Colic has been identified by veterinarians1 and a national survey2,3 as a leading health concern and a major cause of death in horses. Strangulating large-colon volvulus can account for 11% to 27% of surgical cases of colic,4 and fatality of affected horses can approach 34% to 65.3% without resection.5 Even resection does not remove all nonviable colon, and integrity of the remaining mucosa can determine outcome.6 Probability of survival can be influenced by loss of the epithelial barrier, which allows transmucosal leakage of endotoxin, bacterial chemotactic peptides, and bacteria.7 Rapid repair of the epithelium is important for recovery and involves 2 processes that are usually completed within hours: mucosal restitution and tightening of paracellular pathways between remaining cells.8,9 Restitution involves sealing the mucosal defect with remaining viable cells before final repair through cell division and proliferation.10
The effects of prostaglandins and NSAIDs on repair of intestinal mucosa in horses are important because most horses with gastrointestinal diseases are routinely treated with NSAIDs such as FM, and these drugs can be toxic to equine gastrointestinal mucosa.11 In a model of ischemia-induced injury in equine jejunum, FM inhibited mucosal repair in vitro but did not increase mucosal permeability to fluorescent-labeled LPS in ischemic tissues.12 After 2 hours of ischemia followed by 18 hours of recovery before in vitro evaluation, FM, which is more selective for COX-1 in equine blood,13 and the more COX-2–selective NSAID etodolac14 retarded mucosal repair in equine jejunum in vitro.15 In a similar model, recovery of transepithelial barrier function in ischemia-injured equine jejunum was inhibited by FM but not by COX-2–selective meloxicam.16 In contrast to these findings in equine jejunum, FM did not significantly affect repair of chemically damaged equine colon in vitro,17 and other NSAIDs, specifically phenylbutazone and indomethacin, did not affect recovery of oxidant-injured equine colon in vitro.18 Understanding of the effects of NSAIDS on intestinal mucosa in horses is additionally complicated by the finding that NSAIDs can jeopardize barrier integrity in intact equine colonic mucosa in vitro.17,19,a
Conflicting findings regarding the effects of NSAIDs on mucosal repair could be explained by the relative activities of the 2 isoforms of COX, COX-1 and COX-2, and differential effects of NSAIDs on this relationship. The traditional concept is that COX-1 is the predominant isoform in healthy mucosa and is responsible for preserving tissue function and integrity, whereas expression of COX-2 is upregulated during intestinal inflammation.20 Nonspecific COX inhibitors, such as FM, phenylbutazone, and indomethacin, may have detrimental effects in healthy gastrointestinal mucosa through their effect on COX-1 activity, whereas they may be beneficial in inflamed intestine though their effect on COX-2.17 However, both isoforms of COX contribute to many aspects of mucosal defense.21 Furthermore, different patterns of COX-1 and COX-2 expression have been detected in horses, compared with patterns in other species.b Cyclooxygenase-1 and COX-2 are expressed in healthy equine colon, and ischemic injury increases expression of both isoforms.b
The purpose of the study reported here was to examine the functional and morphologic recovery of equine colonic mucosa that was subjected to 2 hours of ischemia followed by 18 hours of recovery in vivo. Our hypothesis was that FM, an inexpensive and popular NSAID for equine practitioners, would not adversely affect recovery of equine colonic mucosa after this ischemic injury.
Materials and Methods
Animals and tissue preparation—Fourteen healthy horses, aged 2.5 to 20.5 years and weighing 390 to 574 kg, were used in the study. Inclusion criteria were that horses be free of gastrointestinal tract disease and require euthanasia for conditions that rendered them unsuitable for use. Horses underwent a 1-week quarantine period, during which time they were fed grass hay (2% of their body weight/d) and were provided with water ad libitum. The 14 horses were assigned to 2 groups of 7 each: one that would receive saline (0.9% NaCl) solution and the other that would receive FM after induction of ischemia. The University of Florida Institutional Animal Care and Use Committee approved the experimental protocol.
Surgical procedures—Horses were sedated with xylazine (1.1 mg/kg, IV), and a 14-gauge, 13.3-cm coated catheter was placed in a left jugular vein for administration of anesthetic drugs and fluids. Anesthesia was induced with ketamine (2.2 mg/kg, IV) and diazepam (0.1 mg/kg, IV). Horses were orotracheally intubated, positioned in dorsal recumbency, and a surgical plane of anesthesia was maintained with isoflurane vaporized in oxygen. Physiologic polyionic fluids were infused continuously IV at 2.5 mL/kg/h. Mean arterial blood pressure was monitored through a 20-gauge, 5.1-cm coated catheter in a facial artery and was maintained at or > 60 mm Hg. Physiologic monitoring during anesthesia included electrocardiography, blood gas analysis, and measurement of end-tidal partial pressure of CO2.
The ventral abdomen was prepared for aseptic surgery and draped, a ventral midline celiotomy was performed, and the pelvic flexure was exteriorized onto a sterile drape. To induce ischemia, intestinal clamps were applied at each end of the selected segment of pelvic flexure to compress mural vessels, and colonic arteries and veins to the same segment were ligated with umbilical tape. A pulse oximeterc was used to detect pulsatile flow and to measure oxygen saturation on the serosal surface to confirm segmental ischemia and to ensure that adjacent segments were perfused.22 The colon was then replaced in the abdomen until the end of the ischemic period, and the abdominal incision was closed temporarily with towel clamps.
