白介素-1受体拮抗剂对大鼠急性脊髓损伤后神经细胞凋亡和caspase-3表达的影响

目的：观察白介素-1受体拮抗剂（IL—1Ra）对脊髓损伤（SCI）后继发性神经细胞凋亡和caspase-3表达的影响。方法：60只SD大鼠，随机分为假手术组、脊髓损伤组、IL-1Ra治疗组和生理盐水对照组，每组15只。采用Allen’s法建立大鼠急性SCI动物模型．IL—1Ra治疗组和生理盐水对照组于SCI后立即硬膜下腔内分别注射IL-1Ra（10μg／10μl）和等量生理盐水，于伤后24h以损伤为中心（假手术组取相应部位），切取长约8mm脊髓组织．用蛋白印迹法和免疫组化染色法检测caspase-3表达的变化：采用实时定量PCR法检测caspase-3mRNA的表达情况；用原位末端标记法（TUNEL）检测SCI后神经细胞凋亡情况。结果：SCI后24h，SCI组与假手术组比较受损伤的脊髓组织中caspase-3mRNA和蛋白表达水平均显著升高（P〈O．05），且TUNEL阳性细胞明显增多（P〈O．01）：IL—1Ra治疗组大鼠受损伤节段脊髓组织caspase-3表达及TUNEL阳性细胞数较生理盐水对照组）均明显减少（P〈O．01）。结论：IL-1Ra治疗可减少急性SCI后脊髓组织中caspase-3的表达和神经细胞凋亡的发生。     

甲基强的松龙对大鼠脊髓损伤后神经细胞凋亡的影响及其机理

目的研究甲基强的松龙(MP)对大鼠横断性脊髓损伤(SCI)后神经细胞凋亡的影响及其作用机理.方法60只成年Wistar大鼠随机分成正常对照组、脊髓损伤组和MP治疗组,每组20只,MP治疗组在横断T10脊髓组织后30min经尾静脉给予MP治疗,损伤组和对照组未予任何治疗.MP治疗组和脊髓损伤组大鼠于脊髓损伤后8h、24h、3d和1周取材,采用透射电镜、TUNEL染色观察细胞凋亡情况,采用免疫组织化学染色观察Fas、半胱氨酸蛋白酶(caspase)-8和caspase-3在SCI前后的变化情况,采用改良Tarlov评分方法观察大鼠后肢运动功能.结果SCI后24h大鼠后肢运动功能逐渐恢复,MP治疗组大鼠的后肢运动功能恢复优于损伤组,7d后尤其明显.TUNEL染色和电镜检查证实SCI后8h即有凋亡细胞出现,其中既有神经元也有胶质细胞3d凋亡细胞数达到高峰;7d仍有凋亡细胞存在,但已明显减少;各时间点治疗组凋亡细胞数量明显少于损伤组(P＜0.05).治疗组和损伤组在SCI后8h可以检测到少量的Fas和caspase-8阳性神经元及胶质细胞,Fas和caspase-8的表达在SCI后3d达到高峰,7d表达下降,各时间点治疗组的Fas和caspase-8灰度值明显大于损伤组,二者有显著性差异(P＜0.05).治疗组和损伤组caspase-3的表达在SCI后8h均逐渐增加(与对照组相比),7d达到高峰,治疗组灰度值大于损伤组,但二者无显著性差异.结论MP能抑制大鼠脊髓横断性脊髓损伤后的神经细胞凋亡,但不能推迟细胞凋亡出现的高峰时间;MP抑制细胞凋亡的途径可能通过非特异性抑制Fas和caspase-8的表达来实现.     

