研究虫草素对大脑中动脉局灶性脑缺血模型大鼠氧化应激指标和脑组织Caspase-3和p53表达的影响。 方法　首先，给药组大鼠每天分别腹腔注射虫草素5、10、20 mg/kg，连续10 d；然后，采用改良Zea Longa线栓法制备大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）模型；造模24 h后，盲法进行神经功能评分，称重法检测脑含水量，HE染色观察脑组织病理损伤，Tunnel染色检测脑细胞凋亡，RT-PCR检测Bcl-2、Bax、Caspase-3和p53 mRNA表达，Western blotting检测Bcl-2、Bax、Caspase-3和p53蛋白表达，试剂盒检测SOD，MDA，GSH水平。 结果　给药组与MCAO组相比，神经功能评分显著降低，脑含水量显著减少，细胞损伤减轻，细胞凋亡率显著减少，Bax mRNA及蛋白表达显著下调，Bcl-2 mRNA及蛋白表达显著上调，MDA含量显著下降，SOD和GSH含量显著上升，Caspase-3和p53 mRNA及蛋白表达显著下调，且这些效果随着虫草素给药量的增加更加显著。 结论　虫草素能够缓解大脑中动脉局灶性脑缺血引起的神经功能障碍和降低脑缺血引起的脑含水量升高，并能抑制大脑中动脉局灶性脑缺血模型大鼠氧化应激和细胞凋亡，从而减缓大脑中动脉局灶性脑缺血造成的损伤。     

磁共振成像及氧化铁粒子标记在干细胞治疗脑缺血大鼠中的应用

目的 探讨磁共振成像及氧化铁粒子标记在干细胞治疗脑卒中模型中的应用价值.方法 线栓法建立9只大鼠大脑中动脉缺血模型,进行神经行为学评分、磁共振影像学和病理学评价,并对上述3项指标进行相关性分析.用超顺磁性氧化铁体外标记骨髓间充质干细胞,并另筛选18只脑缺血大鼠,分别接受缺血对侧皮层(n.6)、缺血同侧纹状体(n=6)的标记干细胞移植,并设对照组(n=6).分别在脑缺血后1 d、细胞移植后第1天、第7天及第14天进行磁共振扫描,观察各时间点的标记干细胞显示情况,并对各组之间梗死体积的变化进行统计学分析.结果 神经行为学评分、磁共振影像学和病理学评价对大鼠脑缺血模型评价的相关性好.磁共振各序列均可显示局部移植的标记干细胞,梯度回波序列最敏感,T2像空间分辨力高.磁共振成像可显示局部移植标记干细胞随时间推移而产生的分布变化.干细胞治疗各组之间脑梗死体积变化无明显差异.结论 磁共振成像是大鼠脑缺血模型评价的理想工具,配合氧化铁粒子标记干细胞可活体状态下了解干细胞移植后的分布变化.     

Neuroprotective properties of prostaglandin I2 IP receptor in focal cerebral ischemia

We and others have identified that inhibition of cyclooxygenase might not be the optimal approach to limiting brain damage after stroke. Now we are investigating the unique properties of the various prostaglandin receptors to determine whether blocking those that mediate toxicity or stimulating those that reduce toxicity will improve neurological outcomes. Here, we determined the respective contribution of the prostaglandin I₂ (PGI₂) receptor in transient middle cerebral artery (MCA) occlusion (tMCAO) and permanent MCAO (pMCAO) preclinical stroke models by using male wildtype (WT) and IP receptor knockout (IP^−/−) C57Bl/6 mice. In addition, we investigated the putative preventive and therapeutic effects of the IP receptor agonist beraprost. The infarct volumes and neurological deficit scores (NDS) were significantly greater in IP^−/− than in WT mice after both tMCAO and pMCAO. Interestingly, beraprost pretreatment (50 or 100 μg/kg p.o.) 30 min before tMCAO and post-treatment (100 μg/kg p.o.) at 2 or 4.5 h of reperfusion significantly reduced the neurological deficit score and infarct volume in WT mice. Post-treatment with beraprost (100 μg/kg p.o.) 4.5 h after pMCAO also significantly decreased neurological deficits and infarct volume in WT mice. Together, these novel findings suggest for the first time that PGI₂ IP receptor activation can attenuate anatomical and functional damage following ischemic stroke.     

