【作者】 郭昌茂；

【导师】 徐小虎；

【作者基本信息】 汕头大学 ， 法医病理学， 2003， 硕士

【摘要】 背景与目的：脑震荡(concussive brain injury,CBI)属于轻型脑外伤(mild traumatic brain injury)，是最常见的脑损伤形式之一，多见于交通事故和各种暴力案件。脑震荡的临床特征是脑外伤后出现短暂的意识丧失不伴有明显的脑内结构损害，常可遗留包括学习记忆功能障碍在内的一系列症状，这些症群可长期存在，被称为脑震荡后综合征(post-concussion syndrome，PCS)。在人类脑震荡中学习记忆障碍是PCS中最持久和对人们健康生活影响最严重的问题之一，然而这并未引起人们的足够重视，这方面的研究也缺乏系统性。相当一部分脑震荡病例的发生都要涉及到法律诉讼问题，而基于上述原因，对脑震荡引起的学习记忆障碍的法医学鉴定尚缺乏科学依据；对脑震荡的临床治疗也还有待于进一步的提高认识。所以，建立脑震荡的动物模型，系统研究脑震荡对学习记忆的影响及其神经机制，对脑震荡的法医学鉴定、脑震荡的临床治疗等诸多方面都具有重要意义。材料和方法：84只大鼠随机分成10组，A、C、E、G、I、J组为脑震荡打击组，每组10只：B、D、F、H组为非打击组，每组6只。为观察脑震荡对大鼠的学习记忆的影响，A组于打击前行水迷宫训练，打击后检测其记忆保持情况，B为对照组；C组打击后检测其空间学习能力、参照记忆和工作记忆能力，D组为对照组。 为探讨脑震荡对大鼠空间学习记忆能力影响的神经机制，E组于打击后72小时处死，取脑组织检测海马CA3区锥体细胞凋亡情况，F组为对照组。G组、I组和J组分别于打击后给予生理盐水、纳洛酮和多巴胺D2受体拮抗剂舒必利腹腔注射，H组给予生理盐水腹腔注射作为对照组，7天后用水迷宫技术检测上汕头大学医学院硕士学位论文述四组动物的空间学习记忆能力。打击后14天动物被处死后取脑组织，一侧脑组织用于激光共聚焦显微镜检测海马cA3区锥体细胞内c扩十积聚情况，另一侧脑组织制成组织蜡块后，切片进行海马CA3锥体细胞计数。结果 l)打击后24小时A组动物探索试验穿越原平台位置水域次数明显比B组动物少(P<0.05)，打击后48、72小时两组动物探索试验无差异(P>0.05)。提示动物脑震荡后出现了一过性逆行性遗忘，48小时后恢复。 2)打击后第8至13天C组动物隐匿平台逃避潜伏期明显比D组动物长 (尸<0.05)，第13天进行的探索试验C组动物穿越原平台位置水域的次数明显比D组动物少(尸<0.05)，可见平台试验无差异(P>0.05)，提示脑震荡对动物参照记忆的获得和保持均有影响，并排除了脑震荡后动物感觉和运动功能改变的影响。第巧至18天的工作记忆检测，C组动物每天的第一次尝试与第二次尝试的逃避潜伏期的差异无统计学意义(P>0.05)，D组动物每天的第一次尝试与第二次尝试的逃避潜伏期的差异有统计学意义(尸<0.05)。提示脑震荡损害了动物的工作记忆。 3)G组(脑震荡十生理盐水组)、H组(假打击十生理盐水组)、I组(脑震荡+纳洛酮组)和J组(脑震荡+舒必利组)各组动物水迷宫行为学检测结果:从打击后第8至13天，每天比较，G组动物的隐匿平台逃避潜伏期明显比H、LJ组动物长(P<0.01)，H、I、J组之间无统计学差异(P>0.05)。第14天探索实验结果:H、I、J组动物的穿越次数明显比G组多(尸<0 .01)。H、I、J组之间无统计学差异(P>0.05)。以上结果说明，脑震荡损害了大鼠的学习记忆能力;纳洛酮以及舒必利均能明显改善脑震荡后大鼠的空间学习记忆能力。 4)E组(脑震荡组)海马CA3区锥体细胞层见大量TUNEL阳性细胞，F组(假打击组)海马CA3区锥体细胞层偶见TUNEL阳性细胞，病理图像分析结果显示，E组的海马CA3区凋亡细胞数、凋亡细胞平均光密度、凋亡细胞面密度以及数密度均明显高于F组(尸<0.01)。E组凋亡细胞平均灰度值明显低于F组(p<0.01)。E组动物海马CA3区锥体细胞的超微结构发生以下变化:线粒体汕头大学医学院硕士学位论文肿胀，峭减少，空泡样变性;核膜皱缩，染色质边集等。F组动物未见上述改变。E组动物海马CA3区锥体细胞的超微结构改变符合凋亡特征。说明脑震荡能引起大鼠海马CA3区锥体细胞凋亡，凋亡是脑震荡大鼠海马CA3区锥体细胞选择性丢失的主要机制之一。 5)G组动物海马CA3区锥体细胞内游离钙荧光探针nu。一3的荧光量组明显高于H、I、J组(p<0.01)，说明脑震荡导致大鼠海马CA3区锥体细胞内游离钙积聚，提示脑震荡后大鼠海马内谷氨酸释放增多，发生了ca2+一兴奋偶联，纳洛酮和舒必利两种药物均能减少脑震荡动物海马CA3区锥体细胞内C扩十积聚。H、I、J组之间无明显差异(P>0.05)。 6)G、I、J组动物海马CA3区锥体细胞数平均值明显比H组低(尸<0.01)，说明脑震荡导致了大鼠海马CA3区锥体细胞的丢失;I、J组动物海马CA3区锥体细胞计数平均值明显比G组高(尸<0.01)，说明脑震荡后纳洛酮和舒必利两种药物均对动物海马CA3区锥体细胞有保护作用。I组动物海马CA3区锥体细胞计数平均值明显比J组高(尸<0.01)，说明纳洛酮对脑震荡后大鼠海马CA3区锥体细胞的保护作用效果比舒必利好。结论: 1)脑震荡打击后，大鼠出现了类似人类脑震荡的一过性逆行性?更多还原

