Micro-computed tomography reveals high-density mineralised protrusions and microstructural lesions in equine stifle joint articular cartilage.

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.

Radiographic analysis in Thoroughbreds reveals morphological changes in healthy maturing stifle joints and possible association between subchondral lesions and femoral condyle width.

OBJECTIVE: Assess femorotibial features in foals with and without medial femoral condyle (MFC) subchondral radiolucencies (SR+ and SR-). METHODS: 3 independent, sequential radiographic studies were performed. Study 1 retrospectively measured femorotibial morphological parameters in repository radiographs (SR- and SR+). Study 2 qualitatively compared drawings of intercondylar notch shape in postmortem radiographs (SR-). Study 3 prospectively measured femorotibial parameters in 1-month-old foals (SR-). In studies 1 and 3, 13 morphologic parameters were measured. Limb directional asymmetry was assessed in 2 age groups (< 7 or ≥ 7 months). RESULTS: Study 1 (SR- group; n = 183 radiographs) showed increased femoral measurements with maturation, except the distal femoral intercondylar notch width (FINwal), which decreased. In contrast, in SR+ stifles (53 radiographs), 3 femoral parameters (MFC width [MFCwpf], MFC height, or FINwal) showed no changes. Tibial plateau width alone increased with maturation in both groups. Interobserver reliability was good to excellent. Study 2 (n = 53 radiographs) confirmed a distal FINw decrease in SR- foals. In study 1, left SR- stifles in greater than or equal to 7-month-old fillies had significantly larger femoral bicondylar width and FINw, while right SR+ stifles in fillies greater than or equal to 7 months had a significantly larger MFCw. In study 3 of 1-month-old foals (n = 94 SR- radiographs), the MFCw, femoral condyle bicondylar width, and lateral femoral condyle height were all greater on the left, whereas the intercondylar intereminence space width was larger on the right. CLINICAL RELEVANCE: In SR+ stifles, the distal femur exhibited divergent maturation, indicating a wider MFC in the right stifle in older foals. As SR lesions are more common on the right, this suggests a potential association with MFC morphology.

Characterization and quantification of in-vitro equine bone resorption in 3D using µCT and deep learning-aided feature segmentation.

High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in µCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with µCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture µCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy.

Vascularity evaluation of the equine tarsocrural and proximal intertarsal joint septum, and comparative analysis of two arthroscopic transection techniques.

OBJECTIVE: To evaluate vascularity of the synovial membrane covered septum (SMS) separating the tarsocrural (TC) and proximal intertarsal (PIT) joints (Part 1) and compare two methods of transection, electrosurgical or Ferris Smith rongeur (FS rongeur) (Part 2). STUDY DESIGN: Experimental study. SAMPLE POPULATION: Part 1, 10 SMS (n = 5 horses). Part 2, six horses (n = 12 tarsi). METHODS: In part 1, SMS harvested postmortem were each divided into eight regions of interest (ROIs), processed for histology, and immunostained with anti-α-actin antibody for blood vessel identification. Vascular density was calculated for each ROI. Data was compared within and between horses. In part 2, six horses underwent TC arthroscopy. Each limb was randomly assigned to undergo either electrosurgical or FS rongeur SMS transection. SMS transection and total operative time were recorded. Intraoperative hemorrhage was scored. Data was compared between both techniques. RESULTS: Significant interindividual variations in SMS vascular density were detected (p = .02), but there were no differences among ROIs. No differences in the transection time were detected between electrosurgery (4.83 ± 0.54 min) and FS rongeur (4.33 ± 0.67 min). No differences were found in intraoperative hemorrhage scores between techniques. CONCLUSION: Vascularity within the SMS varies among horses but not within its regions. Electrosurgical or FS rongeur transection of the medial SMS during tarsocrural arthroscopy is a rapid technique and improves surgical access to the dorsal compartment of the PIT.

An in vitro model for discovery of osteoclast specific biomarkers towards identification of racehorses at risk for catastrophic fractures.

