Cyt-C Bcl-XL和Bcl-XS在大鼠创伤性脑损伤模型中的表达意义

目的 观察大鼠创伤性脑损伤(TBI)后细胞色素C释放及凋亡因子Bcl-XL和Bcl-XS的表达意义.方法 采用大鼠自由落体建立脑损伤模型,分别于伤后2h、6h、12 h、24 h、72 h、120 h、168 h检测大鼠脑挫裂伤侧海马CA2-CA3区细胞色素C染色阳性细胞(免疫组化法),应用Bcl-XL和Bcl-XS原位杂交法检测Bcl-XL和Bcl-XS mRNA表达.结果 脑挫裂伤后,海马CA2-CA3区Cyt-C阳性染色的神经细胞出现具有明显时相性,脑损伤后大鼠脑组织内Bcl-XL、Bcl-XS mRNA出现相应改变.结论 大鼠脑挫裂伤后海马CA2-CA3区细胞有相应变化并出现细胞凋亡.     

腺病毒介导BDNF基因转移对大鼠创伤性脑损伤后iNOS表达及细胞凋亡的影响

研究重组腺病毒介导的脑源性神经营养因子（brain derived neurotrophic factor,BDNF)基因转移对创伤性脑损伤（TBI）后诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS)表达及细胞凋亡的影响。将重组腺病毒载体4μl注入承受单侧大脑皮质重锤打击的大鼠海马,对照组注射病毒缓冲液。伤后3h及1,3,7,14d利用免疫组化单标／双标染色。原位杂交／组化染色及DNA末端原位标记等方法,检测伤侧大脑皮质和海马各多区iNOS、BDNF及凋亡相关信号表达的改变。与对照组相比,同伤组大脑皮质及海马各区iNOS阳性细胞于伤后3h开始显著增多,7d达高峰。多数iNOS阳性细胞同时呈现凋亡相关蛋白阳性反应或TUNEL阳性反应,但很少同时表达BDNF mRNA。注射病毒载体组后3,7d,海马CA1区和DH区表达iNOS、凋亡相关蛋白的细胞及凋亡细胞显著减少（P均＜0．01）,而表达BDNF mRNA的神经元显著增多。提示,TBI诱导海马细胞表达iNOS及诱导海马细胞凋亡;腺病毒介导的BDNF基因转移通过抑制iNOS表达、增加BDNF表达及减少细胞凋亡的机制保护海马神经元。     

大鼠脑创伤后海马CA3区细胞凋亡及相关基因表达研究

目的研究弥漫性脑损伤后不同时间,大鼠海马CA3区细胞凋亡及相关基因Bcl-2、Bax和Caspase-3蛋白的表达情况,探讨脑创伤后神经细胞凋亡的分子生物学机制.方法应用流式细胞仪和免疫组化法,分别检测脑创伤后不同时间海马CA3区细胞凋亡率及Bcl-2,Bax和Caspase-3基因在蛋白质水平的表达情况.结果脑创伤后海马CA3区存在不同程度细胞凋亡,Bcl-2在脑损伤后表达下降,而Bax和Caspase-3在脑创伤后表达升高;Caspase-3表达的峰值时间(72 h)出现在Bax之后(48 h).结论弥漫性脑损伤后,大鼠海马CA3区存在细胞凋亡及Bcl-2,Bax和Caspase-3的表达变化.Bcl-2/Bax表达比值下降早于Caspase-3的上升,Bcl-2/Bax表达比值改变可能与Caspase-3活化有关,进而启动并加重脑损伤后神经细胞凋亡.     

亚低温对大鼠创伤性脑损伤后海马CA3区细胞凋亡及其相关蛋白表达的影响

本研究采用流式细胞仪 (flowcytometer ,FCM )及免疫组化法 ,分别观察亚低温对大鼠创伤性脑损伤 (TBI)后脑海马CA3区细胞凋亡率及Bcl 2、Bax和Caspase 3蛋白表达的影响 ,探讨亚低温抗细胞凋亡的脑保护机制。1　材料和方法1 1　研究对象 :雄性SD大鼠 4 8只 ,体重 350～ 375g ,随机分成空白对照、假手术、单纯脑损伤及脑损伤后亚低温治疗 4组 ,每组1 2只 ,6只用于检测细胞凋亡率 ,另 6只用来观察Bcl 2、Bax及Caspase 3蛋白表达。空白对照组不作任何处理 ,假手术组除了不给予头部打击外 ,其余操作同单纯脑损伤组 ,亚低温治疗组于伤后立…     

