Project description:Traumatic brain injury occasionally causes posttraumatic epilepsy. To elucidate the molecular events responsible for posttraumatic epilepsy, we established a rodent model that involved the injection of microliter quantities of FeCl3 solution into the amygdalar nuclear complex. We previously compared hippocampal gene expression profiles in the traumatic epilepsy model and normal rats at 5 days after brain injury (acute phase) and observed the role of inflammation. In this study, we focused on later stages of epileptogenesis. We compared gene expression profiles at 5, 15 (sub-chronic phase), and 30 days (chronic phase) after brain injury to identify temporal changes in molecular networks involved in epileptogenesis. A total of 81 genes was significantly (at least 2-fold) up- or downregulated over the course of disease progression. We found that genes related to lipid metabolism, namely, Apoa1, Gh, Mc4r, Oprk1, and Pdk4, were temporarily upregulated in the sub-chronic phase. Changes in lipid metabolism regulation might be related to seizure propagation during epileptogenesis. This temporal description of hippocampal gene expression profiles throughout epileptogenesis provides clues to potential markers of disease phases and new therapeutic targets. We employed amygdalar FeCl3 injection to induce chronic, recurrent limbic-type partial seizures with spontaneous secondarily generalized seizures in rats . Sixteen male Wistar rats were kept in hanging cages with unlimited access to food and water and 12-h light-dark cycles. Surgical procedures were conducted following anesthesia with intraperitoneal (i.p.) sodium pentobarbital injections (37.5 mg/kg) at 5 weeks of age. Stereotaxic coordinates were determined with the rat brain atlas. The incisor bar was set 3.3 mm below the interaural line. While under anesthesia, a polyethylene tube containing a stylet to serve as an external guide cannula (1.09-mm outer diameter (o.d.), 0.55-mm inner diameter (i.d.), 2.5 cm in length) was stereotaxically implanted and anchored to the skull with miniature screws and dental cement. The cannula was fixed 5.6 mm anterior and 4.8 mm to the right of the lambda and 8.5 mm below the surface of the skull, positioning it at the right amygdaloid body. Randomly selected rats for in vivo microdialysis to estimate redox had guide cannula placed but were prepared without dental cement on the skull where the microdialysis guide cannula was to be placed. Five days later, the stylet was replaced with an internal delivery cannula (0.5 mm o.d., 0.25 mm i.d.). FeCl3 was dissolved in saline solution (100 mM, pH 2.2). FeCl3 solution (1.0 M-NM-<l) was injected through the inner cannula by means of a microinfusion pump (EP-60; Eicom, Tokyo, Japan) set at a rate of 1.0 M-NM-<l/min (Fe group; n = 12). The external guide cannula was used for electroencephalogram (EEG) recording with an electroencephalograph (type 1A63; SAN-EI, Tokyo, Japan). Rats in the control group (n = 4) were each injected with 1.0 M-NM-<l saline (pH 2.2). Both EEG and behavior were observed for at least 6 h after the injection. While we did not measure the rate or frequency of seizures, we did confirm that the animals had recurrent seizures. These observations and the acute recording confirmed the accuracy of the amygdalar injection. No seizure activity was observed in control group rats. Animals in the control group were sacrificed by cervical dislocation 15 days after amygdalar injection. Animals in the Fe group were subdivided into 3 groups and were sacrificed at 5 (acute phase), 15 (sub-chronic phase of injury), and 30 days (chronic phase of injury) after amygdalar injection. The stages of disease development were categorized according to the EEGs of the model rats; within 5 days after amygdalar injection, epileptiform discharges were recorded in the contralateral and ipsilateral amygdalae; by 15 days after injection, interictal spike discharges were more consistently observed in the contralateral uninjected amygdala; at 30 days after injection bilateral interictal spike discharges continued to be observed. After sacrifice, the right hippocampi were immediately removed and placed in ice-cold phosphate-buffered saline and homogenized (Polytron PT 3000; Brinkmann Instruments, Inc., Westbury, NY, USA) for RNA extraction.

Project description:Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity. Four condition experiment with five samples per condition. The samples include right dentate gyrus from animals with seizures, left dentate gyrus from animals with seizures, right dentate gyrus from animals without seizures, and left dentate gyrus from animals without seizures. A dye swap was included.

Project description:Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity. Four condition experiment with five samples per condition. The samples include right dentate gyrus from animals with seizures, left dentate gyrus from animals with seizures, right dentate gyrus from animals without seizures, and left dentate gyrus from animals without seizures.

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Changes in the gene expression in the inferior colliculus of the sound stimulated rats both Wistar and the strain susceptible to audiogenic seizures WAR

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