Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Stem cells and platelet-rich plasma for the treatment of naturally occurring equine tendon and ligament injuries: a systematic review and meta-analysis.

BACKGROUND: Platelet-rich plasma (PRP) and mesenchymal stromal or stem cells (MSCs) have been investigated as treatments for equine tendon and ligament injuries, but little consensus exists on the efficacy of these treatments. The study sought to evaluate the efficacy of PRP and MSC treatments by systematic review and meta-analysis. METHODS: A systematic review was performed using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. Inclusion criteria required an original, peer-reviewed study where horses were administered MSCs or PRP (or both), and a comparator group was described. Studies were assessed for risk of bias and study quality. Random effects meta-analysis with inverse variance weighting was used to calculate pooled estimates of the ORs for the primary outcomes of return to performance and reinjury. RESULTS: The search criteria identified 764 unique studies, and 21 studies met the inclusion criteria for the systematic review. Seventeen studies were further assessed for the primary outcomes of return to performance and reinjury rate within a meta-analysis. Meta-analyses revealed no increase in the likelihood of a return to performance with any of the biologic treatments. However, MSCs and MSCs administered concurrently with PRP provide a reduced risk of reinjury. CLINICAL RELEVANCE: The current study identified a decrease in reinjury rate in horses administered MSCs or a combination of MSCs and PRP for tendinopathy and desmopathy. However, results should be interpreted with consideration of the heterogeneity of findings, poor study quality, and high risk of bias in the majority of studies.

Lumbar vertebral bone density is decreased in horses with pituitary pars intermedia dysfunction.

BACKGROUND: Pathological fractures have been reported in equids with pituitary pars intermedia dysfunction (PPID) but their prevalence and pathogenesis is unknown. OBJECTIVES: To compare: (1) bone mineral density (BMD) in weight bearing and nonweight bearing bones in PPID+ equids and aged and young PPID- controls; and (2) biomechanical properties of the fourth lumbar vertebral body in PPID+ equids and aged PPID- equids. STUDY DESIGN: Case-control study: five PPID+ equids and six aged and four young PPID- control horses. METHODS: PPID status was based on clinical signs and necropsy examination of the pituitary gland (PG). The lumbar vertebral column, right front third metacarpus (MC3), left hind third metatarsus (MT3), and PG were removed after euthanasia. BMD was determined by quantitative computed tomography of regions of interest (ROI) in each bone and biomechanical testing was performed on the fourth lumbar vertebral body. Serum concentrations of parathormone (PTH), ionised Ca++ , 25-hydroxyvitamin D, and osteocalcin (OC) were also measured. Data were analysed using one-way ANOVA and correlation analyses. RESULTS: BMD of trabecular and cortical regions of interest (ROI) of the third, fourth (L4), and fifth lumbar vertebrae were significantly lower in PPID+ equids as compared with aged (p < 0. 001) and young (p < 0.01) PPID- controls. In contrast, no differences were found in BMD of trabecular or cortical ROIs of MC3 and MT3 between groups. No differences were detected in force at fracture, displacement at fracture, Young's modulus or strain of L4 between PPID+ and aged PPID- horses. No differences were found in serum PTH, ionised Ca++ , 25-hydroxyvitamin D, or OC concentrations between groups. MAIN LIMITATIONS: Limited number of equids studied and variation in test results. CONCLUSIONS: BMD of nonweight bearing bones can be decreased with PPID and could increase risk of developing pathological fractures.

Exercising sheep as a preclinical model for musculoskeletal research.

OBJECTIVE: To establish an orthopedic, preclinical, ovine model of controlled exercise using an equine walker. ANIMALS: 20 Dorset-Polypay sheep. PROCEDURES: Sheep underwent 11 weeks of exercise, 4 days per week. Exercise duration and intensity increased until sheep performed 25 minutes at 1.3 m/s and 5 min at 2.0 m/s. Physical/lameness examinations were conducted every 14 days. Blood was collected every 28 days for analysis of serum bone biomarkers (SBB): bone alkaline phosphatase (BALP), procollagen type I amino-terminal propeptide (PINP), carboxy-telopeptide of type I collagen cross-links (CTX-I), tartrate-resistant acid phosphatase 5b (TRAP5b), and receptor activator of nuclear factor-kß ligand (RANKL). RESULTS: Sheep adapted easily to group exercise. Animals grew taller (P = .006) but had a 4% weight loss (P = .003). RANKL was reduced on days 28 and 84 compared to day 56 (P < .05), CTX-1 was reduced on days 28 and 84 compared to days 0 and 56 (P < .05), and TRAP5b was greater on day 28 compared to day 0 (P = .009). BALP and PINP did not change. CLINICAL RELEVANCE: The described preclinical model of exercising sheep has distinct advantages including ease of handling, an established lameness scale, commercially available ovine SBB assays, and the ability to alter footing characteristics and complete circular exercise. Decreasing CTX-I and RANKL with no change in BALP and PINP suggests reduced bone resorption over the study period. Future studies may include a sedentary group or utilize adult animals to alleviate any influence of growth on SBB.

