骨髓间充质干细胞静脉注射移植对脑缺血大鼠脑内神经细胞增殖和分化的影响

目的观察骨髓间充质干细胞（bone marrow stromal cells,BMSCs）经静脉注射移植对缺血/再灌注脑内神经细胞增殖和分化的影响。方法体外培养和扩增成年雄性大鼠BMSCs;以“四管阻断法”制作大鼠前脑缺血/再灌注模型;造模后3、5、7d通过尾静脉注射Hoechst33342标记的BMSCs（2×10^6/1ml/只）,另设对照组于相同时间点通过尾静脉注射载体溶液。于造模后4周处死大鼠,取脑切片,HE染色观察大鼠海马缺血性损伤情况;BrdU/GFAP、BrdU/MAP2和BrdU/Hoechst33342免疫荧光双标染色,观察大鼠脑内神经细胞的增殖及分化情况。结果BMSCs移植组海马CA1区存活锥体细胞数显著多于载体溶液对照组（P〈0.05）。BMSCs移植大鼠脑内海马结构BrdU阳性细胞数显著多于载体溶液对照组（P〈0.05）,BrdU/MAP-2双标阳性细胞占BrdU阳性细胞的比例显著高于载体溶液对照组（P〈0.05）。结论BMSCs经静脉注射移植能够减轻缺血性脑损伤,促进宿主脑内自体神经细胞增殖,并提高其分化为神经元样细胞的比例。     

骨髓基质细胞移植治疗大鼠实验性脑梗死的疗效分析及组织病理学观察

目的 观察大脑中动脉闭塞(MCAO)模型大鼠脑内移植骨髓基质细胞fBMSCs)的治疗作用,分析植入梗死灶不同区域的BMSCs的存活、迁移情况以及植入细胞的行为与脑内微环境中GFAP阳性细胞的形态关系.方法 75只成年SD大鼠采用随机数字表法分为MCAO组(n=50)和BMSCs移植组(n=25),所有动物均采用线栓法制作MCAO 1 h模型,24 h后BMSCs移植组脑内注射BrdU标记的同种异体BMSCs(2x106个),MCAO组注射等量PBS.MCAO前及MCAO后第1(移植前)、3、5、7、10、14天应用加速转轮试验和贴纸去除试验检测神经功能缺损情况;第14天处死动物,取脑组织切片应用HE染色观察两组的缺血病灶范围,行BrdU和GFAP免疫组化染色观察BMSCs在不同区域和不同胶质细胞环境下的存活和迁移情况.结果 BMSCs移植组MCAO后7 d加速转轮试验结果优于MCAO组,差异有统计学意义(P＜0.05);组织学观察发现植入缺血半暗带区的细胞存活数量最多,且向病变方向放射状迁移,植入缺血病灶核心的细胞甚少存活,且无迁移现象.结论 BMSCs脑内移植可改善MCAO后大鼠神经运动功能;活化的星形胶质细胞构成适合植入细胞存活、迁移的环境,而胶质瘢痕阻碍了细胞的迁移.     

胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋白损伤大鼠海马后的靶向迁移与分化

目的观察小鼠胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋（amyloidβpeptide,Aβ）损伤大鼠海马后的靶向迁移及在体分化。方法采用无血清培养法将表达绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的小鼠胚胎干细胞定向诱导为神经前体细胞,移植至Aβ1-40单侧损伤的大鼠海马;免疫荧光观察移植细胞的迁移距离、迁移方向与分化情况;对移植细胞的平均迁移距离与平均分化率作相关性分析。结果胚胎干细胞经改良的无血清培养法生长可分化为nestin阳性神经前体细胞。移植后第16周,神经前体细胞平均迁移距离比第4周增长了约5倍。移植细胞中,胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）阳性细胞的平均分化率从（30．41±1．45）％增长到（49．25±1．23）％;神经丝蛋白200（neurofilament200,NF200）细胞的平均分化率从（16．68±0．95）％增长到（27．94±1．21）％;靶向迁移至Aβ斑块同侧的GFAP阳性细胞的比例从60．2％增长到81．3％,靶向迁移至Aβ斑块的NF200阳性细胞的比例从61．3％增至84．1％。相关性分析结果显示平均迁移距离与神经元分化率之间存在显著线性相关（r=0．991）,与胶质细胞分化率之间也存在显著线性相关（r=0．953）。结论胚胎干细胞来源的神经前体细胞移植Aβ损伤模型大鼠海马后能够靶向迁移至Aβ损伤区并分化为胶质细胞和神经元。     

