Astrid Grosche

Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine

The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.

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.

Cardiovascular effects of N‐butylscopolammonium bromide and xylazine in horses

REASONS FOR PERFORMING STUDY N-butylscopolammonium bromide (NBB) and xylazine are commonly used medications for the treatment of spasmodic colic and other forms of abdominal pain in horses. Both NBB and xylazine exert significant effects on the cardiovascular system and other vital systems of horses. OBJECTIVE To evaluate the effects of i.v. administration of NBB, xylazine, and the combination of NBB and xylazine on heart rate, other commonly measured physiological parameters, cardiac rhythm and blood pressure. METHODS Six mature horses of mixed breed were used. In a random cross-over design, each horse was given 0.3 mg/kg bwt of NBB i.v., 0.25 mg/kg bwt xylazine i.v., and a combination of 0.3 mg/kg bwt NBB and 0.25 mg/kg bwt xylazine. Heart rate, physiological parameters, cardiac rhythm and indirect blood pressure were recorded at timed intervals before and 60 min following administration. RESULTS Heart rate and blood pressure were significantly elevated immediately following administration of NBB or NBB with xylazine. Administration of NBB with xylazine resulted in significantly greater initial and peak blood pressure values than with NBB alone. Administration of xylazine resulted in a decrease in heart rate, with an initial increase in blood pressure followed by a decrease in blood pressure. Sinus tachycardia was seen with NBB, and NBB and xylazine administration. First and second degree atrioventricular block was identified with xylazine administration. Ventricular tachycardia was identified in one horse following NBB and xylazine administration. CONCLUSIONS Results of this study suggest that the effects of administration of NBB alone or in combination with xylazine to horses with colic, especially to those with systemic cardiovascular compromise, should be considered carefully to assess condition and predict prognosis accurately, and to avoid potential adverse effects.

Expression of cyclooxygenase-1 and -2 in the left dorsal colon after different durations of ischemia and reperfusion in horses.

OBJECTIVE-To identify expression and localization of cyclooxygenase (COX)-1 and COX-2 in healthy and ischemic-injured left dorsal colon of horses. SAMPLE POPULATION-Left dorsal colon tissue samples from 40 horses. PROCEDURES-Tissue samples that were used in several related studies on ischemia and reperfusion were evaluated. Samples were collected during anesthesia, before induction of ischemia, and following 1 hour of ischemia, 1 hour of ischemia and 30 minutes of reperfusion, 2 hours of ischemia, 2 hours of ischemia and 30 minutes of reperfusion, and 2 hours of ischemia and 18 hours of reperfusion. Histomorphometric analyses were performed to characterize morphological injury. Immunohistochemical analyses were performed to characterize expression and localization of COX-1 and COX-2. RESULTS-COX-1 and COX-2 were expressed in control tissues before ischemia was induced, predominantly in cells in the lamina propria. Ischemic injury significantly increased expression of COX-2 in epithelial cells on the colonic surface and in crypts. A similar significant increase of COX-1 expression was seen in the epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE-On the basis of information on the role of COX-2, upregulation of COX-2 in surface epithelium and crypt cells following ischemic injury in equine colon may represent an early step in the repair process.

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.

An amino acid-based oral rehydration solution (AA-ORS) enhanced intestinal epithelial proliferation in mice exposed to radiation.

Destruction of clonogenic cells in the crypt following irradiation are thought to cause altered gastrointestinal function. Previously, we found that an amino acid-based oral rehydration solution (AA-ORS) improved gastrointestinal function in irradiated mice. However, the exact mechanisms were unknown. Electrophysiology, immunohistochemistry, qPCR, and Western blot analysis were used to determine that AA-ORS increased proliferation, maturation, and differentiation and improved electrolyte and nutrient absorption in irradiated mice. A single-hit, multi-target crypt survival curve showed a significant increase in crypt progenitors in irradiated mice treated with AA-ORS for six days (8.8 ± 0.4) compared to the saline-treated group (6.1 ± 0.3; P < 0.001) without a change in D0 (4.8 ± 0.1 Gy). The Dq values increased from 8.8 ± 0.4 Gy to 10.5 ± 0.5 Gy with AA-ORS treatment (P < 0.01), indicating an increased radiation tolerance of 1.7 Gy. We also found that AA-ORS treatment (1) increased Lgr5+, without altering Bmi1 positive cells; (2) increased levels of proliferation markers (Ki-67, p-Erk, p-Akt and PCNA); (3) decreased apoptosis markers, such as cleaved caspase-3 and Bcl-2; and (4) increased expression and protein levels of NHE3 and SGLT1 in the brush border membrane. This study shows that AA-ORS increased villus height and improved electrolyte and nutrient absorption.