大鼠颅脑损伤后神经细胞的凋亡

目的观察大鼠颅脑损伤后不同时间损伤周边区额皮质及海马CA1区神经细胞的凋亡现象,为颅脑外伤的临床治疗提供理论依据。方法将SD大鼠分为正常对照组(30只)、假手术对照组(30只)和损伤组(80只),采用侧位液压冲击法制成颅脑损伤动物模型,损伤组按冲击时脑组织所承受力量大小,进一步分为轻、中、重度3组,采用免疫组织化学技术和原位末端标记法(TUNEL)检测各组大鼠损伤后4、8、24、72、96h和7d大鼠伤灶周边额皮质及海马CA1区的凋亡细胞。结果轻、中、重度损伤组大鼠损伤区周边额皮质和海马CA1区各时间点凋亡阳性细胞数均高于正常对照组和假手术对照组,重度损伤组高于轻度和中度损伤组,且差异均有统计学意义。损伤4h后,各损伤组伤灶边缘额皮质和海马CA1区开始出现凋亡细胞,随后逐渐增加,分别于72和24h达高峰,之后开始下降。结论颅脑损伤可导致损伤周边区大脑额皮质及海马CA1区神经细胞凋亡,凋亡高峰时间分别为损伤后72和24h。     

糖尿病大鼠脑缺血再灌注海马CA1区Fas蛋白的表达

目的观察糖尿病大鼠脑缺血再灌注海马CA1区病理变化及Fas蛋白的表达。方法采用链脲佐菌素(STZ)诱导和线栓法建立糖尿病大鼠大脑中动脉闭塞模型,应用HE染色和免疫组化方法,比较糖尿病大鼠脑缺血再灌注组与缺血再灌注组海马CA1区神经元损伤程度及Fas蛋白表达变化。结果糖尿病大鼠脑缺血再灌注组海马CA1区神经元缺失,且Fas蛋白表达上调,明显高于缺血再灌注组。结论糖尿病大鼠脑缺血再灌注损伤后,海马CA1区神经元发生严重缺失,Fas介导的细胞凋亡机制可能是糖尿病加重脑缺血再灌注海马区神经元损伤的机制之一。     

迷走神经刺激对癫痫大鼠海马缝隙连接蛋白43表达的影响

目的观察迷走神经刺激(vagus nerve stimulation,VNS)对海人酸(kainic acid,KA)致癫痫大鼠海马星形胶质细胞缝隙连接蛋白43(Connexin 43,Cx43)表达的影响,探讨Cx43与癫痫之间的关系及其在VNS治疗癫痫中的作用。方法将SD大鼠随机分为对照组、KA组和VNS+KA组,每组10只;KA组和VNS+KA组大鼠左侧脑室注射3μl 0.05%KA溶液,制备癫痫模型,对照组注射等量生理盐水;VNS+KA组行VNS。观察各组大鼠行为变化,记录皮层脑电图(electrocorticogram,ECoG);采用免疫组织化学法和Western blot法检测各组大鼠海马星形胶质细胞Cx43的表达。结果大鼠左侧脑室注射KA 3⁵ min后,KA组大鼠为RacineⅣ5 min后,KA组大鼠为RacineⅣ^Ⅴ级重型癫痫发作,脑电图可见棘波、棘慢波及簇状高尖波等异常脑电发放;VNS+KA组大鼠为RacineⅠⅤ级重型癫痫发作,脑电图可见棘波、棘慢波及簇状高尖波等异常脑电发放;VNS+KA组大鼠为RacineⅠ^Ⅲ级轻型发作,脑电发放较KA组减轻,对照组无癫痫发作及异常脑电发放。KA组大鼠海马Cx43阳性细胞数和相对表达量均明显高于对照组和VNS+KA组(P均<0.01)。结论癫痫大鼠海马Cx43的表达与癫痫发病机制密切相关,VNS可降低癫痫大鼠海马Cx43的表达,抑制癫痫发作。     