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-1ß 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.

Cannabinoid receptors are expressed in equine synovium and upregulated with synovitis.

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.

Microstructural features of subchondral radiolucent lesions in the medial femoral condyle of juvenile Thoroughbreds: A microcomputed tomography and histological analysis.

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.

Osteoclast density is not increased in bone adjacent to radiolucencies (cysts) in juvenile equine medial femoral condyles.

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: ROI1 (0-1 mm), ROI2 (1-3 mm) and ROI3 (3-6 mm). Osteoclasts were counted and the bone area was measured in each ROI to calculate their density. Chondroclasts were counted in ROI1 . RESULTS: Sections were studied from L and IC (n = 6) and CC sites (n = 5). Osteoclast density was significantly higher in ROI1 when compared with ROI3 in all groups. Although higher osteoclast density was measured in ROI1 in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI1 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.

Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept).

BACKGROUND: There is a growing interest in the scientific community to use computer-based software programs for the quantification of cells during physiological and pathophysiological processes. Drawbacks of computer-based methods currently used to quantify immunohistochemical staining are the complexity of use, expense of software and overly-simplified descriptions of protocol thereby limiting reproducibility. The precise role of mast cells in equine cutaneous wound healing is unknown. Given the contribution of mast cells to the chronic inflammation observed in human keloid, a pathology similar to exuberant granulation tissue (EGT) in horses, mast cells might be present in high numbers in equine limb wounds predisposed to EGT. The main goal of this study was to develop a reliable and reproducible quantification method for immunostained tissues using a computer software that is widely available, at no cost, to the scientific community. A secondary goal was to conduct a proof of concept using the newly-established method to quantify mast cells during wound healing at different anatomical sites (body and limb) in horses to see if a different pattern is observed in limb wounds, which are predisposed to EGT. RESULTS: A good intraclass correlation coefficient (ICC, 0.67 p < 0.05) was found between the computer-based ImageJ method and manual counting. An excellent intra-operator ICC of 0.90 (p < 0.01) was found for the ImageJ quantification method while a good interoperator ICC of 0.69 (p < 0.01) was measured. No significant difference was observed between the variation of the ImageJ and that of the manual counting method. Mast cells were localized below the epidermis, around cutaneous appendages and blood vessels. Mast cell numbers did not differ significantly in relation to anatomical location or time of healing. CONCLUSIONS: The computer-based quantification method developed is reliable, reproducible, available, cost-free and could be used to study different physiological and pathological processes using immunohistochemistry.

Abundant osteoclasts in the subchondral bone of the juvenile Thoroughbred metacarpus suggest an important role in joint maturation.

BACKGROUND: The administration of bisphosphonate medications, which target osteoclastic-bone remodelling, to juvenile and adult racehorses is a matter of debate owing to concerns that these molecules remain bound to the bone-mineralised matrix and may interfere with subsequent bone growth, adaptation to exercise and healing of bone microdamage in equine athletes. Osteoclasts participate in endochondral ossification, subchondral bone remodelling and bone repair. There is a knowledge gap on the role of equine osteoclast biology in the growth and maturation of joint surfaces and this information is important to inform judicious bisphosphonate use. OBJECTIVES: Measure and compare the osteoclast density in the subchondral bone of Thoroughbred (TB) distal third metacarpi (McIII) at different sites, varying depths from the articular surface and with age (0-84 months). STUDY DESIGN: Ex vivo cadaveric study. METHODS: McIIIs from foals, yearlings and adults were collected, fixed in formaldehyde and stored at 4°C. Sections were cut from the lateral hemi-metacarpus, stained and scored for cartilage degeneration. Osteoclasts were counted on immunohistochemically (Cathepsin K) stained sections. Osteoclast density was compared in regions of interest (ROIs-the sagittal ridge, axial and abaxial condyle) and also at two depths (0-3 mm and 3-6 mm) into the subchondral bone below the osteochondral junction. RESULTS: The osteoclast density was consistently highest in the subchondral cortical bone plate (0-3 mm) when compared with the deeper trabecular bone in all age groups. Furthermore, the osteoclast density was significantly higher in juvenile Thoroughbreds (foals and yearlings) within both sites in the subchondral bone when compared with adults. MAIN LIMITATIONS: The number of specimens available for study was restricted. CONCLUSIONS: Osteoclasts are important in normal McIII epiphyseal and articular surface maturation and have a propensity to localise at the osteochondral junction and subchondral cortical bone plate zone in juvenile Thoroughbreds.

Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy.

We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies.

Biomarkers for equine joint injury and osteoarthritis.

We report the results of a symposium aimed at identifying validated biomarkers that can be used to complement clinical observations for diagnosis and prognosis of joint injury leading to equine osteoarthritis (OA). Biomarkers might also predict pre-fracture change that could lead to catastrophic bone failure in equine athletes. The workshop was attended by leading scientists in the fields of equine and human musculoskeletal biomarkers to enable cross-disciplinary exchange and improve knowledge in both. Detailed proceedings with strategic planning was written, added to, edited and referenced to develop this manuscript. The most recent information from work in equine and human osteoarthritic biomarkers was accumulated, including the use of personalized healthcare to stratify OA phenotypes, transcriptome analysis of anterior cruciate ligament (ACL) and meniscal injuries in the human knee. The spectrum of "wet" biomarker assays that are antibody based that have achieved usefulness in both humans and horses, imaging biomarkers and the role they can play in equine and human OA was discussed. Prediction of musculoskeletal injury in the horse remains a challenge, and the potential usefulness of spectroscopy, metabolomics, proteomics, and development of biobanks to classify biomarkers in different stages of equine and human OA were reviewed. The participants concluded that new information and studies in equine musculoskeletal biomarkers have potential translational value for humans and vice versa. OA is equally important in humans and horses, and the welfare issues associated with catastrophic musculoskeletal injury in horses add further emphasis to the need for good validated biomarkers in the horse. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:823-831, 2018.

Clinical indications, complications, and long-term outcome of esophageal surgeries in 27 horses.

The main objective of this retrospective study was to describe clinical findings, management, and short- and long-term outcome in 27 horses that underwent various surgical techniques for esophageal disease. Surgical techniques (sometimes concurrently) performed were: esophagostomy (n = 14), esophagotomy with primary closure (n = 6), esophagomyotomy (n = 3), and esophagoplasty (n = 2). Esophageal perforation in 5 horses was treated by ventral drainage; 3 horses had the esophageal defect sutured (n = 3). Feeding tubes were placed in 15 horses. Postoperative complications occurred in 52% (14/27) with a median of 3 complications/horse (range: 1 to 7). Significantly more complications occurred in horses with a perforated esophagus. Eighteen horses (18/27; 67%) were discharged. Most horses (8/9; 89%) with a lesion located in the proximal esophagus were discharged. Horses with a higher number of postoperative complications, particularly postoperative infection, were more likely to be euthanized. One year after surgery, 41% of the horses were alive and free of complications.

