| Objective: Intracerebral hemorrhage(ICH) is a common clinical disease with high morbidity and mortality. Up to now,we can't find the effective therapy for ICH because the exact pathogenesis of neuronal death following ICH is still unknown. Recent evidence suggests that cell death in the perihematoma region may involve apoptosis. Apoptosis is a voluntary death of the cells in the normal physiological or pathological processes , which called programmed cell death(PCD). Apoptosis is characterized by morphological changes that include DNA fragmentation(180-200bp),chromatin condensation, cell shrinkage, finally forming apoptotic body packed of membrane. Apoptosis by gene controlled is a process withered itself away. It was reported that apoptosis is correlated with the inflammatory response in the perihematoma, cytokine and complement participating, calcium over loading, the toxic role of excitatory amino acid and so on , but the exact mechanism is unknown. Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor and a member of a family of proteins that are critical regulators of a variety of responses, This results in the transcriptional induction of genes for many proinflammatory substances, such as TNF-α, IL-2,3,6 and IL-8 , transforming growth factor ,ICAM-1, the iNOS, epoxide enzyme -2 and so on. Its activation has been played a key role in the inflammatory response in the cerebral diseases, In this study ,The rules of apoptotic cells and immersion of inflammatory cells and NF-κB expression in rat brain after ICH were observed in order to explore correlation between NF-κB and apoptosis after ICH. Methods: 48 rats adult male Sprague-Dawley were randomly divided into 6 groups. According to the methods of Hua,Lee et al, the rats were subjected to stereotactic frame, and a cranial burr hole (1.0 mm) was drilled in the skull (1 mm anterior, 4.0 mm right to the bregma). 50 μl autologous caudate artery blood was infused into right caudate nucleus of the rats, and the shams served as controls. After ICH for 6 h,24 h,48 h,72 h,7 d , the successful ICH models of each group were measured by behavioral tests and then part of the successful ICH models were anesthetized with pentobarbital and perfused through the left ventricle with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered (pH7.4). The removed brain were cut from the hole coronally, and the hematoma can be seen in the right caudate nucleus .We cut the brain into 4mm thick and kept them in 4% paraformaldehyde for 24~48 hours at 4℃, through dehydration, transparency, immersed in wax, embed, finally kept by wax mass. For another part, the successful ICH models were sacrificed by injecting overdose pentobarbital intravenously without perfusingand their brains were removed. The fresh brains were frozen by the liquid nitrogen-isopentane method in embedding compound and then cut into in 20 μm segments with a cryostat section cutter. We can observe inflammatory cells immersing by HE staining and NF-κB expression immunohistochemistory staining and TUNEL-positive by terminal deoxynucleotidy1 transferase mediated dUTP-nick-end-labeling. Results: HE staining showed that there were scattered inflammatory cells infiltrating in perihematoma at 6 h after ICH, inflammatory cells especially neutrophils infiltrating peaked at 48 h after ICH , gliosis and capillary hyperplasia were observed around perihematoma at 72 h after ICH. The volume of hematoma reduced markedly at 7 d after ICH, accompanied by distinct glial and capillary hyperplasia. There was no inflammatory cell infiltrating in the sham group. Immunohistochemistry demonstrated that NF-κB expressed very little in sham group, but in the model group NF-κB expressed increasingly at 6 h, reached maximum at 48 h, continued to 7 d. NF-κB mostly expressed in glial , the positive cells'cytoplasm or nucleus was stained brown yellow. There was no statistic difference about NF-κB expressing between ipsihemotoma and contralhemotoma,P>0. 05;but there was statistic difference about NF-κB expre... |
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