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Large Colon Ischemia and Reperfusion in Horses

Permanent Link: http://ufdc.ufl.edu/UFE0043696/00001

Material Information

Title: Large Colon Ischemia and Reperfusion in Horses Histological and Functional Alterations, and Response of the Innate Immune System
Physical Description: 1 online resource (231 p.)
Language: english
Creator: Grosche, Astrid
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: colon -- eosinophils -- horse -- ischemia -- macrophages -- neutrophils -- reperfusion -- ultrastructure
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Large colon volvulus is the most devastating cause of intestinal ischemia in horses and results in severe mucosal damage, barrier dysfunction, toxic shock and possibly death. Although essential for regeneration, reperfusion can exacerbate mucosal injury after ischemia. Responses to I/R involve a series of synchronized biochemical, cellular and structural changes characterized by generation of radicals, activation of immune cells, and epithelial cell degeneration and death. The objective of the study was to assess the effect of colonic I/R on functional and histological alterations, reaction of neutrophils, eosinophils, macrophages and mast cells, expression of nitrotyrosine, COX and calprotectin, and clinicopathological changes in horses. A segment of the pelvic flexure was submitted to 1hI or 2hI followed by 30minR, 4hR, or 18hR. Mucosal biopsies before and after ischemia, and after reperfusion were processed for H&E-, Luna-, TB-staining, IHC (calprotectin, CD163, nitrotyrosine), TUNEL method, and TEM. Mucosal (H&E) and epithelial damage (TB, TEM) were described and quantified. The number and distribution of mucosal neutrophils (H&E, calprotectin), eosinophils (Luna), macrophages (CD163) and mast cells (TB) were assessed. Mucosal nitrotyrosine and COX-1/-2 expression, and apoptotic cell death (TB, TUNEL) were identified. To assess mucosal barrier integrity, TER and mannitol flux were determined before and after 1hI, and after 4hR. Calprotectin (ELISA), and clinicopathological variables were evaluated in JB and CB before and after 1hI, and after 1hR, 2hR and 4hR. Ischemia caused degeneration and detachment of epithelial cells, early apoptosis, and opening of TJ resulting in decreased TER and increased mannitol flux. Autophagy was a prominent feature in epithelial cells after 1hI. Reperfusion was characterized by apoptosis, epithelial regeneration, and restoration of TJ resulting in recovery of epithelial barrier integrity. Neutrophils infiltrated colonic mucosa after reperfusion, and macrophages, mast cells and eosinophils were activated during I/R. Ischemia caused metabolic acidosis, increased lactate, K+ and CPK, and decreased glucose in colonic venous blood. But they returned to normal after reperfusion despite activation of an inflammatory response characterized by increased neutrophil cell turn over and release of calprotectin after I/R. Equine colonic mucosa subjected to ischemia can repair during reperfusion, despite increased mucosal inflammation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Astrid Grosche.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Freeman, David E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043696:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043696/00001

Material Information

Title: Large Colon Ischemia and Reperfusion in Horses Histological and Functional Alterations, and Response of the Innate Immune System
Physical Description: 1 online resource (231 p.)
Language: english
Creator: Grosche, Astrid
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: colon -- eosinophils -- horse -- ischemia -- macrophages -- neutrophils -- reperfusion -- ultrastructure
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Large colon volvulus is the most devastating cause of intestinal ischemia in horses and results in severe mucosal damage, barrier dysfunction, toxic shock and possibly death. Although essential for regeneration, reperfusion can exacerbate mucosal injury after ischemia. Responses to I/R involve a series of synchronized biochemical, cellular and structural changes characterized by generation of radicals, activation of immune cells, and epithelial cell degeneration and death. The objective of the study was to assess the effect of colonic I/R on functional and histological alterations, reaction of neutrophils, eosinophils, macrophages and mast cells, expression of nitrotyrosine, COX and calprotectin, and clinicopathological changes in horses. A segment of the pelvic flexure was submitted to 1hI or 2hI followed by 30minR, 4hR, or 18hR. Mucosal biopsies before and after ischemia, and after reperfusion were processed for H&E-, Luna-, TB-staining, IHC (calprotectin, CD163, nitrotyrosine), TUNEL method, and TEM. Mucosal (H&E) and epithelial damage (TB, TEM) were described and quantified. The number and distribution of mucosal neutrophils (H&E, calprotectin), eosinophils (Luna), macrophages (CD163) and mast cells (TB) were assessed. Mucosal nitrotyrosine and COX-1/-2 expression, and apoptotic cell death (TB, TUNEL) were identified. To assess mucosal barrier integrity, TER and mannitol flux were determined before and after 1hI, and after 4hR. Calprotectin (ELISA), and clinicopathological variables were evaluated in JB and CB before and after 1hI, and after 1hR, 2hR and 4hR. Ischemia caused degeneration and detachment of epithelial cells, early apoptosis, and opening of TJ resulting in decreased TER and increased mannitol flux. Autophagy was a prominent feature in epithelial cells after 1hI. Reperfusion was characterized by apoptosis, epithelial regeneration, and restoration of TJ resulting in recovery of epithelial barrier integrity. Neutrophils infiltrated colonic mucosa after reperfusion, and macrophages, mast cells and eosinophils were activated during I/R. Ischemia caused metabolic acidosis, increased lactate, K+ and CPK, and decreased glucose in colonic venous blood. But they returned to normal after reperfusion despite activation of an inflammatory response characterized by increased neutrophil cell turn over and release of calprotectin after I/R. Equine colonic mucosa subjected to ischemia can repair during reperfusion, despite increased mucosal inflammation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Astrid Grosche.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Freeman, David E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043696:00001


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1 LARGE COLON ISCHEMIA AND REPERFUSION IN HORSES: HISTOLOGICAL AND FUNCTIONAL ALTERATIONS, AND RESPONSE OF THE INNATE IMMUNE SYSTEM By ASTRID GROSCHE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Astrid Grosche

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3 Fr Mutti

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4 ACKNOWLEDGMENTS I would like to thank my mentor and supervisor, Dr. David E. Freeman for his boundless support and priceless help throughout the years of my studies Many t hank s to Dr. L. Chris Sanchez member of my Supervisory Committee for many help ful discussions about clinical studies and experimental design s related to gastrointestinal ischemia and reperfusion. T hanks to Dr. Lori Warren, member of my Supervisory Committee for her stimulating discussions about experimental design and statistical analy ses. Thanks to Dr. Jeffrey Abbott, member of my Supervisory Committee for navigating me through problems about pathophysiology and histological implications of intestinal ischemia and reperfusion. I would also like to thank Dr. John Valentine, a special member of my Supervisory Committee for his assistance with questions about mucosal immunology and inflammation and a perspective from human medicine Sincere thanks to my colleague and best friend Dr. Alison Morton wh o was always there for me when I needed her T hanks to Karen Kelley from the Bioimaging and Electron Microscopy Lab, and Linda Green from the Hybridoma Core Laboratory of the Interdisciplinary Center for Biotechnology University of Florida f or their outstanding technical and scientific support with transmission electron microscopy and ELISA Thank to Deedie Wrigley Hancock for financial support, and Dr. Charles Courtney, Dr. Sheilah Rob ert son and Sally OConnell for their support and guidance.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 page LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 16 1 INTRODUCTION .................................................................................................... 18 Large Colon Volvulus .............................................................................................. 18 Pathophysiol ogy of Intestinal I/R ............................................................................. 20 Ischemia ........................................................................................................... 20 Reperfusion ...................................................................................................... 22 Intestinal I/R in Horses ..................................................................................... 25 Immune C ells and Inflammatory Response A fter Intestinal I/R ............................... 27 Macrophages .................................................................................................... 27 Neutrophils ....................................................................................................... 29 Eosinophils ....................................................................................................... 31 Mast Cells ......................................................................................................... 32 Morphology, Ultrastructure, Cell Death, and Mucosal Repair A fter Intestinal I/R .... 34 Intestinal Morphology and Ultrastructure .......................................................... 34 Epithelial Cell Death ......................................................................................... 36 Resolving Inflammation and Epithelial Repair .................................................. 38 Clinicopathological Changes A fter Intestinal I/R ..................................................... 41 Hematological and Biochemical Variables ........................................................ 41 Calprotectin ...................................................................................................... 43 2 IN VITRO AND IN VIVO RESPONSES OF MUCOSA FROM THE LARGE COLON OF HORSES TO ISCHEMIA AND REPERFUSION ................................. 49 Materials and Methods ............................................................................................ 51 Horses .............................................................................................................. 51 Surgical Procedure ........................................................................................... 52 Ussing Chamber Experiments .......................................................................... 53 Histological Evaluation and Morphometric Measurements ............................... 55 Statistical Analysis ............................................................................................ 56 Results .................................................................................................................... 57 Ussing Chamber Experiments .......................................................................... 57 Morphological Examinations ............................................................................. 57 Histomorphometric Examination of In Vivo Tissue Samples ............................ 58 Histomorphometric Examinations of Ussing Chamber Tissues ........................ 58

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6 Discussion .............................................................................................................. 59 3 ULTRASTRUCTURAL CHANGES IN THE EQUINE COLONIC MUCOSA AFTER ISCHEMIA AND REPERFUSION .............................................................. 71 Material and Methods ............................................................................................. 72 Animals ............................................................................................................. 72 Experimental Procedures ................................................................................. 72 Sample Preparation .......................................................................................... 74 Results .................................................................................................................... 75 Morphological Changes .................................................................................... 75 Ultrastructural Changes .................................................................................... 76 Ischemia ..................................................................................................... 76 Reperfusion ................................................................................................ 77 Discussion .............................................................................................................. 78 Morphological Changes .................................................................................... 79 Ultrastructural Changes .................................................................................... 80 Ischemia ..................................................................................................... 80 Reperfusion ................................................................................................ 81 4 DETECTION OF CALPROTECTIN AND ITS CORRELATION TO THE ACCUMULATION OF NEUTROPHILS WITHIN EQUINE LARGE COLON AFTER ISCHEMIA AND REPERFUSION .............................................................. 88 Materials and Methods ............................................................................................ 91 Horses .............................................................................................................. 91 Procedures ....................................................................................................... 91 Histology ........................................................................................................... 93 Immunohistochemistry ...................................................................................... 93 Statistical Analysis ............................................................................................ 96 Results .................................................................................................................... 96 Discussion .............................................................................................................. 98 5 MUCOSAL INJURY AND INFLAMMATORY CELLS IN RESPONSE TO BRIEF ISCHEMIA AND REPERFUSION IN THE EQUINE LARGE COLON ................... 106 Material and Methods ........................................................................................... 107 Animals ........................................................................................................... 107 Experimental Procedures ............................................................................... 108 Sample Preparation ........................................................................................ 109 Immunohistoc hemistry .................................................................................... 110 Histomorphometry .......................................................................................... 110 Neutrophils, Eosinophils, Mast Cells and Macrophages ................................. 111 COX 1/ 2 Expression ..................................................................................... 112 Statistical Analysis .......................................................................................... 112 Results .................................................................................................................. 112 Histomorphometry .......................................................................................... 112

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7 Neutrophils, Eosinophils, Mast Cells, Macrophages, and COX 1/ 2 Expression .................................................................................................. 113 Discussion ............................................................................................................ 114 6 EFFECT OF ISCHEMIA AND REPERFUSION ON PRODUCTION OF NITROTYROSINE, ACTIVATION OF EOSINOPHILS AND APOPTOSIS IN EQUINE LARGE COLONIC MUCOSA ................................................................. 127 Materials and Methods .......................................................................................... 129 Animals ........................................................................................................... 129 Study Design .................................................................................................. 130 Anesthesia and Monitoring ............................................................................. 131 Surgical Procedures ....................................................................................... 131 Sample Collection .......................................................................................... 133 Histological Examinations ............................................................................... 133 Immunohistochemical Analysis and TUNEL Staining ..................................... 135 Statis tical Analysis .......................................................................................... 137 Results .................................................................................................................. 137 Discussion ............................................................................................................ 140 7 CALPROTECTIN, AND HEMATOLOGIC AND BIOCHEMICAL CHANGES IN SYSTEMIC AND COLONIC VENOUS BLOOD AFTER COLONIC ISCHEMIA AND REPERFUSION IN HORSES ....................................................................... 148 Materi al and Methods ........................................................................................... 150 Animals ........................................................................................................... 150 Experimental Procedures ............................................................................... 151 Calprotectin Analysis ELISA ........................................................................ 152 Development and a nalytic v alidation of the ELISA ................................... 153 ELISA p rotocol ......................................................................................... 154 Blood Gas, Hemat ological and Biochemical Analyses ................................... 155 Statistical Analysis .......................................................................................... 156 Results .................................................................................................................. 156 Discussion ............................................................................................................ 158 8 CONCLUSIONS ................................................................................................... 170 Mucosal Structure, Function, and Repair A fter Colonic I/R ................................... 170 Morphological Changes .................................................................................. 170 Ultrastructural Changes .................................................................................. 173 Cell Dea th ....................................................................................................... 175 Inflammatory Response ........................................................................................ 178 Nitrotyrosine ................................................................................................... 178 Cyclooxygenase ............................................................................................. 180 Inflammatory Cells .......................................................................................... 182 Mast Cells ................................................................................................ 182 Eosinophils ............................................................................................... 183

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8 Macrophages ........................................................................................... 184 Neutrophils ............................................................................................... 185 Calprotectin and O ther Clinicopathological Variables ........................................... 188 LIST OF REFERENCES ............................................................................................. 193 BIOGRAPHICAL SKETCH .......................................................................................... 231

