局灶性脑缺血预适应后大鼠血浆皮质醇含量变化

目的探讨脑缺血预适应后糖皮质激素与皮层神经元损伤的关系。方法利用线栓法建立大鼠大脑中动脉脑缺血再灌注模型，通过放射免疫方法测定脑缺血再灌注后各组大鼠再灌注3h、12h、24h和48h各时点的血浆皮质醇含量变化。结果缺血组和预缺血组在再灌注4个时点糖皮质激素含量均有增高，同对照组、假手术组相比有显著差异（P〈0．01）。预缺血组与缺血组相比，预缺血组在再灌注4个时点糖皮质激素含量低于缺血组（P〈0．05）。HE染色皮层神经元损伤显著，预缺血组明显轻于缺血组。结论脑缺血预适应导致缺血再灌注后血浆糖皮质激素含量降低，这可能是其减轻皮层神经元损伤的原因之一。     

缺血再灌注对大鼠海马CA1及DG神经元内19S蛋白酶体影响的激光扫描共聚焦显微镜研究

目的探讨缺血再灌注对大鼠海马CA1及海马齿状核(DG)神经元内19S蛋白酶体的影响。方法采用20min全脑缺血的大鼠模型,20只大鼠分为5组,分别为假手术组及按照再灌注时间分为30min组,4h组,24h组,72h组,每组4只。采用含有4%多聚甲醛的PBS液体进行灌注,取出脑组织,放于多聚甲醛中固定24h后行冠状切片,应用免疫组织化学法标记抗19S蛋白酶体抗体,应用激光共聚焦显微镜对组织切片进行观察。结果大鼠海马区CA1神经元内19S蛋白酶体在缺血再灌注30min后开始减少,4h略增高,然后逐渐减少,直至72h细胞大部份死亡;DG神经元内的19S蛋白酶体也于再灌注30min后减少,4h略增高,然后逐渐减少,至24h程度最重,72h则有所恢复。结论全脑缺血再灌注后,海马CA1及DG神经元内19S蛋白酶体的变化影响了神经元内蛋白的降解,是导致缺血后神经元死亡的一个因素。     

预缺血诱导大鼠CA1神经元中蛋白伴侣hsp70的表达并抑制蛋白聚集物的形成

目的 研究预缺血对蛋白伴侣hsp70表达和蛋白聚集物形成的影响,探讨其可能的脑保护机制.方法 采用大鼠双侧颈总动脉暂时夹闭法建立全脑缺血模型.大鼠分为3min缺血组,10min缺血组以及预缺血组.苏木素-伊红染色,光镜下随机计数分析预缺血后海马CA1区死亡神经元数量变化.免疫组织化学及激光扫描共聚焦显微镜法观察蛋白伴侣hsp70在CAI区神经元内的分布.差速离心分离细胞浆、细胞核及蛋白聚集物.蛋白印迹法检测不同缺血状态下海马CA1神经元内蛋白聚集物含量的变化,以及胞浆、胞核及蛋白聚集物内蛋白伴侣hsp70含量的变化.结果 组织学检查显示预缺血能够显著减少海马CA1区神经元死亡数量.预缺血诱导海马CA1区神经元内蛋白伴侣hsp70在再灌注后24h表达.预缺血处理后,海马CA1区神经元内蛋白聚集物显著减少.预缺血诱导的蛋白伴侣hsp70与再缺血形成的异常蛋白结合在一起并防止其聚集.结论 预缺血可能通过诱导蛋白伴侣hsp70的表达和抑制再缺血后蛋白聚集物的形成,减少再缺血引起的神经元死亡.     

