Immunohistochemical evaluation of cyclooxygenase expression in corneal squamous cell carcinoma in horses

OBJECTIVE: To evaluate expression of cyclooxygenase (COX)-1 and COX-2 in the cornea, eyelid, and third eyelid of healthy horses and those affected with squamous cell carcinoma (SCC) by use of immunohistochemical techniques. ANIMALS: 15 horses with SCC involving ocular tissues and 5 unaffected control horses. PROCEDURES: SCC-affected tissues were obtained from the cornea (n = 5 horses), eyelid (5), and third eyelid (5). Site-matched control tissues were obtained from 5 horses unaffected with SCC. Tissue sections of affected and control cornea, eyelid, and third eyelid were stained immunohistochemically for COX-1 and COX-2 via standard techniques. Stain uptake was quantified by use of computer-assisted image analysis of digital photomicrographs. RESULTS: Immunoreactivity for both COX-1 and COX-2 was significantly greater in equine corneas with SCC than in control corneas. No significant differences in COX-1 or COX-2 immunoreactivity were detected in eyelid and third-eyelid SCC, compared with site-matched control tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Immunoreactivity for COX-1 and COX-2 is high in equine corneal SCC, possibly indicating that COX plays a role in oncogenesis or progression of this tumor type at this site. Pharmacologic inhibition of COX may represent a useful adjunctive treatment for corneal SCC in horses.     

Effects of flunixin meglumine or etodolac treatment on mucosal recovery of equine jejunum after ischemia

OBJECTIVE: To examine the effects of flunixin meglumine and etodolac treatment on recovery of ischemic-injured equine jejunal mucosa after 18 hours of reperfusion. ANIMALS: 24 horses. PROCEDURE: Jejunum was exposed to 2 hours of ischemia during anesthesia. Horses received saline (0.9% NaCl) solution (12 mL, i.v., q 12 h), flunixin meglumine (1.1 mg/kg, i.v., q 12 h), or etodolac (23 mg/kg, i.v., q 12 h). Tissue specimens were obtained from ischemic-injured and nonischemic jejunum immediately after ischemia and 18 hours after recovery from ischemia. Transepithelial electric resistance (TER) and transepithelial flux of tritium-labeled mannitol measured mucosal permeability. Denuded villous surface area and mean epithelial neutrophil count per mm2 were calculated. Western blot analysis for cyclooxygenase (COX)-1 and -2 was performed. Pharmacokinetics of flunixin and etodolac and eicosanoid concentrations were determined. RESULTS: Ischemic-injured tissue from horses treated with flunixin and etodolac had significantly lower TER and increased permeability to mannitol, compared with that from horses treated with saline solution. Epithelial denudation after ischemia and 18 hours after recovery was not significantly different among treatments. Both COX-1 and -2 were expressed in ischemic-injured and nonischemic tissues. Ischemia caused significant upregulation of both COX isoforms. Eicosanoid concentrations were significantly lower in tissues from flunixin and etodolac-treated horses, compared with that from horses treated with saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Flunixin and etodolac treatment retarded recovery of intestinal barrier function in jejunal mucosa after 18 hours of reperfusion, whereas tissues from horses treated with saline solution recovered baseline values of TER and permeability to mannitol.     

Determination of expression of cyclooxygenase-1 and -2 isozymes in canine tissues and their differential sensitivity to nonsteroidal anti-inflammatory drugs

OBJECTIVE: To evaluate cyclooxygenase isozyme distribution in tissues from dogs and determine the differential sensitivity of canine cyclooxygenase (COX)-1 and -2 isozymes to nonsteroidal anti-inflammatory drugs (NSAIDs). SAMPLE POPULATION: Canine tissue samples (stomach, duodenum, ileum, jejunum, colon, spleen, cerebral cortex, lung, ovary, kidney, and liver) were obtained from 2 dogs for northern and western blot analyses, and blood for whole blood COX assays was obtained from 15 dogs. PROCEDURE: 11 NSAIDs were evaluated to determine their COX-2 selectivity in whole blood assays. The concentrations of the drug needed to inhibit 50% of enzyme activity (IC50) were then calculated for comparison. Expression and tissue distribution of COX isozymes were determined by northern and western blot analysis. RESULTS: Aspirin, diclofenac, indomethacin, ketoprofen, meclofenamic acid, and piroxicam had little selectivity toward COX isozymes, whereas NS398, carprofen, tolfenamic acid, nimesulide, and etodolac had more than 5 times greater preference for inhibiting COX-2 than COX-1. All canine tissues examined, including those from the gastrointestinal tract, coexpressed COX-1 and -2 mRNA, although protein expression was observed only for COX-1. CONCLUSIONS AND CLINICAL RELEVANCE: Canine COX-2 was selectively inhibited by etodolac, nimesulide, and NS398; tolfenamic acid and carprofen also appeared to be preferential COX-2 inhibitors in dogs. The roles of COX-1 as a constitutive housekeeping enzyme and COX-2 as a proinflammatory inducible enzyme (as determined in humans) appear to apply to dogs; therefore, COX-2-selective inhibitors should prove useful in reducing the adverse effects associated with nonselective NSAIDs.     

