【作者】 方文华；

【导师】 刘运生；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 研究背景重型颅脑外伤是严重危害人类健康和生命的多发疾病,不仅给患者及其家庭带来沉重的精神和生活负担,而且容易引发一系列社会和经济问题。在医学不断进步的今天,重型颅脑外伤的致残、致死率却一直居高不下,至今仍没有一项十分有效的治疗方法。上个世纪末本世纪初,在多项重型颅脑外伤药物临床试验结果不尽人意时,低温在治疗心脏停搏相关脑损伤和新生儿缺血缺氧性脑病方面所取得的积极效果,又使人们把目光聚焦到重型颅脑外伤低温脑保护的研究中。低温治疗的实施可有多种方法,目前应用较广的是全身降温,但有研究发现全身降温可能存在寒战、电解质紊乱、心律失常、出血倾向、易感染、皮肤损害等并发症。设计和实施一种既能有效低温治疗而又可避免全身降温并发症的降温技术,对低温脑保护的研究和应用具有重大意义。目前,低温发挥脑保护作用的机制尚不完全清楚,进一步研究其对重型颅脑外伤病理生理演变和预后转归的影响,有利于降低重型颅脑外伤的致残率和死亡率。第一章硬膜外局部低温方法的降温效能、安全性和并发症研究目的应用自制降温线圈实施硬膜外局部低温,通过与全身降温方法对比,研究该局部低温方法的降温效能、安全性和并发症情况。方法SD大鼠随机分为常温对照组(Nor组)、硬膜外局部低温组(LH组)和全身低温组(SH组),LH组以降温线圈贴附于一侧硬脑膜外实施局部低温,SH组以冰袋和酒精全身降温,两组均使脑温降至31.0～32.0℃并持续10小时。实验中观察大鼠寒战反应,监测同侧脑温、对侧脑温、肛温及呼吸、心率、血压变化,于降温末采血进行血气分析、电解质、血常规及血液流变学检测,并在降温后24 h以改良NSS评分方法评测其神经功能,取脑组织行光镜、电镜检查,以及脑组织水、钠、钾含量和血脑屏障通透性检测。结果降温后,LH组大鼠降温侧脑温在数分钟内从(36.5±0.3)℃下降到(31.4±0.4)℃并维持稳定,其对侧脑温、肛温以及R、HR、MABP无明显变化;SH组的局部脑温也下降到目标范围,但其对侧脑温、肛温同时出现降低。降温过程中,SH组寒战发生率为58.8%,并出现心率下降,血小板降低,高切变率下全血粘度和血浆粘度增高,LH组未观察到如上变化。实施低温后,LH组和SH组大鼠NSS评分正常,光镜和电镜下未发现脑组织损伤表现,两组的脑组织水、钠、钾含量和伊文思蓝含量与Nor组比较无统计学差异。结论该种硬膜外局部低温方法的降温效果与全身降温相似,且没有对脑组织造成急性损害。同时,与全身低温相比,该种硬膜外局部低温的并发症较少。硬膜外局部低温是一项有效、安全、并发症少的低温脑保护技术。第二章硬膜外局部低温治疗重型颅脑外伤及与全身低温疗法的比较目的探索硬膜外局部低温治疗对重型颅脑外伤实验大鼠死亡率、体重、神经功能、脑水肿和组织病理学的影响,并与全身低温方法对比,观察两者的治疗效果。方法SD大鼠随机分为正常对照组(Norm组)、假手术组(Sham组)、重型颅脑外伤组(sTBI组)、硬膜外局部低温治疗组(LHT组)和全身低温治疗组(SHT组)。采用Feeney’s自由落体致伤方法制备重型颅脑外伤模型(25 g,60 cm)。在伤后30 min开始局部或全身低温治疗,持续10 h。在伤后不同时间点测量体重,采用改良NSS评分、平衡木和平衡行走试验以及Morris水迷宫进行神经功能评测,并取脑组织进行水、钠、钾含量检测和HE染色,观察脑水肿情况和组织病理学改变。结果sTBI组、LHT组和SHT组大鼠的死亡率分别为32.32%、15.19%和19.28%。LHT组和SHT组伤后体重下降的幅度比sTBI组小。在伤后相应时间点进行的NSS评分、平衡木、平衡行走试验和Morris水迷宫等评测结果显示,LHT组和SHT组大鼠相应的神经功能障碍均较sTBI组轻。在伤后12 h、1 d和3 d,sTBI组的脑组织水、钠含量增加而钾含量降低,提示脑水肿,而这三个时间点LHT组和SHT组的脑水肿程度均比sTBI组轻。重型颅脑外伤后,脑组织病理形态改变明显,而LHT组和SHT组的脑组织病理损害程度较轻。此外,在死亡率、体重变化、神经功能评分和脑水肿程度方面,LHT组和SHT组之间比较未见统计学差异。结论硬膜外局部低温治疗和全身低温相似,均可减缓大鼠重型颅脑外伤后的体重下降,改善神经功能障碍,减轻伤后脑水肿和脑组织病理改变程度等。该两种低温方法对重型颅脑外伤均具有明显的治疗作用,且在效果上无明显差异。第三章硬膜外局部低温对重型颅脑外伤大鼠脑组织PKC-δ、突触素和GAP-43表达的影响目的探索硬膜外局部低温治疗对重型颅脑外伤实验大鼠脑组织PKC-δ、突触素和GAP-43表达的影响,以研究低温脑保护作用的可能机制。方法SD大鼠随机分为正常对照组(Norm组)、假手术组(Sham组)、重型颅脑外伤组(sTBI组)和硬膜外局部低温治疗组(LHT组)。伤后30 min开始局部低温治疗,持续10小时。透射电镜下观察脑组织的超微形态结构改变,并取伤后12 h、1 d、3 d的脑组织进行PKC-δ免疫印迹和TUNEL染色,伤后7 d、14 d和28 d进行突触素、GAP-43的免疫印迹和免疫组化检测。结果sTBI组大鼠脑组织电镜下超微形态结构改变严重,而局部低温治疗后,细胞核、线粒体、血脑屏障等结构有所改善。sTBI组脑组织全细胞成分中PKC-δ表达下降,而PKC-δ具有活性的裂解片段CF-PKC-δ增加;此外,sTBI组细胞浆中PKC-δ表达减少而细胞膜中PKC-δ增加,提示伴发PKC-δ从胞浆向胞膜的转运。实施硬膜外局部低温后,PKC-δ的裂解和胞膜转位均减少,提示低温可抑制PKC-δ的激活。在伤后12 h、1 d、3 d,sTBI组脑组织TUNEL染色阳性凋亡细胞数增多,而LHT组凋亡细胞数减少,提示低温可能抑制细胞凋亡。在伤后7 d、14 d、28 d,可观察到sTBI组脑组织中突触素和GAP-43的蛋白表达和免疫活性均下降;给予硬膜外局部低温治疗后,LHT组的突触素、GAP-43表达和免疫活性均较sTBI组增加。结论硬膜外局部低温可减轻重型颅脑外伤所导致的神经组织、细胞超微形态结构损害。其可能通过抑制重型颅脑外伤伤后PKC-δ的裂解和胞膜转位,降低PKC-δ的有害活化,并可进一步调节、抑制细胞凋亡,从而发挥脑保护作用,减少急性期神经组织和细胞的损伤。同时,低温还可能通过增加脑组织突触素和GAP-43的表达,促进重型颅脑外伤伤后晚期神经再生和重塑,改善神经功能。综上所述,本研究结论如下:1.硬膜外局部低温方法有效、安全;与全身低温相比,其在实验动物身上观察到的并发症较少。2.硬膜外局部低温治疗可减缓大鼠伤后的体重下降,改善神经功能障碍,减轻伤后脑水肿,改善伤后脑组织病理改变等。其和全身低温相似,对重型颅脑外伤具有明显治疗作用,且二者在效果上无明显差异。3.硬膜外局部低温可能通过抑制伤后脑组织中PKC-δ的裂解和胞膜转位,降低PKC-δ的有害活化,进而调节、减少细胞凋亡,发挥脑保护作用。4.硬膜外局部低温可能通过增加脑组织突触素和神经生长相关蛋白-43(GAP-43)的表达,促进伤后晚期神经再生和重塑,改善神经功能。更多还原

