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.
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.
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.
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.
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
Objectives—To study the functional and structural
responses of the right dorsal colon (RDC) of ponies to
phenylbutazone (PBZ) in vitro at a concentration that
could be achieved in vivo.
Animals—8 adult ponies.
Procedure—Short circuit current and conductance
were measured in mucosa from the RDC. Tissues
incubated with and without HCO3– were exposed to
PBZ, bumetanide, or indomethacin. Bidirectional Cl–
fluxes were determined. After a baseline flux period,
prostaglandin E2 (PGE2) was added to the serosal surfaces
and a second flux period followed. Light and
transmission electron microscopy were performed.
Results—Baseline short circuit current was diminished
significantly by PBZ and indomethacin, and
increased significantly after addictions of PGE2. After
PGE2 was added, Cl– secretion increased significantly
in tissues in HCO3-–-free solutions and solutions
with anti-inflammatory drugs, compared with corresponding
baseline measurements and with control
tissues exposed to PGE2. Bumetanide did not affect
baseline short circuit current and Cl– fluxes. The predominant
histologic change was apoptosis of surface
epithelial cells treated with PBZ and to a lesser extent
in those treated with indomethacin.
Conclusions and Clinical Relevance—Prostaglandin-
induced Cl– secretion appeared to involve a
transporter that might also secrete HCO3–. Both PBZ
and indomethacin altered ion transport in RDC and
caused apoptosis; PBZ can damage mucosa through
a mechanism that could be important in vivo. The clinically
harmful effect of PBZ on equine RDC in vivo
could be mediated through its effects on Cl– and
HCO3– secretion. (Am J Vet Res 2002;220:934–941)
OBJECTIVE To examine effects of continuous rate infusion of lidocaine on transmural neutrophil infiltration in equine intestine subjected to manipulation only and remote to ischemic intestine.
ANIMALS 14 healthy horses.
PROCEDURES Ventral midline celiotomy was performed (time 0). Mild ischemia was induced in segments of jejunum and large colon. A 1-m segment of jejunum was manipulated by massaging the jejunal wall 10 times. Horses received lidocaine (n = 7) or saline (0.9% NaCl) solution (7) throughout anesthesia. Biopsy specimens were collected and used to assess tissue injury, neutrophil influx, cyclooxygenase expression, and hypoxia-inducible factor 1α (HIF-1α) expression at 0, 1, and 4 hours after manipulation and ischemia. Transepithelial resistance (TER) and mannitol flux were measured by use of Ussing chambers.
RESULTS Lidocaine did not consistently decrease neutrophil infiltration in ischemic, manipulated, or control tissues at 4 hours. Lidocaine significantly reduced circular muscle and overall scores for cyclooxygenase-2 expression in manipulated tissues. Manipulated tissues had significantly less HIF-1α expression at 4 hours than did control tissues. Mucosa from manipulated and control segments obtained at 4 hours had lower TER and greater mannitol flux than did control tissues at 0 hours. Lidocaine did not significantly decrease calprotectin expression. Severity of neutrophil infiltration was similar in control, ischemic, and manipulated tissues at 4 hours.
CONCLUSIONS AND CLINICAL RELEVANCE Manipulated jejunum did not have a significantly greater increase in neutrophil infiltration, compared with 4-hour control (nonmanipulated) jejunum remote to sites of manipulation, ischemia, and reperfusion. Lidocaine did not consistently reduce neutrophil infiltration in jejunum.
Objective—To assess the effects of ischemia and reperfusion on indicators of oxidative stress, activation of eosinophils, and apoptosis in the large colonic mucosa of horses.
Procedures—In 1 or two 20-cm-long segments of the pelvic flexure, ischemia was induced for 1 or 2 hours followed by no reperfusion or 30 minutes and 18 hours of reperfusion in anesthetized horses. Mucosal specimens were collected before (controls; n = 20 horses) and after each period of ischemia, and full-thickness tissue samples were collected after each period of reperfusion. Sections of colonic tissues were stained for histomorphometric analysis or assessment of eosinophil accumulation. Nitrotyrosine was identified immunohistochemically, and severity of apoptosis was determined via the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling method.
Results—Numbers of mucosal eosinophils were similar before induction of ischemia, after ischemia, and after ischemia-reperfusion. Eosinophil nitrotyrosine production increased significantly during ischemia and continued through 30 minutes of reperfusion; production was decreased at 18 hours of reperfusion but remained greater than that of the controls. In other leukocytes, nitrotyrosine generation peaked at 1 hour of ischemia and again at 18 hours of reperfusion. Compared with control findings, epithelial apoptosis increased gradually at 1 through 2 hours of ischemia with no further progression after reperfusion.
Conclusions and Clinical Relevance—Results suggested that resident eosinophils in the large colon of horses react to mucosal injury from ischemia and reperfusion and may undergo oxidative stress under those conditions. Epithelial apoptosis could contribute to tissue damage.
Objective—To determine the effect of large colon ischemia and reperfusion on concentrations of the inflammatory neutrophilic protein calprotectin and other clinicopathologic variables in jugular and colonic venous blood in horses.
Animals—6 healthy horses.
Procedures—Horses were anesthetized, and ischemia was induced for 1 hour followed by 4 hours of reperfusion in a segment of the pelvic flexure of the large colon. Blood samples were obtained before anesthesia, before induction of ischemia, 1 hour after the start of ischemia, and 1, 2, and 4 hours after the start of reperfusion from jugular veins and veins of the segment of the large colon that underwent ischemia and reperfusion. A sandwich ELISA was developed for detection of equine calprotectin. Serum calprotectin concentrations and values of blood gas, hematologic, and biochemical analysis variables were determined.
Results—Large colon ischemia caused metabolic acidosis, a significant increase in lactate and potassium concentrations and creatine kinase activities, and a nonsignificant decrease in glucose concentrations in colonic venous blood samples. Values of these variables after reperfusion were similar to values before ischemia. Ischemia and reperfusion induced activation of an inflammatory response characterized by an increase in neutrophil cell turnover rate in jugular and colonic venous blood samples and calprotectin concentrations in colonic venous blood samples.
Conclusions and Clinical Relevance—Results of this study suggested that large colon ischemia and reperfusion caused local and systemic inflammation in horses. Serum calprotectin concentration may be useful as a marker of this inflammatory response.