Outline of the Completed Research

Clinical Science Pathobiology


Effect of low-intensity pulsed ultrasound (LIPUS) on bone remodeling in Thoroughbred horses (2013−2015)

 Low-intensity pulsed ultrasound (LIPUS) has been reported to increase fracture fusion rates in humans, but there have been few reports of this phenomenon in horses. We investigated the effects of LIPUS on bone remodeling in horses by using a fourth metacarpal bone defect model. A 5-mm defect was produced in both fourth metacarpal bones under general anesthesia in 10 Thoroughbred horses. LIPUS was applied to one forelimb from 3 days after surgery until 12 (5 horses) or 15 (5 horses) weeks after surgery, and the contralateral limb was used as a control. LIPUS was performed at a 1000-Hz pulse frequency with 30 mW/cm2 power. Horses received 20 min of treatment once a day 6 days a week. Both fourth metacarpal bones were collected 12 or 15 weeks after surgery, and specimens of the new bone were evaluated histologically. In samples taken at 12 or 15 weeks, the number of osteoblasts and the osteoid surface as a percentage of the whole surface of new bone did not differ significantly between LIPUS treatment and the control. In contrast, the number of osteoclasts and the percentage eroded bone surface were significantly lower in the LIPUS-treated bone than in the control bone after 12 or 15 weeks of treatment. Moreover, the bone volume to tissue volume ratio was significantly higher in LIPUS-treated bone after 15 weeks of treatment. We concluded that LIPUS treatment increased bone volume by inhibiting bone resorption by osteoclasts in the bone remodeling phase after fracture.

Analysis of tenogenic differentiation of equine bone marrow derived-mesenchymal stem cells in vitro (2012-2014)

 We investigated methods of tenogenic differentiation of equine bone marrow derived-mesenchymal stem cells (BM-MSCs) in vitro. Comparison of gene expression in BM-MSCs and tendon tissue revealed that collagen XIV, collagen XXIV, decorin, fibromodulin, and cartilage oligomeric matrix protein, like tenomodulin, could be useful novel tendon-related markers. BM-MSCs in three-dimensional collagen gel culture were exposed with or without mechanical stress to various cytokines and inhibitors of signaling pathways related to differentiation; we then investigated the changes in mRNA expression of the tendon-related markers. We found that an environment that mimicked tendon tissuenamely, one in which the cells were surrounded by collagen under tensional loadwas useful for inducing tenogenic differentiation of BM-MSCs. Furthermore, an analysis of signaling pathways associated with MSC differentiation suggested that activation of integrin signaling and GSK3/β-catenin signaling might play an important role in tenogenic differentiation of equine BM-MSCs.

Clinical and pathobiological research into exercise-related injuries and diseases in racehorses (2008–2012)

 We used pathological, statistical, and clinical medicine techniques to examine ways to prevent and treat not only various exercise-related injuries but also diseases influencing racehorse performance. This diverse research included studies of the characteristics of the new Polytrack training course, the relationship between amount of training and accident rate during races, and the relationship between running speed and accident rate in training. Furthermore, we used statistical calculations to derive the fiducial limits of the accident rate at each racetrack. We also investigated the time-dependent residual hoof concentrations of an aminoglycoside antimicrobial agent (gentamicin) given by regional limb perfusion, so as to establish precise administration intervals for this treatment. In other research, we used biomechanical techniques to investigate the drawbacks of the glue-on horseshoe, and we studied and established a regenerative medical treatment for corneal disease. Finally, we reported internationally on the pathology and clinical features of exudative canker of the hoof (gidoh in Japanese) and of T-cell lymphoma associated with ataxia.

Introduction and establishment of regenerative medicine techniques in clinical racehorse practice (2010–2012)

 In this research project, we introduced the latest regenerative medicine techniques―stem cell therapy for tendon injury―to clinical practice at the Japan Racing Association (JRA). Through collaboration between researchers at our equine research institute and practitioners in racehorse hospitals at training centers, 47 racehorses suffering from tendon injury were treated with stem cells at racehorse hospitals (28 at the Miho Training Center and 19 at the Ritto Training Center) over a 3-year period. As a result of these treatments, we established the most efficient system of cell therapy at the JRA. It consists of collection of bone marrow at the training centers, culture of cells at the institute, and implantation of cells in the hospital. As part of the basic research under this project, we established techniques for artificially creating an equine-tendon-injury model and for verifying the efficacy of stem cell therapy. Additionally, we verified the efficacy of a new type of cell-free regenerative medical treatment called PRP (platelet-rich plasma) therapy. The results suggested that this treatment could be of practical use in treating horses.

