B细胞和T细胞亚型在海人藻酸引起的鼠海马神经元损伤过程中起到了不同的作用

目的 应用海人藻酸(Kainic acid，KA)在C57BL／6免疫缺陷小鼠建立了神经退行性病变并观察了免疫活性B细胞和T细胞亚型在病变过程中的作用。方法 经鼻滴入海人藻酸观察其临床和病理变化，细胞流式仪检测和分析脾细胞表面标记。结果 海人藻酸引起了CD4(CD4-／-)、CD8（CD8-／-)、CD48(CD48-／-)和B细胞(Igh6-／-)基因敲除鼠的临床抽搐症状和海马损伤。其临床症状在CD4(-／-)鼠最重，CD8(-／-)、Igh6(-／-)以及野生型鼠次之，而CD4CD8双重基因缺陷鼠最轻。病理变化大约和临床症状相平行，脾细胞表面标记的表达也证实了上述发现。结论 获得性免疫反应参与了海人藻酸引起的海马损伤，CD4T细胞和B细胞在病变过程中可能起到了保护作用，而CD8T细胞则加重神经退行性病变。     

经鼻滴入海人藻酸引起C57BL/6小鼠嗅球和海马区神经元的退行性病变

目的 应用海人藻酸(KA)在C57BL／6小鼠建立神经退行性病变动物模型并观察其对嗅球神经元的影响。方法 经鼻滴人KA应用尼氏和嗜银染色观察海马及嗅球的病理变化，免疫组化检测Cyclooxygenase2(COX-2)的表达。结果 经鼻滴入KA成功地在C57BL／6小鼠建立了神经退行性病变动物模型，KA通过嗅神经引起双侧嗅球和海马损伤，其病变程度与小鼠体重和滴入KA剂量有关，同时KA引起了脑内明显的胶质细胞增生和炎症因子COX-2在嗅球部的表达。结论 经鼻滴入KA能够引起嗅球和海马的损伤。     

神经免疫调节网络上传信号的细胞来源

目的通过比较不同免疫状态的Balb/c鼠和裸鼠中枢神经免疫调节相关脑区功能的差异,探讨外周免疫系统释放的神经免疫调节网络上传信号的细胞来源。方法 Balb/c鼠和裸鼠各50只,每种动物随机分成免疫2、4、6、8d组和健康对照组。应用免疫组织化学技术检测不同免疫时间点动物的神经免疫调节相关脑区下丘脑外侧区(lateral hypothalamic area,LH)和杏仁核前区(anterior amygdaloid area,AA)功能活化信号白细胞介素-1β(IL-1β)的表达水平,分析T细胞功能缺陷对神经免疫调节功能的影响。结果与健康对照组比较,Balb/c鼠免疫2、4、6d组LH、AA脑区IL-1β表达明显增高(P<0.05),且免疫后2d其表达即增高,免疫后4d达高峰,免疫后6d开始下降(均P<0.05),免疫后8d其表达量降至基础生理水平(P>0.05)。裸鼠免疫组LH、AA脑区IL-1β表达趋势与Balb/c鼠结果相似。裸鼠免疫组LH、AA脑区IL-1β表达水平与Balb/c鼠比较无统计学差异(P>0.05)。结论 Balb/c鼠与裸鼠经抗原免疫注射后均可启动LH、AA脑区的神经免疫调节功能,提示免疫系统功能活化后,向中枢神经系统上传的神经免疫调节功能启动信号由树突状细胞和T辅助细胞共同提呈,但主要来自树突状细胞。     

