RIPK1/RIPK3-Mediated Necroptosis is Involved in Sevoflurane-Induced Neonatal Neurotoxicity in the Rat Hippocampus

Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. "Necroptosis" is a novel pathway of necrosis. We introduced the caspase-specific inhibitor Z-VAD in addition to the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1, to ascertain the existence and importance of necroptosis. Sprague-Dawley rat pups postnatal day 7 were randomly assigned into one of five groups: control, sevoflurane + Z-VAD, sevoflurane + Nec-1, sevoflurane + Z-VAD + Nec-1 and 3% sevoflurane group. Neuronal apoptosis was evaluated by hematoxylin and eosin staining. The MTT assay was performed to evaluate cell viability. Immunofluorescence was employed to measure expression of RIPK1 and RIPK3. Western blots showing expression of RIPK1, RIPK3 and phosphorylation of mixed lineage kinase domain-like (p-MLKL) were used to explore the role of necroptosis. Binding of RIPK1/RIPK3 was detected via co-immunoprecipitation. Finally, the Morris water maze test was used to determine cognitive function. Exposure to 3% sevoflurane for 6 h induced neurotoxicity and inhibited cell viability. Neuron viability was low in the SEV, SEV + Z-VAD and SEV + Nec-1 groups. The study revealed that RIPK1 and RIPK3 protein expression increased significantly, but there was no significant differences between the SEV and SEV + Z-VAD groups. The expression of p-MLKL significantly increased in the SEV and SEV + Z-VAD groups, but not in the SEV + Nec-1 group or SEV + Z-VAD + Nec-1 group compared to the control group. Co-immunoprecipitation results showed that sevoflurane exposure enhanced binding of RIPK1/RIPK3 protein significantly. Blockade of apoptosis and necroptosis alleviated sevoflurane-induced cognitive impairment. Sevoflurane exposure elicited neurotoxicity within neonatal hippocampal neurons and tissues. Blockade of apoptosis or necroptosis alone did not attenuate sevoflurane-induced neurotoxicity (SIN). RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons. SIN could be attenuated only by inhibiting both apoptosis and necroptosis.