Indications cliniques, complications et résultat à long terme de chirurgies œsophagiennes chez 27 chevaux. L'objectif principal de cette étude rétrospective a été de décrire les résultats cliniques, la gestion et les résultats à court et à long terme chez 27 chevaux qui ont subi diverses techniques de chirurgie pour le traitement de la maladie œsophagienne. Les techniques chirurgicales réalisées (parfois de manière concomitante) étaient : l'œsophagostomie (n = 14), l'œsophagotomie avec fermeture primaire (n = 6), l'œsophagomyotomie (n = 3) et l'œsophagoplastie (n = 2). La perforation œsophagienne chez 5 chevaux a été traitée par drainage ventral; 3 chevaux ont subi une suture du défaut œsophagien (n = 3). Des sondes d'alimentation ont été placées chez 15 chevaux. Les complications postopératoires se sont produites chez 52 % des chevaux (14/27) avec une médiane de 3 complications/cheval (fourchette : 1 à 7). Un nombre significativement supérieur de complications se sont produits chez les chevaux ayant une perforation de l'œsophage. Dix-huit chevaux (18/27; 67 %) ont reçu leur congé. La plupart des chevaux (8/9; 89 %) ayant une lésion situé dans l'œsophage proximal ont reçu leur congé. Il était plus probable que les chevaux avec un nombre supérieur de complications postopératoires, particulièrement une infection postopératoire, soient euthanasiés. Un an après la chirurgie, 41 % des chevaux étaient vivants et libres de complications.(Traduit par Isabelle Vallières).

Femoral epiphyseal cartilage matrix changes at predilection sites of equine osteochondrosis: Quantitative MRI, second-harmonic microscopy, and histological findings.

Osteochondrosis is an ischemic chondronecrosis of epiphyseal growth cartilage that results in focal failure of endochondral ossification and osteochondritis dissecans at specific sites in the epiphyses of humans and animals, including horses. The upstream events leading to the focal ischemia remain unknown. The epiphyseal growth cartilage matrix is composed of proteoglycan and collagen macromolecules and encases its vascular tree in canals. The matrix undergoes major dynamic changes in early life that could weaken it biomechanically and predispose it to focal trauma and vascular failure. Subregions in neonatal foal femoral epiphyses (n = 10 osteochondrosis predisposed; n = 6 control) were assessed for proteoglycan and collagen structure/content employing 3T quantitative MRI (3T qMRI: T1ρ and T2 maps). Site-matched validations were made with histology, immunohistochemistry, and second-harmonic microscopy. Growth cartilage T1ρ and T2 relaxation times were significantly increased (p < 0.002) within the proximal third of the trochlea, a site predisposed to osteochondrosis, when compared with other regions. However, this was observed in both control and osteochondrosis predisposed specimens. Microscopic evaluation of this region revealed an expansive area with low proteoglycan content and a hypertrophic-like appearance on second-harmonic microscopy. We speculate that this matrix structure and composition, though physiological, may weaken the epiphyseal growth cartilage biomechanically in focal regions and could enhance the risk of vascular failure with trauma leading to osteochondrosis. However, additional investigations are now required to confirm this. 3T qMRI will be useful for future non-invasive longitudinal studies to track the osteochondrosis disease trajectory in animals and humans. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1743-1752, 2016.

Frequency of Undetected Glove Perforation and Associated Risk Factors in Equine Surgery.

OBJECTIVE: To estimate the frequency of undetected perforations in surgical gloves during equine surgery and to identify risk factors associated with occurrence. STUDY DESIGN: Observational cohort study. SAMPLE POPULATION: Surgical gloves-292 pairs. METHODS: Water leak tests were performed on gloves after equine surgery to detect perforations. Fifty pairs of unused gloves were also tested. Potential risk factors were recorded. Logistic regression was used to explore associations between putative risk factors and perforation. RESULTS: No perforations were detected in the unused gloves. Of 292 pairs of used gloves tested, 80 (27%) had at least one glove perforation per pair. The frequency of perforations was not different between surgery diplomates and residents (P=.69). The length and type of surgery were significantly associated with the likelihood of undetected glove perforation with surgeries longer than 60 minutes approximately 2.5 times more likely to result in glove perforation (P=.005). Surgery classified as soft tissue, orthopedic or exploratory celiotomy was 3 times more likely to result in glove perforation than minimally invasive surgery. The perforations occurred significantly more frequently in the nondominant hand (19%) than the dominant hand (11%) (P=.009). CONCLUSION: There is a high occurrence of undetected glove perforation in equine surgery. Precautions can be suggested based on this study although further investigation is required to assess whether glove perforations are associated with surgical site infections.

