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Hippocampal gene expression profiling in a rat model of posttraumatic epilepsy reveals temporal upregulation of lipid metabolism-related genes

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE40490 | GEO | 2012/12/31

SECONDARY ACCESSION(S): PRJNA174180

REPOSITORIES: GEO

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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:Epilepsy is a chronic disorder characterized by recurring seizures, and results from excessive, synchronized discharge in populations of cells. Epilepsy affects 1% of the population and results from a variety of CNS insults. Traumatic physical brain injury causes 4% of cases. Posttraumatic epilepsy (PTE) is characterized by a delay of weeks to years between the trauma and the first seizures. Proper prophylactic intervention during this incubation time might prevent PTE. Many PTE patients are resistant to anticonvulsant medications and suffer from negative side effects. Better knowledge of the cell biology underlying injury-induced epileptogenesis should lead to better therapeutic strategies for interrupting the events occurring between the injury and the development of epilepsy. The aim of my experiments is to identify cellular factors underlying PTE using an in vitro model system to study two key aspects of brain injury in isolation: deafferentation and axonal transection. My aim is to perform a non-biased comparison of the expression of genes in area CA1, and separately in area CA3, in unlesioned cultures and in cultures lesioned 1, 3 and 7 days earlier. Changes in gene expression due to neuronal denervation will be apparent in area CA1, whereas changes in expression due to axonal transection will be apparent in area CA3. In performing these experiments, I hope to gain insight into the molecular underpinnings of the electrophysiological and immunocytochemical effects that we have already detected, as well as to generate new hypotheses that we can test further using our own expertise and techniques. We use cultured slices of rat hippocampus to study the effects of brain injury. Our model is to lesion the Schaffer collateral projection pathway between areas CA1 and CA3, producing a selective injury of the axons of CA3 cells and selective denervation of CA1 cells. Our preliminary electrophysiological and immunocytochemical data indicate that axonal sprouting is triggered in area CA3 and that the dendritic excitability is increased in CA1 pyramidal cells. Both effects occur only after about 7 days, suggesting they are mediated by changes in gene expression. My hypothesis is that the expression of numerous genes changes after Schaffer collateral transection. I predict that neurotrophins and their receptors change in area CA3 to trigger axonal sprouting, along with genes for proteins mediated axonal extension, and that changes in the expression of genes for glutamate receptors and voltage-dependent ion channels occur in area CA1 to account for their increased excitability. Hippocampal slice cultures prepared from male Sprague-Dawley rats at PN day 6. Slices will be maintained in vitro for 14 days in serum-containing medium. Cultures will then be examined and unhealthy cultures discarded. Remaining sister cultures will be divided into two groups: cultures to be lesioned and cultures remaining unlesioned. Lesioned cultures will have a sterile transection of the Schaffer collateral pathway using a razor blade shard. All cultures then returned to the incubator. At various times , cultures will be removed from the incubator. Individual cultures will be soaked in RNAlater buffer(Ambion), and a razor blade shard will be used to subdissect the CA3 and CA1 segments. Segments are sucked up with a sterile pipette-tip containing RNAlater buffer and transferred into a sterile tube. Total RNA will be prepared by homogenization in TRIzol reagent (GibcoBRL) and precipitation in isopropyl alcohol. Rneasy Kit (Qiagen) will further isolate RNA. Spectrographic analysis of RNA prepared indicates 260nm:280nm ratios above 1.6, and insignificant amounts of either degraded RNA or DNA contaminants detected on a 2% denaturing gel. We have found that it is necessary to pool 15 CA3 or CA1 segments to obtain 5+ ug of total RNA. Total RNA samples will be sent to the consortium for additional quality control, labeling, hybridizations using the Affymetrix Neuro U34 chip, scanning, and preliminary analyses. The gene array data will then be compared in several pairwise manners (lesion vs. age matched control and region vs. region, for each time point), as well as time sequence comparisons for gene expression in each region.

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Hippocampal gene expression profiling in a rat model of posttraumatic epilepsy reveals temporal upregulation of lipid metabolism-related genes

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