依达拉奉对肺泡Ⅱ型上皮细胞凋亡的保护作用

目的 探讨依达拉奉(EDA)对氧化应激诱导的肺泡Ⅱ型上皮细胞凋亡的影响及可能机制.方法 用过氧化氢氧化应激建立氧化应激肺泡Ⅱ型上皮细胞凋亡模型细胞.将细胞分为:过氧化氢处理组(H组)、依达拉奉-过氧化氢处理组(E组)和对照组(C组).采用脂质过氧化物(MDA)、谷胱甘肽(GSH)试剂盒检测A549细胞MDA、GSH的含量,同时测定三组细胞生长的抑制率、凋亡率、半胱氨酸天冬氨酸蛋白酶(caspase)-3与caspase-9 mRNA及蛋白表达.结果 与C组比较,H组细胞MDA、细胞生长抑制率、凋亡指数、caspase-3与caspase-9 mRNA相对表达及蛋白表达量明显升高;而GSH明显降低(P＜0.01).与H组比较,E组细胞MDA、细胞生长抑制率、凋亡指数、caspase-3与caspase-9 mRNA相对表达及蛋白表达量明显降低;而GSH明显增高(P＜0.05).结论 依达拉奉可通过抗氧化及抗caspase-3,caspase-9引起的凋亡以保护肺泡Ⅱ型上皮细胞对抗氧化应激.     

丙酮酸钠在脑缺血-再灌注损伤中的神经保护作用

目的 探讨丙酮酸钠（SP）对脑缺血-再灌注（IR）损伤的影响。方法 雄性SD大鼠24只,体重200~250g,随机分为三组：假手术+生理盐水组（S组）、脑IR+生理盐水组（M组）、脑IR+丙酮酸钠组（P组）。S组行假手术处理,M组、P组建立大脑中动脉梗死（MCAO）模型,阻断血流2 h,分别于血流再灌注时经尾静脉注射生理盐水或丙酮酸钠600 mg/kg。血流再灌注后24 h,采用TTC法测定脑梗死体积,采用mNSS评分测定神经功能缺损评分,采用Western blot法测定cleaved caspase-3、Bax、cleaved PARP-1蛋白含量以及细胞核内AIF蛋白含量,微板法测定谷胱甘肽（GSH）、超氧化物歧化酶（SOD）以及丙二醛（MDA）水平。结果 与S组比较,M组和P组脑梗死体积明显增加,mNSS评分明显升高;M组cleaved caspase-3、Bax、cleaved PARP-1蛋白含量和AIF细胞核内蛋白含量明显升高,GSH与SOD水平明显降低,MDA水平明显升高（P＜0.05）。与M组比较,P组脑梗死体积明显减小,mNSS评分明显降低,cleaved caspase-3、Bax、cleaved PARP-1蛋白含量和AIF细胞核内蛋白含量明显降低,GSH与SOD水平明显升高,MDA水平明显降低（P＜0.05）。结论 丙酮酸钠能够减轻大鼠脑IR损伤,其神经保护作用可能与减少细胞凋亡以及抑制氧化应激水平相关。     

富血小板血浆通过Nrf2信号通路对脊髓损伤小鼠的抗炎抗氧化作用及其机制

目的探讨富血小板血浆通过Nrf2信号通路对脊髓损伤小鼠的抗炎抗氧化作用及其作用机制。方法采用改良Allen’s击打法制备小鼠脊髓损伤模型, 采用随机数字表法将小鼠分为对照组(SHAM组)、脊髓损伤组(SCI组)、富血小板血浆处理组(PRP组)。术后7 d, 小鼠麻醉处死, 酶联免疫吸附试验(ELISA)法检测白细胞介素(IL)-1β、IL-10的表达, 氧化应激试剂盒检测超氧化物歧化酶(SOD)、丙二醛(MDA)和还原型谷胱甘肽(GSH)的含量, 蛋白质印迹法(Western blot)检测Nrf2蛋白和Keap1蛋白的表达, 尼氏染色观察损伤组织中尼氏体的数量, 并使用小鼠BMS评分于损伤后1、3、7、14、21、28 d评估运动功能。组间比较采用t检验和单因素方差分析。结果 (1)PRP组尼氏体数量[(28.50±5.68)个]明显高于SCI组[(12.83±5.04)个], 差异有统计学意义(t=5.053, P<0.05)。(2)PRP组SOD[(131.07±15.45) U/g]和GSH[(5.60±0.98) μg/g]含量高于SCI组[(102.51±24.00)...     

