Effect of large colon ischemia and reperfusion on concentrations of calprotectin and other clinicopathologic variables in jugular and colonic venous blood in horses.

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.

Effects of ischemia and reperfusion on production of nitrotyrosine, activation of eosinophils, and apoptosis in the large colonic mucosa of horses.

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. ANIMALS: 40 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.

In vitro and in vivo responses of mucosa from the large colon of horses to ischemia and reperfusion.

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.

Comparison of 5 surgical techniques for partial liver lobectomy in the dog for intraoperative blood loss and surgical time.

OBJECTIVE: To compare surgical time and intraoperative blood loss for 5 partial liver lobectomy techniques in the dog. STUDY DESIGN: Experimental in vivo study. ANIMALS: Dogs (n=10). METHODS: Five surgical techniques (SurgiTie(™) ; LigaSure(™) ; Ultracision(®) Harmonic Scalpel [UAS]; Suction+Clip; Suction+thoracoabdominal stapler [TA]) for partial liver lobectomy in dogs were evaluated and compared for total surgical time and intraoperative blood loss. Body weight, activated clotting time (ACT), heart rate, and intraoperative blood pressure (BP) were recorded. Blood loss was determined by adding the weight of the blood soaked sponges during surgery (1 g=1 mL) to the amount of suctioned blood (mL). Surgical time (in seconds) was determined from the start of the lobectomy until cessation of bleeding from the stump. Mean surgical time and mean blood loss for each technique were compared using a Tukey's multiple comparison test. RESULTS: No significant differences were found between dogs for weight, ACT, heart rate, and intraoperative BP. No complications were seen with the SurgiTie(™) technique in 9 of 10 cases. There was no significant difference in surgical time between techniques however there was a significant difference for blood loss; the Suction+Clip method had significantly more blood loss than the other techniques. CONCLUSIONS: Skeletonization of the lobar vessels before individually clipping them (Suction+Clip) resulted in a higher blood loss than using Suction+TA, UAS, SurgiTie(™) or the LigaSure(™) device. The SurgiTie(™) appears to be an acceptable method for partial liver lobectomy. CLINICAL RELEVANCE: Although skeletonization and individually clipping the vessels had the highest blood loss, it still was <7.5% of total blood volume. All 5 techniques should be safe for clinical use in small to medium sized dogs up to 26 kg.

Postmortem evaluation of surgery site leakage by use of in situ isolated pulsatile perfusion after partial liver lobectomy in dogs.

OBJECTIVE: To evaluate postmortem surgery site leakage by use of in situ isolated pulsatile perfusion after partial liver lobectomies. ANIMALS: 10 healthy mixed-breed male dogs. PROCEDURES: Dogs were anesthetized, and 5 surgical techniques (pretied suture loop, energy-based sealer-divider, harmonic scalpel, suction with clip application, or suction with use of a thoracoabdominal stapler) were used to perform 5 partial liver lobectomies in each dog. Dogs were euthanatized, and the portal vein and hepatic artery were cannulated and perfused with a modified kidney perfusion machine (pulsatile flow for arterial perfusion and nonpulsatile flow for portal perfusion). Lobectomy sites were inspected for leakage of perfusate, and time until detection of leakage was recorded. The techniques in each dog were ranked on the basis of time until leakage. Time until leakage and rankings for each surgical technique were analyzed by use of an ANOVA. RESULTS: Leakage of perfusate was recorded in 44 lobes at supraphysiologic pressures. Of the 6 lobes without leakage, a pretied suture loop procedure was performed in 5 and a harmonic scalpel procedure was performed in 1. Time until leakage and the ranking differed significantly between the pretied suture loop and the other techniques. Time until leakage and ranking did not differ significantly among the other techniques. CONCLUSIONS AND CLINICAL RELEVANCE: Time until leakage of perfusate was greater for the pretied suture loop technique than for the other techniques, and that technique did not fail in 5 of 10 lobes. However, all techniques appeared to be safe for clinical use.

Effects of flunixin meglumine on recovery of colonic mucosa from ischemia in horses.

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.

Comparison of Vasosol and University of Wisconsin solutions on early kidney function after 24 hours of cold ischemia in a canine autotransplantation model.

INTRODUCTION: Ischemia/reperfusion (I/R) injury is a significant cause of graft dysfunction in donor kidney transplantation. It has been suggested that improvements in organ preservation solutions can ameliorate some of deleterious effects of I/R on the transplanted graft. We evaluated herein the influence of Vasosol (VAS), a solution that is designed to target specific pathways of I/R injury, and University of Wisconsin (UW) solution on early graft status of donor kidneys in a canine autotransplant model. MATERIALS AND METHODS: Left kidneys were recovered from 12 dogs, exsanguinated with either VAS or UW and cooled to 4 degrees C for 24 h. Kidneys were autotransplanted and the right kidneys were nephrectomized. Indices of post-transplant renal function were measured serially for seven days. All animals were euthanized at postoperative day 7. Kidney biopsies were taken at 1, 4, and 24 h postreperfusion for evaluation of tissue myeloperoxidase concentration. RESULTS: All dogs survived the transplant surgery. Post-transplant serum creatinine (mg/dL) and blood urea nitrogen (mg/dL) were significantly elevated in the UW group compared with the VAS group in each of the postoperative days. Moreover, myeloperoxidase tissue levels were significantly elevated in the UW-treated group compared with the VAS-treated group. CONCLUSIONS: Our data suggest that a cold storage preserving solution designed to target several modes of I/R injury can improve the function of the autotransplanted canine kidney compared with the current gold standard solution.

The influence of a novel organ perfusion solution on early graft function in canine renal autotransplantation.

OBJECTIVE: To evaluate the influence of a kidney perfusion solution on early graft function in dogs. STUDY DESIGN: Experimental, randomized study. ANIMALS: Intact adult male mongrel dogs (n=12). METHODS: Dogs had renal autograft transplantation without ureteroneocystotomy with contralateral nephrectomy. Kidney graft flushing with a novel organ perfusion solution was compared with flushing with saline (0.9% NaCl) solution. Serum creatinine (Cr) and blood urea nitrogen (BUN) concentrations were measured daily posttransplant for 7 days. Ultrasound-guided renal biopsy was performed on postoperative day 1 for electron microscopic evaluation. Dogs were euthanatized on day 7. RESULTS: All dogs completed the study. Cr and BUN concentrations of the saline group were significantly greater than the organ perfusion solution group on each postoperative day (P=.01 for S Cr; P=.001 for BUN). Electron micrographs of nuclei and mitochondria from convoluted proximal tubule cells indicated profound ultrastructural disruptions in the saline group and mild ultrastructural disruptions in the organ perfusion solution group. CONCLUSION: Flushing solution composition can influence early graft function in live donor kidney transplantation. CLINICAL RELEVANCE: Use of a specialized flushing solution can improve early graft function in canine kidney transplantation, independent of antigen-mediated events.