Browsing by Author "Jody Gookin, Committee Member"

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Cyclooxygenase Inhibitors Affect Recovery of Ischemic-injured Jejunum
(2005-03-24) Tomlinson, Julia Elizabeth; Malcolm_Roberts@ncsu.edu, Committee Member; Anthony T Blikslager, Committee Chair; Jody Gookin, Committee Member; Mathew P Gerard, Committee Member
he cyclooxygenase (COX) enzyme produces prostaglandins. There are 3 isoforms of COX (including COX-1 and COX-2). As prostaglandins are involved in intestinal repair, the objective of this study was to examine the effects of COX inhibitors on healing of jejunal mucosa following ischemia, with the following specific aims. Aim 1: Assess effects of in vivo administration of flunixin (non-selective inhibitor) and etodolac (COX-2 preferential inhibitor) on barrier function of ischemic-injured equine jejunum after 18hrs recovery. Aim 2: Assess effects of in vitro treatment with a selective COX-2 inhibitor, deracoxib on the permeability of ischemic equine jejunum. Aim 3: Assess effects of ischemia and flunixin on absorption of lipopolysaccharide (LPS) in vitro. Aim 4: Examine the role of COX-2 in jejunal recovery using COX-2 knockout mice. Ussing chambers were used to assess the barrier function of ischemic and normal jejunal mucosa when exposed to the different inhibitors mentioned above. Transepithelial electrical resistance (TER), a sensitive index of barrier function, was calculated and the permeability of the mucosa assessed using mannitol. The amount of epithelial denudation following ischemia was determined and compared between treatments. Levels of expression of the COX-1 and ?2 enzymes before and after ischemia were assessed by Western blot. For aim 3, permeability to lipopolysaccharide (LPS) was measured and the passage of fluorescent-labeled LPS into mucosa assessed with histology. Statistical significance level for all tests was chosen at p < 0.05. Both flunixin and etodolac treatment retarded in vivo recovery of jejunal barrier function after ischemia. These drugs act on the paracellular space as treatments did not alter epithelial denudation or restitution. Normal equine jejunal mucosa expressed both COX-1 and COX-2 and ischemia upregulated both isoforms. The selective COX-2 inhibitor deracoxib also adversely affected mucosal recovery in vitro but to a lesser extent; deracoxib did not significantly increase permeability to mannitol. The increased permeability of mucosa to mannitol after flunixin treatment did not reflect increased LPS absorption despite a trend toward this finding. Ischemia alone increased the absorption of LPS. Although the precise route of LPS across the mucosa was not determined, it was postulated to be mostly through the defects from epithelial cell loss. Attempts to clarify the role of COX-2 in mucosal recovery from ischemia using COX-2 knockout mice produced conflicting results. COX-1 inhibition increased permeability to mannitol in both COX-2 knockout and wild-type mice. There was a trend towards increased permeability due to COX-2 inhibition in wild-type mice. For knockout mice, the COX-2 inhibitor also unexpectedly increased permeability. COX-1 protein was present in all mice and COX-2 was present in normal mucosa of wild-type mice. Both isoforms were not upregulated by ischemia. Untreated horses recovered baseline levels of TER and permeability by 18hrs after ischemia. Both flunixin and etodolac did not allow sufficient prostaglandin production for recovery. In vitro treatment with the selective COX-2 inhibitor deracoxib also adversely affected recovery but to a lesser extent than the other drugs tested. The increased permeability due to flunixin did not exacerbate LPS absorption in this model. The clinical significance of the effects of COX inhibitors on permeability will require further in vivo testing. COX-1 and '2 are both present in normal jejunal mucosa from both horses and mice, thus it is possible that the action of both enzymes is required for complete recovery from ischemia.
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.
Effects of Diesel Exhaust Particle Exposure on Inflamed Murine Lung Epithelial Cells
(2009-10-20) Manzo, Nicholas David; Janice Dye, Committee Co-Chair; Jerry Law, Committee Co-Chair; Philip Sannes, Committee Member; Jody Gookin, Committee Member
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.