内源性CO和NO在大鼠脑缺血-再灌注损伤中的作用

目的 研究血红素氧合酶-1(heme oxygenase-1,HO-1)/一氧化碳(CO)体系和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)/一氧化氮(NO)体系在大鼠脑缺血-再灌注损伤中的相互作用,探讨脑保护策略.方法 24只Wistar大鼠随机均分为四组:脑缺血-再灌注+锌原踮啉组(Z组)、脑缺血-再灌注+氨基胍组(A组)、脑缺血-再灌注组(IR组)、假手术组(C组).Z组和A组夹闭两侧颈总动脉前30 min腹腔分别注射锌原卟啉45 μmol/kg或氨基胍500 mg/kg,C组和IR组腹腔注射等量生理盐水.缺血20 min再灌注6h后处死大鼠取海马,检测大鼠海马组织中CO、NO、SOD、MDA量的变化及HO-1-mRNA和iNOS-mRNA表达水平;电镜观察海马线粒体的变化.结果 与C组相比,IR组CO、NO、MDA的含量增高,SOD降低,HO-1-mRNA和iNOS-mRNA表达增高(P<0.01),电镜观察线粒体受损.与IR组相比,Z组CO和SOD的量降低,NO、MDA的量增高,HO-1-mRNA的表达降低(P<0.01),电镜观察线粒体受损加重;A组NO、MDA的量降低,SOD的量增高,iNOS-mRNA表达降低(P<0.01),电镜观察线粒体受损减轻.结论 脑缺血-再灌注损伤过程中,iNOS/NO体系介导了神经细胞的损伤,而HO-1/CO体系具有抗损伤的作用;iNOS抑制剂在脑缺血-再灌注损伤过程中对神经细胞有保护作用.     

大鼠脊髓损伤节段线粒体自噬蛋白及凋亡因子的表达

目的 :观察脊髓损伤节段线粒体自噬蛋白与凋亡蛋白的表达情况。方法 :将50只清洁级SD雄性大鼠随机分为损伤组(39只)和对照组(11只),损伤组应用Allen′s法建立大鼠脊髓损伤模型,并分为5个不同时间点(损伤后1h、6h、24h、48h、72h),每个时间点7只,在损伤不同时间点处死大鼠,并截取损伤区域约1cm长脊髓节段,通过Western blot检测线粒体自噬蛋白(LC3、NIX)及凋亡蛋白(BNIP3、cleaved caspase-3、cytochrome c)表达水平;另取4只大鼠在损伤后24h截取损伤组织,用透射电镜观察损伤后神经元自噬体结构。对照组不做任何处理,其中7只大鼠用于Western blot分析,4只用于电镜检测。结果:对照组及损伤后1h、6h、24h、48h、72h脊髓损伤组大鼠LC3表达水平分别为0.2893±0.0325、0.3002±0.0474、0.3943±0.1154、0.4818±0.0426、0.5430±0.0865、0.5790±0.0892;NIX表达分别为0.1392±0.0171、0.1431±0.0325、0.1955±0.1379、0.3841±0.1136、0.4043±0.1059、0.4506±0.0174;BNIP3表达水平分别为0.1354±0.0547、0.1896±0.1264、0.2654±0.1341、0.7220±0.1030、0.4713±0.1041、0.3975±0.1505;cleaved caspase-3表达水平分别为0.2806±0.0999、0.4158±0.1137、0.7865±0.4056、0.9354±0.2659、1.0152±0.3441、1.1608±0.2488;细胞色素C表达水平分别为0.1489±0.0300、0.2196±0.0762、0.3162±0.1656、0.4456±0.1180、0.5407±0.1029、0.5812±0.1388。LC3、NIX、cleaved caspase-3和细胞色素C在24h、48h、72h与对照组比较明显增加(P0.05);BNIP3在24h、48h与对照组比较明显增加(P0.05)。进一步通过透射电镜观察损伤后24h细胞超微结构,可观察到双层膜结构自噬体,其内包裹有受损线粒体等细胞器结构。结论:大鼠急性脊髓损伤后可能通过促进NIX蛋白与LC3结合诱导线粒体自噬,减少凋亡水平,从而在脊髓损伤修复中发挥保护作用。     

