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The OARSI histopathology initiative – recommendations for histological assessments of osteoarthritis in the horse

      Summary

      Objective

      Equine models of osteoarthritis (OA) have been used to investigate pathogenic pathways of OA and evaluate therapeutic candidates for naturally occurring equine OA which is a significant clinical disease in the horse. This review focuses on the macroscopic and microscopic criteria for assessing naturally occurring OA in the equine metacarpophalangeal joint as well as the osteochondral fragment-exercise model of OA in the equine middle carpal joint.

      Methods

      A review was conducted of all published OA studies using horses and the most common macroscopic and microscopic scoring systems were summarized. Recommendations regarding methods of OA assessment in the horse have been made based on published studies.

      Results

      A modified Mankin scoring system is recommended for semi-quantitative histological assessment of OA in horses due to its already widespread use and similarity to other scoring systems. Recommendations are also provided for histological scoring of synovitis and macroscopic lesions of OA as well as changes in the calcified cartilage and subchondral bone of naturally occurring OA.

      Conclusions

      The proposed system for assessment of equine articular tissues provides a useful method to quantify OA change. It is believed that addition of quantitative tracing onto plastic and macroscopic measurement as recently described would be an improvement for overall assessment of articular cartilage change.

      Keywords

      Introduction

      Spontaneous osteoarthritis (OA) is a common problem in the horse. Equine degenerative arthritis was reported as an entity and the pathologic changes were compared to human OA in 1930
      • Callender G.R.
      • Kelser R.A.
      Degenerative arthritis: a comparison of the pathological changes in man and equines.
      . Equine OA was reviewed in 1962
      • Mackay-Smith M.P.
      Pathogenesis and pathology of equine osteoarthritis.
      and macroscopic and microscopic changes were described for OA in the distal limb joints of the horse in 1973
      • Nilsson G.
      • Olsson S.C.
      Radiologic and patho-anatomic changes in the distal joints and phalanges of the Standardbred horse.
      . OA can occur early in equine athletes, or later in older horses
      • McIlwraith C.W.
      Current concepts in equine degenerative joint disease.
      • Riggs C.M.
      • Whitehouse G.H.
      • Boyde A.
      Pathology of the distal condyles of the third metacarpal and third metatarsal bones.
      • Kawcak C.E.
      • McIlwraith C.W.
      • Norrdin R.W.
      • Park R.D.
      • James S.P.
      The role of subchondral bone in joint disease: a review.
      . An arthroscopic grading system has also been used for clinical disease in the carpus
      • McIlwraith C.W.
      • Yovich J.V.
      • Martin G.S.
      Arthroscopic surgery for the treatment of osteochondral chip fractures in the equine carpus.
      and fetlock joints
      • Kawcak C.E.
      • McIlwraith C.W.
      Proximal dorsal first phalanx osteochondral chip fragmentation in 336 horses.
      and biomarkers related to macroscopic and histologic cartilage lesions have been evaluated
      • Frisbie D.D.
      • Ray C.S.
      • Ionescu M.
      • Poole A.R.
      • Chapman P.L.
      • McIlwraith C.W.
      Measurement of synovial fluid and serum concentrations of the 846 epitope of chondroitin sulfate and of carboxy propeptides of type II procollagen for diagnosis of osteochondral fragmentation in horses.
      • Fuller C.J.
      • Barr A.R.
      • Sharif M.
      • Dieppe P.A.
      Cross-sectional comparison of synovial fluid biochemical markers in equine osteoarthritis and the correlation of these markers with articular cartilage damage.
      .
      A number of experimental models of OA have been developed and used in the horse
      • McIlwraith C.W.
      • Fessler J.F.
      • Van Sickle D.C.
      • Blevins W.E.
      • Page H.
      • Rebar A.H.
      • et al.
      Experimentally induced arthritis of the equine carpus. Clinical parameters.
      • McIlwraith C.W.
      • Van Sickle D.C.
      Experimentally induced arthritis of the equine carpus: histological and histochemical changes in the articular cartilage.
      • Gustafson S.B.
      • Trotter G.W.
      • Norrdin R.W.
      Evaluation of intra-articularly administered sodium monoiodoacetate induced chemical injury to articular cartilage of horses.
      • Yovich J.V.
      • Trotter G.W.
      • McIlwraith C.W.
      • Norrdin R.W.
      Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.
      • Trotter G.W.
      • Yovich J.V.
      • McIlwraith C.W.
      • Norrdin R.W.
      Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.
      • Peloso J.G.
      • Stick J.A.
      • Caron J.P.
      • Peloso P.M.
      • Soutas-Little R.W.
      Effects of hylan on amphotercin-induced carpal lameness in equids.
      • Firth E.C.
      • Wensing Th
      • Seurin F.
      An induced synovitis model in ponies.
      • Todhunter P.G.
      • Kincaid S.A.
      • Todhunter R.J.
      • Kammermann J.R.
      • Johnstone B.
      • Baird A.N.
      • et al.
      Immunohistochemical analysis of an equine model of synovitis-induced arthritis.[erratum appears in.
      • Palmer J.L.
      • Bertone A.L.
      • Malemud C.J.
      • Mansour J.
      Biochemical and biomechanical alterations in equine articular cartilage following an experimentally-induced synovitis.
      • Hardy J.
      • Bertone A.L.
      • Weisbrode S.E.
      • Muir W.W.
      • O’Dorisio T.M.
      • Masty J.
      Cell trafficking, mediator release, and articular metabolism in acute inflammation of innervated or denervated isolated equine joints.
      • Cornelissen B.P.
      • Rijkenhuizen A.B.
      • van den Hoogen B.M.
      • Rutten V.P.
      • Barneveld A.
      Experimental model of synovitis/capsulitis in the equine metacarpophalangeal joint.
      • Owens J.G.
      • Kamerling S.G.
      • Stanton S.R.
      • Keowen M.L.
      • Prescott-Mathews J.S.
      Effects of pretreatment with ketoprofen and phenylbutazone on experimentally induced synovitis in horses.
      • Auer J.A.
      • Fackelman G.E.
      • Gingerich D.A.
      • Fetter A.W.
      Effect of hyaluronic acid in naturally occurring and experimental osteoarthritis.
      • Simmons E.J.
      • Bertone A.L.
      • Weisbrode S.E.
      Instability-induced osteoarthritis in the metacarpophalangeal joint of horses.
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      • Richardson D.W.
      • Clark C.C.
      Effects of short-term cast immobilization on equine articular cartilage.
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Gaughan E.M.
      • Mclaughlin R.M.
      • Debowes R.M.
      Clinical evaluation of the effects of immobilization followed by remobilization and exercise on the metacarpophalangeal joint in horses.
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Turner A.S.
      • Norrdin R.W.
      Effects of immobilization followed by remobilization on mineral density, histomorphometric features, and formation of the bones of the metacarpophalangeal joint in horses.
      (Table I). Unlike the dog, complete surgical transection of the anterior cruciate ligament (ACL) alone arthroscopically in horses did not result in progressive OA in two cases (Trotter GW, unpublished data, 1996). However in a published study involving one of the authors (MH) arthroscopic and radiographic assessment of naturally occurring ACL deficient horses resulted in severe OA and the horses were Grade 2 or more lame
      • Desjardins M.R.
      • Hurtig M.B.
      Diagnosis of equine stifle joint disorders: three cases.
      . The most commonly published model is the arthroscopically created osteochondral fragment-exercise model developed at Colorado State University (CSU)
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      . This model evolved with two major changes since its inception: (1) burring back an osteochondral lesion that was larger than the fragment; (2) not flushing tissue debris from the joint post-defect creation. This model is clinically relevant to horses and has relevance to human OA. Unlike many other in vivo models, no instability is created. The model induces progressive OA, yet it does not produce a severe lameness
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      • Anonymous
      Definition and Classification of Lameness. Guide for Veterinary Service and Judging of Equestrian Events.
      . Macroscopic and histological evaluations are presented in this paper.
      Table IExperimental models of OA that have been described in the horse
      Type of modelSpecific name
      Intra-articular injection of chemicalsFilipin
      • McIlwraith C.W.
      • Fessler J.F.
      • Van Sickle D.C.
      • Blevins W.E.
      • Page H.
      • Rebar A.H.
      • et al.
      Experimentally induced arthritis of the equine carpus. Clinical parameters.
      • McIlwraith C.W.
      • Van Sickle D.C.
