Sheila Laverty

Investigate meniscal extracellular matrix degradation. Equine menisci (n = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; n = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively). Picrosirius red and second harmonic generation microscopy were performed to investigate collagen ultrastructure. A total of 42 ROIs met the inclusion criteria and were included in the final analysis. The median (range) ROI histological score was 3 (0–9), providing a large spectrum of pathology. The median (range) proteoglycan score was 1 (0–3), representing superficial and central meniscal loss. The median (range) of DIPEN, NITEGE, and C1,2C scores was 1 (0–3), revealing immunostaining of the femoral and tibial surfaces. The proteoglycan scores exhibited significant positive associations with both histologic evaluation (p = 0.03) and DIPEN scores (p = 0.02). Additionally, a robust positive association (p = 0.007) was observed between the two aggrecanolysis indicators, NITEGE and DIPEN scores. A negative association (p = 0.008) was identified between NITEGE and histological scores. The C1,2C scores were not associated with any other scores. Picrosirius red and second harmonic generation microscopy (SHGM) illustrated the loss of the collagen matrix and structure centrally. Proteoglycan and collagen degradation commonly occur superficially in menisci and less frequently centrally. The identification of central meniscal proteoglycan and collagen degradation provides novel insight into central meniscal degeneration. However, further research is needed to elucidate the etiology and sequence of degradative events.

Summary: Background: A screening ultrasonographic examination of yearling Thoroughbred horses has been described which can be performed on farm, as an adjunct to pre‐sale yearling radiography. Objectives: To compare ultrasonographic and radiographic findings in young Thoroughbred horses in the detection of osteochondrosis. Study design: Descriptive and comparative study using ultrasonography and radiography. Methods: Forty‐four horses with a mean age of 10 months underwent an ultrasonographic screening protocol of joint groups including the bilateral stifle, tarsal, carpal and metacarpophalangeal/metatarsophalangeal joints. Concurrent standardised radiographic projections of all the joints were also undertaken. A cohort of 26 of these horses had a second set of radiographs taken for yearling sales approximately 6 months later. Two reviewers reviewed all images for presence or absence of osteochondrosis lesions. Comparison of the ultrasonographic and radiographic findings was undertaken using Gwet's AC1 and the Landis and Kock scale. Results: The principal observer identified 48 ultrasonographic lesions and 15 radiographic lesions on the initial examination. Overall, there was agreement in screening for osteochondrosis using ultrasonographic and radiographic lesions, however, with varying levels of agreement for different joint groups. Agreement for individual joint groups was almost perfect for the carpal (0.98) and metatarsophalangeal joints (0.81), substantial for the stifle joints (0.67), moderate for the metacarpophalangeal joint (0.59) with only fair agreement of the tarsal joints (0.30). Six osteochondral lesions were recorded on the second set of radiographs. There was overall substantial agreement between the ultrasonographic findings and the second set of radiographs. A second observer reviewed the ultrasonographic and radiographic images. There was moderate interobserver agreement comparing ultrasonographic images, and almost perfect interobserver agreement comparing radiographic images. Main limitations: Small number of horses who presented for yearling sale radiography for comparison. One ultrasonographer acquired all images, and two observers reviewed the images. Conclusion: Ultrasonographic screening of yearling horses with a relatively low number of lesions showed agreement with concurrent and yearling sale radiographs.

Background: Stifle osteoarthritis (OA) lesions are most common in the medial femorotibial (MFT) compartment. Their characterisation and mapping will inform equine veterinarians towards an accurate diagnosis of OA.Objectives: Investigate and map micro‐CT (μCT) changes in the hyaline articular cartilage (HAC) in the medial femoral condyle (MFC) and medial tibial plateau (MTP).Study design: Ex vivo cadaveric.Methods: Stifles (n = 7 OA and 17 control [CO]) were retrieved from a tissue bank. The MFC and MFT were imaged with μCT. Regions of interest (ROIs) were cranial (MFCcr; MTPcr) and caudal (MFCca; MTPca) sites. In each ROI, μCT images were scored for HAC fibrillation, surface mineralisation and for the presence of high‐density mineralised protrusions (HDMP). The lesions were mapped, and site‐matched histology was performed.Results: The microstructure of healthy and abnormal HAC was discernible on μCT images and confirmed with histology. HAC fibrillation was more prevalent (p = 0.019) in the MFCcr of the OA group (n = 7/7, 100%) when compared with the CO group (n = 7/17, 41%). Score 1 HAC surface mineralisation was more prevalent (p = 0.038) in the OA MFCca (n = 4/7, 57%) when compared with the CO group (n = 2/17, 12%). HDMP were heterogenous and hyperdense mineralised material protruding into the HAC and were more frequent (p = 0.033) in MFCs (n = 12/24, 50%) compared with MTPs (n = 5/24, 20%). Score 3 HDMPs were also more prevalent (p = 0.003) in the MFCcr (n = 7/24, 29%) compared with MFCca (n = 0/24, 0%) and in MFCs (n = 7/24, 29%) compared with MTPs (n = 3/24, 12.5%) (p = 0.046).Main limitations: Clinical history was not available for all specimens.Conclusions: Equine HDMP and HAC surface mineralisation are imaged for the first time in the MFT joint. HAC fibrillation and erosion and HDMP are more frequent in the cranial aspect of the MFC. μCT images of OA in equine stifle joints provide a novel perspective of lesions and improve understanding of OA.

