【作者】 徐书刚；

【导师】 潘蔚然；

【作者基本信息】 中国医科大学 ， 外科学， 2008， 硕士

【摘要】 前言随着经济和交通手段的日益发展,颅脑损伤的发生率和因伤致残率、致死率均逐年增加。通过药物治疗对颅脑损伤后可能存在的细胞因子变化进行研究,有助于进一步阐明颅脑损伤的机理,从而指导临床治疗,提高颅脑损伤的治愈率、降低死亡率和致残率。肿瘤坏死因子-α属于促炎性细胞因子,在颅脑损伤后表达明显增加,在颅脑外伤后继发性脑损伤中起着重要作用。近年来,大量研究显示了镁剂和纳洛酮在治疗创伤性脑损伤中具有较为肯定的保护作用,但其机制尚未完全阐明。本课题通过Feeney法自由落体动物模型致伤,并给予硫酸镁和纳洛酮治疗,观察大鼠脑创伤后脑组织TNF-α的浓度及脑水肿的变化,探讨两者的脑保护作用机制及其联合用药作用效果。材料与方法Wister雄性大鼠60只,体重270～300g,随机分为致伤对照组、纳洛酮组、硫酸镁组及联合用药组,每组再分伤后6h、12h、24h共3个时间点,各时间点每组动物5只。实验大鼠腹腔注射10%水合氯醛(3ml·kg^-1)麻醉,于中线右侧旁开约2mm,冠状缝后约2mm处钻一直径约5mm骨窗。使用Feeney自由落体损伤装置致伤。致伤成功后各治疗组立即分别给予腹腔注射纳洛酮1mg·kg^-1、12.5%的硫酸镁300 mg·kg^-1、12.5%的硫酸镁300 mg·kg^-1+纳洛酮1mg·kg^-1,致伤对照组给予等量生理盐水。分别于伤后6h、12h、24h处死大鼠并完整取脑,部分脑组织立即放入10℃甲醛中,固定后制成腊块标本;部分脑组织于-70℃冰箱冰冻保存。按Elliotl公式计算致伤对照组及各治疗组大鼠挫伤灶边缘脑皮质含水量、采用酶联免疫方法（ELISA）测定TNF-α含量,同时取标本做病理学检查。采用SPSS13.0统计软件进行数据处理,计量资料以均数±标准差（（?）±s）表示,组间比较采用重复测量数据的方差分析,两两比较用多重比较t检验,P＜0.05为差异有统计学意义。结果光镜下发现脑损伤后存在明显炎症反应,细胞水肿及炎症细胞浸润。各组大鼠脑致伤后6h、12h、24h脑组织含水量比较,明显呈增高趋势（各组三时间点间两两比较,均P＜0.05）,以致伤后24h脑组织含水量最高。三治疗组与致伤对照组比较,脑组织含水量均有所降低（单一用药组P＜0.05,联合用药组P＜0.01）,以联合用药组最明显,且联合用药组与单一用药比较均有显著差异（P＜0.05）。提示:大鼠颅脑致伤后24小时内,脑水肿呈加重趋势,且硫酸镁联合纳洛酮减轻脑水肿作用最明显。损伤组TNF-α水平在损伤后6h、12h、24h三时间点呈上升趋势（P＜0.05）。各治疗组均能抑制TNF-α上升,大鼠致伤后6h、12h、24h脑组织TNF-α的水平与致伤组对照组比较均有差异（P＜0.05,联合用药组P＜0.01）,且联合用药组与单一用药组比较亦有统计学意义（P＜0.05）,提示:纳洛酮和硫酸镁均能抑制TNF-α水平,且联合用药较单一用药更有效。结论大鼠脑损伤后急性期（24小时内）TNF-α的浓度呈增高趋势,随着TNF-α含量的增高,脑水肿也随之明显加重;早期行硫酸镁和纳洛酮治疗能降低脑组织TNF-α的浓度和减轻脑水肿程度,联合用药效果最明显,表明硫酸镁、纳洛酮均对脑创伤后脑损伤有保护作用,并且联合用药效果最佳。更多还原

