大鼠急性脊髓损伤后白细胞介素-1β表达及其与神经细胞凋亡的关系

目的探讨脊髓损伤(SCI)后白细胞介素(IL)1β表达的变化及其与神经细胞凋亡的关系。方法采用Allen’s法建立大鼠急性SCI模型,用实时定量聚合酶链反应(PCR)和免疫组织化学染色检测SCI后IL1β表达,用原位末端标记法(TUNEL)检测SCI后神经细胞凋亡。结果对照组IL1βmRNA为4.87±0.49,SCI后早期IL1βmRNA明显升高,于伤后6h达高峰为6.95±0.95,两组间差异有统计学意义(P<0.01);免疫组织化学染色也显示伤后6hIL1β染色强度明显增加。TUNEL染色显示,对照组大鼠脊髓组织TUNEL阳性细胞极少(1.82±0.64),伤后6h有少许凋亡细胞出现,24hTUNEL阳性细胞增多最明显(32.38±4.75)。IL1β表达升高与TUNEL阳性细胞增多有明显的关系。结论SCI后IL1β表达升高,它可能参与了诱导神经细胞凋亡。     

大鼠脊髓损伤致截瘫后肠道细菌移位的实验研究

目的：探讨大鼠脊髓损伤致截瘫后是否发生肠道细菌移位。方法：建立大鼠脊髓损伤性截瘫模型，以脊髓损伤性截瘫后12h、24h、48h大白鼠为实验组，未损伤脊髓的正常大白鼠为对照组。在无菌条件下，采集动物下腔静脉血进行内毒素定量测定和细菌培养，采集肝、脾、肠系膜淋巴结、肠腔内容物作细菌培养并进行菌种鉴定。取实验组和对照组各动物的肝、脾、肠系膜淋巴结、空肠、回肠进行病理切片HE染色检查，取空、回肠进行电镜检查。结果：大鼠脊髓损伤致截瘫后24h开始出现内毒素血症，截瘫后48h出现细菌移位。结论：大鼠脊髓损伤致截瘫后将发生肠道细菌移位，提示脊髓损伤截瘫的病人应尽早给予抗生素治疗。     

Effect of nerve growth factor on neuronal apoptosis after spinal cord injury in rats

OBJECTIVE: To explore the molecular mechanism of the protective effect of nerve growth factor (NGF) on injured spinal cord. METHODS: The posterior T(8) (the 8th thoracic segment) spinal cords of 60 Wistar rats were injured by impacts caused by objects (weighing 10 g) falling from a height of 2.5 cm with Allen's way. Solution with nerve growth factors (NGF) was given to 30 rats (the NGF group) through a microtubule inserted into the subarachnoid cavity immediately, and at 2, 4, 8, 12 and 24 hours after spinal cord injury (SCI) respectively. Normal saline (NS) with same volume was given to the other 30 rats (the NS group) with the same method. And 5 normal rats were taken as the normal controls. The expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry. The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) staining. RESULTS: The positive expression of bcl-2 protein was strong in the normal controls, but decreased in the NS group, and increased significantly in the NGF group as compared with that of the NS group (P<0.01). The positive expression of bax protein was also strong in the normal controls, but increased in the NS group, and decreased significantly in the NGF group as compared with that of the NS group (P<0.01). Apoptotic neurons were found in the NS group, and they decreased significantly in the NGF group as compared with that of the NS group (P<0.01). CONCLUSIONS: NGF can protect the injured nerve tissues through stimulating the expression of bcl-2 protein, inhibiting the expression of bax protein and inhibiting the neuronal apoptosis after SCI.     