B淋巴细胞瘤-2蛋白多位点磷酸化在大鼠脊髓损伤后细胞自噬中的表达研究

目的探讨大鼠脊髓损伤(spinal cord injury,SCI)后细胞自噬的变化及其与B淋巴细胞瘤-2(Bcell lymphoma-2,Bcl-2)蛋白多位点磷酸化的关系。方法取40只8周龄雄性SD大鼠,采用改良Allen法制备SCI模型;将造模成功的36只大鼠随机分为SCI组、自噬抑制剂组、自噬促进剂组,每组12只。另取12只大鼠仅切除椎板、不损伤脊髓,作为假手术组。造模结束后,自噬抑制剂组及自噬促进剂组分别于脊髓鞘内注射20μL600 nmol/L 3-甲基腺嘌呤、25 nmol/L雷帕霉素,假手术组及SCI组仅注射20μL生理盐水;每天1次,连续4周。造模后1 d及1、2、4周,采用BBB评分法评价各组大鼠后肢运动功能。末次注射后24 h处死各组大鼠并取脊髓组织,ELISA法检测脊髓组织中过氧化物酶(myeloperoxidase,MPO)活性及TNF-α、IL-1β水平;HE染色观察脊髓组织形态学变化;透射电镜观察脊髓组织中线粒体超微结构变化;免疫荧光染色检测自噬相关蛋白(Beclin1)、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)蛋白表达;TUNEL染色观察脊髓组织神经细胞凋亡;免疫荧光双染检测LC3/TUNEL阳性细胞表达;Western blot检测细胞Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Bcl-2及p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达。结果与假手术组相比,SCI组各时间点BBB评分降低,MPO活性、TNF-α、IL-1β水平升高;神经细胞周围间隙增大,细胞肿胀、出现空泡,线粒体中出现自噬小体;Beclin1及LC3蛋白阳性率、神经细胞凋亡率显著升高;LC3、TUNEL阳性细胞增多;Bax、p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达升高,Bcl-2蛋白表达降低;以上指标比较差异均有统计学意义(P0.05)。与SCI组相比,自噬抑制剂组各时间点BBB评分降低,MPO活性、TNF-α、IL-1β水平升高;线粒体中出现少量自噬囊泡;Beclin1及LC3蛋白阳性率降低,神经细胞凋亡率显著升高;LC3阳性细胞减少、TUNEL阳性细胞增多;Bax、p-Bcl-2(Ser87)、p-Bcl-2(Ser70)蛋白表达升高,Bcl-2蛋白表达降低。而自噬促进剂组结果与自噬抑制剂组相反;以上指标组间比较差异均有统计学意义(P0.05)。结论大鼠SCI后通过诱导细胞自噬可降低神经细胞凋亡,保护脊髓功能,其机制可能与抑制Bcl-2蛋白多位点磷酸化有关。     

原花青素对大鼠急性脊髓损伤的保护作用及机制研究

[目的]探讨原花青素对大鼠急性脊髓损伤(spinal cord injury,SCI)的保护作用.[方法]SD大鼠随机分为假手术组(A组)、脊髓打击损伤组(B组)、原花青素治疗组(C组).A组行T8椎板切除术,不伤及脊髓.B、C组采用改良Allens撞击法制备大鼠T8急性SCI模型.C组术后立即腹腔注射原花青素100 mg/kg,A、B组术后立即腹腔注射等量的生理盐水.分别于术后不同时间点评价大鼠后肢运动功能,检测脊髓匀浆超氧化物歧化酶(superoxide dismutase,SOD)、髓过氧化物酶(myeloperoxidase,MPO)活性、丙二醛(malondialdehyde,MDA)含量改变,观察脊髓组织病理学变化.[结果]术后A组大鼠后肢运动功能恢复.术后C组大鼠后肢运动功能较B组均有所改善,72 h后肢运动功能恢复更为显著(P＜0.01).术后48 hB组脊髓组织SOD活性较A组明显降低(P＜0.01),而MDA含量较A组明显增加(P＜0.01);应用GSPE后,术后48hC组脊髓组织SOD活性较B组明显升高(P＜0.01),脊髓组织MDA水平较B明显降低(P＜0.01);SCI后脊髓组织TNF-α含量、MPO活性均随时间推移逐渐增高;各时间点C组脊髓TNF-α含量、MPO活性较B组均有统计学意义(P＜0.01);A组大鼠各检测点脊髓组织形态结构均正常;B、C两组72 h切片变化最为显著;B组可见神经组织多灶性出血、大量神经元坏死、尼氏体溶解消失、核固缩变小及炎性细胞浸润;C组可见局部出血点、细胞轻度肿胀,胞质均匀及少量炎性细胞浸润.[结论] GSPE可明显改善大鼠运动功能,抑制氧化应激、炎症反应和细胞凋亡等,对SCI有神经保护作用.     

