Three steps of neural stem cells development in gerbil dentate gyrus after transient ischemia.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate detailed relations between these three steps after ischemia, the authors evaluated the three steps in the adult gerbil dentate gyrus (DG) after 5 minutes of transient global ischemia using bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), and neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) as markers for proliferation, migration, and differentiation, respectively. Bromodeoxyuridine-labeled cells increased approximately sevenfold, and PSA-NCAM-positive cells increased approximately threefold in the subgranular zone (SGZ) with a peak 10 days after ischemia. Bromodeoxyuridine-labeled cells with PSA-NCAM expression were first detected both in the SGZ and the granule cell layer (GCL) 20 days after ischemia and gradually decreased after that, whereas BrdU-labeled cells with NeuN gradually increased in the GCL until 60 days after ischemia. A few BrdU-labeled cells with GFAP expression were detected in DG after ischemia; no PSA-NCAM-positive cells with GFAP expression were detected, but the radial processes of glial cells were partly in contact with PSA-NCAM-positive cell bodies and dendrites. These results suggest that neural stem cell proliferation begins at the SGZ, and that the cells then migrate into the GCL and differentiate mainly into neuronal cells. The majority of these three steps finished in 2 months after transient global ischemia.     

Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate their detailed relations after ischemia, the three steps were comprehensively evaluated, in the subventricular zone (SVZ) through the rostral migratory stream (RMS) to the olfactory bulb (OB), in adult gerbil brain after 5 minutes of transient forebrain ischemia. Bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP) were used as markers for proliferation, migration, and differentiation, respectively. The number of BrdU-labeled cells that coexpressed PSA-NCAM and the size of PSA-NCAM-positive cell colony increased in the SVZ with a peak at 10 d after transient ischemia. In the RMS, the number of BrdU-labeled cells that coexpressed PSA-NCAM increased, with a delayed peak at 30 d, when the size of RMS itself became larger and the number of surrounding GFAP-positive cells increased. In the OB, BrdU + NeuN double positive cells were detected at 30 and 60 d. NeuN staining and terminal deoxynucleotidyl dUTP nick-end labeling staining showed no neuronal cell loss around the SVZ, and in the RMS and the OB after transient ischemia. These findings indicate that transient forebrain ischemia enhances neural stem cell proliferation in the SVZ without evident neuronal cell loss, and has potential neuronal precursor migration with activation of GFAP-positive cells through the RMS to the OB.     

匹罗卡品诱导癫痫大鼠内源性神经干细胞的可塑性改变

目的探讨匹罗卡品诱导癫痫模型的内源性神经干细胞的增殖、迁移,从而研究癫痫发生后的神经发生及可塑性改变.方法氯化锂和匹罗卡品联合诱导大鼠癫痫模型,分为对照组、癫痫发作后3、7、14、28 d组,用免疫组织化学方法动态检测大鼠海马多唾液酸神经细胞粘附分子(polysiolylated neural cell adhesion molecule,PSA-NCAM)及PSA-NCAM/5-溴脱氧尿苷嘧啶(bromodeoxyuridine,BrdU)的表达.结果与对照组比较,海马齿状回(dentate gyrus,DG)PSA-NCAM及PSA-NCAM/BrdU的阳性细胞在癫痫后3 d开始增加(P＜0.05),14 d达到高峰(P＜0.05),28 d后开始下降,但仍高于对照组(P＜0.05).结论癫痫发生后出现神经干细胞的增殖、迁移,也即出现了神经发生和可塑性改变.     

鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性

目的研究成年大鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性。方法雄性Wistar大鼠共90只,分对照组(n=10)、脑梗死后1d组(n=16)、脑梗死后3d组(n=16)、脑梗死后7d组(n=16)、脑梗死后14d组(n=16)、脑梗死后28d组(n=16)。用免疫组织化学方法动态检测BrdU、GFAP、NeuN、PSA-NCAM的表达。BrdU确定神经干细胞的增殖,GFAP、NeuN确定神经干细胞的分化,PSA-NCAM确定神经干细胞的可塑性。结果与对照组相比,大鼠海马BrdU 细胞数在脑梗死后1d组开始增加,7d组达到高峰,28d组接近正常水平;BrdU /GFAP 细胞数在脑梗死前后无明显变化;BrdU /NeuN 细胞数在脑梗死后14d组开始增加,28d组最多;BrdU /PSA-NCAM 细胞数在脑梗死后7d组开始增加,14d组达到高峰,28d组开始下降,但仍高于对照组,大约占同期BrdU阳性细胞数60%。结论大鼠脑梗死激活自体神经干细胞原位增殖,大多数增殖的神经干细胞分化成神经元并且具有可塑性。     

Changes of localization of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in rat hippocampus with exposure to repeated kindled seizures

Highly polysialylated neural cell adhesion molecules (PSA-NCAMs) are involved in migration of neural stem cells as well as neural plasticity. Immunoreactive PSA-NCAM expression was examined in rat with repeated exposure to amygdaloid kindled generalized seizures (GS). The number of PSA-NCAM positive cells in bilateral dentate gyrus (DG) increased significantly at GS. Although total positive cell number was not significantly different between 3 times GS (3 GS) and 30 times GS (30 GS) groups, a greater number of positive cells was located in the outer granule cell layer (GCL), and the immunopositive dendrite greatly extended to the molecular layer in the 30 GS group. These observations indicate that increased migration of newly generated cells as well as plastic change of originally-existed neural cells may occur in response to the recurrent GS, which may contribute to abnormal reconstruction of synaptic network in hippocampus and epileptogenisity in kindling.     

大鼠脑缺血再灌注诱导自体神经干细胞原位增殖的研究

目的研究缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法参照Pulsinelli-Brierley法制作短暂性全脑缺血动物模型,全脑缺血10min后再灌注,采用SABC免疫组化染色显示5'-溴脱氧尿嘧啶(BrdU)阳性细胞和神经巢蛋白(Nestin)阳性细胞,光镜下观察并统计分析脑缺血损伤后内源性神经干细胞增殖、迁移的变化过程。结果脑缺血再灌流24h后,海马、齿状回和室管膜下区的BrdU阳性细胞和Nestin阳性细胞增多,7～10d达到高峰,术后20d仍有表达;在室管膜下区,BrdU阳性细胞和Nestin阳性细胞有向皮质、海马迁移的现象。结论①成年大鼠全脑缺血后7～10d,内源性神经干细胞的增殖达到高峰。②增殖的内源性神经干细胞存在由增殖区向靶区迁移的现象。     

Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production

Neurogenesis continues throughout life in the hippocampus. To study postnatal neurogenesis in vitro, hippocampal slices from rats on postnatal day 5 (P5) were cultured on a porous membrane for 14 or 21 days. In the initial experiments, precursor cells were labeled with bromodeoxyuridine (BrdU) after 7 days in culture because hippocampal slices are generally used in experiments after 1-2 weeks in culture. Fourteen days after labeling, however, only about 10% of BrdU-labeled cells expressed neuronal markers, although in living rats, about 80% of cells labeled with BrdU on P5 had become neurons by P19. Next, rats were injected with BrdU 30 min before culture, after which hippocampal slices were cultured for 14 days to examine the capacity of in vivo-labeled neural precursors to differentiate into neurons in vitro. In this case, more than two-thirds of BrdU-labeled cells expressed neuronal markers, such as Hu, NeuN, and PSA-NCAM. Furthermore, precursor cells underwent early in vitro labeling by incubation with BrdU or a modified retrovirus vector carrying EGFP for 30 min from the beginning of the culture. This procedure resulted in a similar high rate of neuronal differentiation and normal development into granule cells. In addition, time-lapse imaging with retrovirus-EGFP revealed migration of neural precursors from the hilus to the granule cell layer. These results indicate that in vivo- and early in vitro-labeled cultures are readily available ex vivo models for studying postnatal neurogenesis and suggest that the capacity of neural precursors to differentiate into neurons is reduced during the culture period.     

