Abstract
Numerous animal studies have demonstrated that commonly used general anesthetics may result in cognitive impairment in the immature brain. The prevailing theory is that general anesthetics could induce developmental neurotoxicity via enhanced apoptosis. In addition, inhibited proliferation induced by anesthetics has also been reported. So far, whether autophagy, a well-conserved cellular process that is critical for cell fate, also participates in anesthesia-induced neurotoxicity remains elusive. Here, we first examined autophagy-related changes after sevoflurane exposure and the effect of autophagy on apoptosis and proliferation, and we also explored the underlying mechanisms of autophagy activation. Pregnant rats were exposed to 2 or 3.5% sevoflurane for 2 h on gestational day 14 (G14); then, markers of autophagy and expression of autophagy pathway components were measured in fetal brains 2, 12, 24, and 48 h after anesthesia. Changes in neural stem cell (NSC) apoptosis, neurogenesis, neuron quantity and learning and memory function were examined after administration of an autophagy or PTEN inhibitor. The expression of microtubule-associated protein 1 light chain 3 (LC3)-II, Beclin-1 and phosphatase and tensin homolog on chromosome 10 (PTEN) were increased in the 3.5% sevoflurane group, while Sequestosome 1 (P62/SQSTM1), phospho-protein kinase B/protein kinase B (p-Akt/Akt) and mammalian target of rapamycin (mTOR) were decreased. 3-methyladenine (3-MA), an inhibitor of autophagy, or dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate (V) (bpV), a PTEN inhibitor, significantly attenuated the activation of autophagy, reversed the decreased expression of B-cell lymphoma-2 (Bcl-2) and reduced the number of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) positive cells, ameliorated the decline of Nestin expression, Ki67 positive cell rate, neuron quantity and cross platform times, and shortened the prolonged escape latency. Our results demonstrated that 2 h 3.5% sevoflurane exposure at G14 induced excessive autophagy in the fetal brain via the PTEN/Akt/mTOR pathway. Autophagy inhibition reversed anesthesia-induced NSC apoptosis, proliferation decline and memory deficits.
Highlights
Advanced surgical technologies have made it possible to conduct more fetal intervention operations
In the brain sections from the 3.5%SEV group, LC3B co-localized with Nestin presenting punctate pattern, whereas brain sections from both the CON group and 2% sevoflurane (2%SEV) group showed diffused green light labeling. These results indicated that sevoflurane activated autophagy in neural stem cell (NSC) after exposure to 3.5% sevoflurane; and there were no differences observed between the 2%SEV group and CON group (Figure 2C)
Besides the role of regulating autophagy, we observed a decline in the number of TUNEL positive cells and an increase in both Ki67 positive cell rate and Nestin expression in the 3.5%SEV + bpV group compared with the 3.5%SEV group (Figure 5, TUNEL: P < 0.05; Ki67: P < 0.001; Nestin: FIGURE 3 | 3.5% sevoflurane exposure upregualted autophagy via phosphatase and tensin homolog on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway. 3.5% sevoflurane upregualted PTEN expression and inhibited p-Akt/Akt and mTOR expression in NSCs 2, 12, 24, 48 after anesthesia (A,B)
Summary
Advanced surgical technologies have made it possible to conduct more fetal intervention operations. The results of the study by Wang et al suggested that exposure of G14 rats to 3 or 4% sevoflurane anesthesia showed a negative effect on the learning and memory abilities of their offspring, and both inhibited proliferation and increased apoptosis of nerve cells were revealed (Wang et al, 2017). To gain insight into the role of autophagy in the anesthestic neurotoxicity during the early stage of brain development, we utilized the embryonic day 14 (E14) rat model since the midpregnancy is considered safe for fetal intervention (Palanisamy, 2012). We hypothesized that 3.5% sevoflurane could lead to overactive autophagy, which is responsible for the increased apoptosis, decreased proliferation and impaired neurocognitive function during this period of pregnancy
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