Rat forebrain neurogenesis and striatal neuron replacement after focal stroke

The persistence of neurogenesis in the forebrain subventricular zone (SVZ) of adult mammals suggests that the mature brain maintains the potential for neuronal replacement after injury. We examined whether focal ischemic injury in adult rat would increase SVZ neurogenesis and direct migration and neuronal differentiation of endogenous precursors in damaged regions. Focal stroke was induced in adult rats by 90-minute right middle cerebral artery occlusion (tMCAO). Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunostaining for cell type-specific markers. Brains examined 10-21 days after stroke showed markedly increased SVZ neurogenesis and chains of neuroblasts extending from the SVZ to the peri-infarct striatum. Many BrdU-labeled cells persisted in the striatum and cortex adjacent to infarcts, but at 35 days after tMCAO only BrdU-labeled cells in the neostriatum expressed neuronal markers. Newly generated cells in the injured neostriatum expressed markers of medium spiny neurons, which characterize most neostriatal neurons lost after tMCAO. These findings indicate that focal ischemic injury increases SVZ neurogenesis and directs neuroblast migration to sites of damage. Moreover, neuroblasts in the injured neostriatum appear to differentiate into a region-appropriate phenotype, which suggests that the mature brain is capable of replacing some neurons lost after ischemic injury.     

Cell proliferation in ependymal/subventricular zone and nNOS expression following focal cerebral ischemia in adult rats

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in normal developing brain, but the role of nNOS in neurogenesis in the ischemic brain remains unclear. To investigate the temporal and spatial relationship between cell proliferation of the ependymal/subventricular zone (SVZ), a principal neuroproliferative region in the adult brain, and nNOS expression, the male Sprague-Dawley rats weighing 250-350 g were used. The focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). 10 microl of 0.2% fluorescence dye DiI was injected into the right lateral ventricle to prelabel ependymal/subventricular zone cells before ischemia. The rats were killed immediately after ischemia and days 1, 3, 7, 11, 14, 21 and 28 after ischemia. DiI-labeled cell counting was employed to assess cell proliferation. Immunohistochemistry and grayscale analysis were performed to determine nNOS localization and its quantity in the specific regions. Compared with control, the density of DiI-labeled cells in the ipsilateral ependyma/SVZ was significantly higher at days 1, 3, 7 and 11 after ischemia, whereas the quantity of nNOS expression in the ependyma/SVZ adjacent regions was significantly lower at the above time points. Additionally, nNOS positive cells were largely excluded from SVZ, and their long processes did not enter the ependyma/SVZ. Our results indicate that after focal cerebral ischemia, decreased nNOS expression in the ipsilateral ependymal/SVZ adjacent regions might be related to cell proliferation in the ependymal/SVZ.     

Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia

Adult neurogenesis is modulated by growth factors, physical conditions, and other alterations in the physical microenvironment. We studied the effects of focal ischemia on neurogenesis in the subventricular zone (SVZ), olfactory bulb (OB), and hippocampal dentate gyrus (DG) (known to be persistent neurogenic regions) in the adult non-human primate, the cynomolgus monkey. Three monkeys underwent middle cerebral artery occlusion-induced focal ischemia and were given multiple BrdU injections during the first 2 weeks after ischemia. Twenty-eight days later, the animals were perfused. The number of new neurons (3182 +/- 408/mm3) in the ipsilateral DG of ischemic monkeys was 4.7-fold that in the DG of non-operated monkeys. The number of new neurons (9176 +/- 2295/mm3) in the ipsilateral olfactory bulb of ischemic monkeys was 18.0-fold that in normal olfactory bulb. These observations suggest an increase in the number of new OB neurons, as well as new DG neurons, after focal ischemia in a primate. This substantial increase in new neurons after focal ischemia could result from the enhancement of cell proliferation rather than a change in the rate of cell commitment. Of the three monkeys subjected to ischemia, only one animal possessed a unique progenitor cell type at the most anterior aspect of the ipsilateral SVZ. Within this region, a short migration (approximately 500 microm) of doublecortin-expressing immature neuronal progenitor cells was observed.     

