脑白质低灌注损伤后星形胶质细胞缝隙连接蛋白43的表达变化

目的研究慢性脑白质缺血后星形胶质细胞和缝隙连接蛋白Connexin43(Cx43)的变化。方法原代培养星形胶质细胞,建立体外慢性缺氧模型;双侧颈总动脉狭窄法,建立慢性低灌注脑白质损伤小鼠模型;免疫荧光共染观察星形胶质细胞活化与Cx43表达。Western蛋白定量分析髓鞘相关指标髓鞘相关糖蛋白MAG,星形胶质细胞标记物GFAP和Cx43的表达。结果与对照组相比,细胞慢性缺氧7d后,星形胶质细胞明显增生活化,伴随Cx43表达水平明显上调。Western blot发现,在慢性脑白质缺血过程中,MAG的表达逐渐降低,GFAP持续增高,Cx43表达明显上调。免疫荧光共标记可见,星形胶质细胞中Cx43表达上调,主要分布于胼胝体中央区。结论慢性脑白质缺血损伤过程伴随星形胶质细胞Cx43表达增加,Cx43可能成为临床治疗血管性认知障碍的新靶点。     

重组人红细胞生成素干预与脑白质损伤的修复

动物实验证实,红细胞生成素具有神经保护作用。采用4日龄SD大鼠双侧颈总动脉结扎法建立新生大鼠缺氧缺血性脑白质损伤动物模型,给予重组人红细胞生成素,观察干预前后大鼠脑病理形态学和远期神经行为功能的改变。结果表明,大鼠脑白质损伤后7,26 d,红细胞生成素干预的大鼠体质量明显增高。脑白质损伤后72 h,7 d 红细胞生成素干预的大鼠脑室周围白质中出现可反映脑白质少突胶质细胞髓鞘损伤程度的髓鞘碱性蛋白阳性表达细胞显著增多,可反映轴索损伤淀粉样前体蛋白阳性表达细胞显著减少。脑白质损伤后48 h,72 h,7 d,26 d大鼠脑室周围白质中可反映脑白质星形胶质细胞损伤程度的神经胶质原纤维酸性蛋白阳性表达细胞明显减少。红细胞生成素干预30日龄大鼠,斜坡试验转头时间缩短,旷场试验检测分数增高。结果说明,缺氧缺血损伤后尽早给予红细胞生成素干预能够促进大鼠的生长发育和脑白质损伤的修复,改善其神经行为功能。     

高同型半胱氨酸血症对大鼠周围神经结构和功能的影响

目的 观察高同型半胱氨酸血症(hyperhomocysteinemia,Hhcy)对大鼠周围神经组织结构和传导功能的影响.方法 通过高蛋氨酸饮食建立Hhcy大鼠模型.应用电生理技术检测大鼠尾神经传导功能;光镜下计数大鼠腓肠神经单位面积有髓神经纤维密度(fiber density,FD)、平均髓鞘面积(mean myelin area,MMA);免疫组化方法分析腓肠神经髓鞘碱性蛋白(myelin basic protein,MBP)、神经丝蛋白(neurofilament protein,NF)表达;电镜观察腓肠神经超微结构.结果 Hhcy组大鼠尾神经运动神经传导速度(motor nerve conduction velocity,MCV)、感觉神经传导速度(sensory nerve conduction velocity,SCV)与复合肌肉动作电位(compound muscle action potential,CMAP)、感觉神经动作电位(sensory nerve action potential,SNAP)波幅均较对照组减慢和下降(均P＜0.05);Hhcy组大鼠腓肠神经FD、MMA与MBP、NF积分光密度均较对照组减小(均P＜0.05);Hhcy组大鼠有髓神经纤维出现了髓鞘脱失、轴突肿胀、间质水肿等超微结构改变.结论 Hhcy可造成大鼠周围神经组织结构与传导功能的损害.     

