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


CONCLUSIONS
We conclude that a combined steroid treatment inhibits ischemia-induced neuronal apoptosis through the regulation of intrinsic pathways.
a Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.
Zeinab
Z
Vahidinia
a Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.
Nasim
N
Alipour
a Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.
Mohammad Ali
MA
Atlasi
a Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.
Homayoun
H
Naderian
b Faculty of Medicine , Institute of Neuroanatomy, RWTH Aachen University , Aachen , Germany.
Cordian
C
Beyer
a Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.|c Department of Anatomy, School of Medicine , Kashan University of Medical Sciences , Kashan , Iran.
Abolfazl
A
Azami Tameh
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020520:Brain Infarction / Q000188:drug therapy / Q000209:etiology; D002154:Calpain / Q000378:metabolism*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D002560:Cerebrovascular Circulation / Q000187:drug effects; D004195:Disease Models, Animal; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D020244:Infarction, Middle Cerebral Artery / Q000150:complications / Q000188:drug therapy / Q000473:pathology; D017078:Laser-Doppler Flowmetry; D008297:Male; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D018709:Statistics, Nonparametric; D013256:Steroids / Q000627:therapeutic use*
D005904:Glial Fibrillary Acidic Protein; D013256:Steroids; D002154:Calpain; C000606071:Capn1 protein, rat; D010751:Phosphopyruvate Hydratase
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