Intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells affects microRNA expression in experimental post-traumatic osteoarthritis in horses.

Osteoarthritis (OA) remains a major cause of lameness in horses, which leads to lost days of training and early retirement. Still, the underlying pathological processes are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that serve as regulators of many biological processes including OA. Analysis of miRNA expression in diseased joint tissues such as cartilage and synovial membrane may help to elucidate OA pathology. Since integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) have shown mitigating effect on equine OA we here investigated the effect of integrin α10-MSCs on miRNA expression. Cartilage and synovial membrane was harvested from the middle carpal joint of horses with experimentally induced, untreated OA, horses with experimentally induced OA treated with allogeneic adipose-derived MSCs selected for the marker integrin α10-MSCs, and from healthy control joints. miRNA expression in cartilage and synovial membrane was established by quantifying 70 pre-determined miRNAs by qPCR. Differential expression of the miRNAs was evaluated by comparing untreated OA and control, untreated OA and MSC-treated OA, and joints with high and low pathology score. A total of 60 miRNAs were successfully quantified in the cartilage samples and 55 miRNAs were quantified in the synovial membrane samples. In cartilage, miR-146a, miR-150 and miR-409 had significantly higher expression in untreated OA joints than in control joints. Expression of miR-125a-3p, miR-150, miR-200c, and miR-499-5p was significantly reduced in cartilage from MSC-treated OA joints compared to the untreated OA joints. Expression of miR-139-5p, miR-150, miR-182-5p, miR-200a, miR-378, miR-409-3p, and miR-7177b in articular cartilage reflected pathology score. Several of these miRNAs are known from research in human patients with OA and from murine OA models. Our study shows that these miRNAs are also differentially expressed in experimental equine OA, and that expression depends on OA severity. Moreover, MSC treatment, which resulted in less severe OA, also affected miRNA expression in cartilage.

Pros and cons of using autologous versus allogenic stem cells for the treatment of osteoarthritis.

Intraarticular treatment of osteoarthritis with mesenchymal stem cells (MSCs) has shown promising results and is being increasingly implemented in the clinic. Autologous MSCs are the primary source of therapy but issues related to cell expansion, patient age, and acute therapies have opened a need for allogenic MSCs. Problematic immunological reactions such as pain, joint swelling, urticarial, and MSC destruction are, however, reported when using allogenic MSCs at the first to second treatment. Multiple factors need to be considered when deciding on autologous or allogenic MSC treatment, as argued in this review.

Integrin α10ß1-Selected Mesenchymal Stem Cells Reduce Pain and Cartilage Degradation and Increase Immunomodulation in an Equine Osteoarthritis Model.

OBJECTIVE: Integrin α10ß1-selected mesenchymal stem cells (integrin α10-MSCs) have previously shown potential in treating cartilage damage and osteoarthritis (OA) in vitro and in animal models in vivo. The aim of this study was to further investigate disease-modifying effects of integrin α10-MSCs. DESIGN: OA was surgically induced in 17 horses. Eighteen days after surgery, horses received 2 × 107 integrin α10-MSCs intra-articularly or were left untreated. Lameness and response to carpal flexion was assessed weekly along with synovial fluid (SF) analysis. On day 52 after treatment, horses were euthanized, and carpi were evaluated by computed tomography (CT), MRI, histology, and for macroscopic pathology and integrin α10-MSCs were traced in the joint tissues. RESULTS: Lameness and response to carpal flexion significantly improved over time following integrin α10-MSC treatment. Treated horses had milder macroscopic cartilage pathology and lower cartilage histology scores than the untreated group. Prostaglandin E2 and interleukin-10 increased in the SF after integrin α10-MSC injection. Integrin α10-MSCs were found in SF from treated horses up to day 17 after treatment, and in the articular cartilage and subchondral bone from 5 of 8 treated horses after euthanasia at 52 days after treatment. The integrin α10-MSC injection did not cause joint flare. CONCLUSION: This study demonstrates that intra-articular (IA) injection of integrin α10-MSCs appears to be safe, alleviate pathological changes in the joint, and improve joint function in an equine post-traumatic osteoarthritis (PTOA) model. The results suggest that integrin α10-MSCs hold promise as a disease-modifying osteoarthritis drug (DMOAD).

