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。     

不同时间点应用甲氨蝶呤对大鼠脊髓损伤后神经细胞凋亡的影响

目的探讨不同时间点应用低剂量甲氨蝶呤(methotrexate,MTX)对大鼠脊髓损伤(spinal cord injury,SCI)后神经细胞凋亡的作用,探讨其潜在的神经保护机制与合适的给药时机。方法取成年雄性SD大鼠120只,体质量247~286 g,随机分为4组(n=30),分别为假手术组(A组)、对照组(B组)、MTX治疗组(C组)和MTX预防组(D组)。A组仅行椎板切除,B、C、D组采用改良Allen法制备SCI模型;C组于术后1、6、12、18、24 h,D组于术前30 min及术后6、12、18、24 h,经尾静脉注射MTX(0.5 mg/kg);A、B组于术后1、6、12、18、24 h注射等量生理盐水。术后观察大鼠一般情况,于1、3、7、14、21 d采用BBB评分进行神经功能评估;取材行组织学观察脊髓形态学变化,免疫组织化学染色观察半胱氨酸天冬氨酸蛋白酶3(Caspase-3)表达,TUNEL标记观察细胞凋亡水平。结果 B、C、D组实验过程中共死亡10只大鼠,最终各组纳入25只大鼠进行观察。各时间点A组BBB评分均高于B、C、D组(P0.05),从3 d开始C、D组评分明显高于B组(P0.05);D组从3 d开始均高于C组,除21 d(P0.05)外,其余各时间点比较差异均有统计学意义(P0.05)。组织学观察示,A组术后各时间点脊髓组织为正常结构;D组SCI程度轻于B、C组,C组轻于B组;从14 d开始,B、C、D组病变范围基本固定。各时间点B、C、D组Caspase-3及TUNEL阳性细胞数均显著多于A组,B组多于C、D组,比较差异有统计学意义(P0.05);C组Caspase-3阳性细胞数均多于D组,其中3、7、14 d比较差异有统计学意义(P0.05),而TUNEL阳性细胞数仅3、7 d显著多于D组(P0.05)。术后1、3、7、14、21 d,B、C、D组组内Caspase-3、TUNEL阳性细胞数均成正相关(P0.05)。结论低剂量MTX能够通过抑制神经细胞凋亡有效阻止SCI的继发性损伤;预防性应用MTX效果优于单纯治疗性应用。     

大鼠脊髓损伤后不同时期移植人脐带间充质干细胞的修复效果观察

目的:观察大鼠脊髓损伤后不同时期局部移植人脐带间充质干细胞(human umbilical cord mesenchy-mal stem cells,hUCMSCs)修复脊髓损伤的效果,探讨hUCMSCs局部移植治疗脊髓损伤(spianl cord injury,SCI)的最佳移植时机。方法:雌性Wistar大鼠80只,随机均分为A、B、C、D组,以Impactor Model-Ⅱ打击器制备脊髓胸10(T10)损伤模型。A组为单纯损伤组,B组损伤后3d移植hUCMSCs,C组损伤后1w移植hUCM-SCs,D组损伤后3w移植hUCMSCs,分别于造模后24h、移植前1d及移植后8w(A组与D组损伤后相应时间点相同)每周对各组大鼠后肢功能进行行为学评分(BBB评分);移植后8周免疫组化染色观察移植细胞的存活、分化和血管再生情况,10周时用生物素葡聚糖胺(biotin dextran amine,BDA)示踪皮质脊髓束(corticospinaltract,CST)观察轴突的再生情况。结果:SCI后24h各组大鼠BBB评分无显著性差异(P0.05),D组在移植前和移植后1周与A组相应时间点的BBB评分无显著性差异(P0.05),移植后2w起各时间点评分均明显高于A组相应时间点,差异有统计学意义(P0.05);A组在SCI后9w时到达平台期,与实验结束时相比差异无统计学意义(P0.05)。移植各组移植后BBB评分逐渐上升,后一个时间点与同组前一时间点比较差异有显著性意义(P0.05),移植后6w开始C组明显高于B、D组(P0.05)。免疫组化染色A组脊髓中未见Brdu阳性细胞,损伤区血管形态欠完整,未见新生血管;损伤周围星形胶质细胞大量增生,细胞肥大、突起增多、排列不规则,形成胶质瘢痕;未见BDA标记的皮质脊髓束通过损伤区。B、C、D组脊髓中均有Brdu阳性标记的细胞存活,C、D组中细胞数量明显多于B组,但三组中均未见到移植细胞向神经元或神经胶质样细胞分化;B、D组损伤周围的星形胶质细胞较A组减少,但排列仍欠规则;C组损伤周围星形胶质细胞形态趋于正常,排列规则,且有大量星形胶质细胞通过损伤区,血管再生数目及通过损伤区的皮质脊髓束神经纤维也明显多于B、D组。结论:大鼠SCI后局部移植的hUCMSCs能长期存活,抑制胶质瘢痕形成,改善后肢运动功能;SCI后1w移植的效果优于SCI后3d和3w时移植的效果。     

