大鼠脑缺血后神经元中PYK2及 p38MAPK的活化及其意义

目的观察大鼠局部脑缺血后缺血区神经元中PYK2及p38MAPK的表达并探讨其意义。方法建立大鼠局部脑缺血模型,在缺血后15、30和60min时处死大鼠,采用免疫组化方法观察大鼠缺血区神经元中PYK2及p38MAPK的表达变化。结果正常大鼠皮层仅有微量活化型PYK2和活化型p38MAPK表达。缺血15min后,皮层神经元可见活化型PYK2强免疫阳性标记,这些有活化型PYK2表达的神经元亦呈p38MAPK免疫阳性。缺血区神经元中活化型PYK2和活化型p38MAPK免疫标记在缺血30min时达到最强,60min时开始减退。结论缺血可以诱发神经元中PYK2活化,既而通过p38MAPK信号转导通路介导神经元对缺血的应急反应。     

潜艇脱险中极快速减压后大鼠小胶质细胞与神经元凋亡的变化

目的 研究成年大鼠极快速减压致中枢神经损伤后脑组织内小胶质细胞的变化及其与损伤后神经元凋亡的关系。方法 采用1MPa暴露5．5min、50s快速减压制备SD大鼠中枢减压损伤模型,在减压后6、24、48、72h取材,分别用FITC标记的凝集素B4（IB4）标记小胶质细胞和原位末端TUNEL。法标记凋亡神经元细胞。结果 快速减压后6h组可见少量IB4阳性小胶质细胞;24h组达高峰（P〈0．01）;48h组阳性小胶质细胞数量有所下降,但仍高于6h组（P〈0．01）;72h组阳性小胶质细胞数量明显下降。6h组仅见少量散在TUNEL,阳性细胞;48h组达高峰（P〈0．01）;72组阳性细胞数量有所下降,但仍高于6h组（P〈0．01）。凝集素阳性小胶质细胞分布区域与神经元凋亡分布区域一致,达到高峰的时间前者先于后者,两者的变化趋势相同（r=O．645,P〈0．01）。结论 不安全极快速减压致中枢型减压病的中枢神经损伤中存在神经元凋亡和小胶质细胞的活化,激活的小胶质细胞可能参与了快速减压后的神经元凋亡过程。     

实验性癫痫大鼠海马损害与磁共振波谱对照研究

目的 :观察癫痫持续不同时间氢质子磁共振波谱 (1 H MRS)检测的致痫灶 NAA和 Cho异常与海马结构损害的相关性。方法 :2 1只 Wistar大鼠 ,实验组 15只 ,10 % Pilocarpine 35 0 mg/ kg腹腔注射 ,诱发反复全面强直阵挛发作 ,光镜观察发作持续 5min至 6 0 h大鼠海马区神经元和胶质细胞变化 ,并计数海马 CA1 和 CA3段残存正常神经元数量 ;离体1 H MRS测定代谢产物 NAA和 Cho含量变化。结果 :癫痫持续 (SE) 5～ 30 min,光镜观察到神经元变性 ,1 HMRS检测出 NAA值降低 ;SE30 min～ 3h部分神经元坏死、少许丢失 ,轻度胶质增生 ,NAA明显降低 ;SE3～ 6 h后 ,大部分神经元坏死、丢失 ,胶质增生明显 ,形成典型的海马硬化 ;除NAA值降低外 ,1 HMRS还检测出 Cho值升高。结论 :1 HMRS检出的 NAA可反映癫痫脑损害程度 ,并且癫痫发作持续时间与 NAA值和残存正常神经元数量呈显著的负相关。Cho值升高在海马硬化形成后检出 ,可能反映胶质增生     

