The Mechanism Research Of Neuroprotection Induced By Intermittent Hypoxia Preconditioning On Rats With Seizure Evoked By Lithium-pilocarpine

An epileptic seizure is defined as“a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain ” The reasons why a group of neurons suddenly and temporarily synchronize their activity are not well understood. Many researches suggest that perturbations in the brain energy metabolism are involved in the pathophysiology of temporal lobe epilepsy(TLE), while the most important function of astrocytes is to maintain brain energy homoeostasis. A better understanding of the condition will provide neuroscientists and physicians with crucial knowledge, which will enable the development of new therapeutic approaches with benefits to patients.Preconditioning refers to a paradigm whereby exposing cells or an organ or organism to a sublethal insult provides protection against a subsequent insult that would normally produce injury. Noxious stimuli applied at doses close to but below the threshold of cell injury induce adaptative responses that protect the organ against additional stress from the same(tolerance) or other(cross-tolerance) stimuli. Although it is found that epilepticpreconditioning, a mild epileptic insult that protects neurons against status epilepticus, also protects neurons against ischemia. Few studies focused on whether the hypoxia/ischemic preconditioning can also provide protection to neurons against epilepsy. During hypoxia/ischaemia, lactate release from astrocytes can be taken up by neurons as fuel during energy crises. The physiological role of the monocarboxylate transporter 4(MCT4) which mainly found in astrocytes is mostly associated with this process. Hypoxia-inducible factor 1α(HIF-1α) is one of important transcription factors in mammals, throughing response to hypoxia/ischaemia to maintain the oxygen balance in the body, now regulation more than 70 target genes. Tissue hypoxia/ischaemia can promote many downstream target genes by up-regulating expression of HIF-1α, it’s promoted erythropoiesis, angiogenesis and improve brain tissue blood supply, to reduce brain tissue ischemia and hypoxia injury. MCT4 gene is precisely one of the many target genes of HIF-1α.The purpose of the present study was to test the hypothesis that hypoxia preconditioning could provide protection to neurons against epilepsy by up-regulating MCT4 expression in the astrocytes. The effects of hypoxia manipulations on primary culture astrocytes of rats and intermittent hypoxia preconditioning(IHP) manipulations on lithium-pilocarpine(Li-pilo) induced epilepsy of rats were compared from the perspective of morphology, molecular biology and behavioral test. Results showed that hypoxia increased the expression of astrocytic MCT4 and IHP for 5 days can afford the greatest anti-epileptic functions to epileptic model rats. The protective effect was most prominent by 3 days after standardized IHP manipulations. This experiment is divided into four parts as below:PartⅠ:The effects of OGD manipulations on MCT4 expression of primary culture astrocytes of rats in vitroObjective:Western blot and immunocytochemistry were performed on cultured primary astrocytes taken from hippocampus of Sprague Dawley(SD) rats, to compare the expression of MCT4 on the astrocytes in normal or hypoxic condition. Methods:Cultures astrocytes were randomly assigned to control and hypoxia group. The hypoxia group cellsunderwent the oxygen–glucose deprivation(OGD) treatment for 8h. After recovered in normal conditions for the next 24 h, the cells planted in dishes were harvested for MCT4 western blot detection, the cells adhere to coverlips were double immunofluorescence labelled MCT4 and astrocytic marker glial fibrillary acidic protein(GFAP). Results: Both western blot and double immunofluorescence labelling shown that the expression level of MCT4 in hypoxia group was increasing greatly after ODG treatment for 8h by comparison with the control groups(P<0.05). Conclution:Hypoxia manipulations significantly induced up-regulation of MCT4 on cultured primary astrocytes.PartⅡ:The effects of intermittent hypoxia preconditioning manipulations on MCT4 and HIF-1α expression in hippocampus of rats in vivo.Objective:To determine the standardized IHP manipulations, the expression of MCT4, HIF-1α, GFAP and neuronal nuclei(Neu N) in hippocampus of SD rats underwent different period of IHP manipulations were detected by western blot, double immunofluorescence labeling and immumohistochemical staining. Methods:36 rats were randomly divided into control group and five IHP groups. The rats of IHP groups were respectively treated with IHP for 1 day(IHP 1d group), 3 days(IHP 3d group), 5 day(IHP 5d group), 7day(IHP 7d group) or 14 days(IHP 14 d group). At 24 h after the end of IHP manipulations, three rats from each group were harvested for western blot detection of MCT4 and HIF-1α. the brains of next three rats were taken for double immunofluorescence labelling of MCT4, GFAP and Neu N. Results: Western blot analysis shown that the peak level of HIF-1α expression in IHP groups was occurrenced in IHP 3d time point, the peak level of MCT4 was just lagged in IHP 5d time point, then both the expression were gradually decreased. Compared with control group and IHP 14 d group, the expression of HIF-1α most obviously increased in IHP 3d group(P<0.05) and the expression of MCT4 most obviously increased in IHP 5d group(P < 0.05). Theimmunofluorescence labeling of MCT4 in the hippocampal CA1 of rat brain was consistent with the variation tendency in western blot analysis. However, the immumohistochemical staining of GFAP in the hippocampal CA1 and Neu N in temporal lobe cortex were not affected by the IHP. Conclution : Intermittent hypoxia preconditioning manipulations significantly induced up-regulation of MCT4 and HIF-1α in the hippocampus, but couldn’t cause injury in the brain. So IHP for 5 days was confirmed as the period