大鼠脑损伤后脑内移植骨髓基质细胞的研究

目的研究大鼠脑损伤后骨髓基质细胞(BMSCs)颅内移植对内源性神经干细胞(NSCs)的作用。方法 48只雄性Wistar大鼠随机分为3组:正常对照组、移植组、非移植组。大鼠脑损伤后24 h立体定向下局部注射BMSCs,然后每天腹腔注射5-溴脱氧尿嘧啶核苷(BrdU)2次。损伤后14 d和28 d随机处死取脑,行BrdU免疫组化染色以及BrdU和神经元特异性烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)免疫组化双染色。结果局部注射BMSCs的移植组大鼠表达BrdU/NSE阳性的细胞较非移植组为多。结论大鼠脑损伤后BMSCs对内源性NSCs的分化有促进作用。     

骨髓基质干细胞治疗创伤性脑损伤实验研究

目的观察移植于脑内的骨髓基质干细胞(BMSCs)对创伤脑组织的神经修复作用。方法流式细胞术鉴定原代培养的大鼠BMSCs,免疫荧光技术检测5-溴脱氧尿苷(BrdU)标记BMSCs的比例;建立大鼠颅脑创伤模型后在创伤灶周边进行BMSCs移植,免疫组织化学方法检测BMSCs在脑创伤灶内的分布和神经分化情况;实验动物予以神经功能评分。结果流式细胞术检测结果符合大鼠BMSCs特征,体外BrdU标记的BMSCs呈现强的红色胞核荧光;体内移植显示BMSCs在脑内成功存活,主要分布于创伤灶内或其边缘,并部分表达神经标志蛋白;BMSCs移植组实验动物神经功能评分明显优于对照组。结论脑内移植的BMSCs经过短距离迁移定位于创伤灶区域,并通过向神经细胞方向的分化实现部分神经修复和功能代偿。     

神经生长因子基因修饰骨髓间充质干细胞移植治疗脑梗死大鼠的实验研究

目的观察神经生长因子基因(NGF)修饰的骨髓间充质干细胞(BMSCs)移植治疗脑梗死的作用及可能机制。方法采用线栓法制备大鼠脑梗死模型,将符合条件的30只脑梗死模型大鼠按随机数字表法分为模型组(n=10),BMSCs移植组(n=10)和NGF-BMSCs移植组(n=10),分别经尾静脉注射PBS、BrdU标记的BMSCs和NGF-BMSCs各1mL。分别于术后1d、7d和14d采用改良神经功能损害评分(mNSS)对各组大鼠进行神经功能评估,于术后14d应用HE染色观察脑组织病理情况,应用免疫荧光组织化学检测BrdU标记的移植细胞存活状况和TUNEL法检测脑组织中细胞凋亡情况。结果NGFBMSCs组和BMSCs组mNSS评分和TUNEL阳性细胞数较Model组减低(P0.05),且NGF-BMSCs组较BMSCs组更低(P0.05);HE染色显示NGF-BMSCs组和BMSCs组较Model组脑组织损伤及细胞丢失较轻,NGF-BMSCs组更明显;并且NGF-BMSCs组中的BrdU阳性细胞数较BMSCs组增多(P0.05)。结论 NGF基因修饰的BMSCs移植较单纯BMSCs移植能进一步改善脑梗死大鼠的神经功能,其机制为能够促进植入的BMSCs在脑内存活和减轻神经细胞凋亡。     

大鼠骨髓基质细胞经枕大池移植后在损伤脑组织中的存活和分化

目的:探讨MSCs经枕大池移植到创伤性脑损伤模型大鼠的蛛网膜下腔后迁移到损伤脑组织存活并分化为神经细胞的能力。方法:采用Feeney’s自由落体脑创伤模型,建立23个大鼠脑创伤模型。从SD大鼠的后肢骨髓分离培养得MSCs,将第三代的MSCs以BrdU在体外标记。随机取15个脑创伤大鼠模型接受MSCa枕大池注射(脑创伤细胞移植组);随机取6个脑创伤大鼠模型接受枕大池注射生理盐水(脑创伤生理盐水组);取6个正常大鼠接受枕大池注射MSCs(正常大鼠细胞移植组)。定期杀死大鼠制作脑组织石蜡切片,行BrdU、BrdU-GFAP、BrdU-MAP2的免疫组织细胞化学染色。另取两个脑创伤大鼠在创伤1周后脑石蜡切片行HE染色。结果:观察到脑创伤细胞移植组石蜡切片BrdU染色可见BrdU阳性细胞,细胞进入皮质下的最大距离为3 mm。双标记染色可见部分BrdU阳性细胞胞质呈现GFAP或MAP_2染色阳性。而另外两组均未见阳性细胞。结论:大鼠MSCs具有从蛛网膜下腔迁移入损伤脑组织存活一定时期并分化为神经细胞的能力。     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