      Experimentally induced arthritis of the equine carpus: histological and histochemical changes in the articular cartilage.
      Sodium monoiodoacetate
      • Gustafson S.B.
      • Trotter G.W.
      • Norrdin R.W.
      Evaluation of intra-articularly administered sodium monoiodoacetate induced chemical injury to articular cartilage of horses.
      • Yovich J.V.
      • Trotter G.W.
      • McIlwraith C.W.
      • Norrdin R.W.
      Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.
      • Trotter G.W.
      • Yovich J.V.
      • McIlwraith C.W.
      • Norrdin R.W.
      Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.
      Amphotericin
      • Peloso J.G.
      • Stick J.A.
      • Caron J.P.
      • Peloso P.M.
      • Soutas-Little R.W.
      Effects of hylan on amphotercin-induced carpal lameness in equids.
      E. coli lipopolysaccharide
      • Firth E.C.
      • Wensing Th
      • Seurin F.
      An induced synovitis model in ponies.
      • Todhunter P.G.
      • Kincaid S.A.
      • Todhunter R.J.
      • Kammermann J.R.
      • Johnstone B.
      • Baird A.N.
      • et al.
      Immunohistochemical analysis of an equine model of synovitis-induced arthritis.[erratum appears in.
      • Palmer J.L.
      • Bertone A.L.
      • Malemud C.J.
      • Mansour J.
      Biochemical and biomechanical alterations in equine articular cartilage following an experimentally-induced synovitis.
      Interleukin-1
      • Hardy J.
      • Bertone A.L.
      • Weisbrode S.E.
      • Muir W.W.
      • O’Dorisio T.M.
      • Masty J.
      Cell trafficking, mediator release, and articular metabolism in acute inflammation of innervated or denervated isolated equine joints.
      Polyvinylalcohol foam particles
      • Cornelissen B.P.
      • Rijkenhuizen A.B.
      • van den Hoogen B.M.
      • Rutten V.P.
      • Barneveld A.
      Experimental model of synovitis/capsulitis in the equine metacarpophalangeal joint.
      Carrageenan
      • Owens J.G.
      • Kamerling S.G.
      • Stanton S.R.
      • Keowen M.L.
      • Prescott-Mathews J.S.
      Effects of pretreatment with ketoprofen and phenylbutazone on experimentally induced synovitis in horses.
      InstabilityCarpal fracture
      • Auer J.A.
      • Fackelman G.E.
      • Gingerich D.A.
      • Fetter A.W.
      Effect of hyaluronic acid in naturally occurring and experimental osteoarthritis.
      Cutting collateral & collateral sesamoidean ligaments in metacarpophalangeal joint
      • Simmons E.J.
      • Bertone A.L.
      • Weisbrode S.E.
      Instability-induced osteoarthritis in the metacarpophalangeal joint of horses.
      Osteochondral fragmentation and exerciseCarpal osteochondral fragment-exercise model
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      TraumaSingle impact on medial femoral condyle
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      – progresses to focal defects and OA
      DisuseLower limb cast immobilization
      • Richardson D.W.
      • Clark C.C.
      Effects of short-term cast immobilization on equine articular cartilage.
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Gaughan E.M.
      • Mclaughlin R.M.
      • Debowes R.M.
      Clinical evaluation of the effects of immobilization followed by remobilization and exercise on the metacarpophalangeal joint in horses.
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Turner A.S.
      • Norrdin R.W.
      Effects of immobilization followed by remobilization on mineral density, histomorphometric features, and formation of the bones of the metacarpophalangeal joint in horses.
      The various methods of describing macroscopic changes that have been reported are listed in Table II
      • Fuller C.J.
      • Barr A.R.
      • Sharif M.
      • Dieppe P.A.
      Cross-sectional comparison of synovial fluid biochemical markers in equine osteoarthritis and the correlation of these markers with articular cartilage damage.
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      • Drum M.G.
      • Kawcak C.E.
      • Norrdin R.W.
      • Park R.D.
      • Les C.M.
      • McIlwraith C.W.
      Comparison of gross and histopathologic findings with quatitative computed tomographic bone density in the distal third metacarpal bone of racehorses.
      • Cantley C.E.
      • Firth E.C.
      • Delahunt J.W.
      • Pfeiffer D.U.
      • Thompson K.G.
      Naturally occurring osteoarthritis in the metacarpophalangeal joints of wild horses.
      • Brommer H.
      • van Weeren P.R.
      • Brama P.A.J.
      New approach for quantitative assessment of articular cartilage degeneration in horses with osteoarthritis.
      • Pritzker K.P.H.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.-P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      • Cruz A.M.
      • Hurtig M.B.
      Multiple pathways to osteoarthritis and articular fractures: is subchondral bone the culprit?.
      . Histological grading (microscopic changes) systems for articular cartilage
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      • Thomas C.M.
      • Fuller C.J.
      • Whittles C.E.
      • Shari F.M.
      Chondrocyte death by apoptosis is associated with cartilage matrix degradation.
      have used modifications of the Mankin grading system
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      • Moskowitz R.W.
      Osteoarthritis cartilage histopathology: grading and staging.
      and the Osteoarthritis Research Society International (OARSI) scoring system
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      (Table II). It has also been pointed out that validation studies are necessary when developing alternative methods for scoring
      • Custers J.H.
      • Creemers L.B.
      • Ferbout H.A.
      • van Rijen M.H.P.
      • Dhert W.J.A.
      • Saris D.B.F.
      Reliability, reproducibility and variability of the traditional histologic/histochemical grading system vs the new OARSI osteoarthritis cartilage histopathology assessment system.
      .
      Table IIMacroscopic scoring and microscopic grading systems for horse described in the literature
      Macroscopic scoring
      JointDescription
      Middle carpal joint (CSU system)Four grades of damage based on all articular surfaces lesions as well as assessment of synovial membrane for inflammation and hypertrophy which are used to define a total joint pathology score
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      Metacarpophalangeal jointFive grades distal metacarpus based on arthroscopic system in six locations
      • Fuller C.J.
      • Barr A.R.
      • Sharif M.
      • Dieppe P.A.
      Cross-sectional comparison of synovial fluid biochemical markers in equine osteoarthritis and the correlation of these markers with articular cartilage damage.
      Metacarpophalangeal jointThree grades of wear lines, erosion & palmar arthrosis on distal metacarpus
      • Drum M.G.
      • Kawcak C.E.
      • Norrdin R.W.
      • Park R.D.
      • Les C.M.
      • McIlwraith C.W.
      Comparison of gross and histopathologic findings with quatitative computed tomographic bone density in the distal third metacarpal bone of racehorses.
      Metacarpophalangeal jointMacroscopic scoring of proximal first phalanx and using India ink
      • Cantley C.E.
      • Firth E.C.
      • Delahunt J.W.
      • Pfeiffer D.U.
      • Thompson K.G.
      Naturally occurring osteoarthritis in the metacarpophalangeal joints of wild horses.
      • Brommer H.
      • van Weeren P.R.
      • Brama P.A.J.
      New approach for quantitative assessment of articular cartilage degeneration in horses with osteoarthritis.
      Metacarpophalangeal jointUses adaptation of OARSI scoring system
      • Pritzker K.P.H.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.-P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      product of lesion grade, lesion stage and relevance of based on weight bearing/non-weight bearing location (modified by citing score % or area in mm2 distal metacarpus)
      • Pritzker K.P.H.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.-P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      • Cruz A.M.
      • Hurtig M.B.
      Multiple pathways to osteoarthritis and articular fractures: is subchondral bone the culprit?.
      Medial femorotibial jointUses combination of ICRS systems and modification of WORMS
      • Peterfy C.G.
      • Guermazi A.
      • Zaim S.
      • Tirman P.F.
      • Miaux Y.
      • White D.
      • et al.
      Whole-Organ Magnetic Resonance Imaging Score (WORMS) of the knee in osteoarthritis.
      system on medial femoral condyle
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      (also India ink and tracing to map lesions)
      Grading of microscopic changes
      TissueParameters scored
      Synovial membraneFive grades ranging from (normal–severe) for each of the following changes: cellular infiltration, vascularity, intimal hyperplasia, subintimal edema and subintimal fibrosis
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      A cumulative score is also calculated
      Articular cartilageCSU system: Chondrocyte necrosis, chondrone formation, fibrillation, focal cell loss and Safranin O–Fast Green staining and each graded 0–4
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.


