Browsing by Author "Anthony Blikslager, Committee Chair"

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The Effect of Novel Anti-inflammatory Drugs on the Cyclooxygenase Enzymes and Recovery of Mucosal Barrier Function
(2010-04-02) Marshall, John Fraser; Adam Moeser, Committee Member; Jody Gookin, Committee Member; Anthony Blikslager, Committee Chair; Samuel Jones, Committee Member
The non-steroidal anti-inflammatory drugs (NSAIDs) are a large group of drugs that are commonly used for the treatment of pain and inflammation in the veterinary species. The NSAIDs inhibit the action of the cyclooxygenase (COX) enzymes, COX-1 and COX-2, to reduce the production of prostaglandins. However, their use is associated with adverse effects, particularly in the gastrointestinal tract which may be related to the inhibition of COX-1. To reduce the incidence of these effects, NSAIDs have been designed to selectively inhibit COX-2 while allowing physiologic prostaglandin production by COX-1. The first experiments of this thesis aimed to determine the effect of three NSAIDs in the horse. Flunixin meglumine is commonly used to treat pain and endotoxemia associated with colic in the horse. Deracoxib and firocoxib have been shown to be COX-2 selective in the dog. This study used in vitro whole blood assays to determine the effect of flunixin meglumine, deracoxib, and firocoxib on the COX enzymes. Using this model, flunixin meglumine was shown to non-selectively inhibit COX-1 and COX-2 in the horse. In contrast, deracoxib and firocoxib selectively inhibited COX-2 in the horse. Using an in vitro equine whole blood assay, the next project determined that the novel NSAID robenacoxib is COX-2 selective in the horse. The effect of robenacoxib and flunixin meglumine on the recovery of ischemic-injured equine jejunum was then compared using an equine ex vivo model. While flunixin meglumine significantly inhibited the production of prostaglandin E2 (PGE2) and the recovery of barrier function, robenacoxib allowed barrier function to recovery and production of PGE2. The mechanism of action of the novel anti-inflammatory compound AHI-805 is currently unknown. Using an in vitro equine whole blood model, the effect of AHI-805 on COX-1 and COX-2 was determined. While AHI-805 did not inhibit COX-1, it did significantly inhibit the action of COX-2. The effect of AHI-805 on the recovery of mucosal barrier function in ischemic injured equine jejunum was determined using an equine ex vivo model. Treatment of ischemic injured equine jejunum with AHI-805 significantly inhibited the recovery of mucosal barrier function. Furthermore, in this ex vivo model AHI-805 significantly inhibited the action of both COX-1 and COX-2.
Mechanisms of Prostaglandin-Stimulated Recovery of Mucosal Barrier Function in the Ischemia-Injured Porcine Intestine: Role of Intestinal Ion Transport
(2006-05-16) Moeser, Adam James; Anthony Blikslager, Committee Chair; Glen Almond, Committee Member; Jody Gookin, Committee Member; Jack Odle, Committee Member
A series of experiments were conducted to determine physiologic mechanisms of mucosal repair in the ischemia-injured intestine. The first experiment (Chapter III) investigated the contributory role of individual Cl- channels in the recovery of barrier function in ischemia-injured porcine ileum. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (TER) were measured in response to PGE2 and pharmacologic inhibitors of epithelial Cl- channels. Overall, results from these studies demonstrate that ClC-2-mediated intestinal Cl- secretion restores TER in ischemia-injured intestine. Chapter IV entails a study aimed at more directly investigating the role of ClC-2 in mucosal repair by evaluating mucosal repair in ischemia-injured intestinal mucosa mounted on Ussing chambers treated with the selective ClC-2 agonist, lubiprostone. Results from this suggest that activation of ClC-2 with the selective agonist, lubiprostone, stimulated elevations in TER and reduction in mannitol flux in the Ischemia-injured intestine. In Chapter V, experiments focused on the role of individual NHE isoforms in the recovery of barrier function in ischemia-injured porcine ileum. Results from this study demonstrate that inhibition of NHE2 activity, possibly via EBP50, induces recovery of barrier function in ischemic-injured intestine
The Role of Novel Anti-Inflammatory Drugs in the Repair of Ischemic-Injured Equine Jejunum
(2008-10-30) Cook, Vanessa; Anthony Blikslager, Committee Chair; Jody Gookin, Committee Member; Malcolm Roberts, Committee Member; Samuel Jones, Committee Member