Clodronate disodium is neither cytotoxic nor cytoprotective to normal and recombinant equine interleukin-1ß-treated joint tissues in vitro.

OBJECTIVE: To determine the effects of clodronate disodium (CLO) on control and recombinant equine interleukin-1ß (IL-1ß)-treated equine joint tissues. STUDY DESIGN: In vitro experimental study. SAMPLE POPULATION: Cartilage explants, chondrocytes, and synoviocytes (n = 3 horses). METHODS: Monolayer cultures of chondrocytes and synoviocytes from three horses were subjected to: control media (CON), 5 ng/ml CLO (C/low), 50 ng/ml CLO (C/med), 100 ng/ml CLO (C/high), with and without IL-1ß, and 10 ng/ml IL-1ß (IL) alone for 72 hours. Cartilage explants from three horses were subjected to CON, IL, C/low, and C/med with and without IL-1ß for 72 hours. Culture media was analyzed for prostaglandin-E2 (PGE2 ), interleukin-6 (IL-6), and nitric oxide (NO). Explant media was analyzed for glycosaminoglycan (GAG) content and NO. At 72 hours, explant and monolayer culture viability were assessed, and explant GAG content was measured. RESULTS: IL-1ß treatment resulted in higher media concentrations of GAG, NO, PGE2 , and IL-6 compared to the CON treatment (p < .05), demonstrating a catabolic effect of IL-1ß on explants and monolayer cultures. CLO treatments did not increase media concentrations of GAG, NO, PGE2 , or IL-6 compared to CON, indicating no cytotoxic effect. Nevertheless, CLO treatments administered to IL-1ß-treated monolayer cultures and explants did not significantly reduce the inflammatory response regardless of concentration. CONCLUSION: CLO did not demonstrate cytotoxic nor cytoprotective effects in normal and IL-1ß-stimulated chondrocytes, synoviocytes or explants in culture. CLINICAL SIGNIFICANCE: This study does not support the use of CLO as an anti-inflammatory treatment. Further research is necessary to confirm any anti-inflammatory effects of CLO on joint tissues.

Current use of biologic therapies for musculoskeletal disease: A survey of board-certified equine specialists.

OBJECTIVE: To evaluate the use of mesenchymal stem cells (MSCs), autologous conditioned serum (ACS), platelet-rich plasma (PRP), and autologous protein solution (APS) for the treatment of equine musculoskeletal disease by diplomates of the American College of Veterinary Surgery (ACVS), and American College of Veterinary Sports Medicine and Rehabilitation (ACVSMR). STUDY DESIGN: Cross-sectional study. SAMPLE POPULATION: Diplomates (n = 423). METHODS: An email link was sent to ACVS and ACVMR diplomates. A survey contained 59 questions regarding demographics, as well as indications, frequency, adverse effects, and limitations of use. Responses were analyzed using Fisher's exact test. RESULTS: One hundred and fifty four surveys were analyzed. Years in practice and type of practice were not associated with biologic therapy use. PRP was the most used therapy (120/137; 87.5%). PRP and MSCs were most often administered intralesionally while ACS and APS were most often administered intra-articularly. ACS (50/104; 48.1%) treatment was repeated commonly within 2 weeks of initial injection. MSCs (39/90; 43.3%) and PRP (38/100; 38%) were commonly repeated 1-2 months after initial injection and APS was typically repeated >4 months after initial injection (21/53; 39.6%). Local inflammation and expense were the most common adverse effect and limitation of use. CONCLUSION: Diplomates most commonly utilized PRP and MSC intralesionally for soft-tissue injuries, and ACS and ACP intra-articularly for joint injury. Protocols for repeated administration varied widely. Local inflammation was a clinical concern with the use of biologics. CLINICAL SIGNIFICANCE: Biologic therapies are used commonly by ACVS and ACVSMR diplomates for soft tissue and joint disease.

