次声对成年大鼠齿状回神经前体细胞增殖的影响

目的研究次声对成年大鼠海马齿状回神经前体细胞增殖的影响。方法大鼠随机等分为正常对照组、假次声组和次声组（每组16只）。次声组暴露于8Hz、130dB次声环境7d（2h／d），暴露结束后第1、3、7、14d处死，采用抗5-溴脱氧尿嘧啶尿苷（BrdU）免疫组化方法，观察齿状回BrdU阳性细胞数的变化。结果次声作用结束后第1d，齿状回BrdU阳性细胞数与假次声组和正常对照组相比均无统计学差异；第3d及第7d，BrdU阳性细胞数减少（P〈0．05），第14d恢复正常水平。结论8Hz、130dB次声可抑制正常成年大鼠海马神经前体细胞增殖，可能与次声引起大鼠脑内微环境改变有关。     

成体脑室管膜下区神经发生的研究进展

室管膜下区(SVZ)是指沿着整个脑室侧壁分布的区域,在成年哺乳动物终生存在着神经发生。许多神经退行性疾病的病理过程可促进该区域的神经发生,这些新生的SVZ区神经细胞可以迁移到病灶处,以取代或修复死亡或损伤的细胞。本文介绍了成体SVZ的起源、结构特征和近几年来SVZ神经发生与神经退行性疾病相关性研究的进展。     

大鼠脑皮质电凝损伤对室下区细胞增殖的影响

作为已知的神经发生区，室下区邻近大脑皮质和一些具有重要功能的前脑神经核，室下区的神经发生为脑损伤后的神经修复带来了希望，有资料研究了单侧大脑皮质抽吸损伤和液体冲压伤对室下区的神经发生的影响。本实验的目的在于研究大鼠大脑皮质电凝伤后内源性神经干细胞的增殖性反应和增殖的时间过程。     

嗅球电刺激对成年大鼠室管膜下区神经发生的影响

目的 探讨嗅球电刺激对成年大鼠室管膜下区(SVZ)神经前体细胞(NPC)增殖及其向嗅球迁移的影响,并对其相关机制进行初步探讨. 方法 SD大鼠80只按随机数字表法分为正常对照组、假刺激组、电刺激ld组、电刺激3d组、电刺激1周组、电刺激2周组、电刺激3周组、电刺激4周组,每组10只,后6组大鼠制备嗅球电刺激模型,以5-溴脱氧尿嘧啶(Brdu)标记新生细胞,免疫组化染色观察大鼠SVZ区内源性NPC的增殖,RT-PCR检测嗅球内Prokineticin 2 (prk2)mRNA的表达；另取大鼠15只,按随机数字表法分为正常对照组、假刺激组和电刺激4周组,每组5只,免疫组化染色观察嗅球内Brdu阳性细胞的表达. 结果 8组大鼠SVZ区Brdu阳性细胞数不同,差异有统计学意义(F=51.475,P=0.000).与正常对照组和假刺激组比较,电刺激ld、3d、l周、2周组大鼠刺激侧Brdu阳性细胞增高,差异有统计学意义(P＜0.05)；8组大鼠嗅球内prk2 mRNA的表达不同,差异有统计学意义(F=154.067,P=0.000).与正常对照组和假刺激组比较,电刺激ld、3d、l周、2周、3周、4周组prk2 mRNA增加,差异有统计学意义(P＜0.05)；3组大鼠嗅球内Brdu阳性细胞数不同,差异有统计学意义(F=36.472,P=0.000).电刺激4周组嗅球内Brdu阳性细胞数较正常对照组和假刺激组明显增多,差异有统计学意义(P＜0.05). 结论 电刺激嗅球可促进SVZ区NPC的增殖并向嗅球迁移,这可能与电刺激提高嗅球内Prk 2的表达水平有关.     

N-甲基-D-天冬氨酸受体亚单位2B特异性拮抗剂对缺氧缺血性脑损伤新生大鼠脑室下区神经干细胞增殖的影响

目的:研究N-甲基-D-天冬氨酸受体(NMDAR)亚单位2B(NR2B)特异性拮抗剂(Ro 25-6981)对缺氧缺血性脑损伤(HIBD)新生大鼠脑室下区(SVZ)神经干细胞(NSCs)增殖的影响。方法:7 d龄新生SD大鼠随机分为Ro 25-6981组(HIBD前2 h,腹腔注射Ro 25-6981 10 mg.kg-1)、HIBD组(HIBD前2 h,腹腔注射等剂量生理盐水)和假手术组(仅游离右侧颈总动脉,不结扎)。采用免疫组化学染色检测SVZ Nestin表达量及BrdU阳性细胞数的变化。结果:HIBD组12h后Nestin表达量开始增多,48 h达峰值,之后缓慢下降;与其相比,Ro 25-6981组在12 h和24 h时下降明显(P<0.05)。HIBD组BrdU阳性细胞数在缺氧缺血3 h后缓慢上升,72 h达高峰;与其相比,Ro 25-6981组在各时间点BrdU阳性细胞表达均有所下降,以24、48和72 h减少明显,尤以72 h为著(P<0.05)。结论:Ro 25-6981能够降低HIBD新生大鼠SVZ Nestin的表达及Brdu阳性细胞数,对SVZ NSCs增殖起抑制作用,提示NR2B参与并促进HIBD引起的SVZ NSCs的增殖。     

