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Abstract

Objective—To determine effects of reactive oxygen metabolites (ROMs), with and without flunixin meglumine, on equine right ventral colon (RVC) in vitro.

Animals—18 healthy horses and ponies.

Procedures—In 3 groups of 6 animals each, short-circuit current and conductance were measured in RVC mucosa in Ussing chambers. The 3 groups received physiologic saline (0.9% NaCl) solution, IV, 10 minutes before euthanasia and tissue incubation in Krebs-Ringer-bicarbonate (KRB) solution; flunixin meglumine (1.1 mg/kg, IV) 10 minutes before euthanasia and tissue incubation in KRB solution; or physiologic saline solution, IV, 10 minutes before euthanasia and incubation in KRB solution with 2.7 × 10−5M flunixin meglumine. Incubation conditions included control (no addition) and ROM systems, including addition of 1mM xanthine and 80 mU of xanthine oxidase (to produce the superoxide radical), 1mM H2O2, and 1mM H2O2 and 0.5mM ferrous sulfate (to produce the hydroxyl radical).

Results—All ROMs that were added or generated significantly increased the short-circuit current except in tissues coincubated with flunixin meglumine, and they induced mild epithelial vacuolation and apoptosis, but did not disrupt the epithelium nor change conductance, lactate dehydrogenase release, or [3H]mannitol flux.

Conclusions and Clinical Relevance—Responses to ROMs could be attributed to increased chloride secretion and inhibited neutral NaCl absorption in equine RVC, possibly by stimulating prostaglandin production. The ROMs examined under conditions of this study could play a role in prostaglandin-mediated colonic secretion in horses with enterocolitis without causing direct mucosal injury.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To examine bicarbonate (HCO3) secretion ex vivo in the equine large colon to determine any differences between the right dorsal colon (RDC) and right ventral colon (RVC). The effect of phenylbutazone (PBZ) on HCO3 secretion was examined in the RDC.

ANIMALS

14 healthy horses.

PROCEDURES

In anesthetized horses (n = 10), segments of mucosa from RDC and RVC were harvested to measure HCO3 secretion ex vivo with the pH Stat method. The effect of PBZ on HCO3 secretion in the RDC was studied in 4 additional horses.

RESULTS

Three distinct mechanisms of HCO3 secretion previously described in a murine model were confirmed in the equine colon. The RDC had a greater capacity for electrogenic, Cl-independent HCO3 secretion than the RVC (P = 0.04). In the RDC, all HCO3 secretion was decreased by PBZ (P < 0.02) but was not studied in the RVC because of low baseline secretion.

CLINICAL RELEVANCE

Secretion of HCO3 by the RDC could play a pivotal role in equine colon physiology, because intense microbial fermentation in this site could require HCO3 secretion to buffer short-chain fatty acids. Inhibition of this secretion by PBZ could interfere with mucosal buffering and predispose to changes associated with right dorsal colitis.

Open access
in American Journal of Veterinary Research

Abstract

Objective—To study the effects of phenylbutazone, indomethacin, prostaglandin E2 (PGE2), glutamine, and butyrate on restitution of oxidant-injured right dorsal colon of horses in vitro.

Sample Population—Right dorsal colon from 9 adult horses euthanatized for reasons other than gastrointestinal tract disease.

Procedure—Mucosal segments from the right dorsal colon were injured via exposure to HOCl and incubated in Ussing chambers in solutions containing phenylbutazone, indomethacin, indomethacin and PGE2, glutamine, and butyrate. Transepithelial resistance and mucosal permeability to mannitol were measured, and all mucosal segments were examined histologically.

Results—The HOCl-injured mucosa had lower resistance and higher permeability to mannitol, compared with control tissue. Histologic changes were also evident. Resistance of HOCl-injured mucosa recovered partially during the incubation period, and glutamine improved recovery. Phenylbutazone and indomethacin increased resistance, but these increases were not significant. Butyrate and PGE2 had no effects, compared with nontreated HOCl-injured tissues. Mucosal permeability to mannitol was lower in glutamine-treated tissue, compared with nontreated tissue. Histologic changes reflected the resistance and permeability changes.

Conclusions and Clinical Relevance—According to our findings, phenylbutazone and indomethacin do not seem to interfere with restitution of oxidant-injured mucosa of equine colon in vitro, and glutamine could facilitate mucosal restitution. (Am J Vet Res 2004;65:1589–1595)

Full access
in American Journal of Veterinary Research

Abstract

Objectives—To establish reference values for the range of the number of eosinophils found in equine gastrointestinal mucosa and to describe the distribution of this cell within the equine gastrointestinal mucosa.

Sample Population—Gastrointestinal mucosal specimens from 14 adult horses euthanatized for reasons other than gastrointestinal disease.

