选择性线粒体分裂抑制剂对大鼠急性脊髓损伤后神经细胞的保护作用及其机制

【摘要】目的 检测选择性线粒体分裂抑制剂-1（Mdivi-1）对大鼠急性脊髓损伤（ASCI）后神经细胞线粒体中丙二醛（MDA）、谷胱甘肽（GSH）、细胞色素C（Cyt-C）及神经细胞凋亡的影响。方法成年雌性SD大鼠36只,体质量 250~300g,随机分为假手术组（Sham组）、单纯脊髓损伤组（SCI组）、Mdivi-1预处理组（1.20mg/kg,Mdivi-1组）,各12 只。Sham组只暴露脊髓,不打击。SCI组和Mdivi-1组采用Allen’s方法制备脊髓损伤模型。Mdivi-1组在脊髓打击之前15min尾静脉给予Mdivi-1,而SCI组给予等量二甲基亚砜（DMSO）。Sham组在暴露脊髓8h后立即处死,SCI组和Mdivi-1组均于脊髓损伤后8h处死;然后取出脊髓节段T9~T11,用分光光度计检测各组脊髓组织线粒体中MDA 和GSH的含量,Western Blot法检测线粒体及胞浆内Cyt-C表达情况,荧光TUNEL法观察神经细胞凋亡情况。结果与Sham组相比,SCI组线粒体中Cyt-C和GSH明显减少,但线粒体中的MDA,胞浆中Cyt-C及神经细胞凋亡数目明显增多（P<0.01）;与SCI组相比,Mdivi-1组线粒体中Cyt-C和GSH明显增多,但线粒体中MDA,胞浆中Cyt-C以及神经细胞凋亡数目明显减少（P<0.01）。结论Mdivi-1具有减轻ASCI后神经细胞线粒体氧化损伤,抑制线粒体中Cyt-C 的释放及神经细胞凋亡的作用,促进了脊髓功能恢复。     

Exendin-4 对大鼠脊髓损伤后氧化应激和神经细胞凋亡的影响

目的研究Exendin-4 对大鼠脊髓损伤后氧化应激和神经细胞凋亡的作用。方法成年雄性SD 大鼠36 只（体质量200~250 g）随机分为3 组（n=12）：假手术组（Sham 组）、单纯脊髓损伤组（SCI 组）、Exendin-4 组（Ex-4 组）。Sham 组只暴露脊髓,不打击;SCI 组和Ex-4 组均采用Allen′s 重物打击法制作脊髓损伤模型;Ex-4 组在脊髓损伤后立即腹腔内注射Exendin-4 溶液,剂量10 μg/只;SCI 组和Sham 组均注射等体积生理盐水。在24 h 后检测各组脊髓组织中丙二醛（MDA）含量和过氧化氢酶（CAT）活性,TUNEL 法检测神经细胞凋亡情况,Western blot 检测 caspase-9 和凋亡诱导因子（AIF）的表达情况。结果与Sham 组相比,SCI 组脊髓组织中的MDA 含量明显增加, caspase-9 和AIF 表达水平明显增加,神经细胞凋亡比例明显升高,CAT 活性明显降低（P < 0.01）。与SCI 组相比, Ex-4 组脊髓组织中MDA 含量明显降低,caspase-9 和AIF 表达水平明显降低,神经细胞凋亡比例明显降低,CAT 活性明显升高（P < 0.01）。结论大鼠脊髓损伤后,Exendin-4 可通过减轻氧化损伤抑制神经细胞凋亡。     