COMPARISONS AMONG RADIOGRAPHY, ULTRASONOGRAPHY AND COMPUTED TOMOGRAPHY FOR EX VIVO CHARACTERIZATION OF STIFLE OSTEOARTHRITIS IN THE HORSE.

A better understanding of imaging characteristics of equine stifle osteoarthritis (OA) may allow earlier detection and improve prognosis. Objectives of this ex vivo, prospective, methods comparison study were to (1) describe the location and severity of naturally acquired OA lesions in the equine stifle using ultrasound (US), radiography (XR), computed tomography (CT), and macroscopic evaluation (ME); (2) compare the diagnostic performance of each imaging modality with ME; and (3) describe subchondral bone mineral density (BMD) in equine stifle joints with OA using CT. Radiographic, CT, and US evaluations were performed on 23 equine cadaver stifles and compared with ME. Significant associations were found between osteophyte global scores for all imaging modalities (CT, P ˂ 0.0001; XR, P = 0.005; US, P = 0.04) vs. ME osteophyte global scores. Osteophytes were detected most frequently in the medial femorotibial (MFT) joint. A specific pattern of osteophytes was observed, with a long ridge of new bone at the insertion of the MFT joint capsule cranially on the medial femoral condyle. A novel caudo-10°proximo-5°lateral-cranio-disto-medial oblique radiographic projection was helpful for detection of intercondylar osteophytes. Multiplanar CT reformatted images were helpful for characterizing all osteophytes. Osteophyte grades at most sites did not differ among modalities. Low sensitivity/specificity for subchondral bone sclerosis and flattening of femoral condyles suggested that these signs may not be reliable radiographic and CT indicators of equine stifle OA. Equine stifle OA was associated with a decrease in BMD and specific sites of focal subchondral bone resorption/cyst formation were found in some specimens.

Radiographic evaluation of the width of the femorotibial joint space in horses.

OBJECTIVE: To measure the minimal joint space width (mJSW) in caudocranial radiographic views of orthopedically normal femorotibial joints of horses, to compare the accuracy of measurements with those of a software program designed for humans, and to identify the ideal caudocranial radiographic projection angle for mJSW measurement. ANIMALS 12 healthy mares (22 femorotibial joints) and 3 equine cadavers (6 stifle joints). PROCEDURES: Caudocranial views of femorotibial joints were acquired in the proximodistal plane at 5°, 10°, and 15° (caudo-5°-proximal-craniodistal oblique, 10°, and 15°) and lateromedial plane (caudo-10°-proximo-5°-lateral-craniodistomedial oblique and caudo-10°-proximo-5°-medial-craniodistolateral oblique). The mJSWs of medial and lateral femorotibial joint compartments were measured manually by 2 evaluators and automatically by a digital analysis software program. Interevaluator reproducibility was assessed. Post hoc tests were used to identify the projection angle that provided the largest measurements. Validation of mJSW measurements was performed by evaluation of 6 stifle joints ex vivo. RESULTS: Excellent agreement was achieved between the 2 evaluators and between the veterinary radiologist and the analysis software for the medial and lateral compartments of femorotibial joints. Angle of caudocranial view in the proximodistal but not lateromedial plane had a significant effect on the medial compartment mJSW measurements. Mean mJSW for the medial compartment was significantly higher for the caudoproximal-craniodistal oblique projection made at 10° from the horizontal than for other angles. Angle had no significant effect on mean mJSW for the lateral compartment. Agreement between automated measurements of mJSW in the medial compartment and thickness of nonmineralized cartilage in histologic preparations of associated tissues was excellent. CONCLUSIONS AND CLINICAL RELEVANCE: Measurements of mJSW in the medial compartment of femorotibial joints, the most common site of osteoarthritis in horses, were reproducible and optimal with a caudoproximal-craniodistal oblique radiographic projection made at 10° from the horizontal.

Comparison of an alcohol-based hand rub and water-based chlorhexidine gluconate scrub technique for hand antisepsis prior to elective surgery in horses.

