Intrahippocampal Infusion Of Lipopolysaccharide Induces Seizures And The Associated Mechanisms In Rats

At present, there is not an antiepileptic drug which can prevent the epilepsy-susceptible individuals from getting epilepsy after suffering the central nervous system injury involving intracranial infection, traumatic brain injury and febrile convulsions. Numerous studies discovered that obvious inflammatory reaction existed in the initial and transmission brain region of the epileptic seizure. However, the causal relationship between the epilepsy and the inflammatory reaction, as well as the relevant molecule regulatory mechanism were still unknown. The recent study confirmed that the intracranial inflammation could decrease the seizure thresholds of the experimental animals. Rodgers, et al. reported that cortical innate immune responses alone could produce profound increases of brain excitability resulting in focal seizures. These studies suggested that the epileptic seizure might be facilitated or triggered by the immune reaction of the brain. But it has not yet been clear whether inflammatory reaction could result in acute and chronic epileptic seizure as an independent factor in vivo, and result in the hippocampal sclerosis.LPS (lipopolysaccharide, endotoxin of gram negative bacteria) is a major component of the cell walls of gram negative bacteria, which can bind to the Toll-like receptor4in the glial cells, lead to the immune reaction and release cytokine. It is the most classic and commonly used inflammatory agent currently.Cell factors are a group of polypeptide or glycoprotein, which typical function is to mediate and adjust immune response, stimulate hematopoietic functions, participate in tissue repair. But research indicates that immune factors also play an important role in nervous system in recent years. Neurons and glial cells all have cytokine receptors and can synthetize and secrete various cell factors. IL-1β (Interleukin-1β,IL-1β and TNF-a(Tumor necrosis factor-alpha, TNF-a)are two important cell factors, the raised contents of them in serum of epilepsy patients can be predicted in relation with epilepsy. Giving LPS to astrocyte culture can stimulate astrocytes to generate IL-1βand TNFa. However, the role of activating TLR4on astrocyte hasn’t still been confirmed in vivo.In this experiment, we adopted LPS hippocampal injection to trigger the inflammatory reaction of the central nervous system, mimiced the inflammatory reaction of the central nervous system on the injury and infection, so as to determine whether the inflammation could be solely as the independent epileptogenic factor to result in the epileptic seizure in vivo, and explore the role of activating TLR4on the generation of IL-1βand TNF-a in vivo by detecting the change of concentration of IL-1βand TNF-ain hippocampus after LPS intrahippocampus injection in rats.PART I Intrahippocampal Infusion of Lipopolysaccharide induces seizures in ratsObjective:To investigate whether intrahippocampal infusion of lipopolysaccharide(LPS) could induce epileptic seizures and hippocampal sclerosis in rates.Methods:36male Wistar rats were randomly divided into3groups, and1.5μl LPS (5ug/ul), normal saline (NS) or kainate (0.4ug/ul) was injected into the right dorsal hippocampus of the rats respectively. All rats were then recorded the behavior characteristics and electroencephalography. The morphological features of hippocampus sections were observed by HE staining; the expression of glial fibrillary acidic protein(GFAP) was determined by immunohistochemical technique.Results:The rats of NS group did not have seizure and their electroencephalographic (EEG) showed basic rhythm, while the rats of LPS and KA groups demonstrated different grades of seizures, and EEG showed seizure activities(sharp waves or spikes), The seizure grade of KA group was significant higher than that of LPS group. Seizures could be observed in some rats of LPS and KA group3-4weeks after injection. Compared with the NS group, astrocytes of the LPS and KA group were activated. While in the chronic phase, the astrocytes proliferated and the number of neurons decreased.Conclusion:Intrahippocampal infusion of lipopolysaccharide(LPS) can activate glial cells, induce epileptic seizures and hippocampal sclerosis in rates. PART Ⅱ The effect of Intrahippocampal Infusion of Lipopolysaccharide on nNOS in ratsObjectiveTo explore the effect of intrahippocampal infusion of lipopolysaccharide on neuronal nitric-oxide synthase (nNOS) in rats.Methods36male Wistar rats were randomly divided into3groups, and1.5μl LPS (5ug/ul), normal saline (NS) or kainate (0.4ug/ul) was injected into the right dorsal hippocampus of the rats respectively. The expression of neuronal nitric-oxide synthase (nNOS) was determined by immunohistochemical technique.ResultsCompared with the NS group, the expression of nNOS in neurons was obviously increased in the acute phase.ConclusionIntrahippocampal infusion of LPS can improve the expression of nNOS, and cause nerve injury. PART Ⅲ Change of IL-1β and TNF-ain hippocampus of rats with epilepsy induced by LPSObjectiveTo explore the role of LPS intrahippocampus injection on the concentration of IL-1βand TNF-α in rats.Methods84male Wistar rats were randomly divided into2groups, and1.5μl LPS (5ug/ul), normal saline (NS) or kainate (0.4ug/ul) was injected into the right dorsal hippocampus of the rats respectively. Rats were sacrificed at0.5h,2h,6h,18h,24h,36h after intra-hippocampus injection, Concentration of IL-1βand TNF-ain Hippocampus was detected by adopting ELISA method in accordance with instructions.Results1. Concentration of IL-1βand TNF-aof LPS group were all higher than that of NS group (P<0.05) at1h,2h,6h,12h,18h,24h and36h after hippocampus injection in rats.2. Concentration of IL-1βincreased gradually and reached the peak at18h after hippocampus injection and then started to decrease in LPS group.3Concentration of TNF-aincreased rapidly and reached the peak at2h after hippocampus injection and then started to decrease.ConclusionIL-1and TNF-aincrease obviously after intrahippocampal infusion of LPS, and they probably play a significant role in seizures induced by LPS, which still needs to be confirmed in the later work.