      Bristol system: Modified HHGS Structure (Grades 0–4), cells (0–3) and staining with Haematoxylin and Safranin O (0–4)
      • Thomas C.M.
      • Fuller C.J.
      • Whittles C.E.
      • Shari F.M.
      Chondrocyte death by apoptosis is associated with cartilage matrix degradation.


      Guelph system: Modified OARSI system with six grades (surface intact, surface discontinuity, vertical clefts, erosion, denudation and deformation with histologic subgrades)
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      Bone (osteochondral sections)Osteochondral lesions (graded 0–4), subchondral bone remodeling (0–3) and osteochondral splitting (0–3)

      Anatomy and joint pathology

      The metacarpophalangeal (MCP) joint is the most common site for spontaneous OA followed by the carpal joints. The most published equine model is the osteochondral chip fragment-exercise model in the middle carpal joint. A major difference in these joints compared to the human knee is that the articular cartilage is only 1 mm thick compared to human knee articular cartilage being 2–3 mm. Other aspects pertinent to the horse are that the metacarpophalangeal and carpal joints have close fitting articular surfaces that can quickly develop linear erosions and wear lines in association with osteochondral fragmentation. Some of these are referred to as “kissing” lesions and are directly mechanical whereas others are presumably associated with articular debris being trapped. In addition, when osteochondral defects are created adjacent to the synovial membrane in the carpus and metacarpophalangeal joint, synovial adhesions occur and can result in replacement of the superficial cartilage surface with layers of connective tissue, obscuring the lamina splendens and interfering with cartilage nutrition and homeostasis. Subchondral bone sclerosis and subsequent remodeling creating focal osteonecrosis are also common in this species as OA progresses. The most recently described post-traumatic OA model
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      uses the medial femorotibial joint where the articular cartilage thickness is similar to the human knee
      • Frisbie D.D.
      • Cross M.W.
      • McIlwraith C.W.
      A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee.
      .

