U0126调节体外培养神经细胞缺氧性损伤后凋亡相关蛋白的表达

目的研究体外培养的神经细胞缺血缺氧性损伤后凋亡相关蛋白Bcl-2和Bax的表达,及应用MAPKs信号通路阻断剂U0126对其表达的调节作用。方法取出生1d的SD大鼠,断头处死后迅速解剖分离其皮层组织进行培养,3d后进行缺血缺氧处理致细胞损伤。并分为加药组和缺氧组,前者给予U0126,后者给予空白的DMSO溶剂作为对照。采用MTT法、免疫细胞化学、免疫蛋白印迹(western blot)技术,检测在缺氧性损伤急性期神经细胞的活力、凋亡相关蛋白Bcl-2和Bax的表达情况,及给予U0126后对神经细胞的活力、凋亡相关蛋白Bcl-2和Bax表达的影响。结果缺氧损伤后Bcl-2和Bax表达升高,细胞活力不佳,死亡较多。给予U0126后ERK1/2和ELK1磷酸化水平降低,同时Bax的表达水平降低,Bcl-2表达增高,细胞的状态和活力亦明显改善。结论给予MAPKs阻断剂U0126可以调节缺氧损伤后凋亡相关蛋白Bcl-2和Bax的表达．抑制细胞死亡,改善细胞状态。     

U0126对缺血性脑损伤大鼠脑内水通道蛋白-4表达的影响

目的研究水通道蛋白-4(aquaporin4,AQP4)在缺血性脑损伤大鼠脑内的表达,及丝裂原活化蛋白激酶(MAPKs)信号转导通路抑制剂U0126对其表达的影响。方法用线栓法建立缺血性脑损伤大鼠模型,测定脑组织含水量及伊文斯蓝含量,并采用免疫组织化学、Westernblot和逆转录-聚合酶链反应技术,检测AQP4的表达。测定预先经侧脑室给予U0126后MAPKs信号通路关键蛋白ERK1/2和ELK1磷酸化水平,同时观察U0126对脑水肿和AQP4表达的影响。结果正常组AQP4表达较低[蛋白(吸光度值,下同):123·1±1·0,mRNA(吸光度比值,下同):0·173±0·017],在缺血损伤后表达升高(蛋白:153·6±0·8,mRNA:0·400±0·015),脑组织含水量及伊文斯蓝含量增加,给予U0126后AQP4的表达和脑组织含水量降低(蛋白:149·0±1·1,mRNA:0·328±0·010,P<0·01),同时ERK1/2和ELK1磷酸化水平降低。结论缺血性损伤后AQP4表达上升,脑水肿明显,预先给予U0126可抑制AQP4的表达,减轻脑水肿。     

ERK1/2信号途径对大鼠弥漫性脑创伤后神经细胞凋亡的调控机制

目的 探讨脑创伤后ERK1/2信号调控神经细胞凋亡的分子机制.方法 SD大鼠分为正常对照组、模型组、抑制剂U0126高、低剂量组.Marmarou's法制作弥漫性脑创伤模型.光镜下观察伤后神经细胞形态变化;免疫组化和Western Blot法检测伤后磷酸化ERK1/2水平和Bax表达;TUNEL法检测凋亡细胞.结果 与正常对照组比较,模型组海马区部分神经细胞出现变性坏死和凋亡改变,磷酸化ERK1/2、Bax表达增高,神经细胞凋亡数目增多(P<0.05);U0126治疗后,脑组织形态损伤程度、磷酸化ERK1/2和Bax表达、神经细胞凋亡数目回降,上述变化在U0126高剂量组中更为显著.结论 脑创伤后活化的ERK1/2信号通过调控Bax表达在神经细胞凋亡过程中发挥重要作用.     