After 2 hours of ischemia, mucosal biopsy specimens were obtained from the nonischemic colon (proximal or distal to the ischemic segment) and from the ischemic segment of 3 horses from each treatment group. The clamps and ligatures were removed, and at that time, 1 group of horses received saline solution (12 mL, IV) through the left jugular catheter, and the other group received FM (1.1 mg/kg). Biopsy sites and the celiotomy were closed routinely, and horses recovered from surgery in a padded recovery stall. After recovery from anesthesia, each horse was moved to a stall and monitored for signs of pain, and pain score was recorded every 4 hours according to an established behavioral scoring system.15,23 Horses in both treatment groups received butorphanol (0.05 mg/kg, IM, q 4 h) for the first 8 hours after surgery to alleviate pain.24 Physical examination findings, including rectal temperature, heart rate, respiratory rate, mucous membrane color, capillary refill time, intestinal sounds, and digital pulses, were recorded for each horse every 4 hours. Times when each horse defecated and urinated were also recorded.
Each horse received 1 more treatment of FM or saline solution at 12 hours after the first dose was administered. At 18 hours after blood flow had been reestablished to the ischemic colon and with horses anesthetized as described for the first surgery, full-thickness colon wall was harvested from ischemic-injured and adjacent nonischemic colon for histologic evaluations, western blot analysis, and in vitro experiments in Ussing chambers. After tissues were harvested, horses were euthanatized with an overdose of sodium pentobarbital (88 mg/kg, IV) while anesthetized.
Ussing chamber experiments—A solution of KRB (pH, 7.4) was prepared from 112mM NaCl, 25mM NaHCO3, 10mM glucose, 5mM KCl, 3mM sodium acetate, 3mM sodium butyrate, 2.5mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, and 0.01mM mannitol. Sheets of full-thickness colon wall from the ischemic segment and an adjacent nonischemic (control) segment from each horse were immediately transported to the laboratory at 4°C in KRB solution.25 Each tissue segment was pinned on a rubber surface, with the mucosal surface facing up, and submerged in a fresh batch of KRB solution at 4°C. Sharp dissection was used to remove mucosal sheets to mount in Ussing chambers with an aperture of 1.13 cm2, and 10 mL of KRB solution was applied to each tissue surface. The KRB solution was maintained at a pH of 7.4 by constant perfusion with 95% O2 and 5% CO2 and at 37°C by circulating the solution with a gas lift through water-jacketed reservoirs.
The value for the short-circuit current (MA/cm2) was recorded in each chamber on voltage clampsd through Ag-AgCl2 electrodes connected to 4% agar bridges in KRB solution. Junction potentials of electrodes and fluid resistance were measured before mounting the tissues to allow continuous correction for any effects that these factors would have had on the low potential difference readings generated by the tissue.26,27 When tissues were mounted in chambers, the voltage clamp could then automatically correct for junction potentials of electrodes and fluid resistance, so these effects on recordings were reduced.d Throughout incubation, the tissues were continuously short-circuited, except at 15-minute intervals when the spontaneous potential difference of tissue was measured. The TER was calculated by use of Ohm's law (potential difference divided by short-circuit current). Resistance was used as a measure of integrity of the colonic mucosa and permeability of the paracellular pathway to ions.26–28 The unidirectional flux of tritium labeled (3H-) mannitole from the mucosal to the serosal solution was measured to detect changes in tissue permeability.28 Interval from tissue collection to mounting tissue in the first chamber was approximately 12 minutes and to first recording of TER and addition of 3H-mannitol was 42 minutes. Total incubation time in Ussing chambers was 240 minutes.
Histomorphometric measurements—All tissues were fixed in formalin for light microscopy, embedded in paraffin, and cut into 5-μm-thick sections on silanecoated glass slides. Slides were stained with H&E in a routine manner. For the histomorphometric assessment via light microscopy, a computer-based imaging analysis programf was used, and 3 fields of each tissue were examined as described elsewhere.28 One investigator unaware of the identity of the treatment group performed all histologic evaluations.28 Mucosal height was expressed as the mean vertical distance between tracings of the muscularis mucosae and the epithelial surface in micrometers. Epithelial height was expressed in micrometers as the mean vertical perpendicular distance between the basement membrane and the cell apex. Width of 5 clearly identifiable epithelial cells was measured in 3 different areas in each field. The length of mucosal surface denuded of epithelium was measured and expressed as a percentage of the total surface length of the mucosa in the section. Lifted epithelium was defined as a group of at least 5 epithelial cells that were separated from the basement membrane but were still attached to adjacent epithelial cells that held them in place. The length of lifted epithelium was expressed as a percentage of the total surface length of the mucosa in the section. Detached cells were defined as cells that were morphologically similar to healthy cells but were separated from the basement membrane in groups of at least 5 cells and were completely detached from adjacent epithelium. The length of detached cells was measured and expressed as a percentage of the total surface length of the mucosa in the section. Sloughed cells were defined as cells undergoing necrosis and sloughing one at a time from the epithelial surface. The number of sloughed cells was counted in each field, and the mean number/0.1 mm of mucosal surface length was then calculated.