Background: Osteoarthritis (OA) is a major cause of equine lameness. Cannabinoid (CB) receptors are now considered to be promising therapeutic targets in human rheumatology for pain and inflammation, however, little is known about the equine endocannabinoid system.Objectives: The primary goal was to assess the presence and expression pattern of CB1 and CB2 in the synovium of healthy joints. A secondary goal was to explore the relationship between the CB expression, degree of synovitis and OA pathology.Study design: Ex vivo experimental study.Methods: Metacarpophalangeal joints (n = 25) from a tissue bank were studied. The joints were dissected, and the articular cartilage lesions were scored. Synovial membrane specimens (n = 45) were harvested, fixed and the degree of synovitis was graded on histological sections. Colocalised synovial sections were also immunostained with antibodies to CB1 and CB2. Five regions of interest were randomly selected from digital images of manually segmented synovial intima and scored blindly for positive cellular immunoreactive staining by two independent observers. Interobserver agreement was calculated with an intraclass correlation coefficient (ICC). Relationships between CB1 and CB2 immunoreactive scores and synovitis or joint OA grade were explored with mixed linear models.Results: CB1 was expressed in synovial intimal cells in all specimens studied whereas CB2 expression was identified in 94%. Both receptors were also expressed in the subintimal blood vessel walls. ICCs were 84.6% (CB1) and 92.9% (CB2) for the immunoreactivity scores. Both CB1 and CB2 expression were significantly upregulated (p = 0.04 and p = 0.03, respectively) with increasing degree of synovitis. Conversely, CB1 expression significantly decreased (p = 0.03) with increasing severity of OA.Main limitations: The type of synovial cell expressing CB1 or CB2 was not investigated.Conclusion: Equine synovial intimal cells constitutively express both CB1 and CB2 receptors that are upregulated with synovitis and may have a role in joint pain. They are potential targets for therapy with cannabinoid molecules or their derivatives.

Background: Focal bone microcracks with osteoclast recruitment and bone lysis, may reduce fracture resistance in racehorses. As current imaging does not detect all horses at risk for fracture, the discovery of novel serum biomarkers of bone resorption or osteoclast activity could potentially address this unmet clinical need. The biology of equine osteoclasts on their natural substrate, equine bone, has never been studied in vitro and may permit identification of specific biomarkers of their activity.Objectives: (1) Establish osteoclast cultures on equine bone, (2) Measure biomarkers (tartrate resistant acid phosphatase isoform 5b [TRACP‐5b] and C‐terminal telopeptide of type I collagen [CTX‐I]) in vitro and (3) Study the effects of inflammation.Study design: In vitro experiments.Methods: Haematopoietic stem cells, from five equine sternal bone marrow aspirates, were differentiated into osteoclasts and cultured either alone or on equine bone slices, with or without a pro‐inflammatory stimulus (IL‐1β or LPS). CTX‐I and TRACP‐5b were immunoassayed in the media. Osteoclast numbers and bone resorption area were assessed.Results: TRACP‐5b increased over time in osteoclast cultures without bone (p < 0.0001) and correlated with osteoclast number (r = 0.63, p < 0.001). CTX‐I and TRACP‐5b increased with time for cultures with bone (p = 0.002; p = 0.02 respectively), correlated with each other (r = 0.64, p < 0.002) and correlated with bone resorption (r = 0.85, p < 0.001; r = 0.82, p < 0.001 respectively). Inflammation had no measurable effects.Main limitations: Specimen numbers limited.Conclusions: Equine osteoclasts were successfully cultured on equine bone slices and their bone resorption quantified. TRACP‐5b was shown to be a biomarker of equine osteoclast number and bone resorption for the first time; CTX‐I was also confirmed to be a biomarker of equine bone resorption in vitro. This robust equine specific in vitro assay will help the study of osteoclast biology.

