In the present study, we explored the roles of GABAAR and NKCC1 in myelin development and nerve injury in young mice treated with sevo. Our data suggest that blockade of GABAAR and NKCC1 has good effects on myelin dysfunction and neuronal damage in neonatal mice exposed to sevo, along with an effect of BDNF in this process.
The long-term effects of repeated exposure of infants and young children to sevo in the early postnatal period are of general interest, and many previous preclinical studies have found that sevo can induce neurotoxicity by means of neuronal apoptosis, neuroinflammatory changes, changes in synaptic plasticity, and myelin damage(Feng et al 2012, Liang et al 2021, Wang et al 2013, Yu et al 2020a, Zhu et al 2021). Consistent with previous findings, we found that sevo caused neuronal injury and impaired myelin development in young mice. In this study, we investigated the beneficial effects of blocking GABAAR and NKCC1 to reduce sevo-induced neurotoxicity by TUNEL assay, electron microscopy, and immunofluorescence.
We used a GABAAR antagonist Bic and a NKCC1 antagonist Bum to examine their roles in the neurotoxicity of sevo to the brain. As expected, sevo caused neuronal injury and disturbs myelin development that could be blocked by Bic and Bum, suggesting the presence of GABAAR and NKCC1 mediated effects. The mechanism of sevo-induced neurotoxicity remains unclear at present. Some studies have found that convulsive waves and spikes appeared on EEG during the anesthesia of neonatal rats with sevo(Li et al 2020, Xu et al 2015), while application of the NKCC1 antagonist, Bum, not only caused a significant decrease in the number and duration of convulsive waves and the frequency of spikes but also decreased the level of activated Caspase-3 and restored long-term cognitive impairment induced by neonatal sevo exposure(Edwards et al 2010). One study found that propofol-induced EEG seizures required increased corticosteroid- and GABAAR-mediated excitation(Willis et al 2015). Propofol, with properties similar to sevoflurane, a GABAAR agonist commonly used as an intravenous anesthetic(Sahinovic et al 2018). This suggests that the effects of sevo on developing nerves, after activation of GABAAR, lead to neuronal depolarization/excitation. These findings are consistent with ours and support the notion that GABAAR and NKCC1 play important roles in sevo-induced neurotoxicity.
Oligodendrocytes play a crucial role in myelin formation in the central nervous system (CNS)(Kuhn et al 2019, Nishiyama et al 2021), and sevo exposure during early childhood inhibits oligodendrocyte proliferation and interferes with oligodendrocyte maturation and myelination(Liang et al 2021, Wu et al 2020). Our study showed that bum and Bum reversed sevo-induced decreases in the number of oligodendrocyte precursor cells and oligodendrocytes in young mice, and promoted myelination.
BDNF, a member of the neurotrophin family, is one of the key trophic factors in neurodevelopment and is mainly expressed in the cortex and hippocampus(Wu et al 2021). It has been previously documented that BDNF can act directly on oligodendrocytes to promote myelination(Xiao et al 2010). It has also been demonstrated that blocking GABAAR by Bic in neonatal rats causes an increase in BDNF expression in the adult rat cerebral cortex(Naderipoor et al 2021). At the same time, other studies have shown that exposure to sevo during the early postnatal period induces a significant decrease in BDNF expression levels(Cui et al 2021, Ozer et al 2017, Zhao et al 2022, Zhao et al 2021). We therefore speculated that BDNF is a downstream target of GABAAR and NKCC1. The results were consistent with our speculation that both Bic and Bum ameliorate the sevo-induced decrease in BDNF expression level in the mouse cerebral cortex.
This study has some limitations. First, sex is an important biological variable, and we did not classify mice based on sex, because it is difficult to discern the sex of newborn mice. Second, we only studied the neurotoxicity changes at P14 after multiple sevo exposures in young mice, and did not study the behavioral changes such as long-term neurotoxicity and cognitive function, which we plan to analyze in future research.