Targeting C/EBP homologous protein with siRNA attenuates cerebral vasospasm after experimental subarachnoid hemorrhage

Abstract

Endothelial apoptosis plays a major role in the development of cerebral vascular spasm after subarachnoid hemorrhage (SAH). C/EBP homologous protein (CHOP) orchestrates apoptosis in a variety of cell types in response to endoplasmic reticulum (ER) stress, implicated in the brain injury after SAH. However, the role of CHOP in the mechanism of cerebral vasospasm (CVS) after SAH remains unexplored. The aim of this study was to evaluate the effect of CHOP silencing on endothelial apoptosis and CVS following subarachnoid hemorrhage in the rat. The study was conducted on 65 rats and employed endovascular perforation model of SAH. CHOP siRNAs were injected 24 h prior to the hemorrhage. At 72 h after SAH brains with basilar arteries (BA) were collected from euthanized rats for laboratory investigations. Triple fluorescence stain revealed expression of CHOP in cerebral vascular endothelia after SAH. Marked reduction of CHOP protein and the reduction of its downstream signaling effectors, bim and caspase-3, were found in BA with Western blot analysis. CHOP silencing reduced number of apoptotic endothelial cells in BA, and increased BA diameter after SAH. The amelioration of CVS was associated with reduced neuronal injury in cerebral tissues. In conclusion, CHOP siRNA treatment can effectively combat apoptotic mechanisms of cerebral vasospasm set in motion by subarachnoid bleeding.

Figure 1

Western blot analysis of apoptotic proteins in the vascular tissues of basilar artery. CHOP protein was effectively eliminated with siRNA (panel A). Suppressed bim protein level accompanied CHOP decrease (panel B). Bcl-2 protein level showed increase in the group treated with CHOP siRNAs (panel C). Reduction of cleaved caspase-3 in BA of rats treated with CHOP siRNAs (panel D). Panels A-D: n=6 in each group (^&p<0.05 vs. sham; ^#p<0.05 vs. SAH, ANOVA).

Figure 2

Triple fluorescence of TUNEL (panel A), VWF (panel B) and CHOP (panel C) in the basilar artery after SAH. Merged images (panel D) demonstrate that CHOP colocalizes with TUNEL in endothelial cells of the basilar artery. (n=4 sham group; n=6 in other groups).

Figure 3

CHOP siRNA ameliorates narrowing of the basilar artery and reduces its thickness after SAH. Top panels show representative photographs of BA cross sections. The graph in the panel B shows a statistically significant reduction of BA diameter after CHOP silencing. Middle panels and a graph in the panel C illustrate reduced thickness of BA with either of siRNA treatments. Bottom row of histological panels show reduction of endothelial apoptosis consequent to CHOP silencing. Cell count analysis confirmed a significant reduction in the number of apoptotic endothelial cells with CHOP elimination (panel D) (^&p<0.05 vs. sham; ^#p<0.05 vs. SAH, ANOVA). (n=4 sham group; n=6 in other groups).

Figure 4

Reduced neuronal injury accompanies reduced vasospasm with CHOP siRNA treatment. Representative photographs (panel A) and quantitative cell count in the hippocampus (panel B) and in the subcortex (panel C) show reduced number of damaged neurons with the treatment (^&p<0.05 vs. sham; ^#p<0.05 vs. SAH, ANOVA). (n=4 sham group; n=6 in other groups).