Standing equine cheek tooth extraction: A multivariate analysis of the effect of antibiotics on the risk of post-operative complications.

BACKGROUND: Commonly, cheek tooth extraction performed in standing horses using perioperative prophylactic antibiotics, results in low post-operative complication rates. However, no studies have documented the relevance of perioperative antibiotics to the risk of post-operative complications. OBJECTIVES: To examine the association between perioperative antibiotics and post-operative complications after standing cheek tooth extraction. STUDY DESIGN: Retrospective cohort study. METHODS: Information from clinical records and follow-up questionnaires relating to horses subjected to cheek tooth extractions between September 2016 and May 2020 was obtained. Post-operative complications and associations with perioperative antibiotics, age, sex, breed, diagnosis, tooth position, and extraction method were analysed using multivariate logistic regression. RESULTS: A total of 305 horses were included, and of these 71 (23.3%) received perioperative antibiotics. Antibiotics were not associated with the risk of complications in 264 horses that underwent standard oral extraction; 9/49 (18.4%) that received antibiotics and 35/215 (16.3%) that did not receive antibiotics experienced postoperative complications (P = 1, RR = 0.89, OR = 1, OR CI = [0.41; 2.46]). Of 41 horses that had cheek tooth extraction through minimally invasive transbuccal cheek tooth extraction (MTE), 5/22 (22.7%) that received antibiotics and 10/19 (52.6%) that did not receive antibiotics, experienced postoperative complications. Although not statistically significant when adjusting for multiple comparisons (naïve P = 0.04, adjusted P = 0.26, RR = 2.32, OR = 4.48, OR CI = [1.05; 19.11]), this finding is clinically relevant. Younger age was also significantly associated with development of complications (P = 0.02, OR = 0.92 per year, OR CI = [0.87; 1.36]). MAIN LIMITATIONS: The retrospective nature of the study leads to uncontrollable potential confounders and there is a relatively low number of MTE cases. CONCLUSION: Perioperative antibiotics were not associated with a lower complication rate in horses subjected to standard standing cheek tooth extraction. Use of perioperative antibiotics in conjunction with MTE may be merited, although further investigations are needed.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells in equine osteoarthritis.

Introduction: Equine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Methods: Adipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics. Results: A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs. Discussion: To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

A detailed macroscopic scoring system for experimental post-traumatic Osteoarthritis in the equine middle carpal joint.

OBJECTIVE: Osteoarthritis (OA) is a significant health issue in humans as well as horses. Experimental models of equine carpal OA have been used to investigate OA pathogenesis and potential therapeutic candidates. A 5-scale scoring system (OARSI) for macroscopic pathological cartilage changes already exists, but there is a need for a scoring system with better differentiation of severity. The aim of this study was therefore to develop and validate such a scoring system. RESULTS: New scoring system were developed for cartilage erosions (Copenhagen Equine Total Cartilage Score, CEqTCS) along with synovial membrane pathology and osteochondral fragment healing (Copenhagen Equine Carpal Osteoarthritis Score, CEqCOAS). For the CEqTCS there was excellent intraclass agreement (ICC = 0.993; CI 0.985-0.996; p = 1.08e-31) and consistency (ICC = 0.992; CI 0.985-0.996; p = 4.61e-31), as well as excellent interclass agreement (ICC = 0.974; CI 0.948-0.987, p = 2e-22) and consistency (ICC = 0.973; CI 0.946-0.987; p = 2.77e-22), while the OARSI system had moderate (κ = 0.47) and weak (κ = 0.28) inter- and intra-class agreement, respectively. The OARSI score and the CEqTCS correlated excellently, but every OARSI grade encompassed a wide range of CEqTCS grades. The new score for assessment of equine OA provides means to a better differentiation of OA changes than the existing OARSI system.