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Mesenchymal stem cells for treatment of musculoskeletal disease in horses: Relative merits of allogeneic versus autologous stem cells.

Mesenchymal stem cells (MSCs) are widely used for treatment of musculoskeletal diseases in horses, but there is ongoing debate regarding the relative safety and efficacy of allogeneic MSCs, compared with autologous equine MSCs. This review summarises the currently available published data regarding the therapeutic use of autologous and allogeneic MSCs in horses. Arguments that have been advanced against the use of allogeneic MSCs include higher risk of immunological reactions and shorter cell survival times following injection. Arguments favouring the use of allogeneic MSCs include the ability to bank cells and reduce the time to treatment, to collect MSCs from younger donor animals and the ability to manipulate banked cells prior to administration. In vitro studies and a limited set of experimental in vivo studies have indicated that adverse immunological reactions may occur when allogeneic MSCs are administered to horses. However, newer studies lack evidence of inflammatory reactions or adverse clinical responses when allogeneic MSCs are administered and compared with autologous MSCs. Thus, while the relative merits of allogeneic vs autologous MSCs for treatment of musculoskeletal injuries in horses have not been fully established, accumulating evidence from studies in horses suggests that allogeneic MSCs maybe a safe alternative to autologous MSCs. Large, properly designed, randomised trials in addition to careful immunological evaluation of short-term and long-term, local and systemic immune responses are needed to more fully resolve the issue.

Single and repeated intra-articular injections in the tarsocrural joint with allogeneic and autologous equine bone marrow-derived mesenchymal stem cells are safe, but did not reduce acute inflammation in an experimental interleukin-1ß model of synovitis.

BACKGROUND: Allogeneic and autologous bone marrow-derived mesenchymal stem cells (BMDMSCs) have been administered in equine joints for their anti-inflammatory effects. However, allogeneic BMDMSC offer multiple clinical and practical advantages. Therefore, it is important to determine the relative effectiveness of allogeneic vs autologous BMDMSCs. OBJECTIVES: The objective of the study was to compare the inflamed joint response to autologous vs allogeneic BMDMSCs injections, and to determine if either treatment generated an anti-inflammatory effect. STUDY DESIGN: Randomised controlled study. METHOD: Bone marrow was harvested from eight horses. Autologous BMDMSCs and pooled allogeneic BMDMSCs were culture expanded, cryopreserved and thawed immediately prior to administration. Ten million autologous BMDMSCs were administered with 75 ng rIL-1ß into one tarsocrural joint and the contralateral tarsocrural joint received allogeneic BMDMSC plus 75 ng rIL-1ß. Repeat injections were performed with the same treatment administered into the same joint. Four additional horses received 75 ng rIL-1ß alone in a single tarsocrural joint. Clinical parameters (lameness, joint circumference and joint effusion) and synovial fluid parameters, including nucleated cell count (NCC), differential cell count, total protein (TP), prostaglandin E2 (PGE2 ) and C-reactive protein (CRP), were measured at baseline, 6, 12, 24, 72, 168 and 336 hours post-injection. RESULTS: No difference was detected between autologous and allogeneic treatment groups with respect to subjective lameness, joint effusion, joint circumference, NCC, TP, differential cell count, CRP or PGE2 . Neither autologous nor allogeneic treatments resulted in an improvement in clinical or cytological parameters over that elicited by rIL-1ß alone. MAIN LIMITATIONS: A single dose of rIL-1ß was evaluated and resulted in a severe synovitis which may have been too severe to observe a BMDMSC-mediated effect. CONCLUSIONS: This study revealed that allogeneic and autologous BMDMSCs resulted in an equivalent clinical and cytological response. Allogeneic and autologous BMDMSCs were equally ineffective in reducing the inflammatory response from acute rIL-1ß-induced joint inflammation in horses.

Can Extracorporeal Shockwave Promote Osteogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells In Vitro?

