Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of rat in vitro model of post traumatic epilepsy using brain area CA1 and CA3 in lesioned and non lesioned culture

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Elizabeth Salomon

PROVIDER: E-GEOD-5034 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

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. Keywords: time-course

Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory.

Dataset Information

Transcription profiling of rat in vitro model of post traumatic epilepsy using brain area CA1 and CA3 in lesioned and non lesioned culture

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets