Botulinum neurotoxin type A does not exert concentration-dependent effects on equine articular cartilage in vitro.

OBJECTIVE: To determine whether Botulinum neurotoxin type A (BoNT-A) ameliorates the effects of interleukin 1 (IL-1) on equine articular cartilage, or exerts negative effects on normal equine articular cartilage homeostasis in vitro. SAMPLE: Articular cartilage explants from 6 healthy femoropatellar joints of 3 adult horses. METHODS: Explants were allocated to the IL-1 challenged or unchallenged group, then exposed to 1 of 6 concentrations of BoNT-A (0, 1, 10, 50, 100, or 500 pg/mL) for 96 hours. To assess BoNT-A's effects on inflammation, prostaglandin E2 (PGE2) was measured in media via ELISA. Matrix degradation was determined as the percentage of sulfated glycosaminoglycans (sGAG) released from explants via dimethylmethylene blue assay. Aggrecan synthesis was estimated using CS846 ELISA and collagen type II degradation was estimated using C2C ELISA on media. Chondrocyte apoptosis was assessed via in-situ TUNEL assay. Generalized linear mixed models were fitted to determine treatment effects using α = 0.05. RESULTS: The challenge with IL-1 resulted in increased concentrations of PGE2 and CS846 in media and increased release of sGAG from explants. BoNT-A did not significantly impact PGE2 or CS846 concentration in media, percentage of sGAG released, or chondrocyte apoptosis in IL-1 challenged or unchallenged cartilage explants. The concentration of C2C in media was below the quantifiable limit of the ELISA in all samples. CLINICAL RELEVANCE: BoNT-A did not show chondroprotective effects or have negative effects on cartilage homeostasis in vitro at the concentrations tested. While chondroprotective effects were not observed, BoNT-A may be safe for intraarticular use. In vivo testing is warranted before clinical use.

Bilateral dorsal rotation of the talus with tibiotarsal, talocalcaneal, and proximal intertarsal joint subluxation in an alpaca.

CASE DESCRIPTION: A 14-month-old female alpaca presented with a 3-week history of acute left hind limb lameness and swelling of the left tarsal region. CLINICAL FINDINGS: Radiography revealed intermittent dorsal rotation of the talus with tibiotarsal, talocalcaneal, and proximal intertarsal joint subluxation. TREATMENT AND OUTCOME: In an attempt to stabilize the talus, screws were placed in the distomedial aspect of the talus and the plantaromedial aspect of the central tarsal bone, and a stainless-steel wire was placed around the screws in a figure-eight pattern. The screw head of the proximal screw broke within 4 weeks after surgery, but subluxation did not recur, and the lameness resolved. Seven months later, the same condition was diagnosed in the opposite hind limb and was treated similarly. Implants remained intact on this side, but the animal started to show signs of pain and inability to flex the tarsal joint, prompting removal of the distal screw. Subsequently, the animal became sound and produced 2 healthy crias, but was euthanatized 4 years after the second surgery because of coccidiosis. CLINICAL RELEVANCE: Dorsal rotation of the talus with tibiotarsal, talocalcaneal, and proximal intertarsal joint subluxation is a sporadic condition in New World camelids. This report provides the first account of successful treatment by surgical stabilization of the medial aspect of the proximal intertarsal joint.

Outcome of bilateral equid laparoscopic ovariectomies.

OBJECTIVE: To assess outcomes and behavior changes associated with bilateral laparoscopic ovariectomies. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Fifty-one equids. METHODS: Medical records were evaluated from equid bilateral laparoscopic ovariectomies from January 2012 to October 2018 with a potential of 6 months follow-up. Follow-up information obtained by telephone interviews included behavior before and after surgery. Likelihood ratio chi-square tests and odds ratios (OR) with 95% CI were calculated where applicable, with statistical significance at p < .05. RESULTS: Bilateral ovariectomy was performed in 51 cases, with elective (no pathologic ovaries) ovariectomies performed in 41/51 cases. Occasional estrus-like behavior was observed postoperatively in 14/51 (27%) mares, but the behavior was mild and manageable in all cases. There was no age effect on outcome in all bilateral (p = .56) or elective only (p = .36) cases. In 37/41 (90%) elective cases, improvement was observed in the reason for presentation. Some response to altrenogest administration for behavior modification was observed preoperatively in 12/18 (67%) elective cases. Response to altrenogest was not associated with (p = .31) or able to predict a beneficial response to surgery (OR = 5.5; 95% CI = 0.38-78.57; p = .21). CONCLUSION: Response to altrenogest in elective cases may not predict behavioral outcome with ovariectomy. Occasional estrus-like behavior in mares postoperatively was not problematic for any owners. Bilateral ovariectomy is a viable treatment option for owners seeking to alleviate undesirable behavior in mares. CLINICAL SIGNIFICANCE: This study should aid veterinarians and horse owners in case selection for bilateral ovariectomy.