Lithium reduced neural progenitor apoptosis in the hippocampus and ameliorated functional deficits after irradiation to the immature mouse brain

Lithium was recently shown to inhibit apoptosis and promote survival of neural progenitor cells after hypoxia-ischemia in the immature rat brain. Our aim was to evaluate the effects of lithium on cell death and proliferation in the hippocampus after irradiation (IR) to the immature brain. Male mice were injected with 2mmol/kg lithium chloride i.p. on postnatal day 9 (P9) and additional lithium injections, 1mmol/kg, were administered at 24h intervals for up to 7days. BrdU was injected 4h after lithium injections on P9 and P10. The left hemisphere received a single dose of 8Gy (MV photons) on P11. The animals were euthanized 6h or 7weeks after IR. The number of BrdU-labeled cells in the subgranular zone (SGZ) of the granule cell layer (GCL) 6h after IR was 24% higher in the lithium-treated mice. The number of proliferating, phospho-histone H3-positive cells in the SGZ 7weeks after IR was 59% higher in the lithium group, so the effect was long-lasting. The number of apoptotic cells in the SGZ 6h after IR was lower in the lithium group, as judged by 3 different parameters, pyknosis, staining for active caspase-3 and TUNEL. Newly formed cells (BrdU-labeled 1 or 2days before IR) showed the greatest degree of protection, as judged by 50% fewer TUNEL-positive cells, whereas non-BrdU-labeled cells showed 38% fewer TUNEL-positive cells 6h after IR. Consequently, the growth retardation of the GCL was less pronounced in the lithium group. The number and size of microglia in the DG were also lower in the lithium group, indicating reduced inflammation. Learning was facilitated after lithium treatment, as judged by improved context-dependent fear conditioning, and improved place learning, as judged by assessment in the IntelliCage platform. In summary, lithium administration could decrease IR-induced neural progenitor cell apoptosis in the GCL of the hippocampus and ameliorate learning impairments. It remains to be shown if lithium can be used to prevent the debilitating cognitive late effects seen in children treated with cranial radiotherapy.     

Chronic ethanol consumption transiently reduces adult neural progenitor cell proliferation

Adult neural stem/progenitor cells proliferate throughout the life of the animal in the subependymal zone and the subgranular zone of the dentate gyrus (DG). Treatments such as enriched environment, dietary restriction, running and anti-depressants increase proliferation, however, stress and opiates have been shown to decrease proliferation. While models of binge ethanol drinking decreases proliferation, few studies have characterized the effect chronic ethanol usage has on progenitor cell proliferation. In this study, we have examined changes in the progenitor cell proliferation rate following chronic ethanol consumption. Animals were given a nutritionally balanced liquid diet containing 6.5% v/v ethanol or an isocalorically balanced liquid diet. Bromodeoxyuridine (BrdU) was administered (150 mg/kg x 3) and the animals sacrificed 2 h after the last injection on days 3, 10 or 30 of the ethanol diet. Coronal brain blocks were paraffin embedded and 6 microm sections sliced and immunohistochemically stained for BrdU. Quantitation of the number of BrdU-labeled cells in the subgranular zone of the DG revealed a significant decrease only at the 3-day time-point, with recovery by the 10- and 30-day time-points. Thus, the progenitor cell proliferation rate is transiently decreased by chronic ethanol usage. This data suggests that chronic alcohol use results in a compensatory response that restores the progenitor cell proliferation rate.     

脑缺血再灌注神经干细胞原位激活的研究

目的 探讨大鼠局灶性脑缺血再灌注后不同时间、不同脑部位神经干细胞(NSC)原位激活的变化规律。方法 建立一侧大脑中动脉闭塞缺血再灌注模型,用5-溴脱氧尿苷(BrdU)标记脑内增殖细胞,免疫组化(SP法)技术检测大鼠缺血再灌注不同时间段缺血周边皮质、双侧侧脑室下区(SVZ)、海马齿状回下区(SGZ)具有增殖能力细胞BrdU表达变化。结果 对照组脑组织双侧SVZ、SGZ可见少量BrdU阳性标记细胞;单纯脑缺血2 h组脑组织BrdU阳性细胞数未见明显增加;再灌注3天组,缺血侧皮质、双侧SVZ和SGZBrdU阳性细胞明显增多(P<0.05),7天时达到高峰(P<0.001),11天时下降,且缺血侧与对侧相比,双侧SVZ呈对称分布,而SGZ以缺血侧增加为主(P<0.05)。结论 一侧大脑中动脉闭塞缺血再灌注可致成年大鼠脑内NSC原位激活,且NSC增殖于缺血后1周达到高峰,激活部位以双侧SVZ、缺血侧海马和周边皮质为主。     