慢性癫(癎)鼠海马内移植神经干细胞的存活、迁移、分化及与宿主整合的研究

目的 研究神经干细胞(NSC)移植到慢性海人酸癫(癎)鼠海马CA3区后细胞的存活、迁移、分化及与宿主细胞的整合情况.方法 将增强型绿色荧光(EGFP)标记的原代培养NSC移植到慢性癫(癎)鼠的海马CA3区.分别在移植后第2、4、8、12周4个时间点取脑冷冻切片,在倒置荧光显微镜下观察移植细胞的存活和迁移.采用免疫荧光染色观察移植细胞的分化和整合情况.结果 NSC在移植后2周未见迁移;移植后4周,可见移植细胞迁移到距移植区最近的齿状回;8周和12周,移植细胞可迁移到齿状回和海马各区.12周仍有大量移植细胞(65 045.00±881.72)存活.NSC在移植区以分化为胶质细胞为主,在齿状回和海马各区以分化为神经元为主;γ-氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比例较高.突触素免疫荧光染色示移植细胞与宿主细胞间产生了有机的整合.结论 NSC移植于慢性癫(癎)鼠的海马CA3区后,能够迁移到齿状回和海马各区并长期存活,且可分化为神经元,特别是GABA能神经元,并能与宿主细胞有机整合.     

慢性癫鼠海马内移植神经干细胞的存活、迁移、分化及与宿主整合的研究

目的研究神经干细胞(NSC)移植到慢性海人酸癫疒间鼠海马CA3区后细胞的存活、迁移、分化及与宿主细胞的整合情况。方法将增强型绿色荧光(EGFP)标记的原代培养NSC移植到慢性癫疒间鼠的海马CA3区。分别在移植后第2、4、8、12周4个时间点取脑冷冻切片,在倒置荧光显微镜下观察移植细胞的存活和迁移。采用免疫荧光染色观察移植细胞的分化和整合情况。结果NSC在移植后2周未见迁移;移植后4周,可见移植细胞迁移到距移植区最近的齿状回;8周和12周,移植细胞可迁移到齿状回和海马各区。12周仍有大量移植细胞(65 045.00±881.72)存活。NSC在移植区以分化为胶质细胞为主,在齿状回和海马各区以分化为神经元为主;-γ氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比例较高。突触素免疫荧光染色示移植细胞与宿主细胞间产生了有机的整合。结论NSC移植于慢性癫疒间鼠的海马CA3区后,能够迁移到齿状回和海马各区并长期存活,且可分化为神经元,特别是GABA能神经元,并能与宿主细胞有机整合。     

胚胎大鼠神经干细胞培养及分化鉴定的实验研究

目的建立胚胎大鼠神经干细胞体外培养及分化鉴定的方法。方法分离不问孕龄(13- 18d)SD胎鼠脑室下区、中脑及海马等部位组织,在含有表皮生长因子和碱性成纤维细胞生长因子及N-2 添加剂的DMEM培养基中悬浮培养,形成神经球体后撤除生长因子,经消化传代于含胎牛血清培养基中贴壁生长。应用免疫荧光方法检测原代细胞特异性标记巢蛋白、神经元标记微管蛋白-β、胶质细胞标记胶质纤维酸性蛋白,以及少突胶质细胞标记半乳糖脑苷的表达。结果胎鼠脑组织不同部位均可分离出神经干细胞,体外培养可以向神经元和神经胶质细胞分化,孕龄13d与18d胎鼠产生神经干细胞球体的能力不同,前者优于后者。结论 (1)胚胎大鼠脑内不同部位来源的组织生成神经干细胞球体数量和质量基本相同,但是不同胎龄的鼠脑组织产生神经干细胞球体的能力有所差异。(2)神经干细胞具有向神经元和神经胶质细胞分化的潜能。     

