关键词: anti-epilepsy, apoptosis, autophagy inhibitors, epilepsy, rapamycin, xenon inhalation

Abstract: Objective：Xenon is an inhalation anesthetic with a favorable safety profile，and previous studies have demonstrated the neuroprotective efficacy of xenon in Alzheimer’s disease，spinal cord ischemia/reperfusion injury，intrauterine asphyxia and neonatal asphyxia. Further studies confirmed the inhibition of uptake and efflux of glutamate and mediating the neuroprotective effect through the inhibition of excessive excitation and anti-apoptosis. However， whether xenon plays a role in epilepsy remains unclear. This study aimed to investigate the role of xenon treatment in kainic acid（ KA）-induced acute generalized epileptic seizures in male Sprague-Dawley rats. Methods：All rats were treated with KA （1 mg/0.8 ml，0.65 μl/rat），which was injected into the lateral ventricle through the cannula. Seizure severity was staged as 1~5 based on Racine’s criteria. Rats in the xenon group were treated with xenon mixture （70% xenon/30% oxygen） for 1 h immediately after KA injection，while rats in the control group were treated with 70% nitrogen/30% oxygen. In order to assess the role of autophagy in the antiepileptic effects induced by xenon，the inhibitors of autophagy （3-methyladenine，3-MA or bafilomycin A1，BafA1） and the inducer of autophagy （rapamycin） were administered before xenon inhalation respectively. Western blot was used to investigate that the level of autophagy and apoptosis in the different group. Results：① After KA administration，almost all rats showed immediate and continuous epileptic seizures （mainly stage 4 or stage 5）. Behavioral and electroencephalography （EEG） results indicated that 70% xenon/30% oxygen inhalation significantly reduced the severity of epileptic seizures，such as decreased accumulated time in generalized seizure （6 min versus 49 min，P<0.001），accumulative afterdischarge duration（ 8 min versus 53 min，P<0.001） and increased accumulated time in stage 0 （no epileptic seizure stage，48 min versus 0 min，P<0.001） compared with the control group. Moreover，mortality in the xenon treatment group was 0，while 3 out of 13 rats died in the KA group. The differences in stages were further compared every 5 min in xenon and control groups to assess the onset time of the anti-epileptic effect of xenon. The results
showed that from the second 5 min measurement，the seizure intensity in the xenon group was significantly weaker than that in the KA group （stage 1.8 versus stage 4.7，P<0.001）. Inhalation of 70% xenon/30% oxygen for 60 min was not enough to induce anesthesia and the rats kept sober and free activities during the entire experiment. Western blot results indicate that 70% xenon/30% oxygen treatment significantly upregulated the level of autophagy and attenuated apoptosis compared with the control group treated with 70% nitrogen/30% oxygen. ② The significant antiepileptic effect of xenon disappeared after treatment with 3-MA 6 h before xenon inhalation. Sixty minutes after KA administration，the 3-MA+KA+Xenon group was at a similar seizure stage in every time period compared to the Saline+KA group treated with 30% oxygen/70% nitrogen mixture instead of xenon mixture，and the reduced seizure stages from the second 5 min were reversed when xenon was administered. The accumulated time in every stage analysis showed that the time spent in stages 4 and 5 was significantly longer in the 3-MA+KA+Xenon group than in the Saline+KA+Xenon group （P<0.001 versus 0.001，respectively） and was similar that in the Saline+KA group （P>0.05）. Moreover，3-MA treatment significantly prolonged the decreased cumulative afterdischarge duration and cumulative generalized seizures duration，which is induced by xenon treatment （P<0.001 and 0.001，respectively）. Three rats died in the 3-MA+KA+Xenon group. Western blot results indicate that 3-MA treatment reversed the increased autophagy and weakened apoptosis induced by xenon inhalation. ③ The autophagic inhibitor BafA1 reversed the antiepileptic effect of xenon. BafA1 reversed the weakened epileptic seizure stage induced by xenon inhalation from the second 5 min. Furthermore，the reduced time spent in stages 2~5 induced by xenon disappeared in rats treated with BafA1. Moreover，the cumulative afterdischarge
duration and generalized seizures duration in the BafA1-treated rats were significantly longer than those in the xenon-treated rats （P<0.001 and 0.001，respectively） and was similar to those in the non-xenon group treated with KA only （P>0.05）. Two rats died in the BafA1-treated group. Moreover，BafA1treatment reduced the level of autophagy and strengthened apoptosis，which is significantly weakened after xenon treatment. ④ Rapamycin，which is used as an inducer of autophagy，was administered 1 h before KA administration to simulate the effect of xenon to strengthen the level of autophagy. The results showed that rapamycin treatment significantly reduced the epileptic seizure stages from the first 5 min and this effect was maintained for 60 min after KA administration （P<0.001） compared with the control group treated with DMSO （DMSO+KA group） instead of rapamycin. Rapamycin significantly reduced the time in generalized seizure （stage 4，P<0.05；stage 5，P<0.001） and prolonged the cumulative time of no behavioral seizure（ stage 0，P<0.001）. EEG results indicated that the cumulated afterdischarge duration in the rapamycin-treated group was significantly shorter than that in the control group treated with DMSO instead of rapamycin before KA administration （P<0.001）. Importantly，the total time in stage 0 （no seizure stage was observed） was about 18 min in Rapamycin+KA group，whereas it was 0 min in the DMSO+KA group. No rats died in the rapamycin-treated group. Western blot results showed that rapamycin treatment increased the autophagic level and attenuated apoptosis. Conclusion：This study confirmed that a 70% xenon mixture has strong antiepileptic effects in KA-induced acute generalized seizures in male Sprague-Dawley rats. Because of the lack of toxic side effects，appropriate xenon inhalation may be a safe method to inhibit epileptic seizures. Furthermore，our results indicate that an increase in autophagy level may be the main mechanism underlying the antiepileptic effect of xenon，and may therefore be clinically useful as a therapeutic option for epilepsy. However，further studies are necessary to confirm the effectiveness of xenon， and confirm the optimal effective time-window and the appropriate xenon ratio.

Key words: anti-epilepsy, apoptosis, autophagy inhibitors, epilepsy, rapamycin, xenon inhalation