Following colic surgery, ischemic-injured intestine may remain which must recover for the horse to survive. However, the commonly used analgesic, flunixin meglumine, a non selective cyclooxygenase (COX) inhibitor, may retard the repair of ischemic-injured jejunum. Therefore, we investigated alternative anti-inflammatory drugs which may allow recovery of ischemic-injured jejunum whilst providing effective analgesia. The effect of 0.9% saline 1ml/50kg, flunixin meglumine 1mg/kg IV every 12 hours, lidocaine 1.3mg/kg loading dose and 0.05mg/kg/minute constant rate infusion IV, or the two drugs combined, was evaluated on recovery of mucosal barrier function in equine jejunum following 2 hours of ischemia and 18 hours of recovery (n=6 horses/group). Flunixin meglumine inhibited the recovery of mucosal barrier function as evidenced by a lower transepithelial resistance (TER) and increased LPS flux across ischemic-injured mucosa from horses in that treatment group. When treatment with flunixin meglumine was combined with lidocaine, recovery of mucosal barrier function was not retarded. The mucosal influx of neutrophils seen with flunixin meglumine treatment was ameliorated by treatment with lidocaine. Lidocaine inhibited upregulation of COX-2 in ischemic-injured jejunum. The same model was used to evaluate the effect of a COX-2 preferential inhibitor, firocoxib at 0.09mg/kg IV. Pain scores did not increase after surgery in horses treated with flunixin meglumine or firocoxib. Unlike flunixin meglumine, firocoxib allowed recovery of TER and did not increase LPS flux across ischemic-injured jejunum. Analyses of plasma prostanoids suggested that firocoxib is COX-2 selective in horses. The effect of lidocaine on neutrophils was evaluated by incubating isolated equine neutrophils with 0.1-1000ï g/ml of lidocaine in vitro. Neutrophil adhesion and migration in response to stimulants was subsequently evaluated. LTB4 and IL-8 induced adhesion were increased at 1mg/ml of lidocaine. Migration increased with increasing concentration of lidocaine, in response to the same stimulants. Therefore, the use of firocoxib, or lidocaine in combination with flunixin meglumine, may be advantageous for horses recovering from ischemic intestinal injury, compared to treatment with a non-selective COX inhibitor, such as flunixin meglumine, alone.
The Role of the Enzyme Cyclooxygenase and Bile in the Damage and Repair of Intestinal Epithelium
(2003-11-06) Campbell, Nigel; Anthony Blikslager, Committee Chair
Colic describes poorly localized abdominal pain in horses and accounts for the majority of emergency calls received by equine veterinarians. The main drugs used to treat colic are the nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the enzyme cyclooxygenase (COX), and prevent endotoxin-induced elaboration of prostaglandins. There are 2 isoforms of cyclooxygenase: COX-1, which constitutively produces prostaglandins and COX-2, which is induced by inflammation. Study 1: We hypothesized that the non-specific cyclooxygenase inhibitor flunixin would retard repair of ischemic intestinal injury by preventing production of reparative prostaglandins whereas the selective COX-2 inhibitor, etodolac, would permit repair as a result of continued COX-1 prostaglandin production. Equine jejunum was subjected to ischemia for 1 hour, and recovered for 4 hours in Ussing chambers. In ischemic tissue treated with flunixin, production of prostaglandins was inhibited, and there was no evidence of recovery based on measurements of transepithelial resistance (TER). Conversely, untreated ischemic tissues or tissues treated with etodolac had significant elevations in prostaglandins, and significant recovery of TER. These studies suggest that specific COX-2 inhibitors may provide an advantageous alternative to non-specific cyclooxygenase inhibitors in horses with colic. Study 2: A potential adverse effect of NSAIDS in horses is colitis. It was hypothesized that the non-selective 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. Equine colon was exposed to 1.5mM deoxycholate for 30-minutes, after which they were recovered for 4 hours 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 non-selective 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. Study 3: It has been shown that rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, is attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of normal luminal contents, such as bile salts, which, according to our preliminary studies, reach concentrations as high as 10⁻⁵M in the porcine ileum. The objective of this study was to evaluate the role of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45-minutes of ischemia, after which mucosa was mounted in Ussing chambers, and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in TER, which recovered control levels of TER within 2-hours, associated with significant reductions in mucosal-to-serosal ³H-mannitol flux. However, treatment of ischemic-injured tissues with 10⁻⁵M deoxycholic acid fully inhibited recovery of TER with significant increases in mucosal-to-serosal 3H-mannitol flux, whereas 10⁻⁶M deoxycholic acid had no effect. Histologic evaluation at 2 hours revealed complete restitution regardless of treatment, indicating the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with application of 10⁻⁵M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca²⁺ mobilizing agent thapsigargin. Deoxycholic acid at physiologic concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca²⁺-dependent.