      Scoring of alterations in joint structures

      Macroscopic scoring

      Macroscopic scoring systems are described here for both spontaneous OA in the MCP joint OA as well as the experimental carpal joint OA model. These models have been the most frequently reported and, therefore, scoring recommendations are presented for these two entities. Macroscopic examination is usually done without any ink staining of the surface though its use has been reported in spontaneous OA
      • Cruz A.M.
      • Hurtig M.B.
      Multiple pathways to osteoarthritis and articular fractures: is subchondral bone the culprit?.
      as well as a stifle model
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      . The macroscopic scoring technique recommended for spontaneous OA is depicted in Table III and illustrated in Fig. 1.
      Table IIIMacroscopic staging technique to describe gross changes on the third metacarpal condyle of horses
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Turner A.S.
      • Norrdin R.W.
      Effects of immobilization followed by remobilization on mineral density, histomorphometric features, and formation of the bones of the metacarpophalangeal joint in horses.
      Overall macroscopic change
      LesionGradeDescription
      Wear lines0None
      11 or 2 partial-thickness wear lines/joint surface
      23–5 partial-thickness or 1–2 full-thickness wear lines/joint surface
      3>5 partial-thickness or >2 full-thickness wear lines/joint surface
      Erosions0None
      1Partial-thickness erosion, <5 mm in diameter
      2Partial-thickness erosion, >5 mm in diameter
      3Full-thickness erosion
      Palmar arthrosis (osteochondral lesions distal palmar aspect of metacarpus
      • Norrdin R.W.
      • Kawcak C.E.
      • Capwell B.A.
      • McIlwraith C.W.
      Subchondral bone failure in an equine model of overload arthrosis.
      ) [Fig. 1(C)]
      0None
      1Partial-thickness erosion, <5 mm in diameter
      2Partial-thickness erosion, purple discoloration, >5 mm in diameter
      3Full-thickness erosion, purple discoloration, >5 mm in diameter
      Figure thumbnail gr1
      Fig. 1Gross images of the distal third metacarpus showing the spectrum of gross changes with spontaneous OA of the metacarpophalangeal joint. (A) Demonstration of the spectrum of wear line damage in the third metacarpal condyle. Images for Scores 1 and 2 show partial-thickness wear lines (arrows) and the Score 3 image shows full-thickness wear lines (arrows). Note in Score 3 there are multiple partial-thickness wear lines in addition to the full-thickness wear lines. (B) Arrows demonstrate the progression and severity of articular cartilage erosion on third metacarpal condyles. (C) Arrows demonstrate the severity of palmar arthrosis on the third metacarpal condyle.
      The macroscopic scoring system uses the descriptive terms partial- and/or full-thickness erosion, wear lines and palmar arthrosis. Wear lines have been documented to be vertical clefts within the articular cartilage that can be present even in mildly osteoarthritic joints
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      . They have recently been shown to reduce the mechanical integrity of the articular cartilage surface
      • Bae W.C.
      • Wong V.W.
      • Hwang J.
      • Antonacci J.M.
      • Nugent-Derfus G.E.
      • Blewis M.E.
      • et al.
      Wear-lines and split-lines of human patellar cartilage: relation to tensile biomechanical properties.
      , thus leading many to the conclusion that they are due to a weakening of the collagen matrix complex, ultimately resulting in randomly-oriented articular cartilage fibrillation. Wear lines have also been correlated with reduced prognosis in cases of osteochondral fragmentation in racehorses
      • Kawcak C.E.
      • McIlwraith C.W.
      Proximal dorsal first phalanx osteochondral chip fragmentation in 336 horses.
      . The wear lines were scored, and horses with increased scores had a significantly reduced chance of returning to performance at their previous level. It is therefore presumed that the presence of wear lines indicates early pathologic change that is worth noting and grading in gross assessment of joint surfaces.
      Palmar arthrosis lesions occur on the distal palmar aspect of the third metacarpal condyle at the site of maximal articulation with the proximal sesamoid bones, and represent a spectrum of osteochondral damage
      • Norrdin R.W.
      • Kawcak C.E.
      • Capwell B.A.
      • McIlwraith C.W.
      Subchondral bone failure in an equine model of overload arthrosis.
      . Change in the subchondral bone often precedes macroscopic changes in the cartilage. This area is distinct in its rapid progression of damage compared to other sites within the joint, and represents a consistent spectrum of change in molecular, cellular, matrical and tissue-level properties
      • Kawcak C.E.
      • McIlwraith C.W.
      • Norrdin R.W.
      • Park R.D.
      • James S.P.
      The role of subchondral bone in joint disease: a review.
      • Drum M.G.
      • Kawcak C.E.
      • Norrdin R.W.
      • Park R.D.
      • Les C.M.
      • McIlwraith C.W.
      Comparison of gross and histopathologic findings with quatitative computed tomographic bone density in the distal third metacarpal bone of racehorses.
      • Norrdin R.W.
      • Kawcak C.E.
      • Capwell B.A.
      • McIlwraith C.W.
      Subchondral bone failure in an equine model of overload arthrosis.
      • Dykgraaf S.
      • Firth E.C.
      • Rogers C.W.
      • Kawcak C.E.
      Effects of exercise on chondrocyte viability and subchondral bone sclerosis in the distal third metacarpal and metatarsal bones of young horses.
      • Easton K.L.
      • Kawcak C.E.
      Evaluation of increased subchondral bone density in areas of contact in the metacarpophalangeal joint during joint loading in horses.
      • Kawcak C.E.
      • McIlwraith C.W.
      • Norrdin R.W.
      • Park R.D.
      • Steyn P.S.
      Clinical effects of exercise on subchondral bone of carpal and metacarpophalangeal joints in horses.
      • Norrdin R.W.
      • Kawcak C.E.
      • Capwell B.A.
      • McIlwraith C.W.
      Calcified cartilage morphometry and its relation to subchondral bone remodeling in equine arthrosis.
      • Nugent G.E.
      • Law A.W.
      • Wong E.G.
      • Temple M.M.
      • Bae W.C.
      • Chen A.C.
      • et al.
      Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal condyle.
      • Smith K.J.
      • Bertone A.L.
      • Weisbrode S.E.
      • Radmacher M.
      Gross, histologic, and gene expression characteristics of osteoarthritic articular cartilage of the metacarpal condyle of horses.
      . Since palmar arthrosis lesions are the earliest events to occur in MCP joints undergoing athletic work, their gross characterization is necessary in any study. Although the scoring of these lesions is similar to gross scoring of articular cartilage erosion in other locations in the joint, these lesions are scored separately since they represent a specific pathologic process within the joint. It is also critical that scoring is done in a consistent location.
      A different scoring system for the induced osteochondral chip fragment-exercise OA model is used (Table IV and Fig. 2)
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      . Both middle carpal joints are aseptically prepared and opened. The articular surface is examined for location of lesions as well as lesion character. Pitting, ulcerations and discoloration of the articular cartilage of each carpal bone are scored grossly on a subjective ordinal scale of 0–4 (where 0 is normal and 4 is severe change) for partial- and full-thickness cartilage erosions. A total erosion score is also assigned using the same subjective ordinal scale; this score accounts for the overall gross degeneration of the entire articular surface. The condition of the synovium is evaluated for hypertrophy and inflammation again using a 0–4 scale where 0 represents normal and 4 represents severe pathologic change. Details are recorded and photographs taken.
      Table IVMacroscopic staging system to describe gross changes in the induced osteochondral chip fragment-exercise OA model
      Macroscopic scoring of total erosion (Fig. 2)
      ScoreDescription
      0No gross fibrillation/fissuring
      1Very superficial erosion with articular cartilage swelling
      2Partial-thickness erosion
      3Partial- and full-thickness erosion
      4Extensive full-thickness erosion to the level of subchondral bone
      Figure thumbnail gr2
      Fig. 2Examples of erosion scores based on gross observation from the osteochondral fragment model. Score 0, shows no gross fibrillation; Score 1, shows very superficial erosion with articular cartilage swelling indicated by arrow; Score 2, shows gross partial-thickness erosions depicted by an arrow; Score 3, shows gross full-thickness erosion depicted by an arrow; Score 4 shows both gross full-thickness erosion and cartilage fibrillation lines depicted by arrows.
      Articular cartilage pieces, 5 mm2, are obtained from the radial carpal bone and the radial facet of the third carpal bone as well as osteochondral sections obtained from the radial carpal and third bone for histological processing as described
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      . A synovial membrane specimen that is approximately 3×4 mm is harvested from the middle carpal joint dorsal to the radial carpal bone, with efforts not to include the fibrous joint capsule. This scoring system is illustrated in Fig. 2 and described in Table II.
      A technique utilizing India ink to detect spontaneous osteoarthritic cartilage changes has been described
      • Cruz A.M.
      • Hurtig M.B.
      Multiple pathways to osteoarthritis and articular fractures: is subchondral bone the culprit?.
      and could be used for macroscopic evaluation; however it has not been used in the models presented here. In the stifle joint India ink staining has been used
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      to delineate subtle surface fissures, matrix depletion and thinning as described by others
      • Peterfy C.G.
      • Guermazi A.
      • Zaim S.
      • Tirman P.F.
      • Miaux Y.
      • White D.
      • et al.
      Whole-Organ Magnetic Resonance Imaging Score (WORMS) of the knee in osteoarthritis.
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      . India ink particles are prevented from entering in intact cartilage surface with an unaffected proteoglycan-rich matrix but have a high affinity for articular cartilage with surface fibrillation and proteoglycan depletion
      • Peterfy C.G.
      • Guermazi A.
      • Zaim S.
      • Tirman P.F.
      • Miaux Y.
      • White D.
      • et al.
      Whole-Organ Magnetic Resonance Imaging Score (WORMS) of the knee in osteoarthritis.
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      . The technique used by Cantley et al. was semi-quantitative. A quantitative technique for evaluating OA on the basis of India ink staining and digital imaging techniques has been described in the equine metacarpophalangeal joint but is applicable to the proximal aspect of the first phalanx only
      • Brommer H.
      • van Weeren P.R.
      • Brama P.A.J.
      New approach for quantitative assessment of articular cartilage degeneration in horses with osteoarthritis.
      . Tracing India ink stained area onto plastic applied to the medial femoral condyle was used by Bolam et al.
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      to overcome the problems associated with area measurements digitally of a curved surface and provides staging of progression. A similar technique could be used in the equine carpus and is currently being explored.
      • We recommend different macroscopic scoring systems for spontaneous OA in the MCP joint as well as experimental carpal joint OA; these are presented in Table II, Table III. Future consideration of tracing India ink stained areas onto plastic as described in the medial femoral condyle
        • Bolam C.J.
        • Hurtig M.B.
        • Cruz A.
        • McEwen B.J.
        Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
        is recommended.

