【作者】 徐殿国；

【导师】 史中立；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2005， 硕士

【摘要】 脑本身的缺血-再灌注(I-R)损伤是全球的研究热点,但外周器官I-R 对脑的影响一直未被重视。肢体I-R 是肢体挤压伤、断肢再植术等走向康复的必由转归过程,长时间、严重缺血的肢体再灌注后可引发多器官功能障碍综合症(MODS)。对其处置不当,可导致多器官功能衰竭(MOF)。MOF 是此类患者最严重的并发症,其死亡率占重症监护患者死亡率的30-40%。因此,探讨肢体I-R 引发多器官损伤的机制,一直是创伤外科及重症医学共同关注的重大课题。既往的研究多集中在肾、肺等脏器上,本课题组的研究则发现,大鼠肢体I-R 后其海马区锥体层神经元也呈现明显的病理改变。海马是与学习、记忆及免疫调节密切相关的中枢部位,其损伤可能是外周创伤导致记忆缺失及免疫功能低下的重要原因。因此,在救治缺血肢体的同时,如何加强对脑的保护是一个不容忽视的问题,而阐明肢体I-R 引发海马神经元损伤的机制,具有重要的理论意义和临床应用价值。在缺血后神经元损伤的机制中,NMDA 受体(NR)介导的兴奋毒性倍受关注。NR 有功能亚单位(NR1)和调节亚单位(NR2)两部分组成,NR2 有A、B、C、D 四种类型,不同类型NR2 与NR1 结合后赋予NR 不同的生理及药理学特性。资料显示,成年大鼠海马区主要分布有NR2A、B 两种类型,NR2B 是突触后致密体中最主要的酪氨酸磷酸化蛋白更多还原

【Abstract】 The injury of brain ischemia-reperfusion(I-R) per se hasbeen a world-wide study focus, but it was neglected that I-R ofthe periphery organ effected on the function of brain. I-R oflimbs is a patho-turnover procedure of crush injury of limbs,replantation of severed limbs, and so on. The reperfusion afterserious ischemia is frequently accompanied by multiple organfailure (MOF), the severest complication of this kind ofpatients, its mortality amounting to 30-40 percent of theintensive care cases. Therefore the impaired mechanism of I-Rof limbs is an important topic of traumatic surgery and severecase medical science.Previous studies focused on kidney and lung. But thisstudy revealed that I-R of limbs led to patho-change of stratumpyramidale neuron of hippocampus, the central point relatingto learning, memory and immunological regulation. Lesion ofhippocampus is an important reason for memory deficit andhypo-immunologic function. So the impaired mechanism ofI-R of limbs is of both theoretical and clinical importance.Of the mechanism of neuron injury after limbs ischemia,the exitotoxicity induced by N-methyl-D-aspartate receptor(NR) has been paid much attention. NR is composed of thefunctional subunit NR1 and regulatory subunit NR2. NR2family includes four distinct subunits (A,B, C,D). The bindingof NR1 and distinct NR2 assigns many physiological andpharmacological properties of the receptor. There areNR1/NR2A subtype and NR1/NR2B subtype in adult rathippocampus. The major tyrosine-phosphorylated protein inpost-synapse dense body is NR2B, and its level ofphosphorylation exceeds NR2A extremely. The bondingcapability of NR1/NR2B with glutamate is more efficientbecause the activity of NR is determined on the level ofphosphorylation. Now considering that the hippocampalneuron injury resulting from forebrain ischemia is mainlyrelevant to the receptor of NR1/NR2B subtype. Thehippocampal injury resulting from I-R of limbs and brainischemia per se is different, and it is uncertain that whetherthe injury is induced by the receptor of NR1/NR2B subtype.We will investigate the changes in expression of NMDAreceptor subunit NR2B in rat hippocampal subfileds followingI-R of limbs by immunohistochemistry in this experiment, andfurther, it could accumulate documents for elucidating themechanism of hippocampal neuron injury initiated by I-R oflimbs ultimately.Objective: To explore the changes in expression ofNMDA receptor subunit NR2B in rat hippocampal subfiledsand find the preliminary evidence of hippocampal neuroninjury mediated by the receptor of NR1/NR2B subtype afterI-R of limbs.Methods: Sixty six healthy SD male rats were randomlydivided into three groups: normal(N) group, sham-operated(S)group and I-R6-72h group. After the occlusion of the doublearterial cruralis for 4h, the bulldog clamp was removed, andafter the reperfusion for 6-72h the models were made. Ratswere anesthetized, then perfused and fixed through theascending aorta of heart with 300 ml, 4% citromint ( preparedby 0.1M PBS, pH=7.4) . After that, the brain was taken out anddissected coronally at the plane of optic chiasma and mamillarybody. The middle segment contains hippocampus. Afterpostfixed for 48-72h, the targeted segment was dehydrated,transparentized and embedded routinely. Coronal serialsections were cut with 5-6um in thickness. Then, the sectionswith similar shape and area in dorsal hippocampus were pickout (group control) and immunochemistry-stained withantibody specific for NR2B(1:200) according to standardprocedures. The light color reaction product and granulararefaction was defined as the feeble positive signal, on thecontrary, the opacus color reaction product and granulaconcentration was defined as the fortis positive signaling. Afterobservation and photography with Nikon UFX-DX microscope,the images were analyzed and disposed with JD801 imageanalysis system.Results: The immunohistochemical results of antibody sp-ecific for NR2B were that as followed: ①In normal group, thepositive signaling was weak, and showed brown color, further,the positive products distrubuted along the neuron cellmembrane showed brown granula. All the positive cells formedcell band like “helix of ear”. Compared with CA3 subfiled, thepositive signaling in CA1 subfield is stronger. ②In S group,the positive products of rat stratum pyramidale neuron ofhippocampus had the same distribution as that in normal group.The positive signaling intensity was stronger than that in normalgroup, especially in S24h group. ③In I-R6h group, comparedwith N and S group, the distribution and intensity ofimmunohistochemical products in hippocampus had no markedchange; In I-R12h group, neurons showed positive reaction, andcompared with I-R6h group, granula was thicker; brown colorwas deeper. The positive intensity of stratum pyramidale neuronof hippocampus in I-R48h and I-R 72h groups were strongerthan that in N group and corresponding S groups.The results disposed with image analysis system were that:Immunoreactivity gray scales of CA1 and CA3 in N group were14.32+3.38 and 10.07+1.68, respectively. Immunoreactivitygray scales of CA1 and CA3 subfields were 14.43+3.02,14.43+2.36, 15.33+3.45, 15.22+3.36, 15.08+3.48 and 10.17+1.89, 10.24+1.79, 12.70+3.11, 10.68+1.92, 10.30+1.77 in S6, 12,24, 48, 72 groups, in order and respectively. However, immuno-reactivity gray scales of CA1 and CA3 subfields were 14.64±2.71, 18.90±1.40, 24.71±2.89, 22.10±2.62, 21.62±1.98 and更多还原