Strong neurogenesis, angiogenesis, synaptogenesis, and antifibrosis of hepatocyte growth factor in rats brain after transient middle cerebral artery occlusion

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.     

Enforced physical training promotes neurogenesis in the subgranular zone after focal cerebral ischemia

BACKGROUND: Cerebral ischemia increases neurogenesis in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the dentate gyrus, and this might be modulated by an enriched environment including voluntary physical activity. We examined whether enforced physical training (EPT) influences neurogenesis in the SVZ and SGZ after cerebral ischemia. METHODS: Adult male Sprague-Dawley rats were subjected to focal cerebral ischemia for 2 h, and divided into an EPT and a non-EPT group. All rats in the EPT group were trained using a rota-rod for 14 days. 5-bromo-2'-deoxyuridine (BrdU) was injected to determine levels of cell proliferation. Functional recovery was assessed using a set of behavioral test batteries. Extents of endogenous neurogenesis in the SVZ and SGZ were quantified by immunofluorescence staining. Although final infarction volumes were not significantly different in the groups, functional recovery was better in the EPT group at 10 and 17 days after ischemia. In the SVZ, BrdU labeling and double labeling of BrdU/Dcx and of BrdU/NeuN were not significantly different in the two groups. However, in the SGZ, EPT significantly increased the number of BrdU-positive cell numbers (EPT vs. non-EPT: 159.1+/-19.9 vs. 101.8+/-7.8, p=0.04), and the number of BrdU/Dcx double-labeled cells (130.6+/-16.9 vs. 73.6+/-7.2, p=0.01). CONCLUSIONS: The results obtained indicate that EPT promotes neurogenesis in the SGZ of the dentate gyrus after ischemia, but not in the SVZ. The biochemical mechanism that determines the differential effects of EPT remains to be clarified.     

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。     

普伐他汀对脑缺血大鼠的神经保护及神经发生的作用

目的研究中枢神经系统缺血损伤后,普伐他汀的神经保护和促进神经发生作用。方法采用线栓法造成大鼠大脑中动脉的暂时性缺血,在以下时间点给予普伐他汀：伤后6h,伤后每天直至伤后14天。用神经学评分、平衡实验和旋转实验评价伤后神经学恢复情况。检测血清胆固醇和甘油三酯的含量,计算脑梗塞面积。通过三染色法（BrdU, DCX, NeuN染色）研究普伐他汀对神经发生的作用。结果各组间血清胆固醇和甘油三酯无显著性差异;与对照组相比,实验组动物术后旋转实验评分显著性增加,梗塞面积减小;普伐他汀显著增加了齿状回和脑室下区的BrdU阳性细胞数,并增加了齿状回、脑室下区和纹状体中的BrdU/DCX阳性细胞数。结论中枢神经系统损伤早期重复使用低剂量的普伐他汀是相对安全的,并能够显著改善伤后的神经功能恢复,减少梗死面积。普伐他汀能够诱导大鼠齿状回及脑室下区的神经发生并增加纹状体中迁移神经元的数量,这与普伐他汀的降脂作用无关。     

普伐他汀对脑缺血大鼠的神经保护及神经发生的作用(英文)

目的研究中枢神经系统缺血损伤后,普伐他汀的神经保护和促进神经发生作用。方法采用线栓法造成大鼠大脑中动脉的暂时性缺血,在以下时间点给予普伐他汀:伤后6 h,伤后每天直至伤后14 天。用神经学评分、平衡实验和旋转实验评价伤后神经学恢复情况。检测血清胆固醇和甘油三酯的含量,计算脑梗塞面积。通过三染色法(BrdU, DCX, NeuN染色)研究普伐他汀对神经发生的作用。结果各组间血清胆固醇和甘油三酯无显著性差异;与对照组相比,实验组动物术后旋转实验评分显著性增加,梗塞面积减小;普伐他汀显著增加了齿状回和脑室下区的BrdU阳性细胞数,并增加了齿状回、脑室下区和纹状体中的BrdU/DCX阳性细胞数。结论中枢神经系统损伤早期重复使用低剂量的普伐他汀是相对安全的,并能够显著改善伤后的神经功能恢复,减少梗死面积。普伐他汀能够诱导大鼠齿状回及脑室下区的神经发生并增加纹状体中迁移神经元的数量,这与普伐他汀的降脂作用无关。     