Proliferation of neuronal precursor cells in the dentate gyrus is accelerated after transient forebrain ischemia in mice

We investigated the proliferation of neuronal progenitor cells by labeling dividing cells by systemic application of the thymidine analog 5-bromodeoxyuridine (BrdU) during transient forebrain ischemia in mice. At 3 (n=6), 7 (n=6), 10 (n=6), and 17 days (n=6) after reperfusion, BrdU-labeled cells were detected in the dentate gyrus and paraventricle lesion. After ischemia-reperfusion, BrdU-labeled cells in the dentate gyrus significantly increased in number but not in the paraventricle lesion. These observations may help to clarify the mechanism of functional recovery after stroke.     

Enhanced production and dendritic growth of new dentate granule cells in the middle-aged hippocampus following intracerebroventricular FGF-2 infusions

Declined production and diminished dendritic growth of new dentate granule cells in the middle-aged and aged hippocampus are correlated with diminished concentration of fibroblast growth factor-2 (FGF-2). This study examined whether increased FGF-2 concentration in the milieu boosts both production and dendritic growth of new dentate granule cells in the middle-aged hippocampus. The FGF-2 or vehicle was infused into the posterior lateral ventricle of middle-aged Fischer (F)344 rats for 2 weeks using osmotic minipumps. New cells born during the first 12 days of infusions were labeled via daily intraperitoneal injections of 5'-bromodeoxyuridine (BrdU) and analysed at 10 days after the last BrdU injection. Measurement of BrdU(+) cells revealed a considerably enhanced number of new cells in the subgranular zone (SGZ) and granule cell layer (GCL) of the dentate gyrus (DG) ipsilateral to FGF-2 infusions. Characterization of beta-III tubulin(+) neurons among newly born cells suggested an increased addition of new neurons to the SGZ/GCL ipsilateral to FGF-2 infusions. Quantification of DG neurogenesis at 8 days post-infusions via doublecortin (DCX) immunostaining also revealed the presence of an enhanced DG neurogenesis ipsilateral to FGF-2 infusions. Furthermore, DCX(+) neurons in FGF-2-infused rats exhibited enhanced dendritic growth compared with their counterparts in vehicle-infused rats. Thus, subchronic infusion of FGF-2 is efficacious for stimulating an enhanced DG neurogenesis from neural stem/progenitor cells in the middle-aged hippocampus. As dentate neurogenesis is important for hippocampal-dependent learning and memory and DG long-term potentiation, strategies that maintain increased FGF-2 concentration during ageing may be beneficial for thwarting some of the age-related cognitive impairments.     

头针诱导神经干细胞的增殖分化

目的研究脑缺血再灌注大鼠神经干细胞（neural stem cells,NSCs）增殖、分化情况及头针对其影响。方法将Wistar大鼠70只,随机分为假手术组10只、模型组和头针组各30只;模型组和头针组采用线栓法制作大脑中动脉闭阻（middle cerebralartery occlusion,MCAO）模型,各组大鼠又按照缺血时间（7、14、28d）分为3个亚组,每个时相点10只。头针组大鼠于缺血再灌注成功后即行头针治疗,每日1次,直至处死前;各组大鼠处死前1d腹腔注5-溴脱氧尿核苷（bromodeoxyuridine,BrdU）液;对各亚组大鼠行神经功能缺损评分（neurological severity score,NSS）,免疫荧光法计数各组大鼠海马（dentate gyrus,DG）BrdU阳性细胞和BrdU/NeuN阳性双标细胞。结果第28d头针组的NSS显著低于模型组（P〈0.05）。大鼠缺血再灌注后各组阳性细胞有明显表达,而头针组在不同时相的阳性细胞数较同时相模型组有明显增加。结论头针能促进神经干细胞的增殖和分化,改善神经功能缺损评分,从而促进神经功能的修复。     