【Abstract】 Research background Severe traumatic brain injury is a frequently-occurring disease that usually causes significant harm to human health and life. The aftermath of such injuries leaves survivors with permanent disabilities resulting in lifelong medical, financial, emotional, familial and social problems. Today, although the advancement of medical sciences is obvious, the rate of disability and fatality after traumatic brain injury has been high for many years, and there are still not an effective treatment. In the last 1990s and the beginning of this century, the lack of progress in achieving brain protection with several pharmacologic agents, while a positive evidence of the therapeutic efficacy of mild to moderate hypothermia from the studies in post-cardiac arrest survivors and neonatal hypoxic encephalopathy, led to renewed interest in therapeutic hypothermia for the severe traumatic brain injuries. There are many ways to induce the therapeutic hypothermia, of which the systemic cooling method is most widely used at present. However, the systemic side effects of whole-body cooling such as chills, electrolyte disturbances, arrhythmia, bleeding tendency, easy to infected and skin lesion, have been reported in some studies. It is important to design and imply techniques of cooling in order to maximize the therapeutic benefit and minimize the complications. At present, the mechanisms that cause the neuroprotection provided by hypothermia still a matter of debate and are probably multiple. Further understanding the mechanisms underlying hypothermia’s benefits will lead to more effective treatments to reduce the disability and mortality of severe traumatic brain injuries.Chapter 1. The Efficacy, Security and Complication of the Local Brain Hypothermia Induced by Epidural CoolingObjective To develop an epidural cooling method to induce local brain hypothermia using a self-made cooling coil, and to evaluate the efficacy, security and complications of this epidural local hypothermia technique.Methods SD rats were randomly divided into normal temperature group(Nor), local hypothermia group(LH) and systemic hypothermia group(SH). The local and systemic hypothermia were induced by self-made cooling coil and ice bag, alcohol respectively, the brain temperature were reduced to 31.0-32.0℃and maintained for 10 hours. During cooling, the shivering reaction, ipsilateral and contralateral brain temperature, rectal temperature, respiratory, heart rate and blood pressure of rats were assessed. The blood gas and electrolyte analysis, blood routine and hemorheology of rats were detected after cooling. The neurological evaluation and microscopic examination were carried out at 24 h follow cooling, and the brain water, Na~+, K~+ content and blood-brain barrier permeability were also detected.Results In LH group, local brain hypothermia was produced