Delayed treatment with sildenafil enhances neurogenesis and improves functional recovery in aged rats after focal cerebral ischemia

Increasing age decreases the number of new neurons in the dentate gyrus and the subventricular zone (SVZ). Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, enhances neurogenesis in young rats. The present study tested the hypothesis that sildenafil augments neurogenesis in aged rats after focal cerebral ischemia. Nonischemic aged (18 months, n = 6) Wistar rats exhibited a significant reduction of actively proliferating and relatively quiescent cells in the SVZ measured by the number of minichromosome maintenance protein-2-positive (MCM-2+) cells, a marker of the proliferating cells, compared with nonischemic young (3-4 months, n = 8) rats. Occlusion of the middle cerebral artery did not increase the number of MCM-2+ cells in the SVZ of aged rats at 3 months after focal ischemia. However, treatment with sildenafil at a dose of 3 mg/kg (n = 8) daily for 7 consecutive days starting 7 days after focal ischemia significantly increased the number of MCM-2+ cells in the SVZ of aged rats compared with aged rats treated with saline (n = 8). Double immunostaining revealed that substantially more Ki67+ cells (a marker of proliferating cells) were doublecortin+ (a marker of migrating neuroblasts) in sildenafil-treated than in saline-treated aged animals. In addition, treatment with sildenafil significantly improved functional recovery compared with saline-treated rats. These data suggest that inhibition of PDE5 activity by sildenafil augments neurogenesis in the SVZ of aged ischemic rats, although these rats have reduced numbers of neural progenitor and stem cells in the SVZ.     

Temporal profile of neurogenesis in the subventricular zone,dentate gyrus and cerebral cortex following transient focal cerebral ischemia

Abstract

Background: In the adult mammalian brain, it is considered that neurogenesis persists in limited regions such as the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle. On the other hand, neurogenesis in the cortex after cerebral ischemia and its role in post-stroke recovery have not been clarified yet. In this study, we investigated neurogenesis in the cortex and the spatiotemporal profile of neural progenitors in SVZ and DG of rats subjected to transient focal cerebral ischemia.

Materials and methods: Male Sprague–Dawley rats (270–300 g) were subjected to 60 minute middle cerebral artery occlusion. Proliferating cells were labeled by the cumulative administration of BrdU 1, 2, 3, 4, 6 and 8 weeks after ischemia induction (at weeks 1–4, 6 and 8). Double labeling was also performed with antibodies against BrdU and NeuN.

Results: BrdU-positive cells proliferated in DG and SVZ of the bilateral hemispheres, and their proliferation peaked at week 3 in SVZ and at week 4 in DG. In the peri-infarct zone of cerebral cortex, BrdU-positive cells co-expressed NeuN from weeks 3 to 8.

Conclusion: Neurogenesis was observed in the cerebral cortex and proliferation of neural progenitors occurred in SVZ and DG of rats subjected to transient focal cerebral ischemia. Our data might indicate that endogenous dormant neural stem cells residing in the cortex were activated by ischemic insult to induce the proliferation of neural progenitors and differentiation into mature neurons.     

Bilateral changes after neonatal ischemia in the P7 rat brain

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and subgranular zone (SGZ) and increases in the adult after brain injury. In this study, postnatal day 7 rats underwent middle cerebral artery electrocoagulation and transient homolateral common carotid artery occlusion, a lesioning protocol that resulted in ipsilateral (IL) forebrain ischemic injury, leading to a cortical cavity 3 weeks later. The effects of neonatal ischemia on hemispheric damage, cell death, cell proliferation, and neurogenesis were examined 4 hours to 6 weeks later by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and immunohistochemistry of Ki-67 in proliferating cells and of doublecortin, a microtubule-associated protein expressed only by immature neurons. Neonatal ischemic injury resulted in persistent reduced IL and transient reduced contralateral (CL) hemispheric areas, a consequence of sustained and transient cell death in the IL and CL areas, respectively. Ki-67 immunostaining revealed 3 peaks of newly generated cells in the dorsal SVZ and SGZ in the IL side and also in the CL side at 48 hours and 7 and 28 days after ischemia. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 hours. Ischemic injury also stimulated SVZ neurogenesis, based on increased doublecortin immunostaining in both SVZs at 7 to 14 days after injury. Doublecortin-positive neurons remained visible around the lesion at 21 days but displayed an immature shape in discrete chains or clusters. Although unilateral ischemic damage was produced, results indicate successful regenerative changes in the CL hemisphere, allowing anatomical recovery.     