脑缺血再灌注后ICE、Bcl-2的表达及丹参的神经保护作用研究

目的观察凋亡相关基因白介素-lβ转化酶(ICE)、Bcl-2在脑缺血后的表达情况以及丹参(RSM)对其影响。方法健康沙土鼠63只,随机分成三组假手术组、对照组和丹参组。采用沙土鼠前脑缺血再灌注模型,在沙土鼠脑缺血再灌注后的2h、6h、12h、1d、3d、5d及7d分别处死沙土鼠,将脑组织进行免疫组织化学染色观察。结果ICE的蛋白表达主要在海马CA1区、CA4区及皮质,免疫阳性细胞呈褐色,第3天达高峰,第5天后减少;丹参组ICE的蛋白表达明显减少(P＜0.01)。Bcl～2的蛋白表达主要在大脑皮层、纹状体、丘脑及海马等广泛区域,并于6h达高峰,其后减少;丹参组Bcl-2的蛋白表达明显增加,特别是在海马CA1区及大脑皮层区(P＜0.01)。结论ICE在调节脑缺血中可能发挥重要作用;Bcl-2主要通过抑制细胞凋亡的早期环节而发挥作用;丹参能下调脑缺血后的ICE表达、上调脑缺血后的Bcl-2表达进而发挥其神经保护作用。     

FK506对大鼠脑液压伤后神经行为和记忆的影响

目的 研究FK506对大鼠脑液压伤后神经行为和记忆的影响。方法 通过液压损伤法建立大鼠脑损伤模型,随机分为损伤组、治疗组和对照组(每组8只)。采用免疫组化方法和图象分析技术,检测突触素在皮质、海马和基底节的表达,并用流式细胞仪法检测上述部位的凋亡细胞数。结果 治疗组神经行为与记忆实验成绩高于损伤组。治疗组海马CA1、CA3区突触素表达显著增高(P<0.01)。海马、基底节和皮质的凋亡细胞数较损伤组明显减少。结论 FK506能促进突触素表达,减少海马和皮质细胞凋亡,有利于促进脑损伤后神经行为和记忆的恢复。     

亚低温对大鼠脑创伤后海马区凋亡相关蛋白表达的影响

目的观察亚低温对大鼠创伤性脑损伤(TBI)后海马CA3区细胞凋亡及相关蛋白Bcl-2、Bax及Caspase-3表达的影响,探讨亚低温脑保护的分子生物学机制.方法将大鼠随机分成假手术、单纯脑损伤和脑损伤后亚低温治疗3组,应用改良Marmarou方法制作大鼠TBI模型,分别用流式细胞仪(FCM)和免疫组化法检测各组动物脑海马CA3区细胞凋亡率和Bcl-2、Bax及Caspase-3蛋白的表达.结果与假手术组相比,大鼠TBI后海马CA3区细胞凋亡率及Caspase-3表达增高(P<0.05),Bcl-2/Bax表达比下降(P<0.05).亚低温治疗后,大鼠脑海马CA3区细胞凋亡率及Caspase-3表达较单纯脑损伤组降低(P<0.05),而Bcl-2/Bax表达比升高(P<0.05).结论亚低温对TBI的脑保护作用机制可能与干预伤后凋亡相关基因表达并减少神经细胞凋亡有关.     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

亚低温抑制大鼠弥漫性脑损伤后细胞凋亡的研究

目的探讨大鼠不同程度弥漫性脑损伤后脑组织的凋亡变化过程及亚低温治疗对脑细胞凋亡的抑制作用.方法采用大鼠Marmarou颅脑创伤装置制作弥漫性脑损伤模型,然后将128只Wistar大鼠分为未损伤组(对照组)、重度损伤组、轻度损伤组和亚低温治疗组.通过电子显微镜、组织切片原位末端标记DNA片段(TUNEL染色)、琼脂糖凝胶电泳(DNA Ladder法)等方法,观察和比较不同程度脑损伤后,大鼠脑皮层及海马区凋亡细胞的形态、特点和数量.结果(1)损伤后24～48 h,皮层及海马区可见大量细胞皱缩、核碎裂、核不规则等细胞凋亡现象,48 h较24 h更为严重;亚低温治疗后24～48 h,电子显微镜观察皮层及海马区未见细胞皱缩、核碎裂等细胞凋亡现象.(2)TUNEL染色结果显示,随着损伤程度的加重凋亡明显加重,损伤后48 h达高峰,然后逐渐下降.轻度损伤组细胞凋亡主要限于海马CA2和CA3区;重度脑损伤组细胞凋亡涉及整个海马结构,同时还广泛累及额顶区皮质.损伤后第24、48、72 h,皮层及海马区的凋亡细胞数量较同期未治疗组明显减少.(3)重度损伤后48 h,海马和皮层区细胞琼脂糖电泳可见典型的DNA梯状带,其他时间未见梯状带.亚低温治疗组、轻度脑损伤组及未损伤组亦未见梯状带.结论轻度弥漫性脑损伤后,脑细胞凋亡多发生于海马CA2和CA3区;重度脑损伤后皮层及海马区细胞可发生广泛凋亡.细胞凋亡随着损伤程度的加重而加重,高峰位于伤后第2 d.亚低温治疗可有效地抑制大鼠弥漫性脑损伤后的细胞凋亡.     

