Evidence for a rheostat that determines neuronal survival after traumatic brain injury
ABSTRACT: To investigate the determinants of neuronal survival after traumatic brain injury, we compared the transcriptional profiles of dying (Fluoro-Jade-positive) and immediately adjacent surviving (Fluoro-Jade-negative) neurons from the CA3 subfield of the rat hippocampus 24 hours after experimental TBI. We found that hippocampal neurons that survive TBI invariably express high levels of genes that have cellular functions involved in survival, regeneration, development, proliferation, neuronal plasticity such as cAMP response element binding protein (CREB), brain-derived-neurotrophic factor (BDNF) and mitogen-activated protein kinase 1 (MAPK1). Dying neurons express high levels of genes involved in aberrant cell cycle progression, immune response, inflammation, oxidative stress and apoptosis such as Interleukin-1β (IL-1β), caspase 3 and B-cell linker (BLNK). We conclude that shifting the balance between the global levels of these proteins with pharmacotherapeutic drugs that induce expression of cell survival associated genes, is expected to alter the cellular rheostat that determines cell survival or cell death. Replicate pooled samples (approximately 600 laser capture microdissected hippocampal neurons per sample of dying neurons (labeled with Fluoro-Jade, a fluorescent stain for degenerating CNS neurons) and surviving neurons (Fluoro-Jade-negative) were hybridized in duplicate to rat Agilent whole genome arrays.
Project description:To investigate the determinants of neuronal survival after traumatic brain injury, we compared the transcriptional profiles of dying (Fluoro-Jade-positive) and immediately adjacent surviving (Fluoro-Jade-negative) neurons from the CA3 subfield of the rat hippocampus 24 hours after experimental TBI. We found that hippocampal neurons that survive TBI invariably express high levels of genes that have cellular functions involved in survival, regeneration, development, proliferation, neuronal plasticity such as cAMP response element binding protein (CREB), brain-derived-neurotrophic factor (BDNF) and mitogen-activated protein kinase 1 (MAPK1). Dying neurons express high levels of genes involved in aberrant cell cycle progression, immune response, inflammation, oxidative stress and apoptosis such as Interleukin-1β (IL-1β), caspase 3 and B-cell linker (BLNK). We conclude that shifting the balance between the global levels of these proteins with pharmacotherapeutic drugs that induce expression of cell survival associated genes, is expected to alter the cellular rheostat that determines cell survival or cell death. Overall design: Replicate pooled samples (approximately 600 laser capture microdissected hippocampal neurons per sample of dying neurons (labeled with Fluoro-Jade, a fluorescent stain for degenerating CNS neurons) and surviving neurons (Fluoro-Jade-negative) were hybridized in duplicate to rat Agilent whole genome arrays.
Project description:The current lack of proven pharmacological treatment options for traumatic brain injury (TBI) patients reflects the poor translation of successful preclinical studies in clinical trials. This may be due to poor choice of therapeutic agents based on incomplete knowledge of critical elements of neuroprotection. Our goal is to expedite discovery and translation of therapeutic agents that can improve functional outcome by identifying the common molecular profile of neuroprotective drugs. Since damage to the hippocampus is associated with TBI-induced deficits in learning and memory, we analyzed of the hippocampal transcriptional profiles of TBI rats treated with two clinically used drugs metyrapone and carbenoxolone, which have been shown to improve cognitive deficits in previous studies. Despite their different structures, we found that MT and CB have similar effects on several known biological pathways. The neuroprotective effects of these drugs are associated with a distinctive molecular signature which is characterized not by changes in expression of any individual gene but by a common global effect on multiple cell signaling pathways. These data suggest that drug treatments that induce a coordinated attenuation of multiple injury-induced cell signaling networks, both deleterious and protective, have high translational potential. There were 16 samples for array analysis, two biological replicates each for control (4 and 24 hour), TBI (4 and 24 hour), TBI plus MT (4 and 24 hour) and TBI plus CB (4 and 24 hour). Each biological sample, which is a pooled RNA sample (laser captured CA3 pyramidal neurons) from the hippocampus of 3-6 rats, was hybridized to duplicate arrays so that there were 32 gene arrays in this study.
Project description:Traumatic brain injury (TBI) alters and dysregulates the expression of thousands of genes in the brain. Since some of the most common problems in TBI patients are learning and memory deficits, we are studying the effects of TBI on the hippocampus, a region of the brain which is essential for learning and memory and which is known to be particularly vulnerable to TBI. We are interested in understanding how potential neuroprotective drugs alter the TBI-induced gene expression profile. The objective of this study is to elucidate and compare the differential gene expression profiles in the hippocampus of naive, sham-control, TBI and TBI plus drug treated rats. JM6, PMI-006 and E33 are three compounds with neuroprotective, anti-inflammatory and anti-oxidative effects. Our goal is to determine if different neuroprotective compounds have similar effects on common gene targets. These genes and the cell signaling pathways linked to them would then be the target of new therapeutic strategies for TBI. Rats were prepared for fluid percussion traumatic brain injury or sham injury (naïve rats had no anesthesia and were not handled in any way and gene expression in their brains serve as baseline data) and 24 hr post-injury, hippocampi were obtained, and stored in RNA later. Total RNA was isolated, quantitified and used for Agilent microarray analysis at GenUs Biosystems. Each group of naive, sham control, TBI and TBI plus JM6, TBI plus PMI-006 and TBI plus E33 (estrogen) has three biological replicates.
