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 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: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: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:Time dependent-profiles in the gene expression level following lateral moderate fluid percussion injury in the rat brain We used microarray to elucidate relationship between the alteration of gene expression levels and the progression of brain damages following traumatic brain injury. To examine the levels of gene expression in the early phase of traumatic brain injury, we analyzed the gene expression at 3, 6, 12, and 48 h after trauma using the lateral moderate fluid percussion TBI model. The ratios of the gene expression level were compared between chips corresponding to the 3, 6 and 12 h fluid percussion groups and the sham group chips. On the other hand, the rations of gene expression level after 48 h FPI were compared with 48 h sham chip, because the gene expression levels of 48 h sham chip were distinct from sham group chips (3, 6 and 12 h) in the cluster and principal components analyses.
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.
Project description:We inflicted TBI to wildetype (wt) mice in order to establish whether the anti-inflammatory agent cyclophosphamide can be used therapeutically. Cyclophosphamide was found to regulate distinct inflammatory cells such as activated microglia separate from invading phagocytes and dendritic cells. Cyclophosphamide postinjury selectively reduces antigen-presenting dendritic cells. Findings show feasibility of drug development to interfere with brain inflammation. TBI was carried out in injured wt B6 mice for postinjury treatment with cyclophospamide i.p. using saline as a control substance for comparison with injured but untreated mice. Total RNA was prepared from injured cerebral neocortex after three days. RNA samples were also from uninjured wt mice as reference for hybridization on Affymetrix microarrays.
Project description:We compared arginase-1+ macrophages (macrophages were defined by flow cytometry as CD45hi CD11b+ Ly6G-) with arginase-1- brain macrophages following traumatic brain injury (TBI) by isolating these cells from YARG transgenic mice, which express YFP under the arginase-1 promoter. Both cell populations were isolated from YARG brain tissues one day following TBI. We also examined the expression profile of peripheral blood monocytes (monocytes were defined by flow cytometry as CD11bhi F4/80+) from injured YARG mice and from normal YARG mice. Peripheral blood samples were compared to TBI brain macrophages to assess gene expression changes before and after infiltration into the brain. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an IRES inserted at the 3' end of the gene for arginase-1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21±1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by YFP. Gene expression analysis of Arg1+ and Arg1- brain macrophage populations revealed that these populations were distinct from either classically activated (M1) macrophages or M2 macrophages, with features of both. The Arg1+ cells differed from Arg1- cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI involves recruitment of at least two major macrophage subsets. Overall, our data indicate that the macrophage response to TBI is heterogeneous and unique. Four groups (Arg1- brain macrophages post-TBI, Arg1+ brain macrophages post-TBI, normal blood monocytes, blood monocytes post-TBI) were analyzed. Four replicates of each group were analyzed for a total of 16 samples (only 3 replicates of the blood monocyte groups are included in this submission).
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.