Immunohistochemical detection of calprotectin—For immunohistochemical evaluation, mucosal sections were deparaffinized and rehydrated in a routine manner. Immunohistochemical identification of cytosolic calprotectin29 was performed with 1:100 mouse antihuman macrophages monoclonal antibody (MAC387),g which cross-reacts with equine calprotectin, and a commercially available biotin-free detection kitg by means of a modified staining procedure for equine jejunum.23 For antigen retrieval, each tissue underwent heat treatment with a pressure cooker (125°C for 30 seconds and 90°C for 10 seconds) and a retrieval bufferh with a pH of 6.0. The sections were incubated in 0.03% hydrogen peroxide for 15 minutes to quench endogenous peroxidase and with bovine serum albumin for 15 minutes to block nonspecific binding. Slides were then incubated with the primary antibody diluted in PBS solution for 30 minutes at room temperature (21°C). In additional sections, PBS solution alone was applied to serve as a negative control section. Incubation with a biotin-free secondary antibody that was polymerized directly with horseradish peroxidaseg was performed for 30 minutes at room temperature. Sections were then incubated for 3 to 5 minutes in the chromogen 2.5% diaminobenzidine to visualize the antigen. For each procedure, 1 equine lung specimen was stained in a similar manner, and stained alveolar macrophages were used as positive control specimens.30
All sections were counterstained with Mayer hematoxylin for 5 minutes, and color was developed with ammonium hydroxide (2.9mM). After dehydration with 70%, 2X 95%, 2X 100% ethanol, and 3X xylenes, sections were mounted in mounting mediumi and covered with glass coverslips. Tissues were examined via light microscopy with a 40X objective lens and a computer-based image analysis program.f Three fields of view on each slide were randomly selected for examination. Segments measured 866-μm wide and extended the full height along the vertical axis of the tissue from the muscularis mucosae to the luminal surface. Cytoplasm of calprotectin-positive cells appeared brown, and these stained cells were directly counted per square millimeter of mucosa within 3 segments of each tissue. One investigator who was unaware of treatment group allocation performed all histologic evaluations.
Molecular evaluation of expression of COX-1 and COX-2—Mucosal biopsy specimens were harvested from horses of both treatment groups, according to the aforementioned collection protocols, and were snap frozen.31 Tissues were stored at −80°C prior to preparation for SDS-PAGE, at which time they were thawed to 4°C. Tissue portions (1 g) were added to 3 mL of chilled radioimmunoprecipitation assay buffer (0.15M NaCl, 50mM Tris at a pH of 7.2, 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS), including protease inhibitors. The mixture was homogenized on ice and centrifuged at 4°C for 10 minutes at 7,000 × g, and the supernatant was saved. This supernatant was then centrifuged again at 4°C for 10 minutes at 10,500 × g. Protein was quantified in extract aliquots.j Tissue extracts (amounts equalized by protein concentration) were mixed with an equal volume of SDS-PAGE sample buffer (2×) and boiled for 4 minutes. Lysates (25 Mg/well) were loaded in triplicate on a 10% SDS-polyacrylamide gel. A standardized molecular weight markerj was loaded for size determination of positively staining bands, and B-actin was used as a loading control to ensure equivalent loading of protein samples.31
Electrophoresis was carried out according to standard protocols.31 Proteins were transferred to a nitrocellulose membranek with an electroblotting minitransfer apparatusj according to the manufacturer's protocol. Membranes were blocked at room temperature for 60 minutes in TBSST and 5% dry powered milk. Membranes were washed twice with TBSST and incubated for 1 hour in primary antibodyl (COX-1 or COX-2 affinity purified polyclonal goat anti-human IgG). After 3 washes of 10 minutes each with TBSST, membranes were incubated for 60 minutes with donkey anti-goat IgG horseradish peroxidase–conjugated secondary antibody.l After 3 additional washes of 5 minutes each with TBSST, the membranes were developed for visualization of protein with an alkaline phosphatase conjugate substrate kit.j Following development, the blots were stripped by placement in 100mM 2-mercapto-ethanol, 2% SDS, plus 625mM Tris-Cl at 50°C for 30 minutes. The blots were then washed 3 times for 10 minutes with TBSST and probed for B-actin with mouse monoclonal IgG for primary antibody and goat antimouse IgG horseradish peroxidase–conjugated secondary antibodyl as previously described. Western blot quantitation of COX-1 and COX-2 concentrations was performed via densitometric measurements on scanned images by use of specialized software.m
Immunohistochemical analysis for COX-1 and COX-2 localization—Tissues were fixed in neutral-buffered 10% formalin, routinely processed for paraffin embedding, and cut into 5-μm sections. Following placement on slides, sections were deparaffinized and rehydrated. Heat antigen retrieval was performed, and slides were subsequently incubated in 1% H2O2. Slides were washed in PBS solution and incubated with normal goat seruml for 1 hour. Slides were then incubated for 30 minutes with goat anti-human COX-1 polyclonal antibodyl or goat anti-human COX-2 polyclonal antibody.l This step was not performed on negative control slides. Slides were washed 3 times in PBS solution between 30 minute incubations with biotinylated donkey anti-goat antibodyl and streptavidin-labeled peroxidase. lSections were counterstained with Mayer hematoxylin for 3 minutes, and color was developed with 150 ML of ammonium hydroxide (2.9mM). Slides were then washed in distilled water, dehydrated, and mounted in an aqueous mounting medium, and coverslips were applied.
For the evaluation of the images obtained via light microscopy, a computer-based image analysis programf was used and 3 randomly selected fields of view from each tissue with a length of 866 μm (equal to the length of 1 image by use of a 10X objective lens) were examined. The epithelium, upper and lower lamina propria, and crypts of the colonic mucosa were scored from 0 to 3 for evidence of intracellular COX-1 and COX-2 expression (brown cytoplasm with blue counterstaining) by use of a 40X objective lens. Grade 0 was assigned when stained cells were absent or single cells were detected after careful inspection, and grades 1, 2, and 3 were assigned when accumulation of positively staining cells was mild, moderate, or marked, respectively.
Statistical analysis—Tissues from each horse yielded 1 set of observations for each set of experimental conditions. Data are expressed as least squares mean ± SEM. Although ischemic and nonischemic tissue specimens were collected from 3 horses/treatment group (FM or saline solution) following 2 hours of experimentally induced ischemia, biopsy specimens were not obtained from the remaining horses because of concerns about complications related to biopsy sites. Data from these 2-hour specimens were not used in all comparisons because of the low numbers.