亚低温抑制大鼠弥漫性脑损伤后细胞凋亡的研究

目的：探讨大鼠不同程度弥漫性脑损伤后脑组织的凋亡变化过程及亚低温治疗对脑细胞凋亡的抑制作用。方法：采用大鼠Marmarou颅脑创伤装置制作弥漫性脑损伤模型,然后将128只Wistar大鼠分为未损伤组（对照组）、重度损伤组、轻度损伤组和亚低温治疗组。通过电子显微镜、组织切片原位末端标记DNA片段（TUNEL染色）、琼脂糖凝胶电泳（DNA Ladder法）等方法,观察和比较不同程度脑损伤后,大鼠脑皮层及海马区凋亡细胞的形态、特点和数量。结果：（1）损伤后24-48h,皮层及海马区可见大量细胞皱缩、核碎裂、核不规则等细胞凋亡现象,48h较24h更为严重;亚低温治疗后24-48h,电子显微镜观察皮层及海马区未见细胞皱缩、核碎裂等细胞凋亡现象。（2）TUNEL染色结果显示,随着损伤程度的加重凋亡明显加重,损伤后48h达高峰,然后逐渐下降。轻度损伤组细胞凋亡主要限于海马CA2和CA3区;重度脑损伤组细胞凋亡涉及整个海马结构,同时还广泛累及额顶区皮质。损伤后第24、48、72h,皮层及海马区的凋亡细胞数量较同期未治疗组明显减少。（3）重度损伤后48h,海马和皮层区细胞琼脂糖电泳可见典型的DNA梯状带,其他时间未见梯状带。亚低温治疗组、轻度脑损伤组及未损伤组亦未见梯状带。结论：轻度弥漫性脑损伤后,脑细胞凋亡多发生于海马CA2和CA3区;重度脑损伤后皮层及海马区细胞可发生广泛凋亡。细胞调亡随着损伤程度的加重而加重,高峰位于伤后第2d。亚低温治疗可有效地抑制大鼠弥漫性脑损伤的细胞凋亡。     

人参总皂苷对大鼠脑外伤后神经细胞凋亡的影响

目的 研究人参总皂苷对脑外伤大鼠神经细胞凋亡的影响,探讨其抑制创伤性脑损伤(TBI)后继发性损伤的作用机制.方法 采用改良的Feeney自由落体法建立脑外伤模型,将Sprague-Dawley大鼠54只按照随机数字表法分为假手术组、脑外伤组、人参总皂苷治疗组(治疗组),每组18只.模型建立后24h处死大鼠,采用干湿重法测量脑水含量,光镜下观察尼氏染色海马细胞形态,凋亡细胞原位末端标记法(TUNEL)和免疫组织化学方法观察大脑皮质、海马神经细胞凋亡及凋亡基因bax、bcl-2、caspase-3的蛋白表达情况.结果 与脑外伤组比较,人参总皂苷治疗后的治疗组脑含水量明显减低,损伤侧海马病理学改变明显减轻,神经细胞凋亡减少,bcl-2的表达增高,bax、caspase-3的表达减少,差异均有统计学意义(P＜0.05).结论 人参总皂苷可以减轻脑外伤后继发性损伤,其机制可能是升高bcl-2的表达,抑制bax、caspase-3的表达,从而阻滞TBI后神经细胞凋亡.     

Transgenic mice that overexpress the anti-apoptotic Bcl-2 protein have improved histological outcome but unchanged behavioral outcome after traumatic brain injury

Increasing evidence suggests that apoptosis is a contributing factor to neuronal cell death in traumatic brain injury (TBI). There is increased expression, cleavage and activation of caspases as well as other proteins known to regulate apoptosis in neurons after TBI. These proteins include the proto-oncogene Bcl-2 which belongs to a family of proteins with both pro- and anti-apoptotic properties. To investigate the role of apoptosis in TBI and the importance of Bcl-2 protein on the severity and outcome of injury, Bcl-2 overexpressing transgenic and wild-type control mice were subjected to the controlled cortical impact model of TBI. There was no significant difference in the cleavage of caspase-3 or caspase-9 detected by Western blotting of hippocampal samples from transgenic or wild-type mice after TBI. Bcl-2 transgenic mice had smaller contusion volumes and increased numbers of surviving neurons in CA2 but not other regions of hippocampus compared to wild-type controls. By contrast, there was no difference in motor function determined by the round beam balance and wire grip tests between transgenic and wild-type mice after TBI. Cognitive function assessed by the Morris water maze was also not different between groups. These results suggest that overexpression of Bcl-2 is only partially neuroprotective and other members of this protein family may prove to be more important in protecting neurons from cell death.     