Both bone marrow-derived mesenchymal stem cells (BMDMSCs) and extracorporeal shockwave (ESW) have shown promise for enhancing fracture repair. If exposure of BMDMSCs to ESW enhances osteogenic differentiation, these therapies may be combined in vivo or used as a method for preconditioning BMDMSCs. The objective of this study was to determine the effect of ESW on the osteogenic ability of equine BMDMSCs. We hypothesized that ESW would promote osteogenesis evidenced by increased gene expression, alkaline phosphatase (ALPL) expression, slide morphologic score, and protein expression. BMDMSCs were evaluated from six horses. BMDMSCs were culture expanded to passage 3, dissociated, then placed in conical tubes. Treatment cells ("shocked") were exposed to 500 pulses at 0.16 mJ/mm2 energy. Cells were then reseeded and grown in either growth medium or osteogenic medium. Cellular proliferation and trilineage potential were determined. Cellular morphology was scored and cells were harvested at 1, 3, 7, 14, and 21 days for rtPCR gene expression of osteogenic markers [osteonectin (ONT), osteocalcin (OCN), ALPL, collagen type 3 (COL3), and runt-related transcription factor 2 (RUNX2)]. Media supernatants were evaluated for secretion of BMP-2, VEGF, TGFß, and PGE2 and cellular lysates were evaluated for ALPL production. There was no difference between the proliferative ability of shocked cells versus unshocked cells in either growth medium or osteogenic medium. ALPL production was greater in shocked cells maintained in osteogenic medium versus unshocked cells in osteogenic medium at day 3 (P < 0.005). Independent of media type, ESW caused a decrease in VEGF and TGFß production at day 3. No significant increases in gene expression were identified by rtPCR. Exposure of BMDMSCs to ESW does not result in negative effects. An initial significant increase in ALPL was detected but no persistent osteogenic effect was observed with cell expansion.

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self-assembling peptide hydrogel.

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFß). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFß. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ∼75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ∼65-fold higher than positive controls, which was attributed to the absence of TGFß. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ∼7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self-assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFß may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368-1375, 2019.

In Vitro Effects of High-Intensity Laser Photobiomodulation on Equine Bone Marrow-Derived Mesenchymal Stem Cell Viability and Cytokine Expression.

OBJECTIVE: This study aimed to examine the influence of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on equine bone marrow-derived mesenchymal stem cell (MSC) viability, proliferation, and cytokine expression in vitro. BACKGROUND: Photobiomodulation of cells using monochromatic light is a technique designed to influence cellular processes. Previous studies have shown dose-dependent effects of low-level laser irradiation on cell proliferation and cytokine expression in a range of cell types and species. Evidence for the influence of 1064 nm wavelength near-infrared irradiation on MSCs is sparse, and high-energy doses have shown inhibitory effects. METHODS: MSC cultures from six horses were exposed to 1064 nm irradiation with an energy density of 9.77 J/cm2 and a mean output power of 13.0 W for 10 sec. MSC viability and proliferation were evaluated through flow cytometry and real-time live cell analysis. Gene expression and cytokine production in the first 24 h after irradiation were analyzed through polymerase chain reaction (PCR), multiplex assay, and enzyme-linked immunosorbent assay. RESULTS: No difference in viability was detected between irradiated and control MSCs. Irradiated cells demonstrated slightly lower proliferation rates, but remained within 3.5% confluence of control cells. Twenty-four hours after irradiation, irradiated MSCs demonstrated a significant increase in expression of interleukin (IL)-10 and vascular endothelial growth factor (VEGF) compared with control MSCs. CONCLUSIONS: Under these irradiation parameters, equine MSCs remained viable and expressed increased concentrations of IL-10 and VEGF. IL-10 has an anti-inflammatory action by inhibiting the synthesis of proinflammatory cytokines at the transcriptional level. This response to 1064 nm irradiation shows promise in the photobiomodulation of MSCs to enhance their therapeutic properties.

Left paralumbar laparoscopic bilateral ovariectomy in mares.

