In the present study we demonstrated that exposure to 0.75% isoflurane for 6 h can significantly increase the level of activated caspase-3 in the hippocampus and cortex of P7 rats, and cause long-term cognitive impairment when they reached adulthood. However, administration of NaHS (a H2S donor) was found to decrease isoflurane-mediated neuroapoptosis and improve cognitive impairment.
Based on the British Journal of Anaesthesia, Salzburg Seminar, anesthesia neurotoxicity is influenced by many critical factors, such as the time and degree of the developing brain, the specific anesthetic drugs, the health status of the pups and specific procedure used for exposure to anesthetics [
30]. The reason we chose the brain during the growth period is that it is susceptible to acute neural injuries, including by anesthetics. A previous study showed that P14 mice exposed to 1.7% isoflurane for 35 min daily for 4 successive days caused persistent, progressive memory dysfunction and reduced neurogenesis in young rodents, but not in P60 mice [
31]. By contrast, H
2S in the central nervous system is thought to have anti-apoptotic, anti-inflammatory and anti-oxidative benefits in mouse models of ischemia/hypoxia, Alzheimer’s disease and Parkinson’s disease [
32]. Thus, we felt that it would be therapeutically meaningful to investigate whether H
2S is beneficial against the neurotoxic effects of anesthetics such as neuroapoptosis and cognitive impairment.
Our data show that administration of NaHS decreased isoflurane-induced cleaved caspase-3 levels, which is a well-established biomarker of apoptosis, in the developing rat brain. These results suggest that the mechanism for H
2S-induced neuroprotection occurs via activation of intrinsic or extrinsic anti-apoptotic pathways. However, this still needs further investigation. Fortunately, some previous findings seem to support our results. For example, it has been reported that endogenous H
2S is a neuromodulator and it can mediate membrane ion channels, such as ATP-sensitive potassium (K
ATP) channel and voltage-dependent calcium channels (VDCCs). The activation of K
ATP would hyperpolarization of neurons, reduce abnormal excitatory synaptic activity and are neuroprotective [
33]. H
2S can also indirectly inhibits L-type VDCCs currents in isolated mouse pancreatic β-cells [
34] and rat cardiomyocytes [
35], even another study in cerebellar granule neurons reported that H
2S increased intracellular calcium ion concentration by stimulating L-type VDCCs [
36]. Moreover, it can potentiate the effect of GABA and regulate stimulation of synaptic glutamate by astrocytes [
37]. Interestingly, isoflurane can enhance and open GABA
A receptor channels directly [
38] and open VDCCs. This increases intracellular calcium ion concentrations, which promote the activation of caspase-3 [
11] or activate the mitochondrial apoptosis pathway [
39]. Evidence demonstrate that H
2S had anti-apoptotic effects via inhibition the forming and opening of mitochondrial permeability transition pores and the subsequent release of cytochrome C from mitochondria to the cytosol, as well as the activation of the caspase cascades [
40]. H
2S could protect neurons from oxidative stress via scavenging of reactive oxygen and/or nitrogen species (ROS and RNS) [
41] and increasing production of glutathione which is a major and potent intracellular antioxidant [
42]. H
2S may also upregulate endogenous antioxidants through a nuclear-factor- E2-related factor-2 (Nrf2) dependent signaling pathway [
43]. Evidence also finding H
2S may stimulate glutamate transport function to protect brain from oxidative stress [
44]. On the contrary, many anesthetics, even under normoxic conditions, can increase ROS, which cause neuronal lipid peroxidation and neuronal death in vulnerable brain regions such as the subiculum in the hippocampus [
45]. H
2S can also regulation intracellular signaling pathway to inhibit neuronal apoptosis. It can inhibits rotenone induced apoptosis via regulation of p38- and JNK-MAPK signaling pathway [
40] and suppress H
2O
2-induced ERK 1/2 activation in primary cultured astrocytes [
44]. H
2S also acted via cAMP-mediated PI3K/Akt/p70S6K signal transduction pathways to inhibit hippocampal neuronal apoptosis and protect neurons from oxygen glucose deprivation/reoxygenation induced injury [
46]. H
2S also downregulates cytokines, such as tumor necrosis factor-α and interleukin-6 [
47]. However, it has been found that clinically relevant isoflurane levels can increase the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β and thus may cause neuro-inflammation [
48]. However, further research is needed to determine how H
2S is neuroprotective. As mentioned before, H
2S donors have shown beneficial therapeutic effects in neurodegenerative disease models. Some research also found that the brain levels of H
2S in Alzheimer’s disease are lower than age-matched healthy controls [
49], and that H
2S attenuates deficits in cognition induced by ischemic stroke and surgical trauma [
50,
51]. Therefore, H
2S may be beneficial for extended use of anesthetics in maintaining long-term neurocognition. There are some reports on the causal link between anesthesia-induced neuroapoptosis and anesthesia-induced cognitive impairment. Studies have shown neuronal loss and perturbation of synaptic proteins are linked to cognitive ability [
52], and neurogenesis is thought to play an important role in memory and learning in the hippocampus. Thus excessive apoptosis can affect the development of the central nervous system and even affect its physiological function [
53]. In our study, the reduction of neuroapoptosis correlated with more pronounced memory impairment. The neural mechanisms of learning and memory depend not only on the structural plasticity of the central nervous system but also on the integrity of the neural network. Therefore, neuroapoptosis may affect the integrity of the neural network, thus impairing learning and memory [
54,
55]. Our data suggest that reduced neuroapoptosis during brain development is important for cognitive development. However, Zhu et al. showed that P14 rats exposed to isoflurane for 4 consecutive days (35 min a day) did not have increased numbers of TUNEL-positive cells and active caspase-9 compared with the control group, or changes in synapsin I density, but did have impairment in reversal learning in rats [
31]. These findings suggest some unknown mechanism of neurotoxicity by inhalation anesthetics, which cause cognitive deficits or cell death.