We further evaluated the possibility whether
We further evaluated the possibility whether letrozole mediates its effect through diverting the pathway towards 5α-DHT and further to 3α-Diol. For this purpose, we measured the concentrations of 5α-DHT and 3α-Diol in mice hippocampus and found that letrozole elevated both 5α-DHT and 3α-Diol levels in the hippocampus. Further, both finasteride and indomethacin ameliorated letrozole-induced enhancement of 5α-DHT and 3α-Diol. This indicated that aromatase inhibition by letrozole redirected testosterone for the synthesis of 5α-DHT and 3α-Diol as evidenced by the enhanced concentrations of 5α-DHT and 3α-Diol whereas finasteride and indomethacin reversed the elevation of 3α-Diol. Thus, letrozole demonstrates anticonvulsant activity probably by redirecting testosterone to 5α-DHT and 3α-Diol. The later acts as a positive allosteric modulator of synaptic and extrasynaptic GABAA receptors. The activation of GABAA receptors by several ligands leads to an influx of chloride ions; as a result hyperpolarization of the neuronal membrane occurs, which dampens the neuronal excitability (Reddy and Rogawski, 2002). In our study, the effect of letrozole on KA-mediated neurotoxicity was investigated using cresyl violet staining. Many pieces of evidence indicate that androgens influence on neurogenesis. In a study, it has been demonstrated that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism (Spritzer and Galea, 2007). Various studies provide evidence for the neuroprotective action of testosterone, mediated by its conversion to 5α-DHT and 3α-Diol (Frye et al., 1996). On the contrary, there are also reports on the neuroprotective action of 17β-estradiol by increased aromatase expression (Pietranera et al., 2010; Veliskova and Velisek, 2007). Numerous studies have demonstrated that estradiol exerts trophic actions on neurons and glial (+)-Apogossypol sale promote neuronal survival and decreases neurodegenerative damage (Azcoitia et al., 2011). By this logic, reducing estradiol levels in an epileptic patient might enhance seizure-associated neurotoxicity in the brain. To investigate the same, we evaluated the effect of letrozole on KA-induced neuronal degeneration in the hippocampus. KA provokes behavioral seizures and produces lesions in CA1, CA3 and DG region of the hippocampus, evocative of TLE in humans (Ben-Ari, 1985). KA-induce neuronal injury extremely impacts the hippocampus. A large number of kainate receptors exist in the hippocampus, primarily in CA3b sector of pyramidal layer, which makes the hippocampus susceptible to KA-induced neuronal damage (Darstein et al., 2003; Gualtieri et al., 2012; Lee et al., 2009). KA triggered neuronal damage involves an influx of cellular Ca2+, efflux of glutamate, production of reactive oxygen species leading to mitochondrial dysfunction (Lauri et al., 2001), endoplasmic reticulum stress and fragmentation of the endoplasmic reticulum membrane directing to neuronal apoptosis and necrosis (Sokka et al., 2007). KA-treated mice exhibited pyramidal cells with pyknotic nuclei, vacuolation and neuronal loss in CA1, CA3 and DG regions of the hippocampus. We found that letrozole treatment also showed pyknosis, histopathological changes comparable to KA. Letrozole did not improve morphological outcomes in the hippocampus of mice despite protection against KA-induced behavioral seizures. Although, no significant neuroprotection was found in the hippocampus of mice treated with letrozole; however, it also did not enhance neurodegeneration as compared to KA. These results indicate that letrozole doesn't provide primary neuroprotection which might be due to reduced estradiol synthesis in the brain after KA-induced seizures. Our results are congruent with the previous finding by Sato and Wolley where acute aromatase inhibition did not enhance seizure-induced cell death (Sato and Woolley, 2016). While the reasons for letrozole not enhancing KA-induced neurotoxicity (by reducing estradiol) are not clear at present, it is possible that diversion of pathway to 3α-Diol, which plays a role in neuroprotection possibly by acting on GABAA/benzodiazepine receptor complexes (Frye et al., 1996), activating the mitogen-activated protein kinase (MAPK) pathway to increase hippocampal neurogenesis (Ramsden et al., 2003; Spritzer and Galea, 2007). Thus, the neurotoxic effects of reduced estradiol are probably counterbalanced by enhanced levels of neuroprotective 3α-Diol. However, it is speculative, but we cannot exclude the possibility that estradiol is not always neuroprotective in the brain and some studies report the neurotoxic effects of estrogens. One of the metabolites of 17β-estradiol, 2-methoxyestradiol (2MEOHE2) is neurotoxic and suggesting that endogenous metabolism of 17β-estradiol may counteract its neuroprotective effects (Picazo et al., 2003).