Expression of pro-apoptotic markers is increased along the osteochondral junction in naturally occurring osteochondrosis.

Osteochondrosis (OC) is a naturally occurring disease of the articular-epiphyseal cartilage and subchondral bone layers, leading to pain and decreased mobility. The objective of this study was to characterize gene and protein expression of apoptotic markers in chondrocytes surrounding cartilage canals and along the osteochondral junction of osteochondrosis (OC)-affected and normal cartilage, using naturally occurring disease in horses. Paraffin-embedded osteochondral samples (6 OC, 8 normal controls) and cDNA from chondrocytes captured with laser capture microdissection (4 OC, 6 normal controls) were obtained from the lateral trochlear ridge of femoropatellar joints in 14 immature horses (1-6 months of age). Equine-specific caspase-3, caspase-8, caspase-10, Fas, Bcl-2, BAG-1, TNFα, cytochrome C, thymosin-ß10, and 18S mRNA expression levels were evaluated by two-step real-time quantitative PCR. Percentage of cell death was determined using the TUNEL method. Protein expression of caspase-10, Fas, cytochrome C, and thymosin-ß10 was determined following immunohistochemistry. Statistical analysis was performed using the Wilcoxon rank sum test or two-sample t-test (p < 0.05). In OC samples, there was significantly increased gene expression of caspase-10, Fas, cytochrome C, and thymosin-ß10 in chondrocytes along the osteochondral junction and increased Fas gene expression in chondrocytes adjacent to cartilage canals, compared to controls. In OC samples, higher matrix Fas and cytochrome C protein expression, lower mitochondrial cytochrome C protein expression, and a trend for higher cytoplasmic caspase-10 protein expression were found. Collectively, these results suggest that both extrinsic and intrinsic apoptotic pathways are activated in OC cartilage. Increased apoptosis of osteochondral junction chondrocytes may play a role in OC, based on increased gene expression of several pro-apoptotic markers in this location.

Ex vivo penetration of low-level laser light through equine skin and flexor tendons.

OBJECTIVE To measure penetration efficiencies of low-level laser light energy through equine skin and to determine the fraction of laser energy absorbed by equine digital flexor tendons (superficial [SDFT] and deep [DDFT]). SAMPLE Samples of skin, SDFTs, and DDFTs from 1 metacarpal area of each of 19 equine cadavers. PROCEDURES A therapeutic laser with wavelength capabilities of 800 and 970 nm was used. The percentage of energy penetration for each wavelength was determined through skin before and after clipping and then shaving of hair, through shaved skin over SDFTs, and through shaved skin, SDFTs, and DDFTs (positioned in anatomically correct orientation). Influence of hair color; skin preparation, color, and thickness; and wavelength on energy penetration were assessed. RESULTS For haired skin, energy penetration was greatest for light-colored hair and least for dark-colored hair. Clipping or shaving of skin improved energy penetration. Light-colored skin allowed greatest energy penetration, followed by medium-colored skin and dark-colored skin. Greatest penetration of light-colored skin occurred with the 800-nm wavelength, whereas greatest penetration of medium- and dark-colored skin occurred with the 970-nm wavelength. As skin thickness increased, energy penetration of samples decreased. Only 1% to 20% and 0.1% to 4% of energy were absorbed by SDFTs and DDFTs, respectively, depending on skin color, skin thickness, and applied wavelength. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that most laser energy directed through equine skin was absorbed or scattered by the skin. To achieve delivery of energy doses known to positively affect cells in vitro to equine SDFTs and DDFTs, skin preparation, color, and thickness and applied wavelength must be considered.

Tiludronate concentrations and cytologic findings in synovial fluid after intravenous regional limb perfusion with tiludronate in horses.