依达拉奉抗大鼠脊髓损伤后脊髓细胞凋亡的机制初探

摘要： 目的 探讨依达拉奉 （EDA） 对大鼠脊髓损伤 （SCI） 后内质网应激 （ERS） 介导的细胞凋亡的影响。方法 36 只 SD 大鼠随机分为假手术(sham)组、 SCI 组、 EDA 组, 每组 12 只。采用 Allen’ s 法建立 SCI 模型, sham 组仅行椎板切除术。术后 Sham 组和 SCI 组给予与 EDA 组等体积等频次生理盐水处理; EDA 组在 SCI 模型建成以后予以 EDA(10 mg/kg), 每 12 h 腹腔注射给药 1 次。于术后 3 d 取脊髓, 应用 Western blot 检测 C/EBP 同源蛋白 （CHOP）、 Cleaved caspase-12 和 Cleaved caspase-3 的表达水平, 免疫荧光技术检测脊髓组织中 caspase-12、 CHOP 阳性细胞的比率, TUNEL 法检测脊髓细胞凋亡水平。结果 SCI 组较 sham 组 CHOP、 Cleaved caspase-12、 Cleaved caspase-3 的表达升高, Cleaved caspase-12、 CHOP 阳性细胞比率增加, 脊髓组织细胞凋亡比率增加 （均 P < 0.01）。EDA 组较 SCI 组 CHOP、 Cleaved caspase-12 和 Cleaved caspase-3 的表达降低, Cleaved caspase-12、 CHOP 阳性细胞比率减少, 脊髓组织细胞凋亡比率减少 （均 P < 0.01）。结论 EDA 对脊髓损伤有保护作用, 机制可能与其抑制 SCI 后脊髓细胞的 ERS 和脊髓细胞的凋亡有关。     

MK-801对大鼠脊髓损伤后细胞凋亡及Caspase-3、C-fos的影响

目的 探讨MK-801对急性损伤后脊髓细胞凋亡的影响.方法 30只清洁级SD大鼠随机分为假手术(Sham)组、脊髓损伤(SCI)组和MK-801组,每组10只.假手术组仅作椎板切除,其他两组使用Allen's打击法建立脊髓损伤模型,伤后0.5h,脊髓损伤组腹腔注射5ml/kg生理盐水;MK-801组腹腔注射MK-801...     

尿酸对大鼠脊髓损伤后保护作用的实验研究

目的 观察尿酸（uric acid，UA）对大鼠脊髓损伤后继发性损害的保护作用. 方法 成年雌性SD大鼠随机分成3组:空白对照组,脊髓损伤组,UA干预组.UA干预组在脊髓损伤造模前1 h和造模成功后4 h经腹腔给予UA 500 mg/kg（用0.9%氯化钠溶液配成悬浮液），空白对照组和脊髓损伤组在同样时点经腹腔注射0.9%氯化钠溶液。在脊髓损伤后8,24,72 h和7 d,对损伤部位脊髓组织丙二醛（MDA）含量，超氧化物歧化酶（SOD）的活性进行检测及病理形态学观察,并采用原位末端标记法（TUNEL）标记凋亡细胞.结果 与脊髓损伤组比较，UA干预组大鼠各时间点损伤部位脊髓组织中MDA含量较脊髓损伤组明显下降(P＜0.05), SOD的活性较脊髓损伤组有明显升高(P＜0.05).病理形态结构改善,神经细胞凋亡减少.结论 UA可以抑制脊髓损伤后自由基的损伤作用,减少神经细胞凋亡,对损伤脊髓组织具有保护作用.     

依达拉奉对实验性急性脊髓损伤后细胞凋亡的影响

目的观察依达拉奉（MCI-186）对脊髓损伤后神经细胞凋亡的影响。方法选择SD雄性大鼠40只,随机分成对照组和MCI-186组,均采用改良的Allen打击（WD）法制成150gcf（克厘米势能）中度急性脊髓损伤模型,术后实验组给予MCI-186,3mg/kg对照组给予等量生理盐水。分别于术后6h、12h、24h、72h、7d处死动物取材,采用流式细胞仪检测损伤段脊髓细胞凋亡的情况。结果对照组和MCI-186组均发现凋亡细胞,均在1d达到高峰,但MCI-186组凋亡细胞比率显著低于对照组（P〈0．05）。结论MCI-186通过有效地清除氧自由基,可明显抑制急性脊髓损伤后的脂质过氧化反应及降低神经细胞的凋亡,从而达到保护其神经组织的作用。     

硫化氢对大鼠急性脊髓损伤的保护作用

目的 探讨硫化氢 （H2S） 对大鼠急性脊髓损伤后自噬及细胞凋亡的影响。方法 36 只健康雄性 SD 大鼠随机分为假手术组 （Sham 组）、 单纯脊髓损伤组 （模型组）、 H2S 处理组(H2S 组), 每组 12 只。采用 Allen’ s 法构建大鼠脊髓损伤模型, Sham 组只行椎板切除术, 暴露脊髓不打击; H2S 组在脊髓损伤后 1 h 腹腔注射 50 μmol/kg 硫氢化钠;模型组给予等体积生理盐水。各组脊髓损伤 24 h 后取出脊髓组织, Western blot 检测 LC3、 p70S6K 和 Cleaved caspase-3 表达; 免疫荧光法检测 LC3 表达; TUNEL 染色法检测各组细胞凋亡率。结果 与 Sham 组相比, 模型组 LC3Ⅱ/LC3Ⅰ, Cleaved caspase-3 表达升高,p70S6K 表达降低、 细胞凋亡率升高 （P < 0.01）; 与模型组相比, H2S 组 LC3Ⅱ/LC3Ⅰ、 Cleaved caspase-3 表达降低, p70S6K 表达升高, 细胞凋亡率下降（P < 0.01）。结论 H2S 可抑制大鼠急性脊髓损伤后自噬并减少细胞凋亡。     