大鼠早期局灶性脑缺血再灌注损伤中USP10的表达水平变化及其与自噬的关系

目的 探讨早期局灶性脑缺血再灌注损伤大鼠模型中再灌注不同时间点USP10的表达水平变化及其与自噬的关系。方法 将36只成年雄性SD大鼠随机分成4组:假手术组、脑缺血2 h再灌注6 h模型组、脑缺血2 h再灌注12 h模型组、脑缺血2 h再灌注24 h模型组; 采用线栓法致大脑中动脉栓塞(middle cerebral artery occlusion, MCAO)制备大鼠局灶性脑缺血再灌注模型,给予神经行为学评分,用TTC染色法测定脑梗死体积,透射电镜观察梗死周边区皮层神经元自噬,Western blot 法检测梗死周边区皮层USP10和自噬相关蛋白LC3B的表达水平,免疫荧光双标法检测梗死周边区皮层神经元中USP10及LC3B的表达水平变化。结果 免疫荧光双标显示模型组自噬蛋白阳性细胞数与存活神经元数均于再灌注12 h最多(P<0.05),二者某种程度上趋势一致; Western blot免疫荧光双标均显示与假手术组相比,USP10及LC3B蛋白在模型组中表达上调(P<0.01),且于再灌注12 h组达到高峰(P<0.05); 透射电镜显示再灌注不同时间点自噬强度的变化与免疫荧光自噬蛋白表达水平的趋势基本一致。结论 早期脑I/R损伤中自噬的激活某种程度上可减轻神经元的损伤,而USP10可能通过某种机制调控脑I/R中自噬的发生发展。     

促红细胞生成素对大鼠脑缺血再灌注损伤后缺血侧皮层神经元凋亡和Bcl-2表达的影响

目的探讨促红细胞生成素(Erythropoietin，EPO)对大鼠局灶性脑缺血再灌注损伤后的保护作用。方法采用线栓法阻断大鼠一侧大脑中动脉(MCA)血流2h，再灌注24h制成局灶性脑缺血再灌注损伤模型。将32只雄性SD大鼠随机分成EPO组、缺血再灌注组、假手术组和正常组。于缺血开始时EPO组给EPO 3000U／kg腹腔注射；缺血再灌注组和假手术组给予等剂量生理盐水。再灌注24h后断头取脑、切片，进行HE染色、Bcl-2免疫组化染色和细胞凋亡检测。结果缺血2h再灌注24h后，EPO组和缺血再灌注组大鼠缺血侧皮层可检测到凋亡细胞，且EPO组凋亡细胞数明显少于缺血再灌注组，假手术组和正常组未见凋亡细胞；EPO组和缺血再灌注组缺血侧皮层Bcl-2阳性细胞数均高于假手术组和正常组，与缺血再灌注组相比，EPO组Bcl-2蛋白表达显著增高。结论EPO可抑制缺血再灌注损伤后缺血侧皮层的细胞凋亡，其机制可能是通过上调bcl-2基因表达而实现。     

锌对脑外伤后神经元中泛素化蛋白表达的影响

目的 研究脑外伤(TBI)后锌和泛素化蛋白在神经元中的变化,探讨锌在脑外伤后继发性神经损伤中的作用机制.方法 建立重物坠落致脑外伤动物模型,应用ZP4荧光染色、Western blot法分别观察神经元中锌及泛素化蛋白变化,用Nissle染色检测神经元存活数目;最后在脑外伤患者挫伤区神经元中分别用AMG染色、Western blot法分别检测锌及泛素化蛋白变化.结果 ZP4荧光染色、Western blot法和Nissle染色发现脑外伤可诱导大鼠海马神经元中锌聚集、泛素化蛋白升高以及最终导致神经元损伤;而阻断锌聚集可减少神经元中泛素化蛋白的升高,并产生神经保护作用.最后,在人脑外伤挫伤区神经元中,应用AMG染色和Western blot法也检测到了明显锌聚集和泛素化蛋白的升高.结论 脑外伤后锌稳态的失衡可以影响蛋白质的降解并最终导致神经元的损伤.     

