Objective: To evaluate platelet-neutrophil aggregate (PNA) formation and neutrophil shape as indicators of neutrophil activation in dogs with systemic inflammatory diseases and after blood sample incubation with various platelet and neutrophil agonists. Animals: 20 dogs with systemic inflammatory response syndrome (SIRS) and 10 healthy Beagles. Procedures: Neutrophils were isolated from blood samples directly after blood sample collection and after incubation of blood samples with phorbol myristate acetate, collagen, adenosine diphosphate, epinephrine, or various concentrations of lipopolysaccharide or arachidonic acid. CD61+ neutrophils as an indicator of PNA formation were evaluated, and neutrophil size and granularity were assessed via flow cytometry. Results: Dogs with SIRS had more PNA formation, larger neutrophil size, and less granularity relative to control dogs, but no differences were evident when these dogs were grouped by whether they had sepsis (n = 6) or disseminated intravascular coagulation (12). A significant increase in PNA formation occurred after neutrophil incubation with all agonists, and incubation with phorbol myristate acetate elicited the strongest response. Neutrophils increased in size and decreased in granularity after incubation with all agonists except epinephrine. Incubation with lipopolysaccharide or arachidonic acid resulted in a dose-dependent effect on PNA formation and neutrophil shape. Conclusions and Clinical Relevance: SIRS appeared to increase the degree of PNA formation and neutrophil shape change. Similar changes after neutrophil incubation with platelet agonists suggested that platelet activation has a role in PNA formation. Additional studies are necessary to determine the clinical importance and diagnostic value of PNA formation in dogs with SIRS and sepsis.

Objective: To determine whether the method of lipopolysaccharide (LPS) administration (intermittent vs continuous) affects the magnitude and duration of the systemic inflammatory response in horses and whether prolonged (48 hours) endotoxemia induces laminitis. Animals: 12 healthy adult horses (10 mares and 2 geldings). Procedures: Horses were randomly assigned to receive LPS (total dose, 80 μg; n = 4) or saline (0.9% NaCl) solution (80 mL/h; 4) via constant rate infusion or 8 bolus IV injections of LPS (10 μg, q 6 h;4) during a 48-hour period. Physical examinations were performed every 4 hours, inflammatory cytokine gene expression was determined for blood samples obtained every 8 hours, and IV glucose tolerance tests were performed. Results: All LPS-treated horses had signs of depression and mild colic; those signs abated as the study progressed. Administration of LPS increased expression of interleukin-1β, interleukin-6, and interleukin-8, but results were not significantly different between LPS treatment groups. Cytokine expression was significantly higher on the first day versus the second day of LPS treatment. Interleukin-1β expression was positively correlated with rectal temperature and expression of other cytokines. Glucose and insulin dynamics for both LPS groups combined did not differ significantly from those of the saline solution group. Signs of laminitis were not detected in any of the horses. Conclusions and Clinical Relevance: Horses developed LPS tolerance within approximately 24 hours after administration was started, and the method of LPS administration did not affect the magnitude or duration of systemic inflammation. Laminitis was not induced in horses.

Objective: To characterize acute inflammatory and hemostatic surgical stress responses following castration in cats and to evaluate whether the addition of local anesthesia to the anesthetic protocol attenuates these responses. Animals: 39 male cats. Procedures: Cats undergoing castration were randomly assigned to 2 groups: both groups underwent surgery with general anesthesia, and 1 group additionally received a local anesthetic (lidocaine [2.0 mg/kg in total, divided intratesticularly and SC]) prior to incision. Blood samples were collected after anesthetic induction (baseline) and 1, 5, and 24 hours later. Thromboelastography and coagulation variables (activated partial thromboplastin time [aPTT] and prothrombin time [PT]) were analyzed; fibrinolysis was assessed with plasma D-dimer concentrations. The acute-phase response was evaluated via measurement of plasma fibrinogen and serum amyloid A (last time point, 28 hours) concentrations. Hematologic variables were analyzed at baseline and 1, 5, and 24 hours later. Results: Evidence of hemostatic and inflammatory activation after surgery was detected in both groups. Maximum amplitude and G (global clot strength) were significantly increased at 24 hours, and significant, but not clinically relevant, decreases were detected in aPTT at 5 and 24 hours and in PT at 24 hours, compared with baseline values. Serum amyloid A concentrations were significantly higher at 24 and 28 hours than at baseline, and plasma fibrinogen concentration was significantly increased at 24 hours; WBC and RBC counts and Hct were significantly increased at multiple time points. No differences between groups were detected for any variables. Conclusions and Clinical Relevance: Castration appeared to induce hypercoagulability and an acute-phase inflammatory response in cats. Local anesthesia with lidocaine did not attenuate this response.

