Abstract

Introduction

Recent experimental evidence and emerging clinical trial data indicate that extensively-used general anesthetics in pediatric surgery could be detrimental to the developmental brain, especially at the burst of brain growth [1-5]. The neurodegenerative effects are present as acute neuronal apoptosis and long-lasting alteration in synaptogenesis and cognitive impairment [4,6-8]. Therefore, most of the recent investigations are engaged in elucidating the precise mechanism of anesthesia-induced developmental neurotoxicity so that a targeted and efficient prophylactic measurement can be contrived.

Based on published data, mitochondria are likely to be the integrated hub of general anesthetics-induced harmful signals. It seems this organelle is the initial and the most vulnerable organelle impaired by anesthesia in the brain of neonatal rat [7,9,10]. Sole or combined general anesthetics caused excessive mitochondrial fission even fragmentation, which may contribute to anesthetics-induced neurodegenerative alterations in the developmental brain by disrupting the balance between mitochondrial fusion and fission [6,11,12]. Further investigation showed that general anesthetics promoted dynamin-related protein (Drp)1 oligomerization and translocation from cytosol to mitochondria, which is associated with a disturbed mithochondrial shape [4]. It is noteworthy that Drp1-mediated disproportionate mitochondrial fission is widely involved in neuronal damage in stroke, traumatic brain injury, seizure as well as neurodegenerative diseases [13-18]. Based on these evidence, targeting Drp1 is likely to pharmaceutically beneficial in clinical practice.

Theoretically, measurements that can inactivate Drp1 or prevent Drp1 activation and/or translocation might be effective, at least partially, in mitigating anesthesia-induced developmental neurotoxicity. Mitochondrial division inhibitor-1 (mdivi-1) is a highly selective and efficient inhibitor of Drp1. Mdivi-1 bound to an allosteric site of Drp1 and impeded its conformational change that was required for self-assembly and GTP hydrolysis so that prevents mitochondria fission and formed inter-connected net-like mitochondria [19]. It was further demonstrated that mdivi-1 mitigated apoptosis via inhibiting Drp1 activity and mitochondrial outer membrane permeabilization (MOMP) and hence retarded the release of cytochrome C into cytosol. Recent investigations found that mdivi-1 provided widespread protection against various insults to brain, heart and kidney [17,18,20-22]. Furthermore, it is proven that mdivi-1 mitigated mitochondrial dysfunction and synaptic impairment in Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [23-25]. Interestingly, a current study revealed that mdivi-1 inhibited propofol- or sevoflurane-induced neurotoxicity in vitro [6,26]. These evidence paved mdivi-1 an attractively therapeutic avenue for clinical treating cerebral injuries, and also inspire us to examine the influence of mdivi-1 on the long-lasting cognitive dysfunction induced by isoflurane (an inhalational anesthetic, more neurotoxic than sevoflurane to the developmental brain [27]).

Therefore, we hypothesize that mdivi-1 alleviates isoflurane-induced cognitive dysfunction by inhibiting Drp1-mediated excessive mitochondrial fission and subsequently neuroapoptosis and synaptic injury. In this study, we found that isoflurane exposure downregulated the phosphorylation level of Drp1 (ser656) and facilitated the translocation of Drp1 and Bax to mitochondria. However, mdivi-1 pretreatment inhibited Drp1 activation and translocation, mitigated mitochondrial apoptotic pathway and improved the long-term cognitive performance during later life of rats neonatally exposed to isoflurane.

Materials and methods

Results

Mdivi-1 improved spatial memory of rats undergoing neonatal exposure to isoflurane

To examine whether mdivi-1 provided neuroprotective effect against neonatal isoflurene anesthesia, the test of MWM was explored to assess the spatial learning memory at PND60 after drug treatment. As shown in Figure 1, isoflurane exposure damaged spatial as shown by longthening escape latency at training day 3 (Figure 1A, P<0.05), training day 4 (Figure 1A, P<0.05) and training day 5 (Figure 1A, P<0.05) compared with CON group. As expected, mdivi-1 premedication mitigated isoflurane-induced defect in spatial memeory as indicated by shortening escape latency (Figure 1A, P<0.05) at training day 4 (Figure 1A, P<0.05) and training day 5 (Figure 1A, P<0.05) compared with ISO group.

