Figure 7. PAR-1 knockdown mediates neuroprotection in primary cultured neurons subjected to oxygen/glucose deprivation.

Primary cultured neurons infected with PAR-1 misRNA or shRNA for 24 hours and then grown for 5 or 6 days until at least 80% of cells were infected. The cells were divided into OGD (2 hours) or non-OGD groups at random. During OGD or non-OGD 50U/ml thrombin was added to the wells. The media was removed 2 hr and 24 hr after OGD followed by LDH assay to estimate cell death using spectrophotometric analysis at 492 nm. Data is presented as percentage mean±SE from three different experiments, each carried out in triplicate. Cells infected with PAR- 1 shRNA showed decreased neuronal cell death after OGD or without OGD with thrombin treatment in comparison to PAR-1 misRNA infected cells. PAR-1 knockdown significantly rescued the cells exposed to OGD with or without thrombin treatment compared to misRNA. Inset: Primary cultured cells were treated with increasing concentration of thrombin (1 U/ml to 100U/ml) for 2 hours and replaced with fresh medium after treatment. Medium withdrawn at 2 hours and 24 hours was used for LDH assay to determine percentage LDH release. Thrombin dose above 10U/ml showed a significant increase in cell death at 24 hours. Mean±SE. In both experiments the final results were analyzed using two-way ANOVA and the Bonferroni test for post-hoc comparisons. Significant difference misRNA compared to shRNA: * p<0.05, **p<0.01. Significant difference thrombin compared to control: # p<0.05, ### p<0.01, *** p<0.001