TNF-α、Caspase-3在大鼠脊髓损伤后神经细胞凋亡中相关性研究

目的探讨大鼠脊髓损伤后肿瘤坏死因子-α(TNF-α)、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)在神经细胞凋亡中的动态表达,验证TNF-α和Caspase-3之间的相关性。方法将70只成年健康SD大鼠按Nystrom法建立大鼠脊髓(T8、T9)急性压迫损伤模型,HE染色观察脊髓组织病理学变化;免疫组化染色测定各时间点TNF-α、Caspase-3的表达变化;原位末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸(d UTP)标记法(TUNEL法)检测神经细胞的凋亡水平;通过Western blot法检测大鼠脊髓中TNF-α、Caspase-3蛋白的表达水平。结果正常大鼠脊髓神经细胞TNF-α、Caspase-3的阳性细胞表达率较少;脊髓损伤后8 h,脊髓神经细胞中TNF-α、Caspase-3的阳性表达率明显增多,分别为(24.13%±3.02%)及(40.79%±3.43%),1 d达高峰,分别为(51.36%±4.26%)及(70.78%±5.97%),3 d表达减弱,分别为(20.24%±2.93%)及(37.71%±2.88%),与正常大鼠比较,差异有统计学意义(P<0.01)。TUNEL阳性细胞率在大鼠脊髓损伤后8 h明显增多(39.81%±2.27%),1 d达高峰(71.58%±3.87%),3 d表达减弱(40.55%±2.36%),与正常大鼠比较,差异均有统计学意义(P<0.01)。大鼠脊髓损伤后1 d,与损伤组比较,抑制剂组TNF-α、Caspase-3、TUNEL阳性细胞率明显减少,差异有统计学意义(P<0.01)。造模后7 d,与空白对照组比较,损伤组TNF-α、Caspase-3蛋白的表达上升;与损伤组比较,抑制剂组TNF-α、Caspase-3蛋白的表达下降,差异有统计学意义(P<0.05)。结论脊髓损伤后TNF-α、Caspase-3的表达增强,TNF-α抑制剂使Caspase-3表达减弱,且其参与了脊髓损伤细胞凋亡的调节。     

Bcl-xL基因转染对脊髓损伤细胞凋亡的影响

目的观察脂质体介导的Bcl—xL基因体内转染对大鼠脊髓损伤后伤区脊髓细胞凋亡和神经功能恢复的影响。方法制备大鼠胸段脊髓T8.9压迫损伤模型。38只大鼠分成三组：(1)损伤 pSFFV．Bcl—xL转染组(实验组，15只)；(2)损伤 pSFFV．GFP转染组(对照组，15只)；(3)假损伤组(8只)。将阳离子脂质体质粒混合后直接注入大鼠损伤脊髓，伤后3d和7d利用半定量逆转录一聚合酶链式反应(RT—PCR)和免疫组化检测Bcl—xL基因体内表达；原位末端标记(TUNEL)法检测细胞凋亡；采用开放场地试验BBB评分和斜板试验，评价神经功能。结果脊髓损伤后3d和7d损伤局部Bcl—xL mRNA和蛋白较对照组和假损伤组表达明显增多；TUNEL结果显示，实验组损伤节段细胞凋亡数比对照组明显减少，神经功能改善。结论脂质体介导Bcl—xL体内转基因治疗可有效转染脊髓的神经细胞；外源性Bcl—xL在损伤脊髓的过度表达可减少脊髓不完全性损伤后凋亡引起的神经元死亡和增强神经细胞成活，促进神经功能恢复。     

大鼠坐骨神经损伤后脊髓组织中Caspase 3、Bcl-xl表达变化的实验研究

目的：探讨大鼠坐骨神经损伤后脊髓组织中Caspase3、Bcl-xl的表达变化以及与脊髓神经细胞凋亡的关系。为进一步研究坐标神经损伤后的神经再生提供依据。方法：成年Wistar大鼠96只,随机分为对照组（12只）和实验组（84只）,实验组大鼠切除右侧长约6mm的坐骨神经,分为伤后3d和1,2,3,4,5,6周组,采用Caspase3、Bcl-xl免疫组化检测大鼠脊髓组织Caspase3、Bcl-xl表达变化,测定Caspase3的活性;采用流式细胞仪AnnexinⅤ/PI双标检测和原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法（TUNEL）检测脊髓细胞凋亡情况。结果Caspase3、Bcl-xl免疫组化阳性反应主要位于灰质神经元细胞,高表达区在前角。伤后1周实验组伤侧脊髓组织中Caspase3表达轻度升高,2－4周显著升高,5周后下降,伤后1－3周脊髓Bcl-xl表达减弱,4周后明显升高,高水平持续到伤后6周;伤后2－4周在鼠伤侧脊髓有较多TNEL阳性标记细胞;伤后3d伤侧Caspase3活性荧光值开始升高,1－3周明显升高,4周逐渐下降;流式细胞仪检测,伤后1周伤侧脊髓凋亡细胞开始增加,2,3周为高峰,4周伤侧凋亡细胞逐渐减少。实验组末伤侧与对照组比较,各项检查差异无显著性意义。结论：Caspase3的活化是细胞凋亡的早期生化。与细胞凋亡的形态学表现相互关系;Bcl-xl对减轻大鼠坐骨神经损伤后脊髓组织细胞凋亡有重要作用,及早识别细胞凋亡的早期特征,对促进神经再生具有重要意义。     