依达拉奉对大鼠脊髓损伤过氧化物酶增殖物激活受体γ表达的影响

目的：观察依达拉奉对大鼠脊髓损伤过氧化物酶增殖物激活受体γ（PPARγ）表达的影响。方法：将10周龄SPF级SD大鼠60只,随机分为5组：假手术组（对照组,12只）,SCI+生理盐水组（损伤组,12只）,SCI+依达拉奉A组（治疗组A,12只）,SCI+依达拉奉B组（治疗组B,12只）,SCI+依达拉奉C组（治疗组C,12只）。对照组大鼠仅做椎板切除术,损伤组和治疗组采用改良Allen's法,制成中度脊髓损伤动物模型。治疗组按2mg/kg、3mg/kg和4mg/kg的剂量将依达拉奉用等体积的0．9％氯化钠溶液稀释后,每隔12h腹腔注射1次,给药时间为30min,共14d。对照组和损伤组采用同样方式给予等体积的0．9％氯化钠溶液。结果：治疗组A、治疗组B、治疗组C的PPARγ蛋白表达较对照组、损伤组明显增多（P＜0.05）,治疗组A、治疗组B、治疗组C的PPARγ蛋白表达比较有显著差异（P＜0.05）。结论：依达拉奉可通过上调PPARγ的表达降低炎症反应,且随剂量增加上调能力增加,可有效抑制脊髓损伤周围细胞凋亡,显著促进细胞代谢,从而保护脊髓,为脊髓损伤的治疗及药物筛选提供新方向、新靶标。     

五味子酚对氧化低密度脂蛋白引起的血管内皮细胞及神经细胞损伤的保护作用

目的：氧化低密度脂蛋白（ox-LDL）对血管内皮细胞及神经细胞均有毒性作用，与动脉粥样硬化，神经退行性疾病的发展密切相关。本文探讨了具有很强抗氧化作用的五味子有效成分五味子酚（Sal）对ox-LDI。引起的牛主动脉内皮细胞及神经细胞NGl08-15的损伤和凋亡的保护作用及其初步的机制。方法：以细胞形态，MTT法，LDH释放检测ox-LDL对细胞的损伤及Sal的作用；DNA电泳，核染色质荧光染色观察细胞的凋亡，流式细胞术检测凋亡细胞的百分率；自由基（ROS）荧光探针DCF检测细胞内ROS变化，Western-blot法检测线粒体细胞色素C的释放。结果：ox-LDL可引起内皮细胞和NGl08-15细胞的损伤、凋亡，细胞形态发生明显变化，存活率降低，胞内LDH泄漏增加，出现核染色质聚集、DNA梯状条带，流式细胞术检测出现G1亚峰，凋亡细胞百分率显著增加，并可刺激细胞内ROS产生，促进NGl08-15细胞线粒体细胞色素C的释放增加。提前加入Sal对上述细胞损伤具有明显的保护作用，并能显著抑制细胞的凋亡，减少细胞内ROS产生，降低细胞色素C的表达。结论：Sal能抑制ox-LDL引起的血管内皮细胞及神经细胞的毒性损伤、凋亡，其机制可能与减少胞内自由基、抑制线粒体细胞色素C的释放有关。     

金纳多对大鼠急性脊髓损伤后神经细胞的影响

目的探讨银杏叶提取物(金纳多)对脊髓损伤大鼠损伤区凋亡细胞的作用。方法60只大鼠分正常组、损伤组和药物组,用Allen's改良法建立脊髓损伤模型,腹腔注射金纳多,损伤后1,3,7,14和21d取样,同时对大鼠损伤后双下肢功能作评估,对损伤区脊髓HE染色进行形态学观察,并用TUNEL法检测细胞凋亡。结果损伤组开始有片状出血,以后逐渐减少,7d全部消失。损伤组和药物组中均有凋亡细胞,损伤组凋亡率大于药物组。结论金纳多能抑制或保护大鼠脊髓损伤后神经细胞的凋亡,从而减轻脊髓损伤后的继发性损害。     