丙戊酸对大鼠急性脊髓损伤后神经功能恢复的影响及作用机制

目的:探讨丙戊酸(VPA)对大鼠脊髓损伤(SCI)后运动功能恢复的影响及作用机制。方法:60只雄性SD大鼠随机均分为3组:假手术组(C组)、损伤组(SCI组)和丙戊酸保护组(VPA组)。SCI组和VPA组采用改良Allen法制作大鼠T10 SCI模型。VPA组术后即刻及其后每12h皮下注射VPA 300mg/kg,至取材;C组和SCI组在相应时间点注射等体积的生理盐水。伤后6h,每组取5只大鼠处死取材,其余大鼠在伤后24h、48h和72h每组取5只先行后肢运动功能BBB评分,随后处死取材。切片后分别行HE染色观察脊髓组织病理变化,免疫荧光双标法在激光共聚焦显微镜下观察核因子κB(NF-κB)途径的激活状态,免疫组化法检测白介素1β(IL-1β)的表达。结果:C组大鼠各时间点BBB评分均为21分,VPA组和SCI组各时间点的评分均低于C组(P<0.05),但VPA组各时间点的评分均高于同时间点SCI组,在伤后48h和72h两组差异有显著性(P<0.05)。病理检查显示C组脊髓组织形态正常,VPA组和SCI组伤后6h损伤中央区即可见明显出血灶,灰质中神经元肿胀坏死,白质中神经纤维肿胀;伤后24h、48h出血灶界限更明显,并可见空洞形成和炎症细胞浸润;伤后72h上述病理变化仍明显;VPA组各时间点的病理变化与SCI组相似,但炎症细胞浸润减少。C组偶见或未见NF-κB核阳性细胞和IL-1β表达,与C组相比,SCI组和VPA组NF-κB核阳性细胞百分比和IL-1β表达量从伤后6h即显著性增高,24h达高峰,以后逐渐减少,72h仍显著性高于C组(P<0.05);VPA组各时间点NF-κB核阳性细胞百分比和IL-1β表达量均低于同时间点SCI组(P<0.05)。结论:VPA可促进大鼠SCI后运动神经功能恢复,其机制可能与抑制炎症反应有关。     

重组人红细胞生成素治疗大鼠急性脊髓损伤的用药时间窗探讨

目的:探讨腹腔注射重组人红细胞生成素(rHuEPO)治疗大鼠急性脊髓损伤(ASCI)的用药时间窗.方法:采用显微血管夹夹伤雌性SD大鼠T10脊髓建立脊髓急性损伤模型,将造模成功后的40只大鼠随机分为A、B、C、D、E组,每组8只.A、B、C、D组分别于损伤后即刻、1h、3h和6h腹腔注射rHuEPO 50001U/kg,E组损伤后立即腹腔注射等量生理盐水,作为对照组.各组大鼠于术后1d、3d、5d、7d进行BBB运动学评分,并于术后3d、7d用TUNEL染色和caspase-3免疫组化染色法检测各组大鼠脊髓神经细胞凋亡情况.结果:术后1d、3d各组大鼠BBB评分无统计学差异(P>0.05);5d时A、B、C三组BBB评分均高于D、E两组(P<0.01),A、B、C 三组间与D、E组间无统计学差异(P>0.05);7d时D组BBB评分高于E组(P<0.01),但低于A、B、C三组(P<0.01),而A、B、C三组间无统计学差异(P>0.05).损伤后3d、7d,A～D组的TUNEL、caspase-3染色阳性细胞均高于E组,且损伤后7d时D组TUNEL染色阳性细胞数高于A、B、C三组(P<0.05),A、B、C三组间无统计学差异;损伤后3d.D组caspase-3染色细胞阳性率高于A、B、C三组(P<0.05),A、B、C三组间无统计学差异(P>0.05),损伤后7d,A～D组间无统计学差异(P>0.05).结论:大鼠ASCI后3h内给予rHuEPO可以显著改善运动功能恢复情况,并抑制伤后脊髓神经细胞凋亡现象的发生;伤后6h给药效果较差.     