Human integrin α10ß1-selected mesenchymal stem cells home to cartilage defects in the rabbit knee and assume a chondrocyte-like phenotype.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown promising results in stimulating cartilage repair and in the treatment of osteoarthritis (OA). However, the fate of the MSCs after intra-articular injection and their role in cartilage regeneration is not clear. To address these questions, this study investigated (1) homing of labeled human adipose tissue derived integrin α10ß1-selected MSCs (integrin α10-MSCs) to a cartilage defect in a rabbit model and (2) the ability of the integrin α10-MSCs to differentiate to chondrocytes and to produce cartilage matrix molecules in vivo. DESIGN: Integrin α10-MSCs were labeled with superparamagnetic iron oxide nanoparticles (SPIONs) co-conjugated with Rhodamine B to allow visualization by both MRI and fluorescence microscopy. A cartilage defect was created in the articular cartilage of the intertrochlear groove of the femur of rabbits. Seven days post-surgery, labeled integrin α10-MSCs or vehicle were injected into the joint. Migration and distribution of the SPION-labeled integrin α10-MSCs was evaluated by high-field 9.4 T MRI up to 10 days after injection. Tissue sections from the repair tissue in the defects were examined by fluorescence microscopy. RESULTS: In vitro characterization of the labeled integrin α10-MSCs demonstrated maintained viability, proliferation rate and trilineage differentiation capacity compared to unlabeled MSCs. In vivo MRI analysis detected the labeled integrin α10-MSCs in the cartilage defects at all time points from 12 h after injection until day 10 with a peak concentration between day 1 and 4 after injection. The labeled MSCs were also detected lining the synovial membrane at the early time points. Fluorescence analysis confirmed the presence of the labeled integrin α10-MSCs in all layers of the cartilage repair tissue and showed co-localization between the labeled cells and the specific cartilage molecules aggrecan and collagen type II indicating in vivo differentiation of the MSCs to chondrocyte-like cells. No adverse effects of the α10-MSC treatment were detected during the study period. CONCLUSION: Our results demonstrated migration and homing of human integrin α10ß1-selected MSCs to cartilage defects in the rabbit knee after intra-articular administration as well as chondrogenic differentiation of the MSCs in the regenerated cartilage tissue.

Validation of ultrasonography for measurement of cartilage thickness in the equine carpus.

Articular cartilage thinning is an important hallmark of osteoarthritis (OA), and ultrasonography (US) is a clinically accessible tool potentially suitable for repeated evaluation. The aim of the present prospective methods comparison study was to validate US as a tool for measuring cartilage thickness in the carpus of the horse. Eight Standardbred trotters underwent US examination with 9 and 15 MHz linear transducers. Six anatomical locations in the radiocarpal joint (RCJ) and middle carpal joint (MCJ) were examined. The same joints were assessed by ultrahigh field (9.4 Tesla) magnetic resonance imaging (MRI) and histology. Associations between measurements obtained by the different modalities were assessed by ANOVA, Deming regression, Pearson correlation and Bland-Altman plots. Histologically assessed total cartilage thickness (the noncalcified cartilage (NCC) plus the calcified cartilage zone (CCZ)) overestimated thickness compared to MRI (P < 0.01) and US (P < 0.01). US 15 MHz had substantial agreement with MRI and NCC histology, and repeatability was acceptable (coefficient of variation = 8.6-17.9%) when used for assessment of cartilage thickness in the RCJ. In contrast, 9 MHz US showed poorer agreement with MRI and NCC histology, as it overestimated the thickness of thin cartilage and underestimated the thickness of thicker cartilage in the RCJ and MCJ. Moreover, repeatability was suboptimal (coefficient of variation = 10.4-26.3%). A 15 MHz transducer US is recommended for detecting changes in RCJ cartilage thickness or monitoring development over time, and it has the potential for noninvasive assessment of cartilage health in horses.

Long-term athletic performance in sport horses after desmotomy of the accessory ligament of the deep digital flexor tendon.