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9 LIST OF TABLES Table page 2 1 Histomorphometric values for colonic mucosa after 1hI (ischemic tissue), or after 1hI + 4hR (reperfused tissue) ..................................................................... 66 2 2 Histomorphometric values for control ischemic and reper fused colonic tissues after incubation in Ussing chambers ...................................................... 67 4 1 Number of neutrophils and calprotectinpositive cells per mm2 submucosal venule, mucosa, and mucosal zones M1 M5 during I/R ................................... 103 5 1 Histomorphometric measurements during I/R .................................................. 118 5 2 Calprotectinpositive neutrophils, and eosinophils per mm2 submucosal venule, mucosa, and mucosal zones M1M5 during I/R ................................... 119 5 3 Scores for COX positive cells in the mucosa, epith elium, upper lamina propria, lower lamina propria, and crypts during I/R. ........................................ 120 6 1 A poptotic index (apoptotic epithelial cells) and number of apoptotic cells per mm2 mucosa during I/R .................................................................................... 144 7 1 Differentiation of leukocytes in JB and CB of post anesthesia controls and after I/R ............................................................................................................. 165 7 2 Calprotectin concentrations in JB and CB of post anesthesia controls and after I/R ............................................................................................................. 166

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10 LIST OF FIGURES Figure page 2 1 Mean SEM values for TER of colonic mucosa after 1hI (ischemic tissue), or after 1hI + 4hR (reperfused tissue) ..................................................................... 68 2 2 Mean SEM values for permeability of equine colonic mucosa to tritium labeled mannitol for control, ischemic, and reperfused tissues ........................... 69 2 3 Photomicrographs of colonic mucosa obtained at various time points from horses with experimentally induced I/R .............................................................. 70 3 1 Epithelium and subepithelial lamina propri a of the equine colon after I/R .......... 84 3 2 Epithelial cells and subepithelial structures after 1hI .......................................... 85 3 3 Epithelial cells and subepithelial structures after reperfusion ............................. 86 3 4 Schematic model of epithelial cell injury after 1hI, and epithelial recovery after 4hR ............................................................................................................. 87 4 1 Calprotectin IHC to detect equine alveolar macrophages ................................. 104 4 2 Neutrophils and erythrocytes within a submucosal vessel after 2hI and in m ucosal zones M1M5 after 2hI + 18hR .......................................................... 105 4 3 Calprotectinpositive cells within colonic mucosa of control s, and after 2hI and 2hI + 18hR ................................................................................................. 105 5 1 Colonic mucosal tissues of control s, and after 1hI 1hI + 2hR and 1hI + 4hR .. 121 5 2 Characteristic cellular features of the colonic epithelium and subepithelium after 1hI ............................................................................................................ 122 5 3 Characteristic cellular features of the colonic epithelium and subepithelium after 1hI + 2hR .................................................................................................. 123 5 4 Characteristic cellular features of the colonic epithelium and subepithelium after 1hI + 4hR .................................................................................................. 124 5 5 M acrophages and mast cells p er mm2 lower lamina propria and upper lamina propria during I/R .............................................................................................. 125 5 6 COX expression in the colonic epithelium and upper lamina propria during I/R ..................................................................................................................... 126 6 1 ICR and mucosal hemorrhage score after colonic I/R ...................................... 145

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11 6 2 E osinophils per mm2 colonic mucosa, and mucosal zones M1 M5 during I/R 146 6 3 N itrotyrosinepositive eosinophils and other leukocytes within the submucosa, lower lamina propria, and upper lamina propria during I/R .......... 147 6 4 Photomicrographs of apoptotic cell s in colonic tissue s after I/R ....................... 147 7 1 Values for pH, pCO2, HCO3 -, BE and PCV in JB and CB of post anesthesia controls and after I/R ....................................................................................... 167 7 2 Values for TP, albumin, glucose, Na+, K+, Ca2+, creatinine, CPK and ALP in JB and CB of post anesthesia controls and after I/R ....................................... 168 7 3 Concentrations of serum calprotectin after I/R compared to control samples in JB and CB ..................................................................................................... 169

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12 LIST OF ABBREVIATION S 1hI o ne hour of ischemia 1hR one hour of reperfusion 18hR eighteen hours of reperfusion 2hI t wo hours of ischemia 2hR t wo hours of reperfusion 30minR thirty minutes of reperfusion 4hR f our hours of reperfusion ABC a vidin biotin complex ALP alkaline phosphatase ATP a denosine triphosphate BE base excess Ca2+ calcium CaCl2 calcium chloride CB colonic venous blood CD cluster of differentiation CI confidence interval COX cyclooxygenase CPK creatine phosphokinase CRP C reactive protein DAB d iaminobenzidine DNA deoxyribonucleic acid ELISA e nzyme linked immunosorbent assay ER e ndoplasmic reticulum FABP fatty acid binding protein

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13 GM CSF granulocyte macrophage colony stimulatin g factor HCO3 bicarbonate H&E h ematoxylineosin HRP AEC h orseradish peroxidase3 amino 9 ethylcarbazole ICAM intercellular adhesion molecule ICR i nterstitium crypt ratio IHC i mmunohistochemistry IL interleukin iNOS inducible nitric oxide synthase I/R i schemia and reperfusion K+ potassium KRB K rebs r inger bicarbonate LM l ight microscopy LTC leukotriene MadCAM mucosal vascular addressin cell adhesion molecule MAPK mitogenactivated protein kinase MOTS m odified organ transplant solution MPO m yeloperoxidase MRP macrophage inhibitory related protein Na+ sodium NAC N acetylcysteine NADPH nicotinamide adenine dinucleotide phosphate nuclear factor kappa beta NO nitric oxide O.D. optical density

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14 OPS o rgan perfusion solution PAF platelet activating factor PAS p eriodic acidSchiff PBS p hosphate buffered saline PCV packed cell volume PG prostaglandin pCO2 partial pressure of carbon dioxide pO2 partial pressure of oxygen rER r ough endoplasmic reticulum RIA radioimmunoassay RNS reactive nitrogen species ROS r eac tive oxygen species SB systemic venous blood TB toluidine blue TdT terminal deoxynucleotidyl transferase TEM t ransmission electron microscopy TER t ransepithelial electrical resistance TGF transforming growth factor TJ t ight junction TLR tolllike receptor TMB tetramethylbenzidine peroxidase substrate TNF tumor necrosis factor alpha TP total protein TUNEL terminal deoxynucleotidy l transferase mediated dUTP nick end labeling XO xanthine oxidase

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15 ZO 1 zona occludens 1

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16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LARGE COLON ISCHEMIA AND REPERFUSION IN HORSES: HISTOLOGICAL AND FUNCTIONAL ALTERATIONS, AND RESPONSE OF THE INNATE IMMUNE SYSTEM By Astrid Grosche December 2011 Chair: David E. Freeman Major: Veterinary Medical Sciences Large colon volvulus is the most devastating cause of intestinal ischemia in horses and results in severe mucosal damage, barrier dysfunction, toxic shock and possibl y death. Although essential for regeneration, reperfusion can exacerbate mucosal injury after ischemia. R esponse s to I/R involve a series of synchronized biochemical, cellular and struc tural changes characterized by generation of radicals, activation of immune cells, and epithelial cell degeneration and death. The objective of the study was to assess the effect of colonic I/R on functional and histological alterations, reaction of neutro phils, eosinophils, macrophages and mast cells, expression of nitrotyrosine, COX and calprotectin, and clinic o pathological changes in horses. A segment of the pelvic flexure was submitted to 1hI or 2hI followed by 30minR, 4hR or 18hR. Mucosal biopsies bef ore and after ischemia, and after reperfusion were processed for H&E Luna, TB staining, IHC (calprotectin, CD163, nitrotyrosine), TUNEL method, and TEM. Mucosal (H&E) and epithelial damage ( TB TEM) were described and quantified. The number and distribution of mucosal neutrophils (H&E, calprotectin), eosinophils (Luna), macrophages (CD163) and mast cells ( TB ) were

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17 assessed. Mucosal nitrotyrosine and COX 1/ 2 expression, and apoptotic cell death ( TB TUNEL) were identified. To assess mucosal barrier integ rity, TER and mannitol flux were determined before and after 1hI, and after 4hR. Calprotectin (ELISA) and clinic o pathological variables were evaluated in JB and CB before and after 1hI, and after 1hR, 2hR and 4hR. Ischemia caused degeneration and detachme nt of epithelial cells, early apoptosis, and opening of TJ resulting in decreased TER and increased mannitol flux. Autophagy was a prominent feature in epithelial cells after 1hI. Reperfusion was characterized by apoptosis, epithelial regeneration, and res toration of TJ resulting in recovery of epithelial barrier integrity. Neutrophils infiltrated colonic mucosa after reperfusion, and macrophages, mast cells and eosinophils were activated during I/R. Ischemia caused metabolic acidosis, increased lactate, K+ and CPK and decreased glucose in colonic venous blood. B ut they returned to normal after reperfusion despite activation of an inflammatory response characterized by increased neutrophil cell turn over and release of calprotectin after I/R. Equine colonic mucosa subjected to ischemia can repair during reperfusion, despite increased mucosal inflammation.

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18 CHAPTER 1 INTRODUCTION Large Colon Volvulus Large colon volvulus is a rapidly progressive, lifethreatening form of colic with fatality rates between 30 to 80% (Snyder et al. 1989a; Southwood 2004; Mair and Smith 2005; Southwood 2006; Ellis et al. 2008; Munoz et al. 2008). Large colon volvulus is commonly diagnosed in up to 26% of horses that require abdominal surgery (Fisher and Meagher 1985; Mair and Smith 2005; Munoz et al. 2008). Although large colon volvulus can occur in horses of any age, breed and gender, broodmares are more likely to develop this fatal disease 1 to 3 months post foaling (Snyder et al. 1989a; Embertson et al. 1996). The volvulus is typically located at the origin of the right dorsal colon near the mesenteric attachment to the dorsal body wall, where the colon is twisted on its longitudinal axis up to 720 commonly in dorsomedial direction (Harrison 1988; Snyder et al. 1989a; Gibson and Steel 1999). Large colon volvulus has typical features of a hemorrhagic strangulation obstruction, which is characterized by luminal occlusion and compromised venous drainage. Because of continuous entry of arteri al blood into the tissue, these so called low flow ischemic conditions cause severe interstitial hemorrhage and edema accompanied by compartmentalization of large amounts of fluids, followed by gradual disruption of mucosal tissue architecture (Gibson and Steel 1999; Blikslager 2009). Less frequently, large colon volvulus may result in ischemic strangulation due to combined venous and arterial occlusion resulting in sudden loss of oxygen supply to the organ and rapid degeneration of the mucosa and its epith elium (Meschter et al. 1986; Gibson and Steel 1999; Lopes 2009). Experimentally induced

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19 complete ischemia of the colon for 1 hour caused notable epithelial lesions due to lack of sufficient oxygen supply (Snyder et al. 1988) Three to 4 hours of a 360degr ee volvulus resulted in irreversible damage of the entire intestinal wall followed by direct contact of luminal contents with subepithelial structures (Snyder et al. 1988). Thus, death in horses with large colon volvulus is mostly attributed to hypovolemic and toxic shock as a consequence of the abd ominal compartment syndrome and bacterial toxins that enter the blood across the ischemic damaged mucosa within hours (Snyder e t al. 1989a,b; Hardy 2009). At the beginning of the disease, mild signs of colic may occasionally precede signs of severe colic by hours due to nonstrangulated displacement of the colon (Hackett 2002; Hardy 2009). Most horses with large colon volvulus however have an acute onset of severe uncontrollable abdominal pain, marked progressive abdominal distention, compromised respiration and hemodynamic collapse (Hackett 2002; Lopes 2009). With progressive damage of mucosal capillaries and the epithelium, hemoconcentration, lactic acidosis, electrolyte imbalances and s igns of septicemia are typical laboratory findings (Lopes 2009). Although success in treating large colon volvulus is more likely when it is addressed early, surgical correction of the tissue does not always prevent further intestinal damage after reoxygenation (I/R injury), or the extent of the mucosal damage makes a complete resection of the injured colon difficult or impossible (Fisher and Meagher 1985; Wolfman 1989; Hughes and Slone 1998). Thus, evaluation of the intestinal viability during explorative laparotomy, and preoperative laboratory parameters (PCV above 50%, rectal temperature higher than 102F, and heart rate

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20 more than 80 beats per minute) have been used to determine prognosis and optimal surgical treatment of affected horses (Hughes and Slone 1994; Hardy 2009) Pathophysiology of Intestinal I/R Ischemia The I/R syndrome is a complex cascade of precise synchronized cellular and molecular events that could play a fundamental role in the pathophysiology of large colon volvulus (Meschter et al. 19 86; Moore et al. 1995a ; Mallick et al. 2004; McMichael and Moore 2004). Under ischemic conditions interruption of the cellular aerobic metabolism causes a decrease of intracellular ATP and pH levels that result in accumulation of toxic metabolites such as lactate generated by anaerobic glycolysis (Moore et al. 1995a; Rowe and White 2002; McMichael and Moore 2004). A cascade of cellular enzymatic and metabolic changes are initiated, that are characterized by inactivation of transmembrane electron pumps, intracellular accumulation of Ca2+, Na+ and lactate, and disorganization of cytoskeletal elements Consequently, structural changes of the cell and its organelles, and disruption of TJ are typical features after ischemia (Moore et al 1995a; Carden and Granger 2000; Li et al. 2009; Ivanov et al. 2010). Furthermore, hypoxia promotes the catabolism of adenine nucleotides followed by marked accumulation of hypoxanthine within the cell which is subsequently converted into ROS after reoxygenation (Carden and Granger 2000; Collard and Gelman 2001; McMichael and Moore 2004). As a consequence of the profoundly disturbed cellular homeostasis, cells become irreversibl y damaged and die over time (McMichael and Moore 2004). Moreover, activation of results in initiation of a profound inflammatory response stimulated by the synthesis of