      Microscopic scoring of cartilage alterations

      Recommendation of sectioning and staining to be used

      Cartilage sections in the CSU model are harvested from three locations (radial facet of the third carpal bone, fourth carpal bone and distal radial carpal bone) and are fixed in 10% phosphate buffered formalin, embedded in paraffin, cut into 5 μm sections and mounted on coated glass slides. Duplicate sections are stained with haematoxylin and eosin (H&E) or stained with haematoxylin and 0.1% aqueous safranin O for approximately 6 min in counter stain with 0.1% aqueous fast green. Staining procedures must be very consistent between slides especially with the safranin O–fast green (SOFG) stain and normal equine cartilage and trachea are used as a control between batches. Alternatively, frozen sections can be used and are stained with a haematoxylin/safranin O combination at Bristol
      • Desjardins M.R.
      • Hurtig M.B.
      Diagnosis of equine stifle joint disorders: three cases.
      . Osteochondral sections are first decalcified in 10% formic acid (Stevens Scientific Decalcifying Solution). That can be monitored by the oxalate endpoint determination method

      Kang QK, LaBreck JC, Gruber HE, Nan UH. Histological techniques for decalcified bone and cartilage. In: Handbook of Histology Methods for Bone and Cartilage. Yhan and Martin KL, Eds. Totowa, New Jersey: Humana Press.

      . Following decalcification samples are imbedded in paraffin and cut into 5 μm sections and mounted on coated glass slides. H&E staining and safranin O are the same as detailed above.
      • For simple histological scoring of osteochondral sections we recommend the use of formic acid decalcification, embedding in paraffin, and cutting into 5 μm sections with H&E as well as safranin O staining of different sections. Frozen sections of cartilage are an alternative.

