Rehabilitation Monitoring with mRNA Expression Analysis

2. RESEARCH PLAN I. Horse owner description of the proposed research and its potential impact: The consequences of time out of competition for horses following injury continue to take economic and ethical tolls on the equine community. While equine veterinarians use a variety of means to evaluate a horse’s ability to return to competition, and even with the most careful monitoring and re-evaluation, there is inherent uncertainty in a horse’s successful recovery level following injury and rehabilitation. In humans, this type of assessment can involve the measurement of various biomarkers to gauge levels of tissue healing or lack thereof. Our research group seeks to apply a similar methodology for horses by using messenger RNA (mRNA) as a biomarker for recovery. Biomarkers that provide objective insight regarding ongoing healing would shift the paradigm for rehabilitative monitoring. Through serial evaluation of mRNA in horses diagnosed with and receiving professional rehabilitation for suspensory ligament, tendon, or stifle injuries, we seek to define patterns that may ultimately help better understand and maximize healing responses following injury. This type of information would significantly modernize current return to activity timelines for some of the most common orthopedic and soft-tissue injuries in horses. II. Research problem and background information: The focus on preserving orthopedic health, minimizing loss of use, and improving welfare of athletic horses has taken center stage in the current atmosphere of equine sports medicine. The consequences of time out of competition following injury continue to take economic and ethical tolls on the equine community, with significant new and innovative research into both injury prevention and post-injury rehabilitation required to spur further advancement. A significant, yet unaddressed, barrier to this mission are the limited abilities to objectively assess responses to rehabilitation and identify subsequent responders and non-responders. While equine clinicians most commonly utilize clinical soundness, imaging re-evaluation (when possible), and antiquated timelines to gauge a horse’s suitability to return to sport, even with the most careful monitoring and re-evaluation there is inherent uncertainty in a horse’s successful recovery following rehabilitation. Whole-blood biomarkers would provide the equine clinician with regular insight regarding the individual horse’s response to healing, shifting the paradigm for rehabilitative monitoring and significantly modernizing currently utilized return to sport timelines. In elite human athletes, the measurement of pro-inflammatory cytokine production and its modulation during training adaptation is an area of research interest [1-3]. Specifically, exercise- induced inflammation involving high volume/intensity training is known to produce tissue-level trauma resulting in the release of damage associated molecular patterns (DAMPs) [4]. These molecules are released by cells undergoing necrosis and act as endogenous ‘danger signals’ to promote an inflammatory response [5]. DAMPs bind to receptors on the surface of certain cells, leading to the production of pro-inflammatory cytokines [6]. While DAMP-induced cytokines play a key role in exercise-related tissue repair and remodeling [7], chronic inflammation is associated with tissue damage, subsequent poor performance, and possible injury [1]. To this extent, horses undergoing professional, post-injury rehabilitation provide a unique opportunity to monitor the mRNA response during the recovery period, a period in which significant mRNA differences are expected based on the release of DAMPs and other markers. Importantly, the innovative utility of mRNA analysis in horses has recently been demonstrated by our research group through work with endurance horses [8] and catastrophically injured