Effects of flunixin meglumine on the recovery of ischaemic equine colonic mucosa in vitro.

REASONS FOR PERFORMING STUDY: The effects of prostaglandins and nonsteroidal anti-inflammatory drugs (NSAIDs) on repair of equine intestinal mucosa are important since most horses with gastrointestinal diseases are routinely treated with NSAIDs, such as flunixin meglumine (FM), and these drugs can be toxic to equine gastrointestinal mucosa. HYPOTHESIS: Flunixin meglumine would not affect recovery of equine colonic mucosa in vitro, 18 h after a reversible ischaemic injury. METHODS: In 14 anaesthetised horses, a segment of pelvic flexure was subjected to 2 h of ischaemia and the horses were allowed to recover for 18 h. Seven horses received normal saline and 7 received FM, 1.1 mg/kg bwt i.v., at the end of ischaemia and 12 h later. Colonic mucosa was harvested during a second anaesthesia, 18 h after recovery from ischaemia and then horses were subjected to euthanasia. Transepithelial electrical resistance (TER) and transepithelial flux of tritiated mannitol were used to measure mucosal permeability during 4 h of incubation in Ussing chambers, with the following in vitro treatments: 1) no addition, 2) FM 14 µmol/l as powder, 3) FM 14 µmol/l in injectable form and 4) diluent for injectable FM. Histomorphological changes were assessed by light microscopy. RESULTS: There were no significant differences in any of the measurements between saline and FM treated horses. The mucosal height of the ischaemic FM tissues incubated in diluent was significantly decreased compared to the nonischaemic tissues. CONCLUSIONS: Flunixin meglumine did not adversely affect barrier integrity in ischaemic equine colonic mucosa.

Detection of calprotectin and its correlation to the accumulation of neutrophils within equine large colon during ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required. OBJECTIVES: To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R. METHODS: One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells. RESULTS: The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion. CONCLUSIONS: Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.

Effect of a novel solution for organ preservation on equine large colon in an isolated pulsatile perfusion system.

REASONS FOR PERFORMING STUDY: Several therapeutic agents have been tested in models of ischaemia and reperfusion injury (IRI) in equine jejunum, with mixed results. This study was based on the use of an organ perfusion solution (OPS) designed to protect human allografts from IRI. HYPOTHESIS: A modified OPS can preserve the integrity of equine large colon during 12 h of isolated pulsatile perfusion, in the absence of oxygen and blood. METHODS: Segments of large colon were removed from anaesthetised horses, the contents removed and the mucosa rinsed with 0.9% saline. Experimental segments were perfused for 12 h with one litre modified OPS (n = 7) delivered by pulsatile flow through an extracorporeal circuit. Control segments (n = 4) were perfused on the same circuit with one litre of autologous blood. Vascular resistance, flow and pressure were measured serially, and aliquots of OPS and blood drawn hourly for routine biochemical analyses. Mucosal biopsies of the experimental and control segments were taken at 0, 6 and 12 h and in vivo mucosal tissue at 0 h for baseline comparison. All biopsies underwent histomorphometric analysis and immunohistochemical assessment of calprotectin activity. RESULTS: All colon segments were machine perfused without technical complications. Vascular and biochemical indices remained constant over 12 h in the OPS group, and were constant over 6 h in the control group, but deteriorated later. Mucosal integrity, expression of cyclooxygenases-1 and -2, and expression of mucosal calprotectin were unchanged in the OPS group compared with the baseline tissues, and mucosal integrity was superior to the control tissues. CONCLUSIONS: A modified OPS designed to target specific pathways of damage from IRI can preserve colonic mucosal integrity for 12 h in the absence of blood and oxygen.