成年大鼠脑室下区神经干细胞上N-甲基-D-天冬氨酸受体亚单位1的超微分布

以免疫电镜和免疫组化及免疫金-银双重标记技术,从超微结构方面对N-甲基-D-天冬氨酸受体亚单位1在神经干细胞上进行准确定位,结果证实脑室下区存在神经干细胞,是成年哺乳动物中枢神经系统重要的发生区,而N-甲基-D-天冬氨酸受体亚单位1的表达比Nestin高,且分布于神经干细胞胞膜、胞浆、以及粗面内质网、高尔基复合体上。     

硫胺素对MPTP致成年小鼠脑室管膜下区神经发生受损的保护作用

目的：探讨硫胺素对1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）诱导的急性帕金森病（PD）小鼠模型脑室管膜下区（SVZ）神经发生的保护作用。方法：给予C57BL小鼠腹腔注射MPTP,1 d内连续4次,每次注射间隔2 h,每次20 mg.kg-1,制作PD模型（PD组）;在PD组基础上给予小鼠腹腔注射硫胺素100 mg.kg-1.d-1（从MPTP使用前1周开始注射至MPTP使用后1周）设为硫胺素干预PD组（PDT组）。另设正常对照组（Ctrl组）和正常硫胺素组（CtrlT组）。利用免疫荧光染色法检测各组小鼠脑SVZ区5-溴脱氧尿嘧啶核苷（BrdU）和双皮质蛋白（Dcx）阳性细胞的分布和数目。结果：PD组SVZ区BrdU和Dcx阳性细胞与Ctrl组比较均显著减少（P〈0.05）;PDT组SVZ区BrdU和Dcx阳性细胞与PD组比较明显增加（P〈0.05）。结论：MPTP诱导的急性PD小鼠模型脑SVZ区神经发生明显减少,硫胺素对PD小鼠模型脑SVZ区受损的神经发生具有保护作用。     

脑梗死后康复训练对成年大鼠海马结构中神经干细胞的影响

目的　研究成年大鼠局灶性脑缺血损伤(脑梗死)后康复训练对海马结构中内源性神经干细胞增殖的影响,探讨脑梗死后康复训练使神经功能改善的理论基础。方法　采用线栓法造成成年大鼠永久性MCAO(middle cerebral artery occulsion),形成脑梗死动物模型。大鼠脑梗死24小时后,随机分为梗死对照组和梗死后康复训练组。康复训练组每天进行平衡木、转棒、滚笼训练,对照组不进行训练饲养于标准笼中。运用免疫组化方法,通过神经上皮干细胞蛋白即 nestin标记神经干细胞,观察、比较脑梗死后7天、14天、21天时两组大鼠海马结构中 nestin阳性细胞分布和数量的变化及差异。结果　脑梗死后两组大鼠梗死侧海马结构中nestin阳性细胞均较对侧显著增多,海马附近的侧脑室后角与CA1区之间及齿状回中阳性细胞密集,其中脑梗死后7天阳性细胞最多,以后逐渐减少。脑梗死后7天及14天康复训练组大鼠梗死侧海马结构中nestin阳性细胞数较对照组显著增加 (P < 0.01),脑梗死后21天两组无显著性差异(P >0.05)。结论　康复训练可以促进成年大鼠局灶性脑缺血损伤(脑梗死)后海马结构中神经干细胞增殖水平上调,这可能是脑梗死后康复训练有助于缺损的神经功能恢复的一个机制。     

食蟹猴脑室下区成体神经前体细胞培养及鉴定

目的分离、培养成年食蟹猴脑室下区神经前体细胞并研究其生物学特性。方法取成年食蟹猴脑室下区组织,经木瓜蛋白酶和脱氧核糖核酸酶消化后,接种于DMEM-F12无血清培养基中。结果神经前体细胞可以增殖形成神经球,经免疫细胞化学方法检验这些细胞呈现巢蛋白nestin阳性,神经球分化后的细胞表达胶质细胞的标记物及神经元的标记物。脑室下区来源的神经球进行自主分化时可分化成神经元和胶质细胞,但分化成神经元的比例都很少。结论从食蟹猴脑室下区分离培养的细胞可以在体外增殖形成神经球,并可分化为神经元和胶质细胞,符合神经前体细胞的生物学性状。     

