Mitochondrial Biogenesis In Hippocampi Of Rats With Seizures Induced By Lithium-pilocarpine

As one of the most common neurological disorders, epilepsy affects about9million patients in china, in which about25-40%of patients suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) is the most prevalent refractory epilepsy in human and its pathogenesy still remains obscure. To date, there is still no effective therapy to gain significant curative effects through either administration of antiepileptic drugs (AEDs) or operating. Accumulating evidence suggested that the behavior, electroencephalogram (EEG) and the hippocampal neuronal injury of 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 TLE in human. In the lithium-pilocarpine induced epilepsy, a chronic period with spontaneous recurrent seizures (SRSs) is used to study TLE in human. The main features of the SRSs observed during the long-term period resemble those of human TLE and recurs2-3times per week per animal.Epilepsy is a recent addition to the neurological disorders in which mitochondrial oxidative stress and dysfunction have been suggested to be contributing factors. Consistent with other studies, our previous study reported that chronic recurrent seizures led to mitochondrial dysfunction and mtDNA oxidative damage so developing mitochondrial-targeted therapy is meaningful. There are some mitochondrial targeted protecting mechanisms in cell. Firstly, mitochondrial antioxidant system; Secondly, mitochondrial base excision repair mechanism; Thirdly, mitochondrial biogenesis. The mechanisms above can reserve normal quantitition and quantification of mitochondria genome and/or mitochondria, achieving normal mitochondrial function. Little is known about contribution of mitochondrial biogenesis in chronic seizures.Mitochondrial biogenesis is an essential mechanism in normal cell growth and function, but also represents an adaptive strategy induced by mitochondrial dysfunction. Recently, interest in mitochondrial biogenesis has increased because of its possible role in neurological diseases, such as hypoxic-ischemic brain injury, transient brain hypoxia and suatained brain hypoxia, in which mitochondrial biogenesis are activated. Meanwhile, mitochondrial biogenesis is defective in HD. To definitively demonstrate that mitochondrial biogenesis is invovled in the above neurological diseases will require more work to do, but it is reasonable that induced mitochondrial biogenesis in response to the above stimuli is a neuroprotective mechanism to protect neurons from damage by dysfunctional mitochondria and reactive oxidative species and dysregulated mitochondrial biogenesis may contribute to the bioenergetic defects and oxidative damage in disease such as HD. In this study, we detected the response of mitochondrial biogenesis to chronic seizures to verify the underlying mechanisms of mitochondrial dysfunction and extensively study the molecular biological mechanisms of TLE.PARTIA study of behavior and pathobiology in lithium-pilocarpine induced chronic seizures in ratsObjectiveTo investigate the alteration of behavior 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. Seizures were allowed to last for60min and then were terminated by administration of diazepam. Rats were monitored by video recordings to assure development of status epilepticus (SE)and SRS. Study the pathological changes with Nissl at3h and2months after SE induced by pilocarpine.Results1. Rats that showing stage Ⅳ-Ⅴ convulsive seizures according to Racine were considered to develop SE successfully.81.3%of the rats were induced to develop SE after administration of lithium and pilocarpine. The time from pilocarpine injection to the first onset of stage IV SE was39.8±13.2min. The latency phase is about11.5+6.8days.76%showed seizures at least once a week.2. Nissl staining showed the neuronal damage in hippocampal CA1and CA3regions at3h and2months after SE. 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) and SRS according to the alteration of behavior. Epilepsy induced by lithium-pilocarpine caused hippocampal neuronal damage. PART ⅡAlteration of mitochondrial biogenesis in lithium-pilocarpine induced chronic seizures in ratsObjectiveTo detect the alteration of mitochondrial biogenesis in the of hippocampi in rats with chronic seizure, including the level of mtDNA-encoded protein, mtDNA copy number and the transcription and replication of mtDNA, mitochondrial number and ulstrastructure damage.MethodsAdult male Wistar rats were divided randomly into chronic controls and experimental rats. MtDNA-encoded protein was measured by Western bloting. RNA and DNA was isolated from fresh hippocampi. Quantitative real-time PCR was used to evaluate the mtDNA number and mtDNA transcription and replication. Mitochondrial number and ultrastruture damage was evaluated by electron microscope.Results1. Compared with control group, mtDNA-encoded protein decreased in hippocampi of rats with chronic seizures (p<0.05).2. Compared with control group, experimental group showed decreased mtDNA copy number (p<0.05).3. Compared with control rats, mtDNA transcription and replication decreased in hippocampi of rats with chronic seizures (p<0.05).4. Mitochondrial number was consistent in the chronic seizures rats compared with control rats. However, mitochondrial ultrastructure was damaged and it varied from mild to profoundly severe in the hippocampus during experimental epilepsy.ConclusionsMitochondrial biogenesis is impaired in hippocampi of rats with chronic seizures induced by pilocarpine. The significant decreases in the expression of mtDNA-encoded protein and the relative mtDNA copy number may be due to impaired mtDNA transcription and replication. Mitochondrial number did not differ in two groups while mitochondrial presented with different degrees of damage in rats with chronic seizures. Including, recurrent seizures impaired mitochondrial biogenesis. PART ⅢThe mechanism of impaired mitochondrial biogenesis in hippocampi of rats with chronic seizuresObjectiveTo assess the major factors of mitochondrial biogenesis at both mRNA and protein levels and the activity of mitochondrial transcriptional factors, so as to explore the underlying mechanisms of impaired mitochondrial biogenesis and mitochondrial dysfunction in epilepsy.MethodsAdult male Wistar rats were randomly divided into2groups for treatment:chronic control and chronic seizures (60d). Quantitative real-time PCR and western blot were used to determine the level of major factors of mitochondrial biogenesis. Electrophoretic mobility shift assay (EMSA) was used to measure the activity of mitochondrial transcriptional factor.Results1. Real-time PCR analysis demonstrated increased expression of peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-la), nuclear respiratory factor-1(NRF-1) and mitochondrial transcription factor A (Tfam) in rats with chronic seizures compared with control rats (p<0.05).2. The protein levels of PGC-1a and NRF-1in hippocampi were significantly increased in experimental group (p<0.05). The protein level of Tfam in the mitochondria in hippocampi was increased in experimental group (p<0.05).3. Activity of Tfam was markedly reduced in rats with chronic seizures compared with control rats (p<0.05).ConclusionsThe major factors that regulate mitochondrial biogenesis, including PGC-la, NRF-1and Tfam showed increased expression while activity of Tfam significantly reduced in rats with chronic seizures, which may influence the mitochondrial biogenesis. Impaired mitochondrial biogenesis might serve as a potential mechanism for mitochondrial dysfunction in epilepsy. Epilepsy;PGC-1a;NRF-1;Tfam;pilocarpine;...