within minutes from (36.5±0.3)℃to (31.4±0.4)℃in the ipsilateral hemisphere after cooling, while the contralateral brain and rectal temperature, R, HR and MABP were not significantly altered. In SH group, the decrease of bilateral brain and rectal temperature as well as an 57.1% of shivering incidence were observed during cooling. The decrease of HR and platelet count, and the increase of whole blood viscosity under high shear and plasma viscosity were also detected in SH group. There were no significant difference in NSS score, brain water, Na~+, K~+ and Evans Blue content among groups, and no structural alterations were found in brain tissue of both LH and SH group neither.Conclusions This epidural cooling method can achieve the effect of brain hypothermia as well as systemic cooling technique, and dose not cause fluctuations in vital signs and acute neuronal injury. Meanwhile it may developed few complications compared with systemic hypothermia. Chapter 2. Therapeutic Efficacy of Epidural Local Hypothermia on Severe Traumatic Brain Injury and Comparative Study with Systemic Cooling MethodObjective To study the therapeutic efficacy of epidural local hypothermia on mortality, body weight, neurological function, brain edema and histopathological changes on rats after traumatic brain injury, and which were compared with systemic hypothermia at the same time.Methods SD rats were randomly divided into normal control group(Norm), sham operation group(Sham), severe traumatic brain injury group(sTBI), local hypothermia therapy group(LHT) and systemic hypothermia therapy (SHT). Feeney’s weight-drop (25 g, 60 cm) model was used to induce severe traumatic brain injury in rats. The epidural local hypothermia or systemic cooling were conducted 30min post injury and last for 10 hours. The body weight measurement, NSS score, beam balance test, beam walking test and Morris water maze were used in different time point post injury, as well as the brain water, Na~+, K~+ content and HE stain were detected to evaluate the therapeutic efficacy of hypothermia.Results The mortality of rats in sTBI, LHT and SHT group were 32.32%,15.19% and 19.28% respectively. The body weight loss of rats were less obvious in LHT and SHT group than sTBI group. The neurological function observed on rats in LHT and SHT group were better than sTBI group detected by NSS score, beam balance test, beam walking test and Morris water maze. At 12h、1d and 3d post injury, an increase of brain water, Na~+ content while a decrease of brain K~+ content were detected in rats of sTBI group, indicating a brain edema. Both LHT and SHT group developed less extent of brain edema in the same day. Rats developed a severe histopathological damage after severe traumatic brain injury, and both of local hypothermia therapy and systemic hypothermia therapy could reduce the extent of pathological damage of brain tissue in rats. In addition, no significant differences were found in