      Grading criteria

      The histological and histochemical grading system used in the equine osteochondral fragment model is as presented in Table V and illustrated in Fig. 3.
      Table VMicroscopic grading system for articular cartilage histology
      Articular cartilage (Fig. 3)
      Outcome parameterScoreDescription
      Chondrocyte necrosis
      Chondrocyte necrosis is used to grade presence of lacunae with necrotic nuclei still present compared to focal cell loss which is most likely an extension of the pathologic change but lacunae or nuclei are no longer present.
      0Normal section without necrosis
      1No more than one necrotic cell located near the articular surface per 20× objective
      21–2 necrotic cells located near the articular surface per 20× objective
      32–3 necrotic cells located near the articular surface per 20× objective
      43–4 necrotic cells located near the articular surface per 20× objective
      Cluster (complex chondrone) formation0No cluster formation throughout section
      1Two chondrocytes (doublets) within same lacunae along superficial aspect of the articular cartilage section
      22–3 chondrocytes (doublets & triplets) within same lacunae along superficial aspect of the articular cartilage section
      33–4 chondrocytes within same lacunae along superficial aspect of the articular cartilage section
      4Greater than four chondrocytes within same lacunae along superficial aspect of the articular cartilage section
      Fibrillation/fissuring0No fibrillation/fissuring of the articular cartilage surface
      1Fibrillation/fissuring of the articular cartilage restricted to surface and superficial zone
      2Fissuring that extends into the middle zone
      3Fissuring that extends to the level of the deep zone
      4Fissuring that extends into the deep zone
      Focal cell loss
      Chondrocyte necrosis is used to grade presence of lacunae with necrotic nuclei still present compared to focal cell loss which is most likely an extension of the pathologic change but lacunae or nuclei are no longer present.
      0Normal cell population throughout the section
      1A 10–20% area of acellularity per 20× field
      2A 20–30% area of acellularity per 20× field
      3A 40–50% area of acellularity per 20× field
      4A greater than 50% area of acellularity per 20× field
      SOFG stain uptake0Normal staining
      1Less than 25% loss of staining characteristics
      225–50% loss of staining characteristics
      350–75% loss of staining characteristics
      4Greater than 75% loss of staining characteristics
      Chondrocyte necrosis is used to grade presence of lacunae with necrotic nuclei still present compared to focal cell loss which is most likely an extension of the pathologic change but lacunae or nuclei are no longer present.
      Figure thumbnail gr3a
      Fig. 3Examples of H&E and SOFG stained articular cartilage sections that are 5 mm in length for each of the graded outcome parameters used in the carpal osteochondral fragment model. The entire section is graded and the final score represents the entire section taking into account local areas of more severe pathology i.e., change. (A) Chondrocyte necrosis and chondrone formation. Chondrocyte necrosis (40×): Score 0 represents a normal section without chondrocyte necrosis, arrow shows a normal area of eosin uptake (red/pink stained area) that is common close to the surface and should not be confused with a necrotic chondrocyte. Scores 1–4, arrows represent necrotic cells which are typically located near the articular surface. A Score 1 would typically represent 1–2 necrotic cells in the section typically in the superficial layer, where a Score 2 would represent 2–3 necrotic cells typically in the superficial layer, and Scores 3 and 4 would represent increased numbers of necrotic cells that are not only present at the surface but in deeper layers as well. Cluster (complex chondrone) formation (20×): Scores 1 & 2 have arrows representing cluster formation (doublets) more superficial than normal. Scores 3 and 4 have increased numbers of chondrocytes within a single lacuna that extends deep in the cartilage section. (B) Fibrillation, focal cell loss and SOFG stain uptake. Fissuring (20×): Score 1 has fissuring/fibrillation restricted to surface and superficial zone highlighted with an arrow; Score 2 has fissuring that extends into the middle zone; Score 3 has fissuring that extends to the level of the deep zone and Score 4 has fissuring that extends into the deep zone. Focal cell loss (20×): Scores 1–3 have arrows depicting increasing areas that are devoid of chondrocytes to a point where few cells remain as shown in the Score 4 example. SOFG stain uptake (20×): Score 0, has an example of normal loss of staining in the superficial zone depicted by an arrow; Scores 1–3 have arrows depicting areas of abnormal staining. Score 4 has virtually complete loss of staining in all zones.
      Figure thumbnail gr3b
      Fig. 3Examples of H&E and SOFG stained articular cartilage sections that are 5 mm in length for each of the graded outcome parameters used in the carpal osteochondral fragment model. The entire section is graded and the final score represents the entire section taking into account local areas of more severe pathology i.e., change. (A) Chondrocyte necrosis and chondrone formation. Chondrocyte necrosis (40×): Score 0 represents a normal section without chondrocyte necrosis, arrow shows a normal area of eosin uptake (red/pink stained area) that is common close to the surface and should not be confused with a necrotic chondrocyte. Scores 1–4, arrows represent necrotic cells which are typically located near the articular surface. A Score 1 would typically represent 1–2 necrotic cells in the section typically in the superficial layer, where a Score 2 would represent 2–3 necrotic cells typically in the superficial layer, and Scores 3 and 4 would represent increased numbers of necrotic cells that are not only present at the surface but in deeper layers as well. Cluster (complex chondrone) formation (20×): Scores 1 & 2 have arrows representing cluster formation (doublets) more superficial than normal. Scores 3 and 4 have increased numbers of chondrocytes within a single lacuna that extends deep in the cartilage section. (B) Fibrillation, focal cell loss and SOFG stain uptake. Fissuring (20×): Score 1 has fissuring/fibrillation restricted to surface and superficial zone highlighted with an arrow; Score 2 has fissuring that extends into the middle zone; Score 3 has fissuring that extends to the level of the deep zone and Score 4 has fissuring that extends into the deep zone. Focal cell loss (20×): Scores 1–3 have arrows depicting increasing areas that are devoid of chondrocytes to a point where few cells remain as shown in the Score 4 example. SOFG stain uptake (20×): Score 0, has an example of normal loss of staining in the superficial zone depicted by an arrow; Scores 1–3 have arrows depicting areas of abnormal staining. Score 4 has virtually complete loss of staining in all zones.
      The total histologic grade will range from 0 to 16 and SOFG staining can be scored 0–4 for each layer or 0–4 overall (Total score of 20).
      The above system is relevant for the osteochondral fragment model, but when knee injury and OA are modeled in the horse, the system described by Pritzker et al.
      • Pritzker K.P.H.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.-P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      that uses a product of extent (area affected) and Grade (depth of the lesions) has been proposed as an alternative in an equine femorotibial joint model
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      . This system has also been recently validated for the equine stifle joint (M. Hurtig personal communication). For the sake of brevity, only the osteochondral fragment model has been described here.
      • The histological and histochemical grading system depicted in Table V is recommended for use in the equine osteochondral fragment model. Future consideration should be given to the system Pritzker et al.
        • Pritzker K.P.H.
        • Gay S.
        • Jimenez S.A.
        • Ostergaard K.
        • Pelletier J.-P.
        • Revell P.A.
        • et al.
        Osteoarthritis cartilage histopathology: grading and staging.
        as an alternative (recently used in the equine femorotibial joint model).

      Microscopic scoring of synovial alterations (grading of synoviopathy)

      Recommendation of staining and sectioning

      A 3–4 mm of synovial membrane has been harvested at gross examination from a villous area. The harvested tissue is fixed in 10% formalin for 48 h and then embedded in paraffin prior to 5 μm sectioning, mounting and staining with H&E.