A statistical software program# was used for analysis. Data that were not normally distributed were ranked before repeated-measures ANOVAs were performed. The Kruskal-Wallis test was used to compare measurements made by western blot densitometry (COX-1 and COX-2 protein concentrations) in each treatment group and to compare scores for cells that stained positive for calprotectin and for COX distribution within the colonic mucosa. Whenever a significant F test statistic was obtained for treatment, time, or interaction, appropriate Bonferroni-adjusted P values were used for each family of comparisons. For all statistical analyses, a value of P < 0.05 was considered significant.
Results
Pain scores and heart rate—The first pain score, recorded at 4 hours after the ischemic episode, was significantly (P < 0.05) lower for the horses that received FM (mean ± SEM score, 8.1 ± 1.4) than for horses that received saline solution (13.9 ± 2.1), but no significant differences were evident between the 2 treatment groups at any other times (Figure 1). In both groups, pain scores appeared to decline over time. The median pain score for all 14 horses (9) was approximately a third of the maximum score allowed by the scoring system (26); the median pain score was manifested as milder behavioral changes than those typically observed in horses with colic.23 Heart rates recorded during the postischemic period were similar between groups but were significantly (P < 0.05) higher at all times than preoperative heart rates (Figure 2).
Mean ± SEM values for postoperative pain scores of horses treated with saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7; white bars) or FM (1.1 mg/kg, IV, q 12 h; 7; black bars). * Value for horses treated with FM is significantly (P < 0.05) different from that for horses treated with saline solution.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for postoperative pain scores of horses treated with saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7; white bars) or FM (1.1 mg/kg, IV, q 12 h; 7; black bars). * Value for horses treated with FM is significantly (P < 0.05) different from that for horses treated with saline solution.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for postoperative pain scores of horses treated with saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7; white bars) or FM (1.1 mg/kg, IV, q 12 h; 7; black bars). * Value for horses treated with FM is significantly (P < 0.05) different from that for horses treated with saline solution.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for heart rates of horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q12h;7). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for heart rates of horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q12h;7). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for heart rates of horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q12h;7). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Ussing chamber experiments—Time 0 was recorded after a 45-minute equilibration period, and at time 0 and after 15 minutes in Ussing chambers, nonischemic tissues from both treatment groups had significantly (P < 0.05) greater resistance than did ischemic tissues from both treatment groups (Figure 3). After 30 to 75 minutes, the TER in nonischemic tissues was greater than that in ischemic tissues, but there was no significant difference in TER between treatment groups for ischemic tissues and for nonischemic tissues for the remainder of the Ussing chamber incubation. Regardless of treatment received, TER declined significantly (P < 0.05) with time in nonischemic tissues during the incubation period, whereas it increased significantly (P < 0.05) in ischemic tissues. Because of these changes, the TER for ischemic tissues started at approximately a third of the values for nonischemic tissues and reached and even appeared to exceed (P > 0.05) the TER for nonischemic tissues at 240 minutes.
Mean ± SEM values for TER of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; squares) or FM (1.1 mg/kg, IV, q 12 h; diamonds). Each time point represents the mean value for 7 horses. White symbols represent values for uninjured tissue, and black symbols represent values for tissue 18 hours after recovery from 2 hours of experimentally induced ischemia. * Values for nonischemic tissues from both treatment groups are significantly (P < 0.05) different from values for ischemic tissues from both treatment groups. *Values for nonischemic tissues from both treatment groups are significantly (P <0.05) different from values for ischemic-injured tissues from FM-treated horses.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for TER of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; squares) or FM (1.1 mg/kg, IV, q 12 h; diamonds). Each time point represents the mean value for 7 horses. White symbols represent values for uninjured tissue, and black symbols represent values for tissue 18 hours after recovery from 2 hours of experimentally induced ischemia. * Values for nonischemic tissues from both treatment groups are significantly (P < 0.05) different from values for ischemic tissues from both treatment groups. *Values for nonischemic tissues from both treatment groups are significantly (P <0.05) different from values for ischemic-injured tissues from FM-treated horses.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for TER of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; squares) or FM (1.1 mg/kg, IV, q 12 h; diamonds). Each time point represents the mean value for 7 horses. White symbols represent values for uninjured tissue, and black symbols represent values for tissue 18 hours after recovery from 2 hours of experimentally induced ischemia. * Values for nonischemic tissues from both treatment groups are significantly (P < 0.05) different from values for ischemic tissues from both treatment groups. *Values for nonischemic tissues from both treatment groups are significantly (P <0.05) different from values for ischemic-injured tissues from FM-treated horses.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Values for transmucosal mannitol flux in ischemic and nonischemic tissues were not significantly different between treatment groups at any time points, and measurements at all time points up to and including 105 minutes of incubation were similar. However, at 240 minutes of incubation, the transmucosal flux of mannitol was significantly (P < 0.05) different from previous times for all treatments (Figure 4).
