Dynamic Changes And Synaptic Connections Of Calretinin And Parvalbumin Positive Interneurons In The Hippocampus Of Pilocarpine-treated Rats

Objective:Temporal lobe epilepsy is the most common forms of epilepsy. A huge number of factors and compensatory mechanisms have been accompanied with temporal lobe epilepsy. One of these mechanisms in humans and rats models is sprouting of hippocampal principal cell axons, which indicate that axonal sprouting may be a general phenomenon accompanied with temporal lobe epilepsy. Much of current knowledge about the neuronal organization of the Hippocampus comes from studies of these principal neurons. Only late work points to the decisive role of inhibitory interneurons in regulating the complex interactions between these neurons In the pilocarpine model of seizures in temporal lobe epilepsy (TLE), vulnerability of GABAergic interneurons to excitotoxic damage has been reported in the hippocampal regions. However, little is known about the Calretinin (CR)-containing interneurons that degenerate in these regions at different time points.Methods:The study was conducted at Central South University, First Xiangya Hospital, Changsha, Hunan, China between 2008-2010. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic rats. Two hundred twenty rats were divided into two groups:epileptic rats (n=170):received intra peritoneal injection of lithium in addition to pilocarpine, and control rats (n=50):received intraperitoneal injection of normal saline. Epileptic rats in their turn were stratified in two subgroups: preliminary groups(n=50):pilocarpine was administered as a dose of 30 mg/kg followed later by repeated application of low dose pilocarpine 10 mg/kg at 30-min intervals, and epileptic group(n=120):Pilocarpine was injected as initial dose of 25 mg/kg followed by repeated 10 mg/kg Lithium chloride was administrated 24 hours before pilocarpine injection. Pilocarpine-treated rats were scored in each rat by Racine's scale (Racine, 1972), considering only motor seizures for scoring. To determine whether hippocampal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from both age-matched control and epileptic (2h,6h, Id,3d,7d,15 d,30d and 60d post status epilepticus) rats. The pathological changes of hippocampus were observed by Nissl stain.Results:In the preliminary group 60%of rats developed a status epilepticus and 10 of rats were died,30% did not enter into status epilepticus and one rat that developed status epilepticus died during the procedure. In the other group of rats the pilocarpine resulting in 10% mortality and 85% status epilepticus. The hippocampus of the epileptic rats showed a neuronal cell loss pattern with comparable subregions to hippocampus of the normal rats. Our results revealed the most dramatic cell loss to be in hilar, CA1 and CA3 areas in all epileptic rats at one 7 days,15 days,30 days and 60 days. There were significant difference between control and Epileptic rats (P<0.05) Also there were a significant cell loss at one day post SE (P<0.05).Quantitative analysis revealed significant differences between control and Epileptic rats in the number of CR positive interneurons at 1 days,30 days and 60 days (P<0.05). These interneurons were distributed in hilar, CA1 and CA3 areas and in dentate gyrus of both control and Epileptic rats, but was more numerous in hippocampus of normal rats. However transient increase of CR-positive interneurons was observed in CA1 between 7and 15 days post status epilepticus (P>0.05). CR interneurons were mostly located in hilar and CA1 for epileptic and in hilus for control.Conclusions:Our data suggest that intrperitoneal injection of treatment of lithium-pretreated rats with 25mg/kg pilocarpine efficiently induce status epilepticus with lower mortality rates than 30 mg/kg pilocarpine. The different proportion of inhibitory interneurons were observed in the epileptic rat Hippocampus, their numbers differ from controls. These functional alterations of inhibitory circuits in the Hippocampus are likely to be compensatory changes with a role to balance the enhanced excitatory input in the region.ObjectiveBrain regions be composed of basic local circuits and cell congregation specialized to performed discrete computations that eventually give rise to epilepsy. These circuits are built from regionally specific union of limited types of individual neurons. The hippocampal formation is a brain region critically involved in temporal lobe epilepsy epilepsy. Temporal lobe epilepsy is the most common form of epilepsy. A characteristic feature of temporal lobe epilepsy (TLE) is the