Inducible nitric oxide synthase appears and is co-expressed with the neuronal isoform in interneurons of the rat hippocampus after transient ischemia induced by middle cerebral artery occlusion

The hippocampus (dentate gyrus DG plus Cornu Ammonis, CA) is vulnerable to neuropathological events such as ischemia. The DG is a region where neurogenesis takes place and it has been demonstrated that ischemia stimulates neurogenesis. Nitric oxide (NO) plays a major role in ischemic damage evolution and increases in rat hippocampus after ischemia. No information is available on the presence of nNOS-immunoreactive(IR) neurons in the hippocampus of ischemic animals; whereas, the presence of the iNOS protein has been reported in the DG after focal ischemia.We evaluated, immunohistochemically, the cell types expressing nNOS and iNOS in the rat hippocampus by 24 up to 144 h after transient middle cerebral artery occlusion to ascertain whether ischemia induces changes in nNOS or iNOS expression and whether a relationship exists between these changes and the animal survival.nNOS-IR interneurons were detected in control and ischemic rats; in the latter, their number was significantly decreased at all time points. iNOS-IR interneurons appeared in the hippocampus of ischemic rats at 24 h; their number was significantly higher in the animals with longer survival and did not change at later time points. More than 50% of the nNOS-IR interneurons co-expressed iNOS-IR. All these changes were seen both in the ipsilateral and contralateral hippocampus.In conclusion, the focal ischemia affects the hippocampus which responds bilaterally to the injury. We hypothesize that the decrease in the nNOS-IR neurons is likely due to either a neuronal loss or a switching towards the iNOS production which, by inducing neurogenesis, might compensate the neuronal loss.     

Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats.

Recent studies demonstrated that neurogenesis in the adult hippocampus increased after transient global ischemia; however, the molecular mechanism underlying increased neurogenesis after ischemia remains unclear. The finding that proliferation of progenitor cells occurred at least a week after ischemic insult suggests that the stimulus was not an ischemic insult to progenitor cells. To clarify whether focal ischemia increases the rate of neurogenesis in the remote area, the authors examined the contralateral hemisphere in rats subjected to permanent occlusion of the middle cerebral artery. In the subgranular zone of the hippocampal dentate gyrus, the numbers of bromodeoxyuridine (BrdU)-positive cells increased approximately sixfold 7 days after ischemia. In double immunofluorescence staining, more than 80% of newborn cells expressed Musashi1, a marker of neural stem/progenitor cells, but only approximately 10% of BrdU-positive cells expressed glial fibrillary acidic protein (GFAP), a marker of astrocytes. The number of BrdU-positive cells markedly decreased 28 days after BrdU administration after ischemia, but it was still elevated compared with that of sham-operated rats. In double immunofluorescence staining, 80% of newborn cells expressed NeuN, a marker of differentiated neurons, and 10% of BrdU-positive cells expressed GFAP. However, in the other areas of the contralateral hemisphere including the rostral subventricular zone, the number of BrdU-positive cells remained unchanged. These results showed that focal ischemia stimulated the proliferation of neuronal progenitor cells, but did not support survival of newborn cells in the contralateral hippocampus.     