Mean ± SEM values for permeability of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) to tritium-labeled mannitol at 18 hours after recovery. Black bars represent uninjured tissues from saline solution–treated horses, white bars represent uninjured tissues from FM-treated horses, hatched bars represent ischemic tissues from saline solution–treated horses, and gray bars represent ischemic tissues from FM-treated horses. * Values for all types of tissues are significantly (P < 0.05) different from those of respective tissues at previous time points.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for permeability of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) to tritium-labeled mannitol at 18 hours after recovery. Black bars represent uninjured tissues from saline solution–treated horses, white bars represent uninjured tissues from FM-treated horses, hatched bars represent ischemic tissues from saline solution–treated horses, and gray bars represent ischemic tissues from FM-treated horses. * Values for all types of tissues are significantly (P < 0.05) different from those of respective tissues at previous time points.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM values for permeability of colonic mucosa from horses treated with saline solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) to tritium-labeled mannitol at 18 hours after recovery. Black bars represent uninjured tissues from saline solution–treated horses, white bars represent uninjured tissues from FM-treated horses, hatched bars represent ischemic tissues from saline solution–treated horses, and gray bars represent ischemic tissues from FM-treated horses. * Values for all types of tissues are significantly (P < 0.05) different from those of respective tissues at previous time points.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Histomorphometric measurements—Two hours of ischemia caused edema, purple discoloration of the serosa, and mild serosal ecchymoses in the affected segments of colon. The appearance of the serosal surface of the ischemic colon was close to that of adjacent control segments at 15 minutes after ischemia-inducing clamps were removed. Histologic changes after 2 hours of ischemia were predominantly suggestive of cell death and detachment in the surface epithelial cells and upper parts of crypts, which confirmed mucosal disruption after ischemic injury (Figure 5). Tissues from horses that received FM and saline solution had some histologic evidence of restitution at 18 hours after the ischemic episode, with a mucosal surface that was partly or totally covered with short epithelial cells. All histomorphometric measurements for ischemic tissues were significantly (P < 0.05) different from those of nonischemic tissues after 18 hours of recovery, except for mucosal height and percentage lifted epithelium (Table 1). There were no significant differences between treatment groups at 18 hours after the ischemic episode for all histomorphometric variables examined.
Mean ± SEM histomorphometric values for ischemic and nonischemic colonic mucosa obtained from horses treated with saline (0.9% NaCI) solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) at 18 hours after experimentally induced ischemia.
| Variable | Nonischemic tissue* | Ischemic tissue | |
|---|---|---|---|
| SAL | FM | ||
| Mucosal height (μm) | 463.96 ± 28.35 | 376.24 ± 41.38 | 319.77 ± 26.95 |
| Epithelial height (μm) | 37.60 ± 2.55 | 16.75 ± 3.29† | 10.84 ± 1.08† |
| Epithelial width (μm) | 3.55 ± 0.08 | 4.89 ± 0.40† | 5.26 ± 0.35† |
| Denuded epithelium (%) | 0.06 ± 0.04 | 30.87 ± 13.04† | 36.24 ± 7.23† |
| Lifted epithelium (%) | 0.37 ± 0.21 | 4.91 ± 2.14 | 2.94 ± 1.49 |
| Detached epithelium (%) | 0.00 ± 0.00 | 2.91 ± 0.91† | 6.45 ± 1.84† |
| Sloughed cells/0.1 mm | 0.33 ± 0.09 | 12.57 ± 8.80† | 13.44 ± 4.41† |
* Nonischemic tissues (n = 14) from horses treated with saline solution or FM yielded similar results; therefore, results were combined.
† Value is significantly (P < 0.05) different from corresponding value for nonischemic tissue.
SAL = Saline solution.
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced. A—Mucosa from section of uninjured colon. B—Mucosa obtained immediately after 2 hours of ischemia. Notice the loss of epithelium, including epithelial detachment and denudation. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. H&E stain; bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced. A—Mucosa from section of uninjured colon. B—Mucosa obtained immediately after 2 hours of ischemia. Notice the loss of epithelium, including epithelial detachment and denudation. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. H&E stain; bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced. A—Mucosa from section of uninjured colon. B—Mucosa obtained immediately after 2 hours of ischemia. Notice the loss of epithelium, including epithelial detachment and denudation. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. H&E stain; bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Immunohistochemical detection of calprotectin—The protocol for immunohistochemical identification of calprotectin yielded positive staining of variable intensity in alveolar macrophages and randomly distributed neutrophils within control lung tissues. In colonic tissue specimens, the stain intensity for cells that stained positively for calprotectin was pronounced so they could be readily differentiated from surrounding and adjacent cells (Figure 6). Such cells were sparse in nonischemic tissues and after 2 hours of ischemia, but were diffusely distributed throughout the lamina propria at 18 hours after the ischemic episode. Numbers of cells with cytoplasm that stained positively for calprotectin per square millimeter of tissue were significantly (P < 0.05) greater in ischemic tissues from horses that received saline solution (347.73 ± 263.81 cells/mm2) or FM (372.30 ± 185.91 cells/mm2) at 18 hours after the ischemic episode, compared with values for nonischemic tissues that had been collected at the same time (77.82 ± 74.36 cells/mm2 and 27.15 ± 16.84 cells/mm2, respectively). Numbers of cells that stained positive for calprotectin in nonischemic tissues and in ischemic tissues were not significantly different between horses that received saline solution and FM.
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced and which were immunostained to detect calprotectin (brown-stained cells), signifying leukocyte activation. A—Mucosa from a section of uninjured colon that was not exposed to ischemia. B—Mucosa obtained immediately after 2 hours of ischemia. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. Bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced and which were immunostained to detect calprotectin (brown-stained cells), signifying leukocyte activation. A—Mucosa from a section of uninjured colon that was not exposed to ischemia. B—Mucosa obtained immediately after 2 hours of ischemia. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. Bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Representative photomicrographs of sections of colonic mucosa obtained at various time points from horses in which colonic ischemia was experimentally induced and which were immunostained to detect calprotectin (brown-stained cells), signifying leukocyte activation. A—Mucosa from a section of uninjured colon that was not exposed to ischemia. B—Mucosa obtained immediately after 2 hours of ischemia. C—Mucosa from a horse treated with saline solution, obtained after 18 hours of recovery from ischemia. Notice the reestablished epithelial lining. D—Mucosa from a horse treated with FM, obtained after 18 hours of recovery from ischemia. Again, notice the reestablished epithelial lining. Bar = 0.1 mm.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Molecular evaluation of expression of COX-1 and COX-2—In western blot analyses of mucosal scrapings, B-actin was detected as a strong band at 43 kDa and expression was similar across all lanes, which confirmed equivalent protein loading (Figure 7). Cyclooxygenase-1 was evident as a positive band at 70 kDa, and COX-2 was evident as a positive band at 74 kDa. Both COX isoforms were constitutively expressed in nonischemic specimens of colonic mucosa (Figure 8). The 2-hour ischemic episode significantly (P < 0.05) increased expression of COX-1 and COX-2 in ischemic tissues, compared with values for nonischemic specimens. Expression of both isoforms was significantly (P < 0.05) upregulated further in ischemic tissues after 18 hours of recovery. There were no differences in expressions of COX-1 and COX-2 in tissue specimens from horses that received saline solution versus horses that received FM at any time point.