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21 cell adhesion molecules ICAM 1 and P selectin, an essential step for adhesion of leukocytes at the site of injury (Lefer and Lefer 1993; Granger 1997; Olanders et al. 2002; McMichael and Moore 2004; Seal and Gewertz 2005; Souza et al. 2005; Karrasch and Jobin 2008; Spehlmann and Eckmann 2009). Furthermore, i nactivation of endothelial NO and inhibition of the pr ostacyclin pathway favors vasoconstriction, platelet aggregation, and complement activation in postcapillary venules (Carden and Granger 2000; Riedemann and Ward 2003; Vollmar and Menger 2011). Thus ischemia induces a proinflammatory state that can increase tissue vulnerability to further injury after reoxygenation (Collard and Gelman 2001). Similar to endothelial cells, the intestinal epithelium is extremely prone to hypoxia (Kong et al. 1998; Sun et al. 1998; Carden and Granger 2000). Due to their complex structural and functional properties, intestinal epithelial cells are highly energy dependent so that reduced blood supply and decreased oxygenation result in rapid cellular injury and death (Moore et al. 1995a). However, different segments and cell type s within the gastrointestinal tract have a distinct susceptibility to ischemia ( Leung et al. 1992; Rowe and White 2002). Experimentally induced ischemia for 3 hours in dogs caused more changes in electrophysiologica l properties and permeability of the colon compared to the small intestine, but histopathologic al mucosal damage was similar between both (Takeyoshi et al. 1996). However, small intestinal epithelial cells at the tips of the villi are considered to be particularly sensitive to ischemia due to their location at the end of the central arteriole where the arterial oxygen concentration is already lower under normal conditions (Takeyoshi et al. 1996; Kong et al. 1998; Vollmar and Menger 2011). Reversible occlusion of the superior mesenteric ar tery for 30

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22 minutes in rats caused a greater reduction of mucosal perfusion in the colon than in the small intestine, but mucosal damage was significantly less in the former (Leung et al. 1992) I nfiltration of neutrophils was induced in both locations aft er 1hR although mucosal injury was only exacerbated in the small intestine after IR Hinnebusch et al. (2002) assumed that the sensitivity of small intestinal epithelial cells to ischemia could also be associated with their differentiation state. Differentiated enterocytes, located at the tip of the villi are the first to undergo apoptosis in response to ischemia than undifferentiated cells in the crypts probably because these cells are already programmed for apoptosis under normal conditions. Thus they m ight be primed for an apoptotic response to stressful stimuli (Hinnebusch et al. 2002). Reperfusion Although restoration of blood flow to an ischemic organ is essential to prevent irreversible cellular injury, reperfusion can exacerbate tissue injury in ex cess of that produced by ischemia alone (Parks and Granger 1986). Several theories have been established to explain progression of tissue damage after reoxygenation, and all of them seem to depend on biochemical changes that occur during ischemia (Soffler 2007). The most established mechanism of reperfusion injury is based on oxidation of hypoxanthine to xanthine by XO, both generated during the ischemic period. After reperfusion, ROS and RNS such as superoxide anions, hydroxyl radicals, hypochlorous acid, and nitric oxidederived peroxynitrite are generated within 10 to 30 seconds in the presence of oxygen ( McCord and Roy 198 2 ; Zweier et al. 1987; Prichard et al. 1991; Granger and Korthuis 1995; Moore et al. 1995a; Dabareiner et al. 2005). Additionally, activated neutrophils secret e NADPH oxidase and MPO, and both enzymes could contribute to formation of ROS after I/R ( Bhaskar et al. 1995; Granger and Korthuis

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23 1995; Kaminski et al. 2002). Although oxidants were considered to be poten tially harmful by products of cell metabolism, they have lately been recognized as important messengers in numerous intracellular pathways (Kirschvink et al. 2008). Oxidants promote chemotaxis, activate leukocytes, and initiate cytokine gene expression (Granger and Parks 1983; Granger and Korthuis 1995; Dr ge 2002; Kalia et al. 2003; Soffler 2007; Kirschvink et al. 2008). They can also stimulate expression of cellular adhesion molecules, o ne of the crucial steps in I/R that allows leukocytes to enter the injured tissue rapidly Although free radicals are essential for microbial killing, they play a major role in inflammation and I/R injury (McCord and Roy 1982; Granger and Parks 1983; Soffler et al. 2007). P eroxynitrite, a highly active member of RNS, can cause tissue damage by lipid peroxidation, oxidation of protein sulphydryl groups, and nitration of aromatic amino acids including tyrosine (Radi et al. 1991a,b; Beckman and Koppenol 1996; Muijsers et al. 1997). Peroxynitrite formation has been demonstrate d in activated leukocytes (Ischiropoulos et al. 1992 ; Gagnon et al. 1998; Takemoto et al. 2007), endothelial cells (Kooy and Royall 1994), in inflammatory bowel disease (Dijkstra et al. 1998), I/R (Beckman et al. 1990 ; Ma et al. 1995; Mirza et al. 1999) and septic shock (Wizemann et al. 1994 ; Szabo et al. 1995), and it may be an important mediator of cytokine induced epithelial hyperpermeability (Chavez et al. 1999). In addition to the potential contribution of ROS and RNS to intestinal I/R injury, dysfunction of microcirculation and decreased perfusion in submucosal postcapillary venules referred to as no reflow and refl o w paradox are considered as major factors underlying I/R injury in the intestine (Lefer and Lefer 1993; Seal and Gewertz 200 5; Vollmar and Menger 2011). Interactions of various pathological mechanisms have

PAGE 24

24 been proposed to cause intestinal microcirculatory dysfunction (Lefer and Lefer 1993; Seal and Gewertz 2005). The noflow phenomenon has been attributed to a number of events during I/R including intravascular hemoconcentration and thrombosis, leukocyte plugging, endothelial cell swelling, vasomotor dysfunction and capillary narrowing due to edemaassociated intestinal pressure (Moore et al. 1995a; Menger et al 1997; Dabareiner et al. 2005; Seal and Gewertz 2005; Vollmar and Menger 2011). In addition, a reduced red blood cell velocity and red blood cell sludging due to leukocytevessel wall interactions has been associated with post ischemic perfusion failure (Vollmar and Me nger 2011). I ndividual platelets adhere to the capillary endothelium via P Selectin, an event that could also contribute to collapse of intestinal microvascular perfusion (Massberg et al. 1998; Cooper et al. 2003; Vollmar and Menger 2011). However based on histomorphological studies i n intestinal I/R, the inflammatory cell response is restricted to the submucosal layer, while all intestinal layers experience reduced perfusion (Beuk et al. 2000) T hus additional mechanisms might be involved in the pathogenes is of I/R injury including damage by activation of an uncontrolled inflammatory response Intestinal I/R injury has to be considered an inflammatory disease with recruitment of leukocytes as a rate limiting multistep process (Souza et al. 2005) Among infl ammatory cells, attracted activated neutrophils are known to be the most critical element s during I/R (Moore et al. 1994a; Moore 1997; Kalia et al. 2003). Moreover, CD4+ and CD8+ T cells have been shown to play a crucial role during I/R by upregulation of MadCAM 1, an adhesion molecule located in mucosal venules of post ischemic small intestine (Fujimori et al. 2002; Shigematsu et al. 2002 ; Linfert et al

PAGE 25

25 2009). Furthermore, platelets assist leukocytes during intestinal I/R by activation of neutrophil CD11b/CD18 adhesion molecules, and generation of ROS (Cooper et al. 2004; Vollmar and Menger 2011). R ecently, TLR 4 has been implicated as a possible link between the innate and adapt ive immune system s, thereby mediating intestinal inflammation and injury after I/R (Mollen et al. 2006; Moses et al. 2009). Intestinal I / R in Horses Many studies on I/R injury in the horse have been published, and various inconsistencies have been reported, possibl y due to use of different experimental I/R models (low flow [hemorrhagic strangulation] vs. complete ischemia [ischemic strangulation]), different ischemia and reperfusion times, and different intestinal segments (Reeves et al. 1990; Mesch ter et al. 1991; Moore et al. 1994b; Wilkins et al. 1994; Wilson et al 1994; Darien et al. 1995; Laws and Freeman 1995; Moore et al. 1995a; Moore et al. 1996, 1998a,b; Moore 1997; Rowe and White 2002; Soffler 2007). Researchers and clinicians still debate whether deterioration of intestinal injury after reperfusion is clinically relevant in the horse, or simply a result of progression of mechanisms that have been activat ed under previous hypoxic conditions (Snyder et al. 1988; Blikslager et al. 1997a ; Rowe and White 2002). The pathogenesis of intestinal I/R injury in horses appears to involve activation of the xanthine oxidoreductase system during ischemia followed by generation of toxic radicals that contribute to intestinal damage after reoxygenation ( Prichard et al. 1991; Moore et al. 1995a; Soffler 2007). Equine small and large intestines behave differently in response to I/R (Kooreman et al. 1998; Dabareiner et al. 2001; Rowe and White 2002). There is some evidence that reperfusion injury occurs in the equine small intestine ( Dabareiner et al. 1995, 2001; Van Hoogmoed et al. 2001; Moore et al. 1995a),

PAGE 26

26 with low flow ischemic models showing the most compelling evidence o f postischemic damage ( Laws and Freeman 1995; Blikslager et al. 1997a ; Kooreman et al. 1998; Van Hoogmoed et al. 2001). In these cases, the equine small intestine appears to follow the classic pathophysiology of I/R injury characterized by generation of ROS and RNS as described above ( Prichard et al. 1991 ; Soffler 2007). The enzyme, XO is pres ent in the small intestine, and is significantly increased after ischemic strangulation of the jejunum (Prichard et al. 1991). However, other pathomechanisms that c an exacerbate intestinal damage after reperfusion might be involved in small intestinal I/R injury as well (Laws and Freeman 1995; Vatistas et al. 1996; Vatistas et al. 1998; Dabareiner et al. 2001). Despite some distinct similarities between I/R injury of the small and large intestine (Moore et al. 1995a) the true existence of colonic I/R is not clear. Reperfusion injury was demonstrated in the large colon after 3 hours of low flow ischemia followed by 3 hours of reperfusion (Moore et al. 1994b). Furtherm ore, mucosal lesions progressively worsened after twisting the colon for 2 or 3 hours followed by a 2hour reperfusion time period (Meschter et al. 1991; Darien et al. 1995). Moreover, I/R resulted in significant damage to the vasculature in the equine col on after 2 hours of ischemia (Henninger et al. 1992), but remained unchanged after complete ischemic strangulation for 70 minutes followed by 1hR (Dabareiner et al. 1993). In contrast, several studies on equine large colonic I/R failed to detect reperfusion injury (Snyder et al. 1988; Reeves et al. 1990; Wilkins et al. 1994; Dabareiner et al. 2001; Matyjaszek et al. 2007; 2009; Morton et al. 2009; Grosche e t al. 2011a,b). Furthermore, the classic model of I/R injury which depends on XO is not likely to contribute to postischemic damage in the large colon (Kooreman et al. 1998), because the enzyme is not present or in very low

PAGE 27

27 concentrations in the equine colonic mucosa (Wilkins et al. 1994; Moore et al. 1995a; Blikslager et al. 1997 a ; Kooreman et al. 1998). Thus, other mechanisms such as alternative oxidants mitochondrial dysfunction, failure of mucosal postcapillary microcirculation, collateral tissue damage by infiltrating neutrophils, uncontrolled activation of an innate immune response or simpl y a time related continuation of injury initiated during ischemia, might be responsible for colonic mucosal damage after reperfusion (Moore et al. 1994a; Wilkins et al 1994; Granger and Korthuis 1995; Moore et al. 1995a; Blikslager et al. 1997 a ; McAnulty et al. 1997; Rowe and White 2002; Soffler 2007). Immune Cells and Inflammat ory Response A fter Intestinal I / R The intestinal mucosa is replete with many immune cells, i ncluding lymphocytes, macrophages, mast cells, neutrophils, and eosinophils i ntestinal lymphocytes and macrophages represent ing the largest pool in the body under healthy conditions (Lloyd 2000; Schenk and Mueller 2008). Accumulation of these diverse active inflammatory cells requires well coordinated mechanisms to maintain a delicate balance between protection against infectious agents and injury, and tolerance of the abundant antigens in the intestinal lumen (Sansonetti 2004; Artis 2008; Pasparakis 2009; Turner 2009). Thus, in the event of impaired epithelial integrity, a detrimental inflammatory response could play a critical role in the pathogenesis of intestinal I/R injury by uncontrolled activation of immune cells (Wallace and Ma 2001; Platt and Mowat 2008; Schenk and Mueller 2008; Laskin et al. 2011). Macrophages The innate immune system is the first line of defense to provide protection against mucosal injury, translocation of pathogens and toxins, and to support repair (Podolsky

PAGE 28

28 1999; Medzhitov 2001, 2007; Nizet and Johnson 2009). Resident m acrophages and attracted monocytes strategically positioned in the subepithelial region, are the cell population that immediately recognizes microorganisms and damageassociated signals, and that mediate and tightly regulate an innate immune response (Smith et al. 2005; Kono and Rock 2008; Platt and Mowat 2008; Laskin et al. 2011; Smith et al. 2011). When the epithelial barrier fails and bacteria enter the tissue, proinflammatory molecules released by attrac ted monocytes activate and attract more leukocytes to the site of injury (Mahida et al. 1997; Souza et al. 2004; Chin and Parkos 2007; Platt and Mowat 2008; Laskin et al. 2011). Activation of transcription factor NF B followed by increased production of pr o inflammatory enzymes (iNOS, phospholipase A2, COX) and cytokines (TNF 1, IL 6), and expression of adhesion molecules (ICAM 1 and P selectin) play an important role in this process (Grotz et al. 1999; Wallace and Ma 2001; Olanders et al. 2002; Souza et al. 2005; Karrasch and Jobin 2008; Medzhitov 2008; Spehlmann and Eckmann 2009). The ability of activated macrophages to induce tissue injury is well established (Duffield 2003; Chen et al. 2004; Laskin et al. 2011) These cells release an array of mediators with cytotoxic, proand anti inflammatory, angiogenic, fibrogenic and mitogenic activity, which can be deleterious to tissues when macrophages become hyperresponsive (Laskin et al. 2011). On the other hand, macrophages play a pivotal role in res olution of inflammation and in initiation of repair mediated by anti inflammatory cytokines, bioactive lipids and growth factors ( Duffield 2003; Serhan and Savill 2005; Platt and Mowat 2008; Serhan et al. 2008; Laskin et al. 2011). Thus the re sponse of mac rophages could be an important factor in the pathophysiology of intestinal I/R injury and subsequent repair processes