Project description:To address the hypothesis that silencing deleterious or protective injury-induced genes in the rat hippocampus will reduce or increase the numbers of injured hippocampal neurons, alter cellular pathways essential for neuronal function and improve or worsen functional outcome after traumatic brain injury (TBI), we evaluated the effects of silencing neuronal nitric oxide synthase (nNOS) and glutathione peroxidase-1 (GPx-1) expression in the injured rat hippocampus. Overall design: Two weeks post-TBI/post-AAV treatment, hippocampal pyramidal neurons were laser captured for comparative whole genome microarray analysis.
Project description:Genetic factors are believed to be of importance for outcome of traumatic brain injury (TBI). However, so far mainly allelic variation in apolipoprotein E4 has been studied in human TBI. In order to study the role of genetic factors in experimental TBI, we examined parental DA and PVG strains before and after TBI. A standardized weight drop injury was used and the pericontusional area was dissected 1 day after TBI and transcriptional profiling was performed.
Project description:Traumatic brain injury dysregulates microRNA expression in the brain. We hypothesized that injury-induced epigenetic changes contribute to neurodegeneration and learning and memory deficits after TBI. These changes may provide a mechanistic explanation for neuropsychiatric comorbidities in TBI patients. Our objective is to compare and contrast the effects of several neuroprotective drugs (JM6, PMI-006 and E33-estrogen) on the TBI-induced changes in microRNA expression in the hippocampus, a region of the brain that is critical for learning and memory. We will also study if different neuroprotective drugs have similar effects on common microRNAs which may cooperatively regulate a common set of gene targets. 3 biological samples each of Naïve, Sham control, TBI and TBI plus JM6, TI plus PMI-006, and TBI plus E33 rat hippocampi were obtained 24 hr post-sham injury or TBI, stored in RNA later and sent to GenUs Biosystems for microRNA microarray analysis.
Project description:To elucidate the mechanisms underlying epithelial homeostasis, we explored molecules that might serve as “danger” signals in mediating epithelial regeneration with microarray. We hypothesize that soluble factors may have been released from damaged cells to stimulate the proliferation of surviving epithelial cells. In elucidating the mechanism of dying cell-to-surviving cell communication using normal rat kidney NRK-52E epithelial cells, we observed gene expression profiles in these cells after the induction of cell death using hydrogen peroxide. The results demonstrated up-regulation of Interleukin-6, Heme oxygenase-1 and Hypoxia inducible factor-1 alpha in dying cells. Global gene expression changes were measured after induction of cell death in NRK-52E cells after incubation with hydrogen peroxide. Hydrogen peroxide (0, 0.003, 0.006, 0.009% in DMEM) was teated for 1 hour. After wash with PBS, cells were incubated with non-serum DMEM for 12 hours.
Project description:Spinal cord injury leads to impaired motor and sensory functions. After spinal cord injury there is a an initial phase of hypo-reflexia followed by a developing hyper-reflexia, often termed spasticity. Previous studies have suggested a relationship between the reappearance of plateau potentials in motor neurons and the development of spasticity after spinalization. To understand the molecular mechanism behind this phenomena we examined the transcriptional response of the motor neurons after spinal cord injury as it progress over time. We used a rat tail injury model where a complete transection of the caudal (S2) rat spinal cord leads to an immediate flaccid paralysis of the tail and a subsequent appearance of spasticity 2-3 weeks post injury that develops into strong spasticity after 2 months. Gene expression changes were studied in motor neurones 0, 2, 7, 21 and 60 days after complete spinal transection. Tail MNs were retrogradely labelled with Fluoro-Gold injected into the muscle and intra peritoneally. 5-7 days after tracer injections the spinal cord was dissected out, snap-frozen in liquid nitrogen, sliced in 10 um thick slices and fluorescent motor neurons were laser dissected into a collector tube to a total of ca. 50-200 cells pr sample. RNA was then extracted, two round amplified and hybridized to Affymetrix rat 230 2.0 arays. 31 samples were hybridized onto chips, 4 Spi-0 (Control), 6 Spi-2, 5 Spi-7, 8 Spi-21 and 8 Spi-60.
Project description:To assess neuronal expression divergence between mice and rats, we used the Affymetrix array platform to assay the transcriptomes of micro-dissected individual soma and pool of dendrites of hippocampal neurons in dispersed primary cell cultures from rat and mouse. Using microdissected soma and dendrites from primary cultures of hippocampal neurons of two mouse strains (C57BL/6 and Balb/c) and one rat strain (Sprague-Dawley), we investigate via microarrays, subcellular localization of mRNAs in neurons
Project description:We inflicted TBI to chemokine-deficient mouse lines in order to establish involvement of various signalling pathways that may be addressed therapeutically. Interacting chemokine pathways in brain regulate distinct inflammatory cells. Activated microglia are separate from invading phagocytes and dendritic cells. Findings show potential targets to interfere with specific inflammatory responses after brain injury. TBI was carried out in Ccl3-/- and Ccr2-/- mice, total RNA prepared from injured cerebral neocortex after three days. RNA samples were from uninjured Ccl3-/- and Ccr2-/- mice as reference for hybridization on Affymetrix microarrays.