Determination of the mechanism of water infiltration inside the hoof wall and the influence of urine contamination of the hoof wall: Verification of the functions of horn tubules and intertubular horn as moisture regulating structures (2007-2008)
(Commissioned research conducted by Kyoto University)

 To determine the mechanism of hoof wall degeneration caused by water accumulated in bedding material, the process of water infiltration from the hoof wall cross-section was observed by nuclear magnetic resonance microimaging. Samples of unprocessed normal hoof wall and hoof wall pre-immersed in ultra-pure water, horse urine, ammonia solution or urea solution were used. In the unprocessed hoof wall, amplification of signals proceeded from the water absorptive surface towards the inside. There was a correlation between time-lapse changes in signal intensity in the horn tubules and the intertubular horn, indicating that the infiltration of water could occur in both two parts. In the samples pre-immersed in horse urine, observation up to 24 hours after water absorption revealed that signaling was low compared to those pre-immersed in ultra-pure water, but that amplification of the signal was large in the later stage of the water absorption process. Moreover, in the samples pre-immersed in ammonia solution, images were similar to those of hoof walls processed with horse urine. From these findings, it was suggested that immersion in urine can cause changes in the characteristics of distribution and transfer of water in the hoof wall.

Basic study on equine regenerative medicine using cell growth factor
(2007-2008)

 A basic study was conducted on a drug delivery system (DDS) that provides sustained release of the cell growth factor bFGF and its application to equine regenerative medicine. In studies of the restoration effects on a full-thickness skin loss wound model, it was confirmed that blood vessels were quickly induced and the formation of the epithelium was promoted by the use of the DDS. In studies of restoration effects on a model of full-thickness defects of articular cartilage in the equine carpal joint, the expected regeneration of hyaline cartilage was not observed. Conversely, restoration due to granulation tissue of the bone tunnel in carpal bones was superior compared to the control group. Although it became clear from this research that a DDS based on sustained release of bFGF has potential for clinical application, it was thought necessary to reconsider the choice of cell growth factor appropriate for cartilage regeneration.

Clinico-pathological study on sports injuries and other disorders in racehorses2003-2007

Purpose
 Various sports injuries that impair athletic performance occur as a result of the rigorous training and racing of racehorses. The purpose of this study was to contribute to preventing disease and improving the ability of racehorses by elucidating the pathology of sports injuries and other disorders affecting racehorses, and conducting applied research associated with this.

Results
I. Epidemiological study of sports injuries and factors impairing athletic performance

 1. We studied the incidence and risk of injury by superficial digital flexor tendonitis and suspensory desmitis that occurred during racing and training over the twelve months of 1999. As a result, the respective rates of incidence were 11.1% and 3.6%. We demonstrated that the risk of injury caused by both diseases increased with age, and was also higher in males.

 2. We studied the hoof wall growth rate in laminitic hooves over a year. As a result, the hoof wall growth rate in laminitic hooves was lower than that of normal hooves (the lowest rate recorded being 47% of that of normal horses). We also found that a slowed growth rate can be improved through special farrier techniques whereby the bearing border of the laminitic hoof is raised above the ground level.

 3. We studied the impact of differences in bedding under the foot. As a result, it became clear that sawdust and coconut palm husk easily become lodged under the foot when contaminated with urine, and that, as a consequence of this, sawdust and coconut palm husk are more prone to cause hoof wall damage, compared to rice straw.

II. Clinico-pathological diagnosis of horse fatalities and diseases of unknown cause

 1. We conducted a histopathological study of the healing mechanism of laminitis. As a result, we identified factors such as the point of genesis of lamellar wedge, making it possible to estimate the point when laminitis occurs. We also discovered that there are regions in which the basement membrane zone of the regenerated laminar layer in laminitis does not improve for at least three months, and this was thought to be one cause of the recurrency of laminitis.

 2. The pathology of canker was clinically examined. As a result, we proved that bacteria of the genus Treponema are involved in the occurrence of chronic proliferative pododermatitis, and that combined use of surgery with the removal of lesions using sterile maggots is effective in treating canker.

 3. We conducted endoscopic examination and histopathological examination on a young racehorse that had contracted a respiratory disorder of unknown cause. As a result, we found that dynamic pharyngeal collapse based on muscle degeneration of the muscles of the pharynx (particularly the stylopharyngeus muscles) had occurred. From this, we confirmed that endoscopic examination of the pharynx is important when diagnosing young horses that contract respiratory disorders.

 4. To study the pathology of catheter-induced jugular phlebitis, we experimentally retained a catheter in the jugular vein of a horse for several days and observed changes in the vein tissue. At points that contacted the catheter, acute inflammation consisting principally of eosinophils was recognized, leading to the conjecture that a hypersensitivity reaction to the catheter had occurred.