BMSCs移植对大鼠脊髓损伤后VEGF表达及细胞凋亡的影响

[目的]研究骨髓基质细胞(BMSCs)移植对大鼠脊髓损伤(SCI)后血管内皮生长因子(VEGF)蛋白表达及神经细胞凋亡的影响,探讨骨髓基质细胞移植治疗脊髓损伤的机制.[方法]取大鼠股骨和胫骨骨髓培养BM-SCs并传代.参照Taoka's方法制作大鼠脊髓压迫损伤模型.脊髓损伤后30 min,进行BMSCs或DMEM培养液损伤周围注射.术后3、7和14 d,应用Westem Blot法检测损伤脊髓组织内VEGF蛋白的表达,应用TUNEL方法检测脊髓损伤周围神经细胞凋亡情况.[结果]移植术后3、7、14 d,BMSCs组VEGF蛋白的表达水平呈时间依赖性增加,与DMEM组相比,术后3 d两组VEGF蛋白表达水平没有显著性差异(P>0.05),而术后7和14 d,BMSCs组VEGF蛋白表达水平显著高于DMEM组(P<0.01).此外,与DMEM组大鼠相比,移植术后3、7和14 d,BMSCs移植显著减少脊髓损伤周围区凋亡的神经细胞数目(P<0.05).[结论]脊髓损伤后,BMSCs移植能够增加VEGF蛋白的表达并且抑制神经细胞凋亡.     

Preclinical efficacy of Dexmedetomidine on spinal cord injury provoked oxidative renal damage

Abstract

Purpose: Oxidative renal injury is the mainstay in patients with spinal cord injury (SCI) and it may eventuate to chronic renal failure. In our study, we investigated the potential of α2-adrenoreceptor agonist Dexmedetomidine (Dex) in ameliorating SCI provoked oxidative renal assault. Methods: Complete SCI was generated by surgical transaction of the cord at the T10–12 level. Dex administration (50?mcg/kg, b.wt, intraperitoneally) was initiated 12?h after the surgery for 10 days. Then, blood was collected and kidneys were removed to evaluate the efficacy of Dex on post-SCI renal complications. Results: Dex treatment significantly attenuated elevated serum creatinine and blood urea nitrogen in SCI rats to normalcy. Further in SCI rats elevated level of MDA, protein carbonyl and myeloperoxidase (MPO) were observed and Dex treatment significantly attenuated these toxic manifestations to normalcy. Besides in SCI rats, the antioxidants (SOD, CAT, Gpx and GST and GSH) levels were significantly diminished and Dex treated rats significantly restored the antioxidants level in renal tissue to normalcy. Notably, in our study the protein expression of inflammatory cytokines (TNF-α and IL-6) and cleaved caspase-3 were upregulated in renal tissue of SCI rats. Fascinatingly, Dex treatment downregulated the protein expression of TNF-α, IL-6 and cleaved caspase-3 by anti-inflammatory, antiapoptotic mechanism. Furthermore, SCI rats displayed upregulated protein expression of kidney of SCI rats. Dex treatment diminished the renal apoptosis by downregulating the cleaved caspase-3 expression. Conclusion: Taken together, these results accentuate that Dex may be a beneficial clinical agent to combat post-SCI renal complications.     

