Gonadal steroids block the calpain-1-dependent intrinsic pathway of apoptosis in an experimental rat stroke model

Abstract

Objectives: Apoptosis plays an important role in the progression of the ischemic penumbra after reperfusion. Estrogen and progesterone have neuroprotective effects against ischemic brain damage, however the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In this study, we investigated the possible regulatory effects of a combined steroid treatment on extrinsic and intrinsic apoptotic signaling pathways after cerebral ischemia.

Methods: Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAO) using an intraluminal filament technique for 1 h followed by 23 h reperfusion. Estrogen and progesterone were immediately injected after tMCAO subcutaneously. Sensorimotor functional tests and the infarct volume were evaluated 24 h after ischemia. Protein expression of calpain-1 and Fas receptor (FasR), key members of intrinsic and extrinsic apoptosis, were determined in the penumbra region of the ischemic brain using western blot analysis, immunohistochemistry, and TUNEL staining.

Results: Neurological deficits and infarct volume were significantly reduced following hormone therapy. Calpain-1 up-regulation and caspase-3 activation were apparent 24 h after ischemia in the peri-infarct area of the cerebral cortex. Steroid hormone treatment reduced infarct pathology and attenuated the induction of both proteases. FasR protein levels were not affected by ischemia and hormone application.

Conclusion: We conclude that a combined steroid treatment inhibits ischemia-induced neuronal apoptosis through the regulation of intrinsic pathways.

Keywords: Apoptosis; CBF, Cerebral blood flow; CCA, Common carotid artery; CNS, Central nervous system; Calpain-1; DISC, Death inducing signaling complex; Estrogen; FasR, Fas receptor; GFAP, Glial fibrillary acidic protein; HRP, Horseradish peroxidase; I/R, Ischemia/reperfusion; ICA, Internal carotid artery; IHC, Immunohistochemistry; MCA, Middle cerebral artery; MCAO, Middle cerebral artery occlusion; NeuN, Neuronal nuclear antigen; PBS, Phosphate-buffered saline; PU, Perfusion units; PVDF, Polyvinylidene fluoride; Progesterone; RIPA, Radioimmunoprecipitation assay; ROS, Reactive oxygen species; SDS, Sodium dodecyl sulfate; TBI, Traumatic brain ischemia; TNF, Tumor necrosis factor; TTC, Triphenyltetrazolium chloride; TUNEL, Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-end labeling; tMCAO; tMCAO, transient middle cerebral artery occlusion.