PAGE 29

29 Neutrophils Attracted by release of proinflammatory cytokines and chemokines, large numbers of neutrophils adhere to adhesion molecules that have been expressed by activated endothelial cells of postcapillary venules during I/R (Lefer and Lefer 1993; Kurose et al. 1994; Granger 1997; Olanders et al. 2002; Chin and Parkos 2007). In vitro studies revealed that the adhesion molecule P selecti n stored in endothelial Weibel Palade bodies can be translocated within 30 minutes after I/R (Eppihimer et al. 1997; Panes and Granger 1998; Ga yle et al. 2000) and the beginning of neutrophil endothelial cell adhesion was observed 10 minutes after reperfusion in vivo (Granger et al. 1989; Moore et al. 1994 a ). Neutrophils roll along the endothelium mediated by P selectin until firm adhesion facilitated by neutrophil integrins and endothelial ICAM 1 ( Chamoun et al. 2000; Witko Sarsat et al. 2000). Finally neutrophils transmigrate between endothelial cells into the lamina propria, and travel through the extracellular matrix to the source of tissue injury where they assist in recognizing and ingesting cell detritus and/or destroy invading bacteria directly by generation of antimicrobial peptides, proteolytic enzymes and toxic radicals ( Chamoun et al. 2000; Witko Sarsat e t al. 2000; Nathan 2006; Dale et al. 2008). I ntravital microscopy demonstrated pronounced adherence of neutrophils to mesenteric veins followed by extravasation aft er 1hI and 10 minutes of reperfusion in the small intestine in cats (Granger et al. 1989). Moore et al. (1994a) reported neutrophil accumulation in the colonic mucosa during a 3hour period of ischemia, with further progression of neutrophil infiltration after reperfusion. Despite their protective role during an innate immune response (Mantovani et al 2011) neutrophils are thought to be a crucial factor in the development of endothelial injury (Granger et al. 1986; Hernandez et al 1987; Kurose et al. 1994; Cooper et al. 2004)

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30 and mucosal damage during intestinal I/R (Granger 1988; Grisham and Granger 1988; Grisham et al. 1990a/b ; Schoenberg et al. 1991; Kubes et al. 1992; Moore et al. 1994a/1995a; Friedman et al. 1998; Gayle et al 2000, 2002; Chin and Parkos 2007). Neutrophils have an arsenal of proinflammatory factors, enzymes, antibacterial proteins and other toxic molecules that are directed against invading microorganisms and infectious agents but are also capable of tissue damage Neutrophils that sense tissue damage or infection release their weapons into the extracellular space within 15 to 45 minutes, which is thought to be the approximate time it takes for a neutrophil to emigrate from the blood into the extravascular ti ssue (Nathan 2006). Thus, most of the damage mediated by neutrophils is caused by accidental contact of these toxic molecules with surrounding cells (Grisham and Granger 1988; Arndt et al. 1991; De Greef et al. 1998; WitkoSarsat et al. 2000), or by physic al damage while migrating to the site of injury (Milks et al. 1986; Moore et al. 1995a; Gayle et al. 2000; Nathan 2006; Chin and Parkos 2007). As a consequence, this so called collateral or bystander damage can enhance microvascular permeability, edema and thrombosis, provoke further mucosal injury, and exacerbate inflammation (Hernandez et al. 1987; Gayle et al. 2000; McMichael and Moore 2004). I nflammation is essential for wound repair, and neutrophils have a key role in promoting tissue repair after I/R (Nathan 2006; Blikslager et al. 2007; Mantovani et al. 2011). Neutrophils are essential for controlling infection and sterilizing wounds. They can generate proreparative signals (PGE2, IL inflammatory mediators (lipoxins or adenosine) they inhibit their own diapedesis and recruitment, activate their own death by apoptosis, reduce generation of proinflammatory mediators and toxic

PAGE 31

31 radicals, and initiate repair (Serhan and Savill 2005; Nathan 2006; Blikslager et al. 2007; Martin and Wallace 2006; Dale et al. 2008; Serhan et al. 2008). Neutrophils have been shown to augment recovery of ischemic injured porcine intestine by an ILand COX 2 dependent mechanism (Shifflett et al. 2004). Because neutrophils have a passive role in switching off in flammation, they are the key cells that regulate the change from proto anti inflammatory repair promoting conditions (Serhan and Savill 2005; Nathan 2006; Mantovani et al. 2011). Although, the role of neutrophils during I/R in the equine colon is still unclear (Moore et al. 1995a) other resident immune cells such as eosinophils and mast cells could contribute to the inflammatory response (Meschter et al. 1986; McConnico et al. 1999; Pen i ssi et al. 2003; Rtting et al. 2003, 2008a,b ; Galli et al. 2008; SheaDonohue et al. 2010). Eosinophils E osinophils reside in large amounts in the gastrointestinal lamina propria of the equine colon under healthy states, and their role in gastrointestinal disease and injury remains to be discovered (Rtting et al 2008 a ). E osinophils are a much overlooked cell population, although they are widely accepted to be proinflammatory leukocytes that can generate a mixture of destructive mediators (radicals, lipid mediators, and proteases) and toxic granule proteins (maj or basic protein, eosinophil peroxidase, eosinophil derived neurotoxin, and eosinophil cationic protein) (Walsh 1997; Hogan et al. 2008). Eosinophils can affect pathological changes and the severity of tissue damage in patients with asthma, eosinophil myal gia syndrome and other hypereosinophilic diseases (Wardlaw 1996). Equipped with enzymes that cause oxidative damage to biological targets, the respiratory burst occurs in activated eosinophils as in neutrophils, by generat ion of ROS and RNS such as peroxy nitrite

PAGE 32

32 (V an Dalen et al. 2006, Takemoto et al. 2007; Lotfi et al. 2009). Furthermore, eosinophilic major basic protein stimulates IL8 release, a potent chemoattractant and activator of neutrophils (Moy et al. 1990; Page et al. 1999). Major basic protein also promotes neutrophil respiratory burst in eosinophil associated inflammation (Moy et al. 1990; Haskell et al. 1995). Additionally, a number of cytokines are synthesized by eosinophils increasing the range of their potential functions including wound h ealing and fibrosis (TGF 1) and autocrine stimulation (IL5, IL 3, GM CSF) (Rothenberg et al. 2001, Munitz and Levi Schaffer 2004). Inappropriate accumulation of eosinophils and eosinophil derived granules in tissues are considered as biomarkers for monitoring the severity of the pathological event and they also evoke cellular and tissue damage. Furuta et al. (2005) demonstrated the deleterious effect of major basic protein on colonic epithelial barrier function in mice. Moreover increased intestinal accumulation of eosinophils has been described in horses with experimentally induced acute colitis, I/R injury and parasitism (Moore et al. 1994a ; McConnico et al. 1999; Edwards et al. 2000; Archer et al. 2006 ; Rtting et al 2008b ). Mast Cells Eosinophilic granulocytes are frequently seen in close proximity to mucosal mast cells ( Meschter et al. 1986; Armetti et al. 1999; Munitz and Levi Schaffer 2004), and degranulation of mast cells is thought to be triggered by eosinophilic toxic proteins (Piliponsky et al. 1999). Mast cells can display a variety of phenotypic and functional characteristics particularly during the course of an inflammatory response (Galli et al. 1999; SheaDonohue et al. 2010). Mast cells are potent effector and immunomodulatory cells that can promote and increase inflammation, tissue remodelling and tissue injury and they can also play an importa nt protective role in mucosal defense (Kanwar and

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33 Kubes 1994 b ; Pen i ssi et al. 2003; Galli et al. 2008; SheaDonohue et al. 2010). Activated by pathogens, danger signals or inflammatory mediators, mast cells can clear pathogens by phagocytosis or by secreti on of anti microbial peptides, degrade potentially toxic endogenous peptides, and release numerous pro inflammatory cytokines and other mediators (ROS/RNS, histamine, serotonin, proteases, proteoglycans, TNF 2 and PAF). They recruit and activat e other immune cells, regulate intestinal barrier homeostasis and initiate inflammation (Kanwar and Kubes 1994a/b; Galli et al. 1999; Andoh et al. 2001; Marshall 2004; Dawicki and Marshall 2007; Galli et al. 2008; Santen et al 2008; Groschwitz et al. 2009). Although the production of inflammatory mediators by mast cells is highly selective, their response in certain pathologic situations can be detrimental especially to the intestinal barrier integrity (Peni ssi et al. 2003; Marshall 2004). Mast cells are r ecruited into the tissue during colonic I/R in the rat (Sand et al. 2008), and they are thought to be important mediators of I/R induced mucosal and microvascular dysfunction in the mouse intestine (Kanwar et al. 1998). Mast cells are very sensitive to int estinal ischemia (Boros et al. 1995), and they cause mucosal permeability alterations during reperfusion in canine small intestine; however, they might play only a minor role in I/R induced structural changes (Szabo et al. 1997). Mast cell degranulation has an important effect on small intestinal injury after I/R, and release of histamine contributes most to the severity of mucosal damage compared with other toxic mast cell granule metabolites (Kimura et al. 1998; Boros et al. 1999a). However, mast cell degranulation before ischemia may also induce potentially protective mechanisms in the small intestine and can decrease I/R injury in the dog (Boros et al. 1999b)

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34 Morphology, Ultrastructure, Cell Death, and Mucosal Repair A fter Intestinal I/R Intestinal Morphology and Ultrastructure Intestinal I/R injury can be identified early by an increase in capillary and epithelial permeability (Snyder et al. 1992; Darien et al. 1993; Haglund 1994; Darien et al. 1995) with endothelial cel ls affected foremost during ischemia (Meschter et al. 1991; Henninger et al. 1992; Darien et al. 1993; Lefer and Lefer 1993; Darien et al. 1995). When activated and injured by hypoxia, endothelial cells swell with subsequent disruption of TJ and leakage o f erythrocytes and immune cells (Dabareiner et al. 1995). Changes of the cell shape caused by disintegration of cytoskeletal proteins or by lipid peroxidation of the cell membrane increase the capillary permeability and allow fluid and protein to move int o the interstitium which creat es an increased interstitial pressure with capillary collapse (Dabareiner et al. 1995 ; Vollmar and Menger 2011). In combination with endothelial cell swelling, both events decrease blood flow to the tissue, and exacerbate ischemia (Rowe and White 2002). Experimental torsion of the large colon by 720 for 1 hour caused platelet aggregation and erythr odiapedesis in subepithelial capillaries in conjunction with endothelial gap formation (Darien et al. 1995). After 2 hours, the capillary endothelium had separated from the basement membrane, and fibrin, platelets and neutrophils were seen with in and outside the capillary lumen (Darien et al. 1995). The colonic mucosa appear ed thickened and purple after ischemia due to development of an extensive mucosal edema and interstitial hemorrhage (Snyder et al. 1988; Meschter et al. 1991; Reeves et al. 1990; Moore et al. 1994b). Ischemic intestinal injury is a time dependent process. T he f irst histological signs of epithelial damage begin w ithin 20 minutes of total small intestinal ischemia, and are

PAGE 35

35 characterized by lifting of small clusters of epithelial cells, their detachment from the basement membrane and subsequently dea th by apoptosis and necrosis (Snyder et al. 1988; Meschter et al. 1 991; Moore et al. 1995a; Kong et al. 1998; Haglund and Bergqvist 1999). S tudies have shown that ultrastructural changes characterized by cellular degenerative processes have become irreversible before epithelial sloughing occurs (Snyder et al. 1992). E xper imentally induced 1hI in the equine colon caused minor histological alterations but led to severe epithelial barrier failure characterized by decrease of TER (Graham et al. 2011). Possibly degenerative processes and ultrastructural abnormalities of the epithelial barrier that are not visible by LM might be responsible for loss of barrier function. After 4 hours of low flow ischemia the colonic epithelium is completely denuded (Snyder et al. 1988; Moore et al. 1995a). Diverse inflammatory stimuli can increase leakiness of the epithelial barrier integrity by disassembly of apical junctions (Ivanov et al. 2010). Some studies that have focused on colonic I/R injury in horses at the cellular level describe subepithelial cap illary defects following I/R (Meschter et al. 1991; Dabareiner et al. 1993; Darien et al. 1993, 1995; Wilson et al. 1994; Richter et al. 2002). Few studies that emphasized ultrastructural changes of intestinal epithelial cells demonstrated a pattern of cel l blebbing and swelling, mitochondrial swelling and disruption of christae, nuclear alterations and cytoplasmic vacuolization, disruption of TJ between neighboring cells and intercellular gap formation, rupture of cell organelles and accumulation of necrot ic debris after 1 to 6 hours of ischemia. The damage progressed during 1 to 5 hours of reperfusion resulting in irreversible loss of the entire epithelium and subepithelial

PAGE 36

36 structures by cell necrosis or apoptosis (Snyder et al. 1988; Meschter et al. 1991; Dabareiner et al 1993; Moore et al. 1994b; Darien et al. 1995; Rowe and White 2002). Epithelial Cell Death Cell death by necrosis or apoptosis is triggered by mediators released during I/R (Cummings et al. 1997; Ramachandran et al. 2000; Festjens et al. 2006; Zong and Thompson 2006; Rock and Kono 2008). The two classic types of cell death, apoptosis and necrosis, can occur simultaneously in tissues exposed to the same stimulus but the intensity of the stimulus determines which predominates. For example, conditions associated with ATP depletion trigger switches from apoptosis to necrosis (Leist et al. 1997; Nicotera et al. 1998). N ecrosis is a well orchestrated process, and cells can initiate their own demise by necrosis (Golstein and Kroemer 2006; Zong and Thompson 2006). Histologically, n ecrotic cells appear vacuolated and swollen, and the plasma membrane br e akes down to release cell contents. They exhibit nuclear changes that differ from those of apoptotic cells (Edinger and Thompson 2004; Kroemer et al. 2009). Necrotic cells elicit an inflammatory response by actively releasing intracellular danger signals Activation of TLR mediated pathways, the complement system generation of ROS, and finally the system ( Festjens et al. 200 6 ; Frangogiannis 2007; Kono and Rock 2008; Rock and Kono 2008) can cause severe tissue damage and is thought to contribute to the pathogenesis of I/R injury (Rock and Kono 2008). However the characteristic features of acute inflammation, such as hyperemia, leak of plasma protei ns, and recruitment of leukocytes subserve also a number of useful functions ( Rock and Kono 2008) thus contributing to host defence and tissue repair A poptosis is a programmed energy dependent form of cell death that is associated with normal cell tur nover in the gastrointestinal tract (Hall et al. 1994). The