不同年龄大鼠坐骨神经损伤后睫状神经营养因子对脊髓组织中Caspase3表达的影响

目的　观察睫状神经营养因子 (CNTF)对天冬氨酸特异酶切的半胱氨酸蛋白酶 (Caspase3)在坐骨神经损伤后不同年龄大鼠脊髓组织中表达的影响。方法　幼年、成年、老年Wistar大鼠各 2 70只 ,随机分为正常组 (1 8只 )、CNTF组 (1 2 6只 )和生理盐水组 (1 2 6只 )。CNTF组和生理盐水组切出长 2mm右侧坐骨神经 ,用硅胶管连接近、远侧神经残端制作神经再生小室 ,CNTF组再生小室内注入 2 5 μg/ml重组CNTF1 5 μl,生理盐水组再生小室内注入生理盐水 1 5 μl,CNTF组和生理盐水组分为术后 1、3天和 1、2、4、8、1 2周组。利用免疫组织化学、Westernblotting检测脊髓Caspase3的分布与表达强度变化 ,进行Caspase3活性测定。结果　损伤后各年龄组伤侧脊髓Caspase3蛋白表达增强 (P <0 .0 5或 0 .0 1 ) ,以前角运动神经元变化最为明显 ,幼年、成年、老年CNTF组Caspase3表达较生理盐水组减弱 (P <0 .0 5或 0 .0 1 ) ;各组对侧未伤侧与正常组比较 ,差异无显著性意义 (P >0 .0 5 )。幼年、老年及成年生理盐水组于损伤侧脊髓Caspase3活性升高 ,各时相点幼年大鼠Caspase3活性高于老年及成年 (P <0 .0 5或 0 .0 1 ) ,幼年、成年、老年CNTF组Caspase3活性低于生理盐水组 (P <0 .0 5或 0 .0 1 )。生理盐水组及CNTF组对侧未伤侧Ca     

高压氧对大鼠脊髓损伤后Fas/Fas1及Caspase-3表达与细胞凋亡的影响

目的 观察高压氧对大鼠脊髓损伤后Fas/Fas1、Caspase-3和凋亡细胞的影响,探讨高压氧治疗脊髓损伤的机制.方法 按照Allen's打击法制备SD大鼠脊髓损伤模型,将20只大鼠损伤后随机分为高压氧组、常氧高压氮组、常压吸氧组和常压空气组,于损伤后6 h开始治疗;1、2、 3、 7、和11 d取材.采用凋亡细胞检测法检测凋亡细胞,免疫组化法检测Fas/Fas1和Caspase-3表达,光镜下观察并计数分析.结果 Fas/Fas1、Caspase-3和凋亡细胞于损伤后1 d即升高,Fas/Fas1和Caspase-3于伤后2 d即达到高峰,细胞凋亡高峰出现在第3天,高压氧组与各组比较,峰值降低且差异有统计学意义(P<0.01).结论 高压氧对大鼠继发性脊髓损伤有保护作用,其作用与下调Fas/Fas1的表达,减少Caspase-3,从而抑制细胞凋亡有关.     

