The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, we request continued NIH funding for 7 fellows per year for 3 years. NC State University will provide funding for up to 2 fellows per year and 2 pre-T32 positions. Trainees will be degree-seeking fellows in the Comparative Medicine and Translational Research training program established by the faculty in the College of Veterinary Medicine (CVM) and the Comparative Medicine Institute (CMI) at North Carolina State University. This training program specifically targets individuals with the DVM degree who have completed specialty training and is designed to prepare trainees to compete for an early career development award and a rapid transition to independence as a principal investigator or in another research-intensive career. Trainees complete requirements leading to the PhD degree in Comparative Biomedical Sciences (CBS) in one of 7 areas of concentration: 1) Immunology 2) Cell Biology, 3) Pharmacology, 4) Neurosciences, 5) Infectious Diseases, 6) Population Medicine and Global Health, and 7) Pathology. Training faculty are well-funded productive scientists that have a strong training track-record and diverse research expertise. Training faculty are all members of the CBS graduate program and the CMI and represent 5 departments from 3 Colleges. Research projects emphasize comparative and translational themes fostered by the CVM and CMI in functional tissue engineering, translational pharmacology and physiology, and emerging and infectious diseases. Program requirements include: (1) a capstone comparative animal models course; (2) professional development courses and workshops; (3) courses in research ethics and research rigor and reproducibility; (4) a grant writing course and (5) annual research symposia. These requirements are in addition to those associated with the graduate program. Twenty four fellows have completed training. Twenty hold faculty positions in academia, one is a research pathologist, one is a clinical pathologist in industry, and one is a postdoctoral fellow. Fellows were awarded 18 NIH or other federal grants as PI and more than 50 extramural research grants and published 125 papers (84 first author) arising from their research while in training.

Osteoarthritis (OA), the most common form of arthritis, affects ~ 27M Americans and is increasing in incidence. It occurs due to degeneration of tissues comprising joints, and is associated with pain. OA pain is a major contributor to the burden of chronic pain in society. Current treatment options are limited to steroid injections, nonsteroidal anti-inflammatory drugs (NSAIDS), opioids and non-pharmacological approaches (exercise, weight loss). Unfortunately, each of these therapeutic approaches are problematic. Exercise, which helps weight management, is difficult for patients due to ongoing pain. NSAIDS can cause gastrointestinal irritation and bleeding and increase risk of heart attack or stroke, and opioids are associated with addiction and abuse (and can actually worsen chronic pain). Clearly, there is a critical need to identify new therapeutic targets and/or treatments for individuals suffering from OA pain. Here, we propose that a heretofore unrecognized neural pathway is a critical component of OA pain. This pathway involves ARTN, its receptor GFR��������3, and ���������������pain������������������ channels on nerves (transient receptor potential [TRP] channels). Activation of this pathway initiates and maintains OA pain. The central hypothesis (based on preliminary data in multiple species [mouse, dog, cat, human]) is that ARTN, released from synovium of the OA joint in response to injury, results in de novo increase in its receptor, GFR��������3, in local and distant sensory nerves, producing local and widespread pain and hypersensitivity via Proto-oncogene tyrosine-protein kinase receptor (RET)-mediated upregulation of multiple downstream transient receptor potential (TRP) receptors. In this proposal, we will use multiple OA models and clinically relevant outcome measures, and leverage our unique access to dogs with naturally occurring OA, to achieve the following aims: Aim 1: To test the hypothesis that ARTN expression is increased in OA and is responsible for pain. Aim 2: To test the hypothesis that ARTN/GFR��������3 signaling is responsible for behaviorally manifested OA pain both in early and late stage disease. Aim 3: To test the hypothesis that RET-dependent ARTN/GFR��������3 signaling results in changes in multiple TRP channel expression and activation. Aim 4: To test/validate involvement of the above-described key molecules in a naturally occurring large animal model of OA (dog). Overall, this will be the first work investigating the role and mechanisms of ARTN/GFR��������3/TRP channel in OA pain and sensitivity. Based on solid, clinically relevant preliminary data, and leveraging PI expertise from two different and complementary disciplines, successful completion of this proposed work has the potential to identify clinically relevant neural mechanisms leading to the development of novel, effective therapeutics for the treatment of OA-pain in humans."