脑红蛋白在内毒素脑损伤过程中的表达上调

目的探讨内毒素脑损伤模型大鼠的额叶皮质、海马组织、脑脊液(CSF)和血浆中脑红蛋白(NGB)的表达变化及其意义。方法将SD大鼠随机分为内毒素干预组和对照组,内毒素干预组向大鼠第四脑室内注射内毒素0.1mg/kg,对照组注入等剂量的生理盐水。于注射后3、6、12、24、48和72h采血,分离血清,收集CSF,并处死大鼠,留取额叶皮质和海马组织,应用ELISA、Western blot检测NGB含量,免疫组织化学法检测NGB的表达,干燥法检测鼠脑含水量。结果注射6h后,内毒素干扰组鼠脑含水量明显高于对照组,48h达峰值;额叶皮质、海马组织、CSF和血浆中NGB含量显著高于对照组,48h达峰值。结论在内毒素所致脑损伤中,NGB表达上调,且与内毒素的注入时间相关,NGB表达上调是机体内源性神经保护机制之一。     

扶芳藤提取物对局灶性脑缺血大鼠脑组织IL-6含量的影响

目的探讨扶芳藤提取物对大鼠急性脑缺血再灌注损伤过程IL-6表达的影响。方法取Wistar大鼠随机分为假手术组、模型组和用药组,Longa法制作大鼠急性脑缺血再灌注损伤模型,用酶联免疫标记法测定脑组织与血清中IL-6的浓度。结果扶芳藤提取物能降低再灌注3h、6h、12h、24h后大鼠脑组织与血清中IL-6的表达。结论扶芳藤提取物对大鼠急性脑缺血再灌注损伤的保护作用可能与其抑制脑组织与血清中IL-6的过度表达有关。     

早期气管切开在抢救急重症颅脑损伤的临床价值

目的对比早期气管切开与非早期气管切开在抢救重度颅脑损伤病人中的临床价值。方法对估计近期内意识不能恢复的重度颅脑损伤病人行气管切开,根据受伤到行气管切开的时间分为两组,伤后12h内行气管切开者为A组,伤后12h后行气管切开者为B组。结果A组死亡率为39.02%,B组死亡率为57,45%。结论重度颅脑损伤病人早期行气管切开比非早期气管切开更有临床价值。     

红藻氨酸致痫大鼠海马小窝蛋白(Cav-1)和核因子(NF-κB)的变化及灵芝多糖的干预

观察灵芝多糖(GLP)对于红藻氨酸(kainicacidKA)致痫大鼠海马Cav-1和NF-κB的影响。方法:将60只健康雄性Wistar大鼠随机分成正常对照组、假手术组、癫痫模型组、灵芝多糖治疗低浓度组、灵芝多糖治疗中浓度组、灵芝多糖治疗高浓度组每组10只。采用激光共聚焦方法计数各组海马Cav-1和NF-κB阳性细胞的数量。结果:癫痫模型组Cav-1阳性细胞数明显高于正常对照组;灵芝多糖治疗组Cav-1阳性细胞数高于癫痫模型组;NF-κB阳性细胞数癫痫模型组明显高于正常对照组;灵芝多糖治疗各组NF-κB阳性细胞数低于癫痫模型组。同时,高、中浓度组之间Cav-1和NF-κB蛋白光密度未见明显异常。结论:灵芝多糖能增强大鼠海马神经元Cav-1表达和抑制NF-κB的表达,减轻海马神经元的变性、坏死和凋亡,起到抗癫痫作用。     

标准大骨瓣开颅减压与额颞顶部分骨瓣复位治疗重型颅脑损伤效果比较

目的 探讨标准大骨瓣开颅减压与额颞顶部分骨瓣复位治疗重型颅脑损伤效果。方法 采用回顾性分析的方法,分析我院收治的重型颅脑损伤患者临床资料,依据治疗方式不同分为观察组(额颞顶部分骨瓣复位治疗组)和对照组(标准大骨瓣开颅减压治疗组)。结果 观察组治疗后神经功能评分明显优于对照组,观察组术后并发症明显低于对照组,P<0.05,差异有统计学意义。结论 额颞顶部分骨瓣复位治疗重型颅脑损伤效果明显,预后良好,值得临床借鉴应用。     

大鼠脑缺血再灌注后NF-κB的表达及低分子肝素的保护作用

目的探讨低分子肝素在大鼠脑局灶性缺血再灌注损伤后核转录因子NF-κB的表达及其保护作用。方法选择健康雄性SD大鼠,随机分为假手术组、缺血再灌注组、低分子肝素组,采用改良Longa[1]法制作大鼠右侧大脑中动脉闭塞局灶性脑缺血再灌注模型,观察海马CA1区NF-κB的表达情况。结果再灌注后各时间点NF-κB的表达随再灌注时间延长逐渐增加,低分子肝素组核NF-κB的表达量低于脑缺血再灌注组。结论低分子肝素能抑制模型大鼠的核转录因子NF-κB的释放,减少梗死范围,具有脑保护作用。     