大鼠轻型颅脑损伤后星形胶质细胞与神经元的病理改变

目的 探讨轻型颅脑损伤（TBI）后神经元及星形胶质细胞改变的病理生理过程。方法 将24只成年SD大鼠随机分为轻型TBI组（n=18）和假手术组（n=6）,轻型TBI组又分为伤后3 h（n=6）、伤后24 h（n=6）、伤后72 h（n=6）三亚组。采用液压冲击法制作轻型TBI模型。采用胶质纤维酸性蛋白（GFAP）染色检测星形胶质细胞,采用Fluoro-Jade B（FJ-B）荧光染色检测变性神经元。结果 与假手术组相比,轻型TBI后3 h、24 h、72 h邻近顶叶皮质、海马CA2/3区GFAP阳性细胞数量均明显减少（P<0.05）;缺失区周围星形胶质细胞肿胀增生明显。FJ-B阳性神经元在损伤后3 h无明显增加（P>0.05）,伤后24 h皮层区FJ-B阳性神经元显著增加（P<0.05）,伤后72 h海马区FJ-B阳性神经元显著增加（P<0.05）。伤后72 h伤侧皮层区与海马区GFAP阳性细胞数和FJ-B阳性细胞数呈显著负相关（r=-0.8285,P<0.05）。结论 轻型TBI后星形胶质细胞超急性期（3 h）即出现损害和胶质反应,神经元则在急性期（24 h）至亚急性期（72 h）出现明显损害,星形胶质细胞缺失程度可以反应神经元损伤程度。     

Modulation of the cAMP signaling pathway after traumatic brain injury

Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream target, protein kinase A, from 15 min to 48 h after moderate FPI. To determine if preventing hydrolysis of cAMP by administration of a phosphodiesterase (PDE) IV inhibitor would improve outcome after TBI, we treated animals intraperitoneally with rolipram (0.3 or 3.0 mg/kg) 30 min prior to TBI, and then once per day for 3 days. Rolipram treatment restored cAMP to sham levels and significantly reduced cortical contusion volume and improved neuronal cell survival in the parietal cortex and CA3 region of the hippocampus. Traumatic axonal injury, characterized by beta-amyloid precursor protein deposits in the external capsule, was also significantly reduced in rolipram-treated animals. Furthermore, levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), were significantly decreased with rolipram treatment. These results demonstrate that the cAMP-PKA signaling cascade is downregulated after TBI, and that treatment with a PDE IV inhibitor improves histopathological outcome and decreases inflammation after TBI.     

Early loss of astrocytes after experimental traumatic brain injury

Neuronal-glial interactions are important for normal brain function and contribute to the maintenance of the brain's extracellular environment. Damage to glial cells following traumatic brain injury (TBI) could therefore be an important contributing factor to brain dysfunction and neuronal injury. We examined the early fate of astrocytes and neurons after TBI in rats. A total of 27 rats were euthanized at 0.5, 1, 2, 4, or 24 h after moderate lateral fluid percussion TBI or after sham TBI. Ipsilateral and contralateral hippocampi were examined in coronal sections from -2.12 to -4.80 mm relative to bregma. Adjacent sections were processed with markers for either astrocytes or degenerating neurons. Astrocytes were visualized using glial fibrillary acidic protein (GFAP) or glutamine synthetase immunohistochemistry. Neuronal degeneration was visualized using Fluoro-Jade (FJ) histofluorescence. At 30 min, there was a significant loss of GFAP immunoreactivity in ipsilateral hippocampal CA3 with some loss of normal astrocyte morphology in the remaining cells. The number of normal staining astrocytes decreased progressively over time with extensive astrocyte loss at 24 h. At 4 h, lightly stained FJ-positive neurons were scattered in the ipsilateral CA3. The intensity and number of FJ-positive neurons progressively increased over time with moderate numbers of degenerating neurons in the ipsilateral hippocampal CA3 evident at 24 h. We conclude that astrocyte loss occurs in the hippocampus early after TBI. The data suggest that loss of supporting glial cell may contribute to subsequent neuronal degeneration.     

