OBJECTIVE To compare the anticollagenase efficacy of fresh feline, canine, and equine serum and plasma on in vitro corneal degradation. SAMPLE Grossly normal corneas from recently euthanized dogs, cats, and horses and fresh serum and plasma from healthy dogs, cats, and horses. PROCEDURES Serum and plasma were pooled by species and used for in vitro experiments. Corneas were collected and stored at −80°C. Sections of cornea were dried, weighed, and incubated in saline (0.9% NaCl) solution with clostridial collagenase and homologous fresh serum or plasma. Corneal degradation was assessed as the percentage of corneal weight loss and hydroxyproline concentration, compared with results for positive and negative control samples. RESULTS Homologous fresh serum and plasma significantly reduced the percentage of corneal weight loss, compared with results for positive control samples. No significant difference was found in percentage of corneal weight loss between incubation with serum or plasma for feline, canine, and equine corneas. Canine serum and plasma significantly reduced hydroxyproline concentrations, whereas inclusion of feline and equine serum or plasma did not, compared with results for positive control samples. Hydroxyproline concentrations were moderately correlated with percentage of corneal weight loss for feline samples and weakly correlated for equine samples, but they were not correlated for canine samples. CONCLUSIONS AND CLINICAL RELEVANCE In this study, the anticollagenase efficacy of fresh feline, canine, and equine serum was not different from that of plasma. Plasma should be an acceptable substitute for serum in the topical treatment of keratomalacia.

OBJECTIVE To evaluate species differences and effects of storage duration and temperature on the anticollagenase efficacy of canine, feline, and equine serum on in vitro corneal degradation. SAMPLES Corneas and serum from dogs, cats, and horses. PROCEDURES Clinically normal corneas from dogs, cats, and horses were harvested within 2 hours after euthanasia. Serum samples from dogs, cats, and horses were collected and pooled by species. Corneal specimens were incubated with collagenase derived from Clostridium histolyticum, 5mM calcium chloride in saline (0.9% NaCl) solution, and feline, canine, or equine serum that had been stored for 0, 30, 90, or 180 days at −20° or −80°C. Following incubation, the corneal weight loss percentage and hydroxyproline concentration in the incubation fluid were calculated and compared among experimental combinations. RESULTS Feline serum was more effective than canine or equine serum for minimizing corneal weight loss. Incubation with feline or equine, but not canine, serum significantly reduced hydroxyproline production. Serum storage duration did not affect corneal weight loss, but the hydroxyproline concentration was greater for corneal specimens that were incubated with serum that was stored for 90 days, compared with that for corneal specimens incubated with serum that was stored for 0, 30, or 180 days. Serum storage temperature did not affect corneal weight loss or hydroxyproline concentration. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that serum reduced corneal degradation in vitro, and the duration and temperature at which serum was stored did not affect its anticollagenase efficacy.

Objective—To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs. Animals—5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints. Procedures—Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-β1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed. Results—In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells. Conclusions and Clinical Relevance—Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.

Full-thickness skin wounds were created on the dorsum of both metacarpi in 8 horses. Three topical treatment regimens were studied. All wounds were bandaged with a nonadherent dressing, which was held in place with a snug elastic wrap. Group-A wounds were treated with a proprietary topical wound medication that consisted of a spray and an ointment. Group-B wounds were treated with the same regimen, except the putative active ingredients in the ointment were omitted. Group-C wounds were treated with a dry nonadherent bandage only. Wound dressings were changed every day and the limbs were photographed every other day until the wounds were healed. Specimens of normal skin and biopsy specimens of healed wounds were examined histologically and were assayed for hydroxyproline content. Wound healing measurements quantitated for each wound were number of days to healing, maximal wound size attained, day wound contraction commenced, day epithelium first noticed, rate of wound contraction, final wound size, and fraction of the wound that healed by contraction. The cosmetic appearance of the healed wounds was also graded. Significant differences were not noticed in hydroxyproline content, histologic appearance, or any of the wound healing measurements between treatment groups. The cosmetic appearance of healed group-A and -B wounds was significantly better than the appearance of group-C wounds. The topical treatment regimens studied neither enhanced nor inhibited wound healing in this study.

OBJECTIVE To characterize aminoaciduria and plasma amino acid concentrations in dogs with hepatocutaneous syndrome (HCS). ANIMALS 20 client-owned dogs of various breeds and ages. PROCEDURES HCS was definitively diagnosed on the basis of liver biopsy specimens (n = 12), gross and histologic appearance of skin lesions (4), and examination of skin and liver biopsy specimens (2) and presumptively diagnosed on the basis of cutaneous lesions with compatible clinicopathologic and hepatic ultrasonographic (honeycomb or Swiss cheese pattern) findings (2). Amino acid concentrations in heparinized plasma and urine (samples obtained within 8 hours of each other) were measured by use of ion exchange chromatography. Urine creatinine concentration was used to normalize urine amino acid concentrations. Plasma amino acid values were compared relative to mean reference values; urine-corrected amino acid values were compared relative to maximal reference values. RESULTS All dogs had generalized hypoaminoacidemia, with numerous amino acid concentrations < 50% of mean reference values. The most consistent and severe abnormalities involved glutamine, proline, cysteine, and hydroxyproline, and all dogs had marked lysinuria. Urine amino acids exceeding maximum reference values (value > 1.0) included lysine, 1-methylhistidine, and proline. CONCLUSIONS AND CLINICAL RELEVANCE Hypoaminoacidemia in dogs with HCS prominently involved amino acids associated with the urea cycle and synthesis of glutathione and collagen. Marked lysinuria and prolinuria implicated dysfunction of specific amino acid transporters and wasting of amino acids essential for collagen synthesis. These findings may provide a means for tailoring nutritional support and for facilitating HCS diagnosis.

Eight mature horses with no prior signs of joint disease or history of intra-articular therapy were treated with 8 weekly intra-articular injections of methylprednisolone acetate. Treatments were given at a dose of 120 mg/joint into the right radiocarpal and intercarpal joints, with the left joints as untreated controls. Articular cartilage samples were obtained at necropsy 1, 4, and 8 weeks after the last injection. Compared with controls, cartilage from injected joints had loss of hematoxylin basophilia and decreased intensity of staining in safranin O fast green dye. Chondrocyte necrosis and hypocellularity were observed in all samples of cartilage from treated joints. Proteoglycan content and its rate of synthesis were reduced. There was a progressive loss of proteoglycan content, whereas proteoglycan synthesis increased somewhat 4 and 8 weeks after treatment. Collagen content was unchanged, but its rate of synthesis was markedly inhibited. Collagen synthesis did not recover, but remained decreased at 5 to 15% of the values from untreated cartilage. Water percentage was increased, but fibronectin content was not significantly different. A single injection of methylprednisolone acetate was also given into the right metacarpophalangeal joints of 3 of the 8 horses in this group, with the left joints serving as untreated controls. Sixteen weeks after the treatment, cartilage of the treated joints had a loss of histochemical staining and proteoglycan content was reduced to 50% of control values. The mean rate of proteoglycan synthesis and mean fibronectin content were increased, but the differences were not statistically significant (P greater than 0.05). Other variables were essentially unchanged. For control studies, the right carpal joints of 2 additional horses were injected with the drug suspension vehicle. All measurements, compared with those of samples from untreated joints, were unchanged. On the basis of our findings, we concluded that the effects on cartilage of intra-articular injections of methylprednisolone acetate were not ameliorated at 8 weeks after 8 weekly injections or 16 weeks after a single injection. Cartilage remained biochemically and metabolically impaired.