latency between an early neurological insult and eventual expression of intractable spontaneous recurrent complex partial seizures Latency between an early neurological insult and development of spontaneous recurrent seizures suggests aberrant chronological reorganization in patients with mesial temporal sclerosis associated epilepsy. Pilocarpine-induced status similarly results in delayed development of spontaneous recurrent seizures. Mossy fiber sprouting by the dentate granule cells is a well-characterized manifestation of such temporal structural reorganization in both patients and rat models. However, alterations in other components of hippocampal circuitry have not been studied. in the present Calretinin (CR) and the retrograde tracer Fluoro-Gold (FG) were used to identify synaptic connections among pyramidal cells, and the origin and synaptic connections of CR positive interneurons in CA1 area of the rat hippocampus during chronic phase of epilepsy.MethodsRats subjected to the experiment were randomly divided into the following groups:Rats with epilepsy and Control rats. Rats with epilepsy received intra peritoneal injection of Lithium chloridel8-24 hours prior to 30mg/kg pilocarpine administration, followed by 10mg/kg Pilocarpine at 30-min intervals until status epilepticus (SE) developed, while control rats received intra peritoneal injection of normal saline. rats with epilepsy were scored in each rat by Racine's scale.After two months, both groups rats with epilepsy and control rats were placed in a Kopf Stereotaxic Apparatus and Hamilton syringe were used to inject retrograde tracer reproducibly into specific hippocampal target site. In all rats, retrograde tracers, Fluoro-Gold were microinjected into the CA1 area of the hippocampus. after injection of Fluoro-Gold, rats were allowed to survive for five to seven,later the brains were processed for fluorescence microscopic examination to observe the hippocampal labeling cells and Fluoro-Gold labeling calretinin interneurons,ResultsIn the lithium-pilocarpine models,55% of rats developed SE,20% were expired, and 25% did not enter into status epilepticus.25% of rat that developed SE died during the procedure and, therefore, was excluded from the study.This study demonstrate for the first time a abundant FG labeling of CA1 and CA3 pyramidal cell through precise retrograde study in the Pilocarpine epileptic rats, and also showed a prominent FG labeling of CA1 in the control rats. FG labeled CR positive interneurons were observed in two groups. In CA1 area of hippocampus, there were no significant difference between FG labeled CR positive interneurons and CR positive interneurons (P>0.05). FG labeled CR positive interneurons were observed in CA1, CA3 and hilus in the experimental group and in CA1 in Control group.ConclusionOur data suggested the existence of two parallel FG labeled calretinin positive interneurons pathways, the first pathway is CA3 to CA1 inhibitory synaptic connections and hilus to CA1 of rats with epilepsy, and the second pathway arises in the CA1 of controlled rats and targets to the CA1.also we provided evidence that synaptic reorganization among pyramidal cells is an active process in chronic phase of epilepsy. CR neurons may partially originate from axonal sprouting of CR interneurons in remote area of CA1.Objective:The hippocampus has been shown to play an important role in processing of temporal lobe epilepsy.Among epileptogenic mechanisms, the alternation in the fine balance between the excitatory glutamatergic and inhibitory GABAergic activity of hippocampal structures are of critical importance. The subpopulations of inhibitory GABAergic interneurons expressing calcium-binding proteins, in particular parvalbumin, play an important role in the restriction and control of pyramidal cell excitability. Their qualitative or quantitative disturbances can lead to abnormally synchronous and excessive discharges of large populations of neurons, which manifest as epileptic seizures.The hippocampus, which is involved in neural systems related to seizures, is selectively degenerated in temporal lobe epilepsy. Here, we examined neuropathlogical changes in the hippocampal subfields in post status epilepticus rats at different time points starting from two hours to two months.Methods: One hundred Sprague Dawley Rats were randomly stratified into two groups:epileptic rats, seizures were induced by lithium-pilocarpine protocol and control rats treated with intraperitoneal normal saline. The lithium was injected 24 hour prior to 30mg/kg pilocarpine. To abort or interrupt behavioral seizures