Neuroprotective Effect of Ginsenoside Rd in Spinal Cord Injury Rats

In this study, the neuroprotective effects of ginsenoside Rd (GS Rd) were evaluated in a rat model of spinal cord injury (SCI). Rats in SCI groups received a T8 laminectomy and a spinal contusion injury. GS Rd 12.5, 25 and 50 mg/kg were administered intraperitoneally 1 hr before the surgery and once daily for 14 days. Dexamethasone 1 mg/kg was administered as a positive control. Locomotor function was evaluated using the BBB score system. H&E staining and Nissl staining were performed to observe the histological changes in the spinal cord. The levels of MDA and GSH and the activity of SOD were assessed to reflect the oxidative stress state. The production of TNF‐α, IL‐1β and IL‐1 was assessed using ELISA kits to examine the inflammatory responses in the spinal cord. TUNEL staining was used to detect the cell apoptosis in the spinal cord. Western blot analysis was used to examine the expression of apoptosis‐associated proteins and MAPK proteins. The results demonstrated that GS Rd 25 and 50 mg/kg significantly improved the locomotor function of rats after SCI, reduced tissue injury and increased neuron survival in the spinal cord. Mechanically, GS Rd decreased MDA level, increased GSH level and SOD activity, reduced the production of pro‐inflammatory cytokines and prevented cell apoptosis. The effects were equivalent to those of dexamethasone. In addition, GS Rd effectively inhibited the activation of MAPK signalling pathway induced by SCI, which might be involved in the protective effects of GS Rd against SCI. In conclusion, GS Rd attenuates SCI‐induced secondary injury through reversing the redox‐state imbalance, inhibiting the inflammatory response and apoptosis in the spinal cord tissue.     

Neuroprotective effects of Nigella sativa on experimental spinal cord injury in rats

The aim of this study was to investigate the possible beneficial effects of Nigella sativa (NS) in comparison to methylprednisolone on experimental spinal cord injury (SCI) in rats. SCI was performed by placing an aneurysm clip extradurally at the level of T11-12. Rats were neurologically tested over 24 h after trauma and spinal cord tissue samples were harvested for both biochemical and histopathological evaluation. The neurological scores of rats were not found to be different in SCI groups. SCI significantly increased the spinal cord tissue malondialdehyde (MDA) and protein carbonyl (PC) levels, however SCI decreased superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzyme activities compared to the control. Methylprednisolone and NS treatment decreased tissue MDA and PC levels and prevented inhibition of SOD, GSH-Px and CAT enzymes in the tissues. The most significant results were obtained when NS was given. In SCI and placebo groups, the neurons of spinal cord tissue became extensively dark and degenerated with picnotic nuclei. The morphology of neurons in methylprednisolone and NS-treated groups were well protected, however, not as well as the neurons of the control group. The number of neurons in the spinal cord tissue of the SCI and placebo groups was significantly less than the control, laminectomy, methylprednisolone and NS-treated groups. In conclusion, NS treatment might be beneficial in spinal cord tissue damage, and therefore shows potential for clinical implications.     

Effects of resveratrol and methylprednisolone on biochemical, neurobehavioral and histopathological recovery after experimental spinal cord injury

AIM: To investigate the neuroprotective effect of resveratrol in an experimental spinal cord injury (SCI) model in rats. METHODS: Male Wistar albino rats weighing 200-250 g were randomized into six groups. Weight-drop trauma was performed for SCI. Group 1 underwent laminectomy alone. Group 2 underwent laminectomy followed by SCI. Groups 3, 4, 5, and 6 underwent laminectomy followed by SCI and received resveratrol (100 mg/kg), methylprednisolone (MP) (30 mg/kg), resveratrol (100 mg/kg) plus MP (30 mg/kg), and ethanol (2%), respectively. The rats were divided into two subgroups for biochemical analysis (killed at 24 h after surgery) and for neurobehavioral and histopathological evaluation (killed at 6 weeks after surgery). Posttraumatic neurological recovery after surgery was recorded weekly. RESULTS: Groups 3 and 5 revealed significantly lower malon-dialdehyde, nitric oxide, xanthine oxidase, and higher glutathione levels than group 4 (P<0.05). Neurological recovery rates were significantly better in groups 3 and 5 than group 4 (P<0.05). When spinal trauma size ratios were compared, there was no significant difference between treatment groups. CONCLUSION: Resveratrol treatment revealed better biochemical recovery in the acute stage of trauma than MP treatment. Although resveratrol and combined treatment revealed better neurobehavioral recovery than MP treatment; resveratrol, MP, and combined treatment modalities improved histopathological recovery at the same level in the final stage of the experiment. Future studies involving different doses of resveratrol and different doses combinations with MP could promise better results as each drug has a different anti-oxidative mechanism of action.     