 5. We carried out pathological examination on a sick horse that had tumors all over its body, including numerous organs in the thoracic and abdominal cavities. As a result, these tumors were recognized to be extremely rare nephroblastomes that had translocated all over the body. Synovial osteochondromatosis was also observed inside the hock joint.

Establishing a method of evaluating qualitative and structural changes in hoof wall. Collaborative study between Clinical Sciences and Pathobiology Division, Equine Research Institute and John D. REILY, Nottingham University (2004-2005)

 Despite pronounced improvements in shoeing technology, such as the increased use of shoes designed for both training and racing, racehorses are still forced into frequent layoffs by shoeing difficulties due to hoof diseases thought to be caused by hoof wall degeneration (e.g. hoof wall separation). The causes of this are complex and preventive measures extremely difficult to establish, but before identifying the cause, The aim of the research was to establish a method of evaluating dynamic and structural changes in the hoof wall characteristics and, furthermore, to elucidate whether or not imposing restricted training in the growth stage of the hoof causes changes to the tubule density (TD) of horn tubules in the hoof wall or the modulus of elasticity (ME) of the hoof wall. After establishing a method of evaluating the hoof wall using an exercise group subjected to restricted training and a control group permitted to graze freely, we studied the impact of exercise on hoof wall characteristics (morphological and dynamic properties). As a result, we found that restricted training did not change the thickness of the hoof wall but reduced tubule density (TD). Meanwhile, on measuring the modulus of elasticity (ME) of the hoof wall, we found that restricted training had no impact on ME. Moreover, no significant correlation was found between TD and ME. From this, it was thought that exercise does cause changes to the tissue architecture of the hoof wall through a kind of adaptation, but that exercise does not tend to change dynamic characteristics.

Research on markers for locomotive disorders in racehorses: The usefulness of COMP as a marker for locomotive disorders (2004-2005)

 Cartilage Oligometric Matrix Protein (COMP) is a type of glycoprotein that exists specifically in articular cartilage, synovial membrane and tendons. In humans, fluctuations in the serum content of these has already been clinically applied as a disorder marker for arthritis, and in recent years the possibility of screening injuries using the volume ratio of various types of COMP fragments present in synovial fluid has been reported. There is very little knowledge on equine COMP fragments in the field of veterinary science. Therefore, in joint work with a British researcher, we posed the hypothesis that In equine tendinitis and arthritis, different types of COMP fragments are present in serum, and studied the possibility of their clinical application as markers for locomotive disorders in racehorses.
Specifically, through serum diagnosis using COMP as a marker, we studied the possibility of its clinical application as a marker for locomotive disorders in racehorses, but found that these could not be detected in the antibodies that we had prepared. We also studied the possibility of clinical application as a serological marker for healing of musculoskeletal injury in locomotive disorders, but the serum concentration of COMP did not reflect recovery. From these results, we concluded that, at the present time, there is little possibility of using measurements of COMP concentration in serum for clinical application as markers for locomotive disorders or serological markers for healing of musculoskeletal injury.

Elucidation of the functions of tendon cells using in vitro culture: Development of an evaluation model for the healing process of tendinitis using culture tendon cells. Commissioned to Katsuhiko ARAI, Department of Tissue Physiology, Tokyo University of Agriculture and Technology (2003-2005)

 While clinical tests using actual horses are vital for application to diagnosis and treatment, developing an alternative to animal experiments has become an important task for researchers, with so many problems now emerging in the treatment of experimental animals. In this research, as an aid to establishing a method of diagnosis and early cure for tendinitis, we aimed to establish an experimental model using cultured tendon cells that can reproduce the pathological state of tendinitis, and to elucidate the molecular mechanism of occurrence and healing of tendinitis by studying the characteristics of equine cultured tendon cells under various conditions. First, as a basic study aimed at developing a healing evaluation model for tendinitis using cultured tendon cells, we analyzed the mRNA expression of matrix-related protein in tendon tissue on occurrence of acute tendinitis, and found strong expressions of hyaluronidase, MMP-13 matrix metalloproteinase13 and cathepsin D. Meanwhile, we found that, in tendon cells cultured in relax gel, the expression of the same enzyme genes as in the affected tendon tissue is reproduced. From the above, we felt that culture in relax gel is suitable as a cell culture model that can reflect the pathological state of tendinitis. We also demonstrated differences in cell properties arising from the fact that collagen relaxing capacity and the expression of collagen receptors are lower in cultured cells derived from affected tendons than in those derived from normal tendons, and proved that the cytokine reactivity of cultured tendon cells is influenced by the matrix composition used in culture.