LY294002对小鼠体内调节性T细胞的影响

背景：研究已证实,磷酸肌醇3激酶特异性阻滞剂LY294002在体外具有剂量依赖性抑制细胞活性的作用,可以用于诱导肿瘤细胞凋亡,但LY294002对正常免疫系统的影响尚不清楚。 目的：观察抗肿瘤药物LY294002对C57BL/6小鼠体内调节性T细胞的影响。 设计、时间及地点：以调节性T细胞比例为观察对象,分组对比实验,于2008-08/12在广州南方医科大学珠江医院移植免疫研究所完成。 材料：14只8周龄SPF级C57BL/6小鼠随机分成实验组与对照组,每组7只。LY294002购自广州英韦创津公司。 方法：实验组腹腔注射LY294002溶液200 μL(0.1 mg),对照组给予等体积PBS。10 h后后眼眶静脉从采血,并分离脾脏、胸腺,制备单细胞悬液,流式细胞术检测小鼠外周血、脾细胞和胸腺细胞中的CD4+CD25high T 细胞。 主要观察指标：小鼠外周血、脾细胞和胸腺细胞中CD4+CD25high T 细胞的比例。 结果：实验组小鼠外周血、脾脏、胸腺中CD4+CD25high T细胞比例分别为(0.787±0.036)%,(0.921±0.063)%,(1.230± 0.131)%,对照组小鼠外周血、脾脏、胸腺中CD4+CD25high T细胞比例分别为(0.640±0.030)%,(0.639±0.046)%,(0.857±0.077)%,实验组和对照组比较差异有显著性意义(P < 0.01,P < 0.05)。 结论：腹腔注射LY294002可提升小鼠体内调节性T细胞的比例,提示其有可能会抑制机体免疫反应,从而不利于治疗。     

载脂蛋白E对EAE星形胶质细胞的影响

目的探索载脂蛋白E(apolipoprotein E,apo E)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型小鼠病灶星形胶质细胞的影响。方法采用MOG35-55诱导apo E基因敲除的C57BL/6小鼠(apo E-/-EAE组)及野生型C57BL/6小鼠(普通EAE组),建立EAE模型。取免疫后35 d的EAE小鼠的大脑及脊髓切片,行HE染色,免疫组织化学染色法观察各组小鼠水通道蛋白4(AQP4)和胶质纤维酸性蛋白(GFAP)的表达。结果 apo E-/-EAE组和普通EAE组大脑和脊髓AQP4、GFAP的表达量均高于正常对照组,差异均有统计学意义(P<0.05);apo E-/-EAE组大脑和脊髓AQP4、GFAP表达量均高于普通EAE组,差异均有统计学意义(P<0.05)。结论 EAE中存在星形胶质细胞水肿、增生,apo E缺乏可以加重这一病变。     

神经干细胞和GABA能神经元治疗大鼠颞叶癫痫实验研究

目的探讨并比较神经干细胞(NSCs)和γ-氨基丁酸(GABA)能神经元移植治疗大鼠颞叶癫痫的疗效。方法取孕12 d SD大鼠胎鼠脑组织,分离培养NSCs并鉴定,取第3代NSCs定向诱导分化为GABA能神经元。48只SD大鼠随机分为4组,空白对照组、未移植组、NSCs移植组和GABA能神经元移植组,移植细胞用5-溴脱氧尿苷(BrdU)标记,在模型建立后的第4 d将上述两种细胞移植到癫痫大鼠右侧海马。分别在细胞移植后的4 w、8 w、12 w处死大鼠留取脑标本。常规HE染色和Nissl染色观察大鼠右侧海马的损伤与治疗情况并进行评价。结果 NSCs移植组和GABA能神经元移植组均于移植后第8 w时海马CA3(CA3)区神经元计数最多,组内比较时,与另外两个时间点之间的差异具有统计学意义(P<0.05)。进而取第8 w时间点进行组间比较,结果各组海马区神经元计数之间的差异均具有统计学意义(P<0.05)。结论两治疗组在移植后第8 w时海马区神经元计数最多,且NSC移植组的疗效优于GABA能神经元移植组。     