VEGF-overexpressing transgenic mice show enhanced post-ischemic neurogenesis and neuromigration

New neurons are generated continuously in the subventricular zone and dentate gyrus of the adult brain. Neuropathologic processes, including cerebral ischemia, can enhance neurogenesis, as can growth factors and other physiologic stimuli. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can promote neurogenesis, but it is unknown whether VEGF can enhance migration of newborn neurons toward sites of ischemic injury, where they might be able to replace neurons that undergo ischemic death. In the present study we produced permanent focal cerebral ischemia in transgenic (Tg) mice that overexpress VEGF. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (Brdu) labeling and immunostaining for cell type-specific markers. In VEGF-Tg mice, brains examined 7-28 days after cerebral ischemia showed markedly increased subventricular zone (SVZ) neurogenesis, chains of neuroblasts extending from the SVZ to the peri-infarct cortex, and an increase in the number of newly generated cortical neurons at 14-28 days after ischemia. In concert with these effects, VEGF overexpression reduced infarct volume and improved postischemic motor function. These findings provide evidence that VEGF increases SVZ neurogenesis and neuromigration, consistent with a possible role in repair. Our data suggest that in addition to its neuroprotective effects, which are associated with improved outcome in the acute phase after cerebral ischemia, VEGF enhances postischemic neurogenesis, which could provide a therapeutic target for more chronic brain repair.     

Galectin-1 regulates neurogenesis in the subventricular zone and promotes functional recovery after stroke

Galectin-1 (Gal-1) has recently been identified as a key molecule that plays important roles in the regulation of neural progenitor cell proliferation in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. To test the hypothesis that Gal-1 contributes to adult neurogenesis after focal ischemia, we studied the temporal profile of endogenous Gal-1 expression and the effects of human recombinant Gal-1 on neurogenesis and neurological functions in an experimental focal ischemic model. In the normal brain, Gal-1 expression was observed only in the SVZ. In the ischemic brain, Gal-1 expression was markedly upregulated in the SVZ and the area of selective neuronal death around the infarct in the striatum. The temporal profile of Gal-1 expression was correlated with that of neural progenitor cell proliferation in the SVZ of the ischemic hemisphere. Double-labeling studies revealed that Gal-1 was localized predominantly in both reactive astrocytes and SVZ astrocytes. Administration of Gal-1, which is known to have carbohydrate-binding ability, into the lateral ventricle increased neurogenesis in the ipsilateral SVZ and improved sensorimotor dysfunction after focal ischemia. By contrast, blockade of Gal-1 in the SVZ by the administration of anti-Gal-1 neutralizing antibody strongly inhibited neurogenesis and diminished neurological function. These results suggest that Gal-1 is one of the principal regulators of adult SVZ neurogenesis through its carbohydrate-binding ability and provide evidence that Gal-1 protein has a role in the improvement of sensorimotor function after stroke.     

凝血酶预处理对大鼠脑出血后内源性神经再生的影响

目的 探讨凝血酶预处理(thrombin preconditioning,TPC)对大鼠脑出血后内源性的神经细胞再生的影响。方法 SD大鼠随机分为对照组、脑出血(Intracerebral hemorrhage,ICH)组和 TPC组,每组分为3、7、14、21、28 d亚组。应用胶原酶脑内立体定向注射制作脑出血模型。TPC组首先将1U凝血酶立体定向注入右侧纹状体,1 d后再制作脑出血模型。所有大鼠应用BrdU腹腔注射在体标记再生的脑室下带( subventricular zone, SVZ)细胞,应用免疫组织化学技术检测BrdU阳性细胞。结果 脑出血后3 d同侧SVZ 和基底节BrdU阳性细胞开始增加,但与对照组比较差异没有显著性(P>0.05),7 d时BrdU阳性细胞数增加明显,与对照组比较有明显差异(P<0.05),14 d达到高峰,然后逐渐下降,28 d时BrdU阳性细胞虽然仍有增加,但与对照组比较差异没有显著性(P>0.05)。TPC组3 d时BrdU阳性细胞数目开始增加,与脑出血组和对照组比较,差异均具有显著性(P<0.01),14 d达高峰,一直持续至21 d,28 d时BrdU阳性细胞虽然略有下降,但与对照组和脑出血组比较,仍有统计学差异(P<0.05)。结论 凝血酶预处理能够增强脑出血后内源性神经再生,可能为脑出血后内源性神经再生的研究提供新的思路。     

Delayed treatment of 6-Bromoindirubin-3′-oxime stimulates neurogenesis and functional recovery after focal ischemic stroke in mice