细胞外信号调节激酶在蛛网膜下腔出血损伤中的作用及意义

目的研究细胞外信号调节激酶(ERK)途径在蛛网膜下腔出血(SAH)脑损伤中的作用。方法ICR小鼠随机分为假手术组、模型组、U0126干预组,采用非开颅血管内穿线法制备小鼠SAH模型,给予ERK抑制剂U0126,术后行颅底检查,分别在SAH后12、24、48h 3个时相点取右侧大脑动脉标本,在光镜下观察大脑中动脉病理变化,应用免疫印迹法检测各组p-ERK1/2、caspase-8蛋白表达,TUNEL法检测大脑中动脉内皮细胞凋亡。结果随损伤时间延长,模型组小鼠p-ERK1/2、caspase-8蛋白均有不同程度增强,凋亡细胞增多。与模型组比较,U0126干预组小鼠各时相点3项指标的表达均不同程度下调。结论 ERK信号途径参与小鼠SAH病理损伤过程,并在神经细胞凋亡进程中发挥关键作用。     

细胞外信号调节激酶的磷酸化水平对星形胶质细胞增殖及其细胞周期的影响

目的 探讨细胞外信号调节激酶(ERK1/2)的磷酸化水平对体外星形胶质细胞(Ast)增殖及其细胞周期的影响.方法 将培养成熟的大鼠原代Ast分为两组,其中一组用ERK1/2磷酸化的特异性抑制剂U0126进行处理(U0126组),另一组不做处理,作为对照组.通过Western Blot技术、Click-iT Edu技术和流式细胞术分别检测两组Ast中ERK1/2磷酸化水平、Ast增殖细胞百分比及各期细胞百分比.结果 U0126组ERK1/2的磷酸化水平(39.13％±6.71％)明显低于对照组(100％).U0126处理24 h后,U0126组Ast增殖细胞百分比[(2.63±1.14)％]低于对照组[(21.43±3.81)％](t=21.13,P＜0.01).G1期U0126组Ast[(93.67±0.68)％]高于对照组[(84.63±1.00)％](t=12.91,P＜0.01);S期U0126组Ast[(2.90±0.23)％]低于对照组[(14.21±1.14)％] (t=16.87,P＜0.01);G2期U0126组Ast[(3.43±0.88)％]高于对照组[(2.08±0.21％)％](t=4.35,P＜0.05).结论 降低ERK1/2的磷酸化水平可抑制Ast的增殖,并将其阻断在G1期,抑制Ast从G1期向S期的转化,同时抑制其分裂将其阻断在G2期.     

骨髓间充质干细胞旁分泌作用与脑缺血后的细胞凋亡*

背景：骨髓间充质干细胞移植治疗脑缺血的机制之一是骨髓间充质干细胞的旁分泌作用,而目前对于这一机制的研究报道较少。 目的：观察骨髓间充质干细胞旁分泌作用对脑缺血后细胞凋亡的抑制作用并探索相关机制。 方法：体外培养大鼠骨髓间充质干细胞,建立大鼠大脑中动脉缺血模型。24只SD大鼠随机数字表法分为4组,每组6只。细胞移植给药组：大鼠纹状体内移植骨髓间充质干细胞后给予ERK1/2抑制剂U0126;非移植给药组：注射等量的PBS后给予U0126;细胞移植对照组：移植骨髓间充质干细胞后给予溶剂对照;非移植对照组：注射等量的PBS后给予溶剂对照。7 d后通过Western blot检测血管内皮细胞生长因子、磷酸化ERK1/2蛋白的表达;TUNEL染色检测梗死区周围及皮质区细胞凋亡情况。 结果与结论：细胞移植组较非移植组大鼠纹状体内血管内皮细胞生长因子蛋白的表达明显增高,磷酸化ERK1/2表达增强,细胞凋亡数明显减少;经U0126处理后,血管内皮细胞生长因子的表达没有变化,而随着磷酸化ERK1/2的表达受到抑制,细胞凋亡数明显增高。提示骨髓间充质干细胞在大脑纹状体内可以旁分泌血管内皮细胞生长因子,并通过激活ERK1/2抑制了脑梗死区细胞的凋亡。     