短暂前脑缺血再灌注对大鼠海马中脑源性神经营养因子启动子与组蛋白去乙酰化酶3结合的影响及其作用机制

目的 评价短暂前脑缺血再灌注(ischemia-reperfusion,I/R)对大鼠海马中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)启动子与组蛋白去乙酰化酶3(histone deacetylase 3,HDAC3)结合的影响，并探讨其作用机制。方法 采用Pulsinelli四血管夹闭法建立SD大鼠的I/R模型(I/R组)，同时设假手术组(Sham组)。尼氏染色法观察大鼠海马中神经元存活情况；染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)法检测大鼠海马中BDNF启动子(Bdnf-p1、Bdnf-p2、Bdnf-p4和Bdnf-p6)与HDAC3的结合情况；qPCR法检测大鼠海马中反义脑源性神经营养因子(brain derived neurotrophic factor antisense,BDNF-AS)的表达情况。结果 与Sham组比较，I/R组大鼠海马CA1区神经元数目大幅减少，CA3和DG区神经元数目变化较小。I/R组大鼠海马CA1区中Bdnf-p1和Bdnf-p2与HDAC3结合...     

Differential Expression Patterns of TDP-43 in Single Moderate versus Repetitive Mild Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is a disabling disorder and a major cause of death and disability in the world. Both single and repetitive traumas affect the brain acutely but can also lead to chronic neurodegenerative changes. Clinical studies have shown some dissimilarities in transactive response DNA binding protein 43 (TDP-43) expression patterns following single versus repetitive TBI. We explored the acute cortical post-traumatic changes of TDP-43 using the lateral fluid percussion injury (LFPI) model of single moderate TBI in adult male mice and investigated the association of TDP-43 with post-traumatic neuroinflammation and synaptic plasticity. In the ipsilateral cortices of animals following LFPI, we found changes in the cytoplasmic and nuclear levels of TDP-43 and the decreased expression of postsynaptic protein 95 within the first 3 d post-injury. Subacute pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive mild TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma.     

慢性脑缺血致认知功能障碍大鼠神经细胞Caspase-3表达及GDNF给药的影响

目的观察慢性脑缺血后致认知功能障碍大鼠神经细胞半胱氨酸蛋白酶(Caspase-3)表达,并探讨胶质细胞源性神经营养因子(GDNF)对大鼠认知功能障碍和Caspase-3表达的影响。方法采用双侧颈总动脉永久结扎方法制备慢性前脑缺血大鼠模型,随机分为对照组、GDNF组和生理盐水组,分别在术后1月、2月,根据逃避潜伏期对各组大鼠进行记忆功能测定,应用免疫组织化学方法检测Caspase-3表达。结果双侧颈总动脉结扎1、2月组与对照组相比,逃避潜伏期明显延长;GDNF组与生理盐水组比较,逃避潜伏期明显缩短;与对照组相比,缺血组大鼠额叶皮层、海马的Caspase-3阳性细胞数明显增多,与生理盐水组比较,1、2月GDNF组大鼠额叶皮层、海马的Caspase-3阳性细胞数明显减少。结论GDNF可改善认知功能,其机制可能是通过影响Caspase-3凋亡关键蛋白酶表达,抑制神经细胞凋亡。     

Potential Neuroprotective Mechanisms of Methamphetamine Treatment in Traumatic Brain Injury Defined by Large-Scale IonStar-Based Quantitative Proteomics

Although traumatic brain injury (TBI) causes hospitalizations and mortality worldwide, there are no approved neuroprotective treatments, partly due to a poor understanding of the molecular mechanisms underlying TBI neuropathology and neuroprotection. We previously reported that the administration of low-dose methamphetamine (MA) induced significant functional/cognitive improvements following severe TBI in rats. We further demonstrated that MA mediates neuroprotection in part, via dopamine-dependent activation of the PI3K-AKT pathway. Here, we further investigated the proteomic changes within the rat cortex and hippocampus following mild TBI (TM), severe TBI (TS), or severe TBI plus MA treatment (TSm) compared to sham operated controls. We identified 402 and 801 altered proteins (APs) with high confidence in cortical and hippocampal tissues, respectively. The overall profile of APs observed in TSm rats more closely resembled those seen in TM rather than TS rats. Pathway analysis suggested beneficial roles for acute signaling through IL-6, TGFβ, and IL-1β. Moreover, changes in fibrinogen levels observed in TSm rats suggested a potential role for these proteins in reducing/preventing TBI-induced coagulopathies. These data facilitate further investigations to identify specific pathways and proteins that may serve as key targets for the development of neuroprotective therapies.     