间歇性θ爆发式磁刺激对高血压大鼠胼胝体髓鞘及局部炎症反应的影响

目的探究间歇性θ爆发式磁刺激对慢性高血压大鼠胼胝体区域髓鞘脱失、星形胶质细胞增生和小胶质细胞活化的改善作用。方法对雄性Sprague-Dawley大鼠随机进行双肾双夹术,制作易卒中型肾血管性高血压大鼠模型。术后22周,模型制备成功的高血压大鼠随机接受连续14 d间歇性θ爆发式磁刺激治疗(intermittent theta burst stimulation,iTBS)(高血压iTBS组,n=6)或假性刺激(高血压假刺激组,n=6),假手术大鼠接受假性刺激(假手术假刺激组,n=6)。HE染色观察胼胝体小动脉形态。免疫荧光染色观察胼胝体MBP标记的髓鞘脱失情况。免疫荧光染色观察GFAP标记的星形胶质细胞数量和IBa-1标记的小胶质细胞数量和形态,以评价星形胶质细胞增生和小胶质细胞活化。结果高血压iTBS组和高血压假刺激组出现明显小动脉管壁增厚。与假手术假刺激组相比,高血压假刺激组胼胝体MBP阳性面积比例减少(P<0.01),GFAP阳性细胞和IBa-1阳性细胞数量明显增加(P<0.01),IBa-1阳性细胞胞体增大,突起变粗,分枝变少。iTBS治疗明显增加了高血压大鼠胼胝体MBP阳性面积比例,降低GFAP阳性细胞和IBa-1阳性细胞数量(P<0.01),IBa-1阳性细胞胞体变小,突起变细,分枝变多。结论iTBS治疗可减轻高血压大鼠胼胝体区域髓鞘脱失,抑制星形胶质细胞增生和小胶质细胞活化。     

脑出血大鼠锥体束显微和超微结构改变

目的研究脑出血大鼠锥体束病理变化规律及特点。方法使用Ⅳ型胶原酶．肝素诱导大鼠基底节脑出血,采用劳克坚牢蓝（LFB）染色、神经丝蛋白（NF）免疫组化和电镜对内囊后肢进行观察。结果Ⅳ型胶原酶-肝素能成功建立具有典型神经功能缺损的大鼠脑出血模型。光镜发现锥体束髓鞘损伤在脑出血1～3d逐渐加重,7d开始再生修复,1～7d轴突损伤持续加重,14d轴突光度值（0．09±0．01）有所增加,但与7d（0．10±0．02）相比无显著性差异（P〉0．05）。电镜显示脑出血1d锥体束髓鞘松解,轴突水肿,部分无髓轴突崩解坏死消失,3d髓鞘松解、空泡样变性,局部髓鞘消失、厚薄不均,轴突水肿严重,甚至坏死崩解。结论大鼠脑出血后1w内锥体束损伤呈进行性加重,提示应早期和超早期予以干预治疗以减轻损伤和促进修复。     

NSE、S100B蛋白、MBP、GFAP与缺血性脑卒中临床相关性研究进展

神经元特异性烯醇化酶（NSE）、S100B蛋白、髓鞘碱性蛋白（MBP）及胶质纤维酸性蛋白（GFAP）均是脑组织损伤后的生化标志物,可全面反映神经元、胶质细胞、神经髓鞘膜及星形细胞的损害程度,为判断急性缺血性脑卒中后中枢神经系统受损状况提供了定量信息,对判断预后有一定指导意义,并可为早期治疗提供帮助。     

NF155及其在脑白质损伤疾病中的研究

在中枢神经系统（central nervous system,CNS）,髓鞘由少突胶质细胞（oligodendrocyte,OL）形成,神经元轴突髓鞘化完成预示着神经发育成熟。髓鞘具有重要的生理功能,它不仅能对神经元轴突起保护作用,而且使轴突绝缘,以确保神经冲动通过跳跃式传导而高速传递。然而在脑白质损伤中,由于神经细胞脱髓鞘,髓鞘生理功能部分或完全丧失,导致一系列病理生理改变。脑白质损伤引起的神经纤维脱髓鞘包括了原发性脱髓鞘和继发性脱髓鞘。     

脑源性神经营养因子诱导大鼠骨髓基质细胞成为神经干细胞及其分化作用的实验研究

目的探讨脑源性神经营养因子（BDNF）诱导大鼠骨髓基质细胞（BMSCs）成为神经干细胞及其分化作用。方法取大鼠BMSCs。分别以BDNF和BDNF＋RA（维甲酸）作为诱导物诱导，于诱导3d、7d后行巢蛋白（Nestin）、神经元特异烯醇化酶（NSE）、胶质纤维酸性蛋白免疫细胞化学染色。结果后BDNF和BDNF＋RA诱导组均有大量Nestin染色阳性细胞，BDNF＋RA组阳性率高于BDNF组（P〈0．01）。NSE、GFAP免疫阳性细胞在诱导3d后也有少量表达。诱导7d后BDNF和BDNF＋RA诱导组Nestin阳性细胞明显减少．与诱导3d后比较差异有显著性（P〈0．01），而NSE、GFAP阳性细胞敷增多，与诱导3b比较差异有显著性（P〈0．01），且BDNF＋RA组阳性率高于BDNF组（P〈0．01）。结论联合应用BDNF与RA可提高BMscs神经转化．并促进其向神经元及星形胶质细胞细胞分化。     

