Maximilian M. R. Polyak

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 Tukeys 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.

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.

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.

Ultrastructural changes in the equine colonic mucosa after ischaemia and reperfusion

REASON FOR PERFORMING STUDY Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). OBJECTIVES To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. METHODS Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformaldehyde and osmium tetroxide, and embedded in epon. Morphological and ultrastructural changes were evaluated in toluidine blue-stained semithin sections by light microscopy and in thin sections stained with uranyl acetate/lead citrate by transmission electron microscopy. RESULTS Ischaemia caused swelling of epithelial cells and their organelles, opening of tight junctions, detachment from the basement membrane, early apoptosis and single cell necrosis. Autophagy was a prominent feature in epithelial cells after ischaemia. Reperfusion was characterised by apoptosis, epithelial regeneration and restoration of apical cell junctions. Phagocytic-like vacuoles containing cellular debris and bacteria were evident in epithelial cells after reperfusion. Paracellular and subepithelial clefts formed, accompanied by infiltration of neutrophils, lymphocytes and eosinophils into the epithelium. Subepithelial macrophages and luminal neutrophils had increased phagocytic activity. CONCLUSIONS Ischaemia caused ultrastructural damage to the colonic epithelium, but epithelial cells recovered during reperfusion. POTENTIAL RELEVANCE Transmission electron microscopy can demonstrate subtle ultrastructural damage to epithelial cells and evidence of recovery after I/R in equine colon.

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.

Mucosal injury and inflammatory cells in response to brief ischaemia and reperfusion in the equine large colon.

REASON FOR PERFORMING STUDY Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. OBJECTIVES To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. METHODS Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. Semithin sections (500 nm) of epon-embedded biopsies were stained with toluidine blue for histomorphometric evaluation. The number and distribution of mucosal macrophages (CD163), neutrophils (calprotectin), eosinophils (LUNA) and mast cells (toluidine blue) were determined, and mucosal COX-1 and -2 expression was identified. RESULTS Ischaemia caused epithelial cell and nuclear swelling (mean ± s.e. nuclear width; control: 2.7 ± 0.2 µm vs. 1hI: 4.2 ± 0.2 µm; P<0.01), subepithelial oedema (control: 0.2 ± 0.1 µm vs. 1hI: 3.2 ± 0.2 µm; P<0.01) and increased epithelial apoptosis (control: 14.3 ± 4.1 apoptotic cells/mm mucosa vs. 1hI: 60.4 ± 14.0 apoptotic cells/mm mucosa; P<0.01). COX-2 expression (P<0.01) was evident after ischaemia. Reperfusion caused paracellular fluid accumulation (control: 0.9 ± 0.1 µm vs. 1hI: 0.6 ± 0.6 µm vs. 1hI + 4hR: 1.6 ± 0.2 µm; P<0.05). Epithelial repair started at 1 h of reperfusion (P<0.001), followed by migration of neutrophils into the mucosa after 2 h (control: 72.3 ± 18.4 cells/mm(2) mucosa vs. 1hI + 2hR: 1149.9 ± 220.6 cells/mm(2) mucosa; P<0.01). Mucosal eosinophils, mast cells and macrophages did not increase in numbers but were activated. CONCLUSIONS Epithelial injury and COX-2 expression caused by short-term hypoxia were followed by intense inflammation associated with epithelial repair during reperfusion. POTENTIAL RELEVANCE Equine colonic mucosa subjected to a brief period of ischaemia can repair during reperfusion, despite increased mucosal inflammation.

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.

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.

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.

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.