Photograph of a representative western blot analysis of nonischemic equine colonic mucosa obtained at 2 hours after start of experimentally induced ischemia (2HC) and after 18 hours of recovery from ischemia (18HC) and ischemic-injured colonic mucosa after 2 hours of ischemia (2HI) and after 18 hours of recovery from ischemia (18HR) from 1 horse treated with saline solution (SAL) and another treated with FM. Cyclooxygenase-1 and COX-2 were detected as distinct bands at 70 kDa and 74 kDa, respectively. B-Actin yielded a strong band at 43 kDa and was used as a protein loading control specimen. Both COX enzymes were expressed constitutively as suggested their detection in nonischemic tissue.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Photograph of a representative western blot analysis of nonischemic equine colonic mucosa obtained at 2 hours after start of experimentally induced ischemia (2HC) and after 18 hours of recovery from ischemia (18HC) and ischemic-injured colonic mucosa after 2 hours of ischemia (2HI) and after 18 hours of recovery from ischemia (18HR) from 1 horse treated with saline solution (SAL) and another treated with FM. Cyclooxygenase-1 and COX-2 were detected as distinct bands at 70 kDa and 74 kDa, respectively. B-Actin yielded a strong band at 43 kDa and was used as a protein loading control specimen. Both COX enzymes were expressed constitutively as suggested their detection in nonischemic tissue.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Photograph of a representative western blot analysis of nonischemic equine colonic mucosa obtained at 2 hours after start of experimentally induced ischemia (2HC) and after 18 hours of recovery from ischemia (18HC) and ischemic-injured colonic mucosa after 2 hours of ischemia (2HI) and after 18 hours of recovery from ischemia (18HR) from 1 horse treated with saline solution (SAL) and another treated with FM. Cyclooxygenase-1 and COX-2 were detected as distinct bands at 70 kDa and 74 kDa, respectively. B-Actin yielded a strong band at 43 kDa and was used as a protein loading control specimen. Both COX enzymes were expressed constitutively as suggested their detection in nonischemic tissue.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM densitometry readings for expression of COX-1 (A) and COX-2 (B) in colonic mucosa obtained from adjacent nonischemic tissue after 2 hours of ischemia (control 2h) and at 18 hours after recovery from ischemia (control 18h) and from ischemic tissues after 2 hours of ischemia (ischemia 2h) and at 18 hours after recovery from ischemia (recovery 18h) in horses treated with saline solution (white bars) or FM (black bars). There were no significant differences between tissues from horses treated with saline solution or FM at each time point. * Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours. *Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours and ischemia at 2 hours.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM densitometry readings for expression of COX-1 (A) and COX-2 (B) in colonic mucosa obtained from adjacent nonischemic tissue after 2 hours of ischemia (control 2h) and at 18 hours after recovery from ischemia (control 18h) and from ischemic tissues after 2 hours of ischemia (ischemia 2h) and at 18 hours after recovery from ischemia (recovery 18h) in horses treated with saline solution (white bars) or FM (black bars). There were no significant differences between tissues from horses treated with saline solution or FM at each time point. * Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours. *Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours and ischemia at 2 hours.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Mean ± SEM densitometry readings for expression of COX-1 (A) and COX-2 (B) in colonic mucosa obtained from adjacent nonischemic tissue after 2 hours of ischemia (control 2h) and at 18 hours after recovery from ischemia (control 18h) and from ischemic tissues after 2 hours of ischemia (ischemia 2h) and at 18 hours after recovery from ischemia (recovery 18h) in horses treated with saline solution (white bars) or FM (black bars). There were no significant differences between tissues from horses treated with saline solution or FM at each time point. * Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours. *Value is significantly (P < 0.05) different from those of control tissues at 2 hours and 18 hours and ischemia at 2 hours.
Citation: American Journal of Veterinary Research 70, 2; 10.2460/ajvr.70.2.236
Immunohistochemical analysis for COX-1 and COX-2 localization—Cyclooxygenase-1 was constitutively expressed in cells of the lamina propria of nonischemic tissues, whereas COX-2 was constitutively expressed in cells of the crypts, lamina propria, and surface epithelium. Expression of both enzymes was upregulated at 18 hours after the ischemic episode in various parts of the mucosa (Table 2). There was no significant difference in expression of COX-1 in the total mucosa, regardless of treatment and time. The apparent increase in expression of COX-2 in total mucosa at 18 hours after recovery from the ischemic episode in both treatment groups was not significantly different from that of corresponding control specimens. Significant upregulation of COX-1 expression was detected in the surface epithelium from both treatment groups at 18 hours after recovery from the ischemic episode, compared with COX-1 expression in respective nonischemic tissues. In addition, expression of COX-2 was significantly upregulated in the epithelium from both treatment groups at 18 hours after the ischemic episode, compared with expression in respective nonischemic tissues. At that same time point, expression of COX-2 was significantly upregulated in the colonic crypts of horses that received saline solution, compared with expression of COX-2 in crypt cells of nonischemic tissues from horses that received FM. There was no significant difference in expression of COX-1 or COX-2 between treatment groups.
