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。     

硫胺素缺乏下调与学习相关的成体海马神经发生

目的 研究硫胺素缺乏(TD)对成年小鼠认知功能及其海马神经发生的影响.方法 TD小鼠模型按照TD处理时间分为TD 7、9、14、16、25 d组和正常对照组,每组4～6只小鼠.Y型迷宫检测学习记忆能力,5-溴脱氧尿嘧啶核苷(BrdU)体内掺入及Doublecortin(Dcx)等免疫组化染色检测海马齿状回颗粒下层(DGSG)神经发生.结果 在无病理损害及胆碱能神经变性的TD 9 d组,小鼠学会正确反应的总训练次数(22.3±2.2)与对照组(13.5±3.5)相比差异有统计学意义,而海马DGSG BrdU标记的阳性细胞数(个/DGSG)19.8±0.4及Dcx标记免疫活性(像素/mm2)1537.2±50.2也较对照组(分别为23.9±0.3和2688.9±127.9)显著降低.此时补充硫胺素可上调BrdU阳性细胞数(个/DGSG)23.6±1.9及Dcx免疫活性(像素/mm2)2052.3±269.6,并减少其学会正确反应的训练总次数(16.8±0.5).结论 在病理损害前期,TD导致的学习功能障碍可能与海马神经发生功能下调有关.     

次声对成年大鼠脑室下区神经前体细胞增殖的影响

目的探讨次声作用对成年大鼠脑室下区（SVZ）神经前体细胞增殖的影响。方法成年雄性Sprague-Dawley大鼠（n=24）随机分为正常对照组、对照组和次声组,次声组动物置于次声压力仓（16Hz、130dB）内连续作用7d（2h/d）。照射结束后,分别于3d、7d、10d和14d处死。处死前各组大鼠腹腔注射5-溴脱氧尿嘧啶尿苷（BrdU）3次,以BrdU免疫组织化学法检测各组SVZ内增殖细胞数目的变化。结果次声照射结束后3d,次声处理组动物SVZ区的BrdU阳性细胞数目较对照组显著减少（P〈0.01）,7d时阳性细胞数继续降低（P〈0.01）,但10d时开始逐渐恢复,14d时达到正常水平（P〉0.05）。结论16Hz、130dB次声可抑制成年大鼠SVZ神经前体细胞增殖。     

血管性认知障碍小鼠脑内少突胶质细胞再生分化障碍特点研究

目的 观察血管性认知障碍小鼠模型中,缺血性炎性损伤对室管膜下区及海马齿状回少突胶质细胞 再生分化的影响,为血管性认知障碍的缺血性炎症机制提出新的损伤途径。 方法 成年雄性CD1小鼠随机分为模型组和假手术组,每组24只,模型组采用双侧颈动脉反复缺 血再灌注法制备血管性认知障碍小鼠模型。造模后4～6 d连续腹腔注射5 -溴脱氧尿嘧啶核苷 （bromodeoxyuridine,BrdU）（150 mg/kg）标记新生细胞,分别于术后14 d和28 d每组随机取一半小鼠脑 组织进行脑切片免疫组化、免疫荧光双标共聚焦检测,标记脑组织室管膜下区和海马区的少突胶质 细胞、星形胶质细胞及神经元,观察新生少突胶质细胞增殖及分化情况,并观察星形胶质细胞的增 生活化情况。 结果 造模后14 d和28 d室管膜下区新生细胞（BrdU阳性细胞）在模型组较假手术组明显增加（P均 ＜0.001),造模28 d模型组新生神经元（BrdU/NeuN阳性细胞）较假手术组显著增加（P＜0.001)。与假 手术组相比较,术后28 d模型组海马齿状回少突胶质细胞祖细胞显著增多（P＜0.001）;少突胶质细 胞前体细胞显著减少（P =0.006）。造模后28 d模型组海马齿状回新生星形胶质细胞（BrdU/GFAP阳性 细胞）较假手术组显著增加（P =0.015）。 结论 血管性认知障碍小鼠内源性新生细胞增殖区室管膜下区与海马齿状回区均存在新生细胞反 应性增生的情况。新生细胞区分化的主要细胞为星形胶质细胞,而少突胶质细胞分化障碍,可能是血 管性认知障碍患者影像学常见皮层下白质病变的重要原因。     