Gypenosides pre-treatment protects the brain against cerebral ischemia and increases neural stem cells/progenitors in the subventricular zone

Gypenosides (GPs) have been reported to have neuroprotective effects in addition to other bioactivities. The protective activity of GPs during stroke and their effects on neural stem cells (NSCs) in the ischemic brain have not been fully elucidated. Here, we test the effects of GPs during stroke and on the NSCs within the subventricular zone (SVZ) of middle cerebral artery occlusion (MCAO) rats. Our results show that pre-treatment with GPs can reduce infarct volume and improve motor function following MCAO. Pre-treatment with GPs significantly increased the number of BrdU-positive cells in the ipsilateral and contralateral SVZ of MCAO rats. The proliferating cells in both sides of the SVZ were glial fibrillary acidic protein (GFAP)/nestin-positive type B cells and doublecortin (DCX)/nestin-positive type A cells. Our data indicate that GPs have neuroprotective effects during stroke which might be mediated through the enhancement of neurogenesis within the SVZ. These findings provide new evidence for a potential therapy involving GPs for the treatment of stroke.     

电刺激嗅球对脑室下区神经干细胞增殖、迁移和分化的影响

目的 探讨电刺激嗅球对脑室下区(SVZ)神经干细胞(NSC)增殖、迁移及其分化的影响.方法 取SD大鼠48只,随机分为正常对照组、假刺激组、电刺激1 d、3 d、7 d、14 d、21 d、28 d组,以5-溴脱氧尿嘧啶(BrdU)标记NSC,免疫组化法检测SVZ区NSC的增殖情况.另取大鼠18只,随机分为正常对照组、假刺激组和电刺激组,以免疫组化法观察NSC向嗅球迁移情况,荧光双标法检测NSC分化情况.结果 电刺激1 d,SVZ区BrdU阳性细胞开始增高,第7天达高峰,第3周达对照组水平.注射BrdU28 d后,电刺激组喙侧迁移流(RMS)及嗅球内的BrdU阳性细胞数较正常对照组明显增多,但两组NSC分化情况没有明显差别.结论 电刺激嗅球可促进SVZ区NSC增殖及迁移,但对其分化没有影响.

Abstract：

Objective To investigate the effects of electrical stimulation of olfactory bulb( OB) on the proliferation, migration and differentiation of neural stem cell ( NSC ) in subventricular zone. Method Forty - eight adult female Sprague - Dawley(SD) rats were randomly divided into control group, sham stimulation group and stimulation group including 6 time points(1 day,3 day,7 day, 14 day,21 day, 28 day). The rats were injected intraperitoneally with bromodeoxyuridine( BrdU) to detect the proliferation of NSC by immunohistochemistry staining. Another 18 rats were randomly divided into control group, sham stimulation group and stimulation group. Four weeks after BrdU injection, the rats were sacrificed and immunohistochemistry and immunofluorescence were used to investigate the migration and differentiation of NSC in OB. Results In SVZ, BrdU - positive cells began to increase 1 d after stimulation (17. 67 ± 1.03, P <0.01) .reached to the maximum level at 1 week(28. 50 ± 1. 87, P <0. 01) ,then decreased to normal at 3 week. Four weeks after injection of BrdU,the BrdU -positive cells significantly increased in RMS(67. 33 ±3.50, P <0.01) and OB(44.33 ±5.47, P <0.01) in stimulation group. Fluorescence double staining showed that the stimulation of OB had no effect on the differentiation of NSC into neurons or gliocytes. Conclusions Electrical stimulation of OB could promote proliferation, migration of NSC in SVZ, but it has no effect on the differentiation of NSC into neurons or gliocytes.     

新生大鼠海马区及室管膜下区神经干细胞分离、培养和分化特性

目的建立大鼠神经干细胞（NSCs）分离、培养方法，观察其生长、增殖和分化特点。方法利用无血清培养技术，从新生大鼠海马、室管膜下区分离NSCs，进行体外扩增培养、传代观察。采用荧光免疫细胞化学检测技术，观察鉴定NSCs及其分化结果。结果分离获取的细胞具有自我更新和增殖能力，原代及传代培养均可形成细胞克隆，克隆中的细胞巢蛋白（nestin）表达阳性，显微镜下观察见典型的干细胞特征，诱导后可分化神经元和星形胶质细胞。结论上法分离培养的细胞为具有自我更新和增殖能力的NSCs，可诱导分化为终末神经细胞。     