Amitriptyline pharmacokinetics in experimental spinal cord injury in the rabbit

Previous studies have demonstrated that pharmacokinetic behavior of several drugs such as paracetamol, theophylline, and aminoglycosides are significantly altered in spinal cord injured patients. No pharmacokinetic study of amitriptyline has been performed in patients and experimental models of spinal cord injury. Pharmacokinetic parameters of amitriptyline in orally treated rabbits subjected to laminectomy and spinal cord injury compared with those underwent laminectomy alone. Among twenty four male rabbits were included in this study, nine of them subjected to spinal cord injury at the 8(th) thoracic level by knife severance method and six rabbits underwent laminectomy alone (sham group) and nine rabbits treated as control. All received a single oral dose of amitriptyline (20 mg/kg) 24 h after injury. Blood sampling were done at predetermined times to 36 h after drug administration. Amitriptyline concentration in serum samples was determined by high-performance liquid chromatography. Pharmacokinetic parameters including maximum concentration (C(max)), time to reach maximum concentration (T(max)), half life, and the area under the curve to last detectable concentration time point (AUC(0-t)) were directly determined from the concentration-time curve. Maximum concentration was observed at 6.5 h after administration in sham group with a concentration of 439.6 ng/ml, whereas in SCI group T(max) was at 2.7 h with a concentration of 2763.9 ng/ml. In control group it was 3.3 h and 396 ng/ml, respectively. In SCI group, AUC was 9465.6 ng.h/ml and half life was 6 h and for control group it was 2817.4 ng.h/ml and 6.4 h, respectively. Statistical analysis of data showed that SCI didn't induce significant changes in amitriptyline pharmacokinetic parameters.     

Efficacy of treatment with verbascoside, biotechnologically produced by Syringa vulgaris plant cell cultures in an experimental mice model of spinal cord trauma

In this study we evaluated the effect of glycosylated phenylpropanoid verbascoside (VB), isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae) in experimental animal model of spinal cord injury (SCI). SCI was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis. At 1 and 6 h after injury, the mice were treated with VB extract, administered at the dose of 2 mg/kg with intraperitoneal administration. Immunohistochemical examination demonstrated a marked increase on expression for nitrotyrosine, inducible nitric oxide synthase, poly(ADP-ribose), and apoptosis events (increase of Bax and Bcl-2 expression) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the cytokines expression (TNF-α and IL-1β), neutrophil infiltration (myeloperoxidase), and activation of NF-κB. In contrast, all of these parameters of inflammation were attenuated by treatment with VB. In a separate set of experiment, we have clearly demonstrated that VB treatment significantly ameliorated the recovery of function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with VB extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.     

雷帕霉素增强大鼠脊髓损伤后神经元自噬对凋亡影响的观察

目的探讨雷帕霉素(Rapamycin)对急性脊髓损伤后神经元的保护作用及其可能机制。方法 150只SD大鼠按随机分成假手术组、损伤组和治疗组,Allen法[1]建立大鼠脊髓损伤模型,于损伤后8h、24h、3d、7d、14d取损伤脊髓组织,免疫荧光双标观察神经元LC3表达及凋亡情况,Western blot检测LC3及Caspase-3表达,电镜观察神经元显微结构变化。结果与损伤组相比,治疗组相同时相点LC3阳性神经元增多,凋亡细胞减少(P<0.05);Western blot示LC3Ⅱ损伤后第3天达高峰,治疗组表达较明显,Caspase-3损伤第7天后开始下降,治疗组表达较少(P<0.05);电子显微镜下损伤组及治疗组神经元内均可见自噬小体及自噬溶酶体。结论雷帕霉素增强大鼠脊髓损伤后神经元自噬,减少神经元细胞凋亡,对神经元具有保护作用。