Analysis Of Dynamic Changes And Synaptic Connections Of GABAergic Interneurons In The Hippocampus Of Pilocarpine-treated Rats

PART ONE:NUMBER CHANGES AND AXONAL SPROUTING OF GABAergic INTERNEURONS IN THE HIPPOCAMPUS OF PILOCARPINE-TREATED RATSObjective:Temporal lobe epilepsy(TLE)is one of the most common refractory epilepsy in clinical,but so far the epileptogenesis still is not clear.The enhancement of excitatory circuit and decrease of inhibitory circuit in the hippocampus play an important role in temporal lobe epilepsy. Researches revealed although the foundation of excitatory circuit participated in the generation of temporal lobe epilepsy,it still couldn't fully explain the reasons of spontaneous seizures.So currently GABAergic interneurons which are deeply associated with hippocampal inhibitory circuit have gradually been the research hotspot.Loss and axonal sprouting of GABAergic inhibirory interneurons are the main reasons of decease,recovery or enhancement of GABAergic inhibits. Great arguments still exist on GABAergic transmissions in temporal lobe epilepsy,and different interneuronal subtypes have different sensitivities to injuries induced by seizures in different models and different domains, so further studies are needed about the loss and axonal sprouting of GABAergic interneurons in temporal lobe epilepsy.In this research,we observed the number changes and axonal sprouting of GABAergic interneurons(SS,NPY,PV)at different time points in the hippocampus of rats induced by pilocarpine,and detected the degeneration of interneurons and their neurophils,in order to reveal the roles of GABAergic interneurons in the generation and compensation of temporal lobe epilepsy,provide more theoretical evidence for clinical treatments of refractory epilepsy。 Methods:1)120 healthy male SD rats were divided randomly into epilepsy groups(n=80)and control groups(n=40).Two groups were divided randomly into 8 subsets at 2,6hr and 1,3,7,15,30,60d after pilocarpine or normal sodium(NS)intraperitoneal injection.The models of epilepsy were established by intraperitoneal injection of pilocarpine and lithium, while the controls were injected with NS.The degree of seizure was judged according to Racine standard.2)Nissl stain was used to observe the pathological changes of hippocampus;FJB stain was used to detect the degeneration of hippocampal neurons and their neurophils.3)Immunohistochemistry method was used to detect number changes and axonal sprouting of SS\NPY\PV positive interneurons in different domains of the hippocampus at different time points.4)Degeneration of SS\NPY\PV positive interneurons and their neurophils on 7d and 60d after epileptic status(SE)was detected by the technique of double immunofluorescence combined with FJB.Results:1)After lithium-chloride and pilocarpine administration,92.5%rats were induced SE successfully,and the mortality rate was 21.25%.2)Nissl stain revealed,in the experimental group,loss of hippocampal neurons was most evident on 7d and 60d after SE. significant loss of hilar neurons and pyramidal neurons was present in area CA1(P＜0.01),while the loss of granular cells in the dentate gyrus was relatively slight(P＜0.05).3)FJB stain revealed,in the experimental group,the degeneration of hippocampal neurons was most evident on 7d and 60d after SE,mainly concentrated on pyramidal layer and hilar area. Degeneration of neurophils could be seen in the lacunosummoleculare (lm)layer on 7d after SE.4)In the experimental group,the number of SS positive neurons decreased in each hippocampal domain,and was least on 7d after SE(P＜0.01).In chronic phase,the number of SS neurons partially recovered,and even exceeded the control group(P＜0.01),while the number of SS neurons in the hilus and CA3 area was still less than normal levels(P＜0.01);Abundant SS positive fibers were found in the lm layer and outer molecular layer of dentate gyrus on 30d after SE,and numerous SS positive fibers could be seen throughout the layers of area CA1 on 60d after SE.5)In the experimental group,the number of NPY positive neurons decreased in the hilus of the hippocampus,and was least on 7d after SE(P＜0.01),In chronic phase,the number of hilar NPY neurons partially recovered,but was still less than the number in the control group on 60d after SE(P＜0.05);No evident changes of the number of NPY neurons existed in CA domains(P＞0.05)except the loss of them in CA3 area on 7d after SE(P＞0.05).Increased NPY positive fibers could be seen in the lm layer and molecular layer of the dentate gyrus on 30d after SE.6)There were no evident changes of PV positive neurons in early phase in the hippocampal hilus(P＞0.05),the number of PV neurons decreased in chronic phase(P＜0.01);PV positive neurons in CA1 area increased significantly after 15d after SE(P＜0.01), increased neurophils also could be seen;PV neurons in CA3 area increased as early as the phase after SE,and increased to maximum on 60d(P＜0.01),correspondingly increased neurophils also could be seen.7)Double immunofluorescence revealed,a few SS positive