Nitric oxide synthesis inhibition increases proliferation of neural precursors isolated from the postnatal mouse subventricular zone

The subventricular zone (SVZ) of rodents retains the capacity to generate new neurons throughout the entire life of the animal. Neural progenitors of the SVZ survive and proliferate in vitro in the presence of epidermal growth factor (EGF). Nitric oxide (NO) has been shown to participate in neural tissue formation during development and to have antiproliferative actions, mediated in part by inhibition of the EGF receptor. Based on these findings, we have investigated the possible effects of endogenously produced and exogenously added NO on SVZ cell proliferation and differentiation. Explants were obtained from postnatal mouse SVZ and cultured in the presence of EGF. Cells migrated out of the explants and proliferated in culture, as assessed by bromodeoxyuridine (BrdU) incorporation. After 72 h in vitro, the colonies formed around the explants were constituted by cells of neuronal or glial lineages, as well as undifferentiated progenitors. Immunoreactivity for the neuronal isoform of NO synthase was observed in neuronal cells with long varicose processes. Cultures treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed an increase in the percentage of BrdU-immunoreactive cells, whereas treatment with the NO donor diethylenetriamine-nitric oxide adduct (DETA-NO) led to a decrease in cell proliferation, without affecting apoptosis. The differentiation pattern was also altered by L-NAME treatment resulting in an enlargement of the neuronal population. The results suggest that endogenous NO may contribute to postnatal neurogenesis by modulating the proliferation and fate of SVZ progenitor cells.     

Moderate fetal alcohol exposure impairs neurogenic capacity of murine neural stem cells isolated from the adult subventricular zone

Gestational alcohol exposure leads to a spectrum of neurological symptoms which range from severe mental retardation caused by high dose exposure, to subtle cognitive and neuropsychiatric symptoms caused by low-to-moderate doses. We and other investigators have demonstrated that exposure to moderate levels of alcohol throughout gestation leads to impaired neurogenesis in the adult hippocampus, although the mechanisms by which this occurs are not known. To begin to distinguish cell-intrinsic from microenvironmental contributions to impaired adult neurogenesis, we isolated neural stem progenitor cells (NSPCs) from the adult SVZ of mice exposed to moderate levels of alcohol throughout gestation. We found that NSPCs isolated from fetal alcohol exposed (FAE) mice displayed reduced neurosphere formation in culture, as assessed by a serial passage neurosphere assay, and reduced neuronal differentiation upon growth factor withdrawal. These studies suggest that gestational alcohol exposure leads to long-lasting NSPC-intrinsic dysregulation, which may underlie in vivo neurogenic deficits.     

Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors from the subventricular zone in adult mice

The neuropeptide Y (NPY) is widely expressed in the central nervous system and has been shown to stimulate neurogenesis in the hippocampus and the olfactory epithelium. Here, we demonstrate that intracerebroventricular injection of NPY stimulates proliferation of neural precursors in the mice subventricular zone (SVZ), one the most neurogenic areas of the brain. Newly generated neuroblasts migrate through the rostral migratory stream to the olfactory bulb and also directly to the striatum, as evidenced by BrdU labelling and cell phenotyping. Using knock-out mice, specific NPY receptor agonists and antagonists, we report that this neuroproliferative effect is mediated by the Y1 receptor subtype that we found to be highly expressed in the SVZ both at the mRNA and protein levels. Our data suggest that stimulating endogenous SVZ neural stem cells by NPY may be of a potential interest in cell replacement based therapies of neurodegenerative diseases affecting the striatum such as Huntington's disease.     

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats (7 days old) subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2′-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2′- deoxyuridine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.     

Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone

An understanding of the regulators of neurogenesis in the normal and diseased brain is necessary in order to recruit endogenously produced neural precursors for cell replacement in neurodegenerative disorders such as Parkinson's disease. The location of dopaminergic projections from the midbrain to the neostriatum and nucleus accumbens overlaps with the most active region of neurogenesis in the adult brain, the subventricular zone of the anterior lateral ventricle. This suggests that dopamine may contribute to regulation of the subventricular niche of adult neurogenesis. Here, we show in adult mice that destruction of the dopaminergic neurons in the substantia nigra and ventral tegmental area in a 6-hydroxydopamine model of Parkinson's disease reduced the number of proliferating neural precursors in the subventricular zone of the anterior lateral ventricle by approximately 40%. The effect on neural precursor proliferation correlated with the extent of dopaminergic denervation in the neighboring neostriatum. This identifies dopamine as one of the few known endogenous regulators of adult neurogenesis with implications for the potential use of endogenous neural precursors in cell replacement strategies for Parkinson's disease.