In humans, persistent osteoarthritis (OA) pain not only negatively impacts mobility and function but also causes affective and cognitive deficits that exacerbate the chronic pain experience, leading to a diminished perceived quality of life. OA in pet dogs is common and characterized by similar physical changes, however the impact of OA-pain on affect and cognition has not been evaluated. This is a significant gap that limits the development of novel therapeutic approaches for canines. To address this gap, we need a clinically-relevant way to measure and define the negative emotional and cognitive effects of OA-pain. The overall purpose of the proposed research is to develop a standardized, operational Dog Pain-Related Emotion and Cognition Test Battery (DECTB-P) to evaluate the impact of chronic pain in dogs on these domains. We will achieve this goal in two aims: 1) develop novel emotional and cognitive tasks for domains not currently assessed in dogs, and 2) combine these novel and already-established cognitive and emotional tasks together into a battery that is practical, and feasible for both healthy dogs and dogs with chronic pain. Once complete, we will perform preliminary analysis on the discriminatory ability of our task to distinguish between groups of dogs. We hypothesize that chronic pain causes changes in cognition and emotion in dogs and these changes can be reliably measured. This research will benefit canine health and well-being by providing a rationale for novel treatments for pain and a tool by which to evaluate their efficacy and impact.

While osteoarthritis (OA) and associated pain is a major and growing public health problem that results in decreased mobility and compromised activity, its profound and debilitating effects on emotions, cognition, and quality of life are poorly characterized and often overlooked in most animal models. Dogs, who develop naturally-occurring osteoarthritis and associated pain, have similar emotional and cognitive features to humans that can be readily measured, offering a transformative opportunity to model these domains in a clinically relevant manner. We will leverage our unique combined expertise to develop and validate a feasible test battery to assess the emotional and cognitive domains in dogs with OA pain thereby optimizing this naturally-occurring pain model and providing endpoints for development of novel and non-opioid therapeutics that better capture the multidimensionality of the human chronic pain experience.

"SPECIFIC AIMS: This project will provide insights into mechanisms of multiple joint osteoarthritis (MJOA) and build the foundation for therapeutic development by developing and analyzing an integrated dataset in both humans and dogs including characterization of intestinal permeability (IP), the microbiome, inflammatory and OA-related biomarkers and development or worsening of MJOA. Osteoarthritis (OA) is highly prevalent, a frequent cause of disability, and lacks highly effective therapies in both humans1, 2 and pet dogs3. Multiple joint OA (MJOA), referring to OA in more than one joint site within an individual, is common in both species4-6. Performing a systematic review that included ~30,000 people, we found a high frequency of MJOA based on most definitions (25-74%)7, 8. MJOA has a significant detrimental effect on sufferers with increased OA burden being associated with increased mortality and disability, poorer health and function7, 9-11. Despite its frequency and significant negative impact, MJOA remains poorly understood7, 8. Given the similar manifestations and therapies available for humans and dogs with MJOA3, pet dogs represent an ideal naturally occurring model of MJOA for study and mutual benefit. It is critical to determine underlying mechanism(s) driving MJOA in order to develop more effective therapies for the millions of human and canine patients afflicted. There are few modifiable risk factors for OA in general, and none specifically for MJOA. Although systemic factors such as soluble biomarkers, genetics and metabolism, are related to the overall body burden of OA (i.e., MJOA) rather than OA at single sites of OA, this is not often taken into account. These and other risk factors may act mechanistically, at least in part, by effects on the gut microbiome12, the impact of which is related to intestinal permeability (IP). Increased IP leading to low-level systemic inflammation has been associated with obesity, diabetes, fatty liver, and inflammatory bowel conditions13, 14. Systemic markers indirectly associated with IP and reflecting increased exposure to microbial products that may promote OA include lipopolysaccharide (LPS) and LPS-binding protein (LBP). IP can also be directly assessed using the lactulose-mannitol dual sugar test (DST, humans), iohexol absorption (dogs) or in gut biopsies. Our group has demonstrated associations between LPS, LBP, disease severity, and clinical signs of knee OA15, 16, likely via LPS-driven low-grade inflammation. Fecal transplant from OA patients into germ-free mice with and without meniscal injury resulted in colonic mucosa bacterial invasion in mice transplanted with OA but not control microbiota, establishing a ����������������gut microbiome-OA connection15.��������������� To further elucidate the role of IP as a mechanism in MJOA, the proposed work will leverage two well-characterized observational human cohorts and an animal study: The JoCoOA, a longitudinal study of over 4000 Black and White men and women aged 45 and older and ongoing since 1991; The Johnston County Health Study (JoCoHS), an actively enrolling cohort (2019-, n~2000) with the same infrastructure as the JoCoOA with the addition of younger (35-70 years) and Hispanic individuals; naturally occurring MJOA in pet dogs17-22. Data from all three cohorts will be used to address the following 3 aims: 1. Determine cross-sectional associations between altered IP, systemic inflammation, and radiographic and symptomatic MJOA in humans and pet dogs by: a) assessing the frequency of increased IP both directly by the lactulose-mannitol DST and indirectly through determination of plasma LPS and LBP, along with assessment of inflammation and the microbiome, in 100 JoCoHS participants with and without MJOA, and b) determining the frequency of increased IP by directly analyzing intestinal biopsies using a 2-dimensional organoid approach and measuring iohexol absorption, and indirectly by measuring LPS and LBP, and characterizing inflammation and the microbiome in a cohort of pet dogs (n=?) with and without MJOA and not receiving c

