Vanessa L. Cook

Attenuation of ischaemic injury in the equine jejunum by administration of systemic lidocaine

REASONS FOR PERFORMING STUDY Absorption of endotoxin across ischaemic-injured mucosa is a major cause of mortality after colic surgery. Recent studies have shown that flunixin meglumine retards mucosal repair. Systemic lidocaine has been used to treat post operative ileus, but it also has novel anti-inflammatory effects that could improve mucosal recovery after ischaemic injury. HYPOTHESIS Systemic lidocaine ameliorates the deleterious negative effects of flunixin meglumine on recovery of mucosal barrier function. METHODS Horses were treated i.v. immediately before anaesthesia with either 0.9% saline 1 ml/50 kg bwt, flunixin meglumine 1 mg/kg bwt every 12 h or lidocaine 1.3 mg/kg bwt loading dose followed by 0.05 mg/kg bwt/min constant rate infusion, or both flunixin meglumine and lidocaine, with 6 horses allocated randomly to each group. Two sections of jejunum were subjected to 2 h of ischaemia by temporary occlusion of the local blood supply, via a midline celiotomy. Horses were monitored with a behavioural pain score and were subjected to euthanasia 18 h after reversal of ischaemia. Ischaemic-injured and control jejunum was mounted in Ussing chambers for measurement of transepithelial electrical resistance (TER) and permeability to lipopolysaccharide (LPS). RESULTS In ischaemic-injured jejunum TER was significantly higher in horses treated with saline, lidocaine or lidocaine and flunixin meglumine combined, compared to horses treated with flunixin meglumine. In ischaemic-injured jejunum LPS permeability was significantly increased in horses treated with flunixin meglumine alone. Behavioural pain scores did not increase significantly after surgery in horses treated with flunixin meglumine. CONCLUSIONS Treatment with systemic lidocaine ameliorated the inhibitory effects of flunixin meglumine on recovery of the mucosal barrier from ischaemic injury, when the 2 treatments were combined. The mechanism of lidocaine in improving mucosal repair has not yet been elucidated.

Anti-inflammatory effects of intravenously administered lidocaine hydrochloride on ischemia-injured jejunum in horses

OBJECTIVE To investigate effects of lidocaine hydrochloride administered IV on mucosal inflammation in ischemia-injured jejunum of horses treated with flunixin meglumine. ANIMALS 24 horses. PROCEDURES Horses received saline (0.9% NaCl) solution (SS; 1 mL/50 kg, IV [1 dose]), flunixin meglumine (1 mg/kg, IV, q 12 h), lidocaine (bolus [1.3 mg/kg] and constant rate infusion [0.05 mg/kg/min], IV, during and after recovery from surgery), or both flunixin and lidocaine (n = 6/group). During surgery, blood flow was occluded for 2 hours in 2 sections of jejunum in each horse. Uninjured and ischemia-injured jejunal specimens were collected after the ischemic period and after euthanasia 18 hours later for histologic assessment and determination of cyclooxygenase (COX) expression (via western blot procedures). Plasma samples collected prior to (baseline) and 8 hours after the ischemic period were analyzed for prostanoid concentrations. RESULTS Immediately after the ischemic period, COX-2 expression in horses treated with lidocaine alone was significantly less than expression in horses treated with SS or flunixin alone. Eighteen hours after the ischemic period, mucosal neutrophil counts in horses treated with flunixin alone were significantly higher than counts in other treatment groups. Compared with baseline plasma concentrations, postischemia prostaglandin E(2) metabolite and thromboxane B(2) concentrations increased in horses treated with SS and in horses treated with SS or lidocaine alone, respectively. CONCLUSIONS AND CLINICAL RELEVANCE In horses with ischemia-injured jejunum, lidocaine administered IV reduced plasma prostaglandin E(2) metabolite concentration and mucosal COX-2 expression. Coadministration of lidocaine with flunixin ameliorated the flunixin-induced increase in mucosal neutrophil counts.

The effect of lidocaine on in vitro adhesion and migration of equine neutrophils.