NMDA受体拮抗剂MK-801对新生大鼠室管膜下区胶质纤维酸性蛋白表达的影响

目的研究N-甲基-D-天冬氨酸(NMDA)受体拮抗剂MK-801对新生7 d大鼠室管膜下区(SVZ)胶质纤维酸性蛋白(GFAP)表达的影响。方法将40只新生SD大鼠分成对照组和MK-801组,各组按出生后(P)时间点再随机分成4个亚组:P7 d、P14 d、P21 d、P28 d组。新生大鼠均于生后第3天给药,MK-801组腹腔注射MK-801 10 mg.kg-1;对照组腹腔注射同量生理盐水。通过免疫组化学方法观察大鼠SVZ区GFAP阳性细胞数。结果①对照组GFAP阳性细胞数于P14 d开始增加,至P21 d达最大值;但P28 d时阳性细胞迅速下降;②MK-801组GFAP阳性细胞数与对照组相比,P7 d和P28 d无明显差异,P14 d(65.40±6.11)和P21 d(239.60±12.92)细胞数明显减少;而对照组P14 d(79.20±5.26)、P21 d(265.20±7.40)GFAP阳性细胞数明显增多,差异有显著统计学意义(P<0.01)。结论 NMDA受体拮抗剂MK-801对正常新生大鼠SVZ区GFAP的表达有抑制作用,能够抑制SVZ区神经干细胞的增殖和分化。     

实验性自身免疫性脑脊髓炎对大脑神经干细胞增殖的影响

目的探讨实验性自身免疫性脑脊髓炎(EAE)埘大脑神经干细胞(NSC)增殖能力的影响。方法将C57BL/6小鼠随机分为模型组、佐剂组和对照组,采用少突胶质细胞糖蛋白(MOG)抗原联合完全福氏佐剂(CFA)免疫模型组小鼠,佐剂组小鼠仅接受CFA免疫注射,对照组小鼠仅接受生理盐水注射。观察各组小鼠EAE症状评分,采用HE染色以及劳克坚牢蓝(LFB)髓鞘染色观察其脑和脊髓病理学改变,采用免疫荧光染色检测小鼠海马齿状回(DG)和侧脑室下区(SVZ)BrdU阳性细胞数量,以评价NSC增殖情况。结果模型组有14只(14/15)小鼠被成功诱导EAE,其症状达到高峰后短暂缓解,随后进入慢性持续期;而佐剂组和对照组小鼠均未出现任何EAE症状。模型组中枢神经系统存在大量炎性反应细胞浸润,在白质区可见多处LFB未着色的髓鞘脱失区;而佐剂组和对照组无明显病理学改变。佐剂组与对照组小鼠DG区和SVZ区BrdU阳性细胞数比较差异均无统计学意义。与佐剂组和对照组比较,模型组小鼠大脑SVZ区和DG颗粒下区BrdU阳性细胞均显著降低(均P0.05)。结论慢性持续型EAE可导致小鼠大脑NSC增殖能力下降。     

三重转基因阿尔茨海默病动物模型的神经再生研究

目的 探讨不同鼠龄APP/PS1/Tau三重转基因阿尔茨海默病小鼠(3xTg-AD)室管膜下层(Subventricular zone, SVZ)及海马齿状回(dentate gyrus, DG)神经细胞增殖变化及与β淀粉样斑块的关系.方法 采用3月龄和12月龄A PP /PS1/Tau三重转基因小鼠(3 x T g-AD)作为实验组,同龄同种系野生型小鼠作为对照组,BrdU标记增殖细胞.Doublecortin(DCX),BrdU免疫荧光双标染色未成熟神经细胞,计数室管膜下层和海马齿状回BrdU阳性细胞数和DCX/BrdU双标阳性细胞数.免疫组化法检测β淀粉样斑块.结果 12月龄组3 x Tg-AD小鼠与对照组野生型小鼠相比,室管膜下区及海马齿状回BrdU阳性细胞明显减少(P<0.05),3月龄组无明显差异(P＞0.05).3月龄组3 x Tg-AD小鼠未见β淀粉样斑块形成,12月龄组3xTg-AD小鼠有明显的β淀粉样斑块形成.结论 12月龄3 x Tg-AD小鼠室管膜下层及海马齿状回脑内神经再生能力明显下降,与β淀粉样斑块形成可能相关.     