【Abstract】 PrefaceThe incidence of brain injury and accompanied mutilation and mortality rates have increased year by year with the development of the economy and transportation.The abnormal expression of cytokines in traumatic brain injury after drug treatment was investigated to illuminate the mechanism of cerebral injury,and thus to direct the clinical treatment,which produces the elevated recovery rate and decreased mortality and mutilation rates.The tumor necrosis factor-alpha belongs to pro-inflammatory cytokine,which increases obviously after brain injury and plays an important role in the secondary brain injury,the magnesia mixture and naloxone currently have certain protection for traumatic brain injury demonstrated in many researches,but the mechanism has not been illuminated completely.In this study,the animal model for brain injury by the Feeney method of free falling object was established,and given magnesium sulfate and naloxone.The changing in the concentration of TNF-αand brain edema in rats’ brain tissue was observed to investigate their brain protective mechanism and the effect of combined medication.Material and MethodsTotal 60 Wister masculinity rats were included in this study,whose body mass were from 270g to 300g,and randomly divided into four groups:vulnerate control group、naloxone group、magnesium sulfate group and combined medication group. Each group was subdivided into three time points:6h、12h、24h after injury,and there were 5 rats in every time point.Experimental rats were anaesthetized through injecting 10%chloral hydrate(3mg/kg)into abdominal cavities,and a diameter of about 5mm bony window was drilled in every rats in about 2mm to the right of the median line and about 2mm to the post of sutura coronalis.The device of Feeney free falling body was used to injury the rats.After injury,every group was immediately given peritoneal injection naloxone(1mg/kg)、12.5%magnesium sulfate(300mg/kg)、12.5%magnesium sulfate(300mg/kg)+naloxone(1mg/kg),and equivalent normal sodium to the control group respectively.The rats were put into death at the time of 6h、12h、24h respectively after injury,and to get brain tissues.Partial brain tissues were immediately put into 10%formaldehyde and made into wax sample after fixing;and partial brain tissues were reserved in refrigerator with temperature70℃below zero.The moisture in the pallium at the edge of contusion foci of rats was calculated according to Elliotl formula in every therapeutic group and the control group、the contents of TNF-αwere determined through ELISA method,and the sample was obtained for pathologic examination.Statistical analysis:all measurement data were expressed as(?)±s,and statistical significance was evaluated by multiple comparison of test and ANOVA.A P value of 0.05 or less was considered to indicate statistical significance.All statistical processes were completed via SPSS(V13.0).ResultsUnder the macroscope,obvious inflammatory reaction、cellular edema and inflammatory cell infiltrating were found after cerebral injury.The moisture of brain tissues at 6h、12h、24h after brain injury in rats was compared,and the increasing trend was obvious(every two time points was compared in the three points,P＜0.05),and the moisture of brain tissue at 24h after injury was highest.Comparing the three therapeutic groups and control group,the moisture of brain tissues were all decreased (medication alone P＜0.05,combined medication P＜0.01),most obviously in combined group,and the combined group was more obvious different than medication alone(P＜0.05),these results indicated that during 24h after brain injury of rats,brain edema was increased trend,and combined magnesium sulfate with naloxone was most obvious in relieving brain edema.TNF-αin the injury group was in an upgrading trend at the time of 6h、12h、24h after injury(P＜0.05).Each therapeutic group could inhibit the increasing of TNF-α,and the level of TNF-αat the time point of 6h、12h、24h after injury of rats compared with control group(P＜0.05),which indicated that magnesium sulfate with naloxone can both inhibit the level of TNF-α,and combined medication was better than medication alone. ConclusionIn the acute phase of brain injury of rats in 24 h,the concentration of TNF-αwas in an increasing trend.As TNF-αincreased,the brain edema was also aggravated obviously.Magnesium sulfate and naloxone were used early to decrease the concentration of TNF-αand brain edema,combined medication was most obvious, which indicated that magnesium sulfate and naloxone can both protect the brain injury after trauma,and combined medication was better.更多还原