基于TNFR/RIPK1通路探索紫草素对大鼠脊髓损伤的作用及机制

目的:初步探讨紫草素对大鼠急性脊髓损伤(spinal cord injury,SCI)后神经功能恢复的影响及作用机制。方法:将96只Sprague-Dawley(SD)雄性大鼠分为4组:假手术组,即A组;假手术+紫草素组,即B组;脊髓损伤+二甲基亚砜(dimethyl sulfoxide,DMSO)组,即C组;脊髓损伤+紫草素组,即D组;每组24只。C、D组采用钳夹法制作大鼠急性SCI 模型。所有大鼠硬膜下置管,A 组不给药,B组和D组造模后30 min 经导管注射紫草素100 mg·kg^-1,C组注射等量 DMSO,每日1次,至取材时间点。各组分别于造模后6、12 h和3 d 每组取8只大鼠,行 Basso-Beattie-Bresnahan(BBB)评分及造模后1、3、7、14、21 d行斜板实验,再处死动物取脊髓组织。造模后1 h 每组大鼠腹腔注射碘化丙啶(propidine iodide,PI)1 mg·kg^-1,术后24 h取材检测脊髓组织 PI 红染细胞数;24 h 时取材采用苏木素-伊红(haematoxylin eosin,HE)染色观察脊髓损伤情况,尼氏(Nissl)染色观察神经元存活数量,使用Western-blot技术检测 B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)蛋白及凋亡相关蛋白受体相互作用蛋白激酶1(receptor-interacting protein kinase 1,RIPK1)的表达水平。结果:造模后A组和B组各时间点的 BBB 评分均正常,C、D组各时间点均低于A、B组,D组造模后12 h和3 d的 BBB 评分高于同时间点C组(P<0.05)。造模后12 h,D组PI 红染细胞较C 组明显减少,神经元崩解减轻(P<0.05)。造模后24 h,A 组和 B 组脊髓组织 HE 和 Nissl 染色正常,D 组脊髓组织损伤程度和存活神经元数量均优于 C 组(P<0.05)。Bcl-2、RIPK1蛋白在A 组、B 组表达很低; RIPK1 在C组表达明显增高,在D组表达明显下降,差异有统计学意义(P<0.05);Bcl-2蛋白在D 组表达高于C 组(P<0.05)。结论:紫草素可减轻大鼠急性SCI后的病理变化,改善行为学评分,促进脊髓神经功能恢复。其具体机制可能与抑制TNFR/RIPK1信号通路介导的坏死性凋亡有关。     

大鼠脊髓损伤后线粒体代谢功能和还原型谷胱甘肽水平的变化

目的 探讨大鼠脊髓损伤后伤段脊髓线粒体代谢功能和还原型谷胱甘肽(GSH)水平的变化.方法 取48只SD大鼠,随机分为假手术组(对照组)和脊髓损伤组(SCI组),每组又分为处理后6、12、24 h 3个时相组,每个时相组8只,分别提取伤段脊髓的线粒体,测定线粒体呼吸功能[呼吸Ⅲ态(R3)、呼吸Ⅳ态(R4)、呼吸控制率(RCR)及磷氧比(P/O)值]、三磷酸腺苷酶(ATPase)活性(Na+、K+-ATP酶和Ca2+、Mg2+-ATP酶活性)及GSH的变化.结果 SCI组在伤后6、12、24 h伤段脊髓线粒体的R3、RCR及P/O均显著低于对照组,R4显著高于对照组,差异有统计学意义(P＜0.05).SCl组Na+、K+-ATP酶和Ca2+、Mg2+-ATP酶较对照组明显降低,伤后6 h急剧下降,12 h后稍有代偿性升高,24 h后又下降,与对照组比较差异有统计学意义(P＜0.05).GSH水平SCI组与对照组相比明显降低,以伤后12 h最为明显,差异有统计学意义(P＜0.05).结论 脊髓损伤后伤段脊髓线粒体的呼吸功能、ATPase酶活性及GSH水平明显下降,说明脊髓损伤后线粒体能量代谢功能和自由基清除能力均受到明显损害.     