Procedures—Gastrointestinal mucosal specimens were collected and grouped according to their anatomic regions. For histologic examination slides were stained with Luna's eosinophil stain to determine eosinophil accumulation and distribution. The mucosa was divided into 5 sections for each anatomic location, and the percentage of eosinophils in each of the 5 sections relative to the total eosinophil count in all sections was determined. Additionally, the number of eosinophils per square millimeter of mucosa was calculated as a measure of the degree of eosinophil accumulation.

Results—Lowest numbers of eosinophils were found in the stomach, and numbers increased from there to the cecum, then decreased from the ascending colon (right ventral colon, left ventral colon, pelvic flexure, left dorsal colon, and right dorsal colon) to small colon. In all gastrointestinal sections, most eosinophils were located near the muscularis mucosae and were rarely found near or on the luminal surface of the mucosa.

Conclusions and Clinical Relevance—The distribution of eosinophils in the gastrointestinal tract of horses followed a pattern within the mucosa and between different sections of the gastrointestinal tract. The derived reference values and distribution data could be used to detect changes in eosinophil response in the equine gastrointestinal mucosa caused by diseases states.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To induce ischemia and reperfusion injury in the large colon mucosa of horses in vivo and evaluate the recovery and effects of components of an organ transplant solution on mucosal recovery in vitro.

Animals—6 healthy horses.

Procedures—Horses were anesthetized, and ischemia was induced for 60 minutes in the pelvic flexure, which was followed by reperfusion for 240 minutes. Ischemic (n = 4 horses), reperfused (6), and adjacent control (6) colonic mucosae were isolated for in vitro testing and histologic examinations. Tissues were mounted in Ussing chambers with plain Krebs Ringer bicarbonate (KRB), KRB with N-acetylcysteine (NAC), or KRB with a modified organ transplant solution (MOTS). Transepithelial electrical resistance (TER) and mannitol flux were used to assess mucosal integrity. Data were analyzed by use of ANOVA and Kruskal-Wallis tests.

Results—The TER in reperfused tissues was similar to the TER in control tissues and greater than the TER in ischemic tissues, which was consistent with morphological evidence of recovery in reperfused tissues. Mannitol flux was greater in ischemic tissues than in reperfused tissues. The TER and mannitol flux were not significantly affected by incubation of mucosa with NAC or MOTS.

Conclusions and Clinical Relevance—Ischemia induced during the brief period allowed rapid mucosal repair and complete recovery of tissue barrier properties during reperfusion. Therefore, reperfusion injury was not observed for this method of ischemic damage in equine colonic mucosa.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To examine the effects of flunixin meglumine (FM) on recovery of colonic mucosa from experimentally induced ischemia in horses.

Animals—14 research horses.

Procedures—Ischemia was induced in the colons of anesthetized horses for 2 hours. Afterward, horses received saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) and were allowed to recover for 18 hours after termination of the ischemic event. Postoperative pain scores were recorded every 4 hours throughout the recovery period. At the end of the recovery period, horses were anesthetized, and ischemic and nonischemic segments of colonic mucosa were harvested for histologic evaluation, western blot analysis, and in vitro assessment of transepithelial electric resistance (TER) and transmucosal flux of tritium-labeled (3H-) mannitol. Horses were then euthanatized.

Results—Flunixin meglumine significantly lowered pain scores at the first postoperative recording. There were no significant differences between treatment with saline solution and FM in any of the measurements for TER, 3H-mannitol flux, histomorphometric variables, neutrophil infiltration (detected via calprotectin immunostaining), and expressions of cyclooxygenase-1 and -2. After both treatments, TER declined significantly in nonischemic tissues in vitro, whereas it increased significantly in ischemic-injured tissues.

Conclusions and Clinical Relevance—Flunixin meglumine did not affect recovery of equine colonic mucosa from ischemic injury, and continued use in horses with colonic ischemia is therefore justified.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine characteristics of the inflammatory reaction in the jejunum of horses in response to various mechanical manipulations.

Animals—12 adult warmblood horses without gastrointestinal tract disorders.

Procedures—The proximal aspect of the jejunum in each horse was divided into 5 segments, and the following manipulations were performed: manual emptying, placement of Doyen forceps, enterotomy alone, enterotomy with mucosal abrasion, and serosal abrasion. Jejunum samples were collected before (control), immediately after, and 30 minutes after the end of manipulations and histologically evaluated to determine distribution of neutrophils and eosinophils.

Results—Macroscopically, all manipulations resulted in jejunal hemorrhage and edema. Compared with control samples, neutrophil numbers were significantly higher after manipulations in the serosa (after all manipulation types), circular muscle layer (after manual emptying), submucosa (after placement of Doyen forceps), and mucosa (after all manipulations except enterotomy alone). Eosinophil numbers were significantly higher in the submucosa after mechanical abrasion of the serosa and manual emptying versus control samples.