联合应用PARP-1与Caspase-3抑制剂对脊髓损伤大鼠神经细胞凋亡的影响

目的:探讨联合应用多聚腺嘌呤二核苷酸核糖聚合酶-1(PARP-1)抑制剂3-氨基苯甲酰胺(3-aminobenzamide,3-AB)和Caspase-3抑制剂Z-DEVD-FMK对脊髓损伤大鼠神经细胞凋亡的影响。方法:120只成年健康SD大鼠随机分为假手术组(A组)、模型组(B组)、PARP-1抑制剂组(C组)和联合用药组(D组),每组30只。以Allen′s打击法制备大鼠脊髓损伤模型,每组分别于造模后1d、3d、7d取5只大鼠行BBB评分,处死后利用免疫组化方法检测损伤部位脊髓内PARP-1、凋亡诱导因子(AIF)、Caspase-3及Bcl-2的表达;各时间点各组剩余大鼠处死后利用Western blotting检测PARP-1、Caspase-3蛋白表达水平,实时荧光定量PCR检测PARP-1、AIF、Caspase-3及Bcl-2的m RNA水平,采用原位末端标记(TUNEL)法检测神经细胞凋亡情况。结果:造模后1d时B、C、D组大鼠BBB评分均为0分;3d时三组间的评分无统计学差异;7d时D组及C组明显高于B组,且D组最高(P0.05)。免疫组化及Western blotting结果显示,脊髓损伤后1~7d,B组脊髓组织中PARP-1、AIF及Caspase-3表达逐渐增强,Bcl-2表达逐渐减弱(P0.05);与B组比较,D组及C组的PARP-1、AIF、Caspase-3表达均显著降低,且D组最低(P0.05);而D组及C组的Bcl-2表达显著高于B组,且D组最高(P0.05)。实时荧光定量PCR检测各目的基因表达水平与其蛋白水平一致。TUNEL结果显示,B组脊髓损伤后3d凋亡细胞最多,7d时数量减少,但仍保持在较高水平(P0.05);D组及C组凋亡细胞指数均显著低于B组,且D组最低(P0.05)。结论:联合应用PARP-1抑制剂3-AB和Caspase-3抑制剂Z-DEVD-FMK可有效抑制大鼠脊髓损伤后神经细胞凋亡,其机制可能与PARP-1、AIF、Caspase-3的表达抑制及Bcl-2的表达上调有关。     

Brain-derived neurotrophic factor suppresses delayed apoptosis of oligodendrocytes after spinal cord injury in rats

We evaluated the effect of brain-derived neurotrophic factor (BDNF) on cell death after spinal cord injury. A rat spinal cord injury model was produced by static load, and continuous intrathecal BDNF or vehicle infusion was carried out either immediately or 3 days after the injury. Cell death was examined by nuclear staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After injury, typical apoptotic cells were observed. Double staining with TUNEL and specific cell markers revealed that, soon after the injury, the apoptotic or necrotic cells at the injury site were neurons and microglia. One week after the injury, apoptotic oligodendrocytes, but not apoptotic astrocytes, were observed in the white matter rostral and caudal to the injury site, whereas few apoptotic cells were found in the gray matter. The immediate BDNF treatment significantly reduced the number of TUNEL-positive cells in the adjacent rostral site 1 and 2 weeks after the injury, and in the adjacent caudal site 3 days and 1 week after the injury, even though there was no significant difference between BDNF-treated and control rats at the injury site itself. In addition, similar antiapoptotic effects were observed in these regions 1 week after injury in rats that received BDNF treatment from the third day after injury. These findings suggest that BDNF suppresses delayed apoptosis of oligodendrocytes after spinal cord injury, for which even delayed injections are effective. BDNF administration may therefore be useful for the clinical treatment of spinal cord injury through the suppression of secondary events.     

大鼠脊髓急性损伤后神经细胞凋亡及相关基因表达△

目的研究脊髓急性损伤后神经细胞的凋亡及相关基因的表达.方法大鼠脊髓(T8、T9)经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h、72h、7d、14d和21d处死取材(各时间组n=4).应用HE染色、免疫组化及凋亡细胞原位末端标记法对脊髓组织进行标记.结果损伤4h后,在损伤段及邻近段可见末端标记阳性神经元,损伤段灰质中阳性细胞数8h达高峰,24h白质中阳性胶质细胞数量达高峰.相邻节段阳性细胞数72h达高峰.损伤后P53及Bax大量表达,而Bcl-2仅少量表达.结论脊髓损伤后神经细胞的凋亡是继发损伤期的重要病理变化.     