大鼠脑缺血再灌注后海马组织中GFAP和p－STAT3表达研究

目的：检测脑缺血再灌注（I／R）后海马齿状回区（dentate gyrus, DG）星形胶质细胞活化增殖及其磷酸化信号转导与转录激活子3（phosphorylated STAT3,p－STAT3）表达情况,为调控星形胶质细胞异常活化增殖提供理论依据。方法线栓法制作大鼠脑局部I／R模型,48只Sprague－Dawley（SD）大鼠随机分为假手术（Sham）组、I／R－24 h组、I／R－72 h组及I／R－7 d组。免疫荧光法及Western 印迹检测缺血后不同时间点患侧海马区胶质原纤维酸性蛋白（ glial fibrillary acidic protein , GFAP）标记阳性细胞及p－STAT3表达情况。结果与Sham组比较,I／R－72 h和I／R－7 d组海马区GFAP蛋白表达增加（P＜0．05）;随缺血时间延长,GFAP蛋白表达增加呈上升趋势,且24 h、72 h和7 d各时间点之间两两比较差异均具有统计学意义（P＜0．05）。与Sham组比较,脑I／R后海马区各时间点p－STAT3表达量均显著增加（P＜0．05）;p－STAT3表达在24 h最高,72 h开始下降,7 d仍有一定量表达,与I／R－24 h组比较, I／R－72 h组和I／R－7 d组p－STAT3表达量均显著降低（ P＜0．05）。结论脑I／R损伤后,海马区星形胶质细胞大量增殖及其p－STAT3表达增加;且p－STAT3的表达高峰在时间上早于星形胶质细胞的大量增殖。     

红藻氨酸诱导癫痫发作大鼠脑内GLAST表达的动态研究

目的：观察红藻氨酸（kainc acid,KA)诱导大鼠癫痫发作时脑内谷氨酸转运蛋白亚型GLAST表达的变化。方法：用豚鼠抗GLAST多克隆体及免疫细胞化学ABC法观察KA注射后不同时间脑内GLAST的表达,结果：KA注射后1h,小脑分子层和薄肯野氏细胞层的GLAST免疫反应强度开始增加,至3h达高峰（P＜0．01）,随后呈下降趋势,12h时低于发作前水平（P＜0．05）,72h恢复正常,海马区表达的GLAST阳性星形胶质细胞于KA注射后3－6h内亦有相应的升高,以CA3区改变最明显＊（P＜0．05）,结论：KA诱导大鼠癫痫发作时脑内GLAST的表达早期呈现快速上调,其机理可能是细胞对损伤的一种保护性反应,有助于清除细胞外过量的谷氨酸,预防其对神经元的兴奋性毒性作用,并对控制边缘环路的兴奋性有重要作用。     

大鼠急性脑损伤后神经元类凋亡的观察

目的：观察急性脑损伤后神经元凋亡现象。方法：以大鼠急性弥漫性脑创伤模型为研究对象，采用光镜、电镜观察方法对模型的损伤情况进行组织、细胞形态观察；以DNA－梯形凝胶电泳、凋亡细胞原位末端标记法（TUNEL）对急性脑损伤后鼠脑皮层、海马区神经元DNA损伤情况进行观察。结果：大鼠致伤后，光镜下观察到神经元出现皱缩变性；电锏 上观察发现，伤后2h可观察到神经元类凋亡，24h最为严重，持续到7d；DNA－梯形凝胶电泳显示伤后24h海马及皮层区出现DNA梯状电泳；神经元TUNEL染色伤后2h即可见，24h最为明显，7d时仍高于正常。结论：大鼠急性脑创伤后脑组织中存在神经元类凋亡现象，并在创伤后较长时间内持续存在。     

无镁损伤培养海马神经元后ERK1/2核转移的动态变化

目的 动态观察培养的大鼠海马神经元经无镁损伤后不同时间细胞外信号调节激酶磷酸化(p-ERK1/2)及其核转移,探讨体外培养海马神经元受致痫性损伤后ERK1/2信号通路的动态变化. 方法 选24 h内新生Wistar大鼠,取双侧海马神经元,用NB培养基加B-27培养9 d,换成无镁细胞外液模拟"癫痫微环境",使得神经元受到致痫性损伤.采用免疫荧光标记,在激光共聚焦显微镜下定位无镁损伤前后p-ERK1/2的表达部位;运用Western blot技术检测无镁损伤不同时间p-ERK1/2表达强度的变化. 结果 无镁损伤前,p-ERK1/2主要在神经元胞浆和轴浆内表达,胞核表达不明显,而在损伤之后,p-ERK1/2在神经元胞浆、轴浆以及胞核内都有表达,强度接近;在损伤后1 h,p-ERK1/2表达就增强,3 h达到高峰(p-ERK1:2.2 838±0.1 186;p-ERK2:4.1 273±0.0 927),与对照组比较,差异有统计学意义(P<0.05). 结论无镁细胞外液损伤可引起培养的神经元p-ERK1/2的大量表达,培养神经元致痫性损伤后ERK1/2信号通路的过度激活可能与其反复癫痫样放电有关.     