Conclusions and Clinical Relevance—Results indicated mechanical manipulation of the jejunum resulted in local inflammatory reactions characterized predominantly by infiltration of neutrophils. This could contribute to the development of postoperative ileus or adhesions in horses without macroscopically detectable injury of the jejunum during surgery.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To identify expression and localization of cyclooxygenase (COX)-1 and COX-2 in healthy and ischemic-injured left dorsal colon of horses.

Sample Population—Left dorsal colon tissue samples from 40 horses.

Procedures—Tissue samples that were used in several related studies on ischemia and reperfusion were evaluated. Samples were collected during anesthesia, before induction of ischemia, and following 1 hour of ischemia, 1 hour of ischemia and 30 minutes of reperfusion, 2 hours of ischemia, 2 hours of ischemia and 30 minutes of reperfusion, and 2 hours of ischemia and 18 hours of reperfusion. Histomorphometric analyses were performed to characterize morphological injury. Immunohistochemical analyses were performed to characterize expression and localization of COX-1 and COX-2.

Results—COX-1 and COX-2 were expressed in control tissues before ischemia was induced, predominantly in cells in the lamina propria. Ischemic injury significantly increased expression of COX-2 in epithelial cells on the colonic surface and in crypts. A similar significant increase of COX-1 expression was seen in the epithelial cells.

Conclusions and Clinical Relevance—On the basis of information on the role of COX-2, upregulation of COX-2 in surface epithelium and crypt cells following ischemic injury in equine colon may represent an early step in the repair process.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate the eosinophilic response in intestinal mucosa of horses with intestinal ischemia and reperfusion or with strangulation of the jejunum or colon.

SAMPLE Mucosal samples from horses with naturally occurring strangulation (n = 24 horses) or distention (n = 6) of the jejunum or colon (11), with experimentally induced ischemia and reperfusion of the jejunum (6) or colon (15), or that were euthanized for reasons other than gastrointestinal tract disease (13).

PROCEDURES Mucosal samples were collected and grouped by type of intestinal injury. Slides were stained with Luna eosinophil stain and histologically examined to determine eosinophil accumulation and distribution. Number of eosinophils per mm2 of mucosa was calculated as a measure of eosinophil accumulation. Additionally, mucosa was categorized into 5 regions; the percentage of eosinophils in each of the 5 regions, relative to the total eosinophil count in all regions, was determined.

RESULTS Eosinophil migration toward and onto the luminal surface was evident in tissues after ischemia and reperfusion and after naturally occurring strangulating disease of the jejunum and colon, as indicated by a decrease in the number of eosinophils near the muscularis mucosa and an increase in the number of eosinophils on or near the luminal surface. Ischemia alone did not change eosinophil distribution in the jejunum or colon.

CONCLUSIONS AND CLINICAL RELEVANCE Eosinophils responded to mucosal damage evoked by ischemia and reperfusion by migration toward and onto the luminal surface. This migration could represent an important component of the inflammatory response to injury in equine gastrointestinal mucosa.

Full access
in American Journal of Veterinary Research

Abstract

Objectives—To evaluate the in vitro protective effects of acetylcysteine and response of resident mucosal eosinophils in oxidant-induced injury to tissues of right dorsal colon of horses.

Animals—9 adult horses.

Procedure—Gastrointestinal mucosa was damaged in vitro with 3mM hypochlorous acid (HOCl), with and without prior exposure to 6mM acetylcysteine. Control tissues were not exposed to HOCl or acetylcysteine. Control and damaged tissues were incubated in Krebs-Ringer-bicarbonate solution and tissue resistance measured during 240 minutes. Tissue permeability to radiolabeled mannitol was also used to assess mucosal barrier integrity. Tissues were examined by light microscopy before and after HOCl exposure and during and after incubation.

Results—Exposure to HOCl caused tissue damage and decreased tissue resistance. Restitution did occur during the incubation period. Eosinophils were located near the muscularis mucosae in freshly harvested tissues and migrated towards the luminal surface in response to HOCl-induced injury. Compared with tissues treated with HOCl without acetylcysteine, pretreatment with acetylcysteine prevented HOCl-induced tissue damage, changes in resistance, and histologically detectable eosinophil migration. The permeability to mannitol increased to the same extent in tissues treated with HOCl alone or with acetylcysteine and HOCl.

Conclusions and Clinical Relevance—Eosinophils migrated toward the mucosal surface in equine colon in response to oxidant-induced damage in vitro. This novel finding could be relevant to inflammation in equine colon and a pathophysiologic feature of many colonic diseases. Acetylcysteine protected the mucosa against oxidant-induced injury and may be useful as a treatment option for various gastrointestinal tract disorders in horses. (Am J Vet Res 2003;64:1205–1212)

Full access
in American Journal of Veterinary Research