| 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...
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