细胞外信号调节激酶对大鼠局灶性脑缺血后细胞周期调控的影响

目的 研究脑缺血后细胞外信号调节激酶(ERKs)对细胞周期调控的影响.方法 建立光化学法诱导大鼠局灶性脑缺血模型,分为脑缺血组(对照组及治疗组)和假手术组.治疗组于缺血前30min尾静脉注入U0126溶液,对照组尾静脉注入相同体积不含U0126的DMSO稀释溶液.应用免疫组织荧光化学法观察细胞周期蛋白D1(CyclinD1)和细胞周期蛋白E(CyclinE)阳性细胞表达:免疫印迹(Weaem blot)检测磷酸化ERK1/2(pERK1/2)、CyclinD1和CyclinE的蛋白表达;半定量逆转录-聚合酶链反应(PT-PCR)观察转录因子E2F mRNA的表达.结果 治疗组CyclinD1和CyclinE阳性表达的细胞数较对照组显著减少(P<0.05);缺血对照组pERK1/2蛋白表达显著强于治疗组(P<0.05),4h时间点表达最为明显,12h时间点恢复到基线水平,CyclinD1和CyelinE表达6h开始升高,12h表达最为明显,治疗组较对照组显著减弱(P<0.05);缺血对照组E2F mRNA的表达显著强于治疗组和假手术组(P<0.05),以7d表达最为明显.结论 ERKs在大鼠脑缺血中发挥重要作用,抑制脑缺血引起的ERK1/2磷酸化,可降低细胞周期蛋白CyelinD1、CyclinE和E2F的表达.即ERKs可影响细胞周期的调控.     

促红细胞生成素抑制大鼠创伤性脑水肿形成

目的探讨促红细胞生成素(erythropoietin,EPO)对大鼠创伤性脑水肿的影响及其的潜在分子机制。方法取SD大鼠90只,随机分为假手术组,创伤组和EPO组。创伤组:制作改进式Feeney's脑创伤模型;EPO组:伤后给大鼠腹腔注射重组人促红细胞生成素(5000 IU/kg)。伤后24 h,72 h和120 h,使用平衡木法评定各组大鼠行为学评分。伤后72 h时,检测各组大鼠脑含水量,脑组织胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)的磷酸化水平、水通道蛋白4(aquaporin 4,AQP4)mRNA和蛋白表达水平。结果在伤后各时间点,EPO组大鼠神经功能障碍的行为学评分也较创伤组有明显降低(均P<0.05)。伤后脑组织含水量由假手术组的78.76%±0.65%上升至创伤组的81.26%±0.40%(P<0.01),EPO组脑含水量则降低至79.71%±0.59%(与创伤组比较P<0.01)。与假手术组比较,创伤组ERK磷酸化的水平在伤后72 h明显上升(P<0.01),EPO组伤后ERK磷酸化水平则明显低于创伤组(0.369±0.046 vs.0.815±0.127,P<0.01);AQP4 mRNA和蛋白在伤后的表达水平均较假手术组明显增高(均P<0.01),EPO组AQP4 mRNA和蛋白的表达水平较创伤组均显著下降(均P<0.01)。结论EPO可抑制大鼠脑创伤后ERK信号通路的过度激活及下游AQP4的过表达,减轻大鼠的创伤性脑水肿。     

大鼠蛛网膜下腔出血后细胞外调节蛋白激酶1/2激活介导海马区神经细胞自噬

目的探讨大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后海马区细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK1/2)激活与神经细胞自噬的关系。方法 120只雄性SD大鼠随机分成假手术组、SAH组、ERK1/2抑制剂U0126组、自噬诱导剂雷帕霉素(rapamycin,Rap)组。采用枕大池二次注血法制作SAH大鼠模型;U0126组和Rap组分别于造模前30min侧脑室注射U0126(5μg/μL)和Rap(10nmol/μL)。光镜观察海马区神经细胞形态结构;免疫组化法和实时荧光定量PCR法检测海马区磷酸化ERK1/2(p-ERK1/2)、ERK1/2 mRNA和自噬标志蛋白(Beclin-1和Beclin-1mRNA、LC3-Ⅱ和LC3mRNA)表达水平。结果与假手术组比较,SAH组神经细胞死亡率增加(14.9%±5.7%,28.3%±9.8%,44.2%±10.9%)(q值依次为27.56、35.65、44.81;均P0.05),ERK1/2 mRNA、Beclin-1 mRNA和LC3 mRNA水平增加(1.83±0.01,2.82±0.06,1.34±0.04;1.46±0.02,1.76±0.02,1.35±0.02;1.52±0.04,1.89±0.01,1.31±0.04)(q值依次为42.99、60.66、48.08,71.26、72.46、49.50,48.49、82.40、41.18;均P0.05),p-ERK1/2、Beclin-1和LC3–Ⅱ蛋白水平增加(均P0.05);与SAH组比较,U0126组神经细胞死亡率增加(19.6±6.5%,36.2±7.7%,58.2±12.7%)(q值依次为9.59、10.43、14.66;均P0.05),U0126组ERK1/2 mRNA、Beclin-1 mRNA和LC3 mRNA表达降低(1.23±0.02,1.40±0.02,1.12±0.02;1.22±0.04,1.48±0.06,1.24±0.03;1.34±0.04,1.33±0.02,1.14±0.04)(q值依次为75.66、65.35、31.11,37.18、26.70、15.56,16.79、51.85、22.58;P0.05),p-ERK1/2、Beclin-1和LC3–Ⅱ蛋白水平降低(P0.05);与SAH组比较,Rap组神经细胞死亡率降低(9.1%±4.6%,18.8%±8.6%,28.21%±9.2%)(q值依次为11.86、12.54、16.74;均P0.05),Rap组Beclin-1mRNA和LC3mRNA增加(1.78±0.02,2.27±0.05,1.86±0.04;1.97±0.06,2.31±0.08,1.85±0.00)(q值依次为49.57、48.63、72.12、41.96、38.88、71.73;P0.05),Beclin-1和LC3-Ⅱ蛋白增加(P0.05),ERK1/2 mRNA变化差异无统计学意义(q值依次为2.63、2.65、2.83,P0.05),p-ERK1/2蛋白变化差异无统计学意义(P0.05)。结论 SAH后激活的ERK1/2激活,可促进Beclin-1和LC3-Ⅱ表达介导神经细胞丢失。     

