Intracerebral transplantation of human adipose tissue stromal cells after middle cerebral artery occlusion in rats

The transplantation of cells capable of neuronal differentiation has great potential for the treatment of neurological conditions. I examined whether human adipose tissue stromal cells (hATSCs) can be induced to undergo neuronal differentiation. I isolated hATSCs from human liposuction tissue and induced neuronal differentiation using azacytidine. After neuronal induction, the hATSCs adopted a more neuronal morphology. These hATSCs were injected into the lateral ventricle of the rat brain, after which they migrated to various parts of the brain. After ischemic brain injury induced by middle cerebral artery occlusion (MCAO), a large number of cells migrated to the injured cortex. Intracerebral grafting of hATSCs significantly enhanced the recovery of functional motor deficits in MCAO rats. These data indicate that transplanted hATSCs survive, migrate and differentiate in the ischemic microenvironment and improve neurological recovery after stroke in rats.     

Monocyte chemoattractant protein-1 plays a critical role in neuroblast migration after focal cerebral ischemia.

Transient focal ischemia is known to induce proliferation of neural progenitors in adult rodent brain. We presently report that doublecortin positive neuroblasts formed in the subventricular zone (SVZ) and the posterior peri-ventricle region migrate towards the cortical and striatal penumbra after transient middle cerebral artery occlusion (MCAO) in adult rodents. Cultured neural progenitor cells grafted into the non-infarcted area of the ipsilateral cortex migrated preferentially towards the infarct. As chemokines are known to induce cell migration, we investigated if monocyte chemoattractant protein-1 (MCP-1) has a role in post-ischemic neuroblast migration. Transient MCAO induced an increased expression of MCP-1 mRNA in the ipsilateral cortex and striatum. Immunostaining showed that the expression of MCP-1 was localized in the activated microglia and astrocytes present in the ischemic areas between days 1 and 3 of reperfusion. Furthermore, infusion of MCP-1 into the normal striatum induced neuroblast migration to the infusion site. The migrating neuroblasts expressed the MCP-1 receptor CCR2. In knockout mice that lacked either MCP-1 or its receptor CCR2, there was a significant decrease in the number of migrating neuroblasts from the ipsilateral SVZ to the ischemic striatum. These results show that MCP-1 is one of the factors that attract the migration of newly formed neuroblasts from neurogenic regions to the damaged regions of brain after focal ischemia.     

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.     

Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival.

Astrocytes are intimately involved in both glutamate and gamma-aminobutyric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2- 13C]acetate combined with ex vivo 13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.     

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 × 10⁵) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.     

Migrating neurons cross a reelin-rich territory to form an organized tissue out of embryonic cortical slices

In this study we show that the radial migration of neuronal precursors out of cerebral cortex of embryonic brain slices cultured for 4-7 days gives rise to an organized tissue that forms de novo off developing slices. In our in vitro preparations, migrating neuronal precursors overshot the marginal zone, as did the elongation of radial glial processes out of the slices. These cells detached from radial glia at a distance from the cortex and differentiated into pyramidal and nonpyramidal profiles that expressed different neuronal markers. Glial precursors were shown to proliferate in the slice and in the neotissue, and to differentiate into astrocytes. We show that cells expressing reelin in the marginal zone of embryonic cortical slices persist after a week in culture, which implies that neuronal migration is not necessarily hindered by the presumed stop signals provided by reelin in the marginal zone. Furthermore, our results provide a new model for in vitro studies of migration and differentiation during cortical development.     

bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury*☆

BACKGROUND： Basal cell lymphoma-extra large （bcl-xl） can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE： This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/repcrfusion model by middle cerebral artery occlusion （MCAO） in transgenic and wild-type mice, and observed changes in the number and distribution of apoptotic neural cells, differences in cerebral infarct volume, in neurological function score, and in cytochrome C expression in the ischemic cerebral cortex, at different time points, DESIGN AND SETTING： The present gene engineering and cell biology experiment was performed at the Laboratory of Biology, Hubei Academy of Agricultural Sciences and at the Laboratory of Immunology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： Male bcl-xl over-expression Kunming mice aged 8 weeks and age-matched male wild-type mice were used for this study. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling （TUNEL） kits were purchased from Boliman, France. Cytochrome C antibody and Bcl-x immunohistochemical kit were purchased from PharMingen, USA and Santa Cruz Biotechnology, USA, respectively. METHODS： Following MCAO and reperfusion, apoptosis in the ischemic cerebral cortex was detected by the TUNEL assay. Prior to MCAO and 3 hours after reperfusion, the Bcl-xl protein level in the ischemic cerebral cortex was measured by immunohistochemistry. At 3, 6, 12 and 24 hours after reperfusion, the level of cytochrome C in the ischemic cerebral cortex was examined by western blot analysis. Subsequent to MCAO, cerebral infarct volume measurement and neurological examination were performed. MAIN OUTCOME MEASURES： Neural cell apoptosis and cytochrome C expression in the ischemic cerebral cortex; cerebral infarct volume and neurological function score. RESULTS： Twenty-four hours after reperfusion, cerebral inf     

Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum

Pathological processes, including cerebral ischemia, can enhance neurogenesis in the adult brain, but the fate of the newborn neurons that are produced and their role in brain repair are obscure. To determine if ischemia-induced neuronal proliferation is associated with migration of nascent neurons toward ischemic lesions, we mapped the migration of cells labeled by cell proliferation markers and antibodies against neuronal marker proteins, for up to 2 weeks after a 90-min episode of focal cerebral ischemia caused by occlusion of the middle cerebral artery. Doublecortin-immunoreactive cells in the rostral subventricular zone, but not the dentate gyrus, migrated into the ischemic penumbra of the adjacent striatum and, via the rostral migratory stream and lateral cortical stream, into the penumbra of ischemic cortex. These results indicate that after cerebral ischemia, new neurons are directed toward sites of brain injury, where they might be in a position to participate in brain repair and functional recovery.     

bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury

BACKGROUND:Basal cell lymphoma-extra large (bcl-xl) can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE: This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/reperfusion model by middle cerebral artery occlusion (MCAO) in transgenic and wild-type mice,and observed changes in the number and distribution of apoptotic neural cells,differences in cerebral infa...     

Cortical neurogenesis in adult rats after ischemic brain injury: most new neurons fail to mature

The present study examines the hypothesis that endogenous neural progenitor cells isolated from the neocortex of ischemic brain can differentiate into neurons or glial cells and contribute to neural regeneration. We performed middle cerebral artery occlusion to establish a model of cerebral ischemia/reperfusion injury in adult rats. Immunohistochemical staining of the cortex 1, 3, 7, 14 or 28 days after injury revealed that neural progenitor cells double-positive for nestin and sox-2 appeared in the injured cortex 1 and 3 days post-injury, and were also positive for glial fibrillary acidic protein. New neurons were labeled using bromodeoxyuridine and different stages of maturity were identified using doublecortin, microtubule-associated protein 2 and neuronal nuclei antigen immunohistochemistry. Immature new neurons coexpressing doublecortin and bromodeoxyuridine were observed in the cortex at 3 and 7 days post-injury, and semi-mature and mature new neurons double-positive for microtubule-associated protein 2 and bromodeoxyuridine were found at 14 days post-injury. A few mature new neurons coexpressing neuronal nuclei antigen and bromodeoxyuridine were observed in the injured cortex 28 days post-injury. Glial fibrillary acidic protein/bromodeoxyuridine double-positive astrocytes were also found in the injured cortex. Our findings suggest that neural progenitor cells are present in the damaged cortex of adult rats with cerebral ischemic brain injury, and that they differentiate into astrocytes and immature neurons, but most neurons fail to reach the mature stage.     

The effect of hypothermia on transient middle cerebral artery occlusion in the rat.

We investigated the effect of moderate whole body hypothermia (30 degrees C) on transient middle cerebral artery occlusion (MCAO) in the rat. Male Wistar rats were subjected to 2 h of ischemia by inserting a suture into the lumen of the internal carotid artery and occluding the origin of the MCA. Experimental groups were (a) MCAO induced at 37 degrees C body temperature (n = 15); (b) 30 degrees C body temperature induced prior to ischemia and maintained for 2 h of MCAO and 1 h of reperfusion (n = 12); and (c) MCAO with regional brain and body temperatures measured in normothermic (n = 3) and hypothermic MCAO rats (n = 2). Histopathological evaluation was performed 96 h after reperfusion. All normothermic MCAO animals exhibited ischemic infarct involving the ipsilateral cortex and basal ganglia with infiltration of neutrophils, macrophages, and microvascular proliferation. Hypothermic MCAO animals exhibited minor ischemic damage ranging from selective neuronal injury to small focal areas of infarct with minimal inflammatory response. Our data demonstrate that transient ischemia induced by using the intra-arterial suture method to occlude the MCA results in a reproducible brain lesion and that moderate hypothermia has a profound protective effect on the brain injury after transient MCAO.     

Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex-derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin-positive cells in the post-stroke cerebral cortex, but not in the non-ischemic cortex. Cells obtained from the ischemic core of the post-stroke cerebral cortex formed neurosphere-like cell clusters expressing nestin; such cells had the capacity for self-renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin-producing oligodendrocytes. Nestin-positive cells from the stroke-affected cortex migrated into the peri-infarct area and differentiated into glial cells in vivo . Although we could not detect differentiation of nestin-positive cells into neurons in vivo , our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post-stroke cerebral cortex.     

小鼠局灶性脑缺血再灌注后IL-33及其受体的时程表达

目的检测白细胞介素-33(IL-33)及其膜受体ST2和可溶型受体sST2在小鼠局灶性脑缺血再灌注后不同时程的表达特征。方法利用线栓法闭塞大脑中动脉(MCAO)30 min诱导建立小鼠可逆性局灶性脑缺血再灌注损伤模型,通过半定量RT-PCR检测脑缺血再灌注后6 h、24 h和3 d缺血脑组织中IL-33及其膜受体ST2、凋亡相关蛋白Caspase-8和Caspase-3的mRNA表达水平,并通过免疫组织化学染色观察了IL-33在不同缺血脑区(运动皮质、感觉皮质、海马和纹状体)的时程表达情况;ELISA法检测了小鼠MCAO模型再灌注后不同时间点血清中IL-33及其可溶型受体sST2的表达水平。结果 IL-33 mRNA在缺血后6 h和3 d表达减少,但在24 h无明显改变;凋亡相关蛋白Caspase-3和Caspase-8在缺血后3个时间点均显著增高,且Caspase-3在6 h和3 d的mRNA表达水平较24 h高;ST2 mRNA在缺血后6 h无减少,但在24 h和3 d有明显减少;除了MCAO 24 h组运动皮质和纹状体阳性染色增加外,IL-33阳性细胞数在缺血后不同时程各脑区均有不同程度减少;缺血后外周血中IL-33的表达量无明显升高或降低,而sST2的表达水平在缺血后6 h即已显著升高。结论脑缺血再灌注后IL-33/ST2信号通路被下调,其与sST2表达增多的效应发挥和神经元凋亡有关。     

缺血脑组织内皮素含量变化的实验研究

为探讨内皮素含量变化是否为缺血神经元损伤的危险因素及Ca~(2+)拮抗剂氟桂嗪对脑缺血的保护作用,采用放射免疫分析法,我们测定兔大脑中动脉阻断48小时后缺血区脑组织内皮素含量变化及氟桂嗪对其变化的影响。结果显示,缺血48小时后梗塞区脑组织内皮素含量明显升高(P<0.01),为对照组的10倍,而氟桂嗪能明显降低缺血区脑组织水、内皮素含量(P<0.05)。上述结果提示:缺血脑组织内皮素含量升高是导致缺血神经元损伤的重要因素,氟桂嗪对脑缺血有保护作用。     

Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study

As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain–blood gradient. In this regard, the ability of glutamate oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.     

Transplantation of neural stem cells expressing hypoxia-inducible factor-1α (HIF-1α) improves behavioral recovery in a rat stroke model

We explored the possibility that hypoxia-inducible factor-1α (HIF-1α) might contribute to the therapeutic effect of neural stem cell (NSC) transplantation in cerebral ischemia. The relative efficacy of modified NSC to promote behavioral recovery was investigated in a rat model of stroke induced by a transient middle cerebral artery occlusion (MCAO). A recombinant adenovirus (Ad-HIF-1α) was engineered to express HIF-1α. Control NSC infected with control adenovirus (NSC-Ad), recombinant adenovirus Ad-HIF-1α, or NSC infected by Ad-HIF-1α (NSC-Ad-HIF-1α), were used for intraventricular transplantion into rat brain 24 hours after MCAO. Neurological deficits were assessed over 4 weeks using the modified neurological severity scale (NSS) score. Long-term in vivo expression of HIF-1α was demonstrated by Western blotting and immunocytochemistry, and derivatives of nestin-positive transplanted cells contributed to both neuronal (neurofilament-positive) and astroglial (glial fibrillary acidic protein-positive) lineages. All animals showed functional improvement. Improvement was accelerated in animals receiving either NSC-Ad or Ad-HIF-1α, while improvement at all times between 7 days and 28 days post MCAO was significantly greater in animals transplanted with NSC-Ad-HIF-1α than for other treated animals. NSC-Ad-HIF-1α cells also increased the number of factor VIII-positive cells in the region of ischemic injury, indicating that HIF-1α expression can promote angiogenesis. Gene-modified NSC expressing HIF-1α have therapeutic potential in ischemic stroke.     

