The Neuroprotective Effect And Related Mechanism Of Sevoflurane Preconditiong And Hydrogen Gas Treatment

BackgroundThe perioperative cerebral or spinal ischemia-reperfusion (I/R) injury is not rare due to the patients'factors like a history of cardiac and cerebrovascular disease or other objective factors such as operative and anesthetic stimulations. Data shows that the incidence of the cerebral ischemia-reperfusion injury in patients undergoing cerebrovascular malformation operation is as high as 42.9%. Once the patients suffer'perioerative stroke', the mortality is between 33 to 60%. Meanwhile, paraplegia induced by spinal cord ischemia-reperfusion injury remains a major devastating complication after thoracic aorta and spine surgery. It is reported that the incidence of residual permanent injury (acute and/or delayed paraplegia) is over 10%. Unlike those'common stroke'or accidentally injured spine, such cerebral and spinal ischemic injuries are to some extent predictable, which offers us not only an opportunity but also a challenge. Therefore, the research on the prevention and treatment of perioperative cerebral and spinal ischemic injuries in early stage is feasible and of great significance. Inhalational anesthetics are the most commonly used anesthetics in clinic. Besides its anesthetic effect, researchers have noticed its neuroprotection effect. The previous studies of our lab demonstrated that the inhalational anesthetic sevoflurane when used as a preconditioned drug can obviously attenuate the spinal ischemic injury and improve the neurological behavior scores of the animals. However, the molecular mechanism of the neuroprotection induced by sevoflurane preconditioning is poorly understood.Tandem p domain k1 (k2p) channels, a new class of potassium channel subtypes, have been found in recent years, among which TREK-1 channels distribute widely in mammalian and express highly in the central nervous system. They participate in many processes such as the normal electrical activity of neurons, the formation of pain, neurotransmitter release, emotion regulation and other physiological activities. Recent studies have found that TREK-1 channels are closely related to the neuroprotective effects in some situations. Yet their roles in the neuroprotection induced by inhalational anesthetic preconditioning demand further study. This study will investigate the neuroprotective effect induced by sevoflurane preconditioning in rats focal cerebral ischemia-reperfusion injury model and seek to explore the relationship between the TREK-1 and the neuroprotective effect, thus we hope to find the key " target point " of the preconditioning.We are struggling to find drugs and measures to prevent ischemic injury, but we never forget that the same important thing is to treat it as early as possible when it happens, since we all know the effective early treatment helps much more than the rehabilitation therapy to the patients'prognosis. Recently, early hydrogen gas treatment exerts protective effects in many key organs such as brain, lung, liver and kidney when they suffer I/R. Furthermore, some studies have found that hydrogen-rich saline or hydrogen-rich water can reduce spinal cord injury induced by trauma. In addition, our recent studies have shown that H2 inhalation significantly improved the survival rate and decreased multiple organ damage of septic mice in a concentration and time-dependent manner. These findings strongly indicate that H2 treatment may be beneficial to spinal cord I/R injury. So far, there are no studies attempting to describe the protective effect of H2 on spinal cord ischemia. Therefore the primary objectives of the present study were to determine: (1) Whether hydrogen gas treatment could decrease spinal cord ischemic injury? (2) Whether it is mediated by inhibiting oxidative stress and inflammation?PartⅠThe mechanism of TREK-1 involved in the neuroprotective effects induced by sevoflurane preconditioningExperiment I: The study on the neuroprotective effects induced by sevoflurane preconditioning on focal cerebral ischemia-reperfusion in rats. Objective To investigate the neuroprotective effects induced by sevoflurane preconditioning in the focal cerebral ischemia-reperfusion in rats. Methods A total of 24 male SD rats were randomly assigned to three groups (sevoflurane preconditioning ischemia-reperfusion group, control group, and sham group, n=8 in each). Rats in the sevoflurane preconditioning ischemia-reperfusion (Sevo PC) group and sham group were placed in a chamber and inhaled sevoflurane (2.7% sevoflurane, 97% O2, 45min). Rats in the control (Con) group were placed in the same chamber but pretreated with oxygen only (97% O2, 45min). Sixty minutes after the preconditioning, middle cerebral artery occlusion (MCAO) was performed and the artery was oclluded for 90 min in the Sevo PC group and Con group. Neurological behavior scores (NBS) were evaluated in three consecutive days after reperfusion and TTC staining was used to analysis brain infarct volum at 72h after reperfusion. Another part of the animals were divided into three groups (n=12), which at 4/24/72-hour after reperfusion were killed for the observation of the expression changes of TREK-1 in the brain at different time points, respectively. Results 1. NBS in the Sevo PC group were higer than that of the Con group at the same time points (P<0.05). 