      Grading criteria

      These sections are evaluated and graded blindly for cellular infiltration, vascularity, intimal hyperplasia, subintimal edema and subintimal fibrosis
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      (Table VI). These changes are illustrated in Fig. 4.
      • For evaluation of synovial membrane changes we recommend 5 μm thick paraffin embedded sections stained with H&E and graded blindly for cellular infiltration, vascularity, intimal hyperplasia, subintimal edema and subintimal fibrosis as detailed in Table VI.
      Table VIMicroscopic grading system for synovial membrane histology in the carpal osteochondral fragment model
      Synovial membrane (Fig. 4)
      Outcome parameterGradeDescription
      Cellular infiltration (lymphocytes and some plasma cells)0No mononuclear cells in the section
      1Occasional small areas of mononuclear cells throughout the section
      2Mild presence of mononuclear cells in 25% of the section
      3Moderate presence of mononuclear cells in 25–50% of the section
      4Marked presence of mononuclear cells in greater than 50% of the section
      Vascularity0Normal
      1Slight increase in vessels in focal locations throughout the section
      2Mild increase in number and dilatation of vessels in focal locations throughout the section
      3Moderate increase in number and dilatation of vessels in up to 50% of the section
      4Marked increase in number and dilatation of vessels in greater than 50% of the section
      Intimal hyperplasia0None
      1Villi with 2–4 rows of intimal cells within the section
      2Villi with 4–5 rows of intimal cells over 25–50% of the section
      3Villi with 4–5 rows of intimal cells over 50% of the section
      4Villi with 5 or greater rows of intimal cells over 50% of the section
      Subintimal edema0No edema
      1Slight edema detected within section
      2Mild edema within 25% of the section
      3Moderate edema within 25–50% of the section
      4Marked edema in greater than 50% of the section
      Subintimal fibrosis0Normal
      1Slight increase in fibrosis within the section
      2Mild increase in fibrosis in 25% of the section
      3Moderate increase in fibrosis in 25–50% of the section
      4Marked increase in fibrosis in greater than 50% of the section
      Figure thumbnail gr4a
      Fig. 4Examples of H&E stained sections representing a similar surface area of synovial membrane for each scored outcome parameter used in the carpal osteochondral fragment model. (A) Cellular infiltration and vascularity. Cellular infiltration (10×): Score 0, no inflammatory cells are observed within the section; Score 1, slight inflammatory infiltrates are detected and consist of mononuclear and segmented cells. An example of such an area is depicted by the arrow; Score 2, a mild area of mixed inflammatory infiltrates is highlighted by the arrow. Higher scores represent greater concentration of infiltrates over a larger surface area. Vascularity (10×): Score 1 has examples of increased vessels depicted using arrows; Score 2 and higher also have increases in vessel numbers as well as dilatation of some vessels depicted by arrows. Higher scores represent greater involvement throughout the section. (B) Intimal hyperplasia, subintimal edema and subintimal fibrosis. Intimal hyperplasia (10×): Score 0 represents an example of no intimal hyperplasia depicted by white arrow, Score 1 shows villi that have areas ≈4 rows of intimal cells and 2 rows depicted by arrows; Scores 3 & 4 have increasing amount of intimal cells. Subintimal edema (10×): Scores 1–4 have examples of edema often recognized by the ‘gun blue’ appearance (arrows) of the tissue as well as the lack of subintimal organization which is especially prominent in the Score 4 example. Subintimal fibrosis (10×): Scores 1 & 2 have arrows depicting areas of increased fibrosis which often begins around the vasculature; the higher Grades have evident fibrosis over a greater region of the section.
      Figure thumbnail gr4b
      Fig. 4Examples of H&E stained sections representing a similar surface area of synovial membrane for each scored outcome parameter used in the carpal osteochondral fragment model. (A) Cellular infiltration and vascularity. Cellular infiltration (10×): Score 0, no inflammatory cells are observed within the section; Score 1, slight inflammatory infiltrates are detected and consist of mononuclear and segmented cells. An example of such an area is depicted by the arrow; Score 2, a mild area of mixed inflammatory infiltrates is highlighted by the arrow. Higher scores represent greater concentration of infiltrates over a larger surface area. Vascularity (10×): Score 1 has examples of increased vessels depicted using arrows; Score 2 and higher also have increases in vessel numbers as well as dilatation of some vessels depicted by arrows. Higher scores represent greater involvement throughout the section. (B) Intimal hyperplasia, subintimal edema and subintimal fibrosis. Intimal hyperplasia (10×): Score 0 represents an example of no intimal hyperplasia depicted by white arrow, Score 1 shows villi that have areas ≈4 rows of intimal cells and 2 rows depicted by arrows; Scores 3 & 4 have increasing amount of intimal cells. Subintimal edema (10×): Scores 1–4 have examples of edema often recognized by the ‘gun blue’ appearance (arrows) of the tissue as well as the lack of subintimal organization which is especially prominent in the Score 4 example. Subintimal fibrosis (10×): Scores 1 & 2 have arrows depicting areas of increased fibrosis which often begins around the vasculature; the higher Grades have evident fibrosis over a greater region of the section.

      Microscopic scoring of bone alterations

      Recommendation of sectioning and staining

      Osteochondral sections are decalcified and embedded in paraffin and cut into 5 μm sections and mounted on coated glass slides. Sections are stained with H&E.

      Grading criteria

      Description of microscopic features in osteochondral sections has been limited to studies with spontaneous OA in the equine metacarpophalangeal joints
      • van Harreveld P.D.
      • Lillich J.D.
      • Kawcak C.E.
      • Gaughan E.M.
      • Mclaughlin R.M.
      • Debowes R.M.
      Clinical evaluation of the effects of immobilization followed by remobilization and exercise on the metacarpophalangeal joint in horses.
      • Moskowitz R.W.
      Osteoarthritis cartilage histopathology: grading and staging.
      . Microscopic grading of osteochondral lesions is depicted in Table VII and illustrated in Fig. 5.
      • A proposal for microscopic grading of osteochondral lesions in spontaneous OA of the metacarpophalangeal joint is presented and input for modification relevant to other joints is anticipated.
      Table VIIProposal for microscopic grading system for osteochondral lesions in spontaneous OA of the metacarpophalangeal joint
      GradeDescription
      Osteochondral lesions [Fig. 5(A)]
      0No visible changes in cartilage or bone
      1Minor disruption of subchondral bone matrix. Lesion occupies <25% of histologic condylar surface, and extends no more than 1–2 mm deep to the normal chondrosseous junction. Some of the matrix within the lesion is pale staining and occasional marrow spaces contain debris. Tidemark is reduplicated. Fibrin may be present in the subchondral bone layer. No apparent superficial cartilage fibrillation
      2More severe disruption of subchondral bone matrix. Areas of matrix are fragmented to comminuted. Lesion occupies 25–50% of histologic condylar surface and extends approximately 2–4 mm deep to the normal osteochondral junction. A significant amount of the subchondral bone matrix is pale staining, and there is significant debris within marrow spaces. The tidemark is reduplicated and often disrupted. Moderate amounts of fibrin present in the subchondral bone layer. Cartilage overlying lesion is thickened with superficial cartilage erosion and fibrillation. Reparative fibrocartilage is also apparent in the superficial cartilage layers
      3Complete collapse of osteochondral tissue. Lesion occupies >50% of histologic condylar surface and extends 43 mm deep to the normal osteochondral junction. Pale staining subchondral bone matrix and fibrin are abundant. The tidemark is reduplicated and often disrupted. Superficial thickening of cartilage and/or fibrocartilage present and may be completely detached from thickened deeper cartilage
      4Obvious loss of osteochondral tissue leaving an ulcer (not observed)
      Subchondral bone remodeling [Fig. 5(B)]
      0No visible changes in cartilage or bone
      1Advancement of the subchondral bone into the calcified cartilage layer with scalloped subchondral bone margins (arrowheads), but not crossing any tidemarks
      2Subchondral bone advancement through the calcified cartilage layer, crossing one or more tidemarks, but below the most superficial tidemark front
      3Subchondral bone advancement through the calcified cartilage layer and disruption of the tidemark front
      Osteochondral splitting [Fig. 5(C)]
      0No visible changes in cartilage or bone
      1Involve the tidemark and subchondral bone, but are simple linear defects
      2Have fragments and debris within the split and connections between splits
      3Involve articular cartilage and displacement of calcified cartilage fragments into the underlying subchondral bone
      Figure thumbnail gr5
      Fig. 5Illustration of histopathologic grading of osteochondral lesions in spontaneous OA in equine metacarpophalangeal joints. Microscopic images showing the spectrum of damage in the osteochondral area of the third metacarpal condyle. (A) Various osteochondral lesions that rank in severity from mild disruption in the subchondral bone (arrow in Grade 1) all the way up to subchondral bone collapse and complete articular cartilage erosion with fibrosis in the subchondral bone (Grade 4). (B) Microscopic images showing advancement of subchondral bone remodeling through the calcified cartilage (star). (C) The progression of osteochondral splitting which starts in the calcified cartilage and is demonstrated by the arrows.

