ABSTRACT
OBJECTIVE: Several studies reported that the levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells. METHOD: The effects of TNF-alpha, IL-1beta, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity. RESULTS: TNF-alpha and IL-1beta, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF-alpha (200 pg/ml) and IL-1beta (150 pg/ml) produced DNA ladders at 24-72 h. TNF-alpha but not IL-1beta cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24-72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 micromol/l significantly prevented TNF-alpha-induced cell detachment (p<0.05). DISCUSSION: TNF-alpha induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1beta decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1beta may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-alpha.
Subject(s)
Cerebrovascular Circulation/drug effects , Cytokines/toxicity , Endothelial Cells/drug effects , Animals , Blotting, Western , Capillaries/cytology , Capillaries/drug effects , Caspase 3 , Caspase Inhibitors , Caspases/metabolism , Cattle , Cell Count , Cell Line , Cell Survival/drug effects , DNA Fragmentation/drug effects , Enzyme Inhibitors/pharmacology , Interleukin-1/toxicity , Interleukin-6/toxicity , Interleukin-8/toxicity , Poly(ADP-ribose) Polymerases/metabolism , Subarachnoid Hemorrhage/pathology , Tumor Necrosis Factor-alpha/toxicityABSTRACT
A substantial number of rat models have been used to research subarachnoid hemorrhage-induced cerebral vasospasm; however, controversy exists regarding which method of selection is appropriate for this species. This study was designed to provide extensive information about the three most popular subarachnoid hemorrhage rat models: the endovascular puncture model, the single-hemorrhage model, and the double-hemorrhage model. In this study, the basilar artery and posterior communicating artery were chosen for histopathological examination and morphometric analysis. Both the endovascular puncture model and single-hemorrhage model developed significant degrees of vasospasm, which were less severe when compared with the double-hemorrhage model. The endovascular puncture model and double-hemorrhage model both developed more vasospasms in the posterior communicating artery than in the basilar artery. The endovascular puncture model has a markedly high mortality rate and high variability in bleeding volume. Overall, the present study showed that the double-hemorrhage model in rats is a more suitable tool with which to investigate mechanism and therapeutic approaches because it accurately correlates with the time courses for vasospasm in humans.
