Mitochondrial DNA Damage And The Involvement Of Antioxidant Defense And Repair System In Hippocampi Of Rats With Seizures Induced By Lithium-pilocarpine

Epilepsy is one of the most common neurological disorders affecting about 9 million patients in china. About 25-40% of patients suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) presents the most prevalent refractory epilepsy and its pathogenesy still remains obscure. It is difficult to gain significant curative effects through administration of antiepileptic drugs (AEDs) and operating. The alteration of behavior, electroencephalogram (EEG) and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients, so pilocarpine-induced seizures have been one of the most frequently used models to research SE and TLE.Mitochondrial dysfunction has been implicated as one of the main mechanisms underlying neuronal injury. Our previous study revealed that seizure-induced mitochondrial DNA (mtDNA) damage might be one of the factors contributing to mitochondrial dysfunction. Compared with nuclear DNA (nDNA), mtDNA is more vulnerable. It has been proved that recurrent seizures can result in the accumulation of free radicals in mitochondrion. It is plausible that elevated oxidative stress may be the cause of mtDNA damage and mitochondrial dysfunction.mtDNA is close to the source of reactive oxygen species (ROS) and sensitive to oxidative damage, but there are endogenous antioxidant systems to protect mtDNA. Free radical scavengers, the primary antioxidant system, have been confirmed to be a protective pathway against seizure-induced oxidative damage. Once DNA damage has been generated, it is the role of repair systems to prevent its accumulation. But little is known about contribution of mtDNA repair pathway. Compared with various nuclear DNA repair pathways, only the base excision repair (BER) pathway has been shown to function in mitochondria and thus is critical in the defense of mitochondrial oxidative stress.In this study, we detected the response of antioxidant defense system and mitochondrial base excision repair (mtBER) pathway to verify the underlying mechanisms of mtDNA impairment and extensively study the molecular biological mechanisms of TLE.PARTⅠA study of behavior, electroencephalogram and pathobiology in lithium-pilocarpine induced seizures in ratsObjectiveTo investigate the alteration of behavior, electrophysiology and pathology in lithium-pilocarpine-induced seizures in rats, and to explore the neuronal injury of hippocampus.MethodsAdult male Wistar rats were given lithium-pilocarpine intraperitoneally to induce SE. The change of behavior in rats was observed. Seizures were allowed to last for 60 min and then were terminated by administration of diazepam. Rats were monitored by video recordings to assure development of seizures, record EEG at 3h, study the pathological changes with Nissl at 72h and 2 months after SE induced by pilocarpine.Results 1.83.1% of the rats were induced to develop SE after administration of lithium and pilocarpine (according to Racine, the rats showing stageⅣ-Ⅴconvulsive seizures were considered to develop SE successfully). The time from pilocarpine injection to the first onset of stageⅣSE was 38.5±18.7min, and the death rate within 72h was 23.2%. The latency phase is about 12.7±6.2 days.74% showed seizures at least once a week.2. EEG in cortex and hippocampus of rats showed accumulated spike waves at 3 h after SE.3. Nissl staining showed the neuronal damage in hippocampal CA1 and CA3 regions at 72 h and 2 months after pilocarpine-induced seizures. The surviving neurons showed round and palely stained nuclei, meanwhile, the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.ConclusionsLithium-pilocarpine could induce acute seizures (SE) according to the alteration of behavior and EEG. Seizures induced by lithium-pilocarpine caused hippocampal neuronal damage.PARTⅡAlteration of mitochondrial ultrastructure and mtDNA damage in hippocampi of temporal lobe epilepsy modelObjectiveTo detect the alteration of the mtDNA copy number and the level of mtDNA impairment of hippocampi in rats with seizure, and to explore the molecular mechanisms of mitochondrial dysfunction in epilepsy.MethodsAdult male Wistar rats were divided randomly into acute control,25h, chronic control,60d. Mitochondrial ultrastruture damage was evaluated by electron microscope. DNA was isolated from fresh hippocampi. The ratio of mtDNA to nDNA was determined by quantitative real-time PCR to evaluate the mtDNA number. We designed three pairs of primers specific to deferent regions of mtDNA in case of the interference of mtDNA depletion. Fpg was used to specifically remove oxidized bases and mtDNA damage can be determined from the ratio of intact PCR products in cleaved versus uncleaved