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Figure 1

Mdivi-1 (20 mg/kg, i.p.) pretreatment mitigated cognitive deficit induced by neonatal exposure to isoflurane (2% for 2 h). A. Mean latency plotted against the training days in the spatial acquisition trials. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test (n=10 per group). ISO VS MDV+ISO, P<0.05. B. Percentage of time spent in target quadrant in probe test. Data are presented as mean ± SEM (n=10 per group) CON: control group; ISO: isoflurane group; MDV+ISO: mdivi-1+isoflurane group; MDV: mivi-1 group. *denotes P<0.05 CON vs ISO, #denotes P<0.05 ISO vs ISO+MDV or **denotes P<0.01 CON vs ISO.

In the probe trial, isoflurane exposure decreased the percentaget ime in the target quadrant (Figure 1B, P<0.01) compared with CON group. However, mdivi-1 pretreatment significantly improved spatial cognition performance as revealed by greater percentage time in the target quadrant (Figure 1B, P<0.05) compared with ISO group. Taken together, our results suggested that mdivi-1 pretreatment attenuated isoflurane-induced long-time cognitive injury.

Mdivi-1 inhibited Drp1 phosphorylation and translocation from cytoplasm to mitochondria

Previous reports found that de phosphoryaliton of Drp1 at ser656 resulted in its activation while phosphorylation inhibited mitochondrial fission [24]. In our study, we determined the effect of isoflurane on Drp1 phosphorylation. Western blot analysis demonstrated that isoflurane anesthesia decreased the level of ser656 phosphorylation of cytoplasmic Drp1 but did not affect the protein level of total Drp1 (Figure 2A, P<0.01). Importantly, isoflurane exposure promoted translocation of Drp1 and Bax from cytosol to mitochondria (Figure 2B and ​and2C,2C, P<0.01). However, mdivi-1 pretreatment increased the level of Drp1 phosphorylation and inhibited its translocation while did not induce any change of basic level of Drp1 in cytoplasm. In summary, mdivi-1 pretreatment reversed isoflurane-induced Drp1 dephosphorylation and migration.

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Figure 2

Mdivi-1 pretreatment inhibited isoflurane-induced Drp1 dephosphorylation (ser656) and mitochondrial translocation of Drp1 and Bax. (A) A representative graph of western blot analysis of p-Drp1 expressions in hippocampi. The relative protein level of cytosolic p-Drp1 (ser656) in hippocampi was normalized to the total cytosolic Drp1. The statistical analysis showed that application of mdivi-1 (20 mg/kg, i.p.) inhibited the isoflurane-induced dephosphorylation of cytoplasmic Drp1 in the hippocampi of neonatal rats. Relative levels of mitochondrial Drp1 (B) and Bax (C) in hippocampi of developmental rats, normalized to the internal reference Porin. The statistical analysis showed that administration of mdivi-1 (20 mg/kg, i.p.) significantly attenuated isoflurane-induced up-regulation of mitochondrial Drp-1 and Bax in hippocampi of neonatal rats. Data are presented as mean ± SEM (n=5 per group) *denotes P<0.05, **denotes P<0.01.

Mdivi-1 pretreatment attenuated isoflrane-induced the release of cytochrome C and activation of the mitochondrial apoptotic cascade

Based on previous findings that cytochrome C release was an indicator of permeability of the mitochondrial membrane, we attempted to examine whether mdivi-1 pretreatment would mitigate the change. As displayed in Figure 3A (P<0.05), isoflurane resulted in burgeoning release of cytochrome C into cytoplasm, but mdivi-1 pretreatment reduced the leak of cytochrome C from mitochondria. Moreover, mdivi-1 pretreatment curbed the activation of caspase9 (Figure 3B, P<0.05) and the executioner of mitochondrial apoptotic pathway active caspase3 (Figure 3C, P<0.001).

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Figure 3

Mdivi-1 pretreatment attenuated isoflurane-induced the release of cytochrome C and activation of mitochondrial apoptotic cascade. A. The representative immunoblots (the upper) and quantitative graphs (the lower) of cytochrome C. Pretreatment with mdivi-1 (20 mg/kg, i.p.) significantly attenuated isoflurane-induced the release of cytochrome C in the hippocampus of neonatal rats. B. Pretreatment with mdivi-1 (20 mg/kg, i.p.) significantly reduced isoflurane-induced up-regulation of active caspase9 in the hippocampus of neonatal rats. C. Application of mdivi-1 (20 mg/kg, i.p.) significantly reduced isoflurane-induced enhancement of active caspase3 in hippocampi of neonatal rats. Data are presented as mean ± SEM (n=5 per group) *denotes P<0.05 or **denotes P<0.01.

Midiv-1 restored isoflurane-induced excessive mitochondrial fission

Several delicate studies showed that anesthetics-triggered Drp1 activation led to excessive mitochondrial division even fragmentation. Therefore, we set out to determine whether mdivi-1 pretreatment circumvented the pathological morphological changes. As shown in Figure 4A (P<0.01), mitochondria in ISO group was markedly condensed and round-like compared to that in CON group and MDV group, while mdivi-1 pretreatment inhibited these changes in MDV+ISO group. As shown in Figure 4B (P<0.05) and Figure 4C (P<0.05), mdivi-1 pretreatment increased mitochondrial length and size that was reduced by isoflurane exposure.

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Figure 4

Mdivi-1 pretreatment alleviated isoflurane-induced mitochondrial morphological changes. A. The representative images of mitochondrial morphological changes of each group in hippocampi of developmental rats. B. Isoflurane exposure induced a significant decrease of the mitochondria length in hippocampi, whereas the decrease was reversed by the delivery of mdivi-1 (20 mg/kg, i.p.). C. Pretreatment with mdivi-1 (20 mg/kg, i.p.) significantly alleviated isoflurane-induced decrease of the mitochondrial average size in hippocampi of neonatal rats. Data are presented as mean ± SEM (n=5 per group), five neurons in each rats was scanned by transmission electron microscopy. *denotes P<0.05 or **denotes P<0.01.

Mdivi-1 mitigated isoflurane-induced synaptic protein loss

Anesthetics induced synaptic disturbance contributed to the long-term cognitive lesion. Hence we attempted to examine whether mdivi-1 pretreatment mitigated isoflurane-induced synaptic protein loss. Our data showed that isoflurane anesthesia downregulated the expression of PSD95 (Figure 5A, P<0.05) and synapsin I (Figure 5B, P<0.05) in hippocmapi. However, mdivi-1 pretreatment significantly inhibited downregulated levels of PSD95 and synapsin I, as shown in Figure 5A (P<0.05) and Figure 5B (P<0.05).

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Figure 5

Mdivi-1 pretreatment mitigated isoflurane-induced synaptic protein loss in hippocampi of neonatal rat. A. Pretreatment with mdivi-1 (20 mg/kg, i.p.) increased the hippocampal expression of synapsin I that was reduced by isoflurane exposure. B. Prophylactic application with mdivi-1 (20 mg/kg, i.p.) upregulated the expression of PSD95 that was weakened by isoflurane anesthesia in hippocampi. Data are represented as mean ± SEM (n=5 per group), *denotes P<0.05.

Discussion

This study indicated that neonatal isoflurane exposure promoted Drp1 dephosphorylation, an indispensable step in Drp1 conformational change and activation, and translocation from cytoplasm to mitochondria. Furthermore, isoflurane anesthesia facilitated Drp1 and Bax translocation to mitochondria. This alteration resulted in increased MOMP and promoted cytochrome C leakage into cytosol and initiated the mitochondria-dependent apoptosis. Importantly, pretreatment with mdivi-1, a highly efficient and selective blocker of Drp1, attenuated these acute conversions and long-lasting cognitive impairment of rats exposed to isoflurane. The neuroprotective effect is associated with preventing dephosphorylation and translocation of Drp1 and the following uncontrolled mitochondrial division and increased MOMP.