慢性癫痫大鼠脑组织GAT-1的表达变化及其与星形胶质细胞的相关性

目的 研究γ-氨基丁酸转运体-1(GAT-1)与胶质纤维酸性蛋白(GFAP)在戊四氮点燃的慢性癫痫大鼠脑中的变化.方法 采用免疫组化技术分别标记正常SD大鼠和戊四氮点燃的慢性癫痫大鼠发作后海马及外侧皮质区GAT-1和GFAP,结合Western blot实验结果分析其变化关系.结果 GAT-1和GFAP在慢性癫痫发作后,均有不同程度的表达增高,GAT-1增高更明显.结论 GAT-1和GFAP的同方向变化证明,在慢性癫痫发作后,GAT-1的表达增高并不只是由于星形胶质细胞的增生所致.     

过表达脑源性神经营养因子的神经干细胞移植对受照海马内神经营养因子的影响

目的 探讨过表达脑源性神经营养因子（BDNF）的神经干细胞（NSCs）移植入放射性脑损伤大鼠模型后,对海马内神经营养因子水平及小胶质细胞活化的影响。方法 从胎鼠脑中分离海马神经干细胞并进行培养。选用绿色荧光蛋白（GFP）-慢病毒、GFP-BDNF-慢病毒感染神经干细胞。将SD大鼠按随机数表法分为4组：健康对照组、单纯照射组（R组）、照射后GFP修饰的神经干细胞移植组（R+NSCs组）、照射后GFP-BDNF修饰的神经干细胞移植组（R+BDNF-NSCs组）。全脑单次20 Gy照射后1个月将神经干细胞移植入大鼠双侧海马内。移植后2和8周检测海马组织中BDNF、胶质源性神经营养因子（GDNF）、神经生长因子（NGF）的表达情况;免疫荧光染色观察小胶质细胞活化情况。结果 移植后2和8周时,与R组相比,R+BDNF-NSCs组海马组织中BDNF、NGF蛋白表达均水平明显增高（P<0.05）;移植后8周R+NSCs组和R+BDNF-NSCs组活化的小胶质细胞与R组相比并未显著减少（P>0.05）。结论 过表达BDNF的神经干细胞移植后促进BDNF、NGF的产生,增加了辐射暴露后的海马内神经营养因子水平。     

SP600125对大鼠脑缺血再灌注神经元的保护作用

目的探讨SP600125-JNK特异性抑制剂对大鼠脑缺血再灌注神经元损伤的保护性作用及其作用机制。方法雄性SD大鼠54只,体重230～250g,随机分成假手术组（SH组）,缺血再灌注组（IR组）和JNK抑制剂SP600125组（SP组）,每组根据再灌注时间分为30min、24h和72h3个亚组,每亚组6只动物。采用4-VO法建立SD大鼠脑缺血模型,三组于缺血前30min侧脑室注射DMSO,DMSO及JNK抑制剂SP600125（溶媒采用DMSO）,容积均为10μl;脑缺血再灌注后30min、24h、72h免疫组织化学方法测定各时间点海马CA1区Bcl-2和Bax蛋白表达阳性细胞数量,TUNEL法检测CA1区凋亡细胞。结果缺血再灌注使海马CA1区Bcl-2和Bax阳性锥体细胞数目表达增加,再灌注24h阳性锥体细胞数目表达至高峰（P〈0.01）,再灌注24～72h可见阳性锥体细胞数目表达减少（P〈0.05）。其中SP组Bcl-2阳性锥体细胞数目显著多于IR组（P〈0.05）,而Bax阳性锥体细胞数目显著小于IR组（P〈0.05）。脑缺血再灌注后海马CA1区神经元存活数目SP组高于IR组（P〈0.01）,凋亡细胞数目低于IR组（P〈0.01）。结论 SP600125对大鼠脑缺血再灌注神经元损伤具有保护作用。     