of standardized IHP manipulations in present study.Part Ⅲ:The effects of IHP manipulations on epileptic seizure susceptibility of epileptic rat model induced by lithium-pilocarpine injection.Objective: To further elucidate the relationship between epileptic seizure susceptibility and the change of HIF-1α and MCT4 expression in hippocampus of rats, electrophysiology detection, ethology observation, western blot and double immunofluorescence labelling were performed on epileptic model rats underwent standardized IHP manipulations. Methods:99 rats were randomly divided into nine groups: control group, seizure group(only injected Li-pilo to induce seizure), IHP group(only underwent standardized IHP manipulations) and six IHP-seizure groups. At 1 day(IHP-1d seizure group), 2 days(IHP-2d seizure group), 3 days(IHP-3d seizure group), 5 day(IHP-5d seizure group), 7 day(IHP-7d seizure group) or 14 days(IHP-14 d seizure group) after standardized IHP manipulations, the rats of IHP-seizure groups were respectively induced seizure by Li-pilo injection. After inducing seizure, three rats of seizure group and IHP-seizure group were taken electrophysiology detection for 4h, the next eight rats underwent ethology observation for 4h. Behavioral seizures in rats were scored every 5 min for up to 4h in accordance with a modified version of the Racine scale. The latency period of generalized seizures(stage 4) and the percentage of generalized seizures(times having scored stage 4 and 5 among all scored time points)were alsorecorded. The protein expressions of MCT4, HIF-1α, and GFAP in hippocampus were evaluated 24 h post-seizure induction using western blot and double immunofluorescence labelling. Results: Background electroencephalogram(EEG) recordings in seizure group rats revealed generalized tonic-clonic epileptiform abnormalities that included polyspike abnormalities and spike-wave discharges(SWD), while the background EEG in IHP-3d seizure group rats only arose accidental epileptiform discharges. Modified Racine scales of induced seizure peaked on average between 50–150 min after Li-pilo administration and then slowly decayed. The average modified Racine scale in IHP-3d seizure group was significantly lower than the other groups. The latency of generalized seizures and percentage of generalized seizures in the IHP-3d seizure group rats were different from the parameters in the seizure group rats with statistical significance(P<0.05). Western blot analysis shown that compared with the seizure group, both the expression of MCT4 and HIF-1α was significantly up-regulated in IHP group, IHP-3d seizure group and IHP-5d seizure group(P<0.05). Double immunofluorescence labelling shown that the expression of MCT4 in the hippocampal Dentate Gyrus(DG) of rats were mostly upregulated in the IHP-3d seizure group compared with the seizure group, while the expression of GFAP did not change. Conclution : Standardized IHP manipulations can afford the greatest protection to against epileptic seizure in rats by up-regulation of MCT4 and HIF-1α expression in the hippocampus. The epileptic tolerance effect was most prominent by 3 days after IHP manipulations.Part Ⅳ:The effects of lactate transport function disturbances on the effective protection of epileptic tolerance induced by IHP manipulations in rats.Objective: To further elucidate whether function disturbances of lactate transport can block the protection of epileptic tolerance in rats, electrophysiology detection, ethology observation, western blot and double immunofluorescence labelling were performed onepileptic model rats underwent different treatments. Methods:33 rats were randomly divided into three groups: seizure group(only injected Li-pilo to induce seizure), IHP-seizure group(72h after standardized IHP manipulations, injected Li-pilo to induce seizure) and IHP-CHC-seizure group(rats underwent the same procedure as IHP-seizure group, only injected α-cyano-4-hydroxycinnamate(CHC) which is a specific covalent inhibitor of mitochondrial lactate and pyruvate transport 4h before pilocarpine injection.).After inducing seizure, three rats of each group were taken electrophysiology detection for 4h, the next eight rats underwent ethology observation for 4h. The same ethological parameters as Part Ⅲwere recorded. The protein expressions of MCT4, HIF-1α, and GFAP in hippocampus were also evaluated 24 h post-seizure induction using western blot and double immunofluorescence labelling. Results: Background EEG recordings in IHP-CHC-seizure group rats revealed generalized tonic-clonic epileptiform abnormalities as seizure group, while the background EEG in IHP-seizure group rats only arose accidental epileptiform discharges. Modified Racine scales of induced seizure peaked on average between 50–150 min after Li-pilo administration and then slowly decayed. The average modified Racine scale in IHP-seizure group was significantly lower than the other two groups. However, the performance of rats in IHP-CHC-seizure group and seizure group were similar to some extent. The latency of generalized seizures and percentage of generalized seizures in the IHP-seizure group were different from the other two groups with statistical significance(P<0.05). Western blot analysis shown that compared with the seizure group, the expression of MCT4 was significantly up-regulated in IHP-seizure group and IHP-CHC-seizure group(P<0.05), while no apparent difference was ascertained between IHP-seizure group and IHP-CHC-seizure group(P > 0.05). Double immunofluorescence labelling shown that compared with the seizure group, the expression of MCT4 in the hippocampal CA1 of IHP-seizure group and IHP-CHC-seizure group rats were mostly upregulated. Conclution:The CHC was an inhibitor of lactate and pyruvate transport but can not interfere the expression of MCT4. However, lactate transport function disturbances can block the effective protection of epileptic tolerance induced by IHP manipulations in rats.