Regional expression of Par-4 mRNA and protein after fluid percussion brain injury in the rat.

Regional levels of prostate apoptosis response-4 (Par-4) protein and mRNA were measured after lateral fluid percussion (FP) brain injury in rats. Immunochemical studies indicated that Par-4 immunoreactivity (ir) is present in cortical neurons and hippocampal CA1-CA3 pyramidal neurons in uninjured rats. Increases of Par-4-ir were observed in the CA3 neurons of the ipsilateral hippocampus (IH), but not in injured left cortex (IC) at 48 h after FP brain injury. Levels of the Par-4 mRNA measured by RT-PCR assay and protein measured by Western blot procedure were significantly increased in the injured IC and IH, but not in the contralateral right cortex and hippocampus after brain injury. Levels of both Par-4 protein and mRNA were significantly increased in the IC and IH as early as 2 h and stayed elevated at 24 and 48 h after injury. These data show that the induction of proapoptotic Par-4 mRNA and protein occurs only in the IC and IH that have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Because increased expression of Par-4 has been observed to contribute to apoptosis and cell death in cultured neurons, the present temporal pattern of Par-4 expression is consistent with a role for Par-4 in apoptosis and neuronal cell death after traumatic brain injury.     

自由基清除剂预处理对大鼠脑缺血再灌注损伤后脑组织细胞凋亡及其相关蛋白表达的影响

目的探讨自由基清除剂依达拉奉预处理对大鼠脑缺血再灌注损伤后神经细胞凋亡及其相关蛋白Bcl-2、Bax、热休克蛋白70(HSP70)表达的影响。方法将45只雄性SD大鼠随机分为假手术组、对照组、依达拉奉预处理组,每组15只。采用线栓法制作大鼠缺血2h再灌注24h模型。预处理组大鼠建模前12h腹腔注射依达拉奉(3mg/kg),对照组给予等容量生理盐水。再灌注24h后断头取脑,应用免疫组织化学法检测Bcl-2、Bax、HSP70蛋白表达,末端脱氧核糖核酸转移酶介导的原位缺口末端标记法检测凋亡细胞。结果依达拉奉预处理组和对照组大鼠缺血周围脑组织中凋亡细胞和Bcl-2、Bax及HSP70阳性细胞数比假手术组均明显增加(P<0.01);与对照组比较,其凋亡细胞和Bax阳性细胞数均明显减少(P<0.01),而Bcl-2和HSP70阳性细胞数明显增加(P<0.01)。结论细胞凋亡在缺血再灌注损伤中起着重要作用;依达拉奉可能通过上调Bcl-2、HSP70蛋白表达、下调Bax蛋白表达减轻大鼠脑缺血再灌注后的细胞凋亡,增加脑缺血再灌注损伤耐受性,从而起到神经保护作用。     

Expression of Fas and Fas ligand after experimental traumatic brain injury in the rat.

Apoptotic cell death plays an important role in the cascade of neuronal degeneration after traumatic brain injury (TBI), but the underlying mechanisms are not fully understood. However, increasing evidence suggests that expression of Fas and its ligand (FasL) could play a major role in mediating apoptotic cell death in acute and chronic neurologic disorders. To further investigate the temporal pattern of Fas and FasL expression after experimental TBI in the rat, male Sprague Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for Fas and FasL protein expression and for immunohistologic analysis at intervals from 15 minutes to 14 days after injury. Increased Fas and FasL immunoreactivity was seen in the cortex ipsilateral to the injury site from 15 minutes to 72 hours after the trauma, respectively. Immunohistologic investigation demonstrated a differential pattern of Fas and FasL expression in the cortex, respectively: increased Fas immunoreactivity was seen in cortical astrocytes and neurons from 15 minutes to 72 hours after the injury. In contrast, increased expression of FasL was seen in cortical neurons, astrocytes, and microglia from 15 minutes to 72 hours after impact injury. Concurrent double-labeling examinations using terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling identified Fas- and FasL-immunopositive cells with high frequency in the cortex ipsilateral to the injury site. In contrast, there was no evidence of Fas- and FasL-immunopositive cells in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. Further, Fas and FasL immunoreactivity was absent in the contralateral cortex and hippocampus at all time points investigated. These results reveal induction of Fas and FasL expression in the cortex after TBI in the rat. Further, these data implicate an involvement of Fas and FasL in the pathophysiologic mechanism of apoptotic neurodegeneration after TBI. Last, these data suggest that strategies aimed to repress posttraumatic Fas- and FasL-induced apoptosis may open new perspectives for the treatment of TBI.