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37 defining characteristic of apoptosis is a complete change in cellular morphology characterized by shrinkage, chromatin condensation and margination, membrane blebbing, and segmentation and division into apoptotic bodies that can be phagocytized within several hours (Kerr et al. 1972; Aschoff and Jirikowski 1997; Darzynkiewicz et al. 1997; Jones and Gores 1997; Saikumar et al. 1999; Taylor et al. 2008 ; Kroemer et al. 2009). The highest levels of spontaneous apoptotic cell death is observed in the crypts of the small intestine, specifically located in the proliferative zone above the Paneth cells and at a rate of 1 to 10% (Potten 1997; Anderson 2000). The rate of spontaneous apoptosis is significantly lower in the large intestine, and apoptotic cells are scattered throughout the proliferative compartment of the crypts in this organ. Dysregulated apoptosis is associated with a number of pathological conditions i n the gastrointestinal tract including inflammatory bowel disease, coeliac disease, ulcerative colitis, gastric carcinoma, I/R, and infection (Levine 2000; Ramachandran et al. 2000; Tarnawski and Szabo 2001). Apoptosis is a major cause of cell death in intestinal I/R injury (Ikeda et al. 1998; Noda et al. 1998; Baylor et al. 2003; Rowe et al. 2003; Fujise et al. 2006; Grosche et al. 2011a/b/c). Rowe et al. (2003) determined high numbers of apoptotic cells in equine large and small intestines after obstruct ing and strangulating lesions. However, it is unclear whether increased apoptosis affects intestinal function, and controversial findings complicate interpretation (Abreu et al. 2000; Gitter et al. 2000). A loss of 50% of cells via Fas mediated apoptosis i n colonic cell cultures caused only minor disruption of the intestinal barrier and propagation of inflammation (Abreu et al. 2000). Furthermore, phagocytized apoptotic cells are immunosuppressive, and are

PAGE 38

38 essential for terminating the destructive inflammat ory response and facilitating tissue repair (Sun and Shi 2001; Savill et al. 2002; Maderna and Godson 2003; Erwig and Henson 2007). Death of injured enterocytes by apoptosis is rapid and detachment of these cells into the intestinal lumen was followed by r epair of the epithelial lining within 1 hour after a 30minute ischemic period in the human small intestine (Derikx et al. 2008). Apoptotic cell death was a prominent event during I/R of the equine colon, but tissues recovered during reperfusion despite increased apoptosis (Grosche et al 2011a,b,c).Thus apoptotic cell los s might be designed to control tissue damage, maintain a defensive barrier, regulate inflammation and initiate resolution (Sun and Shi 2001; Maderna and Godson 2003; Serhan and Savill 2005; Maniati et al. 2008). Resolving Inflammation and Epithelial Repair Inflammation is the basic process that preceds repair of structure and recovery of function, and subsequently to maint aining tissue homeostasis (Henson 2005). Acute inflammation is often characterized by rapid influx of neutrophils, followed swiftly by monocytes that mature into inflammatory macrophages. I nflammation can resolve if granulocytes are eliminated, debris is removed from inflamed sites, and tissue macrophages and lymphocytes return to normal preinflammatory numbers and phenotypes (Serhan and Savill 2005). There is emerging evidence that an active, coordinated program of resolution is initiated in the first few hours after the inflammatory response begins (Serhan and Savill 2005). This process is characterized by switching of arachidonic acidderived PGs and LTCs to anti inflammatory mediators such as lipoxins, resolvins and protectins that are generated by neutrophil s and macrophages themselves (Serhan and Savill 2005; Serhan et al. 2008). Thus, neutrophils have a key role in promoting tissue repair, and the signals generated by neutrophils finally inhibit

PAGE 39

39 their accumulation and activation, promote their own death, and attract and program macrophages to stop the damage and orchestrate repair (Nathan 2006; Mantovani et al. 2011). In addition, neutrophils are a rich source of PGE2, a COX derived PG, which has been shown to facilitate mucosal repair in the intestine (Blikslager et al. 1997 b ; Blikslager et al. 1999; Martin and Wallace 2006). Moreover, apoptosis of neutrophils and clearance by inflammatory macrophages is a crucial step in reducing inflammation (Haslett et al. 1994; Savill et al. 2002; Maderna and Godson 2003). Uptake of apoptotic cells stimulates macrophages to release mediators that suppress the inflammatory response and trigger tissue repair such as TGF 10 and various growth factors (Serhan and Savill 2005; Martin and Wallace 2006; Eming et al. 2007). When injury to the intestinal mucosa occurs, translocation of luminal contents across the damaged epithelium rapidly induces multiple proand anti inflammatory signaling events These then affect the essenti al steps of the epithelial cell response to wounding, ie epithelial cell proliferation, migration and apoptosis (Karrasch and Jobin 2008). These processes are governed by numerous cell signaling pathways including induced signaling events (TGF such as bile acids, short chain fatty acids, trefoil factors, and bacteria (Karrasch and Jobin 2008). In general, the healing process and restoration of the epithelial integrity and function can be classified in three phases (Playford and Ghosh 2005). The initial rapid response involves the migration of surviving cells from the wound edge to cover the denuded area, and closure of leaky epithelial intercellular spaces and TJ (Florian et al. 2002). This process, termed restitution, is locally regulated by mediators arising from a complex network of nerves,

PAGE 40

40 effector immune cells, fibroblasts, endothelial cells and subepithelial extracellular matrix, and begins within minutes after injury (Florian et al. 2002; Playford and Ghosh 2005; Blikslager et al. 2007). During restitution, cells bordering the injured mucosal zone undergo major changes of t heir shape and phenotype. They flatten and aquir e a squamoid appearance before they begin to migrate across the denuded basement membrane (Florian et al. 2002). M cNeil and Ito (1989) demonstrated that wounded epithelial cells seem to participate in restitution of the injured mucosa by extending membrane pseudopodia over denuded lamina propria. These allow cells to migrate over the defect until they contact other cells. After reestablishment of cell to cell contacts, junctional complexes between epithelial cells reform, which is critical for restoration o f the epithelial barrier (Mammen and Matthews 2003). Substantial damage of the jejunal epithelium after 30 minutes of ischemia in humans was completely resealed and fully recovered within 60 minutes of reperfusion, as demonstrated by restoration of the TJ protein ZO 1 and FABP containing intact epithelial cells (Derikx et al. 2008). Studies on large colon I/R injury in horses showed restitution and functional recovery of ischemic damaged colonic mucosa after 4hR characterized by normalization of TER and tra nsmucosal mannitol flux (Graham et al. 2011). Upon restoration of intercellular junctional interactions, proliferation of crypt stem cells and their differentiation into mature epithelial cells is stimulated 1 to 2 days after the damage has occurred. The f inal stage of tissue repair is characterized by remodeling processes, whereby the mucosa slowly reestablishes its normal morphology, structure and function (Mammen and Matthews 2003; Playford and Ghosh 2005; Blikslager et al. 2007). In the small intestine, v illus contraction could be considered a fourth event during intestinal repair, because it

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41 is thought to aid in restoration of barrier function before restitution occurs. Contraction of damaged villi is a result of activation of lamina propria smooth musc le cells and myofibroblasts within the villi, and reduces the surface area of denuded basement membrane to facilitate resealing by migrating epithelial cells (Blikslager et al. 2007). Clinic opathological Changes A fter Intestinal I / R Hematological and Biochemical Variables Necrosis has been regarded as a major component of intestinal I/R injury and is triggered by persistent hypoxia. As the cell membranes deteriorate, intracellular homeostasis fails, the cell s swell and cellular elements such as enzym es, proteins and electrolytes leak into the interstitium (Moore et al. 1995a; Rowe and White 2002; Festjens et al. 2006). In addition to the local inflammatory response, translocation of bacterial toxins and release of cell contents from damaged and necrotic cells during intestinal I/R can cause a prominent systemic inflammatory reaction ( Festjens et al. 2006; Rock and Kono 2008). This so called acute phase reaction is characterized by systemic activation of neutrophils and other leukocytes, and generation and release of a large number of pro inflammatory, regulatory and metabolic proteins and enzymes into circulation ( Moore et al. 1995c; Gruys et al. 2005; Vandenplas et al. 2005; Cray et al. 2009). Released cell contents and stimulation of a variety of tar get cells and organs cause a series of metabolic and biochemical alterations that can be measured in the blood including changes in leukocyte numbers, activation of the complement and coagulation system s, and changes in plasma electrolyte, protein and enzyme concentrations (Petersen et al. 2004; Gruys et al. 2005). Serological and hematological parameters are widely used to assess disease activities However the few markers that can be used to evaluate intestinal inflamm ation

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42 and damage are of low sensitivity and specificity (Parry 1987; Niels e n et al. 2000; Latson et al. 2005). Serum CRP, an acute phase protein and nonspecific inflammatory indicator is most commonly used (Takiguchi et al. 1990; Gruys et al. 2005; Desai e t al. 2007). Although increased plasma concentrations were found in horses with pneumonia, enteritis and arthritis, CRP does not reflect the severity of intestinal inflammation, including ulcerative colitis in humans ( Petersen et al. 2004; Vermeire et al. 2004). Many studies have been reported on hemostatic, hematological, biochemical and enzymatic alterations in systemic blood and peritoneal fluid after intestinal I/R in humans and other species (Parry 1987; Harrison 1988; Snyder et al. 1989a,b; Fried et al. 1991; Moore et al. 1994c; Caglayan et al. 2002; Feige et al. 2003; Saulez et al. 2004, 2005; Latson et al. 2005; Grosche et al. 2006; Johnston et al. 2007; Block et al. 2008; Delgado et al. 2009; Evennett et al. 2009). Changes in hydration, oxygenation, coagulation, fibrinolysis, and generation and release of metabolites and intracellular enzymes and proteins such as Llactate, D dimer and ALP have been used for diagnosis and prognosis of intestinal I/R in the horse (Parry et al. 1983; Gibson and Steel 1999; Monreal et al. 2000; Saulez et al. 2004, Latson et al. 2005, Delesalle et al. 2007; Johnston et al. 2007; Delgado et al. 2009; Cesarini et al. 2010; V an den Boom et al. 2010). Only few studies have focused on hematological and biochemical alterations in colonic vs. systemic venous blood in horses with I/R (Moore et al. 1994a; Kawcak et al. 1995; Moore et al. 19 95b,c, 1998a,b ). Moore et al. (1995b) demonstrated local alter ations of oxygenation, lactate and pyruvate concentrations in venous drainage from the equine colon, in the absence of a systemic effect Low glucose concentration and

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43 anion gap in colonic blood after I/R are thought to result from glucose utilization by t he colon as fuel that generates acid anions during low flow ischemia in the horse (Moore et al. 1998a ). Increased phosphorus and K+ in systemic and colonic blood are considered to be more likely a result of leakage from necrotic cells during colonic I/R. A dverse changes in l actate, oxygen saturation and PO2 in colonic venous blood were significantly correlated with the severity of mucosal damage after 3 hours of ischemia and 1hR (Kawcak et al. 1995). In recent years, more relevant variables have been measur ed to improve the characterization and prognostication of intestinal I/R injury (Derikx et al. 2010). Among these neutrophilic proinflammatory molecules and enzymes such as lactoferrin, neutrophil elastase, lysozyme, MPO and calprotectin which are known to correlate with the activation status of neutrophils, were used for evaluation of intestinal I/R and inflammation (Kurose et al. 1994; Martins et al. 1995; Grulke et al. 1999, 2008; Weiss and Evanson 2003; DInca et al. 2007; De La Rebiere De Pouyade et al. 2010). However none of them ha s been p roven to be a reliable measure of severity and prognosis for intestinal I/R. Calprotectin As a key component of the early inflammatory response, neutrophils play an important rol e in intestinal mucosal defense, but can also contribute to loss of mucosal integrity, cell death and tissue damage under certain pathological situations (Gayle et al. 2000, 2002; Nathan 2006). Neutrophils produce a large array of proinflammatory factors, enzymes, antibacterial proteins and other toxic molecules that are originally directed against invading microorganisms and infectious agents. One of these antibacterial proteins is calprotectin, a member of endogenous proinflammatory

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44 molecules of the innate immune system that signals ti ssue and cell damage, and has antimicrobial and antifungal functions ( Johne et al. 1997; Foell et al. 2007; Lotze et al. 2007). Calprotectin is a heterodimeric complex that is highly associated with inflammatory reactions (Foell et al. 2004). The complex belongs to the S100 superfamily of proteins and is also referred to as S100A8/A9, L1, or MRP 8/14. The 36kDa heterodimer consists of one light chain (S100A8; 10.8 kDa) and two heavy chains (S100A9; 13.2 kDa) Calprotectin is a calcium and zinc binding protein that is pri marily present in the cytoplasm and on plasma membranes of neutrophils, monocytes and macrophages (Johne et al. 1997; Kerkhoff et al. 1998). In neutrophils, calprotectin constitutes 5% of total proteins and approximately 60% of the cytoso lic protein fraction (Fagerhol et al. 1980a, 1990). Each neutrophil cell contains 5 to 25 pg calprotectin whereas in monocytes calprotectin accounts for approximately 1.6% of the total protein content (Fagerhol et al. 1980a; Berntzen et al. 1988; Fagerhol et al. 2005). Several research groups have stated the possibility of an extracellular secretion of calprotectin from stimulated neutrophils, but calprotectin is also released as a result of cell disruption or death (Dale et al. 1985; Hetland et al. 1998; Voganatsi et al. 2001). Although the exact biological role of calprotectin is still unknown, available evidence suggests calprotectin can modulate inflammatory reactions through activities that inhibit growth and induce apoptosis in fibroblasts or othe r cell types (Johne et al. 1997; Nisapakultorn et al. 2001 ; Yui et al. 2003). Furthermore, there is strong evidence that calprotectin is a potent stimulator of neutrophils, and it is involved in neutrophil migration to inflamed tissues when released at the site of cell damage (Roth et al. 2003 ; Ryckman et al. 2003; Vandal