Effect of VEGF and CX43 on the promotion of neurological recovery by hyperbaric oxygen treatment in spinal cord–injured rats

Background contextSpinal cord injury (SCI) is a serious health issue that may result in high health care costs, with additional social and psychological burdens. Hyperbaric oxygen (HBO) treatment has been found to be beneficial for neurological recovery; however, the underlying mechanisms are yet to be characterized.PurposeThe aim of this study was to investigate the mechanisms of HBO treatment in SCI by measuring the expression levels of vascular endothelial growth factor (VEGF) and Connexin43 (CX43) in the injured spinal cord tissue.Study design/settingAn experiment animal study of rats undergoing SCI and HBO treatment.MethodsThe spinal cord injury model was established in rats, which were randomly divided into the following four groups: (1) the sham-operated group (SH), (2) the sham-operated and hyperbaric oxygen treatment group (SH+HBO), (3) the spinal cord injury group (SCI), and (4) the spinal cord injury and hyperbaric oxygen treatment group (SCI+HBO). For groups of SH+HBO and SCI+HBO, the animals received 1 hour of HBO at 2.0 ATA in 100% O2 twice per day for 3 days and then daily for the following days consecutively after surgery. After operation, neurological assessments were performed, the spinal cord tissue samples were harvested for histopathological evaluation, Western blot and real-time polymerase chain reaction analysis.ResultsThe Basso-Bettie-Bresnahan scores were significantly improved in the SCI+HBO group compared with the SCI group on the postoperative 7th and 14th days. The histology scores were significantly decreased by HBO treatment compared with that in the SCI group on the postoperative 3rd, 7th, and 14th days. Western blot analysis and real-time polymerase chain reaction revealed that the expression level of vascular endothelial growth factor (VEGF) in the SCI+HBO group was significantly increased compared with the SCI group. The protein expression level of CX43 and its mRNA level in the SCI+HBO group were significantly decreased on the postoperative 3rd and 7th days, whereas its expression was significantly increased by HBO treatment on the postoperative 14th day compared with the SCI group.ConclusionsHBO treatment improved neurological recovery when applied after SCI. The expression level changes of VEGF and CX43 may contribute to the further understanding on the molecular mechanisms of HBO treatment on SCI.     

Necroptosis,a novel type of programmed cell death,contributes to early neural cells damage after spinal cord injury in adult mice

Abstract

Background

Necroptosis is an emerging programmed necrosis other than traditional necrosis and apoptosis. Until recently, there have not been studies that have investigated a relationship between necroptosis and pathogenesis of cell death after spinal cord injury (SCI).

Objective

To investigate whether necroptosis takes part in the early pathophysiological processes of traumatic SCI in mice.

Methods

Female ICR mice were randomized equally into three groups: the sham, the vehicle-treated + SCI group, and the Nec-1-treated + SCI group. To induce SCI, the mice were subjected to a laminectomy at T9 and compression with a vascular clip. After mice were sacrificed 24 hours post-SCI, propidium iodide (PI)-positive cells were detected using in vivo PI labeling. Morphological analyses were performed by hematoxylin and eosin staining and Nissl staining. The samples were evaluated for apoptosis by the in situ TUNEL assay. The expression of caspase-3 was assessed by western blot. Locomotor behavior of hindlimb was evaluated by BMS (Basso mouse scale) score at 1, 3, 5, 7, and 14 days post-injury.

Results

Compared with dimethyl sulfoxide -treated mice, necrostatin-1-treated mice showed decreased PI-positive cells (P < 0.05), alleviated tissue damage, more surviving neuron at 24 hours after SCI (P < 0.05), and improved functional recovery from days 7 to 14 (P < 0.05). Necrostatin-1 did not reduce the expression of caspase-3 and the number of TUNEL-positive cells at 24 hours after SCI (P > 0.05).

Conclusions

Necroptosis contributes to necroptotic cell death and influences functional outcome after SCI in adult mice. The inhibition of necroptosis by necrostatin-1 may have therapeutic potential for patients with SCI.     