脊髓牵拉损伤后七叶皂苷钠对细胞凋亡的抑制作用

目的观察脊髓牵拉损伤后七叶皂苷钠对细胞凋亡的抑制作用。方法实验组大鼠脊髓损伤前尾静脉推注七叶皂苷钠6 mg/kg。将T10脊髓水平方向牵拉1.7 mm,持续5 m in。检测神经细胞bc l-2、bax表达;原位末端脱氧核糖核苷酸转移酶介导dUTP标记技术观察凋亡细胞。结果(1)bc l-2:实验组7 d达高峰为(43.61±5.89)%;对照组7 d时为(26.85±4.61)%,差异具有统计学意义(P<0.01)。(2)bax:实验组3 d达高峰为(36.19±5.42)%;对照组3 d时为(44.15±6.81)%,差异具有统计学意义(P<0.01)。(3)各时间点实验组细胞凋亡指数较对照组低。结论脊髓损伤前应用七叶皂苷钠能抑制因脊髓牵拉损伤引起的神经细胞凋亡。     

雌激素影响急性大鼠脊髓损伤后细胞凋亡的相关性实验研究

目的在建立大鼠脊髓损伤模型基础上,观察不同时间点雌激素对脊髓胶质细胞,神经元凋亡的影响,以期为临床急性脊髓损伤的治疗提供理论依据。方法健康成年大鼠72只,随机分为两组,其中单纯损伤组（单纯打击损伤）36只,雌激素组（手术加雌激素）36只。脊髓损伤动物模型的制备：用自制Allen打击器25gcm致伤力撞击脊髓,制成脊髓损伤动物模型。雌激素组每日肌注雌激素（100μg／kg）直至处死,单纯损伤组仍每日肌注生理盐水0．5ml直至处死。组织切片的制备：脊髓损伤后1d、3d、5d、8d、14d、21d共6个时间段分批处死动物。4％多聚甲醛心肌灌注固定24h,切取每只大鼠T5～T13节段脊髓组织,常规制成切片,给于Bcl－2检测,TUNEL原位末端标记,检测大鼠脊髓损伤后细胞凋亡的情况。脊髓损伤后2周给予Gale评分及斜板维持试验。结果Bcl－2蛋白检测：在脊髓损伤后的第1天,脊髓组织即开始较高表达Bel－2蛋白。单纯损伤组Bcl－2高峰发生在损伤后3天,而雌激素组伤后8天达到高峰,此时Bcl－2蛋白不仅表达在神经细胞中,更多的在胶质细胞中大量表达,且此状态一直维持到伤后14天才开始下降,伤后21天仅少量表达（P〈0．05）。TUNEL原位标记检测：单纯损伤组24小时已出现不少阳性细胞,以胶质细胞为主．3～8天达到高峰,此后逐渐回落,但21天时仍有阳性细胞,应用雌激素治疗后,凋亡细胞明显减少（P〈0．05）。脊髓损伤后2周Gale评分及斜板维持率雌激素组优于单纯损伤组（P〈0．01）。结论雌激素在脊髓损伤中通过改善微循环,促进Bcl－2蛋白的表达,清除氧自由基,抗氧化作用能够抑制脊髓损伤早期神经细胞和胶质细胞的凋亡,从而减轻脊髓继发性损伤．促进脊髓神经功能的恢复。所以脊髓损伤后早期应用雌激素配合其它药物阻止神经细胞和胶质细胞的凋亡可能有助于减轻脊髓损伤的损害程度,促进脊髓神经功能的恢复,为SCI的治疗提供一个新的途径。     

大鼠脑胶质瘤伽玛刀治疗对细胞凋亡和Caspase-3、Fas表达的影响

目的　研究大鼠脑胶质瘤伽玛刀治疗后肿瘤细胞凋亡的时程变化和Caspase 3、Fas的表达。方法　立体定向法制作C6大鼠脑胶质瘤模型 6 0只 ,分为治疗组和对照组 ,治疗组于肿瘤接种后 14d行伽玛刀治疗 ,治疗后不同时间点行流式细胞学检查测定肿瘤细胞的凋亡率以及Caspase 3和Fas基因蛋白表达情况 ,分析凋亡率与两种蛋白的相关性。结果　与对照组相比 ,治疗组肿瘤细胞的凋亡率明显增高 (P <0 0 5 ) ,治疗后 4 8h肿瘤细胞凋亡率达高峰 ,然后逐渐下降 ;Caspase 3和Fas的表达情况与凋亡率的变化呈正相关 (r1 =0 92 8,r2 =0 916 )。结论　肿瘤细胞的凋亡为伽玛刀治疗胶质瘤疗效之一 ,Caspase 3和Fas基因可能参与和调节伽玛刀照射后肿瘤细胞凋亡。     