星形胶质细胞AQP4蛋白在缺氧/复氧条件下表达变化的研究

目的观察缺氧/复氧条件下星形胶质细胞形态和AQP4蛋白的表达变化以及葛根素对其表达变化的影响,探讨脑缺血再灌注损伤与AQP4的关系以及葛根素的干预作用。方法原代培养星形胶质细胞,用5%CO2+95%N2混合气体造成缺氧,以LDH漏出率及MTT降解率作为细胞受损指标,应用Western blot技术检验星形胶质细胞缺氧/复氧各个时间点AQP4蛋白的表达变化及葛根素的干预效果。结果体外培养的星形胶质细胞在缺氧环境下损伤不明显,随着复氧时间的延长细胞损害加重。AQP4蛋白在缺氧时表达与正常对照组无明显差异,复氧后表达升高并随时间延长呈增高趋势(P0.05)。葛根素干预组AQP4蛋白表达丰度与缺氧/复氧组无明显差异(P0.05)。结论星形胶质细胞AQP4蛋白表达变化与细胞损伤有明显相关性,葛根素对星形胶质细胞损伤的保护作用不是通过改变AQP4的表达来实现的。     

Monitoring Biomarkers of Cellular Injury and Death in Acute Brain Injury

Background

Extension of hemorrhage into the subarachnoid space in primary intracerebral hemorrhage (ICH) has recently been associated with poor outcomes, although the mechanisms underlying that association are uncertain. The objectives of this study are to confirm the association between fever and poor outcomes after ICH, and to determine whether subarachnoid hemorrhage extension (SAHE) is associated with fevers.

Methods

Patients with primary ICH were enrolled into a prospective registry between December 2006 and July 2012. SAHE was identified on imaging by blinded expert reviewers. Patient temperature was recorded hourly, and we defined febrile as any recorded temperature >38 °C within the first 14 days. Regression models were developed to test whether fever was associated with poor outcome and whether the occurrence of SAHE was a predictor of fever.

Results

Of the 235 patients studied, 39.7 % had SAHE and 58 % had fever. Fever was associated with higher modified Rankin scores at 3 months (odds ratio, OR 1.8 [1.04–3.12], p = 0.04) after adjustment for ICH score. SAHE was a predictor of fevers (OR 1.82 [95 % confidence interval 1.02–3.24], p = 0.04) after adjustment for ICH score, and remained significant after adjustment for other confounders like pneumonia identified in the univariate analysis.

Conclusions

Our data confirm the deleterious effect of fever on the outcome of patients with ICH and show that SAHE is an independent predictor of fever after ICH. SAHE may provoke dysfunctional thermoregulation similar to what is observed after aneurysmal subarachnoid hemorrhage, creating mechanistic pathway between SAHE and poor functional outcomes.     

