Objective: To assess the in vitro effects of various nalbuphine concentrations on viability and wound healing ability of corneal cells and potential drug transport through the corneal epithelium. Sample: Cultured canine and human corneal epithelial cells (CECs) and cultured canine corneal stromal fibroblasts. Procedures: CECs and stromal fibroblasts were exposed to nalbuphine (concentration of solutions ranged from 0% to 1.2%) for up to 30 minutes, and viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A standard scratch test technique was used. Wound healing of CECs and stromal fibroblasts was evaluated following treatment with nalbuphine solutions < 0.1%. Liquid chromatography–mass spectrometry–mass spectrometry analysis was used to evaluate drug transport across a monolayer and a multilayer of human CECs. Results: A progressive decrease in viability was detected in canine CECs for all nalbuphine treatment groups, whereas treatment with only 0.5% or 1.2% nalbuphine significantly reduced corneal stromal fibroblast viability, compared with results for control cells. Within 24 hours, treatment with 0.1% nalbuphine solution significantly altered the healing rate of both canine CECs and stromal fibroblasts. Continuous increases in transport rates of nalbuphine were detected with time for both the monolayer and multilayer of human CECs. Conclusions and Clinical Relevance: In vitro, nalbuphine potentially could penetrate through corneal tissue, but it may cause damage to the corneal epithelium and stromal fibroblasts. Therefore, nalbuphine potentially may impair corneal wound healing.

Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.

Objective: To describe the immunopathologic characteristics of superficial stromal immune-mediated keratitis (IMMK) immunopathologically by characterizing cellular infiltrate in affected corneas of horses. Animals: 10 client-owned horses with IMMK. Procedures: Immunohistochemical staining was performed on keratectomy samples with equine antibodies against the T-cell marker CD3 and B-cell marker CD79a (10 eyes) and the T-helper cytotoxic marker CD4 and T-cell cytotoxic marker CD8 (6 eyes). Percentage of positively stained cells was scored on a scale from 0 (no cells stained) to 4 (> 75% of cells stained). Equine IgG, IgM, and IgA antibodies were used to detect corneal immunoglobulin via direct immunofluorescence (10 eyes). Serum and aqueous humor (AH) samples from 3 horses with IMMK were used to detect circulating and intraocular IgG against corneal antigens via indirect immunofluorescence on unaffected equine cornea. Results: Percentage scores (scale, 0 to 4) of cells expressing CD3 (median, 2.35 [range, 0.2 to 3.7]; mean ± SD, 2.36 ± 1.08) were significantly greater than scores of cells expressing CD79a (median, 0.55 [range, 0 to 1.5]; mean, 0.69 ± 0.72). All samples stained positively for CD4- and CD8-expressing cells, with no significant difference in scoring. All samples stained positively for IgG, IgM, and IgA. No serum or AH samples collected from horses with IMMK reacted with unaffected equine cornea. Conclusions and Clinical Relevance: Pathogenesis of superficial stromal IMMK included cell-mediated inflammation governed by both cytotoxic and helper T cells. Local immunoglobulins were present in affected corneas; however, corneal-binding immunoglobulins were not detected in the serum or AH from horses with IMMK.