interneurons and fibers were also labeled by FJB in area CA1 on 7d after SE and in CA domain\hilus on 60d after SE;NPY positive interneurons labeled by FJB were only present in the hilus on 60d after SE;double labeled PV positive neurons were not seen. Conclusions:1)Different subtypes have different sensitivities to injuries induced by seizures in different time points and different domains,hilar dentritic inhibitory interneurons are most sensitive to injuries.2)Loss of GABAergic interneurons play an important role in the generation of temporal lobe epilepsy;loss of dentritic interneurons are partially because of the degeneration and death of neurons,while loss of perisomatic neurons may be not due to the cell's degeration and death.3)Axonal sprouting of inhibitory GABAergic interneurons, especially numerous increase of SS positive neurophils within area CA1 in chronic phase,may play a significant role in the generation and compensation of temporal lobe epilepsy.PART TWO:CHANGES OF NEURONAL SYNAPTIC CONNECTIONS IN AREA CA1 OF PILOCARPINE-TREATED RATS OBSERVED BY FLUORO-GOLDBackground and objective:Rearrangements of hippocampal excitatory and inhibitory circuits are deeply related to the generation of temporal lobe epilepsy.Axonal sprouting is the general phenomenon associated with temporal lobe epilepsy,and is the important structure basis of circuitry rearrangements. Nowadays there have been a lot of reports about excitatory circuit rearrangement,but the elucidation of synaptic connections among principal cells in temporal lobe epilepsy remain topics of intensive investigation;and researches about GABAergic inhibitory rearrangement are few,especially studies of synaptic connections among inhibitory interneurons during chronic phase of temporal lobe epilepsy are much more less.Results in part one revealed,numerous SS positive neurophils were present within CA1 area in chronic phase of temporal lobe epilepsy, and might be an important component of aberrant inhibitory circuits.In this study,we used fluorogold(FG)to observe synaptic connections among pyramidal cells in CA1 area of the hippocampus during chronic phase,and used FG tracer technique combined with immunofluorescence to observe the origin of aberrant numerus SS positive fibers and synptic connections among inhibitory interneurons within CA1 area during chronic phase in pilocarpine-treated rats,in order to reveal the important role of circuit rearrangements in the generation and compensation of temporal lobe epilepsy.Methods:1)20 healthy male SD rats were divided randomly into epilepsy groups(n=10)and control groups(n=10).The models of epilepsy were established by intraperitoneal injection of pilocarpine and lithium,while the controls were injected with NS.The degree of seizure was judged according to Racine standard.2)On about 60d after SE,we injected retrograde tracer FG into CA1 area of the hippocampus in vivo by using the stereotaxic apparatus,the same method was performed in control animals.After surgery,animals were allowed to survive for 5～7 days before perfusion-fixation.3)Immunofluorescence about SS was performed,confocal microscopy was used to observe the distribution of FG and double labling of SS and FG in neurons.Results:1)After lithium-chloride and pilocarpine administration,one animal was failed to induce SE,two were dead because of severe seizures,other seven animals survived.2)At the zone of FG-injected site,abundant FG-labeled pyramidal cells could be seen in two groups;additional FG-labeled pyramidal cells could be seen remote from the zone of dye spread in CA1 area in five rats of the experimental group,also additional FG-labeled pyramidal cells could be seen in subiculum in two experimental rats;additional FG-labeled pyramidal cells were not seen in the control group.3)At the zone of FG-injected site,FG-labeled SS positive interneurons could be seen in two groups,and there were no significant differences about the ratio of double labeled neurons to SS neurons between two groups(P＞0.05);Additional FG-labeled SS positive neurons could be seen remote from the zone of dye spread in CA1 area in experimental rats,also a few of additional FG-labeled SS neurons could be seen in CA3 area in two experimental rats;additional double labeled interneurons were not seen in control rats.Conclusions:1)Aberrant synaptic connections among pyramidal cells in CA1 area and aberrant connections of pyramidal cells between CA1 area and subiculum in the hippocampus of temporal lobe epilepsy,may be an anatomy basis which form the aberrant excitatory circuit,and have a promotive effect on the generation of temporal lobe epilepsy.2)Aberrant synaptic connections among SS positive interneurons exist in CA1 area of the hippocampus in temporal lobe epilepsy, increased SS positive neurophils may partially originate from enhanced axonal sprouting of SS interneurons in remote regions of CA1 area.The changes above may lead to the formation of aberrant inhibitory circuit, and play an important role in the generation and compensation of epilepsy.