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.     

早期手术减压对损伤脊髓caspase-3表达的影响

目的探讨大鼠脊髓损伤后不同减压时间对大鼠脊髓细胞caspase-3表达的影响。方法将动物分为:大鼠脊髓挫伤即刻手术减压组(A组),大鼠脊髓挫伤2小时手术减压组(B组),大鼠脊髓挫伤8小时手术减压组(C组),大鼠脊髓挫伤24小时手术减压组(D组)。手术后1、3、7、14、28天对脊髓损伤区进行细胞凋亡caspase-3蛋白表达的测定(免疫组化法),采用计算机图像分析技术,进行定量分析。并用行为学和电生理检查观察大鼠功能恢复情况。结果A、B、C、D四组中均发现凋亡caspase-3蛋白阳性表达细胞,图象分析发现,各组凋亡细胞caspase-3免疫反应阳性细胞表达顺序为D>C>B>A,与大鼠后肢功能恢复有平行的变化趋势。结论大鼠脊髓损伤早期手术减压能抑制脊髓损伤后的细胞凋亡,促进大鼠后肢功能恢复。     

亚低温对脊髓损伤后神经细胞凋亡的影响

目的观察大鼠脊髓损伤(SCI)细胞凋亡现象及亚低温对细胞凋亡的影响。方法大鼠SCI后分别于8h、24h、7d取材，采用常规病理HE染色和末端脱氧核苷酸转移酶(TdT)介导的dutp缺口末端标记技术(TNEUL)，研究亚低温对大鼠SCI后神经细胞凋亡的影响。结果SCI后常温组8h灰质区出现较多凋亡细胞，24h时白质和灰质内均有凋亡细胞分布，7d后凋亡细胞多见于白质；亚低温组凋亡细胞明显减少(P＜0．05)。结论亚低温明显减少SCI后细胞凋亡的发生，从病理上为亚低温脊髓保护提供了可靠的依据。     

Honokiol exerts protective effects on neural myelin sheaths after compressed spinal cord injury by inhibiting oligodendrocyte apoptosis through regulation of ER-mitochondrial interactions

ObjectiveTo investigate the effect of honokiol on demyelination after compressed spinal cord injury (CSCI) and it''s possible mechanism.DesignAnimal experiment study.SettingInstitute of Neuroscience of Chongqing Medical University.InterventionsTotal of 69 Sprague–Dawley (SD) rats were randomly divided into 3 groups: sham group (n=15), honokiol group (n=27) and vehicle group (n=27). After established CSCI model by a custom-made compressor successfully, the rats of sham group were subjected to the limited laminectomy without compression; the rats of honokiol group were subjected to CSCI surgery and intraperitoneal injection of 20 mg/kg honokiol; the rats of vehicle group were subjected to CSCI surgery and intraperitoneal injection of an equivalent volume of saline.Outcome measures: The locomotor function of each group was assessed using the Basso, Beattie and Bresnahan (BBB) rating scale. The pathological changes of myelinated nerve fibers of spinal cord in 3 groups were detected by osmic acid staining and transmission electron microcopy (TME). Immunofluorescence and Western blot were used to research the experessions of active caspase-3, caspase-12, cytochrome C and myelin basic protein (MBP) respectively.ResultsIn the vehicle group, the rats became paralyzed and spastic after injury, and the myelin sheath became swollen and broken down along with decreased number of myelinated nerve fibers. Western blot analysis manifested that active caspase-3, caspase-12 and cytochrome C began to increase 1 d after injury while the expression of MBP decreased gradually. After intervened with honokiol for 6 days, compared with the vehicle group, the locomotor function and the pathomorphological changes of myelin sheath of the CSCD rats were improved with obviously decreased expression of active caspase-3, caspase-12 and cytochrome C.ConclusionsHonokiol may improve locomotor function and protect neural myelin sheat from demyelination via prevention oligodendrocytes (OLs) apoptosis through mediate endoplasmic reticulum (ER)-mitochondria pathway after CSCI.