| IntroductionThe prognosis of hemorrhage lesions in brain is less favorable than that of purely ischemic lesions. The mortality rate at 30 days after intracerebral hemorrhage (ICH) approaches 50% , and many survivors are left with significant neurological deficits. Yet, in comparison with ischemia, relatively few experimental investigations have been devoted to studying the pathophysiology of ICH.Apoptosis depends of to a significant extend on the activation of cysteine proteases, caspase - 3 has received a great of attention because of their key role as a final initiator of cell apoptosis. Recently, attention has been directed to the role of caspase - 3 in neuronal apoptosis. Although caspase - 3 and apoptosis have been implicated in the pathogenesis associated with several models of brain damage, including ischemia^ traumatic brain injury^ and epilepsy禄their role has not been studied in ICH. The present study investigate a relationship between apoptosis and activation caspase - 3 in the blood clot periphery and ipsilateral cortex after ICH in rat.MethodExperimental ICH model in ratsExperimental ICH was induced by the blood injection model in male wistar rats. Autologous blood samples'were drawn form the femoral artery. With the use of a stereotaxical technique, a 1mm diameter burr hole was produced in the skull (0. 2mm anterior and 3.5mm lateral to bregma) , and a 1mm injector wasinserted for blood 100 l injection into the right basal ganglion (5. 0mm deep to bregma). Control animals were subjected to the same manipulations as ICH rats, but infused with 100 l of saline.Experimental groupsFive groups of rats were used ( n = 3 in each group). ICH animals were killed at 6 hours or at 1, 3, or 7 days after injection. Control rats were killed at 3 days.Brain section manufactureThe animals were perfused through the left ventricle with 100ml 0.9% saline followed by 100ml of 4% paraformaldehyde. The brains were removed and postfixed overnight at 4 , and were placed in 30% sucrose for 24 hours. The brain were cutted into 20mm segments with a cryostat microtome. After treatment with 0.3% H202 and 0.3%Triton in PBS solutions, the sections were detected by TUNEL and immunohistochemistry.TUNEL was used to detected apoptosis.The section were incubated with TDT and fluorescein - dUTP for 1 hour at 37 C and washed three times in PBS and incubated with anti -fluorescein perox-idase. The sctions were developed by using diaminobenzidine. TUNEL - positive cells around the blood clot and ipsilateral cortex were counted in section.Immunohistochemical detection of caspase - 3.The sections were incubated by using 10% normal goat serum in PBS for 1 hour at room temperature; the sections were incubated in a 1:100 dilution of rabbit anticaspase -3 antibody for 1 hour at 371 followed by the avidin - biotin complex process. Finally, the sections were exposed to stable diaminobenzidine. TTie integral optical density total of caspase - 3 posititive cells was detected in each section.ResultsTUNEL detect neuronal apoptosis after ICH.TUNEL - positive cells increased significantly at 1 day, peeked at 3 days,and decreased at 7 days ( p < 0.05). TUNEL - positive cells were observed a-round blood clot and ipsilateral cortex. The nuclei of most TUNEL - positive cells were detected in brown with a granular pattern, chromatin margination, or nuclear condensation, indicating that cells with DNA fragmentation were mainly associated with apoptosis. Most TUNEL - positive cells appeared to be neurons, whereas some appeared to be glia and endothelial cells.Immunohistochemical analysis for caspase - 3Caspase - 3 immunoreactivity were observed around the blood clot and ipsilateral cortex. According to the morphology and location, most caspose -3 im-munoreactive cells appeared to be neurous, whereas some appeared to be glia. The cells were detected with brow plasma. Most caspase - 3 immunoreactivity were observed at 1 day or 3 days after ICH. Compared with controls, activation were significantl... |
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