There have been many investigations of anesthesia-induced neurotoxicity in the developing brain of neonates in animals and humans. Sevoflurane is the most commonly used inhalation anesthetic.
Here, it was shown that sevoflurane exposure elicited neurotoxicity in hippocampal neurons and tissues. Also, it was also shown that the blockade of apoptosis did not attenuate SIN and cell viability. Furthermore, it was revealed that RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons, and that the blockade of apoptosis enhanced necroptosis. Finally, it was found that inhibition of apoptosis and necroptosis rescued SIN. These data offer potential new ideas indications for clinical anesthesia regarding the prevention of neurotoxicity caused by anesthesia exposure in neonates.
Infants are administered general anesthesia for diagnostic and surgical procedures. The safety of anesthesia in neonates and infants is controversial. Accumulating evidence from animal and clinical studies (Wilder, et al. 2009; DiMaggio, et al. 2011; Ing, et al. 2012; Hansen, et al. 2011; Sun. 2010.) has suggested that exposure to anesthetics in the neurodevelopmental period can result in neurotoxicity and learning difficulties (Deng, et al. 2014; Zou, et al. 2011.). Sevoflurane exposure has been shown to lead to neuronal apoptosis and pathologic alterations in the hippocampus of neonatal rats (Feng, et al. 2012; Zhang, et al. 2008; Satomoto, et al. 2009.). However, the mechanisms underlying SIN are largely unknown.
Importantly, it was found that, except for apoptosis, necroptosis (a novel manner pathway of cell death) was involved in the mechanisms underlying SIN. It was also found that necroptosis might act as an “insurance policy” if apoptosis is inhibited. To prevent SIN, inhibiting apoptosis alone was demonstrated to be insufficient in our study. Blockade of apoptosis and necroptosis may provide new thinking for preventing SIN in neonates. The "sev + Nec-1 group" was not included in our study because the main object of our research was to study the relationship between necroptosis and SIN. However, in Sevoflurane + Nec-1 group, necroptosis was inhibited, the apoptosis was still induced, it could only prove that the apoptosis was involved in SIN which has already been confirmed in the previous studies (Dong Y, et al. 2009; Lu Y, et al. 2010.).
Necroptosis is a newly confirmed type of programmed cell death. It is essential for normal embryonic development, chronic intestinal inflammation, and T-cell proliferation (Peter. 2011; Kaiser, et al. 2011.). Necroptosis is different from apoptosis, since it is a caspase-independent process. Also, It is different from conventional necrosis because necrosis is a type of unexpected cell death whereas necroptosis is a tightly controlled process of cell death. Necroptosis is largely dependent on RIPK1/RIPK3 and MLKL, and formation of RIPK1/RIPK3 complexes is essential for necroptosis. Necroptosis can be blocked by the RIPK1 inhibitor Nec-1. It was shown that the inhibition of apoptosis significantly increased RIPK1/RIPK3 expression and enhanced the binding of RIPK1/RIPK3, which indirectly demonstrates demonstrated the existence of necroptosis.
Studies have shown that necroptosis may act as an alternate pathway if apoptosis or necrosis are inhibited. For example, Moon and colleagues have investigated on the regulation of necrosis and apoptosis after spinal-cord injury (Moon, et al. 2012). They found that the protection against the death of neurocytes via inhibition of necrosis and apoptosis is limited. Additionally, Liu and colleagues indicated that necroptosis contributed to the death of nerve cells and affected functional outcomes in mice that with suffered spinal-cord injuries (Liu, et al. 2015). Inhibiting necroptosis via Nec-1 administration may serve as a feasible therapeutic strategy to treat spinal-cord injury. In this study, the same situation was applied in SIN.
It was found that apoptosis and necroptosis coexisted in SIN and that necroptosis may act as an alternate cell death pathway if apoptosis is inhibited. It was hypothesized that the inhibition of apoptosis and necroptosis may rescue SIN. Hence, the pan-caspase inhibitor Z-VAD and RIPK1 inhibitor Nec-1 were introduced to test this hypothesis.
The MTT assay in vitro assay showed that neuron viability was rescued to a more closely observed level in the control group compared to the SEV + Z-VAD + Nec-1 group (Fig. 1B). H&E staining of brain tissue in vivo showed that the SEV + Z-VAD + Nec-1 group was similar to control group (Fig. 2D). The number of cells that underwent necroptosis and apoptosis was counted in three microscopic fields: the SEV + Z-VAD + Nec-1 group showed no significant difference compared to the control group (Fig. 2E, Fig. 2F). In terms of expression and binding of RIPK1/RIPK3, the SEV + Z-VAD + Nec-1 group showed obviously low RIPK3 expression compared with that in the SEV + Z-VAD group, but no significant difference in expression of RIPK1 protein was noted. Co-IP showed that the binding capacity of the RIPK1/RIPK3 protein returned to that observed in the control group in the SEV + Z-VAD + Nec-1 group (Figure. 4). Taken together, the results shown above suggest the inhibition of apoptosis alone provided only a limited effect against SIN, whereas inhibition of apoptosis and necroptosis played an important protective role.
The study had four main limitations. First, due to sevoflurane diverse effects, it is difficult to distinguish key factor leading to its neurotoxicity properties. Second, the other effects of sevoflurane were not detected, such as anesthesia-related cerebral anoxia, which may have affected the results. Therefore, further experiments are needed to explore the roles of these effects. Third, other brain regions related to learning and memory (e.g., the amygdala and striatum) were not included in this study, but should can be considered in future research.. Finally, animal experiments cannot be directly converted into clinical conclusions applied to humans.