Abstracts

Rationale: TBI and its residuals including post-traumatic epilepsy (PTE) are major health problems. Genetic factors are known to influence outcome following TBI both in humans and animals. Prior research utilizing a pair of unique and complementary inbred rat strains selected for either increased or decreased rates of perforant path kindling has demonstrated significant differences in sequelae of TBI. The goal of this study is to examine the effects of genetic background on gene expression following TBI. Methods: Perforant path kindling susceptible (PPKS) and perforant path kindling resistant (PPKR) strains were bred from a Sprague-Dawley (SD) background, selecting for fast or slow development of perforant path kindled seizures for >15 generations. PPKS, PPKR, and SD rats (n = 5 for each group at each time point) were maintained under isoflurane anesthesia (2% in O2) for 5 minutes prior to delivery of a CCI (3mm blunt impactor, 6m/s, 500msec dwell time). Rats were allowed to recover and were euthanized at 24 hours, 1 day, and 1 month after injury. PPKS, PPKR, and SD rats (n = 5 for each group) in a non-surgery control group were also included. Brain was dissected into cortex and hippocampus, both ipsilateral and contralateral to the CCI.Tissue was pulverized under cryogenic conditions, RNA was isolated by a TRIzol protocol, and RNA was purified through a column. RNA was sequenced with an Illumina HiSeq2500 platform. Data was analyzed for systematic changes using the DESeq2 method. A cutoff at an adjusted p-value < 0.05 was used to determine statistical significance. Gene functional annotation and classification was performed using the Database of Annotation, Visualization, and Integrated Discovery (DAVID). Results: Approximately 19,000 transcripts were identified from the hippocampus. In the non-surgery control groups, 6400 transcripts were differentially expressed among the PPKS, PPKR, and SD rats, with 615 transcripts were differentially expressed between PPKR and PPKS rats. At the 24 hour time point, PPKS rats demonstrated 7640 transcripts differentially expressed as compared to the non-surgical PPKS rats.  For the PPKR rats, 7973 transcripts were differentially expressed at the 24 hour post-CCI timepoint as compared to the non-surgical control. 36 transcripts were differentially expressed in the hippocampus between the PPKS and PPKR strains at the 24 hours post-CCI time point.Functional annotation and classification of the transcripts identified a variety of genes, including voltage-gated Na channels, K channels, ACh receptors, Ca-binding proteins, cell adhesion molecules. Conclusions: The differences described demonstrate that selective genetic pressures to increase or decrease plasticity in the setting of a model of epileptogenesis also influence gene expression following TBI. Furthermore, as the two inbred strains are known to have differing risks for a variety of post-TBI sequelae including PTE and learning deficits, analysis of gene expression at baseline may identify protective or detrimental factors, while patterns of gene expression at various time points following TBI may identify patterns of response to TBI which lead to favorable or unfavorable outcomes. Analysis of gene expression from both cortex and hippocampus allows for comparisons between these two anatomic sites, while analysis of the tissue ipsilateral to the injury and contralateral allows for segregation of changes directly due to injury from diffuse and global changes. Identification of these patterns in gene expression will be critical for future diagnostic and therapeutic approaches to TBI and sequelae. Funding: RJK: VA CDA-2 (IK2BX002986), TPS: Defense Hypothesis Development Award (DR080424)