This prospective clinical study evaluates the effectiveness of an alcohol-based hand rub (Avagard™) for pre-surgical hand antisepsis in an equine hospital and compares it with traditional scrubbing technique using 4% chlorhexidine gluconate sponges and water. Prior to elective surgery, 3 board-certified surgeons were randomly assigned to hand antisepsis with either technique. Culture samples of each hand were taken at 4 times: before and after neutral soap hand wash, after scrub or rubbing technique, and after surgery. There was no significant difference in mean bacterial colony forming units between scrub and rub techniques over the 3 time periods (P = 0.6), controlling for initial counts. One horse from the scrub group had a skin incision infection following stifle arthroscopy; this was resolved with medical treatment. The alcohol-based hand rub is equivalent in efficacy for pre-surgical hand antisepsis to traditional water-based scrubs in an equine hospital setting.

Comparaison de l'alcool à friction et d'une technique de brossage au gluconate de chlorhexidine à base d'eau pour l'antisepsie des mains avant les chirurgies non urgentes chez les chevaux. Cette étude clinique prospective évalue l'efficacité d'un alcool à friction (AvagardMD) pour l'antisepsie des mains préalable à une chirurgie dans un hôpital équin et elle la compare à la technique de brossage traditionnelle au moyen d'éponges de gluconate de chlorhexidine 4 % et d'eau. Avant une chirurgie non urgente, trois chirurgiens agréés ont été assignés au hasard à l'antisepsie des mains avec l'une ou l'autre technique. Des échantillons de chaque main ont été prélevés quatre fois pour culture : avant et après le lavage des mains avec un savon neutre, après la technique de brossage et de friction et après la chirurgie. Il n'y avait aucune différence significative dans les moyennes des dénombrements bactériens d'unités formatrices de colonies entre les techniques de brossage et de friction pendant les trois périodes de temps (P = 0,6), en tenant compte des témoins pour les dénombrements initiaux. Un cheval du groupe de brossage a développé une infection au site de l'incision après une arthroscopie du grasset qui a été résolue par traitement médical. La friction des mains à l'alcool est équivalente, en matière d'efficacité pour l'antisepsie des mains avant la chirurgie, au brossage traditionnel à l'eau dans un milieu hospitalier équin.(Traduit par Isabelle Vallières).

Differences in the vascular tree of the femoral trochlear growth cartilage at osteochondrosis-susceptible sites in foals revealed by SWI 3T MRI.

Focal ischemic chondronecrosis of epiphyseal growth cartilage (EGC) during endochondral ossification is believed to be a key early event on the pathway to osteochondrosis (OC) in both animals and humans. The lateral ridge of the equine trochlea is a site where severe osteochondritis dissecans lesions frequently arise and is a model for the study of naturally occurring disease. Non-invasive imaging to investigate EGC vascularity may help elucidate why focal ischemia occurs. 3T MRI susceptibility-weighted imaging (SWI) of femoral trochlea of OC predisposed (n = 10) and control (n = 6) day-old foals, with minimal joint loading after birth, was performed. SWI and 3D images revealed the EGC vascular architecture without a contrast agent, and matched histologic observations. No vascular lesions were identified. There was no difference in the vascular density and architecture between control and OC specimens, but a striking difference in vascular pattern was seen at the OC-predilected site in the lateral ridge of the trochlea in all specimens, when compared to the medial ridge of the trochlea, where OC lesions are rarely observed. This site was less ossified with more perichondrial vessels not yet bridging with the subchondral bone. Furthermore, the mean vascular density of all specimens was significantly higher at this site. We speculate that joint morphology and focal internal trauma on this site with a unique vascular architecture may trigger ischemic events at this site. SWI permitted visualization of EGC in young foals with a clinical 3T MRI and paves the way for non-destructive longitudinal studies to improve understanding of OC in all species. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1539-1546, 2016.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy.

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.