Summary: Background: There is a knowledge gap about how equine MFC subchondral radiolucencies (SR) arise and evolve. Osteoclasts are believed to have a role but have not been studied in situ. Objectives: To measure and compare osteoclast density and the percentage of chondroclasts in healthy and MFC SR specimens from juvenile Thoroughbreds. Study design: Cadaveric study. Methods: Medial femoral condyles (MFC) from a tissue bank of equine stifles were studied. Inclusion criteria were MFCs (≤8 months old) with a computed tomography SR lesion and histological focal failure of endochondral ossification (L group). Contralateral, lesion‐free, MFCs were a control group (CC). Osteochondral slabs were cut through the lesion (L), a healthy site immediately caudal to the lesion, (internal control; IC) and the contralateral, site‐matched controls (CC). Histological sections were immunostained with Cathepsin K for osteoclast counting. Osteoclasts in contact with the growth cartilage (chondroclasts) were also counted. The sections were segmented into regions of interest (ROI) at different depths in the subchondral bone: ROI ₁ (0‐1 mm), ROI ₂ (1‐3 mm) and ROI ₃ (3‐6 mm). Osteoclasts were counted and the bone area was measured in each ROI to calculate their density. Chondroclasts were counted in ROI ₁ . Results: Sections were studied from L and IC (n = 6) and CC sites (n = 5). Osteoclast density was significantly higher in ROI ₁ when compared with ROI ₃ in all groups. Although higher osteoclast density was measured in ROI ₁ in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI ₁ was lower in the L sections when compared with controls but no significant differences were detected. Main limitations: Limited sample size. Conclusions: Osteoclasts are important actors in MFC subchondral bone development, digesting both growth cartilage (chondroclasts) and bone, but the pathophysiology of early MFC SRs cannot be explained solely by an increased osteoclast presence in the subchondral bone.

Background: The aetiology of equine medial femoral condyle (MFC) subchondral bone radiolucencies (SR) is unknown. Objectives: Characterise the microstructural structural features of MFC SR in juvenile Thoroughbreds with microcomputed tomography (μCT) and histology. Study design: Cross‐sectional post‐mortem study. Methods: Distal femurs were collected at post‐mortem. Conventional tomodensitometry was employed to scout for MFCs with and without SR lesions (SR+ and SR−, respectively). Group 1 were CT MFC SR+ and Group 2 age‐matched SR− controls. Both underwent μCT and histological analysis. Group 3 CT MFC SR− foals, <6 months, were selected to search for chondronecrosis. Histological sections, processed from the lesion (Group 1) and a corresponding site in Groups 2 and 3, were assessed for chondronecrosis, fibrin, fibroplasia and osteochondral separation. Group 3 sections were surveyed for chondronecrosis alone. Results: A total of 178 femurs from 89 Thoroughbreds were harvested. Of these horses 19.1% (95% CI: 10.9%‐27.3%) were CT MFC SR+ (17/23; 7.46 ± 4.36 months) and met the inclusion criteria for Group 1. Group 2 included 30 CT MFC SR− specimens (5.00 ± 2.73 months) and Group 3 had 44 CT MFC SR− s (2.68 ± 1.74 months). SR were located axially in foals <7 months of age, and centrally thereafter. All SRs had areas of thickened cartilage on histology and separation at the osteochondral junction containing fibrin (acute event) and fibroplasia (chronicity) in 73.9% (17/23; 95% CI: 56%‐91.9%). In Group 1 specimens, chondronecrosis was present in 82.6% (19/23; 95% CI: 67.1%‐98.1%) but four MFC SR+ had no evidence of chondronecrosis. Chondronecrosis was not detected in the Group 3 foal MFCs. Main limitations: No longitudinal follow‐up. Conclusions: The absence of chondronecrosis, pathognomic of osteochondrosis, in four MFC SR+s and in all of the CT MFC SR− foals suggests that osteochondrosis is not the cause, or the only cause, of these lesions and favours trauma as an alternate aetiological hypothesis.