髓鞘碱性蛋白免疫治疗对出血性卒中大鼠的保护作用研究

目的研究髓鞘碱性蛋白(myelin basic protein,MBP)主动免疫,对大鼠出血性卒中的保护作用,探讨其预防继发性脑损害的可能机制。方法SD大鼠84只,采用随机数字表法,分为MBP、卵白蛋白(Ovalbumin,VOA)、PBS和空白对照四组(n=21)。制备脑出血模型后24小时内,用不完全福式佐剂(Incom-plete Freund's Adjuvant,IFA)乳化的MBP、VOA、PBS分别免疫三组大鼠,对照组只做脑出血模型,在相应时间点行为学评分,并标本取材,行胶质纤维酸性蛋白(Glial fibrillary acid protein,GFAP)和神经微丝蛋白200(Neurofilament protein,NF200)免疫染色,电镜观察血肿周围脑组织的超微结构变化。结果(1)MBP组免疫后第1～3天行为学评分明显优于其它三组(P<0.05);(2)出血免疫后第1～5天,MBP组GFAP阳性细胞数与其它三组相比有显著性差异(P(0.05),后三组相比无统计学意义(P>0.05)。(3)MBP组NF200阳性细胞数在免疫后第3～14天与后三组相比有显著性差异(P(0.05),后三组之间相比无明显差异(P>0.05)。(4)免疫后第7天,MBP组神经元亚细胞器较丰富,神经微丝排列整齐,其他三组亚细胞器变形或消失,神经微丝排列紊乱。结论MBP免疫治疗能阻断出血性卒中的继发性脑损害,促进神经症状的改善,即血肿周围神经元的恢复。     

PCNA和GFAP在脑星形胶质细胞瘤中表达的双重染色研究

目的研究人脑星形胶质细胞瘤中增殖细胞核抗原（PCNA）和胶质纤维酸性蛋白（GFAP）的表达及其与肿瘤分级的关系。方法采用免疫组化双重染色法对41例人脑星形胶质细胞瘤进行PCNA和GFAP两重标记检测。结果脑星形胶质细胞瘤中PCNA与GFAP表达率均为100％，PCNA表达水平与肿瘤分级呈正相关（r=-0．627，P〈0．01），GFAP表达水平与肿瘤分级呈负相关（r=-0．568，P〈0．01）；Ⅰ-Ⅱ级与Ⅲ-Ⅳ级胶质瘤间PCNA和GFAP表达均有显著性差异（P〈0．05）；GFAP表达和PCNA表达水平呈负相关（r=-0．332，P〈0．05）。结论PCNA与GFAP的表达有一定的相关性。PCNA与GFAP的双重表达与脑星形胶质细胞瘤的增殖活性和恶性程度有关。     

特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值

目的特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值,并观察其病理的改变。方法按脊髓重量与冰盐水体积之比为1:5的比例制备豚鼠脊髓匀浆抗原,免疫Wistar大鼠,建立EAE模型;组织切片进行HE染色,三色染色,髓鞘碱性蛋白(MBP)及神经微丝(NF)免疫组化,光镜下观察病理改变。结果Wistar大鼠免疫后第16.07±4.25天发病,发病形式多样,除表现EAE经典症状外,尚出现痉挛状态、斜颈等特殊症状。HE染色发现神经组织内炎细胞浸润,血管"袖套"样病灶形成;三色染色可见轴突肿胀,呈串珠状,且不连续,着色不均匀;髓鞘结构层次紊乱,疏松,崩解;MBP及NF免疫组化研究发现病变组织内白质脱髓鞘及轴突损伤。结论三色染色结合MBP、NF免疫组化检测,较常规HE染色更能直接地、准确地显示EAE大鼠的病理变化,可推广应用。     

Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor.

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium-induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady-state mRNA levels (expressed on a "per nerve" basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring.     