The effect of lidocaine on in vitro migration and adhesion of equine neutrophils was evaluated. Neutrophils were isolated from equine whole blood using a Percoll-gradient centrifugation protocol. Purified neutrophils were incubated with lidocaine at concentrations from 0.1 to 1000 microg/ml for 30 min at 37 degrees C, after calcein loading. Neutrophil integrin-mediated adhesion in response to stimulation with 100 nM LTB(4), 100 nM PAF, or 100 ng/ml IL-8, or integrin-mediated migration in response to stimulation with 100 nM LTB(4), 150 nM PAF, or 100 ng/ml IL-8 was assessed. Statistical significance was set at P<0.05. Neutrophil adhesion was significantly increased in response to all three stimulants. IL-8-stimulated adhesion was significantly increased when neutrophils were incubated with 1mg/ml lidocaine, compared to lower lidocaine concentrations. LTB(4)-stimulated adhesion was significantly increased when neutrophils were incubated with 1mg/ml lidocaine compared to that at 5 microg/ml lidocaine. Migration was significantly increased in response to IL-8. IL-8 and LTB(4) stimulated migration was significantly increased when neutrophils were incubated with 1mg/ml lidocaine, compared to lower lidocaine concentrations. In conclusion, lidocaine did not inhibit neutrophil migration or adhesion in vitro at therapeutic concentrations, and increased migration and adhesion at higher concentrations.

Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide infusion in horses

REASONS FOR PERFORMING THE STUDY Endotoxaemia contributes to morbidity and mortality in horses with colic due to inflammatory cascade activation. Effective therapeutic interventions are limited for these horses. Ethyl pyruvate (EP), an anti-inflammatory agent that alters the expression of proinflammatory cytokines, improved survival and organ function in sepsis and gastrointestinal injury in rodents and swine. Therapeutic efficacy of EP is unknown in endotoxaemic horses. OBJECTIVES Determine the effects of EP on signs of endotoxaemia and expression of proinflammatory cytokines following administration of lipopolysaccharide (LPS) in horses. METHODS Horses received 30 ng/kg bwt LPS in saline to induce signs of endotoxaemia. Next, horses received lactated Ringers solution (LRS), (n = 6), 150 mg/kg bwt EP in LRS, (n = 6), or 1.1 mg/kg bwt flunixin meglumine (FM), (n = 6). Controls received saline followed by LRS (n = 6). Physical examinations, behaviour pain scores and blood for clinical pathological testing and gene expression were obtained at predetermined intervals for 24 h. RESULTS Lipopolysaccharide infusion produced clinical and clinicopathological signs of endotoxaemia and increased expression of tumour necrosis factor alpha (TNFα), interleukin 6 (IL-6) and IL-8 (P<0.001) compared with controls. Leucopenia and neutropenia occurred in all horses that received LPS. Horses treated with EP and FM had significantly (P<0.0001) reduced pain scores compared with horses receiving LPS followed by LRS. Flunixin meglumine was significantly more effective at ameliorating fever compared with EP. Both EP and FM significantly diminished TNFα expression. Ethyl pyruvate significantly decreased, but FM significantly increased, IL-6 expression. Neither EP nor FM altered IL-8 expression. CONCLUSIONS AND POTENTIAL RELEVANCE Ethyl pyruvate administered following LPS diminished the clinical effects of endotoxaemia and decreased proinflammatory gene expression in horses. Ethyl pyruvate suppressed expression of proinflammatory cytokines better than FM. However, FM was a superior anti-pyretic compared with EP. Ethyl pyruvate may have therapeutic applications in endotoxaemic horses.

Use of ultrasound to evaluate outcome following colic surgery for equine large colon volvulus