Rapid alterations in diffusion-weighted images with anatomic correlates in a rodent model of status epilepticus

BACKGROUND AND PURPOSE: Diffusion-weighted MR imaging has emerged as a noninvasive tool for the detection of regional neuronal damage. We hypothesize that changes in diffusion-weighted images will correlate with pathophysiologic alterations caused by pilocarpine-induced status epilepticus. METHODS: MR images of brain tissues were examined in vivo by use of T2- and diffusion-weighted imaging at 3, 6, 12, and 24 hours after pilocarpine-induced seizures. Histologic verification of neuronal damage was also performed after imaging to assess the extent and the time course of neuronal cell death. RESULTS: The piriform cortex, amygdala, and retrosplenial (and somatosensory) cortex displayed significant apparent diffusion coefficient (ADC) decreases 12 hours after seizure initiation. In contrast, an ADC rise of 19% was observed in the hippocampus 24 hours after seizure induction. Histologic data from the piriform cortex and amygdala confirmed severe neuronal loss, whereas hippocampal damage was much less pronounced at 12 hours. Interestingly, very little histologic damage was seen in the retrosplenial cortex. CONCLUSION: This study capitalized on diffusion-weighted imaging as a sensitive technique for the early identification of seizure-induced neuronal damage and differentiation of regional severity of these alterations. Hippocampal neuropathology is slower and longer in duration (approximately 7 days), while the piriform cortex and amygdala exhibit very rapid neurodegenerative alterations (approximately 24 hours) after pilocarpine-induced status epilepticus. These histologic changes are reflected in opposing ADC values within these regions.     

Transient MR signal changes in patients with generalized tonicoclonic seizure or status epilepticus: periictal diffusion-weighted imaging.

BACKGROUND AND PURPOSE: Our purpose was to investigate transient MR signal changes on periictal MR images of patients with generalized tonicoclonic seizure or status epilepticus and to evaluate the clinical significance of these findings for differential diagnosis and understanding of the pathophysiology of seizure-induced brain changes. METHODS: Eight patients with MR images that were obtained within 3 days after the onset of generalized tonicoclonic seizure or status epilepticus and that showed seizure-related MR signal changes had their records retrospectively reviewed. T1- and T2-weighted images were obtained of all eight patients. Additional diffusion-weighted images were obtained of five patients during initial examination. After adequate control of the seizure was achieved, follow-up MR imaging was performed. We evaluated the signal changes, location of the lesions, and degree of contrast enhancement on T1- and T2-weighted images and the signal change and apparent diffusion coefficient (ADC) on diffusion-weighted images. We also compared the signal changes of the initial MR images to those of the follow-up MR images. RESULTS: The initial MR images revealed focally increased T2 signal intensity, swelling, and increased volume of the involved cortical gyrus in all eight patients. The lesions were located in the cortical gray matter or subcortical white matter in seven patients and at the right hippocampus in one. T1-weighted images showed decreased signal intensity at exactly the same location (n = 6) and gyral contrast enhancement (n = 4). Diffusion-weighted images revealed increased signal intensity at the same location and focally reduced ADC. The ADC values were reduced by 6% to 28% compared with either the normal structure opposite the lesion or normal control. Follow-up MR imaging revealed the complete resolution of the abnormal T2 signal change and swelling in five patients, whereas resolution of the swelling with residual increased T2 signal intensity at the ipsilateral hippocampus was observed in the other two patients. For one of the two patients, hippocampal sclerosis was diagnosed. For the remaining one patient, newly developed increased T2 signal intensity was shown. CONCLUSION: The MR signal changes that occur after generalized tonicoclonic seizure or status epilepticus are transient increase of signal intensity and swelling at the cortical gray matter, subcortical white matter, or hippocampus on periictal T2-weighted and diffusion-weighted images. These findings reflect transient cytotoxic and vasogenic edema induced by seizure. The reversibility and typical location of lesions can help exclude the epileptogenic structural lesions.     