developed by pilocarpine, chloral hydrate was administered intra peritoneal 1 hour after the onset of status epileptics, which is the time range needed to induce chronic epileptogenesis. The neuropathlogical changes in the hippocampal subfields in post lithium pilocarpine induced epilepsy rats were examined using Nissl stain and parvalbumin immunohistochemistry.Results:In the epileptic rats,55% of rats developed seizure,40% died and 5% of rats did not induce seizure. There were 4 rats with low seizure frequency. The mean interval from first pilocarpine injection to onset of status epilepticus was 15 min. At one day, seven days and 2months post-status spontaneous seizures were evidentHistological stains revealed the most dramatic cell loss to be in pyramidal layer of the CA1 and CA3 subfields. In accordance, immunohistochemical staining showed that neurons and fibres that contain calcium-binding protein (PV) were also more frequently altered in these subfields than in the hilus subfields. Detailed analysis further revealed that parvalbumin positive interneurons showed morphological alterations early in the hippocampal pathology of epilepsy. The degeneration of parvalbumin-immunoreactive neurons networks was observed mainly in straetum oriens. The neurons containing parvalbumin were often significantly decreased in the CA1 and CA3 at one day post status (p<0.05) and gradually decreased at late time points, although morphological alterations were observed. The maximal decrease were observed at 2 months post status epilepticus(P<0.01). There were significant decreases in parvalbumin positive interneurons at two months as compared to 24 hour post status (P<0.05).Conclusion:Our findings indicate that specific subfields of the hippocampus involving neurons that contain distinct calcium-binding protein (PV) are differentially vulnerable in temporal lobe epilepsy. This could have an impact on the topographically organized inputs and outputs of the hippocampus in temporal lobe epilepsyObjective:Pilocarpine is widely used tools for the induction of experimental epilepsy. Since their original descriptions, systemic administration of pilocarpine have been recognized as the animal models which most adequately reflect the clinical and neuropathological features of human temporal lobe epilepsy Understanding the pathophysiogenesis of epilepsy largely rests on the use of models of status epilepticus, as in the case of the lithium-pilocarpine model. Available animal models of epilepsy are produced by acute treatments that often have high mortality rates and/or are accompanied with a low proportion of animals developing spontaneous chronic motor seizures.Our first part of this thesis (neuroscience. 2010-accepted) showed low mortality rate by decreasing the dose of pilocarpine however the ratio of rats developed seizure was not satisfactory. In this study, Sprague Dawley rats were given Lthium 12 hours prior to further decrease dose of intraperitoneal injections of pilocarpine in order to minimize the mortality rate and to increase number of animals developing status epilepticus.Methods:This Study was conducted at Xiangya Medical College, central South University, China and at Al Najah University, Palestine.200 lithium-pretreated rats were divided into two groups.:group A were received Lthium followed 24 hour later by start dose of 30 mg/kg pilocarpine in addition to 10 mg/kg i.p., every 30 min and group B received Lthium followed 12 hour later by initial dose of 20 mg/kg pilocarpine, and then additional 10 mg/kg doses until induction of SE. The time the rats spent in SE was limited by 10%chloral hydrate (3.5ml/kg) at time point 60 minutes.Results:In group A rats, the mean repeated low dose of pilocarpine needed to induce SE was 32 mg/kg, while in group B the mean repeated low dose of pilocarpine was 22 mg/kg. When SE was stopped after 60 min, Group A rats produced SE in approximately 62% of the animals, but 17% of the rats died, 21% did not inter status epilepticus, While Group B rats produced status epilepticus in approximately 100%, mortality rate was zero, which was significantly lower compared to the first group. All rats which had experienced status epileptics of at least 60 min developed chronic epilepsy. Average latency to the first spontaneous seizure was approximately 15-30 days in both groups. The frequency and severity of spontaneous seizures was not significantly different between groups. Conclusion:The present study demonstrates that incidence of convulsions increased and mortality rates decreased when lithium pretreatment time and doses of pilocarpine was decreased.