丙戊酸对大鼠急性脊髓损伤后氧化应激的影响

[目的]探讨丙戊酸(VPA)对大鼠脊髓损伤(SCI)后氧化应激的影响。[方法]72只雄性SD大鼠随机分为3组:假手术组(C组)、损伤组(SCI组)和丙戊酸保护组(VPA组)。采用改良的Allen法制作脊髓损伤动物模型。VPA组术后即刻及其后每12 h皮下注射VPA 300 mg/kg至取材;C组和SCI组在相应时间点注射等体积的生理盐水。大鼠在伤后24、48、72 h和1周先行后肢运动功能BBB评分,随后处死取材。通过石蜡切片HE染色观察脊髓组织病理变化,并用免疫组化法检测诱导型一氧化氮合酶(iNOS)的表达;通过化学比色法测定脊髓组织中丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的含量。[结果]BBB评分显示C组运动功能未受影响,VPA组的BBB评分均高于SCI组,两者相比在伤后48、72 h和1周差异有显著性(P<0.01)。HE染色示C组脊髓组织形态正常,VPA组各时间点的病理变化与SCI组相比没有明显改善。C组偶见或未见iNOS阳性表达细胞。与C组相比,SCI组和VPA组的iNOS表达均明显增加(P<0.05),在伤后72 h达高峰,但VPA组的iNOS表达在各时间点均明显低于SCI组(P<0.05)。SCI组和VPA组脊髓组织的MDA含量明显高于C组,而GSH-Px活性明显低于C组(P<0.05),VPA组和SCI组相比较,MDA含量在各时间点均明显下降,GSH-Px活性均明显升高(P<0.05)。[结论]VPA通过减轻SCI所诱导的氧化应激,从而对SCI发挥保护作用。     

罗西格列酮对脊髓损伤大鼠神经功能恢复的作用及机制

目的:观察罗西格列酮对脊髓损伤(SCI)大鼠后肢运动功能恢复的作用,探讨其作用机制。方法:75只成年SD大鼠,应用Allen改良法制作大鼠T10SCI模型,随机分为A、B、C三组,每组25只,B、C组于损伤后5min、6h、24h腹腔注射罗西格列酮,C组在腹腔注射罗西格列酮前1h给予G3335,A组于相应时间点腹腔注射等体积生理盐水作为对照组。每组取6只大鼠于伤后1d、7d、2w、4w、6w时对后肢运动功能进行BBB评分;伤后3d每组取4只动物脊髓组织行免疫组织化学染色法检测核转录因子κB(nuclear factor kappa-light-chain-enhancer of activated B cells,NF-κB)的表达;伤后1、3、5、7d和2w每组取3只应用Westernblot法检测脊髓组织中凋亡相关蛋白caspase-3和Bcl-2的表达。结果:伤后1d、7d时3组大鼠BBB评分均为0分,伤后2w开始B组BBB评分高于A组和C组,4w和6w时与A、C组比较有显著性差异(P0.05);伤后3d时三组NF-κB表达均为阳性,但B组平均光密度值明显低于A、C组(P0.05),B组与C组比较无显著性差异(P0.05);伤后各时间点B组caspase-3表达量均低于A组和C组(P0.05),而Bcl-2表达均高于A组和C组(P0.05),其差异均在伤后5d达到高峰,A组与C组同时间点比较无显著性差异(P0.05)。结论:罗西格列酮可促进SCI大鼠神经功能恢复,其机制可能与抑制炎症反应及细胞凋亡有关。     