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45 et al. 2003; Simard et al. 2010). I f present in an excess amount for a long period, calprotectin can cause local tissue destruction (Yui e t al. 2003). Calprotectin is one of the proin flammatory proteins that is used as a sensitive marker of acute and chronic inflammatory conditions such as systemic infections, pneumonia, rheumatoid arthritis, lamintitis, dermatitis, inflammatory bowel disease and intestinal I/R in human beings, horses and dogs (Eckert et al. 2004; Foell et al. 2004; Striz and Trebichavsky 2004; Little et al. 2005; Grosche et al. 2008; Faleiros et al. 2009). Because of its distribution in various cells, tissues and body fluids, calprotectin is emerging as a valuable marker for diagnosis, monitoring, and prognosis of gastrointestinal diseases, and it may serve as a marker for any diseases associated with increased neutrophil or monocyte/macrophage activity ( Johne et al. 1997; Poullis et al. 2003). Calprotectin is resistant to bacterial degradation in the gut and is stable in feces for up to 1 week at room temperature (Roseth et al. 1992). The half life of calpr otectin in human plasma is 5 hours (Fagerhol et al. 2005). It is readily quantified by ELISA or RIA, and over the last decade, improved assays have been developed to increase the specificity and sensitivity of its detection (Fagerhol et al. 1980b; Ivanov e t al. 1996; Ton et al 2000; Heilmann et al. 2008b). Normal values of 2 to 897 g/g feces have been determined in humans, with fecal calprotectin concentrations of up to 6850 g/g feces in patients with active inflammatory bowel disease (Konikoff and Denson 2006). The excretion of calprotectin in feces seems to be related to the flux of neutrophils and mononuclear cells into the intestinal wall, their turnover and their migration into the gut lumen as shown by a correlation between excreted labeled neutrophils and fecal

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46 calprotectin (Roseth et al. 1997, 1999; Tibble et al. 2000). Reference concentrations for human plasma calprotectin has been assessed at 0.1 to 0.9 g/L. U p to 40fold increased concentrations have been found in plasma in a variety of inflamm atory conditions which seems to reflect an increased leukocyte turnover or possible release of calprotectin by activation or cell death (Sander et al. 1984; Johne et al. 1997; Striz and Trebichavsky 2004). Serum calprotectin values of up to 11, 15 and 46 g/L were associated with systemic bacterial infections, after major surgeries or rheumatoid arthritis in humans, respectively ( Johne et al. 1997). Plasma and fecal calprotectin have been used routinely to assess disease activity and therapeutic progress or relapse in patients with inflammatory bowel disease (Aadland and Fagerhold 2002; Foell et al. 2004; Konikoff and Denson 2006; Sutherland et al. 2008). Plasma c alprotectin was also increased up to 5 days after abdominal surgeries in humans. This response was related to an increase of CRP and endotoxin following surgery, possible due to translocation of bacterial toxins from the gastrointestinal tract that triggered a post operative acute phase reaction and activation of monocytes and neutrophils (Berger et al. 1997). In horses, c alprotectin expression correlated significantly with neutrophil infiltration in colonic tissues, and calprotectin expression increased during colonic I/R in h orses (Grosche et al. 2008, 2011b; Matyjaszek et al. 2009). Recently, canine calprotectin has been purified and characterized, and a RIA has been developed for quantification of calprotectin in serum and feces from dogs with reference values of 92 to 1121 g/L serum and 29 to 1375 g/g feces (Heilmann et al. 2008a, b). Since there is no immunoassay available for horses an ELISA for

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47 quantification of equine calprotectin in blood and fecal samples is needed for this species, as a clinical marker for inflamm atory conditions in the gastrointestinal tract and for any systemic or local inflammation Despite major progress in understanding intestinal I/R in horses over the last decades, the pathophysiologic processes of equine colonic I/R are not fully elucidat ed. Because most of the horses with large intestinal strangulation obstructions are presented in later stages of the disease, where mucosal damage has already become irreversible, evaluation of epithelial changes at the beginning of hypoxia could be useful for understanding the pathomechanisms involved in disease progress. Thus precise information on morphological, ultrastructural, functional and clinic o pathological alterations i n the equine large colon, and the response of innate immune cells to short term I/R could provide more insi ght into disease mechanisms and complications. The current study includes the following objectives: To evaluate morphological and functional changes of the colonic mucosa caused by short term I/R on the basis of histo morphometric evaluation and determination of TER and transepithelial mannitol flux in horses. To illustrate ultrastructural changes of the colonic epithelium after short term I/R in horses on the basis of LM and TEM. To establish the association between the number of neutrophils and expression of calprotectin in colonic mucosa after I/R in horses. To determine the number and distribution of mucosal neutrophils, eosinophils, macrophages and mast cells, and expression of mucosal COX enzym es after short term I /R of the equine colon. To examine the potential role of oxidative stress on the basis of nitrotyrosine production by mucosal immune cells and to evaluate mucosal apoptosis in the equine colon after I/R. To compare serum calprotectin concentrations, and r outine hematological and biochemical measurements in systemic and colonic venous blood of horses after short term colonic I/R.

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48 The data presented here provide information on functional, morphological, and ultrastructural alterations of the colonic mucosa, systemic and local clinic o pathological changes, and the response of mucosal innate immune cells after I/R of the large colon in horses. Each of the objectives corresponds to one chapter of the dissertation. Each chapter is format ed as a standalone manuscript that has been published or is ready for submission (Chapter 2 : reprinted by permission from Graham et al., Am J. V et Res 2011, pages 982 989 ; Chapter 3: reprinted by permission from Grosche et al., Equine Vet. J. 2011, pages 8 15; Chapter 4: reprinted by permission from Grosche et al., Equine Vet. J. 2008, pages 393399 ; Chapter 5: reprinted by permission from Grosche et al., Equine Vet. J. 2011, pages 1625 ; Chapter 6: reprinted by permission from Grosche et al., Am J. V et Res 2011, in press ; Chapter 7: ready for submission).

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1Reprinted with permission from Grosche et al., Am. J. Vet Res. 2011, pages 982989. 49 CHAPTER 2 IN VITRO AND IN VIVO RESPONSES OF MUCOSA FROM THE LARGE COLON OF HORSES TO ISCHEMIA AND REPERFUSION1 Strangulating volvulus of the large colon is one of the most severe forms of colic and can account for 11 to 27% of horses requiring surgical correction of colic in referral hospitals (Harrison 1988; White 1990; Embertson et al. 1996). Survival after surgery for correction of large colon volvulus is dependent on the degree of ischemic injury to the colon and the severity of the systemic response. The survival rate without surgical resection has been reported to be as low as 34.7% (Harrison 1988) but resection improved the survival rate of affected horses in two studies ( 57.7 % Dri scoll et al. 2008 ; 74% Ellis et al 2008, respectively) In most horses that undergo colon resection, some ischemic tissue remains Thus, survival of these horses can be correlated with loss of the epithelial barrier in the remnant portion, which allows transmucosal leakage of endotoxins, bacterial chemotactic peptides, and bacteria (Snyder 1989b ) Therefore, r apid repair of the ischemic injured epithelium is cruci al to recovery of a horse after large colon volvulus but could be impaired by reperfusion injury (Granger et al. 1986; Moore et al. 1994a 1995a ; Blikslager et al. 1997 a ; McMicheal and Moore 2004; Grosche et al. 2008) The biochemical pathway responsible f or reperfusion injury starts with accumulation of products tha t build up during ischemia and ROS generated on reperfusion (Granger et al. 1986; Moore et al. 1995a ; Blikslager et al. 1997a ; Grosche et al. 2008) Neutrophil infiltration is a time dependent process that occurs mostly during the reperfusion period (Moore et al. 1994 a ; Grosche et al. 2008) and activation of these

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50 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. cells results in degranulation and additional release of inflammatory mediators, such as cytokines, ROS, proteases and other regulatory proteins (McMicheal and Moore 2004) Although ischemia is a major pathophysiological feature of large colon volvulus, exacerbation of the ischemic injury during reperfusion has been detected during experimentally induced low flow ischemia in equine colon (Moore et al. 1995a ) However, the importance of reperfusion injury in the equine colon remains controversial (Blikslager 2006) Studies on reperfusion injury primarily have been directed at de tecting an exacerbation of ischemic damage during reperfusion, with or without protection against that damage via various interventions. A novel OPS used in human kidney transplant patients (P olyak et al. 2000; Guerrera et al. 2004; Agarwal et al. 2005) was able to preserve viability in an isolated segment of equine colon in the absence of oxygen and blood (Polyak et al. 2008) The solution represents a multimodal approach to treating reperfusi on injury but is expensive. However, three of its components are inexpensive and warrant study separately as treatments for reperfusion injury. The amino acid Larginine is an indirect precursor of NO (Polyak et al 2008) and therefore has the potential to hasten tissue healing and modulate the inflammatory response ( Coeffier and Dechelotte 2010) The amino acid L glutamine can influence several metabolic pathways during intestinal stress (Kojima et al 1998) and was protective in an in vitro evaluation of chemical damage to equine colonic mucosa (Rtting et al. 2004) Metabolism of arginine and glutamine is closely related, and these amino acids could result in an additive effect on several biochemical pathways (Coeffier and Dechelotte 2010) Acetylcysteine can protect mucosa through its antioxidant properties (Aruoma et

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51 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. al. 1989; Cotgreave 1997) and by providing sulfhydryl groups required for replenishment of glutathione, which is an important intracellular antioxidant (Cotgreave 1997) In equine colonic mucosa, acetylcysteine can prevent mucosal damage and reduce eosinophil migration after chemical damage in vitro (Rtting et al. 2003) Our hypothesis was that experimentally induced ischemia of short durati on would cause mild to moderate reversible injury t o the equine colonic mucosa and that this injury would be exacerbated during reperfusion. The purpose of the study reported here was to evaluate mucosal damage of the equine colon caused by I/R as determined on the basis of histomorphometric changes; to d etermine TER and permeability to mannitol in tissues subjected to I/R compared with results of control tissues and tissues subjected to ischemia only; and to evaluat e in vitro effects of components of an OPS on functional and morphological measurements of mucosal recovery. Materials and Methods Horses Six healthy adult horses (3 Thoroughbreds, 2 Quarter horses, and 1 warmblood) were included in the study. Horses were 11 to 26 years old (mean 16 years) and weigh ed 440 to 660 kg (mean, 548 kg). Inclusion criteria were that the horses were in good health, were free of gastrointestinal tract disease, and required euthanasia for conditions that rendered them unsuitable for use. Horses were housed on pasture with grass hay and water provided ad libitum and did not receive any medications for the 2week period preceding the study. The Institutional Animal Care and Use Committee of the University of Florida approved the study protocol.

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52 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Surgical P rocedure Horses were sedated with xylazine (1 mg/kg, IV) A 14gauge, 13.3cm Teflon catheter was inserted into each of the jugular veins ; the left jugular vein was used for administration of anesthetic drugs and isotonic fluids and the right jugular vein was used for collection of blood samples. Anesthesia was induced with ketamine (2.2 mg/kg, IV) and di azepam (0.1 mg/kg, IV). Orotracheal intubation was performed and horses were positioned in dorsal recumbency A nesthesia was maintained with isoflurane (1 to 3%) in oxygen via mechanical ventilation. Isotonic polyionic fluids were continuously infused IV at a rate of 2.5 to 5 m L /kg/h. Mean arterial blood pressure was monitored through a 20gauge, 5.1cm Teflon catheter in the facial artery and was maintained at or 60 mm Hg. M onitoring performed dur ing anesthesia included electrocardiography, arterial blood gas analysis, capnography and direct blood pressure measurement The ventral abdomen of each horse was prepared and draped for aseptic surgery and ventral midline celiotomy was performed. The large colon was exteriorized and positioned on a sterile drape on the ventral abdomen. To induce ischemia, a 40cm segment of colon at the pelvic flexure was subjected to transmural compression via intestin al clamps placed at each end, and venous and arterial occlusion was achieved with umbilical tape ligatures. After induction of ischemia, the colon, colonic vasculature and associated mesentery were surgically divided at the pelvic flexure so that two segme nts of colon comparable in size (dorsal and ventral) did not communicate. The colon was then replaced in the abdomen, and the abdominal incision was closed temporarily with towel clamps. After 1hI the colon was again exteriorized and one of the two ischem ic segments (ischemic tissues) was resected for histological evaluations and in vitro experiments in Ussing chambers Ischemic tissues were no t harvested for

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53 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Ussing chamber experiments from the first 2 horses; thus, tissues from only 4 horses were used for this phase of the study. At the same time, the clamps and ligatures were removed from the other segment of ischemic colon; this segment was replaced in the abdomen to allow resumption of blood flow (reperfusion) for 4 hours (horses remained anesthetized d uring reperfusion) Small (1 to 2 cm2) mucosal biops y specimens were collected before ischemia, after 1hI (time 0 for ischemic tissues) and after 1hR, 2hR and 4hR After 4hR the reperfused segment of colon (reperfused tissues) and an adjacent noninjured segment of colon (control tissues) were removed (time 0 for control and reperfused tissues) for histological evaluation and in vitro experiments in Ussing chambers. After control and reperfused tissues were harvested, the anesthetized horses were euthaniz ed with an overdose of sodium pentobarbital (88 mg/kg, IV). Ussing Chamber Experiments A solution of KRB (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 [pH, 7.4]) was prepared After removal, full thickness tissue sections of ischemic, reperfused and control colon were immediately placed in cold (4C) KRB solution and transported to our laboratory (Freeman et al. 1989) The timing of sample collection and mounting of tissues in Ussing chambers for the in vitro experiments was such that experiments were completed on the ischemic tissues before the same experiments were performed on control and reperfused tissues. Mucosal sheets from each colon segment (control, ischemic, and reperfused tissues) were removed and mounted in Ussing chambers as described elsewhere (Rtting et al. 2008; Matyjaszek et al. 2009) Three treatment solutions were used to incubate the mucosal tissues: KRB alone; KRB with 70 mg of NAC (Sigma Chemical Co., St. Louis, MO