      Evaluation of sources of variability

      Intra- and inter-observer validation study

      Validation of synovial membrane and articular cartilage scoring systems (H&E & SOFG) for the CSU osteochondral fragment model was carried out during the first 4 years of model development through the agreement of one board certified pathologist specializing in musculoskeletal tissue and three board certified equine surgeons specializing in orthopaedics. Each of four observers would score a section and then the group would discuss the score and agree on an overall score for the particular section.
      More recently representative sections of synovium and articular cartilage for each level of all outcome parameters were randomly and blindly presented to each of the authors for grading. These results were then analyzed using PROC GENMOD in SAS (www.support.sas.com) to perform linear regression for repeated measures. The analyses investigated differences by tissue (synovium or cartilage), specific outcome variables, observers and their interactions. The results indicated no significant differences with respect to tissue or outcome variables although one of six observers provided scores that were systematically different from the median (for all observers) or the lead observer. Thus differences were not detected between five and six reviewers when individuals were compared to mean values or those of the lead evaluator. The data was also analyzed to calculate an intraclass correlation (ICC) for all the authors. This method of agreement indicated 0.82 [95% confidence interval (CI)=0.77–0.86] ICC value with a value of 1 indicating perfect homogeneity and −1 indicating extreme heterogeneity. When a group (N=4) of untrained individuals were asked to evaluate the slides based on the outlined grading scheme there level of agreement was 0.92 (95% CI=0.84–0.96) when their individual scores were compared to their mean scores. It is clear that independent agreement can be obtained from multiple evaluators using the above described grading criteria even if they have not been tutored.

      Discussion

      It is now accepted that the joint is an organ and, consequently, OA involves all it’s tissues including articular cartilage, subchondral bone, synovium, fibrous capsule and joint fluid and, in the case of the knee, menisci. The task of this paper was to make recommendations on macroscopic scoring and microscopic grading in equine OA. The range of joints and models used by the authors collectively was wide and consequently we initially had a wide range of scoring (staging) and grading systems. The evaluation systems presented here have been necessarily restricted. We chose to present macroscopic evaluation of spontaneous OA in the metacarpophalangeal joint
      • Norrdin R.W.
      • Kawcak C.E.
      • Capwell B.A.
      • McIlwraith C.W.
      Subchondral bone failure in an equine model of overload arthrosis.
      and in the experimental OA model
      • Foland J.W.
      • McIlwraith C.W.
      • Trotter G.W.
      • Powers BE
      • Lamar C.H.
      Effect of betamethasone and exercise on equine carpal joints with osteochondral fragments.
      • Kawcak C.E.
      • Frisbie D.D.
      • Trotter G.W.
      • McIlwraith C.W.
      • Gillette S.M.
      • Powers BE
      • et al.
      Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation.
      • Frisbie D.D.
      • Kawcak C.E.
      • Trotter G.W.
      • Powers BE
      • Walton R.M.
      • McIlwraith C.W.
      Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model.
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      • Frisbie D.D.
      • Kawcak C.E.
      • Baxter G.M.
      • Trotter G.W.
      • Powers BE
      • Lassen E.D.
      • et al.
      Effects of 6alpha-methylprednisolone acetate on an equine osteochondral fragment exercise model.
      • Frisbie D.D.
      • Ghivizzani S.C.
      • Robbins P.D.
      • Evans C.H.
      • McIlwraith C.W.
      Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene.
      • Frisbie D.D.
      • Kawcak C.E.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Clinical, biochemical and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis.
      • Kawcak C.E.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Werpy N.M.
      • Park R.D.
      Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis using an equine model.
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      . We had the most collective experience with spontaneous OA in the metacarpophalangeal joint and the carpal osteochondral fragment-exercise model has been the subject of 10 published studies. It is felt that the evaluation systems described here can be extrapolated to other models. With further experience models such as the recently described femorotibial traumatically induced OA model
      • Bolam C.J.
      • Hurtig M.B.
      • Cruz A.
      • McEwen B.J.
      Characterization of experimentally induced post-traumatic osteoarthritis in the medial femorotibial joint of horses.
      could gain increased usage and modifications of the evaluation system used in that model implemented. It is to be recognized that there are no previous publications giving collective recommendations on macroscopic and microscopic assessments in equine OA. Hopefully, this will serve as a basis for further development and validation of more sophisticated systems.
      The assessment systems are also based on the initial case material provided. Osteochondral evaluation of bone as well as articular cartilage is critical with spontaneous OA in the fetlock joint. The system for osteochondral evaluation is therefore presented from spontaneous disease in this joint. On the other hand articular cartilage has been examined in the experimental carpal OA model without calcified cartilage or subchondral bone being included in the sections. In one study the bone changes have been evaluated with this model and details on subchondral bone change in this model are available from this publication
      • Kawcak C.E.
      • Norrdin R.W.
      • Frisbie D.D.
      • McIlwraith C.W.
      • Trotter G.W.
      Effect of osteochondral fragmentation intra-articular triamcinolone acetonide treatment on subchondral bone in the equine carpus.
      .
      As new techniques develop, both imaging and body fluid biomarkers can add to the information in vivo and prior to (or to the exclusion of) post-mortem. With the equine chip fragment model for instance it has been evaluated using radiography, computed tomography (CT) and magnetic resonance imaging (MRI) as well as synovial fluid and serum biomarkers. The results of these studies have been published recently
      • Frisbie D.D.
      • Al-Sobayil F.
      • Billinghurst R.C.
      • Kawcak C.E.
      • McIlwraith C.W.
      Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses.
      • Kawcak C.E.
      • Frisbie D.D.
      • Werpy N.M.
      • Park R.D.
      • McIlwraith C.W.
      Effects of exercise vs experimental osteoarthritis on imaging outcomes.
      .
      In summary the horse is a suitable model for addressing selected questions related to human OA and there is adequate tissue to harvest for multiple outcome measures. This species requires special facilities as well as trained personnel but control of activity level and other interventions are easily done. This review represents a consensus of the authors to create standardization between laboratories and allow for comparisons to be made between studies in the future.

      Disclosures

      C. Wayne McIlwraith is employed by Colorado State University.
      David D. Frisbie is employed by Colorado State University.
      Christopher E. Kawcak is employed by Colorado State University.
      Cathy J. Fuller is employed by the University of Bristol.
      Mark Hurtig is employed by the University of Guelph.
      Antonio Cruz is employed by the University of Guelph.
      Thomas Aigner is employed by the University of Leipzig.

      Conflict of interest

      No author has any conflict of interest related to this work.

      Acknowledgement

      No external sources of funding were provided for this work except that the printing costs were supported by an unrestricted educational grant to OARSI by Bayer, Expanscience, Genzyme, Lilly, MerckSerono, Novartis, Pfizer, SanofiAventis, Servier, and Wyeth. The work performed was not influenced at any stage by the support provided.

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