Macrophage-depletion induced impairment of experimental CNS remyelination is associated with a reduced oligodendrocyte progenitor cell response and altered growth factor expression

Although macrophages are mediators of CNS demyelination, they are also implicated in remyelination. To examine the role of macrophages in CNS remyelination, adult rats were depleted of monocytes using clodronate liposomes and demyelination induced in the spinal cord white matter using lysolecithin. In situ hybridization for scavenger receptor-B and myelin basic protein (MBP) revealed a transiently impaired macrophage response associated with delayed remyelination in liposome-treated animals. Macrophage reduction corresponded with delayed recruitment of PDGFRalpha+ oligodendrocyte progenitor cells (OPCs), which preceded changes in myelin phagocytosis, indicating a macrophage effect on OPCs independent of myelin debris clearance. Macrophage-depletion induced changes in the mRNA expression of insulin-like growth factor-1 and transforming growth factor beta1, but not platelet-derived growth factor-A and fibroblast growth factor-2. These data suggest that the macrophage response to toxin-induced demyelination influences the growth factor environment, thereby affecting the behavior of OPCs and hence the efficiency of remyelination.     

Induction of recurrent experimental allergic encephalomyelitis with myelin basic protein

Recurrent experimental allergic encephalomyelitis (EAE) was induced in Lewis rats by inoculation of human myelin basic protein (MBP) and complete Freund's adjuvant (CFA). The animals developed clinical disease characterized by unsteadiness, ataxic gait, and abnormal posturing of the limbs. Spontaneous remissions and relapses were noted for periods up to 120 days. Histologically there were perivascular infiltrates of mononuclear cells, especially prominent in the cerebellar white matter. There was little evidence of demyelination. This study demonstrates that relapsing EAE may be induced with MBP in the rat. Sensitization with other myelin antigens is not required, although immunity to such antigens may be necessary to induce demyelination. It is postulated that relapsing EAE may be associated with a defect in suppressor cell function.     

The Distribution of Inflammatory Demyelinated Lesions in the Central Nervous System of Rats with Antibody-Augmented Demyelinating Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) has long been studied as an animal model of the human demyelinating disease Multiple Sclerosis. However, EAE induced in the Lewis rat by injection of myelin basic protein (MBP), or MBP-specific T-lymphocytes, is primarily an inflammatory condition of the central nervous system (CNS) with little or no demyelination. In EAE models in which demyelination does result, it is either not very widespread or is unpredictable in its degree and location. In this study we have produced antibody-augmented demyelinating EAE (ADEAE) in the Lewis rat by injection of activated MBP-specific T-lymphoblasts, followed by injection 4 days later of a monoclonal antibody against myelin/oligodendrocyte glycoprotein, an extrinsic protein of myelin. We have documented the extent and location of inflammatory cell infiltrates and demyelination throughout the CNS using histochemistry, immunofluorescence, and image analysis. Perivascular inflammatory infiltrates were seen in the deep cerebellar white matter and in the folia. Perivascular, periventricular, and subpial inflammation was widespread throughout the pons/medulla and at all levels of the spinal cord. Very little inflammation was apparent in the forebrain. MBP immunofluorescence demonstrated extensive areas of periventricular demyelination in the forebrain around the third ventricle. Both periventricular and perivascular lesions were commonly observed in the cerebellum and pons/medulla. The extent of demyelination in the spinal cord increased caudally with large confluent areas of subpial demyelination seen throughout the lumbar cord. The extensive and reproducible distribution of inflammatory demyelinating lesions in ADEAE provide the possibility to select areas of the CNS for more detailed analysis of the cellular changes that accompany demyelination and remyelination.     

β4 integrin and other Schwann cell markers in axonal neuropathy

Schwann cell gene expression is dynamically regulated after peripheral nerve injury and during regeneration. We hypothesized that the changes in protein expression described after rat peripheral nerve injury could be used to identify single Schwann cell-axon units in human axonal neuropathy. Therefore, we performed immunofluorescence staining on sections of injured rat sciatic nerves compared with sections of neuropathic human sural nerves. We chose the markers β4 integrin, P0 glycoprotein, and glial fibrillary acidic protein (GFAP) to characterize Schwann cells, and neurofilament-heavy (NF-H) to recognize axons. Normal rat or human myelin-forming units demonstrated a sharp ring of β4 staining at their outer surface, P0 staining in the myelin sheath, and NF-H staining in the axon. Acutely denervated rat units transited from broken rings of β4 and P0 staining, to diffuse β4 and absent P0 and NF-H staining. Chronically denervated rat Schwann cells re-expressed β4 more highly, but in a diffuse, non-polarized pattern. In contrast, regenerating units re-expressed β4, P0, and NF-H; β4 staining was polarized to the outer surface of Schwann cells. Finally, GFAP staining increased progressively after injury and decreased during regeneration in the distal nerve stump. In neuropathic human sural nerves, we identified units exhibiting each of these β4, P0, and NF-H staining patterns; the proportion of each pattern correlated best with the extent and chronicity of axonal injury. Thus, synchronous injury of rat sciatic nerve predicts patterns of Schwann cell marker expression in human axonal neuropathy. In addition, the unique changes in the polarity of β4 integrin expression, in combination with changes in P0 and NF-H expression, may distinguish normal from denervated or reinnervated myelin-forming Schwann cells in human sural nerve biopsies. © 1996 Wiley-Liss, Inc.     