Objective: To characterize mucosal gene expression in dogs with chronic enteropathy (CE). Animals: 18 dogs with CE and 6 healthy control dogs. Procedures: Small intestinal mucosal biopsy specimens were endoscopically obtained from dogs. Disease severity in dogs with CE was determined via inflammatory bowel index scores and histologic grading of biopsy specimens. Total RNA was extracted from biopsy specimens and microchip array analysis (approx 43,000 probe sets) and quantitative reverse transcriptase PCR assays were performed. Results: 1,875 genes were differentially expressed between dogs with CE and healthy control dogs; 1,582 (85%) genes were downregulated in dogs with CE, including neurotensin, fatty acid–binding protein 6, fatty acid synthase, aldehyde dehydrogenase 1 family member B1, metallothionein, and claudin 8, whereas few genes were upregulated in dogs with CE, including genes encoding products involved in extracellular matrix degradation (matrix metallopeptidases 1, 3, and 13), inflammation (tumor necrosis factor, interleukin-8, peroxisome proliferator–activated receptor γ, and S100 calcium-binding protein G), iron transport (solute carrier family 40 member 1), and immunity (CD96 and carcinoembryonic antigen–related cell adhesion molecule [CEACAM] 18). Dogs with CE and protein-losing enteropathy had the greatest number of differentially expressed genes. Results of quantitative reverse transcriptase PCR assay for select genes were similar to those for microchip array analysis. Conclusions and Clinical Relevance: Expression of genes encoding products regulating mucosal inflammation was altered in dogs with CE and varied with disease severity. Impact for Human Medicine: Molecular pathogenesis of CE in dogs may be similar to that in humans with inflammatory bowel disease.

This cross-sectional clinical study compared inflammation, including expression of the chemokine interleukin (IL)-8 and intercellular cell adhesion molecule-1 (ICAM-1), in the stifle joints of 4 control dogs and 23 dogs with cranial cruciate ligament rupture (CCLR). The CCL, synovial membrane, meniscus, cartilage, and synovial fluid from the affected stifle joints of all the dogs were examined. Inflammatory cell counts were performed on the synovial fluid, and the tissues were processed for histologic study and immunohistochemical detection of IL-8 and ICAM-1. The synovial fluid from the stifle joints of the dogs with CCLR had an increased percentage of neutrophils (P = 0.054) and a decreased percentage of lymphocytes (P = 0.004) but not macrophages compared with the fluid from the control dogs. There was accumulation of inflammatory cells and increased expression of IL-8 and ICAM-1 in the vascular endothelium of the synovial membrane and the CCL of the dogs with CCLR. The increase in inflammatory cells in the stifle joints of dogs with CCLR may therefore be due to increased expression of IL-8 and ICAM-1 in the synovial membrane and the CCL after the injury. These data may help in understanding the mechanisms of inflammation associated with CCLR.

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.