复方丹参对大鼠脊髓损伤后细胞凋亡及诱导型一氧化氮合酶表达的影响

目的:探讨大鼠脊髓损伤后应用复方丹参对细胞凋亡及诱导型一氧化氮合酶(iNOS)表达的影响。方法:成年大鼠随机分为正常组、脊髓损伤后应用复方丹参组(组)和应用生理盐水对照组(组),损伤后不同AB时间点(8h、1d、3d、7d、14d、28d)处死大鼠,用HE染色观察损伤脊髓组织病理变化,原位末端标记法(TUNEL法)标记凋亡细胞,用免疫组化染色检测iNOS阳性细胞。结果:HE染色镜检发现脊髓组织病理学改变A组明显轻于B组。A、B两组均发现凋亡细胞及iNOS表达,神经细胞凋亡指数及iNOS表达均为B组>A组(P<0.01)。结论:复方丹参能抑制大鼠脊髓损伤后细胞凋亡及iNOS表达。     

Transplantation of viable mitochondria attenuates neurologic injury after spinal cord ischemia

ObjectivesSpinal cord ischemia (SCI) is one of the major concerns of postoperative paraplegia during major vascular or aortic surgery. Since mitochondrial dysfunction develops at the early stage of SCI, this study tested the neuronal protective effect of transplantation of viable mitochondria to the ischemic cord in rats.MethodsSCI was induced by crossclamping of thoracic aorta at T6 level for 25 minutes, followed by release of vascular clip to restore aortic blood flow in the anesthetized rats. Mitochondria (100 μg) were isolated from freshly harvested soleus muscle and delivered via the internal jugular vein before releasing of vascular clip. The motor function was assessed independently up to 7 days after reperfusion. Spinal cords were harvested and analyzed for molecular and histological changes.ResultsWhole-body in vivo images acquired by an in vivo imaging system confirmed the enhancement of MitoTracker fluorescence at the regions below crossclamping and in the ischemic cord. Compared with control vehicles, transplantation of mitochondria significantly improved the lower-limb locomotor function of rats subjected to cord ischemia up to 7 days after surgery. Mitochondrial transplantation suppressed the regional endoplasmic reticulum stress in the ischemic cord by attenuating CCAAT-enhancer-binding protein homologous protein expression and restoring binding immunoglobulin protein levels. In accordance, tissue levels of interleukin-6, tumor necrosis factor-α, and caspase-3 were attenuated in the mitochondrial transplanted group. Histologic examination also showed significant increase in numbers of Nissls bodies in the neurons at the ventral horn of ischemic cord following mitochondrial transplantation.ConclusionsOur study showed that transplantation of freshly isolated mitochondria during the early stage of spinal cord ischemia–reperfusion injury suppressed the oxidative stress in endoplasmic reticulum of the injured cord, thereby reducing neuroapoptosis and improving locomotor function of rats with SCI.     