13例淋巴瘤合并周围神经病变临床分析

目的 对淋巴瘤合并周围神经病变的临床特点、辅助检查结果进行分析,并探讨其发病机制.方法 回顾分析13例淋巴瘤合并周围神经病变患者诊断、治疗经过,排除继发性带状疱疹病毒感染、相关治疗的不良反应及其他导致周围神经病变的病因.其中T细胞性非霍奇金淋巴瘤(NHL-T)3例,B细胞性非霍奇金淋巴瘤(NHL-B)9例,1例具体病理类型不详.对其周围神经受累的类型和部位、脑脊液细胞学和免疫组织化学、影像学、电生理学以及腓肠神经活体组织病理学检查结果等进行分析.结果 13例中10例周围神经系统症状出现于淋巴瘤明确诊断之前,其中8例以周围神经系统症状首发:11例脑神经受累,8例腰骶神经根受累,6例同时合并听力减退及腰骶神经根受累.脑脊液检查主要呈现蛋白定量(13例)、白细胞计数(8例)以及脑脊液压力(5例)升高,葡萄糖水平降低(7例);细胞学检查可见异型淋巴细胞(9例);免疫组织化学染色及基因重排检测主要表现为B细胞标记抗原(CD19、CD20、CD79α)阳性(3例),CD20阳性细胞数日增加(1例),以及CD4阳性细胞数目增加、T细胞(抗原识别)受体阳性和抗体IgH阴性(1例).肌电图检查显示,周围神经轴索病变或合并髓鞘损害(9例).腓肠神经活体组织病理学检查呈轴索变性和(或)脱髓鞘病变且无淋巴瘤细胞浸润(3例).头部及腰骶部MRI检查显示,双侧听神经及马尾神经根增粗、强化或脑膜强化.13例中9例系由淋巴瘤细胞直接浸润脑脊膜神经根所致,1例为淋巴瘤转移和局部肿大淋巴结压迫引起,其余3例无明确肿瘤细胞浸润证据,结合临床特点和实验室检查结果,考虑副肿瘤综合征可能.结论 淋巴瘤可通过肿瘤细胞直接浸润或副肿瘤综合征导致周围神经病变;周围神经系统症状在淋巴瘤明确诊断前即可出现,临床应对此提高认识.     

Kainic acid-induced excitotoxic hippocampal neurodegeneration in C57BL/6 mice: B cell and T cell subsets may contribute differently to the pathogenesis

The roles of T cells and B cells in kainic acid (KA)-induced hippocampal lesions were studied in C57BL/6 mice lacking specific T cell populations (CD4, CD8, and CD4/CD8 cells) and B cells [Igh-6(-/-)]. At 48 mg/kg of KA administrated intranasally, KA-induced convulsions were seen in all groups. However, CD4/CD8(-/-) mice exhibited the mildest seizures; the responses of CD8(-/-), Igh-6(-/-) and wild-type mice were intermediate, whereas CD4(-/-) mice displayed much more severe clinical signs and 100% early mortality, indicating that a deficiency of CD4 T cells obviously increased susceptibility to KA-induced brain damage. Histopathological analysis of the mice that survived 7 days after KA administration revealed that CD4/CD8(-/-) mice had the fewest pathologic changes but Igh-6(-/-) mice showed more severe lesions in area CA3 of the hippocampus than CD8(-/-) and wild-type mice. Reactive astrogliosis were prominent in all KA-treated mice. Locomotor activity as assessed by open-field test increased after KA administration in Igh-6(-/-) and wild-type mice only. These results denote the influence of the adaptive immune response on KA-induced hippocampal neurodegeneration and suggest that B cell and T cell subsets may contribute differently to the pathogenesis.     