Anecdotal accounts of tiludronate administration via intravenous regional limb perfusion (IVRLP) exist despite a lack of information regarding safety for synovial structures in the perfused area. The objective of this study was to determine whether tiludronate concentrations in synovial structures after IVRLP with low dose (0.5 mg, LDT) or high dose (50 mg, HDT) tiludronate remain below a value demonstrated in vitro to be safe for articular cartilage (<19,000 ng/ml), and to determine effects of tiludronate on synovial fluid cytology variables compared to saline perfused control limbs. Using a randomized controlled experimental study design, horses received IVRLP with LDT (n = 6) or HDT (n = 6) in one forelimb and IVRLP with saline in the contralateral limb. Synovial fluid cytology variables and tiludronate concentrations were evaluated in navicular bursae (NB), and distal interphalangeal (DIP) and metacarpophalangeal (MCP) joints one week before and 30-45 min after IVRLP, and in DIP and MCP joints 24 h after IVRLP. Data were analyzed with 2-way rmANOVA (p < 0.05). Highest measured synovial fluid tiludronate concentrations occurred 30-45 min post-perfusion. Mean tiludronate concentrations were lower in LDT limbs (MCP = 39.6 ± 14.3 ng/ml, DIP = 118.1 ± 66.6 ng/ml, NB = 82.1 ± 30.2 ng/ml) than in HDT limbs (MCP = 3,745.1 ± 1,536.6 ng/ml, DIP = 16,274.0 ± 5,460.2 ng/ml, NB = 6,049.3 ± 1,931.7 ng/ml). Tiludronate concentration was >19,000 ng/ml in DIP joints of two HDT limbs. Tiludronate was measurable only in synovial fluid from HDT limbs 24 h post-perfusion. There were no differences in synovial fluid cytology variables between control and treated limbs. Conclusions. In some horses, IVRLP with HDT may result in synovial fluid concentrations of tiludronate that may have adverse effects on articular cartilage, based on in vitro data. IVRLP with LDT is unlikely to promote articular cartilage degradation. Further studies to determine a safe and effective dose for IVRLP with tiludronate are needed.

Wnt/ß-catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis.

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin-embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine-specific Wnt signaling molecule mRNA expression levels were evaluated by two-step real-time qPCR. Spatial tissue protein expression of ß-catenin, Wnt-11, Wnt-4, and Dkk-1 was determined by immunohistochemistry. There was significantly decreased Wnt-11 and increased ß-catenin, Wnt-5b, Dkk-1, Lrp6, Wif-1, Axin1, and SC-PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased ß-catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis.

Carpal valgus in llamas and alpacas: Retrospective evaluation of patient characteristics, radiographic features and outcomes following surgical treatment.

This study evaluated outcomes of surgical treatment for carpal valgus in New World camelids and correlated successful outcome (absence of carpal valgus determined by a veterinarian) with patient characteristics and radiographic features. Univariable and multivariable analyses of retrospective case data in 19 camelids (33 limbs) treated for carpal valgus between 1987 and 2010 revealed that procedures incorporating a distal radial transphyseal bridge were more likely (P = 0.03) to result in success after a single surgical procedure. A greater degree of angulation (> 19°, P = 0.02) and younger age at surgery (< 4 months, P = 0.03) were associated with unsuccessful outcome. Overall, 74% of limbs straightened, 15% overcorrected, and 11% had persistent valgus following surgical intervention. To straighten, 22% of limbs required multiple procedures, not including implant removal. According to owners, valgus returned following implant removal in 4 limbs that had straightened after surgery.

Valgus carpien chez les lamas et les alpagas : évaluation rétrospective des caractéristiques des patients, des radiographies et des résultats après un traitement chirurgical. Cette étude a évalué les résultats du traitement chirurgical pour le valgus carpien chez des camélidés du nouveau monde et a établi une corrélation entre les résultats fructueux (absence de valgus carpien déterminée par un vétérinaire) et les caractéristiques du patient et des radiographies. Des analyses à variable unique et à variables multiples des données de cas rétrospectifs chez 19 camélidés (33 jambes) traités pour le valgus carpien entre 1987 et 2010 ont révélé que les procédures intégrant un pont radial distal transphyséal présentaient une plus grande probabilité de succès (P = 0,03) après une seule intervention chirurgicale. Une déviation supérieure (> 19°, P = 0,02) et un jeune âge à la chirurgie (< 4 mois, P = 0,03) étaient associés à des échecs. Dans l'ensemble, 74 % des jambes ont été redressés, 15 % ont été corrigées et 11 % présentaient un valgus persistant après l'intervention chirurgicale. Pour le redressement, 22 % des jambes ont nécessité des procédures multiples, ce qui n'incluait pas l'enlèvement de l'implant. Selon les propriétaires, le valgus est réapparu après l'enlèvement de l'implant dans 4 jambes qui avaient été redressées après la chirurgie.(Traduit par Isabelle Vallières).