Cyclooxygenase-2 immunoreactivity in equine ocular squamous-cell carcinoma.

Squamous-cell carcinoma (SCC) is the second most common tumor in horses, and 40%-50% may occur in ocular and adnexal structures. Cyclooxygenase (COX)-2 is an inducible enzyme responsible for the production of prostaglandins that control cell growth and the development and progression of cancer. Mechanisms responsible for the initial upregulation of COX-2 in neoplasia are unclear; prolonged sunlight exposure and mutations in the p53 gene may be possibilities. Because the etiopathogenesis of ocular SCC in horses may involve ultraviolet sunlight and p53 mutations, the purpose of this study was to characterize the immunoreactivity of COX-2 in these tumors. Cyclooxygenase-2 expression was found in 6 of 22 (27%) paraffin-embedded equine SCCs. Cyclooxygenase-2 immunoreactivity was associated with the mitotic index (P < 0.001). Strategies to inhibit COX-2 by the use of topical or systemic COX-2 inhibitors might prove to be a safe and economical treatment in some horses with SCC.     

In vitro effects of cyclooxygenase inhibitors in whole blood of horses, dogs, and cats.

OBJECTIVE: To determine potency and selectivity of nonsteroidal anti-inflammatory drugs (NSAID) and cyclooxygenase- (COX-) specific inhibitors in whole blood from horses, dogs, and cats. SAMPLE POPULATION: Blood samples from 30 healthy horses, 48 healthy dogs, and 9 healthy cats. PROCEDURE: Activities of COX-1 and COX-2 were determined by measuring coagulation-induced thromboxane and lipopolysaccharide-induced prostaglandin E2 concentrations, respectively, in whole blood with and without the addition of various concentrations of phenylbutazone, flunixin meglumine, ketoprofen, diclofenac, indomethacin, meloxicam, carprofen, 5-bromo-2[4-fluorophenyl]-3-14-methylsulfonylphenyl]-thiophene (DuP 697), 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furan one (DFU), 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone (MF-tricyclic), and celecoxib. Potency of each test compound was determined by calculating the concentration that resulted in inhibition of 50% of COX activity (IC50). Selectivity was determined by calculating the ratio of IC50 for COX-1 to IC50 for COX-2 (COX-1/COX-2 ratio). RESULTS: The novel compound DFU was the most selective COX-2 inhibitor in equine, canine, and feline blood; COX-1/COX-2 ratios were 775, 74, and 69, respectively. Carprofen was the weakest inhibitor of COX-2, compared with the other COX-2 selective inhibitors, and did not inhibit COX-2 activity in equine blood. In contrast, NSAID such as phenylbutazone and flunixin meglumine were more potent inhibitors of COX-1 than COX-2 in canine and equine blood. CONCLUSIONS AND CLINICAL RELEVANCE: The novel COX-2 inhibitor DFU was more potent and selective in canine, equine, and feline blood, compared with phenylbutazone, flunixin meglumine, and carprofen. Compounds that specifically inhibit COX-2 may result in a lower incidence of adverse effects, compared with NSAID, when administered at therapeutic dosages to horses, dogs, and cats.     

The effects of cyclo-oxygenase inhibitors on bile-injured and normal equine colon

A potential adverse effect of cyclo-oxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs [NSAIDs]) in horses is colitis. In addition, we have previously shown an important role for COX-produced prostanoids in recovery of ischaemic-injured equine jejunum. It was hypothesised that the nonselective COX inhibitor flunixin would retard repair of bile-injured colon by preventing production of reparative prostaglandins, whereas the selective COX-2 inhibitor, etodolac would not inhibit repair as a result of continued COX-1 activity. Segments of the pelvic flexure were exposed to 1.5 mmol/l deoxycholate for 30 min, after which they were recovered for 4 h in Ussing chambers. Contrary to the proposed hypothesis, recovery of bile-injured colonic mucosa was not affected by flunixin or etodolac, despite significantly depressed prostanoid production. However, treatment of control tissue with flunixin led to increases in mucosal permeability, whereas treatment with etodolac had no significant effect. Therefore, although recovery from bile-induced colonic injury maybe independent of COX-elaborated prostanoids, treatment of control tissues with nonselective COX inhibitors may lead to marked increases in permeability. Alternatively, selective inhibition of COX-2 may reduce the incidence of adverse effects in horses requiring NSAID therapy.     

Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses

OBJECTIVE: To compare the levels of mRNA expression of cycooxygenase (COX)-1 and COX-2 in the digital laminae of normal horses and horses in the developmental stages of laminitis experimentally induced by administration of black walnut extract (BWE). SAMPLE POPULATION: Samples of mRNA extracted from the digital laminae of 5 control horses and 5 horses at the onset of leukopenia after administration of BWE. PROCEDURE: Specimens of laminae were collected from anesthetized horses prior to euthanasia. Expression of COX-1 and COX-2 mRNA in laminae of control and affected horses was evaluated via real-time quantitative polymerase chain reaction techniques. RESULTS: Expression of COX-2 mRNA was significantly increased in the BWE-treated group, compared with that in control horses. In contrast to COX-2 regulation, COX-1 mRNA expression was not significantly different between groups. Interestingly, despite consistent clinical signs such as leukopenia in all BWE-treated horses, distinct differences in COX-2 mRNA expression were detected among those 5 horses (compared with values for control horses, the increase in COX-2 mRNA expression ranged from no increase to a 30-fold increase). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was a significant upregulation of COX-2 mRNA expression during the developmental stages of laminitis, with no significant change in expression of the COX-1 isoform. These data appear to provide support for aggressive use of nonsteroidal anti-inflammatory drugs in horses at risk for laminitis; further investigation into the clinical value of selective COX-2 inhibitors for treatment of laminitis in horses appears to be warranted.     

Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin.

OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.     

Evaluation of cyclooxygenase protein expression in traumatized versus normal tissues from eastern box turtles (Terrapene carolina carolina)

This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.     

Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States

OBJECTIVE: To determine the incidence of bovine papillomavirus (BPV) type 1 or 2 in sarcoids and other samples of cutaneous tissues collected from horses in the western United States. ANIMALS: 55 horses with sarcoids and 12 horses without sarcoids. PROCEDURE: Tissue samples (tumor and normal skin from horses with sarcoids and normal skin, papillomas, and nonsarcoid cutaneous neoplasms from horses without sarcoids) were collected. Tissue samples were analyzed for BPV-1 or -2 DNA, using a polymerase chain reaction (PCR) and restriction fragment length polymorphism. The PCR products from 7 sarcoid-affected horses were sequenced to evaluate percentage homology with expected sequences for BPV-1 or-2. RESULTS: Most (94/96, 98%) sarcoids contained BPV DNA. Sixty-two percent of the tumors examined had restriction enzyme patterns consistent with BPV-2. Thirty-one of 49 (63%) samples of normal skin obtained from horses with sarcoids contained BPV DNA. All samples subsequently sequenced had 100% homology with the expected sequences for the specific viral type. All tissues from healthy horses, nonsarcoid neoplasms, and papillomas were negative for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine papillomaviral DNA was detected in essentially all sarcoids examined. There appears to be regional variation in the prevalence of viral types in these tumors. The fact that we detected viral DNA in normal skin samples from horses with sarcoids suggests the possibility of a latent viral phase. Viral latency may be 1 explanation for the high rate of recurrence following surgical excision of sarcoids.     

Preferential and non-selective cyclooxygenase inhibitors reduce inflammation during lipopolysaccharide-induced synovitis

Synovitis in horses is frequently treated by administration of non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase isoforms (COX-1 and COX-2). Constitutively expressed COX-1 is involved in physiologic functions such as maintenance of gastric mucosal integrity, whereas COX-2 is up-regulated at sites of inflammation. Thus, COX-2 inhibitors reduce inflammation with reduced gastrointestinal side effects as compared to non-selective COX inhibitors. The objective of the present study was to compare the anti-inflammatory effects of the preferential COX-2 inhibitor etodolac with the non-selective COX inhibitor phenylbutazone in horses with lipopolysaccharide (LPS)-induced synovitis. Three groups of horses (n=6) received no treatment, phenylbutazone (4.4 mg/kg, IV, q12h), or etodolac (23 mg/kg, IV, q12h), respectively, 2-h following injection of LPS into one middle carpal joint. Synovial fluid was analyzed for white blood cell (WBC) count, and TXB2 and PGE2 levels. Phenylbutazone and etodolac significantly reduced WBC count 6 and 24-h following injection of LPS compared to untreated horses. In addition, both drugs significantly reduced PGE2 levels (P<0.05) 6-h following LPS injection, whereas the probable COX-1 prostanoid TXB2 was significantly reduced by phenylbutazone (P<0.05), but not etodolac. Etodolac may serve as a more selective anti-inflammatory agent than phenylbutazone for treatment of equine synovitis.     