Objective: To evaluate effects of a single dose of enrofloxacin (5 mg/kg, IV) on body temperature and tracheobronchial neutrophil count in healthy Thoroughbreds premedicated with interferon-α and undergoing long-distance transportation. Animals: 32 healthy Thoroughbreds. Procedures: All horses received interferon-α (0.5 U/kg, sublingually, q 24 h) as an immunologic stimulant for 2 days before transportation and on the day of transportation. Horses were randomly assigned to receive enrofloxacin (5 mg/kg, IV, once; enrofloxacin group) or saline (0.9% NaCl) solution (50 mL, IV, once; control group) ≤ 1 hour before being transported 1,210 km via commercial vans (duration, approx 26 hours). Before and after transportation, clinical examination, measurement of temperature per rectum, and hematologic analysis were performed for all horses; a tracheobronchial aspirate was collected for neutrophil quantification in 12 horses (6/group). Horses received antimicrobial treatment after transportation if deemed necessary by the attending clinician. Results: No adverse effects were associated with treatment. After transportation, WBC count and serum amyloid A concentration in peripheral blood samples and neutrophil counts in tracheobronchial aspirates were significantly lower in horses of the enrofloxacin group than in untreated control horses. Fever (rectal temperature, ≥ 38.5°C) after transportation was detected in 3 of 16 enrofloxacin group horses and 9 of 16 control horses; additional antimicrobial treatment was required in 2 horses in the enrofloxacin group and 7 horses in the control group. Conclusions and Clinical Relevance: In horses premedicated with interferon-α, enrofloxacin appeared to provide better protection against fever and lower respiratory tract inflammation than did saline solution.

Objective: To evaluate the analgesic efficacy of ABT-116, a transient receptor potential cation channel vanilloid subfamily V member 1 antagonist, and compare it with that of buprenorphine by measurement of mechanical and thermal nociceptive thresholds in dogs. Animals: Six 7- to 8-month-old dogs (3 males and 3 females). Procedures: In a crossover study design, all dogs received ABT-116 (30 mg/kg, PO) and buprenorphine (0.03 mg/kg, orotransmucosally), with each treatment separated by 1 week. Physiologic variables were recorded prior to and 1, 6, and 24 hours after drug administration. Thermal (thoracic) and mechanical (dorsolateral aspect of the radius [proximal] and dorsopalmar aspect of the forefoot [distal]) nociceptive thresholds were assessed prior to (baseline) and 15 minutes and 1, 2, 4, 6, 12, 18, and 24 hours after treatment. Results: Buprenorphine administration resulted in higher overall thermal and proximal mechanical nociceptive thresholds, compared with ABT-116. Distal mechanical nociceptive thresholds after treatment were higher than baseline values for both treatments, but the magnitude of change was greater for buprenorphine at 1 hour after administration. Whereas HR and RR sporadically differed from baseline values after ABT-116 administration, rectal temperature increased from a baseline value of 39 ± 0.2°C (mean ± SD) to a peak of 40.6 ± 0.2°C at 6 hours. Conclusions and Clinical Relevance: In dogs without inflammation or nerve injury, PO administration of ABT-116 did not consistently result in an increase in nociceptive thresholds. However, clinically relevant increases in rectal temperature were identified after ABT-116 administration.

A number of studies have measured collagen fibers and collagen
deposition in wound healing process with advances imaging techniques. However, these are performed by complicated methods and need specific tools. In search of the easier ways in routine histopathological laboratory, collagen measurement and staining pattern of wound healing process were observed in wounded skin of Sprague Dawley’s rat by using two different stains which are standard haematoxylin and eosin (H&E) and modified Masson’s
trichrome staining (MT). The comparison between these staining in wounded tissues was made to evaluate the advantages and disadvantages of both staining in wound healing study for 21 days postwounding. Tissues which stained with MT staining was then evaluated its collagen re-organization and density by using polarized light microscope with the aid of image analyzer software. Results showed that tissues stained with standard H&E could not be used to measure and differentiate the collagen deposition which is contradictory to MT staining. Wounded tissue stained with MT staining has showed a clear view of collagen fibers deposition
and re-organisation compared to H&E staining. This finding could validate the using of modified MT staining which leads to accurate histopathological analysis and observation in wound healing study.

Corona Discharge Therapy,an evidence-based therapeutic modality, was used on a 40-year-old female Malaysian elephant. The elephant was on systemic therapy for her wounds for 5 days. All therapies were then stopped for 4 days. Corona Discharge Therapy was subsequently applied on both the hind limb joints for 3 sessions. The non-invasive
treatment was made with a multi-patented, electronic therapeutic device called VET Sonotron without the concurrent use of drugs. Corona discharge beam (CDB) of the device was used to relieve the joint pain and inflammation. The skin surface of the elephant joints was scanned with the CDB emanating from a discharge electrode to
which was applied periodic bursts of radio frequency at 0.43 mHz, pulsed at 1 kHz of sound frequency. This therapy showed remarkable anti-inflammatory effects.