锂对慢性铝暴露大鼠学习记忆功能的影响及其机制

目的 探讨锂对慢性铝暴露大鼠学习记忆功能以及脑内淀粉样B蛋白前体（APP）代谢的影响。方法 将24只慢性铝暴露大鼠随机分为锂治疗组和非治疗组，每组12只；另取12只非铝暴露大鼠为正常组。锂治疗组给予氯化锂溶液（200mg·kg^-1·d^-1）灌胃，非治疗组以等量氯化钠溶液灌胃，正常组大鼠不进行干预。6周后Morris水迷宫测试，比较三组大鼠的学习记忆功能；分别采用免疫组织化学法和免疫印迹法检测各组大鼠脑组织内淀粉样B蛋白（AB）和APP含量。结果 （1）锂治疗组大鼠定向航行实验潜伏期[（15．4±8．7）s]短于非治疗组[（26．8±11．2）s；F=-3．8，P〈0．05]，空间搜索中随机式所占比例（11．1％）低于非治疗组（20．7％；P〈0．05），站台停留时间[（47±7）s]和穿台次数[（12．6±2．2）次]多于非治疗组[（35±7）s和（7．9±2．6）次；P〈0．05]；（2）正常组大鼠脑内APP的含量（1．0±0．3）低于其他两组[锂治疗组为（1．8±0．4），非治疗组为（1．8±0．5）；P〈0．01]。非治疗组脑内海马、皮层AB反应阳性数目多于其他两组（P〈0．01）。结论 锂可以改善慢性铝暴露大鼠学习记忆功能，可能是通过抑制B分泌酶裂解途径而影响APP的代谢，减少AB的产生。     

食蟹猴骨髓源性神经干细胞自体脑内移植的研究

目的探讨食蟹猴自体骨髓基质细胞诱导分化成神经干细胞后移植到脑内的生长情况.方法对6只食蟹猴进行骨髓基质干细胞体外培养、诱导分化成神经干细胞,经BrdU标记后进行自体脑移植.动物分为即时移植组和延迟移植组,采用立体定向多点注射的方法将神经干细胞悬液移植到猴皮层创伤灶.结果HE染色显示即时移植和延迟移植的创伤区细胞数量都明显多于假移植治疗对照;免疫组织化学染色显示两组动物在干细胞移植后1～6个月脑皮层创伤灶内均有BrdU阳性表达细胞,移植后半年的动物在移植区邻近的脑白质内也可观察到有BrdU阳性表达的细胞,而创伤对照动物、假移植治疗动物和正常脑组织中则未见BrdU阳性表达. 结论BMSCs体外培养得到的神经干细胞经过自体移植能在脑皮层内存活,并且能增殖、分化和迁移,可成为神经干细胞的替代细胞或来源细胞;干细胞移植到陈旧性的脑皮层损伤灶也具有成活、增殖和迁移能力.     

视频脑电图在小儿癫痫诊断中的应用

目的评价视频脑电图(video-EEG)在小儿癫诊断中的应用价值。方法对126例具有发作性症状的患儿进行连续8h的包括清醒、睡眠、诱发试验及必要的认知测验的视频脑电图监测。结果经发作期视频脑电图证实,39例初诊为癫性发作的患儿中14例(35%)为非癫性发作;15例其他症状发作中13例(86%)为非癫性发作。64例样放电患儿中51例(80%)确定发作类型,22例(34%)确定癫类型。视频脑电图可发现短暂轻微的癫发作及样放电引起的一过性认知损伤。结论视频脑电图在排除非癫性发作、确定癫性发作的类型、评价脑电-临床关系方面可提供准确可靠的证据,进一步提高癫的临床诊断水平。     

Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity

The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.     

Storage of lipofuscin in neurons in mucopolysaccharidosis

Summary A histochemical and ultrastructural study was made on the brain of a 23-year-old man with Sanfilippo's syndrome. In accordance with previous reports the cortical nerve cells contained a PAS-positive lipid storage substance. This showed intense autofluorescence in UV-light and was positive with various stains for lipofuscin. The storage material appeared ultrastructurally as inclusion bodies composed of short lamellated membranes, granular material, and vacuoles. In addition, concentrically and transversely lamellated membranous cytoplasmic bodies were observed in the nerve cells. It is concluded that the PAS-positive lipid storage material in the neurons was composed partly of lipofuscin in addition to other lipids presumably glycosphingolipids.Supported by a grant from the Expressen Prenatal Research Foundation     

Role of vincristine in the inhibition of angiogenesis in glioblastoma

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α).

Methods: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O₂ (hypoxia).

Results: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G₂-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC₅₀), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels.

Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.     

脑电图预测痫性发作研究进展

癫痫(epilepsy)是由脑部神经元高度同步化异常放电所致的临床综合征,系神经系统的常见病,困扰着全世界约1%的人群.每次神经元的阵发性放电或短暂的脑功能异常称为痫性发作(seizures).     

