Deleterious effects of dihydrotestosterone on cerebral ischemic injury

Outcome from cerebral ischemia is sexually dimorphic in many experimental models. Male animals display greater sensitivity to ischemic injury than do their female counterparts, however the underlying mechanism is unclear. The present study determined if the potent and non-aromatizable androgen, dihydrotestosterone (DHT), exacerbates ischemic damage in male rats and alters post-ischemic gene expression after middle cerebral artery occlusion (MCAO). Adult male rats were castrated and hormone-replaced with either testosterone (T) or DHT (50 mg subcutaneous implant) one week before MCAO. 2h MCAO was achieved by intraluminal filament techniques and uniformity of MCAO was confirmed by laser Doppler flowmetry. The effect of DHT on cerebral infarct damage was quantified at 22h reperfusion by 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) histology and compared to that of T. Micropunches (1 mm diameter) from cortical peri-infarct zones and corresponding contralateral tissue were isolated and analyzed for early gene expression at 6 h of reperfusion using DNA microarrays (Affymetrix). Quantitative real-time RT-PCR (qPCR) was used for subsequent confirmation of differential expression pattern of selected gene candidates, including anti-apoptosis gene bcl-2 and pro-apoptosis gene bax. As expected, castration reduced plasma T and DHT to below the level of assay detection (Radioimmunology Assay), while hormone replacement restored total T and DHT plasma levels (37±7 and 3±1 ng/ml, respectively) to the physiologically relevant ranges. Castrated males (CAST) sustained smaller damage than gonadally intact males (Intact) or DHT-treated animals. (Cortex: CAST 15±6% of ipsilateral structure; Intact 34±4%; DHT 42±4%; Striatum: CAST 43±6 %; Intact 75±5%; DHT 88±4%). T increased infarction in striatum (82±5%) as compared to CAST, but not in cortex. Microarray studies identified transcripts that were altered by DHT alone or DHT with MCAO/ 6 h reperfusion. In DHT-treated animals, 422 genes/ ESTs were increased by MCAO and 358 were decreased. In castrated animals, 271 genes/ESTs were increased and 350 were depressed by ischemia. When ischemic cortex was compared between DHT and CAST groups (DI-CI), 421 genes were increased by DHT treatment and 239 genes were decreased. Functional analysis of the genes with differential expression patterns between DI-CI indicated that a larger number of genes were elevated by DHT vs castration after ischemia, many of which could account for cell death through enhanced inflammation, dysregulation of blood-brain barrier and the extracellular matrix, apoptosis, and ionic imbalance. We used qPCR and immunoblotting to confirm the microarray data. Seven of ten gene candidates were confirmed by qPCR and the differential expression pattern of cyclooxygenase-2 was additionally confirmed by immunoblotting. By using qPCR techniques we also found that intact and DHT groups demonstrated higher levels of cortical bax mRNA relative to CAST at 22h reperfusion, but bcl-2 was similar in all groups. Our data suggest that DHT is an important androgenic mediator of ischemic damage in male brain and that transcriptional mechanisms should be considered as we seek to understand innate male sensitivity to cerebral ischemia.