Diazepam neuroprotection in excitotoxic and oxidative stress involves a mitochondrial mechanism additional to the GABAAR and hypothermic effects

The aim of the present investigation was to analyze the molecular mechanism(s) of diazepam neuroprotection in two models of selective neuronal death in CA1 sector of hippocampus: in vivo following transient gerbil brain ischemia and in vitro in rat hippocampal brain slices subjected to glutamatergic (100 μM NMDA) or oxidative (30 μM tertbutyl-hydroksyperoxide (TBH)) stress. In the in vivo model the diazepam treatment (two doses of 10 mg/kg i.p. 30 and 90 min after the insult) resulted in more than 60% of CA1 hippocampal neurons surviving the insult comparing with 15% in untreated animals. To test whether the protective effect of diazepam was due to the postulated drug-induced hypothermia we followed the fluxes of body temperature during postischemic reperfusion: diazepam reduced temperature from 36.6 ± 1 °C to 33.4 ± 2 °C. Equivalent hypothermia induced and maintained in animals after ischemia did not prevent neuronal cell loss to the same extent as diazepam did (42.8 ± 9.2% and 72.4 ± 14.5% of live neurons, respectively). In vitro, under constant temperature conditions, diazepam exerted neuroprotective effects following a “U-shaped” dose–response curve, with concentration efficacy window of 0.5–10 μM. Five micro-molar diazepam showed significant protection by reducing over 50% the number of (dead) propidium iodide labeled cells even in the presence of GABAA receptor antagonist bicuculline. Next, we have shown that diazepam reduced the efflux of cytochrome c out of mitochondria both in compromised CA1 neurons in vitro and in isolated mitochondria treated with 30 μM THB. Our results suggest that the neuroprotective action of diazepam relies on additional mechanism(s) and not solely on its hypothermic effect. We suggest that diazepam evokes neuroprotection through its central receptors located on the GABAA receptor complex and, possibly, through its peripheral receptor, the translocator protein TSPO (previously called the peripheral benzodiazepine receptor) located in the outer mitochondrial membrane.