大鼠局灶脑缺血后钙调神经磷酸酶活性和含量变化

目的 研究大脑中动脉闭塞后钙调神经磷酸酶（Calcineurin,CaN)的活性和含量变化规律。探讨CaN在脑缺血中的作用。方法 制备大鼠大脑中动脉永久性闭塞模型。分别测定缺血后不同时间点病灶侧大脑皮质和海马CA1区CaN的活性和含量。结果 皮质组织在缺血6h及其后各时间点CaN 的含量下降，其活性于缺血后4h,6h和12h短暂性增强。海马CA1区CaN的含量于缺血后24h开始降低且不恢复；CaN的活性在缺血后2h,4h和6h减弱。12h始恢复至正常水平。可见，CaN的活性与含量分离。结论 局灶脑缺血后CaN独特的时间变化规律显示其参与介导缺血性神经元损伤。可能具有毒性作用。     

小鼠真皮多能干细胞在缺血性损伤脑组织移植后的分布与分化

目的探讨小鼠真皮多能干细胞(SKP)经股静脉移植后在缺血性损伤脑组织中的分布及分化情况。方法分离培养绿色荧光蛋白转基因小鼠(C57BL/6-gfp)SKP,随机选取5只同基因型小鼠采用线栓法制作局灶性大脑中动脉栓塞(MCAO)模型,缺血2h后行再灌注,再灌注24h后将C57BL/6-gfp来源的SKP经小鼠股静脉输入动物模型体内,植入后第7天处死小鼠,作冷冻切片,采用免疫组织荧光染色法,检测SKP在脑缺血小鼠体内的分布及分化情况。结果移植SKP7d后,MCAO小鼠脑组织冷冻切片在荧光显微镜下可见移植的SKP主要分布在缺血灶周围,且这些细胞表达胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)。结论经股静脉移植的SKP主要分布在MCAO模型鼠脑缺血损伤区周围,并可向神经细胞分化。     

Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice.

The authors transplanted adult bone marrow nonhematopoietic cells into the striatum after embolic middle cerebral artery occlusion (MCAO). Mice (n = 23; C57BL/6J) were divided into four groups: (1) mice (n = 5) were subjected to MCAO and transplanted with bone marrow nonhematopoietic cells (prelabeled by bromodeoxyuridine, BrdU) into the ischemic striatum, (2) MCAO alone (n = 8), (3) MCAO with injection of phosphate buffered saline (n = 5), and (4) bone marrow nonhematopoietic cells injected into the normal striatum (n = 5). Mice were killed at 28 days after stroke. BrdU reactive cells survived and migrated a distance of approximately 2.2 mm from the grafting areas toward the ischemic areas. BrdU reactive cells expressed the neuronal specific protein NeuN in 1% of BrdU stained cells and the astrocytic specific protein glial fibrillary acidic protein (GFAP) in 8% of the BrdU stained cells. Functional recovery from a rotarod test (P < 0.05) and modified neurologic severity score tests (including motor, sensory, and reflex; P < 0.05) were significantly improved in the mice receiving bone marrow nonhematopoietic cells compared with MCAO alone. The current findings suggest that the intrastriatal transplanted bone marrow nonhematopoietic cells survived in the ischemic brain and improved functional recovery of adult mice even though infarct volumes did not change significantly. Bone marrow nonhematopoietic cells may provide a new avenue to promote recovery of injured brain.     

C—fos反义寡脱氧核苷酸部分阻断MCAO大鼠缺血侧皮质内BDNF的表达

用c fos反义寡脱氧核苷酸侧脑室微量注射和细胞免疫化学等技术和方法 ,探讨大鼠局灶性脑缺血(MCAO)模型中 ,即早反应基因c fos表达与脑源性神经营养因子 (BDNF)表达的关系。结果表明 ,局灶性脑缺血再灌注可引起c fos和BDNF在缺血侧皮质的大量表达。侧脑室微量注射c fos反义寡脱氧核苷酸后 ,脑内BDNF的部分表达明显被阻断 ,脑缺血损伤加重。提示脑缺血损伤后 ,脑内BDNF的表达对脑缺血再灌注损伤起一定的保护作用 ;脑缺血后BDNF的表达可能部分通过c fos调控。