Analysis Of Dynamic Changes And Synaptic Reconstruction Of GABAergic Interneurons In Epilepsy Rats

Background and Objective:Temporal lobe epilepsy(TLE) is one of the most common refractory epilepsy in clinical, but so far the epileptogenesis is still 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 such as mossy fiber sprouting 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. Most studies have showed loss of GABAergic inhibitory interneurons in early period of SE, while some axonal sprouting exist in hippocampus and dentate gyrus in chronic period. But totally the range of research on GABAergic interneurons in temporal lobe epilepsy is incomplete and there are still much great arguments on it, so further studies are needed to be done. We have already observed the dynamic changes and axonal sprouting of SS&NPY by immunohistochemistry technology in our early study, and the aim of this research is to find out transmissions of other subtypes of GABAergic interneurons in temporal lobe epilepsy.Methods:1.180 healthy male SD rats were divided randomly into epilepsy groups(n=105) and control groups(n=75). Two groups were divided randomly into 5 subsets at 1,7,15,30,60d 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 and NeuN immunohistochemistry was used to observe the pathological changes of hippocampus.3. Immunohistochemistry method was used to detect number changes and axonal sprouting of CB\PV positive interneurons in different domains of the hippocampus at different time points.4. Immunohistochemistry method was used to detect number changes of NeuN positive neurons in different domains of the hippocampus at different time points, and coexpression of SS/NPY positive interneurons combined with NeuN was detected by the technique of double immunofluorescence.Results:1. After lithium-chloride and pilocarpine administration,80% rats were induced SE successfully, the mortality rate was 10.7%, and the success rate of the model was 71.4%.2. Nissl stain revealed that 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 CA area(P<0.01), while loss of granular cells in the dentate gyrus was relatively slight(P<0.05).3. CB expression could also be seen in CA1 pyramidal neurons, but weaker than that of CB interneurons. The number of CB positive neurons in CA1 area decreased after SE(P<0.05), but no obvious change in CA3 area. CB neurons in hilus decreased to minimum on 1d(P<0.05), and recoverd gragrully in chronic phase. On 15d after SE, plenty of CB positive neurophils could be seen in stratum radiatum of CA1 area.4. There was no evident changes of PV positive neurons in early phase in 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.5. NeuN immunohistochemistry revealed that in the experimental group, significant loss of hilar neurons, pyramidal neurons in CA area was present (P<0.01), while loss of granular cells in the dentate gyrus, neurons in stratum radiatum and lacunosum-moleculare of CA3 was relatively slight(P<0.05). Changes in outer molecular layer of dentate were not notable. On 60d after SE, neurons of pyramidal layer in CA1 almost disappeared(P<0.01), but in initiation site of CA1 area, part of NeuN positive neurons reserved, even beyond normal in stratum oriens and stratum radiatum.6. Double immunofluorescence revealed that both in experimental and control group, double labeled SS interneurons could be seen at each time point. The number of it was least on 7d in hilus; partially recovered on 15d in stratum oriens of CA1,even exceeded the controls in all layers of CA1 on 60d; decreased to minimum on 7d in CA3 and also recovered gragrully in chronic phase. Similar changes could be seen in double labeled NPY interneurons, and differences were that little change happened in CA3 area.Conclusions:1. Different subtypes of interneurons have different sensitivities to injuries induced by seizures in different time points and different domains. On the whole, there are some features of interneurons after SE:a) the number in CA1 decreases in early phase and recovers in chronic phase, even beyond normal; b) not obvious in CA3; c) decreases in hilus.2. Level of axonal sprouting vary from different subtype of interneurons, especially numerous increase of SS positive neurophils within area CA1 in chronic phase may come from increased interneurons in stratum oriens and stratum radiatum of CA1,and form a pathological inhibitory loop to take in part in generation and compensation of temporal lobe epilepsy. Background and Objective:There are trisynaptic loop in normal hippocampal stuctrue, involving excitatory circuits mediated by pyramidal neurons and granule cells and inhibitory circuits by interneurons. Rearrangements of hippocampal excitatory and inhibitory circuits are deeply related to the generation of temporal lobe epilepsy. 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 incestigation. Meanwhile researches about GABAergic inhibitory rearrangement are few, especially studies of synaptic reconstruction among inhibitory interneurons during chronic phase of temporal lobe epilepsy are much more less. Neuroanatomical tracing can help us to observe the synaptic connections through a total level, and aberrant inhibitory circuits will be revealed through interneurons tracing technique. Results of our previous study showed numerous SS positive neurophils present within CA1 area in chronic phase of temporal lobe epilepsy, and in this study, we hoped to use fluorogold(FG) to observe synaptic reconstruction in different subtypes of interneurons in every area of hippocampus during chronic phase, in order to reveal aberrant formation of inhibitory circuit rearrangements in temporal lobe epilepsy.Methods:1.80 healthy male SD rats were divided randomly into epilepsy groups(n=40) and control groups(n=40). 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 and CA3 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 7-10 days before perfusion-fixation.3. Immunohistochemistry about FG to observe aberrant excitatory circuit rearrangements.4. Confocal microscopy was used to observe the distribution of FG Double immunofluorescence combined with SS/NPY and FG was performed, to observe aberrant inhibitory circuit rearrangements.Results:1. After lithium-chloride and pilocarpine administration, five animals were failed to induce SE, four were dead because of severe seizures. The success rate of the model was 77.5%.2. FG immunohistochemitry showed abundant FG-labeled neurons at the zone of FG-injected site, and the injection site could be divided into two bands. After injected FG into CA1 area, FG-labeled pyramidal cells could be seen remote from the zone of dye spread in CA1 area, CA3 area, and subiculm, while FG-labeled non-principle neurons could be seen in stratum oriens of CA1 and hilus in experimental group. When injection was taken place in CA3 area, FG-labeled pyramidal cells could be seen in the whole CA3 area and hilus in two groups; additional part of pyramidal cells in CA1 in experimental group; additional part of granule cells in dentate in control group; also additional some FG-labeled non-principle cells could be seen in hilus and remote from the zone of dye spread in CA1 area.3. Double immunofluorescence about FG and SS/NPY revealed that, at the zone of FG-injected site, FG-labeled SS/NPY positive interneurons could be seen in two groups, and there were no significant differences about the ratio of double labeled neurons to SS/NPY total neurons between two groups(P>0.05). As FG injection into CA1, double labeled interneurons could be seen remote from the zone of dye spread in CA1 area, CA3 area and hilus in experimental rats. When injection was taken place in CA3 area, FG-labeled SS/NPY neurons could be seen in hilus in experimental rats.Conclusions:1. Aberrant synaptic connections among pyramidal cells in CA1 area, pyramidal cells between CA1 and subiculum and pyramidal cells between CA1 and CA3 in the hippocampus in chronic phase of temporal lobe epilepsy may form an aberrant excitatory circuit arrangement, which will eventually develop into hyperexcitability in hippocampus.2. Aberrant synaptic connections among dendritic interneurons exist in CA1 area, CA3 to CA1, hilus to CA1, and hilus to CA3 area of the hippocampus in chronic phase of temporal lobe epilepsy. The inhibitory circuit arrangement related to axonal sprouting may play an important role in the generation and compensation of epilepsy.