2. TTC staining observations showed that the Sevo PC group had smaller infarct volume than that of the Con group. 3. Both the expression of TREK-1 protein and TREK-1 mRNA transcription level in Sevo PC animals at the same time point were higher than that of the Con group (P<0.05). Conclusion Sevoflurane preconditioning can induce ischemic tolerance in the central nervous system and TREK-1 was up-regulated at both transcriptional and protein levels.Experiment II: The effect of down-regulation of cellular TREK-1 on the neuroprotection induced by sevoflurane preconditioning.Objective To investigate the influence of the down-regulation of TREK-1 protein on sevoflurane preconditioning-induced neuroprotection in cells. Methods (1) Human neuroblastoma cell line (SH-SY5Y) differentiation and identification: The cultured cells differentiating for different periods (1day, 4days, 7days or 10 days) were prepared for the measurements of NeuN andβⅢ-Tublin, and then the optimal differentiating time for getting neural cells was decided according to the result. (2) Cells siRNA interference and identification: The chemical synthesis of small RNA interference TREK-1 product was added to the neural cells. TREK-1 protein was measured for identification. (3) To investigate the impact of siRNA intereference on preconditioning effect, cells were divided into five groups:① Control: differentiated cells without any other treatments;②Pre+siRNA+OGD, differentiated cells by siRNA interference, 24h after preconditioning, oxygen and glucose deprivation was performed for 2 hours;③Pre+OGD, differentiated cells received OGD 24 hours after preconditioning;④Con+siRNA+OGD, differentiated cells with siRNA interference received OGD 24h after preconditioning;⑤Con+OGD, differentiated cells without siRNA interference were exposed to air instead of sevoflurane for 2 hours and OGD were performed 24h. after preconditioning. Protein extraction was performed at 24h after OGD or sham treatment for western-blot analysis and immunofluorescence. Results 1. NeuN andβⅢ-Tublin expressed the highest in 7 days group when compared to the other three groups(P <0.05). 2. Cells siRNA interference and identification: Western-blot results showed that expression of TREK-1 in TREK-1 siRNA group was lower than the control group and TREK-1-control group (P <0.05). 3. The influence of siRNA intereference on preconditioning: No significant difference was found in expression of Capase-3 between Pre+siRNA+OGD group and Con+OGD group,whereas the expression was higher in Pre+siRNA+OGD when compared with Pre+OGD group (P<0.05). MTT results showed lower OD values in Pre+siRNA+OGD group than Pre+OGD group and was not significantly different with Con+OGD. Conclusion In vitro experiment, we confirmed that inhibition of expression of TREK-1 protein reversed the neuroprotective effects induced by sevoflurane preconditioning, indicating that TREK-1 mediated sevoflurane preconditioning induced neuroprotection.Experiment III: The effect of down-regulation of cerebral TREK-1 on the ischemic tolerance induced by sevoflurane preconditioningObjective To investigate the influence of inhibition of TREK-1 protein expression on sevoflurane preconditioning-induced neuroprotection in vivo. Methods (1) Rats received intracerebroventricular injection of small RNA interference TREK-1 product and TREK-1 protein content of the brain was measured for identification of the effect of siRNA interference; (2) Ninety male SD rats were randomly divided into 5 groups (n = 18):①Sham group, rats underwent MCAO surgical operation without occlusion of the artery;②Control group, rats received 97% oxygen inhalation for 45 minutes before underwent MCAO ischemia for 90 minutes;③Pre+MCAO, rats received 97% oxygen and 2.7% sevoflurane inhalation for 45 minutes before underwent MCAO ischemia for 90 minutes;④siRNA+Pre+MCAO, 24 hours after a single intracerebroventricular injection of a siRNA, rats received 2.7% sevoflurane+97% oxygen inhalation for 45 minutes and after 60 minutes underwent MCAO ischemia for 90 minutes;⑤siRNA-c+Pre+MCAO, a siRNA-control instead of a siRNA was given to rats and other treatments were the same as those given to group siRNA+Pre+MCAO. Neurological behavior scores (NBS) observed in three consecutive days after reperfusion. TTC staining was used to analysis brain infarct volum at 72h after reperfusion. Results 1. Neurological behavior scores (NBS) of Pre+MCAO group was better than that of siRNA+Pre+MCAO at 24,48,72 hours after reperfusion respectively(P <0.05), and there is no significant difference between the control and siRNA+Pre+MCAO group at the same points. 