粒细胞集落刺激因子动员BMSCs归巢治疗大鼠脊髓损伤的疗效观察

目的观察粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)动员BMSCs归巢对大鼠脊髓损伤的治疗效果,评估G-CSF动员BMSCs治疗脊髓损伤的可行性。方法将24只成年健康雌性SD大鼠术前12 h于鼠尾静脉注射绿色荧光蛋白(green fluorescence protein,GFP)标记的BMSCs(GFP-BMSCs),并随机分为假手术组(A组)、假手术+G-CSF组(B组)、脊髓损伤组(C组)、脊髓损伤+G-CSF组(D组),每组6只。C、D组采用T10水平脊髓半切法建立脊髓损伤模型,A、B组仅行椎板切除,不损伤脊髓;术后1 h B、D组分别注射G-CSF(10μg/kg·d),连续注射3 d;A、C组注射等量生理盐水。术后1、3、7、14、21、28 d采用BBB评分行大鼠双后肢神经功能评估,并采用ELISA法检测血清TNF-α和基质细胞衍生因子1(stromal cell-derived factor 1,SDF-1)表达。术后28 d处死大鼠取脊髓样品行免疫组织化学染色观察细胞因子SDF-1、BDNF、VEGF和TNF-α表达,免疫荧光染色观察GFP-BMSCs阳性细胞及双染荧光黄色的GFP/神经元核抗原(neuronal nuclei,NeuN)阳性神经元细胞和GFP/胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性神经胶质细胞数;并采用TUNEL法检测细胞凋亡。结果术后各时间点A、B组BBB评分较术前无明显变化;术后1 d,C、D组BBB评分降至最低,后逐渐上升。除术后1 d外,其余各时间点D组BBB评分均显著高于C组(P0.05)。术后3、7、14、21、28 d,C、D组TNF-α和SDF-1含量均明显高于A、B组(P0.05);但D组各时间点TNF-α含量显著低于C组,SDF-1含量显著高于C组(P0.05)。免疫组织化学染色示,术后各时间点C、D组SDF-1、BDNF、VEGF和TNF-α表达均显著高于A、B组(P0.05);D组SDF-1、BDNF、VEGF表达显著高于C组,TNF-α表达显著低于C组(P0.05)。免疫荧光染色示,C、D组GFP-BMSCs、GFP/NeuN、GFP/GFAP阳性细胞数均显著多于A、B组,D组显著多于C组,差异均有统计学意义(P0.05)。TUNEL法检测示,C、D组凋亡细胞数目显著低于A、B组,D组显著低于C组,差异均有统计学意义(P0.05)。结论 G-CSF可以动员BMSCs归巢至大鼠脊髓损伤部位并参与修复,其作用可能与其下调TNF-α减少细胞凋亡,上调SDF-1、BDNF、VEGF促进BMSCs迁移有关。     

BMSCs移植联合督脉电针对大鼠脊髓损伤后神经功能恢复的影响

目的 探讨骨髓间充质干细胞(BMSCs)移植联合督脉电针对大鼠脊髓损伤(SCI)后神经功能恢复的影响. 方法 从2011年3月至7月,采用全骨髓差速贴壁培养法进行SD大鼠BMSCs的体外分离与培养;利用多中心急性脊髓损伤打击器(MASCIS Impactor)建立大鼠T11脊髓损伤模型,建模成功后将大鼠随机分为4组:对照组(A组)、电针组(B组)、BMSCs移植组(C组)、联合组(D组).SCI后1周,C组与D组大鼠自尾静脉注射移植BMSCs悬液0.1 ml,A组与B组自大鼠尾静脉注射无血清的DMEM/F12培养液0.1 ml,B组与D组接受督脉电针治疗,1次/天.于SCI后1、2、4、8周采用BBB评分法进行后肢运动功能评分;于SCI后8周采用SEP检测脊髓传导功能,采用HE染色观察脊髓空洞大小,采用免疫组化染色检测神经丝蛋白(NF200)的表达.实验中获得的数据采用单因素方差分析. 结果 SCI后2、4、8周,与A组比较,B、C、D组BBB评分均显著升高,差异有统计学意义(P＜0.05);与B、C组比较,D组BBB评分升高更明显,差异有统计学意义(P＜0.05).SCI后8周,与A组比较,B、C、D组SEP潜伏期缩短、波幅增高,差异有统计学意义(p＜0.05);与R、C组比较,D组SEP潜伏期更短,波幅更高,差异有统计学意义(P＜0.05).SCI后8周,4组大鼠脊髓组织损伤区均可见组织结构紊乱,细胞肿胀,空泡变性,且损伤区均可见瘢痕组织及空洞形成,其中A组脊髓空洞最大,B组和C组次之,D组最小.SCI后8周,4组大鼠脊髓组织均有NF200阳性表达,与A组比较,B、C、D组NF200阳性表达增强,差异有统计学意义(P＜0.05);与B、C组比较,D组NF200阳性表达更强,差异有统计学意义(P＜0.05). 结论 BMSCs移植联合督脉电针对脊髓损伤大鼠神经功能恢复的促进作用更为显著,优于单纯BMSCs移植和督脉电针治疗.     