Temporal profile of neural stem cell proliferation in the subventricular zone after ischemia/hypoxia in the neonatal rat brain

OBJECTIVES: Ischemia/hypoxia (I/H) causes severe neonatal brain injury, such as periventricular leukomaracia and hypoxic/ischemic encephalopathy. Neural stem cell research could lead to a treatment for such disorders. In order to elucidate the dynamic changes in neural stem cells in the neonatal brain after I/H, we investigated the proliferation of new cells in the subventricular zone (SVZ). METHODS: Seven-day-old Wister rats were subjected to ligation of the left carotid artery followed by 2 hours of hypoxic stress (8% O(2) and 92% N(2), at 33 degrees C). In order to elucidate the dynamic change of neural stem cells in the SVZ, single bromodeoxyuridine (BrdU; 50 mg/kg) was administered 2 hours before death 1, 7, 14 and 21 days after I/H. Immunohistochemical and immunofluorescent studies for BrdU and doublecortin (DCX) were carried out. As a control, a group of rats was subjected to sham surgery (incision of skin, but no ligation of the carotid artery) and no I/H. RESULTS: The numbers of BrdU-labeled cells in the SVZ, for both the ipsilateral side and the contralateral side of the I/H brain, were twice the level of the control at 7 days after I/H, but the numbers for both sides returned to the control level at 21 days. In the ipsilateral side of the I/H brain, the number of BrdU-labeled cells in the SVZb (lining the upper wall of lateral ventricle) was 4-fold at 7 days and 15-fold at 21 days after I/H compared with the control level. This chronological pattern is very similar to the pattern for I/H results of the posterior periventricle (pPV). DCX appeared in most BrdU-labeled cells in the SVZb and pPV. DISCUSSION: These findings indicate that I/H enhances neural stem cell proliferation in the SVZ, and some newborn cells migrate as neural precursors to the SVZb and pPV after I/H in the neonatal rat brain.     

糖皮质激素对大鼠内源性神经前体细胞增殖的影响

目的探讨大剂量糖皮质激素(GCs)对成年大鼠内源性神经前体细胞增殖的影响.方法将25只大鼠随机分为对照组和地塞米松(DEX)作用1、3、7、14 d组,应用免疫组化方法检测神经前体细胞标记物巢蛋白(nestin)的表达,并通过5-溴脱氧尿苷(BrdU)观察神经前体细胞的增殖.结果正常大鼠海马齿状回(DG)和室下区(SVZ)存在神经前体细胞,并且其中一些细胞处于分裂增殖状态.应用大剂量GCs作用3、7、14 d组与对照组相比DG的nestin和BrdU阳性细胞数明显减少,SVZ的nestin和BrdU阳性细胞数在DEX作用7、14 d组与对照组相比明显减少,并且DG与SVZ二者阳性细胞数随着作用时间的延长而减低更为明显.结论大剂量GCs持续作用可抑制大鼠脑内的内源性神经前体细胞的增殖,DG区的神经前体细胞对GCs的反应较SVZ更为敏感.     

Status epilepticus during old age is not associated with enhanced hippocampal neurogenesis