As known, mitochondria are dynamic organelles that keep constant fusion and fission and change their shape and location in response to cellular activity. The fusion event is mediated by Opa1, mitofusin (mfn) 1, and mfn 2. The fission process is mainly mediated by Drp1 [35,36]. The balance of fusion and fission and the proper distribution of mitochondria in axonal growth cones and synapse is critical for neuronal function such as synaptogenesis, while disturbed mitochondrial dynamics and distribution may compromise the synaptic plasticity [14,37-39]. In light of the great importance of mitochondrial fission in neuronal activity, tremendous effort during the last two decades has been made to decipher the physiological role in neuronal development and growth and pathophysiological mechanism in neurodegenerative diseases. It was well documented that Drp1 interacted with hyperphosphorylated-tau, amyloid beta, Parkin, and mutant huntingtin and promoted neuronal damage and synaptic degeneration [13,16,40,41]. This implicates that Drp1-mediated mitochondrial fission may be a crucial event in these neurodegenerative diseases.

Although there is no direct clinical evidence that general anesthesia administered to infant can cause toxic effect on the brain, extensive data gained from rodents and primates experiments showed that neonatal exposure to general anesthetic results in acute neuronal injury in the vulnerable brain area such as hippocampus and long-term learning and memory defect in the later life [1-3,10,42,43]. In fact, it has been demonstrated that general anesthetics-activated mitochondrial apoptotic pathway seemed to be the integrated step in vivo and in vitro [3,7,9,10]. Further investigations found that anesthetics cocktail (isoflurane-nitrous oxide-midazolam) or sole anesthetics (sevoflurane, desflurane, or propofol) administered to the developing neurons generated neuronal ultrastructural changes, including synaptic disturbance, mitochondrial excessive division and abnormal distribution [5,6,11,12,26,31]. This raises the interesting, although speculative, notion whether activation of factors regulating mitochondrial excessive fission could be the initial event in this neuronal injury. In this regard, several recently published investigation indicated that drp1 activation and translocation preceded the initiation of mitochondrial apoptotic cascade. Moreover, it was proposed that general anesthetics induced imbalance of mitochondrial ultrastructure accompanied by Drp1 oligomerization [4,5]. Consistent with previous results, our study found that isoflurane, a clinically used inhalational anesthetic, repressed expression of phosphorylated Drp1 (ser656) and promoted Drp1 and Bax mitochondrial translocation in hippocampi of developing rats. Unexpectedly, we found that Drp1 and Bax formed complex in cytosol using co-immunoprecipitation. We proposed that this combination might facilitate Bax mitochondrial shift as inhibiting Drp1 dephosphorylation decreased Bax translocation. More importantly, we found that mdivi-1 treatment prior to isoflurane anesthesia remarkably inhibited the Drp1 phosphorylation, translocation, and the interaction with Bax. Mdivi-1 pretreatment also reduced the leakage of cytochrome C into cytosol and the expression of active caspase9 and active caspase3. This implied that drp1 dephosphorylation at ser656 was involved in isoflurane-induced neuroapoptosis and inhibition of Drp1 activation may offer neuroprotection against isoflurane-induced developmental neurotoxicity.

Dephosphorylation of Drp1 at serine656 promoted excessive mitochondrial fission even fragmentation and augmented the susceptibility to apoptotic stimuli [44]. Some studies argued that Drp1-mediated mitochondrial fission did not inevitably cause apoptosis, but it had been proven an important step in apoptosis [45,46]. The phosphorylation of Drp1 is regulated by a few kinases in the different context [47]. However, it remains elusive which signaling pathways are involved in isoflurane-induced Drp1 dephosphorylation. The pro-apoptotic factor Bax plays an essential role in execution of apoptotic cascade. However, it was reported that Drp1-dependent mitochondrial fission is dispensable for Bax-dependent apoptosis [48,49], while Drp1-mediated apoptotic mitochondrial fission is independent of Bax translocation to mitochondria [50]. These two factors seem irrelevant to each other in the apoptotic program. In fact, recent studies found that Drp1 is involved in Bax shift to mitochondria in response to apoptotic stress [51,52]. In line with these results, we found that Drp1 bound with Bax in cytosol. This may imply that Drp1 facilitates Bax mitochondrial translocation in hippocampi after isoflurane exposure.