Development of in vitro assays for the evaluation of cyclooxygenase inhibitors and predicting selectivity of nonsteroidal anti-inflammatory drugs in cats

OBJECTIVE: To develop and validate in cats suitable in vitro assays for screening and ranking nonsteroidal antiinflammatory drugs (NSAIDs) on the basis of their inhibitory potencies for cyclooxygenase (COX)-1 and COX-2. ANIMALS: 10 cats. PROCEDURE: COX-1 and COX-2 activities in heparinized whole blood samples were induced with calcium ionophore and lipopolysaccharide, respectively. For the COX-2 assay, blood was pretreated with aspirin. The COX-1 and COX-2 assays were standardized, such that time courses of incubation with the test compounds and conditions of COX expression were as similar as possible in the 2 assays. Inhibition of thromboxane B2 production, measured by use of a radioimmunoassay, was taken as a marker of COX-1 and COX-2 activities. These assays were used to test 10 to 12 concentrations of a COX-1 selective drug (SC-560) and of 2 NSAIDs currently used in feline practice, meloxicam and carprofen. Selectivities of these drugs were compared by use of classic 50% and 80% inhibitory concentration (ie, IC50 and IC80) ratios but also with alternative indices that are more clinically relevant. RESULTS: These assay conditions provide a convenient and robust method for the determination of NSAID selectivity. The S(+) enantiomeric form of carprofen was found to be COX-2 selective in cats, but meloxicam was only slightly preferential for this isoenzyme. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro pharmacodynamic and in vivo pharmacokinetic data predict that the COX-2 selectivity of both drugs for cats will be limited when used at the recommended doses. This study provides new approaches to the selection of COX inhibitors for subsequent clinical testing.     

Pharmacokinetics of etodolac in the horse following oral and intravenous administration

The purpose of this study was to determine the pharmacokinetics of etodolac following oral and intravenous administration to six horses. Additionally, in vitro cyclooxygenase (COX) selectivity assays were performed using equine whole blood. Using a randomized two-way crossover design, horses were administered etodolac (20 mg/kg) orally or intravenously, with a minimum 3-week washout period. Plasma samples were collected after administration for analysis using high pressure liquid chromatography with ultraviolet detection. Following intravenous administration, etodolac had a mean plasma half-life (t(1/2)) of 2.67 h, volume of distribution (Vd) of 0.29 L/kg and clearance (Cl) of 234.87 mL/h kg. Following oral administration, the average maximum plasma concentration (Cmax)) was 32.57 mug/mL with a t(1/2) of 3.02 h. Bioavailability was approximately 77.02%. Results of in vitro COX selectivity assays showed that etodolac was only slightly selective for COX-2 with a COX-1/COX-2 selectivity ratio effective concentration (EC)50 of 4.32 and for EC80 of 4.77. This study showed that etodolac is well absorbed in the horse after oral administration, and may offer a useful alternative for anti-inflammatory treatment of various conditions in the horse.     

Early laminar events involving endothelial activation in horses with black walnut- induced laminitis

OBJECTIVE: To determine proinflammatory gene expression, endothelial adhesion molecule gene expression, and matrix metalloproteinase (MMP) concentrations in laminar specimens at 1.5 hours after administration of black walnut extract (BWE) and to compare these values with later time points. ANIMALS: 25 horses. PROCEDURES: After nasogastric administration of BWE, anesthesia was induced at 1.5 hours in early time point (ETP) horses (n = 5), between 3 and 4 hours in developmental time point horses (5), and between 9 and 10 hours in acute onset of lameness time point horses (5). Anesthesia was induced at 3 and 10 hours after nasogastric administration of water in 2 groups of control horses (3-hour control group, n = 5; 10-hour control group, 5). Real-time quantitative PCR assay was performed on laminar specimens from control and ETP horses for cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6, IL-8, IL-10, MMP-2, and MMP-9 gene expression; and on laminar specimens from all groups for endothelial adhesion molecules, intercellular adhesion molecule (ICAM)-1, and E-selectin gene expression. Leukocyte emigration was assessed via CD13 immunohistochemistry, and gelatinase accumulation was determined by gelatin zymography. RESULTS: Laminar concentrations of IL-1beta, IL-6, IL-8, COX-2, ICAM-1, and E-selectin mRNA were significantly increased in ETP horses, compared with control horses. Concentrations of IL-1beta, IL-8, ICAM-1, and E-selectin mRNA peaked at 1.5 hours. In ETP horses, leukocyte emigration was present in 3 of 5 horses and pro-MMP-9 was detected in 2 of 5 horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that endothelial activation and laminar inflammation are early events in laminitis; MMP accumulation likely is a downstream event.     

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa

OBJECTIVE: To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). ANIMALS: 14 adult horses. PROCEDURE: Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. RESULTS: Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.