Pre-Race Inflammation in Catastrophically Injured Horses: Education Research Services

Abstract Research Plan—Scientific Terms (Do not exceed one page) Over the last two decades, numerous studies and investigators have reported on a multitude of risk factors associated with catastrophic injuries (CI’s) in Thoroughbred racehorses across the world. There has been increased interest in the development of different techniques to identify these horses, including advanced imaging, genetic screening, in-depth risk factor modeling, and serum protein analysis for biomarkers. Despite this work, only moderate progress has been made in preventing CI’s. It has been established that many CI’s occur in horses with underlying or pre-existing musculoskeletal pathology, leading to the theory that acute injury is due to the accumulation of damage over time at a rate that exceeds the healing capacity of the affected tissues. This provides a potential opportunity, as the development of modalities to assist with the early detection of this damage, coupled with further investigation and corrective action, could reduce the incidence of catastrophic injuries. Demonstrating the utility of such a tool is the goal of this project. Based on work with human athletes, we have developed an approach for identifying horses at risk of catastrophic injury through the use of mRNA expression analysis from blood samples. We recently demonstrated that horses with catastrophic injuries have significantly altered expression of IGF-1, IL1RN, and MMP2 when compared to non-injured control horses. While these samples were collected immediately post-injury, we predict that they represent the pre-race status of the horses given that we have shown whole blood mRNA expression changes take hours in response to a localized inflammatory stimulus. We also demonstrated that the expression of these three genes did not change during the immediate post-race period (~45 minutes) in non-injured horses. Though these results are consistent with the assertion that our findings represented pre-race expression of these genes, it is imperative that this be confirmed using only pre-race/pre-injury samples. This would remove any confounding effects on expression by the injury and demonstrate the utility of this innovative tool in the quest to decrease catastrophic injuries in Thoroughbred racing. Hence, we hypothesize Thoroughbreds that experience a catastrophic injury during racing will demonstrate pre-race differences in mRNA expression of multiple genes when compared with non-injured, race-matched control horses. A total of 15,000 pre-race samples will be collected for this project (funding for 12,500 requested) using Tempus™ Blood RNA Tubes from Thoroughbred horses racing in Southern California. All samples will be collected during pre-race TCO2 testing. Based on recent national data, we anticipate at least 17 catastrophically injured horses will be sampled, providing the study with a power of approximately 0.976. Should the number of CI’s decrease to 10, the study will remain appropriately powered (0.8). Samples will be blinded through the use of unique numbers and an identification key prior to storage at -20oC. Quarterly, samples from CI’s and non-injured horses will be sorted by a non-interested party and provided to the investigators for blinded analysis of the samples via RT-qPCR. Samples will be assayed for at least 22 different gene transcripts, which were selected previously based on their role in inflammation, bone repair/remodeling, tissue repair, and response to injury. Additionally, any new gene transcripts we identify via ongoing work utilizing RNA-sequencing will be added to the analysis. Using an endogenous control gene, relative quantities (RQ’s) for each gene of interest will be calculated using the delta-delta-CT method. We will examine IGF-1, IL1RN, and MMP2 expression as these three genes were identified in our original model as being significant markers for increased CI risk. Additionally, we will analyze qPCR expression data from all 22+ genes using t-tests, receiver operating characteristic (ROC) analysis, and Mantel-Haenszel analysis using optimized mRNA expression values from the ROC analysis. Further analysis or different tests/modeling may be utilized based on recommendations from our collaborator, Dr. Tim Parkin. Based on our recently completed work, we readily anticipate that horses with a catastrophic injury during racing will have evidence of significant pre-race expression differences with multiple genes. As all samples will be collected pre-race, the data generated by this study will confirm the utility of mRNA expression analysis as an economical, effective, accessible, and non-invasive means of detecting horses at high risk of catastrophic injuries before they occur.