表鬼臼素对慢性致癫幼鼠海马结构的影响

目的：探讨幼鼠早期癫痫持续状态与成鼠后海马硬化的关系;探讨表鬼臼素对海马硬化的影响。方法：采用锂-匹罗卡品腹腔注射制成幼鼠癫痫持续状态模型,应用Timm组织化学染色方法观察苔藓纤维发芽情况。结果：表鬼臼素干预组海马结构的损害与匹罗卡品对照组一致,苔藓纤维发芽现象较匹罗卡品组轻。结论：幼鼠早期长时间的痫性发作与成鼠后海马硬化有关;表鬼臼素对海马硬化的发生可能有抑制作用。     

大鼠大脑照射后海马区细胞凋亡与病理形态学变化的研究

目的　了解大鼠大脑受电离辐射后早期海马区细胞凋亡、bcl 2表达和病理形态学变化的情况。方法　用流式细胞仪测定凋亡细胞的比例与bcl 2蛋白的含量 ,用电镜和光镜分别作形态学变化的观察。结果　受 2 0Gy照射后第 1天起海马中就出现了凋亡细胞并逐步增加 ,至照射后3个月时恢复正常 ,而bcl 2蛋白的含量则呈相反的改变。在 3 0Gy照射后 3个月组中有坏死灶的存在 ,而其他组别中可以看到血管、胶质细胞和神经元等组织的形态学异常。结论　大鼠大脑受辐射后早期海马区可发生神经细胞凋亡、bcl 2表达的下降 ,以及各组织成分的病理形态学改变 ,这些变化的程度与照射剂量和观测时间有关。     

丹参对持续癫癎幼鼠脑损伤保护作用的实验研究

 目的探讨丹参对持续癫癎发作诱发幼鼠脑神经元损伤是否具有保护作用.方法皮下及腹腔注射贝美格针诱发健康幼龄鼠癫癎持续状态发作.光镜下观察神经元病变情况;电镜观察海马神经元超微结构的改变.结果持续癫癎组幼鼠脑组织光镜下可见明显的神经元病变,电镜下可见海马区神经元的超微结构病变.丹参治疗组神经元病变均轻于持续癫癎组;而正常对照组未见类似病变.结论丹参在组织、细胞和亚细胞水平对持续癫癎幼鼠脑神经元损伤具有一定的保护作用,为临床有效防治小儿惊厥性脑损伤提供了可靠的实验依据.     

Hippocampal shape analysis in status epilepticus associated with acute encephalitis

We performed MR imaging deformation-based hippocampal shape analysis in a 28-year-old woman in whom status epilepticus developed after acute encephalitis. Hippocampal shape analysis revealed severe global bilateral hippocampal atrophy. Regional volume loss was most accentuated in the medial and lateral aspects of the hippocampal head; the loss was similar to shape changes in hippocampal sclerosis of chronic temporal lobe epilepsy. This deformation pattern may reflect a common pathologic process that causes hippocampal volume loss in both of these conditions.     

Thalamus lesions in chronic and acute seizure disorders

Introduction  

Transient signal changes in the pulvinar have been described following status epilepticus. However, we observed persistent thalamus changes after seizures. The purpose of this study was to characterize thalamus changes in patients with seizure disorders and to correlate imaging findings with clinical features.     

应用Fluoro-Jade C评价匹罗卡品模型中新大脑皮质神经变性

目的 应用Fluoro-Jade C(FJC)在小鼠匹罗卡品癫痫模型中探测新大脑皮质结构中神经元的变性情况.方法 雄性昆明种小鼠10只(对照组5只,匹罗卡品处理组5只).对照组给予阿托品、生理盐水腹腔注射;匹罗卡品处理组给予阿托品、匹罗卡品腹腔注射诱发癫痫持续状态.在癫痫持续状态后12 h,通过经心灌注固定处死匹罗卡品处理组小鼠.对照组小鼠处死时间及方法同匹罗卡品处理组.在各新大脑皮质水平切制冠状切片,行FJC染色,在荧光显微镜下,观察FJC阳性细胞的形态和在新大脑皮质中的整体分布情况.结果 在匹罗卡品处理组,许多新大脑皮质出现呈亮黄绿色荧光的FJC阳性细胞,而对照组未见.结论 在小鼠匹罗卡品癫痫模型中,运用FJC染色技术在新大脑皮质中显示发生了大量神经元变性,有利于更好地理解颞叶癫痫的长期病理变化和自发反复发作的癫痫机制.