OBJECTIVE: To develop a novel technique for left paralumbar laparoscopic bilateral ovariectomy in standing, sedated mares. STUDY DESIGN: Prospective descriptive clinical study. ANIMALS: Twelve client-owned mares. METHODS: Healthy mares with normal ovarian palpation and ultrasonographic appearance were enrolled. Horses were restrained in standing stocks and administered a variable rate infusion of sedation. A left paralumbar bilateral ovariectomy was performed with 1 laparoscopic and 3 instrument portals. Access to the right ovary was facilitated by retracting the descending colon and associated mesentery with a fan retractor. Ovaries were removed with traumatic forceps, and a blunt tip vessel sealing device. A laparoscopic portal within the 17th intercostal space provided good visualization of the right ovary and reduced interference between instrumentation. Complications were recorded during hospitalization. In addition, an owner follow-up questionnaire was used to determine complications after discharge from the hospital. RESULTS: Left paralumbar bilateral ovariectomy was successful in all 12 mares, with a mean surgical time of 76 ± 8 minutes. Postoperative complications occurred in 4/12 (33%) mares and included fever, mild colic, incisional swelling, and azotemia. Complications resolved and mares returned to full activity, with complete owner satisfaction. CONCLUSIONS: Bilateral ovariectomy can be successfully achieved with minimal morbidity in standing mares with normal ovarian ultrasonographic appearance via left paralumbar laparoscopy.

Autologous and Allogeneic Equine Mesenchymal Stem Cells Exhibit Equivalent Immunomodulatory Properties In Vitro.

The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared with those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. The immunosuppressive activity of the two cell types was evaluated by adding autologous or allogeneic BMDMSCs to activated lymphocytes and assessing suppression of lymphocyte proliferation and IFNγ production. Fifty-six allogeneic and 12 autologous combinations were evaluated. Studies were also done to elucidate mechanisms by which equine mesenchymal stem cells (MSCs) suppress lymphocyte function. Potential mechanisms evaluated included production of prostaglandin E2 (PGE2), nitric oxide, transforming growth factor-beta, and indoleamine 2,3-dioxygenase. We found that autologous and allogeneic BMDMSCs both induced mild but equivalent levels of spontaneous lymphocyte activation in vitro. In in vitro assays assessing the ability of BMDMSCs to suppress activated lymphocytes, both allogeneic and autologous BMDMSCs suppressed T cell proliferation and IFNγ production to an equal degree. The primary mechanism of equine BMDMSC suppression of T cells was mediated by PGE2. We concluded that allogeneic and autologous BMDMSCs are equivalent in terms of their immunomodulatory properties, and stimulated peripheral blood mononuclear cells appear to trigger the immunosuppressive properties of MSCs. Therefore, both cell types appear to have equal potency in modulating inflammatory processes related to acute or chronic musculoskeletal injuries in the horse.

The Effects of Mepivacaine Hydrochloride on Antimicrobial Activity and Mechanical Nociceptive Threshold During Amikacin Sulfate Regional Limb Perfusion in the Horse.

OBJECTIVE: To determine the effect of intravenous regional limb perfusion (IVRLP) with a combination of mepivacaine hydrochloride and amikacin sulfate on synovial fluid amikacin sulfate concentration, antimicrobial activity, and mechanical nociceptive threshold (MNT). STUDY DESIGN: Experimental study. ANIMALS: Healthy adult horses (n=9). METHODS: One IVRLP treatment was randomly administered by cephalic vein of each limb: amikacin alone (1 g amikacin in 60 mL saline) or amikacin with mepivacaine (1 g amikacin and 500 mg mepivacaine in 60 mL saline). Opposite treatments were repeated after a 24 hour wash-out period. Amikacin concentration and antimicrobial activity were determined for synovial fluid from middle carpal joints at tourniquet removal and 30 minutes following. Zone of inhibition was determined for Staphylococcus aureus and Escherichia coli. MNT was determined at 3 dorsal metacarpal locations prior to and after sedation, after Esmarch tourniquet application, and 30 minutes after IVRLP prior to and after tourniquet removal. RESULTS: Two limbs from each treatment group were removed because of undetectable amikacin concentrations for a total of 14 data sets analyzed. Synovial fluid amikacin concentrations and zone of inhibition were not significantly different between treatments at any time point. MNT were significantly increased 30 minutes after IVRLP prior to and following tourniquet removal using amikacin and mepivacaine (median, range; 40.0 µg/mL, 38.7-40.0 and 40.0, 25.8-40.0, respectively) compared to amikacin alone (19.5 µg/mL, 18.7-25.6 and 15.3, 13.2-20.5, respectively). CONCLUSION: Addition of mepivacaine to amikacin for IVRLP in the horse as a means of providing analgesia without decreasing antimicrobial activity.