Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Population Medicine
Non Technical Summary
Osteoarthritis (OA) is a debilitating disease affecting the athletic performance of horses. Lameness, the symptomatic manifestation of OA, has been described as the most important concern for horse owners, and carries significant economic cost to the equine industry. There is no cure for OA so early identification and approaches to prevent or slow progression of the disease are the best approaches for reducing the pain and prolonging the functional use of the joint. Therefore, there is a need for minimally invasive, reliable, quantitative tests, called biomarkers, for detection of early changes associated with OA.Molecular biomarkers of OA are one of the most used biomarkers at this time. They reflect changes associated with joint metabolism. Two basic types of molecular biomarkers are available to help with early diagnosis of OA in horses, including direct and indirect biomarkers. Direct biomarkers are those bi-products that result directly from altered joint metabolism, whereas indirect biomarkers are those that indirectly affect joint metabolism. Both types of biomakers can be analyzed in blood, joint fluid, or urine.Study of both direct and indirect biomarkers has been performed on horses with naturally-occurring OA and in equine models of OA to help diagnose and monitor progression of joint injury and OA. However, it is important to point out that much of the analyses of these biomarkers has been performed on joint fluid from fetlocks (ankles) and carpi (knees). Both of these areas are common locations for OA in the horse, but there are many other joints that commonly suffer from OA as well, including the coffin joint (DIP), lower hock joints (TMT and DIT), and two stifle joints (mFT and FP). These joints have rarely been studied using biomarkers, even though they are commonly afflicted. One of the main reasons for this is because researchers are not able to collect samples from normal horses since these joints are either difficult to obtain fluid from due to their size or location. Therefore, even if samples are collected from horses with naturally-occurring OA in these joints, it is difficult to document the disease status if there are no known normal concentrations to compare to. This lack of knowledge prevents the use of biomarkers for early identification of OA in these commonly affected joints. This is important to the equine industry because depending upon the discipline of the horse, these joints may actually be affected more often than either ankles or knees. Therefore the rationale for this proposal is that even though direct and indirect biomarkers have been examined in fetlock and carpal joints, little is known about the biomarker concentrations in joint fluid from other commonly afflicted joints. This lack of knowledge prevents researchers from examining biomarkers in other clinically important joints pointing out an urgent need to assimilate this data so that these studies can progress.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Goals / Objectives
Currently, the most accurate method of identifying the presence of arthritis (OA) in a joint is the combination of thorough physical, lameness, and x-ray examinations. However, many horses may have pain localized to a joint with minimal x-ray changes present. This may indicate that the joint is normal or that the disease is in its earliest stages. Therefore, there is a need for minimally invasive, reliable tests, called biomarkers that reflect what happens in the joint at the molecular level. Two basic types of molecular biomarkers are available to help with early diagnosis of OA in horses, including direct and indirect biomarkers. Direct biomarkers are those bi-products that result directly from altered joint metabolism. Indirect biomarkers are those that often cause a change in joint metabolism.Study of both direct and indirect biomarkers has been performed on horses with naturallyoccurring OA and in horse models of OA to help diagnose and monitor progression of joint injury and OA. However, it is important to point out that much of the analyses of these biomarkers has been performed on joint fluid from fetlocks (ankles) and carpi (knees). Both of these areas are common locations for OA in the horse, but there are many other joints that commonly suffer from OA as well, including the coffin joint, 2 lower hock joints, and 2 joints in the stifle. These joints have rarely been studied using biomarkers, even though they are commonly afflicted. One of the main reasons for this is because researchers are not able to collect samples from normal horses since these joints are either difficult to obtain fluid from due to their size or location. Therefore, even if samples are collected from horses with naturally-occurring OA in these joints, it is difficult to document the disease status if there are no known normal concentrations to compare to. This lack of knowledge prevents the use of biomarkers for early identification of OA in these commonly affected joints. This is important to the equine industry because depending upon the discipline of the horse, these joints may actually be affected more often than either ankles or knees. Therefore the rationale for this proposal is that even though direct and indirect biomarkers have been examined in ankles and knees, little is known about the biomarker concentrations in joint fluid from other commonly afflicted joints. This lack of knowledge prevents researchers from examining biomarkers in other clinically important joints pointing out an urgent need to assimilate this data so that these studies can progress.We will collect 12 joint fluid samples from each forelimb coffin joint, and each of the two lower hock joints and 2 stifle joints from horses that are donated to the University. Joints will be examined to determine that they are normal. Twenty-three biomarkers of interest will then be examined in each joint fluid sample using a combination of different technologies. Concentrations will be compared to breed, gender, age, and between joints. We aniticpate that this study will provide pilot data for equine researchers in the form of normal reference ranges for each of these biomarkers and joints, enabling future biomarker studies in these important joints.