神经生长因子对大鼠脊髓损伤后神经元凋亡的影响

目的探讨神经生长因子(NGF)对脊髓损伤保护作用的分子机制.方法采用Allen's法以25 gcm力致伤大鼠T8脊髓,经蛛网膜下腔导管于术后即刻、2、4、8、12、24 h各注入NGF溶液,并与生理盐水组(NS组)和正常对照组作对照,采用免疫组织化学方法和末端单位标记法(TUNEL)原位末端标记法分别检测bcl-2、bax蛋白在脊髓神经元的表达及神经元凋亡情况.结果正常组中脊髓灰质bax蛋白阳性细胞吸光度(A)值为34.51±4.47,NS组中bax蛋白表达增加,以2 h及12 h最为显著,而NGF组与NS组相比,bax蛋白表达明显减少(P＜0.01).正常组中脊髓灰质bcl-2蛋白表达的A值为19.72±2.92,NS组中bcl-2表达下降,而NGF组与NS组相比较,bcl-2蛋白表达明显增多(P＜0.01).TUNEL检测结果显示,正常对照组中未见神经元凋亡,NS组中自2 h后可见神经元凋亡,NGF组与NS组相比,神经元凋亡指数明显减少(P＜0.01).结论NGF能通过抑制bax蛋白的表达,促进bcl-2表达抑制脊髓损伤后神经元凋亡,从而保护损伤的脊髓组织,这可能是NGF对脊髓损伤具有保护作用的机制之一.     

大鼠脊髓损伤后血脊髓屏障通透性变化的观察

目的：探讨脊髓损伤后血脊髓屏障通透性变化的机理。方法：采用美国纽约大学（New York University,NYU)脊髓损伤模型,选用体重300－350克雄性成年Wistar大鼠35只,随机分为对照组5只;脊髓损伤组30只,分为伤后4、6、12、24、48、72h6组,每组5只,应用NYU脊髓损伤模型,采用免疫组织化学方法,观察脊髓损伤后不同时间免疫球蛋白G（immunglobularprotein,G,IgG)、补体3的C片断（c fragment of complement3,C3c)的表达变化。结果：脊髓损伤后C3c、IgG渗入了脊髓损伤区域、损伤区域周边及血管,脊髓损伤不同时间段这些区域的免疫标记不同。结论：脊髓损伤后C3c、IgG参与了血脊髓屏障的破坏,参与了脊髓损伤后神经细胞的继发性损伤。     

Activation of spinal GABA receptors attenuates chronic central neuropathic pain after spinal cord injury

In this study, we investigated the role of the spinal GABAergic system in central neuropathic painlike outcomes following spinal cord injury (SCI) produced by a spinal hemitransection at T13 of the rat. After SCI, mechanical allodynia develops bilaterally in both hind paws of the rat, lasting longer than 40 days, as evidenced by an increase in paw withdrawal frequency in response to a weak von Frey filament. In naive rats, intrathecal (i.t.) administration in the lumbar spinal cord of GABAA and GABAB receptor antagonists, bicuculline (1-5 microg) and phaclofen (0.1-5 microg), respectively, causes a dose-dependent increase in the magnitude of mechanical allodynia. The SCI-induced mechanical allodynia in both hind-paws is attenuated by i.t. administration in the lumbar spinal cord of GABAA or GABAB receptor agonists, muscimol (1 microg) or baclofen (0.5 microg), respectively. In electrophysiological experiments, rats with SCI show a bilateral increase in hyperexcitability in response to natural stimuli in wide dynamic range (WDR) neurons in the lumbar spinal dorsal horn. The topical application of muscimol (1 microg) or baclofen (0.5 microg) onto the lumbar cord surface reduce the SCIinduced increased responsiveness of WDR neurons. Inhibitory effects of muscimol and baclofen on both the behavioral mechanical allodynia and the hyperexcitability in WDR neuron with SCI compared to controls, were antagonized by pre-treatment of bicuculline (10 microg) and phaclofen (5 microg), respectively. This study provides behavioral and electrophysiological evidence for the important role of the loss of spinal inhibitory tone, mediated by activation of both GABAA and GABAB receptors, in the development of central neuropathic pain following SCI.     

IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.     