BACKGROUND: Outcome after desmotomy of the accessory ligament of the deep digital flexor tendon (AL-DDFT) to treat flexural deformity of the distal interphalangeal joint has been reported to be excellent. However, no studies have compared long-term athletic performance of sport horses exposed to desmotomy of the AL-DDFT to that of matched controls. OBJECTIVES: The objective of this study is to compare long-term athletic performance in sports horses subjected to desmotomy of the AL-DDFT with the performance of matched controls. STUDY DESIGN: This is an observational multicentre retrospective matched cohort study. METHODS: Records from horses undergoing desmotomy of the AL-DDFT between 2004 and 2015 were reviewed. Various databases were used to identify age-matched siblings as unexposed controls and data on the horses' athletic careers. RESULTS: Seventy-four exposed and 194 matched unexposed horses were included. Although not significantly different, the proportion of exposed horses entering competition (28%, 95% CI 16%-38%) had a substantial risk difference compared with the proportion of unexposed horses entering competition (38%, 95% CI 26%-44%) (P = .2). Career longevity was significantly better for unexposed (15.6 competitions [95% CI 10.7-22.5] over a median of 570 days [IQR 210-1340]) than for exposed horses (9.7 competitions [95% CI 6.4-14.6] over a median of 219 days [IQR 2-1159] for horses operated in one limb and 6.1 competitions [95% CI 3.6-9.9] over a median of 446 days [IQR 23-603 days] for horses operated in two limbs, P < .001). Age at surgery and whether the condition was unilateral or bilateral did not affect chance of competing. MAIN LIMITATIONS: Small sample size, varying quality of medical records, stage of contraction not noted in many records and missing information on reasons for not entering into competition. CONCLUSIONS: Desmotomy of the AL-DDFT is associated with decreased long-term athletic performance in sport horses compared with matched unexposed horses.

Identification and Quantification of Transient Receptor Potential Vanilloid 1 (TRPV1) in Equine Articular Tissue.

Joint pain and osteoarthritis (OA) are some of the most common causes of lameness in horses, and most of the available treatments focus on symptomatic relief without a disease-modifying effect. TRPV1 is a potential target for treating joint diseases, including OA, and the present study aims to investigate if the TRPV1 receptor is present in equine articular tissue and determine whether the number of receptors is upregulated in joint inflammation. Metacarpo/metatarsophalangeal (MCP/MTP) joints from 15 horses euthanised for reasons unrelated to this study were included. Based on synovial fluid analysis, macroscopic evaluation, and magnetic resonance imaging (MRI), joints were divided into two groups: healthy joints and joints with pathology. ELISA analysis was performed on synovial tissue harvested from all joints. TPRV1 was found in all joints. The mean concentration of TRPV1 compared to total protein in healthy joints (8.4 × 10-7 ng/mL) and joints with pathology (12.9 × 10-7 ng/mL) differed significantly (p = 0.01, t-test with Welch correction). Quantitative real-time reverse transcriptase PCR analysis was performed on RNA isolates from synovial tissue from all joints. TRPV1 mRNA expression ratio normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in healthy joints (0.16 (SD: 0.19)) and joints with pathology (0.24 (SD: 0.14)) did not differ significantly (p = 0.43, t-test with Welch correction). mRNA expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) was very low for both groups. In conclusion, TRPV1 was detected both on mRNA and the protein level, with a higher expression of TRPV1 in samples from joints with pathology. Future studies will determine the clinical potential of equine TRPV1 as a target in the management of joint pain and inflammation.

Arthroscopic approach and intra-articular anatomy of the equine atlanto-occipital joint.

OBJECTIVE: To develop arthroscopic approaches to the atlanto-occipital (A-O) and describe associated arthroscopic anatomy. STUDY DESIGN: Experimental ex vivo study and clinical case report. ANIMALS: Ten equine cadaver joints and 1 clinical case. METHODS: CT arthrograms of 8 A-O joints were performed to determine the placement of an arthroscopic portal. Arthroscopy was performed via dorsal and/or ventral approaches (dorsal or ventral to the longissimus capitis tendon) in 10 cadaveric A-O joints and the A-O joint of a 2-week-old foal with septic arthritis. Accessible cartilage was debrided in 3 cadaver joints. Accessibility and risks were assessed by review of arthroscopic images, postoperative necropsy, and computed tomography (CT). RESULTS: Dorsal and ventral outpouchings of the A-O joint were identified with CT. Arthroscopy of the dorsal pouch provided access to 50% of the dorsocranial occipital condyle and 15% of the dorsocranial atlas articular surfaces. Joint distension caused displacement of the dura. Dura perforation occurred with a blind dorsal approach in 2 of 5 joints. Dura perforation did not occur after ultrasonography-guided approaches. Arthroscopic debridement of septic arthritis and osteomyelitis was successful in 1 clinical case. CONCLUSION: Approaches to the A-O joint were determined from CT examinations. The cranial aspect of the dorsal pouch of the A-O joint was accessed via arthroscopic triangulation in all horses of this study. Ultrasound-guided joint access prevented perforation of vital structures, including the spinal canal. CLINICAL SIGNIFICANCE: Advanced imaging improves the diagnosis of A-O joint pathology. Descriptions of arthroscopic anatomy and accessibility provide important information for surgical intervention.

An equine pain face.

OBJECTIVE: The objective of this study was to investigate the existence of an equine pain face and to describe this in detail. STUDY DESIGN: Semi-randomized, controlled, crossover trial. ANIMALS: Six adult horses. METHODS: Pain was induced with two noxious stimuli, a tourniquet on the antebrachium and topical application of capsaicin. All horses participated in two control trials and received both noxious stimuli twice, once with and once without an observer present. During all sessions their pain state was scored. The horses were filmed and the close-up video recordings of the faces were analysed for alterations in behaviour and facial expressions. Still images from the trials were evaluated for the presence of each of the specific pain face features identified from the video analysis. RESULTS: Both noxious challenges were effective in producing a pain response resulting in significantly increased pain scores. Alterations in facial expressions were observed in all horses during all noxious stimulations. The number of pain face features present on the still images from the noxious challenges were significantly higher than for the control trial (p = 0.0001). Facial expressions representative for control and pain trials were condensed into explanatory illustrations. During pain sessions with an observer present, the horses increased their contact-seeking behavior. CONCLUSIONS AND CLINICAL RELEVANCE: An equine pain face comprising 'low' and/or 'asymmetrical' ears, an angled appearance of the eyes, a withdrawn and/or tense stare, mediolaterally dilated nostrils and tension of the lips, chin and certain facial muscles can be recognized in horses during induced acute pain. This description of an equine pain face may be useful for improving tools for pain recognition in horses with mild to moderate pain.

Intra-articular injection of morphine to the horse: establishment of an in vitro-in vivo relationship.

BACKGROUND: In the area of parenteral depots, a strong need exists for the development of suitable in vitro drug release models that might enable establishment of in vitro-in vivo relations (IVIVRs). AIM: The objective of this study was to investigate the possibility of establishing an IVIVR between morphine disappearance from the joint cavity and in vitro release data obtained employing the rotating dialysis cell model. METHOD: In vitro release experiments were conducted using the rotating dialysis cell model. For establishment of an IVIVR, data from a previous study on pharmacokinetics of intra-articular (IA) morphine in horses with lipopolysaccharide-induced synovitis were used (Lindegaard et al., (2009). Vet Anaesth Analg, 37, 186-195). RESULTS: A rate constant of morphine disappearance from the donor phase of the in vitro model of 1.8 × 10(-2) min(-1) was calculated, independently of the different release media used. The in vivo synovial fluid disappearance rate constants were in the range of 1.0 × 10(-2)-1.7 × 10(-2) min(-1). An IVIVR (R(2) = 0.89) was established between the calculated disappearance data and the joint disappearance data. CONCLUSION: The results indicate that the IA fate of morphine administered in the form of a solution can be predicted from the in vitro release data obtained in the rotating dialysis cell model. Thus, this model might be a valuable tool in the establishment of IVIVRs after IA administration of drugs with similar properties.

On the search for in vitro in vivo correlations in the field of intra-articular drug delivery: administration of sodium diatrizoate to the horse.

Development of suitable in vitro release models for formulation development as well as quality control purposes has to be initiated in the early design phase of injectable depots. Optimally, construction of an in vitro release model may lead to the establishment of in vitro in vivo correlations. By using a model compound (sodium diatrizoate, DTZ), the purpose of this study was to investigate the possibility of establishing in vitro in vivo relations between the DTZ disappearance profile obtained from the donor compartment of the rotating dialysis cell model and the joint disappearance profile following intra-articular administration. In vitro experiments were conducted by applying solutions of DTZ to the donor compartment. In the in vivo experiments, five horses were subjected to both intravenous and intra-articular administration of an aqueous solution of 3.9 mg DTZ/kg. A strong relation (R(2)=0.99) was obtained between the disappearance data from the donor compartment of the in vitro model and the disappearance data from the synovial fluid after intra-articular administration of DTZ. Furthermore, a relation (R(2)=0.91) between the appearance data obtained from the acceptor compartment and the deconvolved appearance serum data upon intra-articular administration of DTZ was obtained. The correlations obtained in this study hold promise that the rotating dialysis cell model has a role in the prediction of the intra-articular fate of drugs injected as solutions.