虎纹捕鸟蛛毒素-Ⅰ对全脑缺血再灌损伤大鼠海马组织中TNF凋亡通路的影响

目的探讨注射虎纹捕鸟蛛毒素-Ⅰ(HWTX-Ⅰ)对全脑缺血模型大鼠神经保护作用的可能分子机制.方法48只SD大鼠随机分为假手术组、非用药组和用药组,采用改良的Pulsinelli大鼠四血管阻断全脑缺血结合蛛网膜下腔置管模型,应用光镜、免疫组化方法观察各组大鼠海马CA1区锥体细胞Nissl染色形态学变化及海马组织中肿瘤坏死因子α(TNFα)、肿瘤坏死因子受体Ⅰ(TNFRI)、TNF相关死亡结构域(TRADD)、Fas相关死亡结构域(FADD)、Caspase8等TNF凋亡通路相关因子蛋白表达水平的变化.结果(1)用药组大鼠锥体神经元排列较整齐,略为疏散分布,而非用药组大鼠锥体神经元排列散乱,层次不完整,稀疏分布;(2)TNFα、TNFRI、TRADD、FADD、Caspase8阳性单位的表达值以非用药组最高,用药组次之,假手术组表达最低.结论HWTX-Ⅰ能明显减轻全脑缺血再灌损伤大鼠海马锥体细胞的损伤,并降低大鼠海马组织TNFα、TNFRI、TRADD、FADD、Caspase8蛋白水平表达,表明HWTX-Ⅰ对全脑缺血再灌损伤大鼠海马神经细胞凋亡具有抑制作用,在一定程度上对全脑缺血再灌损伤有神经细胞保护作用.     

垂体腺苷酸环化酶激活肽对大鼠创伤性脑损伤后神经细胞凋亡的影响

目的观察大鼠创伤性颅脑损伤(TBI)后神经细胞凋亡现象。应用垂体腺苷酸环化酶激活肽(PACAP)观察其对损伤后大鼠神经细胞凋亡的影响。方法采用TBI模型,运用原位末端标记(Tunel)技术,观察大鼠中度脑损伤后2小时～3天伤侧大脑皮层、海马神经细胞凋亡情况。结果(1)Tunel染色:伤侧大脑半球广泛存在细胞凋亡,以受伤区周缘为甚,伤后2小时即可见凋亡细胞,2～3天达高峰。(2)PACAP治疗后12小时～3天,伤侧皮层、海马区细胞凋亡数与损伤组比较明显减少(P<0.05)。结论大鼠TBI后,神经元在发生变性、坏死的同时,存在凋亡现象。PACAP能阻滞大鼠TBI后神经细胞的凋亡,具有脑保护作用。     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

Bronchial stump fistula :treatment with covered retrievable hinged metallic stents-preliminary clinical experience

Objective To evaluate the preliminaily clinical efficacy and retrievability of a retrievable hinged covered metallic stent in the treatment of the bronchial stump fistula （BSF）. Methods Between April 2003 and March 2005, 8 patients with bronchial stump fistula after pneumonectomy or lobectomy were treated with two types （A and B） of retrievable hinged covered metallic stents. Type A stent was placed in 6 patients and type B in 2 under fluoroscopic guidance. The stent was removed with a retrieval set when BSF was healed or complications occurred. Results Stent placement in the bronchial tree was technically successful in all patients, without procedure-related complications. Immediate closure of the BSF was achieved in all patients after the procedure. Stents were removed from all patients but one. Removal of the stents was difficult in two patients due to tissue hyperplasia. Patients were followed up for 6 - 21 months. Placement of the stents remained stable in all patients except one due to severe cough. Permanent closure of BSF was achieved in 7 （87.5%） of 8 patients. Conclusion Use of a retrievable hinged covered expandable metallic stent is a simple, safe, and effective procedure for closure of the BSF. Retrieval of the stent seems to be feasible. （J Intervent Radiol, 2007, 16： 253-257）     

Prevalence and severity of hip and groin pain in sub‐elite male football: a cross‐sectional cohort study of 695 players

The purpose of this study was twofold: (a) to investigate the prevalence of hip and groin pain in sub‐elite male adult football in Denmark and (b) to explore the association between prevalence and duration of hip and groin pain in the previous season with the Copenhagen Hip and Groin Outcome Score (HAGOS) in the beginning of the new season. In total 695 respondents from 40 teams (Division 1–4) were included. Players completed in the beginning of the new season (July–Sept 2011) a self‐reported paper questionnaire on hip and/or groin pain during the previous season and HAGOS. In total 49% (95% CI: 45–52%) reported hip and/or groin pain during the previous season. Of these, 31% (95% CI: 26–36%) reported pain for >6 weeks. Players with the longest duration of pain during the previous season had the lowest HAGOS scores, when assessed at the beginning of the new season, P < 0.001. This study documents that half of sub‐elite male adult football players report pain in the hip and/or groin during a football season. The football players with the longest duration of pain in previous season displayed the lowest HAGOS scores in the beginning of the new season.