Spinal Cord Injury and Traumatic Brain Injury: A Cost-of-Illness Study

Background: Among traumatic injuries, spinal cord injuries (SCI) and traumatic brain injuries (TBI) are of major importance because of their epidemiological and economic impact on society. The overall objective of this study was to estimate the economic cost associated with people with SCI and TBI in Spain in 2007. Methods: A cost-of-illness analysis was performed, considering the perspective of society, using a 1-year time horizon. Medical costs, adaptation costs, material costs, administrative costs, and costs of police, firefighters and roadside assistance, productivity losses due to institutionalization and sick leave, as well as an estimate of productivity losses of carers, and productivity losses due to death were included. Results: The economic cost associated with people with SCI is between EUR 92,087,080.97 and 212,496,196.41 (USD 131 million and 302 million) according to the injury mechanism, and between EUR 1,079,223,688.66 and 3,833,752,692.78 (USD 1,536 million and 5,458 million) for people with TBI. Conclusions: There is an urgent need to develop effective interventions known to prevent SCI and TBI, and to evaluate their effectiveness and efficiency.     

Duty to Avoid Injury to Oneself and Thereby Psychiatric Injury to Others

Tortious liability for causing foreseeable mental harm to others is a complex category of tort law. It continues to evolve as plaintiffs devise novel arguments to test the parameters of what used to be known as pure psychiatric injury law. In Homsi v Homsi [2016] VSC 354, J Forrest J was called upon to rule on whether a mother could sue her deceased son for driving negligently and causing his own death and thereby causing her mental harm. Forrest J declined to permit recovery but in the course of his judgement helpfully analysed the current state of mental harm law in Australia, reviewed the circumstances in which relatives are able to sue one another for mental harm caused by self-injury, and articulated a range of policy considerations in relation to indeterminate liability for defendants. His judgement provides an important opportunity to reflect on the directions in which mental harm law should evolve to arrive at a rapprochement between plaintiffs’ reasonable aspirations for recovery and ensuring that the liability of defendants is not unreasonably wide.     

Acute Kidney Injury in Patients with Severe Traumatic Brain Injury: Implementation of the Acute Kidney Injury Network Stage System

Background  

There is limited information on the incidence and effect of acute kidney injury (AKI) in patients with severe traumatic brain injury (TBI), although AKI may affect outcome. Recently, acute kidney injury network (AKIN) classification has been widely accepted as a consensus definition for AKI. The aim of this study is to estimate the frequency and level of severity of AKI in patients with severe TBI by using AKIN criteria and to study whether AKI affects outcome.     

Head Injury during Childbirth

Head injuries are the most common type of birth injuries. Among them, most of the injuries is limited to the scalp. and the prognosis is good enough to be unnoticed in some cases. Intracranial injuries caused by excessive forces during delivery are rare. However, since some of them can be fatal, it is necessary to suspect it at an early stage and evaluate thoroughly if there are abnormal findings in the patient.     

Brain Injury Biomarkers as Outcome Predictors in Pediatric Severe Traumatic Brain Injury

Background

To systematically review the literature on brain injury biomarkers, defined as any injury biomarker detected in cerebrospinal fluid (CSF) or blood injury biomarkers primarily expressed in the brain parenchyma, to determine outcome prediction in pediatric severe traumatic brain injury (sTBI).

Methods

A search of MEDLINE^®, EMBASE^®, PsycINFO^®, Pubmed^®, and the Cochrane Database, as well as grey literature sources, personal contacts, hand searches, and reference lists. The search terms used were traumatic brain injury, biomarkers, prognosis, and children. No language, publication type, or publication date restrictions were imposed. All articles were critically reviewed by two clinicians independently.

Results

A total of 7,150 articles were identified initially with 16 studies identified for review. Eighteen different biomarkers were examined; 11 in CSF and 7 in blood. Outcomes assessed included either in-hospital mortality or functional state (hospital discharge, 3-months or 6-months; Glasgow Outcome Scale or Pediatric Cerebral Performance Category). Significant correlations were established between sTBI outcomes and various biomarkers in CSF (IL-6, IL-8, IL-1β, S100β, NGF, NSE, DCX, ET-1, HMGB-1, cytochrome C) and blood (GFAP, NF-H, UCH-L1, SBDP-145, leptin). Mixed results were obtained for blood S100β. Outcome did not correlate with several biomarkers in either CSF (BDNF, GDNF, α-Syn) or blood (NSE, MBP). The Class of Evidence was considered II in 1 study and III in the remaining 15 studies.

Conclusions

Based on the status of current sTBI biomarker research, we recommend that future research should be directed at both novel biomarker discovery and validation of biomarker panels in large, well-designed longitudinal studies.