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 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 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 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.
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.
OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease.
ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases.
PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases.
RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses.
CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.