Symmetry indices based on accelerometric data in trotting horses.

Detection and quantification of lameness in horses consists primarily of a subjective assessment, whereby both intra- and inter-observer disagreements exist, especially with low grade lameness. Therefore, clinically applicable methods are needed for reliable, objective assessments. The aim of this study was to describe three symmetry indices derived from a simple accelerometric method and investigate these in sound trotting horses. The indices describe the overall symmetry of the gait, the symmetry of loads placed on the limbs and the symmetry in timing between left and right steps. These symmetry indices were able to quantify the high degree of symmetry of the trot in sound horses that has been described in earlier studies using other gait analysis methods. Also, we have analysed the variances and have found high repeatability for all three indices. This provides a basis for future investigations of the symmetry indices and their potential for objective detection and quantification of lameness in horses.

Analgesic efficacy of intra-articular morphine in experimentally induced radiocarpal synovitis in horses.

OBJECTIVE: To compare the analgesic effect of intra-articular (IA) and intravenous (IV) morphine in horses with experimentally induced synovitis. ANIMALS: Eight adult horses. STUDY DESIGN: Randomized, observer blinded, double dummy trial with sequential crossover design. METHODS: Radiocarpal synovitis was induced by IA injection of lipopolysaccharide on two occasions separated by a 3-week washout period. In one study period horses received treatment IA; morphine IA (0.05 mg kg(-1)) plus saline IV and in the other study period they received treatment IV; saline IA plus morphine IV (0.05 mg kg(-1)). Lameness and pain were evaluated repeatedly by two observers throughout each of the two 168-hour study periods. Pain was evaluated by use of a visual analogue scale of pain intensity (VAS) and a composite measure pain scale (CMPS). Comparison of treatments was performed by analysis of variance with repeated measurements. Significance level was set to p < or = 0.05. Inter-observer agreement and agreement between the VAS and CMPS was assessed by use of the Bland-Altman method. RESULTS: Intra-articular injection of LPS elicited a marked synovitis resulting in lameness and pain. IA morphine resulted in significantly less lameness than IV morphine (p = 0.03). CMPS (p = 0.09) and VAS (p = 0.10) pain scores did not differ significantly between treatments. Inter-observer agreement of the CMPS was classified as good, but only fair for the VAS. Agreement between the two pain scales was considered fair. CONCLUSIONS AND CLINICAL RELEVANCE: An analgesic effect of IA morphine was demonstrated by significantly reduced lameness scores. The results support the common practice of including IA morphine in a multimodal analgesic protocol after arthroscopic surgery, although further studies in clinical cases are needed. The employed CMPS had good reproducibility, and was easy to use, but may have limited sensitivity at mild intensity pain.

Pharmacokinetics of intra-articular morphine in horses with lipopolysaccharide-induced synovitis.