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54 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. USA )/L [ KRB NAC treatment ] ) ; and KRB with 5 mg of L arg i nine (Sigma Chemical Co., St. Louis, MO, USA ) /L, 70 mg NAC/L and 10 mg of L glutamine ( Sigma Chemical Co., St. Louis, MO, USA )/L (KRB MOTS treatment ). Each tissue incubation was examined in duplicate. The short circuit current was recorded in each chamber by use of voltage clamps th r ough silver silver chloride electrodes connected to 4% agar bridges in KRB solution. Junction potentials of electrodes and fluid resist ance were measured before mounting of the tissues to allow continuous correction for any effects that these factors might have on the low potential difference generated by the tissues (Schultz and Zalusky 1964; Kotyk and Janacek 1975) When the tissues wer e mounted in the chambers, the voltage clamp could then automatically correct for junction potentials of electrodes and fluid resistance and reduce t heir effect on the recordings. Throughout incubation, the short circuit current was continuously applied to the tissues except for a brief period at 15minute intervals when the potential difference of the tissue was measured. The TER was calculated by use of Ohms law, whereby resistance is equal to the potential difference of the tissues divided by the short circuit current. Resistance was used as a measure of integrity of colonic mucosa and permeability of the paracellular pathway to ions (Freeman et al. 1989; Rtting et al. 2008; Matyjaszek et al. 2009) The unidirectional flux of tritium labeled mannitol (New England Nuclear Corp. Boston, MA USA ) from the mucosal to the serosal solution was an additional measure of colonic mucosa permeability (Richter et al. 2002; Rtting et al. 2008; Matyjaszek et al. 2009) For scintillation counting, fluid samples were collected from both sides (mucosal and serosal) at 45, 75, 105 and 240 minutes after addition of radiolabeled

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55 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. mannitol to the mucosal side. Transmucosal flux of mannitol was expressed as the percentage of th e initial scintillation counts detected on the mucosal side of the tissue that was detected on the serosal side of the tissue at the 4 time points. The interval from tissue collection to mounting the tissue in the first chamber was approximately 10 minutes and to first recording of TER and addition of tritium labeled mannitol was approximately 40 minutes. Total incubation time for each tissue in the Ussing chamber was sufficient to record TER and mucosal flux for 240 minutes. After Ussing chamber experiment s were completed, the tissues were removed and placed in 10% neutral buffered formalin for histologic evaluation and histomorphometric measurements. Histological Evaluation and Morphometric Measurements B iopsy specimens of colonic mucosa obtained in vivo b efore induction of ischemia (time 0), after 1hI 1hR, 2hR and 4hR and in vitro at the end of U ssing chamber experiments were fixed in 10% neutral buffered formalin Tissues were embedded i n paraffin, and cut into 5 m thick sections, placed on silane coat ed glass slides and stained with H&E for examination via LM For histomorphometric assessment of mucosal damage via LM, a computer b ased imaging analysis program (Image Pro Express Version 5.0; Media Cybernetics Inc Bethesda, MD USA ) was used and three fields from each tissue w ere examined as described elsewhere ( Rtting et al. 2003; Grosche et al. 2008; Matyjaszek et al. 2009) One investigator (AG), who was unaware of the treatment group for each biopsy specimen, performed all histologic evaluati ons (Rtting et al. 2003; Matyjaszek et al. 2009) The length of denuded epithelium was measured and expressed as a percentage of the total mucosal length in the section. The epithelium was defined as lifted when > 5 epithelial cells were separated from the basement membrane but still attached to

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56 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. adjacent cells. The length of epithelium affected by lifting was expressed as a percentage of the total surface length. Detached cells were defined as cells that appeared morphologically normal but were separated f rom the basement membrane in groups of 5 cells and completely detached from adjacent epithelium. The length of detached epithelium was measured and expressed as a percentage of the total surface length of the mucosa. Restituted cells were defined as cell s that were flattened in appearance but had intact attachments to the basement membrane and to adjacent cells. The length of epithelium consisting of restituted epithelial cells was measured and expressed as a percentage of the total surface length of the mucosa. The number of sloughed cells was counted for each field; the mean number of sloughed cells / 0.1 mm of surface length was calculated. The sloughed cells were not counted in tissue samples incubated in the Ussing chambers because the re were no sloughed cells present or the sloughed cells were of undetermined origin. Statistical Analysis The tissues obtained from each horse yielded two sets of observations for each experimental condition in the Ussing chambers. The mean of data w as calculated and ex pressed as least squares mean SEM and a statistical software program (SAS OnlineDoc, V ersion 8.0 ; SAS Institute Inc Cary, NC USA ) was used for analysis. Repeatedmeasures ANOVA was performed on the TER for each of the 3 tissue groups. Whenever there was a significant result of an F test for treatment, time, or the treatment by time interaction, appropriate Bonferroni adjusted P values w ere used for each family of comparisons. To determine the effect of ischemia and reperfusion on tritium labeled mann itol flux after incubation in the Ussing chambers fo r 240 minutes a oneway ANOVA was performed. Statistical analysis of histologic data was performed

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57 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. by use of a Kruskal Wallis test to determine significant differences in histomorphometric measurements b etween the treatment groups. Post hoc analysis was performed by us e of the Mann Whitney U test. V alue s of P < 0.05 w ere considered significant for all statistical analyses. Results Ussing Chamber E xperiments The TER was not significantly changed by the Ussing chamber treatments (KRB KRB MOTS and KRB NAC) within each of the three tissue groups (control tissues [ n = 6 horses], ischemic tissues [ 4 ], and reperfused tissues [ 6 ] ) The TER of the control tissues decreased significantly with time during the in cubation period whereas there was no effect of time on the ischemic or the reperfused tissues. The TER in each control tissue was significantly greater than the TER in each ischemic tissue from 15 minutes until 120 minutes of incubation and from 180 minut es to 225 minutes of incubation ( Figure 21 ). The TER of each reperfused tissue was significantly greater than the TER of each ischemic tissue at 90, 120, and 180 to 240 minutes of incubation in the Ussing chambers. Ussing chamber treatments did not affect the transmucosal flux of mannitol for control, ischemic and reperfused tissues. A fter incubation for 240 minutes the transmucosal flux of tritium labeled mannitol was significantly higher for ischemic tissues incubated in KRB than for reperfused tissues incubated in KRB ( Fig ure 22). Morphological Examinations One hour of ischemia caused edema, purple discoloration of the serosa, and serosal petechiation. By 10 to 15 minutes after the ischemiainducing clamps and

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58 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. ligatures were removed, the appearance of the reperfused segment was similar to that of the adjacent control segment. Histomorphometric Examination of In Vivo Tissue Samples Histologic changes in the in vivo mucosal samples obtained after 1hI were suggestive of cell injury, and lifting of the surf ace epithelial cells confirmed mucosal disruption (Fig ure 23). Mucosa collected after 4hR had evidence of an intact epithelial lining. Significant differences were observed in the mucosa of control tissues and ischemic tissues with respect to mucosal heig ht (P < 0.00 1 ), epithelial height (P < 0.00 1 ), epithelial width (P < 0.001), lifted epithelium (P = 0.006), degenerated epithelium (P = 0.001), and the number of necroti c or sloughed cells (P < 0.00 1 ). There was no significant difference in the amount of d enuded epithelium or the amount of restituted epithelium (Table 2 1 ). There were significant differences between ischemic and reperfused mucosae with respect to mucosal height (P = 0.002), lifted epithelium (P = 0.023), degenerated epithelium (P = 0.006) a nd restituted epithelium (P < 0.001 ). The control and reperfused mucosa differed in that reperfused epithelial cells were wider and flatter than were control epithelial cells, which suggest ed mucosal restitution during reperfusion rather than exacerbation of ischemic injury. Histomorphometric Examinations of Ussing C hamber Tissues H istologic changes observed in the mucosa after incubation in Ussing chambers for 240 minutes were similar to those observed for the in vivo samples (Table 2 2 ). Compared with the results for the control and reperfused tissues, ischemic tissues had a significantly decreased mucosal height (P = 0.002), epithelial height (P < 0.00 1 and P = 0.001 for the control and reperfused tissues, respectively ) and epithelial wi dth (P = 0.001) There was also a significant (P < 0.001) increase in percentage of denuded

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59 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. epithelium for the ischemic tissues compared with the percentage for the c ontrol and reperfused tissues. Compared with results for the control tissues, ischemic ti ssues had a significantly greater percentage of denuded epithelium (P < 0.001 ) and greater epithelial restitution (P = 0. 003). The ischemic tissues also had a greater percentage of epithelial restitution than the reperfused tissues ; however, these values did not differ significantly (P = 0.05). Results for the reperfused tissues were not significantly different from results for the control tissues with respect to any of the variables examined. There was no significant difference in the percentage of lifted epithelium or degenerated epithelium among any of the groups. There was no significant effect of Ussing chamber treatments (KRB, KRB NAC, or KRB MOTS) on the histomorphometric measurements ; therefore, all statistical analyses were performed for the tissues incubated in KRB. When the in vivo mucosal biops y specimens were compared with the in vitro biops y specimens the most notable difference was evidence of epithelial cell restitution in the ischemic tissues after incubation in the Ussing chambers fo r 240 minutes (Fig ure 23 ). Predictably, all tissues had reduced intensity of staining in the lamina propria after incubation in the Ussing chambers for 240 minutes Discussion In present study of experimentally induced ischemia in the equine large colon, reperfusion did not exacerbate the ischemic damage but appeared to allow mucosal recovery, as determined on the basis of histologic and functional measurements of tissue integrity. The ischemia experimentally induced in the colon of horses in the study rep orted here caused mild injury that should have allowed some expression of reperfusion injury over the next 4 hours (Matyjaszek et al. 2009) A more severe ischemic injury m ay not have allowed a sufficient number of viable cells to survive and

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60 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. cause an obvi ous reperfusion injury. However, further studies are required to determine whether 1 hour is an optimum ischemic period to allow generation of ROS and the associated tissue damage in equine colonic mucosa In the original experimental design, responses of interest were to be examined in two separate groups of horses that would be assigned by use of a randomization procedure to an ischemia group and I/R group. However, it was evident after the procedure was performed on the first 2 horses in the I/R group that the experimental procedures allowed sufficient time for investigators to mount the ischemic tissues in the Ussing chambers incubate the tissues, and then empty and rinse the chambers in time for the reperfused tissues to be mounted. In this manner each horse could be used to provide tissues subjected to ischemia and to reperfusion, and the difference bet ween the two tissues was sufficient such that only 4 sets of ischemic tissue were required to yield statistical significance. When the function of t he mucosa was examined in Ussing chambers, the ischemic tissues performed significantly worse than did the control and reperfused tissues, as measured on the basis of TER. There was no significant difference between the TER for the control and reperfused t issues. Similarly the reperfused tissues were less permeable to mannitol than were the ischemic tissues, which indicat ed some restoration of barrier function in the reperfused tissues. Histomorphometric examination also revealed restitution in reperfused tissues with almost complete mucosal recovery after 4 hours in vivo. This early recovery of mucosal epithelium was mainly attributable to migration of surviving epithelial cells over the

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61 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. damaged surface and not by cell proliferation, which would require a considerably longer observation period in vivo ( Park and Haglund 1992) C omplete arteriovenous occlusion for 1 hour was used in the present study to closely mimic large colon volvulus in the horses (Matyjaszek et al. 2009) A period of 4hR was chosen as a reasonable amount of time in which observations could be made wi th horses remaining anesthetized In other studies (Tomlinson et al. 2004; Little et al. 2007a ; Matyjaszek et al. 2009) in which investigators used similar ischemic m odels for the equine colon and jejunum a reperfusion period of 18 hours had been used, which necessitated recovery of the horses from anesthesia. Because mucosal restitution and tightening of paracellul ar pathways are critical for recovery, the time period chosen for examination of the effects of reperfusion should be appropriate to evaluate these aspects of early repair. The findings in the study reported here are also consistent with findings in another study (Matyjaszek et al. 2009) conducted by our research group in which we found that a more severe mucosal injury followed by reperfusion for 18 hours also allowed mucosal recovery, although to a lesser extent than in the present study. Although only 4 horses were used in the ischemic group in the present study, changes were identical to those reported for this duration of ischemia in tissues of the pelvic flexure of horses (Rtting et al. 2008; Morton et al. 2009) S elected components (NAC and MOTS) of a novel OPS solution (Polyak et al. 2008) were added to Ussing chambers to determine whether these components could improve recovery in vitro (Rtting et al. 2003; Rtting et al. 2004) These components were chosen rather than the OPS solution described elsewhere (Polyak et al. 2008) because, if effective, they could be administered systemically in the clinical setting at

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62 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. low cost. Some benefit has been proposed for combining arginine and glutamine ( Kojima et al. 1998) and inclusion of acetylcysteine is warranted on the basis of salutary effects in horses (Rtting et al. 2003) and rat s with chemically induced mucosal injury (Ancha et al. 2009) There was no evidence of any improvement in recovery from mucosal injury in vitro fo r the conditions of the present study, although the changes induced in this reversible ischemia model would have seemed appropriate to test such an effect. The MOTS components were applied directly to the mucosal and serosal surfaces to improve access to epithelial and inflammatory cells in the mucosa. This design could only be expected to detect an effect on epithelial cells and on neutrophils already present in the tissues in vitro, without an opportunity to evaluate any putative antioxidant effect on neutrophil infiltration. There fore, further investigation into the potential clinical use of these components on the inflammatory response in reperfused mucosa might be better conducted in vivo. The rapid recovery of barrier function during reperfusion, as measured on the basis of TER, can be attributed to increased expression of TJ proteins (occludin and ZO 1) during mucosal recovery (Blikslager et al. 2007) Application of PG E2 to ischemia injured ileal mucosa stimulates increases in TER initiated through chloride secretion in restituting epithelium (Moeser et al. 2004) which suggest s that COX enzymes may play an important role in intestinal protection and recovery. The marked upregulation of COX 2 (Morton et al. 2009) expression in the epithelial and crypt cells of the equine colon after ischemia may induce the production of PG E2 and other prostanoids during reperfusion, which could enhance epithelial recovery and restitution of barrier function (Blikslager et al. 1997b )