银杏叶提取物对大鼠急性脊髓损伤后神经功能恢复的作用及其机制

目的:观察银杏叶提取物(EGb)对大鼠实验性脊髓损伤后组织结构及运动功能恢复的作用,探讨其对急性脊髓损伤的作用机制。方法:120只SD雄性大鼠,随机分为假手术组(A组)、损伤对照组(B组)、甲基强的松龙(MP)治疗组(C组)和EGb治疗组(D组),每组30只。B、C、D组用Allen′s法以50g·cm致伤大鼠T9脊髓制作损伤模型,B组为单纯脊髓损伤,不给药;C组为脊髓损伤后30min内,由腹腔注入MP30mg/kg;D组为术后至处死前每天腹腔给予EGb17.5mg/kg;A组只打开T9椎板,不打击脊髓,不给药。术后24h、3d、5d、7d、14d对大鼠进行脊髓运动功能(BBB)评分。于术后24h、3d、5d、7d、14d处死动物(n=6),取T9节段脊髓,切片苏木素-伊红(HE)染色观察脊髓大体组织结构变化,用免疫组织化学方法检测B细胞淋巴瘤/白血病基因-2(Bcl-2)和B细胞淋巴瘤/白血病基因伴随蛋白x(Bax)在脊髓前角运动神经元中的表达变化情况。结果:各时间点B、C、D组大鼠脊髓运动功能(BBB)评分均显著低于A组(P<0.01),伤后7d、14d时C、D组评分显著高于B组(P<0.05),各时间点C组与D组评分比较无统计学意义(P>0.05)。HE染色C组和D组大鼠脊髓损伤区较B组坏死程度轻、形成囊腔少,A组正常;1周后D组较C组片状出血灶少,神经细胞肿胀不明显。各时间点B、C、D组大鼠损伤脊髓前角运动神经元中Bcl-2阳性细胞数均显著高于A组(P<0.01),C、D组显著高于B组(P<0.01),7d、14d时D组显著高于C组(P<0.05);各时间点B、C、D组大鼠损伤脊髓前角运动神经元中Bax阳性细胞数均显著高于A组(P<0.01),C、D组显著低于B组(P<0.01),7d、14d时D组显著低于C组(P<0.01)。结论:EGb可能通过抑制Bax表达、提高Bcl-2表达,抑制脊髓损伤后神经元凋亡,在运动功能恢复、损伤脊髓组织保护上发挥其有益作用,1周后EGb仍能抑制脊髓损害后的继发性损伤。     

Necroptosis in Immunity and Ischemia‐Reperfusion Injury

Transplantation is invariably associated with ischemia–reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best‐studied RN pathway, necroptosis, is triggered by perturbation of caspase‐8‐mediated apoptosis and depends on receptor‐interacting protein kinases 1 and 3 (RIPK1/RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death‐associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN‐specific inhibitors such as necrostatin‐1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second‐generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection‐mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis‐triggering receptor(s).     

Deficiency of receptor-interacting protein kinase 3 (RIPK3) attenuates inflammation and organ injury in neonatal sepsis

Introduction

Sepsis is the third leading cause of morbidity and mortality in neonates. Sepsis in neonates is characterized as the systemic inflammation owing to infection within the first 28 days after birth. The molecular mechanism causing the exaggerated inflammation phenotype in neonates has not been completely elucidated. Receptor interacting protein kinase 3 (RIPK3) is a protein identified as a mediator in programmed necrosis or necroptosis. We hypothesize that RIPK3 could be responsible for the inflammatory response in neonates and that deficiency in the RIPK3 protein attenuates inflammation and organ injury in neonatal sepsis.

RIPK3‐Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase‐8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin‐1 or use of RIPK3^?/? TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase‐8 in donor B6 mouse kidneys increased necroptosis, enhanced high‐mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3^?/? kidneys postischemia. Following transplantation, recipients receiving RIPK3^?/? kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3‐mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

A comparison of the inhibitory effects of four volatile anaesthetics on the metabolism of chlorzoxazone, a substrate for CYP2E1, in rabbits

Background: Halothane inhibits in vitro and in vivo activity of cytochrome P-450 (CYP) 2E1. There are several fluorinated volatile anaesthetics besides halothane, and most of them are defluorinated by CYP2E1. It is unclear whether other fluorinated anaesthetics inhibit the in vivo activity of CYP2E1.
Methods: We compared the inhibitory effects of therapeutic concentrations of four inhalational anaesthetics, halothane, enflurane, isoflurane, and sevoflurane, on chlorzoxazone metabolism in rabbits receiving artificial ventilation.
Results: All four inhalational anaesthetics decreased arterial blood pressure and increased plasma chlorzoxazone concentration. However, no significant differences in the plasma chlorzoxazone concentration were found between the four anaesthetics. The estimated chlorzoxazone clearance increased after beginning inhalation with all four agents, but no significant difference in clearance was noted between agents.
Conclusions: At therapeutic concentrations, the in vivo inhibitory effect on chlorzoxazone metabolism was similar for all four inhalational anaesthetics examined, even though their chemical characteristics and extent of hepatic metabolism differ considerably.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.