REASONS FOR PERFORMING STUDY The post operative response of the large colon wall after a surgically corrected large colon volvulus (LCV) has not been investigated. OBJECTIVES To use transabdominal ultrasound to monitor the post operative change in large colon wall thickness following surgical correction of LCV. HYPOTHESIS A prolonged period to colon wall involution is correlated with an increased rate of post operative morbidity and mortality. METHODS A prospective clinical study including horses that presented to the North Carolina State University Veterinary Teaching Hospital for colic between September 2006 and March, 2008, had surgically diagnosed and corrected LCV (at least 360 degrees ) without resection and recovered from anaesthesia. Ultrasound of the ventral large colon was performed at the time of anaesthetic recovery and every 6-8 h until the colon wall returned to normal thickness (< or = 5 mm). Outcome was evaluated using a one-way ANOVA to compare average time to colon wall involution between: 1) survivors and nonsurvivors; and 2) horses that developed multiple organ dysfunction syndrome (MODS) during the post operative period and those that recovered without evidence of MODS. RESULTS Sixteen horses that recovered without evidence of MODS had a significantly shorter period to colon wall involution (< or = 5 mm) compared to those diagnosed with MODS (mean +/- s.e. 19.6 h +/- 2.5 and 39.7 h +/- 6.7 respectively, P = 0.006). There was no significant difference in mean period to colon wall involution between survivors and nonsurvivors (26.2 +/- 4.9 and 33.2 +/- 7.8 h, respectively). CONCLUSIONS A shorter time to colon wall involution was associated with decreased post operative morbidity in horses presented for surgical correction of large colon volvulus without resection. POTENTIAL RELEVANCE Ultrasonographic monitoring of colon wall involution after surgical correction of LCV may aid in identifying those cases at risk of MODS. Further investigation of colon wall involution time using a larger number of horses is warranted.

The use of nonsteroidal anti‐inflammatory drugs in critically ill horses

Objective To review the physiology of the cyclooxygenase (COX) enzymes with reference to the beneficial effects of nonsteroidal anti-inflammatory drugs (NSAIDs) related to their analgesic and antiendotoxic properties as well as the mechanisms responsible for adverse gastrointestinal, renal, and coagulation effects. Data Sources Human and veterinary peer reviewed literature Veterinary Data Synthesis NSAIDs are frequently administered to critically ill horses for their analgesic and anti-inflammatory effects. However, NSAIDs have significant side effects principally on the gastrointestinal mucosa and kidneys. These side effects may be exacerbated in critically ill horses if they have gastrointestinal damage or are volume depleted Conclusions This review provides important information for equine veterinarians and criticalists on the advantages and disadvantages of using traditional NSAIDs and newer equine COX-2 selective NSAIDs for the management of different conditions in critically ill horses.OBJECTIVE To review the physiology of the cyclooxygenase (COX) enzymes with reference to the beneficial effects of nonsteroidal anti-inflammatory drugs (NSAIDs) related to their analgesic and antiendotoxic properties as well as the mechanisms responsible for adverse gastrointestinal, renal, and coagulation effects. DATA SOURCES Human and veterinary peer reviewed literature VETERINARY DATA SYNTHESIS NSAIDs are frequently administered to critically ill horses for their analgesic and anti-inflammatory effects. However, NSAIDs have significant side effects principally on the gastrointestinal mucosa and kidneys. These side effects may be exacerbated in critically ill horses if they have gastrointestinal damage or are volume depleted CONCLUSIONS This review provides important information for equine veterinarians and criticalists on the advantages and disadvantages of using traditional NSAIDs and newer equine COX-2 selective NSAIDs for the management of different conditions in critically ill horses.

Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses.

REASONS FOR PERFORMING THE STUDY Endotoxaemia causes substantial morbidity and mortality in horses with colic and sepsis. Ethyl pyruvate is a novel anti-inflammatory medication that improved survival in preclinical models of severe sepsis endotoxaemia and intestinal ischaemia and reperfusion in rodents, swine, sheep and dogs and may be a useful medication in horses. HYPOTHESIS Ethyl pyruvate has no adverse effects in normal horses and is biologically active based on suppression of proinflammatory gene expression in endotoxin stimulated whole blood, in vitro. METHODS Physical and neurological examinations, behaviour scores, electrocardiograms and clinicopathological tests were performed on 5 normal healthy horses receiving 4 different doses of ethyl pyruvate. Doses included 0, 50, 100 and 150 mg/kg bwt administered in a randomised crossover design with a 2 week washout period between doses. Biological efficacy was assessed by stimulating whole blood with endotoxin from the horses that received ethyl pyruvate prior to and 1 and 6 h after drug infusion. Gene expression for TNFα, IL-1β and IL-6 was assessed. RESULTS There were no effects of drug or dose (0, 50, 100 or 150 mg/kg bwt) on any of the physical or neurological examination, behaviour factors, electrocardiogram or clinical pathological results collected from any of the horses. All parameters measured remained within the normal reference range. There was a significant reduction in TNFα, IL-1β and IL-6 gene expression in endotoxin stimulated whole blood from horses 6 h after receiving 150 mg/kg bwt ethyl pyruvate. There were no detectable effects on gene expression of any of the other doses of ethyl pyruvate tested. CONCLUSION We were unable to detect any detrimental effects of ethyl pyruvate administration in normal horses. Ethyl pyruvate significantly decreased proinflammatory gene expression in endotoxin stimulated blood 6 h after drug administration. CLINICAL RELEVANCE Ethyl pyruvate may be a safe, effective medication in endotoxaemic horses.