Reduced susceptibility to kainic acid-induced excitoxicity in T-cell deficient CD4/CD8(-/-) and middle-aged C57BL/6 mice

Kainic acid (KA)-induced hippocampal injury is a good model for studying human neurodegenerative diseases. To investigate the roles of immune cells and age related changes in neurodegeneration, we used this model to assess reactions in young and middle-aged wild-type and CD4/CD8(-/-) mice by intranasal administration of KA. We found that CD4/CD8-deficiency resulted in a significant reduction of the severity of clinical signs and pathological changes in KA-treated young, but not in KA-treated middle-aged mice. Middle-aged wild-type mice had a similar reaction to KA insult as young and middle-aged CD4/CD8(-/-) mice. CD4/CD8(-/-) mice exhibited decreased locomotor and rearing activities as they approached to middle-aged state, which was not seen in wild-type mice. In addition, CD4/CD8-deficiency and increased age prevented KA-induced increase of both locomotor and rearing activities. The results suggest that a decline of immunological function is associated with aging, and both of them may contribute to the relative resistance to KA-induced neurotoxicity.     

CD4 and CD8 T cells,but not B cells,are critical to the control of murine experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune disease of the peripheral nervous system that duplicates the clinical, pathological, and electrophysiological features of Guillain-Barré syndrome in humans. However, the molecular pathogenesis of EAN remains controversial. Therefore, for this study, we induced EAN with P0 protein peptide 180-199 in CD4(-/-), CD8(-/-), CD4(-)8(-), and B cell knockout (microMT) mice to further investigate the roles of these cells in EAN. Our results showed that the severity of clinical signs and histopathological manifestations of EAN and the T cell response to P0 peptide 180-199 in CD4(-/-) mice were significantly lower than those in their wild-type counterparts. CD8(-/-) mice also had a milder clinical course, less histopathological change, and a diminished T cell response to P0 peptide 180-199. However, more severe clinical and histopathological manifestations, a stronger T cell response to P0 peptide 180-199, and enhanced IFN-gamma production in the spleen were observed in the EAN of CD4(-)8(-) and microMT mice, but these were not obviously different from those of wild-type mice. Levels of IgG production were similar in sera from CD4(-/-), CD8(-/-), and CD4(-)8(-), and wild-type mice. These findings suggest that the induction and control of murine EAN are dependent on both CD4(+) and CD8(+) T cells and that B cells apparently do not perpetuate the related inflammatory demyelination.     

Overexpression of apolipoprotein E4 increases kainic-acid-induced hippocampal neurodegeneration

Apolipoprotein E (apoE) has an intricate biological function in modulating immune responses and apoE isoforms exhibit diverse effects on neurodegenerative and neuroinflammatory disorders. In the present study, we investigated the individual roles of apoE isoforms in the kainic acid (KA)-induced hippocampal neurodegeneration with focus on immune response and microglia functions. ApoE2, 3 and 4 transgenic mice as well as wild-type (WT) mice were treated with KA by intranasal route. ApoE4 overexpressing mice revealed several peculiarities as compared with other transgenic mice and WT mice, i.e. (1) they had more severe KA-induced seizures than apoE2 and 3 mice, (2) they exhibited neuron loss in hippocampus that was higher than in apoE2, 3 and WT mice, (3) KA administration resulted in higher counts of their head drops in the cross-area of elevated plus-maze, (4) they showed lower KA-induced rearing activity than apoE2 mice in the open-field test, (5) their KA-induced microglial expression of MHC-II and CD86 was elevated compared to apoE3 mice, (6) the KA-induced increase of microglial iNOS was higher than that in the other groups of mice, and (7) the TNF-α and IL-6 expression was decreased 7 days after KA application compared to untreated mice and mice treated 1 day with KA. However, the signaling pathway of NFκB or Akt seemed not to be involved in apoE-isoform dependent susceptibility to KA-induced neurotoxicity. In conclusion, over-expression of apoE4 deteriorated KA-induced hippocampal neurodegeneration in C57BL/6 mice, which might result from a higher up-regulation of microglia activation compared to apoE2 and 3 transgenic mice and WT mice.     