半乳凝素1对脑损伤大鼠室管膜下区内源性神经干细胞原位增殖及迁移的影响

背景：半乳凝素1表达于室管膜下区的星形胶质细胞中并诱导其分化，分化的星形胶质细胞可明显提高脑源性神经生长因子的产生。 目的：观察侧脑室注入半乳凝素1对脑缺血损伤大鼠内源性神经干细胞增殖、迁移的影响。 设计、时间及地点：细胞学体内观察实验，于2007-03/11在佳木斯大学神经科学研究所完成。 材料：纯种清洁级成年Wistar大鼠48只，随机分为模型组、药物组，24只/组。半乳凝素1为北京晶美生物工程公司产品。 方法：两组大鼠均采用线栓法制作局灶性大脑中动脉闭塞模型。造模后24 h，药物组经右侧侧脑室注入10 μL浓度为0.2 g/L的半乳凝素1，模型组注射等量生理盐水。分别于缺血再灌注后3，7，14，28 d处死大鼠，取脑组织制作石蜡切片，处死前1 d腹腔注射BrdU。 主要观察指标：免疫组织化学染色检测大脑室管膜下区BrdU和巢蛋白阳性细胞的表达。 结果：模型组缺血再灌注3 d后大脑室管膜下区BrdU、巢蛋白阳性细胞开始增加，7 d增殖达高峰，14 d后表达开始下降，28 d后下降至最低。药物组缺血再灌注3 d后大脑室管膜下区两种阳性细胞均明显增加；7 d 增殖达高峰，半定量分析BrdU、巢蛋白阳性细胞数分别是模型组的2倍和1.5倍，且BrdU阳性细胞向腹外侧迁移，巢蛋白阳性细胞胞体变大，突起增长，并有向外侧迁移进入脑实质的迹象；14 d后开始下降；28 d降至最低，但仍明显多于模型组（P < 0.05）。 结论：经侧脑室注入半乳凝素1在大鼠脑缺血后可激活内源性神经干细胞原位增殖，并存在由增殖区向外周脑实质迁移的趋势。     

经鼻给予酸性成纤维细胞生长因子对脑梗死大鼠的血管和神经再生作用

目的研究经鼻给予酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）对脑梗死后神经和血管再生及神经功能恢复的影响。方法健康雄性SD大鼠30只,随机分为aFGF组（n=12）、对照组（n=12）和假手术组（n=6）;aFGF组和对照组大鼠建立大脑中动脉闭塞模型,脑缺血再灌注24h后经鼻分别给予10μg aFGF（200μl）和等容积生理盐水,1次／d,连续7d;同时腹腔注射5-溴脱氧尿核苷（Bromodeoxyuridine,BrdU）50mg／kg,1次/d,连续13d;假手术组操作过程和给药与对照组相同,但不用线栓阻塞大脑中动脉;分别在术前和术后第1、7、14d采用改良神经功能评分评价大鼠神经功能改变情况,并于术后第14d经尾静脉注入异硫氰酸荧光素右旋糖酐（FITC—dextran）,采用免疫组化及激光共聚焦方法分别检测梗死灶周、室管膜下区和纹状体BrdU阳性细胞及微血管的数量。结果术后第7及14d aFGF组神经功能评分显著低于对照组;术后第14d aFGF组缺血侧室管膜下区和纹状体BrdU阳性细胞数与对照组相比均显著增高（P〈0.01）,假手术组仅见少量BrdU阳性细胞;激光共聚焦显示aFGF组梗死灶周微血管数较对照组显著增加（P〈0.05）;同时,与对照组相比经鼻给予aFGF能增加Brdu阳性细胞在血管内皮细胞中的百分比,且有统计学意义（P〈0.05）。结论经鼻给予aFGF能有效促进神经和血管再生,改善脑缺血后神经功能评分,对于治疗脑梗死具有潜在的应用前景。     

实验性脑出血SVZ细胞再生的实验研究

目的明确脑出血后是否存在内源性的神经细胞再生现象。方法 SD大鼠随机分为对照组和脑出血组,每组分为1、3、7、14、21、28d亚组。应用胶原酶脑内立体定向注射制作脑出血模型,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7d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(P0.05),脑出血后14d同侧SVZ和基底节BrdU阳性细胞达到高峰,与对照组比较差异具有显著性(P0.01),然后逐渐下降,28d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(P0.05)。结论脑出血后存在内源性神经细胞再生现象。     

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.     

Impaired hippocampal neurogenesis is involved in cognitive dysfunction induced by thiamine deficiency at early pre-pathological lesion stage

It has not been reported whether thiamine deficiency (TD) affects hippocampal neurogenesis or not. Here, we explored the influence of TD at early pre-pathological lesion stage on hippocampal neurogenesis and the correlation between affected hippocampal neurogenesis and cognitive dysfunction. We prepared TD mouse model by feeding a thiamine-depleted diet. Learning and memory functions of TD mice were tested with Y-maze. Hippocampal neurogenesis was studied with BrdU, PCNA, Dcx, and NeuN immunohistochemical staining. The results showed significant decline in learning ability and hippocampal neurogenesis simultaneously since 9-days of treatment when the model mice did not exhibit regular pathological lesion, the loss of cholinergic neurons, decrease of NeuN-positive hippocampal cell, and abnormal long-term potentiation of hippocampal CA1 and CA3. Re-administering thiamine reversed the weakened learning ability as well as the impaired hippocampal neurogenesis induced by TD at early pre-pathological lesion stage. The present study demonstrated that hippocampal neurogenesis was vulnerable to TD and the impaired hippocampal neurogenesis is greatly involved in cognitive dysfunction induced by TD at early pre-pathological lesion stage.     