Increased production of new neurons in the adult dentate gyrus (DG) by neural stem/progenitor cells (NSCs) following acute seizures or status epilepticus (SE) is a well known phenomenon. However, it is unknown whether NSCs in the aged DG have similar ability to upregulate neurogenesis in response to SE. We examined DG neurogenesis after the induction of continuous stages III-V seizures (SE) for over 4 h in both young adult (5-months old) and aged (24-months old) F344 rats. The seizures were induced through 2-4 graded intraperitoneal injections of the excitotoxin kainic acid (KA). Newly born cells in the DG were labeled via daily intraperitoneal injections of the 5'-bromodeoxyuridine (BrdU) for 12 days, which commenced shortly after the induction of SE in KA-treated rats. New cells and neurons in the subgranular zone (SGZ) and the granule cell layer (GCL) were analyzed at 24 h after the last BrdU injection using BrdU and doublecortin (DCX) immunostaining, BrdU-DCX and BrdU-NeuN dual immunofluorescence and confocal microscopy, and stereological cell counting. Status epilepticus enhanced the numbers of newly born cells (BrdU(+) cells) and neurons (DCX(+) neurons) in young adult rats. In contrast, similar seizures in aged rats, though greatly increased the number of newly born cells in the SGZ/GCL, failed to increase neurogenesis due to a greatly declined neuronal fate-choice decision of newly born cells. Only 9% of newly born cells in the SGZ/GCL differentiated into neurons in aged rats that underwent SE, in comparison to the 76% neuronal differentiation observed in age-matched control rats. Moreover, the number of newly born cells that migrate abnormally into the dentate hilus (i.e., ectopic granule cells) after SE in the aged hippocampus is 92% less than that observed in the young adult hippocampus after similar SE. Thus, SE fails to increase the addition of new granule cells to the GCL in the aged DG, despite a considerable upregulation in the production of new cells, and SE during old age leads to much fewer ectopic granule cells. These results have clinical relevance because earlier studies have implied that both increased and abnormal neurogenesis occurring after SE in young animals contributes to chronic epilepsy development.     

脑缺血再灌注海马齿状回神经干细胞Mash-1的表达

背景：Mash-1在中枢神经系统和周围神经系统发育中都有表达,对神经元和神经胶质细胞的分化都有很重要的作用。 目的：检测Mash-1在大鼠脑缺血再灌注海马组织神经干细胞中的表达及其变化。 方法：建立大脑中动脉栓塞制作大鼠脑缺血再灌注模型。实验分为假手术组、缺血再灌注3,7,14,21,28 d组。 结果与结论：免疫组织化学检测结果,随着脑缺血再灌注第3天海马神经元BrdU阳性细胞明显增多,第7天达高峰,然后逐渐减少。Mash-1反应产物随脑缺血再灌注时间延长逐渐增多,第21天表达最强,以后表达逐渐减少。提示Mash-1呈时间依赖性表达,与神经干细胞增殖分化进程相吻合,说明其在神经干细胞晚期分化中起重要调控作用。     

Expression of polysialylated neural cell adhesion molecule in rat brain after transient middle cerebral artery occlusion

The highly polysialylated form of neural cell adhesion molecule (PSA-NCAM) is important for neurite outgrowth. With this molecule as a marker of plastic change in neurons, we investigated its temporal expression in rat brain after transient middle cerebral artery (MCA) occlusion. In sham-control brain, only subependymal neurons showed a positive immunoreactivity for PSA-NCAM. After 90 min of transient MCA occlusion, neurons in the piriform cortex began to be positively stained at 1 h, while neurons in the cortex and caudate of the MCA territory became positive after 8 h. The stainings persisted for 1 and 3 days after reperfusion. The present results indicate that neurons in the cerebral cortex and caudate have the capability of plastic change in the adult brain, and that those in the piriform cortex rapidly undergo plastic change probably in response to transneuronal injury.     

Temporal and spatial differences of PSA-NCAM expression between young-adult and aged rats in normal and ischemic brains

Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is transiently expressed specifically in newly generated cells, and is important for migration and neurite outgrowth. To investigate the effect of aging on the migration of neural stem cell (NSC) after brain ischemia, the spatiotemporal expressions of immunoreactive PSA-NCAM were examined at 4 h or 1, 3 or 7 days after 90 min of middle cerebral artery occlusion (MCAO) in the young-adult or aged rats. In the sham control brain, PSA-NCAM staining was slightly observed both in dorsal and ventral parts of subventricular zone (SVZ) in the aged brain, but only in the dorsal part of SVZ in the young brain. After transient MCAO, immunoreactivity for PSA-NCAM increased in the number and the intensity in SVZ ipsilateral to MCAO in the young-adult brains and became the peak at 1 day, while that was at 3 days in the aged brains. These findings suggest that PSA-NCAM was located in different spatial distribution in normal condition between young and old rats. PSA-NCAM was induced after ischemia, and the temporal expression was also different after transient MCAO between young and older rats.