Comparing the pathogenesis of experimental autoimmune encephalomyelitis in CD4-/- and CD8-/- DBA/1 mice defines qualitative roles of different T cell subsets

Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG(1-125)) in CD4(-/-) and CD8(-/-) DBA/1 mice. Both gene-deleted mice developed clinical signs of EAE, albeit milder than in wild-type mice, suggesting that both CD4(+) and CD8(+) cells participate in disease development. Demyelination and inflammation in the central nervous system was reduced in the absence of CD8(+) T cells. Antibody depletion of CD4(+) cells completely protected CD8(-/-) mice from MOG-induced EAE while depletion of CD8(+) cells in CD4(-/-) mice resulted in fewer EAE incidence compared to that in control antibody-treated mice. Antibody depletion of CD4(+) cells in wild-type mice protected from EAE, but not depletion of CD8(+) cells, although demyelination was reduced on removal of CD8(+) T cells. Immunization with immunodominant MOG(79-96) peptide led to EAE only in the presence of pertussis toxin (PT) in the inoculum. PT also triggered an earlier onset and more severe EAE in CD8(-/-) mice. We interpret our findings such that in an ontogenic lack of CD4(+) T cells, EAE is mediated by CD8(+) and elevated levels of alphabetaCD4(-)CD8(-) cells, and that CNS damage is partly enacted by the activity of CD8(+) T cells.     

Immune cell subtyping in the cerebrospinal fluid of patients with neurological diseases

The analysis of cerebrospinal fluid (CSF) with the assessment of CSF cell counts and proteins is an important method in the diagnostic workup of neurological diseases. As an addition to this standard approach, we here present data on the distribution of CSF immune cell subsets in common neurological diseases, and provide reference values along with cases of rare neurological diseases. CD4+ and CD8+ T cells, the CD4/CD8 ratio, B cells, plasmablasts, monocytes and NK cells in the CSF of 319 patients with inflammatory or non-inflammatory neurological diseases were analysed by seven-color flow cytometry. Diagnoses included headache, idiopathic intracranial hypertension, Guillain–Barré syndrome, multiple sclerosis, Lyme neuroborreliosis, bacterial and viral meningitis, human immunodeficiency virus (HIV) infection, stroke, and CNS malignancies, among others. T cells were the predominant population in the CSF with CD4+ T cells being more prevalent than CD8+ T cells. Mostly in HIV patients, and under other conditions of immunosuppression, CD4+ and CD8+ T cells were significantly altered and the CD4/CD8 ratio reduced. B cells and plasmablasts could hardly be detected in non-inflammatory diseases but were consistently elevated in inflammatory diseases. Monocytes were reduced in neuroinflammation and showed a negative correlation with B cells. NK cells were slightly elevated in neuroinflammation. Both monocytes and NK cells were slightly elevated in CNS malignancies. The analysis of immune cell subsets in the CSF adds valuable information to clinicians and is a promising tool for the differential diagnosis of neurological diseases.     

CD28-B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.     

Immune disorders in depression: higher T helper/T suppressor-cytotoxic cell ratio.

This study investigated the leukocyte T helper and T suppressor-cytotoxic cell (sub)set profile of minor, simple major and melancholic depressives versus normal controls. Using both monoclonal antibody staining and flow cytometry, we determined the absolute numbers and percentages of the following T cell immune subsets: T helper (CD4+), T virgin (CD4+CD45+), T memory (CD4+CD45-), T suppressor/cytotoxic (CD8+), CD8+ T suppressor (CD8+CD57-) and CD8+ T cytotoxic (CD8+CD57+) cells. After computing the CD4+/CD8+ ratio, we detected a significantly increased ratio in depressed patients as compared with healthy controls. Depression per se is characterized by a higher percentage of CD4+ and a lower percentage of CD8+CD57- cells. Melancholic depressed subjects exhibit a significantly increased number of CD4+ and CD4+CD45- cells. The combined use of various percentages of CD4+ and CD8+ (sub)sets yields a high degree of marker positivity for melancholia: through cumulative evaluation of those percentages, the marker positivity increases to 68% (sensitivity) and the specificity is 95%. These results together with our previous reports may refer to a depression-related state of T cell activation.