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Establishment of Immature Rat MTLE Model and the Study of TLR4, IL-1β Expression Changes in MTLE

Author: Camara Aboubacar Damaye（DaMaYi）
Tutor: YinFei
School: Central South University
Course: Pediatrics
Keywords: MTLE developing rats inflammation TLR4 IL-1β
CLC: R742.1
Type: Master's thesis
Year: 2011
Downloads: 67
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Abstract

Epilepsy is one of the most frequent disorders of the central nervous system (CNS). Clinically, the epilepsies are characterized by spontaneous, recurrent epileptic seizures, either convulsive or non-convulsive, which are caused by partial or generalized discharges in the brain. In addition, 50% adult patient of epilepsy has their first seizure before their 15 years old. Epilepsy treatment is successful in the majority of the cases; however, still one third of the epilepsy patients are refractory to treatment. Among the several kinds of epileptic disorders, mesial temporal lobe epilepsy (MTLE) has high incidence, which represents about 10-30% of all childhood epilepsy cases, characterizing by seizure generation from the mesial temporal lobe. Moreover,70% of the patients with MTLE do poorly responds to anti-epileptic drugs (AEDs) and surgically interruption, which is a headachy problem in the world. So, in order to find more effective AEDs and improve the patient prognosis, it is important to make clear the deeply pathogenesis of MTLE. Previous studies indicated hippocampal sclerosis (HS) is the pathological substrate of this disease. HS is characterized by neuron death and aberrant mossy fibers sprouting (MFS), both of which are suspected to contribute to abnormal recurrent excitation or inhibition circuitry and play major roles in epileptogenesis. It is thought that HS and MTLE was secondary to the injuries of hippocampus occurred in childhood, including trauma, febrile convulsion and status epilepticus (SE), nevertheless the deeply mechanism is not clearly till now.Several animal models and clinic samples were used to study the pathogenesis of epilepsy, in which most of them was undertaken in the final stage of this disease, lacking a systematic and consecutive research. However, MTLE is often the result of an early insult that induces changes and reorganization of the hippocampus formation. So it has great significance to determine the nature of injuries that might contribute to the development of epilepsy, to observe and intercede in the disease process subsequent to an injury preceding the onset of spontaneous seizures, and also to study the chronically epileptic brain in detail. Furthermore, in early development of the central nervous system, the structure and function, the neurotransmitters and receptors related to epilepsy had enormous room for growth. The influence of seizures to the immature brain and the effect of the AEDs are various in the different stages of the brain’s development. Thus, it is necessary to establish an ideal immature animal model for the study of the mechanism contributing to epileptogensis.The current molecular researches related to MTLE have been focused on regeneration and synaptic plasticity, neurons damage regulation, cellular metabolism and protein synthesis, neurotransmission and signal transduction, cytoskeleton changes and ion channels changes and ect, For a long time, the relationship between inflammation and MTLE has been ignored, because blood brain barrier made CNS seemed as an inflammation-free organ. However, recently researches suggested inflammation could play a role in the pathogenesis of MTLE. Toll like receptor-4(TLR4) is a key receptor of innate immunity, which is widely investigated in various CNS injury models. It could induce promotion of inflammatory cytokines (such as IL-1βand TNF-a) via NF-κB dependant pathway, leading to staring of inflammation. Recent clinic data showed in the surgically removed hippocampus of epilepsy patient, the expression of TLR4 and IL-1(3 was promoted. It also has been observed that IL-1βwas up-regulated in the hippocampus of febrile convulsion mouse, and a fairly high ratio of MTLE adult patients with HS can be pursued long history of febrile convulsion in childhood. In addition, people find the interaction between LPS and TLR4 promoted the epileptic charge in rat, which could be suppressed by inhibitor of IL-1βreceptor. These data indicated the possibility that the inflammation in hippocampus, especially mediated by TLR4 and IL-1β,could be related to the pathogenesis of early brain which could lead to MTLE. It has a significant meaning to study the dynamic changes of TLR4 and IL-1βin the whole process of MTLE, which could lead to further learning of MTLE occurrence and development mechanism.In our experiment, we employed pilocarpine on immature rats to establish immature rat chronic MTLE model, and by using immunohistochemistry, western blotting and RT-PCR analysis, we examined the dynamic expression of TLR4, IL-1βin the subfield of hippocampus and evaluated whether they had correlation with the epileptogenesis.Section 1 Establishment of immature rat chronic MTLE modelObjective The purpose of our study is to develop an easy, reliable animal model mimicking the human MTLE. Methods 165 healthy 3-week old Sprague-Dawley rats of either sex were divided into pilocarpine epileptogenesis group (experimental group) and control group. In experimental group, the rats were intraperitoneally injected with lithium chloride 3 (mEq/kg) and pilocarpine hydrochloride (30 mg/kg) to induce status epilepticus (SE); 90min after the onset of SE, the animals received a deep intramuscular injection of 10mg/kg diazepam; Survival rats were grouped at 3 subgroups randomly:E1 gourp (sacrificed at 1.5 hours after SE), E2 gourp (sacrificed at 3 weeks after SE) and E3 gourp (sacrificed at 8 weeks after SE); Nissl’s staining (to evaluate neuron loss) and Timm staining (to evaluate MFS) were performed on 2/3 of each subgroup rats, and the rest were executed for EEG scanning (to evaluate epileptic charges); in addition, continuously observed for onset and recurrence of spontaneous seizures were processed every day. In control group, an equivalent volume of saline were used to instead of pilocarpine, and the other treatment were same as which taken on the experimental group. Results In experimental group,94.1% of the rats had successfully induced SE with a high mortality of 68.8%; 75% of the survival rats were observed for spontaneous recurrent seizures (SRSs) which is quite consistent with the features of human MTLE. In control rats, behavioral and EEG manifestations are normal. MFS was observed in group E2 and E3 with the exception of group E1 and controls. Also, the Nissl-stained sections revealed many neurons in DG, CA1 and CA3 regions in the control groups; inversely, the neuron numbers were significantly decreased in the E2 and E3 groups. Conclusions 1.We used lithium chloride and pilocarpine to establish an immature rat chronic MTLE model,75% of the survival model rats occured SRSs.2. The established MTLE animal model is quite consistent with the features of human MTLE in the aspect of seizure style, EEG manifest and hippocampus morphology.Section 2 TLR4, IL-1βdynamic expression in MTLE pathogenesisObjective To detect the expression changes of TLR4 and IL-1(3 in hippocampus of the immature MTLE rats during the development of epilepsy. Method Based on the MTLE rat model established in section 1, both taking advantage of immunohistochemistry and western blot approaches, the expression level of TLR-4 in the subfields of hippocampus were detected at different time-point, and RT-PCR method was applied to detect the expression of IL-1β. Results TLR4 and IL-1βexpression was significantly increased in early stage (1.5 hours after SE) in the MTLE model rat, and this promoted expression was persist in the whole stage of MTLE process, which was positively correlated with the preference of neuron loss and MFS in DG, CA1 and CA3 region of MTLE hippocampus. Conclusion 1.TLR4 and IL-1βpromoted in the very early stage of MTLE, which indicated inflammation could be the initiating factor of MTLE.2. High expression of TLR4 and IL-1βwere related to hippocampal sclerosis.

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