The Effect And Mechanism Of Vascular Endothelial Cells Apoptosis On Vasospasm In Rat Brain After Experimental Subarachnoid Hemorrhage

PURPOSE: 1. To investigate the effect of oxyhaemoglobin (OxyHb) with various concentrations and various incubation time on proliferation and apoptosis of cultured rat brain microvascular endothelial cells(RBMvECs) and the change of Caspase-3 expression during this course. 2. To study the role of mitochondrial membrane potential and cytochrome C on the apoptosis of RBMvECs induced by OxyHb. 3. To investigate the role of mitochondria pathway in regulating the apoptosis of RBMvECs induced by OxyHb when the mitochondria permeability transition pore(MPTP) was blocked. 4. To study the relationship between endothelial cells apoptosis and CVS after SAH through detecting the apoptosis and apoptosis-related proteins in vascular endothelial cells after SAH in rat.METHODS: 1. Experimental study on the apoptosis of RBMvECs induced by OxyHb. (l)Primary cultured RBMvECs were incubated with various concentrations of OxyHb(1μM, 10μM, 100μM and 1000μm) for various times(6h, 12h, and 24h) and cell proliferation ability was assessed by MTT method. (2) RBMvECs were incubated with 10μM and 100μM OxyHb, cell apoptosis was measured by flow cytometry and Hochest33258 fluorescent stain method, Caspase-3 protein was measured by Western blot and spectrophotometric method, mitochondria membrane potential was measured by flow cytometry, and cytochrome C was measured by Western blot, respectively. (3) 10μM and 100μM OxyHb was added when RBMvECs were precultured with CsA for 30mins, cell apoptosis, mitochondria membrane potential and cytochrome C were also measured with the methods mentioned above. 2. Morphology study and apoptosis of endothelial cells of the basilar artery in rat SAH model. Autologous arterial blood without heparinization were injected into male rats cisterna magna at 0.1ml/100g, the same blood injection procedure was repeated 48h later. At different time point the brainstem were embeded in paraffin, serial sections in 5μm carried on hematoxin eosin dyeing. The vessel wall thickness and the cross section area of the basilar artery were measured with Image-Pro-Plus 5.0software. The apoptosis of basilar artery endothelial cells were detected by TUNEL method, and bcl-2 and bax expression were measured by immunohistochemistry.RESULTS: 1. (1) The inhibitory effect of cell proliferation was in proportion to OxyHb incubation concentrations and times. At certain incubation concentrations(10fiM~1000|jM) and incubation times(6~24h), OxyHb could inhibit the proliferation of RBMvECs significantly. (2) At the OxyHb concentrations of \0\iM and lOO^M, obvious apoptosis was observed by Hoschest 33258 staining and flow cytometry. (3) The expression of Caspase-3 and cytochrome C in endothelial cells were increased with the increasing of OxyHb incubation concentrations and times, and there is a positive correlation between the expression of Caspase-3 and the extent of cell apoptosis. On the contrary, mitochondria membrane potential depressed with the increasing of OxyHb incubation concentrations and times, and there is a negative correlation with the extent of cell apoptosis. (4) CsA could significantly inhibit the decreased expression of mitochondria membrane potential and increased expression of cytochrome C, and alleviate the extent of apoptosis of RBMvECs. 2. The basilar artery show vasospasm from Id and the most obvious change was observed in 5d and return to normal state in 14d after SAH. The number of endothelial cells with positive TUNEL staining and bcl-2, bax expression level are increased gradually after SAH and return to normal level in 14d.CONCLUSIONS: 1. The apoptosis of primary cultured RBMvECs induced by OxyHb is concentration-dependent and time-dependent. OxyHb can induce the increasing expression of Caspase-3 and cytochrome C and depressing of mitochondria membrane potential, the expression of Caspase-3 has positive correlation with the extent of cell apoptosis, on the contrary, the expression of mitochondria membrane potential has negative correlation with cell apoptosis. Pretreated with CSA could significantly inhibit the decreased expression of mitochondria membrane potential, the extent of RBMvECs apoptosis and the increased expression of cytochrome C, which suggested that mitochondria pathway could participate in the apoptosis of the RBMvECs. 2. The basilal artery gradually showed vasospasm from Id and reached peak in 5d and resumed normal condition in 14d after SAH model in rat. The positive staining of TUNEL, baxand bcl-2 expression showed a tendency which increases first and then decreases in vascular endothelial cells. There was a positive correlation between positive staining of TUNEL with area of the basilar artery and positive staining of TUNEL with Bax/bcl-2. All of these indicated that apoptosis of RBMvECs could be one of the reasons of CVS after SAH.