Review of current evidence for apoptosis after spinal cord injury

The initial mechanical tissue disruption of spinal cord injury (SCI) is followed by a period of secondary injury that increases the size of the lesion. The secondary injury has long been thought to be due to the continuation of cellular destruction through necrotic (or passive) cell death. Recent evidence from brain injury and ischemia suggested that cellular apoptosis, an active form of programmed cell death seen during development, could play a role in CNS injury in adulthood. Here, we review the evidence that apoptosis may be important in the pathophysiology of SCI. There is now strong morphological and biochemical evidence from a number of laboratories demonstrating the presence of apoptosis after SCI. Apoptosis occurs in populations of neurons, oligodendrocytes, microglia, and, perhaps, astrocytes. The death of oligodendrocytes in white matter tracts continues for many weeks after injury and may contribute to post-injury demyelination. The mediators of apoptosis after SCI are not well understood, but there is a close relationship between microglia and dying oligodendrocytes, suggesting that microglial activation may be involved. There is also evidence for the activation of important intracellular pathways known to be involved in apoptosis in other cells and systems. For example, some members of the caspase family of cysteine proteases are activated after SCI. It appears that the evolution of the lesion after SCI involves both necrosis and apoptosis. It is likely that better understanding of apoptosis after SCI will lead to novel strategies for therapeutic interventions that can diminish secondary injury.     

脊髓损伤后细胞凋亡基因调控研究进展

脊髓损伤后神经细胞发生凋亡与基因调控密切相关,调控过程有多基因参与,包括半胱氨酸天冬氨酸特异性蛋白酶(caspase)家族、血清凋亡相关蛋白Fas及其配体FasL系统、p53基因、细胞凋亡相关基因Bcl-2家族和即刻早基因等,其中caspase家族参与凋亡各个时期,是细胞凋亡途径的核心成分。该文就这些基因在脊髓损伤后神经细胞凋亡调控机制中的作用近期研究进展作一综述。     

米诺环素对大鼠脊髓损伤后线粒体细胞色素C的释放及神经细胞凋亡的影响

目的：探讨大鼠脊髓损伤后应用米诺环素（minocycline）对线粒体细胞色素C的释放及神经细胞凋亡的影响。方法：成年大鼠脊髓损伤后分为应用米诺环素腹腔注射的治疗组（A组）和应用生理盐水的对照组（B组），于损伤后不同时间点取材，采用流式细胞仪磷脂结合蛋白V，碘化丙啶（AnnexinV／PI）双标检测凋亡细胞，HE染色观察损伤脊髓组织病理变化，免疫组化染色检测胞浆中细胞色素C表达阳性的神经细胞以及BBB运动评分观察术后动物行为学。结果：HE染色镜检发现损伤脊髓组织病理学改变A组明显轻于B组；免疫组化染色A、B两组均发现凋亡的神经细胞以及胞浆中细胞色素C的阳性表达，神经细胞凋亡率及细胞色素C表达的阳性细胞率B组均〉A组（P〈0．01）；术后动物行为学观察显示，与B组比较A组大鼠后肢的运动功能显著增强，后肢反射的恢复较快。BBB运动评分B组明显小于A组（P〈0．05）。结论：米诺环素能有效抑制大鼠脊髓损伤后神经细胞凋亡以及线粒体中细胞色素C的释放，促进神经功能的恢复。     

二甲胺四环素对大鼠脊髓损伤后Caspase-3表达及细胞凋亡的影响

[目的]探讨大鼠脊髓损伤后应用二甲胺四环素（minocyc line）对Caspase-3表达及细胞凋亡的影响。[方法]成年大鼠脊髓损伤后应用二甲胺四环素的治疗组（A组）和应用生理盐水对照组（B组），于损伤后不同时点取材，用HE染色观察损伤脊髓组织病理变化，用免疫组化染色检测Caspase-3表达阳性细胞，原位末端标记法（TUNEL法）标记凋亡细胞。[结果]HE染色镜检发现脊髓组织病理学改变A组明显轻于B组。A、B两组均发现凋亡细胞及Caspase-3表达，神经细胞凋亡指数及Caspase-3表达均B组〉A组（P〈0．01）。[结论]二甲胺四环素能抑制大鼠脊髓损伤后Caspase-3表达及细胞凋亡。