Mean ± SEM scores for immunohistochemical distribution of COX in ischemic and nonisch-emic colonic mucosa obtained from horses treated with saline solution or FM at 18 hours after experimentally induced ischemia.
| Variable | Nonischemic tissue | Ischemic tissue | ||
|---|---|---|---|---|
| SAL | FM | SAL | FM | |
| COX-1 | ||||
| Total mucosa | 4.24 ± 0.44 | 4.24 ± 0.44 | 5.33 ± 0.70 | 5.86 ± 0.81 |
| Epithelium | 0.047 ± 0.047a | 0.096 ± 0.096a | 0.62 ± 0.25b | 1.05 ± 0.27b |
| Upper lamina propria | 1.80 ± 0.20 | 1.91 ± 0.19 | 1.90 ± 0.27 | 2.19 ± 0.20 |
| Lower lamina propria | 1.67 ± 0.25 | 1.62 ± 0.15 | 1.80 ± 0.25 | 1.62 ± 0.25 |
| Crypt | 0.67 ± 0.28 | 0.67 ± 0.24 | 1.00 ± 0.23 | 1.00 ± 0.24 |
| COX-2 | ||||
| Total mucosa | 3.52 ± 0.68 | 3.52 ± 0.68 | 6.90 ± 0.70 | 6.67 ± 0.67 |
| Epithelium | 0.62 ± 0.18a | 0.33 ± 0.16a | 2.14 ± 0.30b | 2.57 ± 0.17b |
| Upper lamina propria | 1.33 ± 0.33 | 0.95 ± 0.17 | 1.52 ± 0.16 | 1.67 ± 0.15 |
| Lower lamina propria | 0.86 ± 0.35 | 0.47 ± 0.23 | 0.90 ± 0.19 | 0.62 ± 0.21 |
| Crypt | 1.5 ± 0.33 | 0.95 ± 0.27a | 2.33 ± 0.22b | 1.81 ± 0.11 |
a,bDifferent superscript letters across each row indicate significantly (P < 0.05) different values.
See Table 1 for remainder of key.
Discussion
After ischemia was experimentally induced for 2 hours in the colons of horses, administration of FM did not appear to affect any of the measurements used to assess mucosal recovery at 18 hours after the ischemic episode. These findings are in agreement with results of our other study18 of the effects of phenylbutazone and indomethacin on restitution and tight junction permeability in equine colonic mucosa after damage induced by hypochlorous acid in vitro. However, the results are in contrast to those of similar studies12,15,16 involving equine jejunum, in which FM and etodololac retarded these rapid repair processes. Another difference between jejunum and colon is that TER of ischemic-injured jejunum in horses treated with saline solution is significantly greater than TER in uninjured tissue,16 whereas the reverse is true for equine colon, regardless of treatment. The pain scores for horses that received FM or saline solution were lower or the same as scores reported for each of these treatments in similar studies15,16 involving equine jejunum.
The dosage and treatment intervals for FM (1.1 mg/ kg, q 12 h) were similar to those used after colic surgery in our clinic and by others,32 and similar to those used in another study15 involving equine jejunum with the same type and duration of ischemia and postischemic recovery. A pharmacokinetic study33 revealed that IV administration of FM at 1.1 mg/kg suppresses thromboxane generation as detected in serum for 12 hours. Therefore, this dosing protocol allowed us to find that colonic response to ischemic injury was different than the response that occurs in equine jejunum by use of a similar protocol.15,16 This comparison is relevant to differences in the effects of prostaglandins on recovery of barrier properties between the 2 segments of equine intestine and to the manner in which horses with colonic diseases are treated.
The disparity between changes in TER and mannitol permeability in ischemic tissues and the apparent concordance with the same measurements in nonischemic tissues is consistent with evidence that several populations of sizeand charge-restrictive pores in tight junctions respond to physiologic stimuli in ways that are different from what would be predicted in a static model of paracellular permeability.34,35 In an ischemia-recovery model similar to ours, TER recovered in ischemia-injured jejunum in horses that were not treated with FM, but transmucosal fluxes of inulin and LPS did not, compared with values in nonischemic mucosa.16 The proinflammatory cytokine interferon-γ preferentially increases paracellular permeability to large versus small molecules in T84 colonic monolayers by acting on a specific population of paracellular pores.34 This and related findings led to the conclusion that these cells contain restrictive pores that are traversed predominantly by small molecules and a less abundant, nonrestrictive pathway for permeation by large molecules.34 The nonrestrictive pores apparently are a more important component of junctional permeability in inflammatory diseases,34 which could make them relevant to the colonic ischemia model used in the study reported here. The decline in TER and increased transmucosal flux of mannitol in nonischemic tissues in the present study suggested that these tissues were not influenced by mediators responsible for the divergent effects on TER and mannitol permeability in ischemic tissues. The specific mediators involved and their effects on pores in equine colonic mucosa require additional investigation but, unlike the mediators in equine jejunum, do not appear to involve prostaglandins.15,16
Combined in vivo and in vitro experiments, as used in the present protocol and in similar jejunal studies,15,16 can be complicated by an unavoidable lag between tissue collection and the first recordings of TER and transmucosal mannitol flux. Measurements obtained after this lag period might not be representative of those that prevailed in vivo at the time of tissue collection. Also, handling of tissue to isolate the mucosa could release endogenous prostanoids that can have profound effects on ion transport and intestinal barrier properties in colonic mucosa.9,19,36–38 We processed our colonic segments during the critical transport and dissection phases of our study in an ice-cold solution to retard production of prostanoids and other mediators before we measured permeability. This procedure does not have any lasting effects on intracellular water volume and ionic composition and other measures of colonic function and viability.25
The most critical time for measuring mucosal barrier properties in the present study was in the earlier in vitro incubation periods because of temporal proximity to the in vivo condition of the tissue. Mucosal barrier responses were recorded to 240 minutes of short-circuiting in Ussing chambers to allow comparison with results of another in vitro study18 and because NSAIDs can have a detrimental effect on TER in nonischemic equine colon after 150 minutes.17 In our other studies18,26 involving incubation in Ussing chambers for 240 minutes, control tissues appeared to be intact on histologic examination but, in the present study, clearly declined in viability on the basis of barrier properties. In vitro measures of barrier recovery obviate many of the factors that complicate similar measurements in vivo, such as the expense of prolonged anesthesia necessary to follow passage of permeability markers and difficulty in using radioisotopes and electrophysiologic markers of mucosal integrity in vivo. Also, the combined in vivo and in vitro approach was used in the present study to allow comparison of results with those of published reports15,16 on nonselective NSAIDs such as FM on barrier function in equine jejunum. Nonetheless, in vivo experiments should provide the next step in elucidation of this process.