2. TTC staining observations showed that infarct volume of Pre+MCAO group was smaller than siRNA+Pre+MCAO at 72 hours after reperfusion (P <0.05), and no significant difference between the control and siRNA+Pre+MCAO group was seen at the same time points. Conclusion We confirmed in vivo that inhibition of expression of TREK-1 protein reversed the neuroprotective effects induced by sevoflurane preconditioning, indicating that TREK-1 mediated sevoflurane preconditioning induced cerebral ischemic tolerance.PartⅡRole of hydrogen gas against spinal cord ischemia–reperfusion injury in rabbitsExperimentⅠEffect of early hydrogen gas treatment on spinal cord ischemia-reperfusion injuryObjective The present study is to determine whether hydrogen gas treatment could decrease spinal cord ischemic injury and improve limb motor function after I/R injury. Methods 1%/2%/4% H2 gas was administered to anesthetized rabbits, and H2 gas was supplied through a gas flow meter with the anesthetic gas from 10 min before reperfusion to 60 min after reperfusion. The concentration of H2 gas in the anesthetic gas was continuously monitored. A total of 32 male rabbits were randomly divided into 4 groups (n=8 each group): control (Con) group, 1% H2, 2% H2 and 4% H2 groups. The rabbits in all four groups were exposed to spinal cord I/R surgery. The animals in control group underwent only I/R operation without H2 treatment. The animals in the 1%H2, 2%H2 or 4% H2 groups were treated with H2 inhalation under 1%, 2% or 4% concentration respectively, from 10 min before reperfusion to 60 min after reperfusion (total 70 min). Arterial blood gases and blood glucose were measured at preischemia, 10min before reperfusion, and 60 min after reperfusion, respectively. The neurological assessment was taken at 24h, 48h and 72h after reperfusion. After the last neurological assessment, the histopathology of spinal cord was detected. Results There is no significant differences in pH, PaO2, PaCO2 or blood glucose among all four groups at different time points. Rabbits in Con group were completely paraplegia of hind-limbs at 48h and 72h after reperfusion. Treatment with 2% or 4% H2 gas significantly improved the neurological outcome of spinal cord I/R in animals after reperfusion (P<0.05). However, there is no significant difference between the 1% H2 treatment and control group. In addition, 4% H2 treatment did not further improve the neurological scores of spinal cord I/R animals when compared to 2% H2 treatment group (P>0.05). Conclusion The results indicate that spinal I/R damage can cause significant deficit in hind-limb motor function, which is improved markedly by 2% and 4% H2 treatment.ExperimentⅡHydrogen gas attenuates spinal cord ischemia-reperfusion injury through anti- inflammation and oxidative stress inhibitionObjective The study was to investigate whether the hydrogen gas treatment protects spinal cord from ischemic injury by means of inhibiting inflammation and oxidative stress. Methods 2% H2 gas was used in this part of experiment according to the result of experimentⅠ. Eighteen animals were randomly divided into 3 groups (n=6 each group): Sham, control, and H2 treatment groups. The rabbits in sham group suffered the similar operation without spinal cord I/R. The levels of 8-iso-PGF2αand MDA, the activities of SOD and CAT, TNF-αand HMGB1 in serum and spinal cord of ischemia were measured at preischemia, as well as 6h, 12h, 24h, 48h and 72h after reperfusion. At 72 h after reperfusion, in addition, we detected the same parameters (8-iso-PGF2α, MDA, SOD, CAT, TNF-αand HMGB1), TUNEL staining and caspase-3 activity in spinal cord. Results 1.Oxidative products: The level of MDA and 8-iso-PGF2αin serum and spinal cord of Con group were higher than that of sham group. (P<0.05). When compared with control group, H2 treatment significantly decreased the levels of MDA and 8-iso-PGF2αin serum and spinal cord (P<0.05). 2. Antioxidant enzymes: The activities of superoxide dismutase ( SOD ) and CAT were detected in serum and spinal cord. The results showed that spinal cord I/R injury significantly decreased the activities of SOD and CAT in serum and spinal cord (P<0.05 vs. Sham).3.Inflammatory cytokines: Inflammatory cytokines of TNF-αand HMGB1 were also measured in serum and spinal cord at different time points. Our results showed that spinal cord I/R animals had a significant increase of TNF-αand HMGB1 levels in serum and spinal cord (P<0.05 ). However, H2 treatment significantly decreased the levels of TNF-αand HMGB1 in serum and spinal cord when compared with control (P<0.05). 4. The number of TUNEL positive neuron in spinal cord was different between control and H2 treatment groups(P<0.05). Furthermore, we found that the caspase-3 activity in spinal cord I/R animals was higher than that of sham group (P<0.05), which was significantly decreased by 2% H2 treatment(P<0.05 vs. Control). Conclusion H2 gas treatment can inhibit oxidative stress and inflammation after spinal cord ischemic injury, which might be the key mechanism of the protective effect of H2 treatment.Conclusions1. Sevoflurane preconditioning can induce ischemic tolerance in focal cerebral ischemia-reperfusion model of rats.2. Sevoflurane preconditioning can increase TREK-1 protein expression and gene transcription level compared with ischemia control group.3. Down-regulation of TREK-1 reversed the neuroprotective effect in vitro and attenuated cerebral ischemic tolerance in rats.4. Early H2 gas treatment can inhibit spinal cord ischemia reperfusion injury in a rabbit model, and 2% H2 gas inhalation can make satisfactory beneficial effect.5. The protective mechanism of H2 gas treatment has been mediated though inhibiting early oxidative stress and delayed inflammation response, which is helpful for improving hind-limb motor function.