Mdivi-1 is an efficient and selective inhibitor of Drp1. Evidence suggested that mdivi-1 produced rapid, reversible, and inter-net-like mitochondria in mammalian cells [19,53]. In addition, observation demonstrated that mdivi-1 inhibited the activity of Drp1 by directly binding to an allosteric site so that prevented the conformational change and subsequent Drp1 oligomerization and GTP hydrolysis [19]. The anti-apoptotic characteristic of mdivi-1 makes it an attractive pharmaceutical measurement for neurodegenerative diseases and stroke. For instance, mdivi-1 protected hippocampal neurons from epilepsy-induced apoptosis [22] and traumatic brain injury [54]. Strikingly, it had demonstrated that mdivi-1 prevented neuronal damage in Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [23-25]. Recent studies also found that mdivi-1 provided neuroprotective effect against ischemic brain injuries and reduces the cognitive injuries [18,21,29,55]. Moreover, mdivi-1 offered anti-apoptotic effect in cardiac ischemic models and renal injury models [17,20].

In accordance with these observations, we found that mdivi-1 pretreatment mitigated isoflurane-induced ultrastructural change of mitochondria and inhibited neuroapoptosis in hippocampus. In addition, mdivi-1 inhibited isoflurane-induced release of cytochrome C confined in mitochondrial cristae and downregulated the expression of active caspase9 and active caspase3 in hippocampi. More importantly, we found that mdivi-1 alleviated, not completely inhibited, long-term cognitive impairment in rats receiving isoflurane anesthesia on PND7. As aforementioned, synaptic degeneration is another demonstration of developmental neurotoxicity induced by isoflurane. In our study, the expression of two representative proteins of synaspsin I and PSD95 were both reduced after isoflurane exposure. However, mdivi-1 pretreatment effectively mitigated this change. This indicates that mdivi-1 also can attenuate isoflurane-induced synaptic impairment.

In present study, we found mdivi-1 pretreatment almost completely inhibited Drp1 dephosphorylation and mitochondrial translocation and did mitigate, but not fully restore, isoflurane-induced neuroapoptosis, synaptic injury, and cognitive deficit. In our opinion, this phenomenon might be attributed to several factors. First of all, besides phosphorylation, the activity of Drp1 is regulated in several post-translational modification manners including ubiquitination, SUMOylation, and S-nitrosylation. It is unclear whether mdivi-1 could repress drp1 activity in all ways. We cannot rule out the possibility that isoflurane activates Drp1 in other ways except phosphorylation. An alternative explanation might be that other paralleled components in maintaining the equilibrium between mitochondrial fusion and fission is dysregulated and involved in the neurotoxicity. A recent study found that Drp1 mediated the acute neuronal injury while degradation of mfn2, located on mitochondrial out membrane and involved in mediating the process of mitochondrial fusion, contributed to delayed excitotoxic damage [56]. Previous investigations showed that anesthetic altered the expression of mfn2 in developing brain and cultured neurons [4,26]. However, it needs to be further examined whether the disturbed mfn2 expression is related to neurotoxicity induced by anesthetics in developmental brain.

Conclusion

Data from this study showed that mdivi-1 pretreatment mitigated cognitive impairment of rats exposed to isoflurane at PND7 by preventing Drp1 phosphorylation and translocation to mitochondria and synaptic protein loss. The neuroprotective effect of mdivi-1 is associated with inhibiting activation of mitochondria-dependent apoptotic cascade and synaptic degeneration. Mdivi-1 hold potential to be an efficient prophylactic measurement for isoflurane-induced acutely developmental neurotoxicity and long-lasting cognitive dysfunction.

Acknowledgements

References