DWI检测超急性期兔大脑中动脉梗死模型神经血管单元损伤的初步研究

目的　评价磁共振弥散加权成像（diffused weighted imaging, DWI）对兔脑超急性期脑梗死模型中神经血管单元（neurovascular unit , NVU）损伤的诊断价值。 方法　15只健康新西兰大白兔,随机分为对照组（5只）和实验组（10只）。 其中实验组用微导丝超选择性栓塞法制作兔大脑中动脉阻塞(middle cerebral artery occlusion, MCAO)模型,并根据存活时间分为0.5 h和4 h组。所有动物行磁共振DWI扫描和T2-flair扫描并分析表观弥散系数（apparent diffusion coefficient,ADC）和相对表观弥散系数（relative apparent diffusion coefficient,rADC）,之后立即取全脑固定,切片进行尼氏染色、GFAP（glial fibrillary acidic protein, 星形胶质细胞标记物）和CD31（血管内皮标记物）染色。 结果　和对照组相比, 0.5 h组缺血区DWI成像见高信号,ADC成像见低信号,T2-Flair成像未见异常信号,尼氏染色未见异常,GFAP染色表达增加;4h组缺血区DWI成像见高信号,ADC成像见低信号,二者成像范围和强度较0.5h组扩大增强,T2-Flair成像见高信号,尼氏染色见尼氏体数目显著减少,GFAP染色表达较0.5h组显著增加。与0.5h组比,4h组缺血区的ADC值及rADC值均有显著差异（P<0.01）。实验组rADC值与GFAP荧光强度值呈负直线相关关系（r=－0.672,P<0.001）。 结论　DWI对超急性期兔脑梗死可以及时准确的进行诊断,对应时间点梗死模型的NVU损伤特点符合影像学特征。     

Rapamycin alleviates brain edema after focal cerebral ischemia reperfusion in rats

Brain edema is a major consequence of cerebral ischemia reperfusion. However, few effective therapeutic options are available for retarding the brain edema progression after cerebral ischemia. Recently, rapamycin has been shown to produce neuroprotective effects in rats after cerebral ischemia reperfusion. Whether rapamycin could alleviate this brain edema injury is still unclear. In this study, the rat stroke model was induced by a 1-h left transient middle cerebral artery occlusion using an intraluminal filament, followed by 48?h of reperfusion. The effects of rapamycin (250?μg/kg body weight, intraperitoneal; i.p.) on brain edema progression were evaluated. The results showed that rapamycin treatment significantly reduced the infarct volume, the water content of the brain tissue and the Evans blue extravasation through the blood–brain barrier (BBB). Rapamycin treatment could improve histological appearance of the brain tissue, increased the capillary lumen space and maintain the integrity of BBB. Rapamycin also inhibited matrix metalloproteinase 9 (MMP9) and aquaporin 4 (AQP4) expression. These data imply that rapamycin could improve brain edema progression after reperfusion injury through maintaining BBB integrity and inhibiting MMP9 and AQP4 expression. The data of this study provide a new possible approach for improving brain edema after cerebral ischemia reperfusion by administration of rapamycin.     

缺血后适应对大鼠局灶性脑缺血再灌注后谷氨酸的影响

目的:研究缺血后适应对大鼠局灶性脑缺血再灌注后谷氨酸(Glu)浓度变化的影响。方法:建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型,将36只雄性SD大鼠随机分为假手术(Sham)组、脑缺血再灌注(I/R)组和缺血后适应(I Postcond)组,每组12只。应用高效液相色谱检测缺血脑组织匀浆Glu浓度。结果:局灶脑缺血再灌注6 h后,I Postcond组Glu浓度相较I/R组明显降低(P<0.01),局灶脑缺血再灌注24 h后,I Postcond组Glu浓度较I/R组低,但两者差异亦无显著性(P>0.05)。结论:本研究表明,I Postcond能够减轻MCAO大鼠模型中I/R损伤。细胞间隙Glu清除的加速可能是其重要保护机制之一。     

线栓法大鼠局灶性脑缺血模型制作方法的改进及评价

目的:改进线栓法大鼠大脑中动脉梗死局灶性脑缺血模型的制作方法,并运用多个指标评价该模型的使用价值。方法:采用体质量300～350 g的雄性Wistar大鼠,分成模型组、模型对照组和假手术组。所有动物均采用水合氯醛麻醉,模型组在手术过程中用丝线结扎翼腭动脉,从颈内动脉将直径0.25 mm的渔线插入大脑中动脉起始处,并恢复颈总-颈外动脉血流。结果:模型组成功率为80%,神经缺损症状出现率为100%,24 h存活率为75%。模型对照组成功率为73.9%,神经缺损症状出现率为88.2%,24 h存活率为80%。与假手术组比,模型组及模型对照组大鼠神经缺损评分、脑梗死体积、脑指数、脑含水量及体质量下降率均明显升高。结论:选用体质量均衡的大鼠,采用丝线结扎翼腭动脉,同时不结扎颈总动脉,从而保持颈总-颈内动脉血流畅通,能提高模型成功率和稳定性。     