DNA using quantitative PCR.Results1. Mitochondrial ultrastructure was damaged and it varied from mild to profoundly severe in the hippocampus during experimental epilepsy. Mild damage was characterized by early swelling as manifested by separation of cristae. In the more severe cases, mitochondrial swelling was accompanied by clearing of matrix density and disruption of membrane integrity. The most severe damage of mitochondria was vacuolar plus rupture of inner and outer mitochondrial membranes.2. Compared with acute control group,25h group showed constant mtDNA copy number(p>0.05), and the frequencies of DNA damage in different regions we chose showed no difference (P>0.05).3. After incubation with fpg, still no difference were found between control and 25h group(p>0.05), and no statistical differences were detected in the quantification of the three mtDNA regions(p>0.05).4. The ratio of mtDNA to nDNA decreased about 1/4 in group of 60d compared with control (P<0.05). No statistical differences were detected in the quantification of the three mtDNA regions(p>0.05).5. After incubation with fpg, the percentage of intact mtDNA decreased significantly in group of 60d (p<0.05). The frequencies of DNA damage in different regions we chose showed no difference (P>0.05).ConclusionsReal-time PCR revealed a decreased copy number of mtDNA accompanied with increased amount of oxidized bases in hippocampi of rats with recurrent seizures. The results suggested that recurrent seizures lead to severe mtDNA damage in hippocampi, including oxidative alteration and strand breaks.PARTⅢThe involvement of antioxidant defense system in hippocampi of rats with seizuresObjectiveTo detect the lipid peroxidation and the response of endogenous antioxidants, and to evaluate the redox state in hippocampi of rats with seizures.MethodsAdult male Wistar rats were randomly divided into 5 groups for treatment:control, 3h, and 25h after the onset of SE, chronic control, and chronic seizures (60d). The contents of MDA, SOD1, SOD2 and GSH were measured as the methods described by the commercial assay kits.Results1. MDA was dramatically increased at 3h,25h, and 60d after the onset of SE (p<0.05).2. Compared with control the content of GSH showed no difference at 3h, and decreased at 25h after the onset of SE(p<0.05). Recurrent seizures resulted in a 1/3 decrease in GSH (p<0.05).3. Total SOD and SOD1 activity were constant at 3h and reduced at 25h and 60d. No significant differences were observed in SOD1 activity (p>0.05).ConclusionsSeizures induced lipid peroxidation in hippocampi; the endogenous antioxidants appeared to decrease in experimental groups. The results revealed elevated oxidative stress in hippocampi after seizures, and the balance between free radical producing and scavenging was broken. Besides, the reduction of SOD2 but not SOD1 demonstrated a higher risk of oxidative damage in mitochondria..PARTⅣThe involvement of mitochondrial base excision repair pathway in hippocampi of rats with seizuresObjectiveTo assess the response of enzymes of mitochondrial base excision repair (mtBER) pathway at both mRNA and protein levels, so as to explore the underlying mechanisms of mtDNA damage in epilepsy.MethodsAdult male Wistar rats were randomly divided into 6 groups for treatment:control, 3,9, and 25h after the onset of SE, chronic control, and chronic seizures (60d). Q-PCR and western blot were used to determine the level of mtBER pathway enzymes.Results1. Real-time PCR analysis demonstrated lower expression of both OGG1 and poly in experimental group at 3,9, and 25h after the onset of SE (p<0.05), with no significant difference between experimental and control rats in APE1 level (p>0.05).2. The protein levels of poly and OGG1 were significantly reduced at 3,9, and 25 h after the onset of SE (p<0.05). However, APE1 decreased at 3 and 9h (p<0.05) and then returned to the control level at 25h (p>0.05). The protein level of APE1 in the hippocampi was constant in these group (p>0.05).3. The protein level of APE1 was significantly reduced in rats with epilepsy (p<0.05). Meanwhile DNA polymeraseγ(polγ) was increased compared with the control group (p<0.05).4. The mRNA levels of poly were also raised (p<0.05), which were compatible with the results of western blot. Interestingly APE1 showed no significant changes in mRNA level and hippocampal protein level (p>0,05;t-test).5. Along with the increase of polγ, Tfam was elevated in mRNA and protein level (p<0.05; t-test).ConclusionsmtBER enzymes failed to respond to SE induced by pilocarpine in rat hippocampi, and the mtBER pathway showed unbalanced expression, which may influence the repair of oxidative damage in mtDNA generated by seizures. mtDNA replication might serve as a potential compensatory mechanism for mtDNA damage.