Glycogen synthase kinase 3β (GSK3β) was originally identified as a regulator for glycogen metabolism and is now an important therapeutic target for a variety of brain disorders including neurodegenerative diseases due to it's pivotal role in cellular metabolism, proliferation and differentiation. In the development of stroke therapies focusing on tissue repair and functional recovery, promoting neurogenesis is a main approach in regenerative medicine. In the present investigation, we explored the effects of a GSK3β specific inhibitor, 6-Bromoindirubin-3′-oxime (BIO), on regenerative activities of neuroblasts in the subventricular zone (SVZ) and functional recovery after focal cerebral ischemia. Adult C57/BL mice were subjected to occlusion of distal branches of middle cerebral artery (MCA) supplying the sensorimotor barrel cortex. Three days later, BIO (8.5 μg/kg, i.p.) was administered every 2 days until sacrificed at 14 or 21 days after stroke. The BIO treatment significantly increased generation of neuroblasts labeled with BrdU and BrdU/doublecortin (DCX) in the SVZ. Comparing to vehicle controls, increased number of neuroblasts migrated to the peri-infarct region where they differentiate into mature neurons. Along with the elevated BDNF expression at the peri-infarct area, the number of newly formed neurons was significantly increased. BIO treatment significantly enhanced sensorimotor functional recovery after the focal ischemia. It is suggested that the GSK3 signaling may be a potential therapeutic target for regenerative treatment after ischemic stroke.     

Spatiotemporal changes of apolipoprotein E immunoreactivity and apolipoprotein E mRNA expression after transient middle cerebral artery occlusion in rat brain

Apolipoprotein E (ApoE) is a constituent of lipoprotein and plays an important role in the maintenance of neural networks. However, spatiotemporal differences in ApoE expression and its long-term role in neural process after brain ischemia have not been studied. We investigated changes of ApoE immunoreactivity and ApoE mRNA expression both in the core and in the periischemic area at 1, 7, 21, or 56 days after 90 min of transient middle cerebral artery occlusion. Double stainings for ApoE plus NeuN or plus ED1 were performed in order to identify cell type of ApoE-positive stainings. The maximal increase of ApoE expression was observed at 7 days in the core and at 7 and 21 days in the periischemic area. In the core, ApoE plus NeuN double-positive cells increased at 1 and 7 days, without ApoE mRNA expression, whereas they increased in the periischemic area, with a peak at 21 days, with ApoE mRNA expression in glial cells but not in neurons. On the other hand, ApoE plus ED1 double-positive cells increased only in the core, with a peak in number at 7 and 21 days and marked ApoE mRNA expression in macrophages. The present study suggests that ApoE plays various important roles in different type of cells, reflecting spatiotemporal dissociation between degenerative and regenerative processes after brain ischemia, and that ApoE is profoundly involved in pathological conditions, such as brain ischemia.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri‐infarct angiogenesis after focal cerebral ischemia

Neurogenesis and angiogenesis are two important processes that may contribute to the repair of brain injury after stroke. This study was designed to investigate whether transplantation of human embryonic neural stem cells (NSCs) into cortical peri‐infarction 24 h after ischemia effects cell proliferation in the subventricular zone (SVZ) and angiogenesis in the peri‐infarct zone. NSCs were prepared from embryonic human brains at 8 weeks gestation. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. Animals were randomly divided into two groups (n = 30, each) at 24 h after ischemia: NSC‐grafted and medium‐grafted groups. Toluidine blue staining and 5′‐bromo‐2′‐deoxyuridine (BrdU) or von Willebrand factor (vWF) immunohistochemistry were performed at 7, 14 and 28 days after transplantation. NSC transplantation increased the number of BrdU‐positive cells in the ischemic ipsilateral SVZ compared with the medium control at 7 days (P < 0.01). This difference in SVZ cell proliferation persisted at 14 days (P < 0.01), but was not significant at 28 days (P > 0.05). In addition, angiogenesis, as indicated by BrdU and vWF staining in cortical peri‐infarct regions, was augmented by 46% and 65% in NSC‐grafted rats versus medium‐grafted rats at 7 and 14 days, respectively (P < 0.05). However, this increase became non‐significant at 28 days (P > 0.05). Our results indicate that NSC transplantation enhances endogenous cell proliferation in the SVZ and promotes angiogenesis in the peri‐infarct zone, even if it is performed in the acute phase of ischemic injury.