Transiently impaired neurogenesis in MPTP mouse model of Parkinson's disease

It is still being debated whether neurogenesis in the subventricular zone (SVZ) is enhanced in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injury in the adult mouse brain. Our previous studies provided evidence that MPTP induces apoptosis of migrating neuroblasts (neural progenitor cells, A cells) in the SVZ and rostral migratory stream (RMS). We investigated cellular kinetics in the adult SVZ and olfactory bulb (OB) after MPTP damage. Cells were labeled with bromodeoxyuridine (BrdU), and the effects of MPTP on the survival and fate of migrating and residing neuroblasts were evaluated. Two days after BrdU labeling and MPTP treatment, the number of BrdU-positive cells in the SVZ and OB of MPTP-treated mice was significantly lower than in the SVZ and OB of saline controls. Additionally, fewer BrdU-positive cells migrated to the OB of treated mice than to that of saline controls, and the cells that did migrate diffused radially into the granule cell layer (GCL) when observed at 7, 14, and 28 days. In the OB GCL, the differentiation of BrdU-positive cells into mature neurons significantly attenuated 14 and 28 days after MPTP injury. Moreover, the impaired neurogenesis was followed by a recovery of A cells in the SVZ and OB, suggesting activation of the self-repair process as a result of MPTP-induced depletion of BrdU-positive cells. Our findings clarify the kinetics underlying neurogenesis in MPTP-treated mice and may contribute to the development of an animal model of Parkinson's disease, and the demonstration of cellular kinetics in SVZ may also provide a new insight into assessing neurogenesis in MPTP-treated mouse.     

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。     

Evidence for constitutive neural cell proliferation in the adult murine hypothalamus

Compelling evidence suggests that the mammalian brain is capable of generating new neurons throughout adult life. While neurogenesis can be induced at various brain sites by exogenous cues, constitutive birth of new neurons has been unambiguously demonstrated within the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus. The lack of strong evidence that constitutive neurogenesis occurs elsewhere in the adult brain could be due to its exclusive restriction to the SVZ and SGZ or, for instance, to the inadequacy of the methods used to reveal new-born neurons at other brain sites. By using intracerebroventricular (icv) delivery of the mitotic marker bromodeoxyuridine (BrdU) we demonstrate that new cells are born continuously and in substantial numbers in the adult murine hypothalamus and that many of these cells appear to differentiate into neurons as assessed by the expression of doublecortin (Dcx) and other neuronal fate markers. As compared to intraperitoneal (ip) BrdU injections, central BrdU infusion also uncovers a higher-fold induction of hypothalamic cell proliferation by ciliary neurotrophic factor (CNTF). It appears that new cells are born throughout the hypothalamic parenchyma without an apparent restriction to a specific neurogenic layer, as seen in the SVZ. Thus, we provide evidence that the adult hypothalamus is constitutively neurogenic and that hypothalamic cell proliferation is highly responsive to mitogen action.     

硫胺素缺乏引起脑内β淀粉样蛋白沉积和tau蛋白磷酸化的增加

目的：探讨硫胺素缺乏（TD）对脑内β淀粉样蛋白（Aβ）沉积、tau蛋白磷酸化的影响。方法：硫胺素剥夺饮食结合腹腔注射吡啶硫胺制作TD小鼠模型,正常对照组给予正常饮食及腹腔注射生理盐水。造模13d后取脑,行苏木精-伊红染色观察两组小鼠脑内易损区域病理改变,免疫组织化学染色检测脑内Aβ沉积、tau蛋白磷酸化及β-分泌酶（BACE）的表达情况。结果：苏木精-伊红染色显示TD模型组小鼠内侧丘脑出现典型的对称性针尖样出血;免疫组化显示TD模型组小鼠皮质、海马及丘脑均出现Aβ沉积,且丘脑Aβ沉积较皮质及海马更为明显,tau蛋白磷酸化及BACE的阳性细胞数显著增加。正常对照组小鼠苏木精-伊红染色脑内未见病理损伤,皮质、海马及丘脑均未发现Aβ沉积,tau蛋白磷酸化及表达BACE的阳性细胞数均明显低于TD模型组,两组差异有统计学意义（P〈0．05）。结论：TD可引起Aβ沉积、tau蛋白磷酸化增加等阿尔茨海默病的特征性病理改变,且Aβ沉积可能与上调BACE的表达有关。