Midazolam in treatment of various types of seizures in children

Midazolam is a recently developed water-soluble benzodiazepine that shares anxiolytic, muscle relaxant, hypnotic and anticonvulsant actions with other members of this class. There are limited studies that midazolam can be used successfully to treat seizures in adults and children. In this study, 0.2 mg/kg intramuscular (IM) midazolam was administered to 11 children (eight boys and three girls), aged 3 days to 4 years (mean age 1.8±1.4 years), with seizures of various types. In all but one child, seizures stopped in 15 s–5 min after injection. No side effects were observed. These results suggest that IM administration of midazolam may be useful in a variety of seizures during childhood, especially in case of intravenous (IV) line problem.     

自噬参与帕金森病发病的研究进展

近年来,蛋白质的降解障碍被认为是帕金森病（Parkinson’Sdisease,PD）发病过程中的重要因素,人们已经公认泛素一蛋白酶体系统（ubiquitin--pro—teasomesystem,UPS）功能异常或衰竭能够导致细胞内异常蛋白蓄积、细胞功能障碍,甚至细胞凋亡。与此同时,蛋白降解的另一条途径——自噬-溶酶体途径（autophagy—lysosomepathway,ALP）也已成为了生命科学领域的研究热点,自噬与神经变性疾病,尤其是PD的关系日益受到人们的重视。     

Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons

The diffusible chemical messenger nitric oxide (NO) is involved in neuronal plasticity and it is, therefore, supposed to play a role in brain development. A shortage of NO during the critical period of brain maturation may theoretically have long-lasting consequences on the organization of the adult brain. We have performed in neonatal rats a chronic inhibition of the enzyme responsible for NO production, nitric oxide synthase (NOS), from postnatal day 3 to postnatal day 23, through administration of the competitive antagonist N-nitro-L-arginine methylester (L-NAME). The calcium-dependent catalytic activity resulted almost completely inhibited throughout the period of treatment and it took more than 4 days after its suspension to get a full recovery. The expression of the neuronal isoform of the enzyme (nNOS), revealed by immunoblotting, was unchanged during the treatment and after it. The histochemical reaction for NADPH diaphorase was reduced at the end of the treatment and recovered in concomitance with the recovery of the catalytic NOS activity. No gross structural alterations were detected in brain morphology. The levels of three neurotransmitter-related and one astrocytic marker were unchanged in the cerebellum, hippocampus and cortex of 60-day-old rats which had been neonatally treated. A similar lack of significant effects on neurochemical brain maturation was also noticed in a parallel series of experiments, in which a short pulse of NOS inhibition was performed at a critical prenatal time of brain development, from gestational day 14 to gestational day 19. In vitro, chronic exposure of cerebellar granule cells to L-NAME (500 microM) resulted in slight decrease of surviving neurons after 8 days in culture and in better resistance to the challenge of stressful culture conditions. The present results suggest that the basic plan of brain organization can be achieved despite an almost complete NOS inhibition during the maturation period. In vitro, NOS inhibition may bring to more pronounced consequences on neuronal viability and function.     

The Burden of Agoraphobia in Worsening Quality of Life in a Community Survey in Italy

ObjectiveCurrent nosology redefined agoraphobia as an autonomous diagnosis distinct from panic disorder. We investigated the lifetime prevalence of agoraphobia, its association with other mental disorders, and its impact on the health-related quality of life (HR-QoL). MethodsCommunity survey in 2,338 randomly selected adult subjects. Participants were interviewed with the Advanced Neuropsychiatric Tools and Assessment Schedule (ANTAS), administered by clinicians. The diagnoses were based on the ICD-10 criteria. The Short-Form Health Survey (SF-12) was used to quantify HR-QoL. ResultsIn the sample, 35 subjects met the criteria for agoraphobia (1.5%), with greater prevalence among women (2.0%) than men (0.9%): odds ratio (OR) 2.23; 95% CI: 1.0-5–2. Agoraphobia was more often seen among those with (n=26; 1.1%) than without (n=9; 0.4%) panic disorder: OR=8.3; 2.9–24.4. Co-morbidity with other mental disorders was substantial. The mean score of SF-12 in people with agoraphobia was 35.2±7.8, with similar levels of HR-QoL in people with (35.3±7.9) or without (34.8±7.3) panic disorder: ANOVA: F(1;33)=0.0; p=1.00. ConclusionOne out of seventy people may suffer from agoraphobia in their lifetime. The attributable burden in terms of HR-QoL is substantial and comparable to the one observed for chronic mental disorders such as major depression, post-traumatic stress disorder, or obsessive-compulsive disorder.