Ultrastructural analysis of the paranodal junction of myelinated fibers in 31-month-old-rats

Recent studies have revealed a significant decrease in white matter volume, including loss of myelin, with age but minimal decrease in gray matter volume (Guttmann et al., [1998] Neurology 50:972-978). Myelin is necessary for the rapid conduction of impulses along axons. Myelinated nerve includes various domains, the node of Ranvier, the paranodal region, the juxtaparanodal region and the internode. The paranodal junction may serve to anchor the myelin sheath to the axon. We analyzed the ultrastructure of the paranodal region in myelinated fibers from the aged rat brain. Severe alterations of myelinated fibers were observed in 31-month-old rats, resulting in the appearance of macrophages, splitting of the myelin sheath, myelin balloon formation and separation from the axon. Many paranodal retractions of myelinated axons occurred in the aged rats. It should be noted that the paranodal junction is functionally important, serving to anchor the myelin to the axon and that there is a diffusion barrier in the paranodal region. We analyzed myelin-related proteins from young and aged rat brains. The 21.5-kDa isoform of myelin basic protein (MBP) almost disappeared in the 31-month-old rats, whereas other myelin proteins were not significantly changed between young and aged rats. These results suggest that this isoform, a highly cationic charged major dense component protein that binds lipid bilayer in the membrane, may participate in the formation of a paranodal diffusion barrier at the myelin/noncompact membrane border.     

Demyelination in vitro. Absorption studies demonstrate that galactocerebroside is a major target

Myelinated cultures of mouse spinal cord have been exposed to sera raised in rabbits against whole white matter (anti-WM), myelin basic protein (anti-MBP) and galactocerebroside (anti-GC), the major glycolipid of CNS myelin, to determine which factor in central nervous system (CNS) tissue in vitro is the target of serum demyelinating and myelin swelling antibodies. The sera were tested by radioimmunoassay for activity against MBP and against GC and were also specifically absorbed with MBP, GC and control antigens. Studies were also performed with and without active complement. The findings show that demyelination and myelin swelling in vitro are caused by antibodies against GC and not against MBP. Ultrastructurally, the effects of anti-WM and anti-GC sera with and without complement were indistinguishable. This study demonstrates that GC is a major target in antibody-mediated demyelination.     

HIF-1α基因修饰神经干细胞移植对大鼠损伤脊髓NF200和GFAP表达的影响

【摘要】目的研究低氧诱导因子-1α（HI-1α）基因修饰的神经干细胞移植对大鼠脊髓损伤后神经丝蛋白200（NF200）和胶质纤维酸性蛋白（GFAP）表达的影响及意义。方法采用电控脊髓损伤打击装置制作大鼠脊髓损伤模型。按随机数字表将120只SD大鼠平均分为4组：假手术组（Sham组）,单纯损伤组（SCI组）,神经干细胞组（NSC组）和HIF-1α基因修饰NSC组（HIF—NSC组）。应用免疫组化法检测受伤脊髓中HIF-1α、NF200和GFAP的表达。结果HIF-NSC组中HIF-1αt免疫阳性细胞平均光密度值比其他各组各时间点均高（P〈O．01）,且表达高峰延迟至移植后14d;除第1天外,HIF—NSC组NF200表达比SCI组和NSC组明显增高（P〈0．05）,移植后28dNF200免疫阳性轴突数目也比SCI组和NSC组明显增多（P〈0．01）;移植后7d、14d、28dGFAP免疫阳性细胞面积均比SCI组和NSC组明显减少（P〈0．01）。结论HIF-1α基因修饰NSC移植可引起HIF-1α在损伤脊髓内有效表达,且能明显的促进NF200的表达,并能在脊髓损伤的后期抑制GFAP的表达。这提示HIF-1α基因修饰的NSC移植可减少受伤脊髓中胶质细胞的增生和胶质疤痕的形成,促进轴突再生。