Fibrochondrogenic potential of synoviocytes from osteoarthritic and normal joints cultured as tensioned bioscaffolds for meniscal tissue engineering in dogs.

Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous synoviocytes from the affected stifle is an attractive cell source for tissue engineering replacement fibrocartilage. However, the diseased state of these cells may impede in vitro fibrocartilage formation. Synoviocytes from 12 osteoarthritic ("oaTSB") and 6 normal joints ("nTSB") were cultured as tensioned bioscaffolds and compared for their ability to synthesize fibrocartilage sheets. Gene expression of collagens type I and II were higher and expression of interleukin-6 was lower in oaTSB versus nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth muscle staining and less collagen I and II staining on histologic analysis, whereas collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic joint-origin synoviocytes can produce extracellular matrix components of meniscal fibrocartilage at similar levels to normal joint-origin synoviocytes, which makes them a potential cell source for canine meniscal tissue engineering.

Effects of low and high dose intraarticular tiludronate on synovial fluid and clinical variables in healthy horses-a preliminary investigation.

To determine effects of intraarticularly administered tiludronate on articular cartilage in vivo, eight healthy horses were injected once with tiludronate (low dose tiludronate [LDT] 0.017 mg, n = 4; high dose tiludronate [HDT] 50 mg, n = 4) into one middle carpal joint and with saline into the contralateral joint. Arthrocentesis of both middle carpal joints was performed pre-treatment, and 10 min, 24 h, 48 h, 7 and 14 days after treatment. Synovial nucleated cell counts and total solids, tiludronate, sulfated glycosaminoglycan (sGAG), chondroitin sulfate 846 epitope (CS-846, a measure of aggrecan synthesis), and collagen type II cleavage neoepitope (C2C) concentrations were determined. Histologic analysis of joint tissues and sGAG quantitation in cartilage was performed at 14 days in HDT horses. Data were analyzed by repeated measures non-parametric ANOVA and Wilcoxon signed-rank test. High dose tiludronate administration produced synovial fluid tiludronate concentrations of 2,677,500 ng/mL, exceeding concentrations that were safe for cartilage in vitro, and LDT administration produced synovial fluid concentrations of 1,353 ng/mL, remaining below concentrations considered potentially detrimental to cartilage. With HDT, synovial fluid total solids concentration was higher at 24 h and 7 days and sGAG concentration was higher at 48 h, compared to control joints. Synovial fluid CS-846 concentration was increased over pre-treatment values in HDT control but not in HDT treated joints at 24 and 48 h. All joints (HDT and LDT control and treated) showed a temporary decrease in synovial fluid C2C concentration, compared to pre-treatment values. Histologic features of articular cartilage and synovial membrane did not differ between HDT treated and control joints. High dose tiludronate treatment caused a transient increase in synovial total solids and temporarily increased proteoglycan degradation in cartilage. Although clinical significance of these changes are questionable, as they did not result in articular cartilage damage, further investigation of the safety of intraarticular HDT in a larger number of horses is warranted.

Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog.

Tissue engineering is a promising field of study toward curing the meniscal deficient stifle; however the ideal cell type for this task is not known. We describe here the extraction of synoviocytes and meniscal fibrochondrocytes from arthroscopic debris from six dogs, which were cultured as tensioned bioscaffolds to synthesize meniscal-like fibrocartilage sheets. Despite the diseased status of the original tissues, synoviocytes and meniscal fibrochondrocytes had high viability at the time of removal from the joint. Glycosaminoglycan and collagen content of bioscaffolds did not differ. Meniscal fibrochondrocyte bioscaffolds contained more type II collagen, but collagen deposition was disorganized, with only 30-40% of cells viable. The collagen of synoviocyte bioscaffolds was organized into sheets and bands and 80-90% of cells were viable. Autologous, diseased meniscal fibrochondrocytes and synoviocytes are plausible cell sources for future meniscal tissue engineering research, however cell viability of meniscal fibrochondrocytes in the tensioned bioscaffolds was low.