mortality, body weight loss, neurological dysfunction and brain edema between LHT and SHT group.Conclusions The same as systemic hypothermia, the epidural cooling technique could slow down the weight loss, improve neurological deficits, reduce brain edema and ameliorate the histopathological damage of brain tissue after head injury. Both of local hypothermia and systemic hypothermia provide the same definite therapeutic efficacy on severe traumatic brain injury. Chapter 3. Effect of Epidural Local Hypothermia on Expression of PKC-delta, Synaptophysin and GAP-43 after Severe TraumaticBrain Injury in RatsObjective To study the expression of PKC-delta, after severe traumatic brain injury in rats for an investigation on the mechanisms of neuroprotection by hypothermia.Methods SD rats were randomly divided into normal control group(Norm), sham operation group(Sham), severe traumatic brain injury group(sTBI) and local hypothermia therapy group(LHT). Feeney’s weight-drop model was used. The epidural local cooling were conducted 30 min post injury and last for 10 hours. The ultrastructure changes of brain tissues were observed under transmission electron microscope. The expressions of PKC-δwere detected by western-blotting. TUNEL staining were conducted at 12 h, 1 d and 3 d post injury. The expressions of synaptophysin and GAP-43 were detected at 7 d, 14 d and 28 d post injury by western-blotting and immunohistochemisty.Results Rats in sTBI group developed a serious ultrastructure damage, and the structures of nucleus, mitochondria and blood-brain barrier were improved in LHT group. The amount of constitutively activated C-terminal catalytic fragment(CF-PKC-δ) increased, as well as the translocation to the membrane of PKC-5, both of which were hallmarks of PKC-8 activation, were observed after severe traumatic brain injury. These events were blocked by hypothermia, implicating the inhibition of PKC-δactivation by hypothermia. Meanwhile, an increase of the number of apoptotic cells in brain tissue were observed by TUNEL staining in the acute stage(3 days post injury) of severe traumatic brain injury, and could be reduced by hypothermia. In addition, a reduction of synaptophysin and GAP-43 expressions due to severe traumatic brain injury were investigated in the late stage, and hypothermia could raise the expressions of synaptophysin and GAP-43, which were coincident with the results of Morris Water Maze in rats.Conclusions Hypothermia could reduce the ultrastructure damage caused by severe traumatic brain injury. Hypothermia protects against the traumatic damage in part dues to the suppressing PKC-δactivation by blockage of CF-PKC-δproducing and PKC-δtranslocation to the membrane. Meanwhile, hypothermia could inhibit the apoptosis after injury. In addition, hypothermia might promote nerve regeneration and remodeling in late stage of TBI by the way of increased expressions of synaptophysin and GAP-43 in brain tissue.更多还原