In the United States, roughly 68 million veterinary visits are adversely affected by fearful behavior annually with negative consequences for canine welfare. Dog characteristics account for only a small portion (<7%) of the fear experienced in the clinical environment. It is critical that we address this knowledge gap regarding what drives fearful behavior in the clinic. We postulate that pain catastrophizing may be the missing factor. Pain catastrophizing is a cognitive and emotional response toward actual or anticipated pain, often characterized in humans by verbal reports and behavioral changes. People who are high in pain catastrophizing are at higher risk of pain-related interference in activity, development of chronic pain, and have worse health outcomes across a spectrum of diseases. In pediatric medicine, pain catastrophizing occurs in children and results in higher reported pain intensity scores and greater anxiety. Additionally, childrens��� pain experience is affected by caregivers who catastrophize about their child���s pain. Using the pediatric/caregivers model, we hypothesize that pain catastrophizing can be quantified in dogs using our novel experimental paradigm and is positively associated with owner catastrophizing about their dog���s pain. Establishing a measure of pain catastrophizing in dogs and understanding the owner influence on the phenomenon would be a major advance for veterinary medicine ��� specifically in pain management and behavioral health and welfare. Ultimately, our work will improve health outcomes including surgical recovery and management of chronic pain conditions ��� both of which have been shown in humans to be negatively affected by pain catastrophizing.

Buprenorphine is a partial ��-opioid receptor agonist commonly used for both preoperative and postoperative pain management in dogs. It is favored due to its relatively lower incidence of undesirable side effects such as vomiting, nausea, respiratory depression, and dysphoria, when compared to other opioids. However, its relatively short duration of action can limit its effectiveness. While multiple options exist for managing perioperative pain in companion animals, there remains a need for formulations that can be administered in-clinic and provide sustained pain relief during the critical first few days post-surgery at home. Extended-release buprenorphine (XR-buprenorphine) formulations have been developed to address this gap, offering analgesia of prolonged duration. One such formulation utilizes lipid encapsulation in medium-chain triglyceride (MCT) oil, which reduces buprenorphine diffusion. This allows for the administration of higher drug doses, minimizes toxicity, and extends the opioid's therapeutic effects. Our study aims to evaluate the pharmacokinetics and analgesic effects of a single subcutaneous administration of XR-buprenorphine in a canine stifle arthrotomy model.

In this project, I will work closely with Dr. Shyni Varghese and her team to optimize the methodology around the measurement of pain - sensitivity, activity, limb use. I will provide the apparatus to measure pronograde limb use. I will be present, and work ���������������hands on������������������ to help set up and optimize the behavioral pain assay work, assist with data collection and data analysis.

More than 80% of patients undergoing treatment for head and neck cancer are prescribed opioid pain killers for management of radiation-associated pain, and unfortunately, 6 months after finishing radiotherapy, one-third of head and neck cancer patients will still be opioid-dependent. Chronic opioid use is associated with side effects such as constipation and decreased alertness; it can also lead to opioid abuse, misuse, addiction, and eventual overdose. Safer, more effective and less addictive pain relief strategies are desperately needed, and our proposed research addresses that need by investigating a novel radiation-activated pain signaling pathway that might serve as an excellent target that could be inhibited with new pain medications.

Statement of Work ��� Proposed Supplement As Co-PI in this supplemental project, I will work closely with Dr. Amanda Nelson and her team to ensure the milestones of the supplement are met. This will involve carefully overseeing the recruitment to ensure this is kept on track / accelerated in order to meet enrollment goals outlined in the supplement, and oversight of the additional sample collection. Importantly, I will work closely with the Research Specialist (TBD from supplement funds) that will coordinate all the data to ensure it is complete, checked, set up and has undergone quality control in time for the advanced analysis work described in the supplement. In collaboration with the statistician (Arbeeva Liubov), I will assist with data analysis, and work closely with Co-PI Dr. Amanda Nelson on the interpretation, creating abstracts, writing manuscripts and communication of results to the general public.