Culture of canine synoviocytes on porcine intestinal submucosa scaffolds as a strategy for meniscal tissue engineering for treatment of meniscal injury in dogs.

Meniscal injury is a common cause of canine lameness. Tissue engineered bioscaffolds may be a treatment option for dogs suffering from meniscal damage. The aim of this study was to compare in vitro meniscal-like matrix formation and biomechanical properties of porcine intestinal submucosa sheets (SIS), used in canine meniscal regenerative medicine, to synoviocyte-seeded SIS bioscaffold (SSB), cultured with fetal bovine serum (SSBfbs) or chondrogenic growth factors (SSBgf). Synoviocytes from nine dogs were seeded on SIS and cultured for 30days with 17.7% fetal bovine serum or recombinant chondrogenic growth factors (IGF-1, TGFß1 and bFGF). The effect on fibrochondrogenesis was determined by comparing mRNA expression of collagen types Iα and IIα, aggrecan, and Sry-type homeobox protein-9 (SOX9) as well as protein expression of collagens I and II, glycosaminoglycan (GAG), and hydroxyproline. The effect of synoviocyte seeding and culture conditions on biochemical properties was determined by measuring peak load, tensile stiffness, resilience, and toughness of bioscaffolds. Pre-culture SIS contained 13.6% collagen and 2.9% double-stranded DNA. Chondrogenic growth factor treatment significantly increased SOX9, collagens I and IIα, aggrecan gene expression (P<0.05), and histological deposition of fibrocartilage extracellular matrix (GAG and collagen II). Culture with synoviocytes increased SIS tensile peak load at failure, resilience, and toughness of bioscaffolds (P<0.05). In conclusion, culturing SIS with synoviocytes prior to implantation might provide biomechanical benefits, and chondrogenic growth factor treatment of cultured synoviocytes improves in vitro axial meniscal matrix formation.

Synoviocyte neotissues towards in vitro meniscal tissue engineering.

Meniscal injuries are a common cause of pain and osteoarthritis in dogs. We describe here the production of synoviocyte-derived autologous neotissues for potential application in meniscal tissue engineering, via two different culture techniques: contracted or tensioned synthesis of synoviocyte neotissues. Synoviocytes were obtained during routine stifle arthroscopy and cultured from 14 dogs with naturally occurring osteoarthritis of the stifle. Neotissues were analyzed for meniscal-like matrix components and their gene expression, inflammatory gene expression, and cell viability. Tension improved cell viability, and, independent of cell viability, fibrochondrogenic activity by promoting expression of collagen type 1 and aggrecan genes and attenuating gene expression of IL-6. Through this mechanism tension increased collagen protein content and chondrogenic index of neotissues. Alpha smooth muscle actin was present in all neotissues and was responsible for grossly visible contractile behavior. Application of tension to synoviocytes may be a viable culture method towards in vitro meniscal tissue engineering.

Concentration-dependent effects of tiludronate on equine articular cartilage explants incubated with and without interleukin-1ß.

OBJECTIVE: To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1ß (rEq IL-1). SAMPLE: Articular cartilage explants from the femorotibial joints of 3 young adult horses. PROCEDURES: Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E(2) (PGE(2)) concentrations in culture medium and explant digests were analyzed via PGE(2) enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. RESULTS: rEq IL-1 increased PGE(2) concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1-induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. CONCLUSIONS AND CLINICAL RELEVANCE: Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE(2) secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Equine urolithiasis.

The prevalence of equine urolithiasis has been estimated to be low. In horses with clinical signs of urolithiasis, uroliths are most commonly encountered in the urinary bladder, but it is not uncommon to detect uroliths in more than one location. The most common clinical signs for cystic calculi are urine scalding of the hind limbs, hematuria, tenesmus and dysuria. Numerous surgical techniques and approaches have been described for the treatment of urolithiasis in horses; however, independent of which approach is chosen, the goal should be to remove all calculi completely from the urinary tract, thus decreasing the chance of recurrence of urolithiasis. Laser lithotripsy and shock wave lithotripsy represent means to fragment uroliths with little morbidity, but limited availability of and costs associated with the equipment have precluded these technologies from being used more commonly in horses.