大鼠脑缺血再灌注损伤后皮层Homerla蛋白改变及意义

目的探讨大鼠脑血再灌注损伤后皮层组织Homerla蛋白的表达改变及其意义。方法选择成年雄性SD大鼠59只,随机分为正常对照组、缺血再灌注损伤后10rain、1h、3h、6h、12h、24h、48h及72h共9组,每组6只。免疫组化染色法和Western blot法测定Homerla蛋白含量,T-PCR法测定HomerlaRNA表达。结果在实验过程中5只动物死亡,54只进入结果分析。与对照组比较缺血再灌注损伤后Homerla蛋白和mRNA灰度值表达出现3个峰值,分别在10min、6h和24h,但在72h仍维持较高水平;与对照组Homerla蛋白免疫评分相比,缺血再灌注损伤后出现两个峰值,分别在6h和24h;Homerla蛋白除神经元外,在胶质细胞也表达。结论在缺血再灌注损伤后,Homerla出现3个表达高峰,可能在不同的机制的影响下对脑损伤发生发展起重要作用。     

大鼠局灶性脑缺血再灌注后瞬时感受器电位C1、M7的表达变化

目的 研究大鼠局灶性脑缺血再灌注后瞬时感受器电位(TRP)C1和TRPM7的表达变化,进一步探讨局灶性脑缺血再灌注损伤的分子病理机制.方法 将50只Wistar大鼠按照随机数字表法分为假手术组、缺血2h再灌注3 h组、缺血2h再灌注12h组、缺血2h再灌注24h组、缺血2 h再灌注72 h组,每组10只.线栓法建立大鼠大脑中动脉阻塞再灌注模型,应用RT-PCR、Western blot和免疫组化染色方法分别检测再灌注后不同时相缺血侧皮层TRPC1和TRPM7 mRNA和蛋白水平的表达.结果 假手术组TRPC1和TRPM7均有均有少量表达.脑缺血2 h再灌注3 h TRPC1表达开始增加,随再灌注时间的延长表达逐渐增强,至再灌注12 h达高峰,然后逐渐减弱,再灌注72 h时仍高于假手术组水平,差异均有统计学意义(P<0.05).TRPM7表达也随再灌注时间的延长而增加,高峰在再灌注24h.结论 TRPC1和TRPM7参与了局灶性脑缺血再灌注损伤的病理过程.     

细胞周期调控对大鼠局灶性脑缺血后迟发性神经元死亡的影响

目的探讨细胞周期调控对脑神经细胞凋亡的影响。方法采用光化学方法制作大鼠局灶性缺血模型,术后立即腹腔注射细胞周期抑制剂Olomoucine进行干预。HE染色来测定皮层梗死灶的体积;应用免疫组织化学法观察缺血后第3天假手术组、缺血对照组和干预组大鼠损伤侧皮层病灶周围凋亡神经细胞的表达;聚丙烯酰胺凝胶电泳(Western blot)和半定量逆转录-聚合酶链式反应(RT-PCR)观察Caspase-3蛋白及mRNA的表达。结果 HE染色显示对照组梗死灶明显大于干预组;免疫组织化学可见病灶周围凋亡神经细胞的阳性表达,干预组较假手术组显著增高,但明显低于对照组(P<0．05);Western blot和RT-PcR显示Caspase-3蛋白和mRNA的表达,干预组较假手术组增高(P<0．05),而较对照组明显降低(P<0．05)。结论脑缺血损伤后细胞周期调控参与了迟发性神经元的死亡过程。     

大鼠局灶性脑缺血后LMO4和pCREB的表达

目的 探讨大鼠脑缺血再灌注后核转录因子LMO4和pCREB在缺血半暗带的表达规律及与凋亡抗原的共表达.方法 将雄性SD大鼠随机分为假手术组及缺血再灌注1h、3h、6h、12h、24h、48h组,线栓法制备局灶性大脑中动脉闭塞(MCAO)模型,缺血2h后恢复再灌住,采用Western blot法、免疫荧光法检测LMO4、pCREB在缺血半暗带的表达变化,免疫荧光双标观察LMO4、pCREB的表达定位及与TUNEL阳性细胞之间的共表达情况.结果 与假手术组比较,缺血侧半暗带皮层组织LMO4蛋白水平再灌后3h开始升高,24h达高峰,48h明显下降,pCREB蛋白水平6h开始升高,24h达高峰,48h逐渐下降(P＜0.05或P＜0.01);LMO4、pCREB阳性细胞数再灌后6h开始升高,24h达高峰,48h显著下降(P＜0.05或P＜0.01);免疫荧光双标显示LMO4和pCREB仅表达于神经元,LMO4主要表达于细胞核,少量位于细胞浆,pCREB仅表达于细胞核;LMO4与pCREB完全共表达;LMO4、pCREB均与TUNEL阳性细胞呈分离表达.结论 脑缺血再灌后诱导LMO4、pCREB表达升高,并呈动态变化趋势,可能为神经细胞对缺血再灌注损伤的一种内部适应性保护机制.     