外源性神经生长因子对缺氧脑损伤新生大鼠血脑屏障的通透性及其脑组织分布

目的探索外周应用神经生长因子(NGF)对缺氧脑损伤新生大鼠血脑屏障的通透性及其在脑组织中的吸收分布情况。方法将Wistar大鼠分为成年大鼠组、新生大鼠对照组和新生大鼠缺氧组,分别经腹腔注射125I-βNGF5μg/kg,给药后30min,取各组大鼠血液及脑组织,检测其放射活度及脑组织不同部位的吸收分布情况。结果125I-βNGF注射后,各组大鼠脑组织中125I-βNGF含量差异有统计学意义,新生大鼠缺氧组含量最高;以同一时相血液125I-βNGF含量为参照,脑组织中125I-βNGF含量与血液含量比较,成年大鼠组最低,新生大鼠对照组次之,新生大鼠缺氧组最高,且差异有统计学意义。125I-βNGF在新生大鼠对照组和缺氧组基底前脑、小脑、额皮质、海马、嗅球和丘脑下区均有吸收,且在新生大鼠缺氧组额皮质、海马和小脑的吸收显著高于新生大鼠对照组。结论外周应用NGF可以透过新生缺氧大鼠血脑屏障,并被脑组织吸收。     

CpG-ODN对肾小球肾炎进展的影响

目的观察CpG-ODN对肾小球肾炎进展的影响。方法将Wistar雄性大鼠随机分为N组(正常对照)、M组(模型对照)、CpG组和GpC组。N组大鼠经尾静脉注射生理盐水2.5ml/(kg·d),其余3组大鼠经尾静脉注射抗-thy1.1单克隆抗体2.5ml/(kg·d),隔日1次,共3次。注射抗-thy1.1单克隆抗体后,隔天,CpG组经尾静脉注射CpG-ODN,300μg/只,GpC组经尾静脉注射GpC-ODN,300μg/只,隔日注射,共3次。各组大鼠分别于用药前和第1针注射后1周测定尿蛋白;用药后第8周留取大鼠24h尿、血清及肾脏组织,检测24h尿蛋白定量、血清白蛋白和肾功;肾脏组织用于肾脏病理检查以及RT-PCR法检测TLR-9mRNA的表达。结果CpG组与M组和GpC组相比,血清白蛋白含量明显降低,且差异有显著意义;M组、CpG组和GpC组在给药后1周均出现尿蛋白,在给药后8周,24h尿蛋白含量明显增多;CpG组与M组相比,TLR-9mRNA在肾脏的表达水平明显增加,且差异有显著意义;GpC组与M组相比,差异无显著意义;光镜下可见CpG组肾组织病理改变明显加重。结论天然CpG-ODN可促进肾小球肾炎病情加重,TLR-9可能在其发生机理中起重要作用。     

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex,Striatum, Thalamus and Hippocampus in a Mouse Model

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.     

Lack of Glutamate Receptor Subunit Expression Changes in Hippocampal Dentate Gyrus after Experimental Traumatic Brain Injury in a Rodent Model of Depression

Traumatic brain injury (TBI) affects over 69 million people annually worldwide, and those with pre-existing depression have worse recovery. The molecular mechanisms that may contribute to poor recovery after TBI with co-morbid depression have not been established. TBI and depression have many commonalities including volume changes, myelin disruption, changes in proliferation, and changes in glutamatergic signaling. We used a well-established animal model of depression, the Wistar Kyoto (WKY) rat, to elucidate changes after TBI that may influence the recovery trajectory. We compared the histological and molecular outcomes in the hippocampal dentate gyrus after experimental TBI using the lateral fluid percussion injury (LFPI) in the WKY and the parent Wistar (WIS) strain. We showed that WKY had exaggerated myelin loss after LFPI and baseline deficits in proliferation. In addition, we showed that while after LFPI WIS rats exhibited glutamate receptor subunit changes, namely increased GluN2B, the WKY rats failed to show such injury-related changes. These differential responses to LFPI helped to elucidate the molecular characteristics that influence poor recovery after TBI in those with pre-existing depression and may lead to targets for future therapeutic interventions.