Abstract
Estetrol (E4) has strong antioxidative, neurogenic and angiogenic effects in neural system resulting in the attenuation of neonatal hypoxic–ischemic encephalopathy. We aimed to define the role of estrogen receptors in E4-dependent actions in neuronal cell cultures and prove the promyelinating effect of E4. In vitro the antioxidative and cell survival/proliferating effects of E4 on H2O2-induced oxidative stress in primary hippocampal cell cultures were studied using different combinations of specific inhibitors for ERα (MPP dihydrochloride), ERβ (PHTTP), GPR30 (G15) and palmytoilation (2-BR). LDH activity and cell survival assays were performed. In vivo the promyelinating role of different concentrations of E4 (1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 50 mg/kg/day) was investigated using the hypoxic–ischemic brain damage model in the 7-day-old immature rats before/after the induction of hypoxic–ischemic insult. Myelin basic protein (MBP) immunostaining was performed on brain coronal sections. Our results show that LDH activity is significantly upregulated in cell cultures where the E4’s effect was completely blocked by concomitant treatment either with ERα and ERβ inhibitors (MPP and PHTPP, respectively), or ERα and ERβ inhibitors combined with 2-BR. Cell survival is significantly downregulated in cell cultures where the effect of E4 was blocked by ERβ inhibitor (PHTTP) alone. The blockage of GRP30 receptor did affect neither LDH activity nor cell survival. MBP immunostaining is significantly upregulated in E4-pretreated groups at a concentration of 5 mg/kg/day and 50 mg/kg/day E4, whereas the MBP-positive area OD ratio is significantly increased in all the E4-treated groups. E4’s antioxidative actions mostly depend on ERα and ERβ, whereas neurogenesis and possibly promyelinating activities might be realized through ERβ.
Highlights
Neonatal encephalopathy is mainly triggered by perinatal hypoxic–ischemic brain injury and accompanied by neurodevelopmental deficits such as learning disabilities, mental retardation and hearing and visual impairments
Similar pattern of activities was observed when E4 was used in combination with PHTTP alone, and only the combined use of E4 with MPP and PHTTP completely blocked the E4-dependent effect by increasing the lactate dehydrogenase (LDH) activity to the same levels than that in the H2O2treated cells (Fig. 1B)
In the presence of 2-BR, the LDH activity was significantly lower than that in H2O2-treated cell cultures, in those treated by E4 alone, suggesting that the inhibition of palmitoylation even potentiated the E4-dependent antioxidative effect
Summary
Neonatal encephalopathy is mainly triggered by perinatal hypoxic–ischemic brain injury and accompanied by neurodevelopmental deficits such as learning disabilities, mental retardation and hearing and visual impairments. Two recent clinical trials provided updated information on mortality and neurodevelopmental outcomes in infants with moderate and severe HIE as follows: 23–27% of infant mortality was recorded prior to discharge from the neonatal intensive care unit (NICU) and 37–38% of mortality at follow-up 18–22 months later (Gluckman et al 2005, Shankaran et al 2005). The neurodevelopmental outcome at 18 months included mental and psychomotor development retardation, cerebral palsy (CP), epilepsy, blindness and hearing impairment (Gluckman et al 2005, Shankaran et al 2005). No medical treatment provides important neuroprotection against neonatal HIE
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