大鼠局灶性脑缺血后远隔区域脑损伤及药物干预的研究

目的　观察大鼠局灶性脑缺血后急性期两侧额叶、丘脑、小脑、下丘脑局部脑血流量 (rCBF)和细胞形态学的变化 ;评估川芎嗪对上述变化的干预作用。方法　采用线栓法建立大鼠一侧大脑中动脉闭塞 (middlecerebralarteryocclusion ,MCAO)模型 ;用氢清除法测定两侧额叶、丘脑、小脑、下丘脑在MCAO后 1、3、6和 2 4h的rCBF ;用HE染色和TUNEL法观察两侧额叶、丘脑、小脑MCAO后 6h、2 4h、1周细胞形态学变化。结果　正常对照组各相应部位rCBF无显著差异 (P >0 0 5 ) ;一侧MCAO后 ,两侧额叶皮层、丘脑、下丘脑、对侧小脑半球rCBF下降 ,MCAO后 1h达最低水平 (P <0 0 5或 P <0 0 1) ,随时间延长 ,rCBF逐渐增加 ,除对侧小脑、下丘脑外 ,其余部位rCBF至MCAO后 2 4h基本恢复正常 ;而川芎嗪可明显抑制上述rCBF的下降程度 (P <0 0 5 )。同侧额叶、丘脑在MCAO后 6h开始出现TUNEL阳性细胞 ,2 4h后明显增加 ,至 1周阳性细胞减少 (P <0 0 5 ) ,而川芎嗪可明显抑制上述部位凋亡细胞的产生。结论　局灶性脑缺血后可引起远隔区域有关脑组织rCBF下降 ,并进一步引发细胞凋亡。中药川芎嗪可明显抑制上述变化     

Calcium metabolism of focal and penumbral tissues in rats subjected to transient middle cerebral artery occlusion

The present experiments were undertaken to define changes in tissue calcium metabolism in focal and perifocal (“penumbral”) tissues following 2 h of transient middle cerebral artery occlusion (MCAO) in rats, induced with an intraluminal filament occlusion technique. The extracellular calcium concentration ([Ca²⁺]_e) was measured with ion-selective microelectrodes in neocortical focus and penumbra. For measurement of total tissue calcium content, tissue samples from these areas were collected and analyzed with atomic absorption spectrometry. During MCAO, [Ca²⁺]_e in a neocortical focal area fell from a normal value of about 1.2 mM to values around 0.1 mM, suggesting translocation of virtually all extracellular calcium to intracellular fluids. Recirculation was accompanied by re-extrusion of calcium within 5–7 min; however, [Ca²⁺]_e never returned to normal but stabilized at about 50% of the control value for the first 6 h, and decreased further after 24 h. In penumbral areas, [Ca²⁺]_e showed the expected transient decreases associated with spreading depression-like (or ischemic) depolarization waves. Recirculation was followed by return of [Ca²⁺]_e towards normal values. In the focus, water content increased from about 79% to about 80.4% at the end of the 2-h period of ischemia. After 2 h and 4 h of recirculation, the edema was aggravated (mean values 81.9% and 81.2%, respectively). After 6 h and 24 h, the edema was more pronounced (83.6% and 83.8%, respectively). In the penumbra, no significant edema was observed until 6 h and 24 h of recirculation. The total tissue calcium content in the focus (expressed by unit dry weight) increased at the end of the ischemia period demonstrating calcium translocation from blood to tissue. After 6 h and 24 h, the content increased two- to threefold, compared with control. Changes in the penumbra were qualitatively similar but less pronounced, and a significant increase was not observed until after 6 h of recirculation. The results suggest that 2 h of MCAO leads to a profound perturbation of cell calcium metabolism. In focal areas, cells fail to extrude the calcium that is gradually accumulated during reperfusion and show massive calcium overload after the first 4–6 h of recirculation. Penumbral tissues show a similar increase in calcium concentration after 6 h of recirculation. Received: 2 June 1997 / Accepted: 26 November 1997     

短暂性脑缺血再灌注对大鼠脑PGRN表达的影响

目的检测短暂性脑缺血再灌注早期,大鼠的额颞叶皮质、海马及纹状体中颗粒蛋白前体(progranulin,PGRN)的表达变化,为进一步探讨PGRN对脑缺血的治疗提供实验基础。方法 SPF级成年SD大鼠随机分为2组,假手术组(shamoperation group)和实验组(MCAO group)。用线栓法制作右侧大脑中动脉阻塞(MCAO)1 h,然后再灌注30 min、2 h、12 h、24 h模型,以缺血侧为实验组(ipsilateral group),其对侧为对照组(contralateral group)。假手术组为处理对照。用TTC染色法观察缺血梗死体积。用免疫荧光组织化学法和Western-blot分别检测PGRN的细胞定位及其表达变化。结果 TTC染色表明,脑缺血区域呈现白色,而对照无缺血区为红色。免疫组织化学结果提示,PGRN在神经元中大量表达,在小胶质细胞中有少量表达,而在星形胶质细胞中几乎无表达。Western blot结果提示,与对照组相比,缺血再灌注后2 h大鼠脑额颞叶皮质的缺血侧与非缺血侧PGRN含量均显著上调。在海马区缺血再灌注能瞬间降低PGRN水平,随着时间延长PGRN表达水平在24 h逐步恢复到正常水平。与皮质和海马相比,纹状体在短时间再灌注后PGRN具有较高的表达,特别是在缺血再灌注24 h后。结论短暂性脑缺血再灌注能显著影响PGRN在大鼠脑缺血区及半暗带区的水平,提示PGRN可能参与缺血后脑的调节。