Distribution of AT4 receptors in the Macaca fascicularis brain

Angiotensin IV (Val Tyr lie His Pro Phe), administered centrally, increases memory retrieval and induces c fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT₁ and AT₂ receptors and is known as the AT₄ receptor. These observations suggest that the AT₄ receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [¹²⁵I]angiotensin IV to map AT₄ receptors in the macaca fascicularis brain. The distribution of the AT₄ receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT₄ receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT₄ receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT₄ receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT₄ receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT₄ receptor in the primate brain.     

Osteopontin is an alpha motor neuron marker in the mouse spinal cord

Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN+/ChAT+ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN+/ChAT+ cells was nearly identical to that of NeuN+/ChAT+ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN+/ChAT+ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN+ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.     

Antibody to a soluble protein purified from brain selectively labels layer V corticofugal projection neurons in rat neocortex

An antibody to a soluble protein (protein 36) isolated and purified from rat brain labels the cell bodies and processes of pyramidal cells within layer V of the rat neocortex. We have used the fluorescent retrograde axonal tracer, Fast blue, in combination with FITC immunocytochemistry to determine the projection sites of the cortical neurons detected by this antibody. Retrogradely labeled pyramidal tract neurons and corticotectal neurons are labeled with the protein 36 antibody, but the callosally projecting neurons within layer V are not. Thus within the neocortex the antibody to protein 36 may selectively detect a particular class of neuron, the corticofugal projection neurons of layer V.     

Thermally-induced activities of the mesencephalic reticulospinal and rubrospinal neurons in the rat

Unit activities of 226 midbrain reticulospinal (mRfS) and non-mRfS neurons and 238 rubrospinal (RbS) and non-RbS neurons were investigated during changes in temperatures of midbrain (Tmb), preoptic and anterior hypothalamus (Thyp) and skin (Ts) in the urethane-anesthetized rat. Responsiveness to Tmb, Thyp and Ts were found in 43.5%, 41.6% and 51.5% of neurons of midbrain reticular formation (mRf), and in 35.2%, 32.7% and 17.6% of neurons of red nucleus (Rb). Higher incidence of responsiveness to remote temperatures was found among Tmb responsive neurons than Tmb unresponsive neurons in both mRf and Rb. The mRf contains significantly greater numbers of neurons having such multiple thermal responsiveness and also of neurons which were activated by falls in temperatures (cold-responsive neurons) than the Rb. These characteristics were more conspicuously seen among mRfS neurons, showing a high degree of convergence of cold signals from different sites of body. On the other hand, RbS neurons did not differ from non-RbS neurons regarding thermal characteristics and showed no particular combinations of responsiveness to temperatures of different sites. Microinjection of procaine and glutamate into the mRf just dorsolateral to the Rb, but not into the Rb, decreased and increased cold-induced increase in EMG activity and shivering without changes in cardiovascular and respiratory parameters and pilomotor activity. The results suggest that mRfS neurons are involved in the control of thermoregulatory muscle tone and shivering.     