OBJECTIVE: To describe the pharmacokinetics of intra-articularly (IA) administered morphine. STUDY DESIGN: Experimental randomized, cross-over study. ANIMALS: Eight adult healthy mixed breed horses aged 6.5 +/- 2.3 (mean +/- SD) years and weighing 535 +/- 86 kg. METHODS: Unilateral radiocarpal synovitis was induced by IA injection of 3 microg lipopolysaccharide (LPS) on two occasions (right and left radiocarpal joint, respectively) separated by a 3-week wash-out period. Treatments were administered 4 hours post-LPS-injection: Treatment IA; preservative free morphine IA (0.05 mg kg(-1)) plus saline intravenous (IV) and treatment IV; saline IA plus preservative free morphine IV (0.05 mg kg(-1)). Concentrations of morphine, morphine-3-glucuronide and morphine-6-glucuronide (M6G) were determined repeatedly in serum and synovial fluid (SF) by high-performance liquid chromatography mass spectrometry, at 2 and 4 hours and then at 4 hours intervals until 28 hours post-treatment. RESULTS: Injection of LPS elicited a marked and comparable synovitis in all LPS-injected radiocarpal joints. IA administered morphine was detectable in SF of all eight joints 24 hours post-treatment and in 6/8 joints 28 hours post-treatment. The terminal half-life of morphine in SF was estimated to be 2.6 hours. IA administration of morphine resulted in mean serum concentrations of morphine below 5 ng mL(-1) from 2 to 28 hours after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Intra-articularly administered morphine remained within the joint for at least 24 hours. At the same time only very low serum concentrations of morphine and M6G were detected. The present results suggest that IA morphine at 0.05 mg kg(-1) may be used for IA analgesia lasting at least 24 hours and give strong support to the theory that previously observed analgesic and anti-inflammatory effects of IA morphine in horses are most likely to be mediated peripherally.

Anti-inflammatory effects of intra-articular administration of morphine in horses with experimentally induced synovitis.

OBJECTIVE: To compare the effects of intra-articular (IA) versus IV administration of morphine on local and systemic inflammatory responses in horses with experimentally induced acute synovitis. ANIMALS: 8 horses. PROCEDURES: Each horse received the following 2 treatments 4 hours after synovitis was induced: IA administration of morphine (0.05 mg/kg) with IV administration of 1 mL of saline (0.9% NaCl) solution/100 kg, and IA administration of 1 mL of saline solution/100 kg with IV administration of morphine (0.05 mg/kg). Treatments were administered in randomized order with a washout period of 3 weeks between treatments. Before each treatment, aseptic synovitis was induced by injection of lipopolysaccharide into a radiocarpal joint. For the second treatment, the contralateral radiocarpal joint was selected. Joint swelling and skin temperature over the treated joints were recorded. Clinical examinations were performed, and blood WBC count, serum amyloid A (SAA) concentration, serum cortisol concentration, synovial fluid WBC count, synovial fluid total protein (TP) concentration, and synovial fluid SAA concentration were measured before and repeatedly during each of the two 168-hour study periods. Data were analyzed by use of ANOVA with repeated measures. RESULTS: IA administration of morphine resulted in significantly less joint swelling and lower synovial fluid TP and serum and synovial fluid SAA concentrations, and blood WBC count than did IV administration of morphine. CONCLUSIONS AND CLINICAL RELEVANCE: IA administration of morphine exerted anti-inflammatory properties in horses with experimentally induced acute synovitis, supporting its use as a part of a balanced analgesic protocol.

Evaluation of pain and inflammation associated with hot iron branding and microchip transponder injection in horses.

OBJECTIVE: To compare effects of hot iron branding and microchip transponder injection regarding aversive behavioral reactions indicative of pain and inflammation in horses. ANIMALS: 7 adult horses. PROCEDURES: In a randomized controlled clinical crossover study, behavioral reactions to hot iron branding and microchip transponder injection were scored by 4 observers. Local and systemic inflammation including allodynia were assessed and compared by use of physiologic and biochemical responses obtained repeatedly for the 168-hour study period. Serum cortisol concentration was measured repeatedly throughout the first 24 hours of the study. Sham treatments were performed 1 day before and 7 days after treatments. RESULTS: Hot iron branding elicited a significantly stronger aversive reaction indicative of pain than did microchip transponder injection (odds ratio [OR], 12.83). Allodynia quantified by means of skin sensitivity to von Frey monofilaments was significantly greater after hot iron branding than after microchip transponder injection (OR, 2.59). Neither treatment induced signs of spontaneously occurring pain that were observed during the remaining study period, and neither treatment induced increased serum cortisol concentrations. Comparison with sham treatments indicated no memory of an unpleasant event. The hot iron branding areas had significantly increased skin temperature and swelling (OR, 14.6). Systemic inflammation as measured via serum amyloid A concentration was not detected after any of the treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Microchip transponder injection induced less signs of pain and inflammation and did not seem to pose a higher long-term risk than hot iron branding. Consequently, results indicated that hot iron branding does inflict more pain and should be abandoned where possible.