In the present study, immunohistochemical identification of calprotectin within the neutrophilic cytoplasm was used to measure neutrophil infiltration into injured colonic mucosa,29 although cells in the lamina propria that stained positive for calprotectin could have been neutrophils or macrophages.39 Neutrophils are the cells most likely to predominate in the inflammatory process associated with mucosal ischemia,40–42 and we have confirmed this in ischemia models similar to those used in the present study.29 We could not determine to what extent neutrophil influx altered permeability in the present study, but results suggested that cells containing calprotectin accumulated at the tissue surface within 18 hours after ischemic injury, where they would be poised to physically disrupt barrier function.43 However, in contrast to the situation in equine jejunum, in which FM and meloxicam increase neutrophil infiltration into ischemic mucosa (compared with infiltration in tissues from horses treated with saline solution),16 ischemia-induced infiltration into ischemic colon was similar in tissues from the horses in our study regardless of whether they were treated with FM or saline solution. The difference in methods used to assess neutrophil influx in the present study and that in equine jejunum would be unlikely to explain these differences between colon and jejunum.29
A scoring technique was used to quantitate the immunohistochemical distribution of COX enzymes in the mucosa because counting of single cells was cumbersome and prone to bias, particularly in areas of intense staining and cell overlap. Both COX enzymes were constitutively expressed in nonischemic tissues, although we cannot discount the possibility that both were induced in nonischemic tissues at 18 hours after the ischemic episode in response to ischemia in adjacent tissues.15 Similarly, results of western blot analyses confirmed constitutive expression of COX-1 and COX-2 in nonischemic tissues after the 2-hour ischemic episode (n = 3) and 18 hours afterward (3), and both isoforms were upregulated at the same time points in ischemic tissues. The rapid upregulation of expression of COX-1 and COX-2 that was detected after the ischemic episode was consistent with other findings in equine jejunum,12 equine colon,b and porcine ileum.44 There were no significant differences between expression of COX-1 or COX-2 between treatment groups for densitometry readings and for immunohistochemical scoring.
Significant upregulation of expression of COX-1 was evident in the surface epithelium at 18 hours after the ischemic episode, whereas expression of COX-2 was significantly upregulated in the epithelium and in crypt cells at the same time point. Association of COX-2 with crypt cells after 18 hours of recovery from ischemia is consistent with the concept that a population of pericryptal stromal cells, capable of expressing COX, is repositioned in response to injury in the colon, and that prostaglandin E2 promotes epithelial proliferation and epithelial homeostasis in that tissue.45 Although prostaglandins produced via COX-1 would be expected to play the dominant role in cytoprotection and repair,20 there is growing evidence that prostaglandins produced via COX-2 are also salutary in damaged mucosa.21,46,47
On the basis of our results, FM did not adversely affect the in vivo recovery of colonic mucosa from ischemic injury in horses. Our findings would support continued use of FM in horses with large-colon diseases that are characterized by epithelial damage such as large-colon volvulus and colitis. However, given the conditions of the study, FM did not appear to affect the severity of mucosal inflammation, as measured by an immunohistochemical marker of neutrophil influx.
ABBREVIATIONS
| COX | Cyclooxygenase |
| FM | Flunixin meglumine |
| Ig | Immunoglobulin |
| KRB | Krebs-Ringer-bicarbonate |
| LPS | Lipopolysaccharide |
| PAGE | Polyacrylamide gel electrophoresis |
| TBSST | Tris-buffered saline solution plus 0.05% Tween-20 |
| TER | Transepithelial electric resistance |
Freeman D. Effect of cyclooxygenase inhibitors on transepi-thelial permeability in equine colon (abstr), in Proceedings. 5th Equine Colic Res Symp 1994;40.
Morton AJ, Rotting AK, Freeman DE, et al. Characterization of cyclooxygenase 1 and cyclooxygenase 2 expression in normal and ischemic-injured left dorsal colon (abstr), in Proceedings. 8th Int Equine Colic Res Symp 2005;146.
Nonin Medical Inc, Plymouth, Minn.
World Precision Instruments, Sarasota, Fla.
New England Nuclear, Boston, Mass.
Image Pro Express, version 5.0, Media Cybernetics Inc, Bethesda, Md.
Serotec, Raleigh, NC.
Deloaker RTU buffer, Biocare Medical, Concord, Calif.
Permount mounting medium, Biomeda, Burlingame, Calif.
Bio-Rad Laboratories, Hercules, Calif.
Hybond ECL, Amersham Life Science, Birmingham, England.
Santa Cruz Biotechnology Inc, Santa Cruz, Calif.
Biorad Quantity One Image Analysis Software, version 4.5.2, Hercules, Calif.
SAS, version 8, SAS Institute Inc, Cary, NC.
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