【Abstract】 Background and objective: Concussive brain injury (CBI), seen frequently in traffic accidents and criminal cases, has been reported to be predominating in human traumatic brain injury (TBI) and be characterized by both a transient loss of consciousness in the absence of localized gross anatomical changes in the brain and a post-concussion syndrome including long-lasting cognitive deficits. In human CBI, learning and memory deficits are those of the most persistent disabilities, to which not enough attention has been given, however, and systematic study is needed. Most of CBI cases are involved with lawsuit, lacking of objective evidences, forensic medicine identification of CBI remains difficult. The neural mechanisms of learning and memory deficits following CBI not being established, diagnosis and treatment for CBI remains difficult also. Therefore, it is important and necessary to duplicate the animal model of concussive brain injury, and make a systematic study on the neural mechanisms of learning and memory deficits following CBI.Materials and methods: 84 SD rats (male 40 and female 44) were randomly divided into 10 groups. Animals in group A (n=10), group C (n=10), group E(n=10), group G(n=10), group I(n=10) and group J(n=10) were assigned to receive a concussive brain injury, animals in group B(n=6), group D(n=6), group F(n=6) and group H(n=6) were assigned to receive a sham injury, respectively. To investigate the retrograde amnesia seen frequently in human CBI, animals in group A and in group B were trained in Morris water maze pre-concussion (sham injury in group B), and theirspatial memory abilities were tested P<0st-concussion. To investigate the learning and memory deficits following CBI in rats, rats in group C and in group D were used to test spatial learning, reference memory and working memory abilities P<0st-concussion.To detect the neural mechanisms of learning and memory deficits following CBI, rats in group E and in group F were assigned to sacrifice on day 3 P<0st-concussion to detect whether an aP<0ptosis mechanism was included in the hipP<0campus CA3 pyramidal cells damages. Rats in group G, in group h, in group I and in group J were assigned to receive injury/NS treatment, sham injury/NS treatment, injury/naloxone hydrochloride treatment, and injury/sulpiride treatment, respectively. Learning and memory abilities were tested following injury/treatment, then rats were sacrificed on day!4 P<0st-concussion, the level of intracellular calcium in hipP<0campus pyramidal cells was measured employing the technique of laser confocal scanning microscope, numbers of pyramidal cells in CA3 area were counted with Nissl staining.Results:1 .The number of crossing the former site of the hidden platform was tested 24h, 48h, 72h P<0st-concussion, respectively. The number in rats of group A was reduced significantly compared to that in rats of group B, when tested 24h P<0st-concussion (P<0.05) . While no statistical difference was found when tested 48h and 72h P<0st-concussion (P>0.05) . The results suggested a transient retrograde amnesia happened following CBI in rats.2.Escape latencies in rats of group C and group D were tested day8-13 P<0st-concussion. The mean escape latencies in rats of group C was increased significantly compared to that in rats of group D each day (P<0.05) . The mean number of crossing the former site of the hidden platform in rats of group C was reduced significantly compared to that in rats of group D tested on day 13 P<0st-concussion (P<0.05) . T-test showed a damage effect of CBI on the spatiallearning and memory (reference memory) in rats. Working memory deficits were also observed in CBI rats on day 15-18 P<0st-concussion.3.one-way ANOVA showed a protective effect of naloxone and sulpiride on learning and memory deficits following CBI in rats. The escape latencies in rats of group I and group J were significantly reduced compared to those in rats of group G (P<0.05) . The mean number of crossing the former site of the hidden platform in rats of g更多还原