Distribution of phosphate-activated glutaminase in the human cerebral cortex

Phosphate-activated glutaminase (PAG), which catalyses conversion of glutamine to glutamate, is a potential marker for glutamatergic, and possibly GABA, neurons in the central nervous system. A polyclonal antibody, raised in rabbits against rat brain PAG, was applied to postmortem human brain tissue to reveal the distribution of PAG in the cerebral cortex. PAG immunoreactivity was observed in pyramidal and non-pyramidal neurons but not in glial cells. In the neocortex, large to medium-sized pyramidal neurons in layers III and V were stained most intensely, while the majority of smaller pyramidal cells were labeled either lightly or moderately. Such modified pyramids as the giant Betz cells, the large pyramidal cells of Meynert, and the solitary cells of Ramón y Cajal were also stained intensely. Fusiform cells in layer VI showed moderate to intense labeling. A number of cortical non-pyramidal neurons of various sizes stained moderately to intensely. These included large basket cells which were identified by their characteristic morphology and size in primary cortical areas. Pyramidal cells in the hippocampal formation as well as basket cells of the stratum oriens stained moderately to intensely. Since pyramidal cells are believed to be glutamatergic and large basket cells GABAergic, these results suggest that PAG plays a role in generating not only transmitter glutamate, but also GABA precursor glutamate.     

The hematopoietic cytokine, colony-stimulating factor 1, is also a growth factor in the CNS: Congenital absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury

In this study we used op/op mice, which are deficient in the hematopoietic cytokine, colony-stimulating factor 1 (CSF-1), to determine the effect of CSF-1 on neuronal survival and microglial response in injury. In normal mice microglia express the CSF-1 receptor and are primarily regulated by CSF-1, produced mainly by astrocytes. The CSF-1 deficiency in op/op mice results in a depletion in the number of monocytes and macrophages but does not affect the number of morphology of microglia. We produced an ischemic lesion in the cerebral cortex of mice by disrupting the pia-arachnoid blood vessels in a defined area. Using Nissl stain and strocyte- and microglia-specific antibodies, we determined the number of viable neurons in such injury and the intensity of glial reaction. The cellular response to injury on the operated side of op/op mice was compared to that on the non-operated contralateral side and to the cellular response in similar lesions in CSF-1 producing C₃H/HeJ mice. We found that the systemic lack of CSF-1 in op/op mice results in a significant increase in neuron vulnerability to ischemic injury and considerably reduced microglial response to neuron injury. Remedying the CSF-1 deficiency, either by grafting CSF-1 secreting astroglia into the brain or by implanting encapsulated CSF-1 secreting fibroblast-like cells into the peritoneum, partially restores the microglial response to neuron injury and significantly potentiates neuronal survival in cerebral cortex ischemic lesions. Astroglial reaction was approximately the same in the lesions in op/op mice, grafted annd implanted op/op mice and C₃H/HeJ mice, indicating that CSF-1 modulates microglia, but not the response of astrocytes to injury. The degree of neuronal survival was not correlated to the degree of microglial proliferation and intensity of their reaction. We report some indications that CSF-1, in addition to modulation of microglia, may also act directly on neurons.     

Modification of motor neuron size and position in the central nervous system of adult snapping shrimps

Cell bodies of claw closer motor neurons in snapping shrimp are dimorphic. Snapper claw motor neurons are larger than corresponding pincer claw motor neurons, but the relative sizes of these cells are reversed during claw transformation. An additional neuronal modification occurs early within this period, in that the pincer claw dorsal inhibitor cell body migrates within the nervous system, from a dorsal to a ventral position. These findings are evidence of rapid, reversible changes in the nervous system following the trigger for the transformation process.     

Clinical evolution of pure upper motor neuron disease/dysfunction (PUMMD)

Introduction: PLS is defined as pure upper motor neuron disease/dysfunction (PUMND) beyond 48 months after symptom onset. We know little about its early stages, but such knowledge would help to identify the mechanisms underlying PLS and ALS and determine why PLS patients seem to be protected against lower MND (LMND). Methods: We reviewed 622 MND cases during a 4‐year period and identified 34 patients with PUMND (5.4%). Results: Among 23 cases with follow‐up data/electromyograms (EMGs; 2 had only 1 EMG), 13 (57%) remained classified as PUMND, and 8 (35%) developed LMND (mean, 51.4 months after onset). Of these 8, LMND developed in 3 after 48 months from symptom onset. Patients with PUMND and LMND were more functionally impaired (P = 0.02). Separately, we identified 5 patients with PUMND who developed LMND long after 48 months (range, 50–127 months). Conclusions: PLS belongs to the ALS spectrum, and perhaps all cases eventually develop LMND. Muscle Nerve, 2013