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1β or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.

Evaluation of the relationship between lesions in the gastroduodenal region and cyclooxygenase expression in clinically normal dogs.

OBJECTIVE To determine whether clinically normal dogs have lesions in the pylorus and duodenum and to examine the expression of cyclooxygenase (COX) isoforms in the pylorus and duodenum of these dogs. ANIMALS 27 clinically normal dogs. PROCEDURES Physical examination was performed on clinically normal dogs from animal shelters and research projects; the dogs were then euthanized. After the dogs were euthanized, the pylorus and duodenum were photographed and scored for gross appearance of lesions. Samples were obtained for histologic evaluation and determination of COX expression via western blot analyses. Tissues from the pylorus and duodenum were categorized as normal, inflamed, or eroded on the basis of histologic analysis. Each histologic category of tissue was then evaluated to determine the correlation with gross appearance and COX expression. RESULTS Of the 27 dogs, 5 had unremarkable histologic findings in the pylorus and duodenum. Inflammation was found in the pylorus of 10 dogs and in the duodenum of 5 dogs. Epithelial erosion was detected in the pylorus of 1 dog and in the duodenum of 3 dogs. Gross appearance was not significantly correlated with histologic appearance. Expression of COX-1 was not upregulated by inflammation, whereas COX-2 expression was increased by inflammation or erosion. CONCLUSIONS AND CLINICAL RELEVANCE Dogs that appear to be clinically normal may have underlying gastroduodenal lesions associated with upregulation of COX-2. Because of the inability to determine this during routine physical examination, practitioners should be aware of this potential situation when prescribing COX inhibitors.

Ethyl pyruvate diminishes the endotoxin-induced inflammatory response of bovine mammary endothelial cells

The endotoxin-induced inflammatory response during coliform mastitis is difficult to control with the currently available therapeutics. Endothelial cells are among the first cell type to be engaged in the inflammatory response and can modulate the severity of inflammation by producing proinflammatory mediators upon endotoxin exposure. Ethyl pyruvate, an ethyl ester of pyruvic acid, can ameliorate endotoxin-induced inflammatory responses by inhibiting the production of proinflammatory mediators in several in vitro and in vivo endotoxemia models. The objective of this study was to determine the effect of ethyl pyruvate on the production of vascular proinflammatory mediators that are associated with the pathogenesis of coliform mastitis. The ability of ethyl pyruvate to reduce the expression of proinflammatory mediators was evaluated in cultured bovine mammary endothelial cells (BMEC) stimulated with endotoxin. Treatment of endotoxin-stimulated BMEC with ethyl pyruvate significantly reduced gene expression of IL-6, IL-8, and intercellular adhesion molecule 1 as well as expression of eicosanoid-producing enzymes, including cyclooxygenase 2 and 15-lipoxygenase 1. This is the first time that the effect of ethyl pyruvate was evaluated in an in vitro BMEC model of coliform mastitis. The ability of ethyl pyruvate to effectively inhibit gene and protein expression of potent vascular proinflammatory mediators in vitro warrants further investigations to assess in vivo efficacy. Ethyl pyruvate is safe for human consumption, and it may be an attractive candidate as a therapeutic in ameliorating the severe pathogenesis associated with coliform mastitis.