The Role Of Myelinated Fibers In Epilepsy Induced By Lithium-pilocarpine In Mature And Immature Rats

PARTⅠ CHANGE OF MYELINATED FIBERS INIMMATURE AND MATURE RATS AFTER STATUSCONVULSION INDUCED BY LITHIUM-PILOCARPINEObjective: To study the change of myelinated fibers in hippocampusand corpus callosum of immature and mature rats after status convulsion(SC) induced by lithium-pilocarpine.Methods: Seventy-four immature and seventy mature rats, in whichSC was induced by lithium-pilocarpine (pilo group), and forty-eight foreach immature and mature rats (control group) were evaluated.Morphological changes of myelinated fibers in corpus callosum wereexamined by Gallyas staining, the expression of myelin basic protein (MBP)was determined by Western blot, and the Micro-structural characteristics ofmyelinated fibers were determined by transmission electron microscopy atdifferent time point (acute phase, latency phase, beginning of chronic phaseand chronic phase).Results: The results showed that the myelinated fibers both in the hippocampus and corpus callosum of the pilo group were degenerated, andwith significantly decreased MBP expression, relative to control group ratsat the acute phase lasting to the chronic phase after SC. While myelinatednerve fibers in control rats were normal, with plasma membrane rings ofregular heights and dense structure, the fibers in pilo-treated rats showedstratification, collapse and disruption, as well as disordered arrangement atdifferent time points after SC. There is no age-related difference in theextent of myelinated fibers damge induced by SC.Conclusion: Myelinated fibers in hippocampus and corpus callosumwere damage irreversible after SC, which had no difference betweenmature and immature brain. PARTⅡ ALTERED HIPPOCAMPAL MYELINATED FIBERINTEGRITY IN A LITHIUM-PILOCARPINE MODEL OFTEMPORAL LOBE EPILEPSY: A STEREOLOGICALINVESTIGATIONObjective: The damage of white matter, primarily myelinated fibers,in the central nervous system (CNS) of temporal lobe epilepsy (TLE)patients has been recently reported. However, limited data exist addressingthe types of changes that occur to myelinated fibers inside the hippocampus as a result of TLE. The current study was designed to examine this issue ina rat model.Methods: Eight adult rats, in which TLE was induced bylithium-pilocarpine treatment (pilo group), and seven normal adult rats(control group) were evaluated. Microstructural characteristics ofmyelinated fibers were quantitatively determined by transmission electronmicroscopy and stereology.Results: The results showed that the total volumes of hippocampalformation, myelinated fibers, and myelin sheaths in the hippocampus ofpilo group rats were decreased by20.43%,49.16%, and52.60%,respectively. In addition, pilo group rats showed significantly greater meandiameters of myelinated fibers and axons, whereas the mean thickness ofmyelin sheaths was less, especially for small axons with diameters from0.1to0.8μm, compared to control group rats. Finally, the total length of themyelinated fibers in the hippocampus of pilo group rats was significantlydecreased by56.92%, compared to that of the control group, with thedecreased length most prominent for myelinated fibers with diameters from0.4to0.8μm.Conclusions: Both morphological and molecular changes wereobserved for hippocampal myelinated fibers in TLE rats, which maycontribute to abnormal discharge in patients with TLE. PART Ⅲ EFFECT OF CUPRIZONE-INDUCEDDEMYELINATION ON SEIZURE SUSCEPTIBILITY INIMMATURE RATSObjective: To study the effect of cuprizone-induced demyelinationon seizure susceptibility in immature rats.Methods: Demyelination model was induced in21day immature ratsfed for2weeks (CPZ2week group) and4weeks (CPZ4week group) with0.6%cuprizone. Morphological changes of myelinated fibers wereexamined using Gallyas staining. The expression of myelin basic protein(MBP) was determined by immunohistochemistry and western blot. Thelatency of seizure induced by pilocarpine and the latency of spontaneous ofdischarge induced by Mg2+free artificial cerebrospinal fluid (ACSF) inhippocampus slices were determined. In addition, behavior andepileptiform spikes were recorded by video-EEG monitoring. Theapoptosis cells were detected by TUNEL.Results: The results showed that myelinated fibers of the CPZ groupwere degenerated, and with significantly decreased MBP expression,related to control group rats. The latency of seizure induced by pilocarpinewas decreased in CPZ4weeks group (13.33±3.46min) when compared tothat in control group (19.66±4.33min)(P<0.01). In addition, the latency induced by Mg2+free ACSF in hippocampus slices was significantlydecreased in CPZ2weeks (30.71±5.09min) and4weeks (22.14±4.76min)compared with2week control group(49.86±10.65min)and4week controlroup(51.86±10.78min)(P<0.05, P<0.01, respectively).While nospontaneous epileptiform spikes were observed in control group, EEGabnormal discharge can be seen in66.64%of CPZ2w rats (P<0.01) and100%of CPZ4w rats (P<0.01). Finally, there was no difference in thenumbers of apoptosis cells in hippocampus between CPZ groups andcontrol groups.Conclusion: Immature rats treated with CPZ showed increasedseizure susceptibility that was strongly correlated with the severity ofdemyelination. PART Ⅳ THE RELATION BETWEEN GLUTAMATE ANDMYELINATED FIBERS DAMAGE IN EPILEPSY INDUCED BYLITHIUM-PILOCARPINE IN MATURE AND IMMATURE RATSObjective: In the first part, the damage of myelin was found inhippocampus and corpus callosum at different phase after seizureconvulsion (SC) induced by lithium-pilocarpine treatment. Previous studyhas reported that oligodendrocytes were vulnerable to oxidative damage mediated by glutamic acid and microglia play an important role insustaining the balance of glutamate. However, limited data exist addressingthe relation among glutamic acid, microglia and the damage of myelinduring different phase after SC. The current study was designed to examinethis issue in a rat model.Methods: sixty-two immature rats and fifty-nine adult rats wererandomly respectively divided into control group and pilo group whichstatus convulsion was induced by lithium-pilocarpine treatment. Thechange of glutamate concentrations was measured by high performanceliquid chromatography and microglial changes in hippocampus and corpuscallosum was examined at different period (acute period, latency, earlyphase of chronic and chronic) after SC by analyzing IBA-1.Results: An increased glutamate concentration and significantlygreater IBA-1immunolabeling were observed in hippocampus and corpuscallosum not just in immature rats but in adult rats at acute phase after SCand continuing throughout the chronic phase. In addition, both the area ofIBA-1labeled processes and the number of microglial cells was increasedat different phase after SC in immature and adult rats.Conclusion: The precise temporal and spatial response pattern ofglutamate and microglial cells after SC which was coincident with thechange pattern of myelin, might signify detrimental role in damage ofmyelin through excitotoxic cell death after SC.