Project Methods
Sample Population: We will obtain synovial fluid from horses that are donated to the University of Minneosta Equine Center for euthanasia due to their overall condition or behavior (reasons unrelated to this study).We will collect synovial fluid from the forelimb distal interphalangeal joints, and the hindlimb tarsometatarsal, distal intertarsal, medial femorotibial, and femoropatellar joints immediately after euthanasia (within 30 minutes). The joints will be disarticulated following fluid collection to see if there is any gross indication of OA present (cartilage score lines, synovial hypertrophy/hyperplasia, or osteophytes), or any other questionable pathology (such as old laceration over joint). If so, these samples will not be entered into this study. As determined by our power calculations (see statistical methods section), we need to collect 12 synovial fluid samples from each joint of interest. In addition, even though we will collect fluid from joints bilaterally (ie left and right front distal interphalangeal joints), only one sample from each horse (ie left or right) will be used for analysis so that for each joint, samples will come from 12 different horses. It is expected that within a horse we will not be able to collect samples from all joints of interest because, 1) OA is present in a given joint, or 2) we were not able to obtain fluid (especially in the tarsometatarsal and distal intertarsal joints due to small size). This means that we will need more than 12 horses to collect appropriate samples, but not more than 20 horses will be needed to collect all samples.Sample Collection: All samples will be collected within 30 mintues after euthansia. Standard approaches for arthrocentesis of the distal interphalangeal, tarsometatarsal, distal intertarsal, medial femorotibial, and femoropatellar joints will be used to aspirate as much synovial fluid as possible. In order to complete all assays, it is ideal to collect between 1.0 to 1.5 mL of synovial fluid. This should not be a problem for the medial femorotibial and femoropatellar joints, but may be a problem for some distal interphalangeal, tarsometatarsal and distal intertarsal joints. We anticipate that approxiamtely 50% of these joints sampled will have less than the required amount. After all joints have been sampled, each joint will be disarticulated and grossly examined for evidence of OA (cartilage score lines, synovial hypertrophy/hyperplasia, or osteophytes). All joints will be labelled and photographed. Samples will not be used if any signs of OA are present. Synovial fluid samples will be centrifuged, aliquoted, cataloged, and stored at -80°C until further analysis.Biomarker analysis: Synovial fluid samples will be analyzed using a panel of direct and indirect biomarkers that have been previously validated by the PI for equine use. They will examine the inflammatory and enzymatic responses as well as the bi-products of synthesis and degradation of the extracellular matrix for bone and cartilage. Direct Biomarkers: Direct biomarkers that will be examined include: CTX II, C2C, CPII, CS846, and BAP. CTX II measures degradation of type II collagen (close to the bone cartilage interface) using a sandwich ELISA based on a monoclonal antibody for the type II collagen Ctelopeptide epitope (preclinical CartiLaps®, IDS/Nordic Bioscience Diagnositcs, Herlev, Denmark). C2C measures degradation of type II collagen by identifying the neo-epitope created after collagenase cleavage of the triple helix via a competitive immunoassay that utilizes a mouse polyclonal antibody (IBEX Technologies, Inc., Montreal, CAN). CPII measures the by-product of type II collagen synthesis using a commercially available enzyme linked competitive immunoassay that utilizes a rabbit polyclonal antibody (IBEX Technologies, Inc., Montreal, CAN). CS846 measures large fetal forms of aggrecan with at least one CS846 epitope, which is thought to be synthesized in adult cartilage in an attempt to repair damage. It is measured via a competitive immunoassay that utilizes a mouse monoclonal antibody (IBEX Technologies, Inc., Montreal, CAN). BAP measures bone alkaline phosphatase (BAP), an early osteoblastic marker using a sandwich ELISA (Metra BAP, Quidel Corporation, San Diego, CA). All direct biomarkers will be examined using the commercially available enzyme linked immunoassays listed above. They will be analyzed using a FLUOstar Optima microplate reader (BMG LABTECH GmbH, Offenburg/Germany) at appropriate dilutions determined from previous studies.Indirect Biomarkers: We will measure indirect biomarkers using multiplex assay technology (Luminex® platform), which is a bead based, flow cytometer system that can evaluate up to 100 different analytes attained from a single sample of fluid. Each bead is internally color-coded with varying ratios of 2 fluorescent dyes, and as such, is fashioned to determine an analyte of interest through covalent coupling to specific capture antibodies. One laser identifies the specific bead set and another laser quantifies the fluorescent reaction to the antibody. Multiple cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs) can be quantified and compared. When determining biomarker concentrations in equine synovial fluid, equine specific ELISA kits are unavailable. To best determine which species kit would identify equine analytes of interest, the homology of equine protein sequences for common cytokines, matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases were compared to protein sequences from humans, mice and rats. Results revealed highest homology between human and horse protein sequences, and as such we utilize human antibody kits (R&D Systems). We will measure the following indirect biomarker concentrations using these commercially available human multiplex assay kits: Interleukins (IL) -1β, -2, -4, -5, -6, -8, and -10, tumor necrosis factor alpha (TNF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP) -1, -2, -3, -9, and -13, and tissue inhibitors of metalloproteinases (TIMPs) -1, -2, -3, and -4. All samples will be run in duplicate at two dilutions (determined from previous work by the PI) and analyzed on a Bio-Rad Bio-Plex 200 plate reader that uses Luminex® multiplex technology.