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63 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Anesthetic preconditioning via administration of volatile anesthetics, such as isoflurane before ischemia can diminish the severity of I/R injury in the brain (Zhao et al. 2007; Li and Zuo 2009) heart (Tanaka et al. 2004) kidney s (Kim et al. 2007) l ungs (Liu et al. 1999) and l iver (Beck Schimmer et al. 2008) in human s and other animals. To our knowledge, the use of preconditioning with volatile anesthetics has not been found to reduce I/R injury in the intestines (Annecke et al. 2007) In the present study, isoflurane was used to anesthetize the horses for I/R periods and m ay have reduced the severity of the ischemic injury or attenuated the degree of reperfusion injury through its anti inflammatory effects. However, the clinical relevance of such pr econditioning m ay be questionable because large colon volvulus is typically corrected in horses that are anesthetized by use of an anesthetic regimen similar to that used in the present study. Therefore, all horses undergoing surgery for large colon volvulus will have some anesthetic preconditioning before reperfusion. The TER in reperfused tissues in the present study was sustained throughout the in vitro phase at values close to the maximum values for t he control tissues whereas T ER values in control tissues decreased toward the end of incubation (Fig ure 21 ). Tissues subjected to 1hI had lower TERs in the study reported here and in another study (Rtting et al. 2008) conducted by our research group, wh ich is in marked contrast to responses of equine jejunum to reversible ischemic injury (Little et al. 2007a ; Morton et al. 2009) Ischemic equine jejunum has significantly higher values throughout incubation in vitro than does the control jejunum (Little e t al. 2007a ) These findings can be explained by populations of TJ throughout the jejunum that are more permeable in control conditions but that become recruited into the intense and widespread process of

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64 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. TJ closure after injury (Tomlinson et al. 2004) Th e difference in expression of this process between jejunum and colon could be explained by the importance of a permeable paracellular pathway for nutrient absorption in jejunal epithelium (Turner 2009) This amount of permeability could be lacking in the c olon, which has a limited capacity to absorb nutrients (Freeman et al. 1989) and a greater need to have a tight epithelial barrier against noxious luminal contents (Turner 2009) The lack of reperfusion injury in the present study is consistent with results of other studies (Reeves et al. 1990) in horses and raises concerns about the relevance of this process in equine intestine s (Blikslager et al. 1997a ) However, the results reported for a low flow m odel of ischemia in the equine colon ( Moore et al. 1994a, b) revealed a marked influx of neutrophils associated with continued mucosal degeneration during reperfusion. In clinical ly affected horses with large colon volvulus, the relative roles of low flow ischemia and complete isc hemia are unknown, so the most suitable m ethod for investigation of this disease is not been established. However, the nature of the lesions would suggest that almost complete cessation of blood flow, as was induced in the present study, should be the most likely vascular change (Provost et al. 1991) In equine colonic mucosa subjected to periods of ischemia similar to that used in the present study, but with shorter and longer intervals of reperfusion, rapid and intense neutrophil influx was evident throug hout reperfusion (Grosche et al. 2008) Although neutrophil influx was not measured in the present study, marked increases in neutrophil numbers were observed after 4hR a time point at wh ich tissues had recovered from ischemic damage. For most m odels of intestinal reperfusion injury, influx of neutrophils of such magnitude would be expected to exacerbate the ischemic injury (Moore et al.

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65 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. 1994a, b). The role of neutrophils in reperfusion injury is further complicated by evidence that reperfusioninduced mucosal damage in equine jejunum can be independent of neutrophils (Van Hoogmoed et al. 2001) We concluded for the experimental methods of the study reported here that complete ischemia of equine colonic mucosa for 1 hour followed by reperfusion for 4 hours did not result in functional or morphological evidence of reperfusion injury. Therefore, in horses that undergo surgery for large colon volvulus, efforts to hasten mucosal recovery probably should have precedence over aggressive efforts to treat reperf usion injury

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66 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Table 21. Mean SEM h istomorphometric values for colonic mucosa obtained from anesthetized horses after 1hI ( ischemic tissue [ n = 4 horses]) or after 1hI + 4hR (reperfused tissue [ 6 ] ) or from adjacent control segments before I/R (control tissue [6]). Variable Control Ischemic Reperfused 470.0 12.41a,c 328.48 16.97a,b 426.08 8.29b,c 36.13 1.66a,c 25.643 1.11a 26.21 1.28c 3.72 0.10a,c 4.87 0.11a,b 4.62 0.27b,c Lifted epithelium (%) 0 0a 2.58 0.77a,c 0 0c Denuded epithelium (%) 0 0 0.97 0.47 0 0 Degenerated epithelium (%) 0.56 0.22a 8.78 2.44a,c 0.96 0.45c Restituted epithelium (%) 0 0c 0 0c 10.30 5.23c Necrotic/ sloughed cells/0.1 mm 0.11 0.03a,c 2.97 0.96a 2.20 1.15c Note: a c Within a row, values with different superscript letters differ significantly (P < 0.05)

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67 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Table 22. Mean SEM h istomorphometric values for control (n = 4 horses) ischemic (6) and reperfused (6) colonic tissues after incubation with KRB, KRB MOTS or KRB NAC in Ussing chambers for 240 minutes Variable Control Ischemic Reperfused KRB MOTS NAC KRB MOTS NAC KRB MOTS NAC Mucosal height 401.59 17.34 390.15 16.74 406.61 22.87 260.87 18.79 297.39 19.47 269.96 39.72 375.22 21.65 377.08 32.04 352.42 27.79 Epithelial height 27.23 1.72 25.75 0.94 27.47 1.26 10.49 2.54 14.25 2.91 9.32 1.68 24.65 1.57 23.30 1.37 24.43 0.54 Epithelial width 4.84 0.20 4.86 0.13 5.02 0.22 7.21 0.61 7.40 0.85 7.88 0.68 4.87 0.21 5.02 0.15 4.95 0.23 Denuded epithelium (%) 0.02 0.02 0.05 0.05 0.13 0.13 71.91 7.10 32.97 11.06 67.28 21.13 1.05 0.88 0 0 0 0 Lifted epithelium (%) 0 0 0 0 0 0 4.88 4.88 4.48 4.48 0 0 0.13 0.13 0 0 0 0 Degenerated epithelium (%) 4.41 1.02 14.29 7.11 11.57 1.78 8.02 2.86 8.93 1.33 2.43 1.69 6.55 2.46 10.23 2.37 8.81 2.09 Restituted epithelium (%) 0 0 0 0 0 0 11.30 5.42 21.48 9.81 14.94 6.74 1.90 1.58 0.19 0.19 0.36 0.36 Note: *Values differ significantly (P < 0.05) between all control and all ischemic tissues. Values differ significantly (P < 0.05) between all ischemic and all reperfused tissues

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68 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Figure 21. Mean SEM values for TER of colonic mucosa obtained from anesthetized horses after 1hI (ischemic tissue [ solid line s and black symbols] ) or after 1hI + 4hR (reperfused tissue [ dashed lines and black symbols]) or from adjacent control segments after I/R (control tissue [ solid line s and white symbols] ). Each time point represents the mean value for samples (evaluated in duplicate) obtained 6 horses (control and reperfused tissues) or 4 horses (ischemic tissue) Time 0 = end of 1hI (ischemic tissues) or end of 4hR (control and reperfused tissues) Immediately after collection, tissues were mounted in Ussing chambers and incubated with KRB (circles), KRB MOTS (KRB MOTS treatment [squares]), or KRB NAC (KRB NAC treatment [triangles]) for 240 minutes Values did not differ significant ly (P < 0.05) among treatments at any time within the control, ischemic or reperfused tissue groups. Values for all reperfused tissues differed significantly (P < 0.05) from values for all ischemic tissues except when comparing reperfused and ischemic tissues incubated in KRB MOTS *Wi thin a time point, values for all control tissues (KRB, KRB MOTS, and KRBNAC) differed significantly (P < 0.05) from values for all ischemic tissues Within time point, values for all reperfused tissues (KRB, KRB MOTS, and KRB NAC) differed significantly (P < 0.05) from values for all ischemic tissues. Within time point, values for all control tissues differed significantly (P < 0.05) from values for all ischemic tissues, except when comparing control tissues incubated in KRB or KRB MOTS with values from ischemic tissues incubated in KRB MOTS.

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69 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Figure 22. Mean SEM values for permeability of equine colonic mucosa to tritium labeled mannitol for control ischemic and reperfused tissues after the tissues were incubated in an Ussing chamber for 240 minutes Tissues were incubated in KRB (g rey bars ), KRB MOTS (white bars), or KRB NAC ( black bars ). Transmucosal flux of mannitol was expressed as the percentage of the initial scintillation counts detected on the mucosal side of the tissue that were detected on the serosal side of the tissue at 4 time points (ie, 45, 75, 105, and 240 minutes after addition of radiolabeled mannitol to the mucosal side). Value differ significantly (P<0.05) from the value for reperfused tissues incubated in KRB.

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70 Reprinted with permission from Am. J. Vet. Res. 2011, Pages 982989. Figure 23. P hotomicrographs of representative sections of colonic mucosa obtained at various time points from horses with ex perimentally induced ischemia. A ) Mucosa f rom a control section of colon before adjacent tissues were subjected to 1hI and were rep erfused for 4 hours. B ) Mucosa from a control section of colon after incubation in an Ussing chamber for 240 minutes C ) Mucosa obtained immedi ately after 1hI Notice the decrease in mucosal height, compared with the mucosal height of the control section i n panel A Notice also the loss of epithelium and areas of lifting of epithelial cells and den uded epithelium D ) Mucosa obtained after 1hI and incubation in an Ussing chamber for 240 minutes Notice the evidence of mucosal restitution in the epithelial ce lls that are flattened but attached to adjacent cells and to the basement membrane. E ) Mucosa obtained after 1hI + 4hR Notice the greater mucosal height compared with that for the ischemic mucosa in panel C F ) Mucosa obtained after 1hI and 4hR followed by inc ubation in an Ussing chamber for 240 minutes Notice the intact epithelial barrier (no denuded epithelium is evident).

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1Reprinted by permission from Grosche et al., Equine Vet. J. 2011, pages 815. CHAPTER 3 ULTRASTRUCTURAL CHANGES IN THE EQUINE COLONIC MUCOSA AFTER ISCHEMIA AND REPERFUSION1 Strangulation obstruction of the large colon is the most devastating form of colic in horses. Rapid ischemic degeneration of the colonic epithelium facilitates translocation of bacterial toxins through the damaged epithelial barrier resulting in endotoxic shock and possible death (Snyder et al. 1989 a ; Gibson and Steel 1999). Although prompt restoration of blood flow by surgical correction of the volvulus is essential to prevent irreparable damage, reperfusion can exacerbate epithelial damage (Meschter et al 1991; Moore et al. 1995 a ). However, the importance of colonic I/R injury in horses is not fully understood (Rowe and White 2002). Intestinal I/R injury can be identifie d early by increase in capillary and epithelial permeability (Snyder et al. 1992; Darien et al. 1995). First histological signs of colonic mucosal damage are characterized by lifting of small clusters of epithelial cells, their detachment from the basement membrane and subsequently death by apoptosis or necrosis ( Meschter et al. 1991; Snyder et al. 1992). Although the colonic epithelium is completely denuded after 4 h ours of low flow ischemia, intracellular degenerative processes and abnormalities of the cell structure are apparent long before epithelial detachment occurs (Snyder et al. 1992). One hour of experimentally induced ischemia in the equine colon caused minor mor phological alterations but severe epithelial barrier failure, characterized by decreased TER (Graham et al. 2011). However, this measurement of barrier integrity returned to normal after 4hR without any apparent morphological explanation based on LM (Grah am et al. 2011). Possible degenerative processes and ultrastructural abnormalities of the epithelial barrier not seen by routine

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72 LM might be responsible for loss of the barrier function. The few published studies on colonic I/R injury in horses at the cell ular level (Meschter et al. 1991; Wilson et al. 1994; Darien et al. 1995; Dabareiner et al. 2001) have not demonstrated early ultrastructural changes of the colonic epithelium in horses that could affect its barrier function during I/R (Graham et al. 2011) The purpose of the present study was to illustrate alterations of the equine colonic epithelium after 1hI and during 4hR and to describe ultrastructural abnormalities demonstrated by TEM and morphological changes on semithin sections evaluated by LM. Th e hypothesis was that early ischemic injury results in distinct but reversible ultrastructural alterations of epithelial cells that play a role in barrier dysfunction and recovery. Material and Methods Animals Six horses used in this study were of mixed breeds with a mean age of 16 years and a mean bodyweight of 548 kg. They were donated for research purposes and they were free of gastrointestinal diseases determined by physical, clinic o pathological (white blood cell count and differentiation, TP and albumin) and fecal examinations. The study was performed with approval and under guidelines of the Institutional Animal Care and Use Committee of the University of Florida. Horses were fed grass hay (2% of their bodyweight per day), and water was provided ad l ibitum. Horses were adapted to their diet and environment for at least 1 week before the study. Experimental Procedures A 14 gauge, 13.3cm Teflon catheter was inserted into the left jugular vein for administration of anesthetic drugs and isotonic fluids. Horses were placed under

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73 general anesthesia according to the following protocol: xylazine (1.0 mg/kg, IV) to provide sedation, and then general anesthesia was induced with diazepam (0.1 mg/kg, IV) to effect followed by ketamine (2.2 mg/kg, IV) as a bolus injection. General anesthesia was maintained with isoflurane (1 to 3%) in 100% oxygen. Horses were mechanically ventilated at 6 breaths/min. Isotonic polyionic fluids were infused IV continuously at 2.5 to 5 m L /kg/ h. Mean arterial blood pressure was monitored through a 20gauge, 5.1cmTeflon catheter in the facial artery, and was maintained at or above 60 mmHg. Other monitoring tools used during anesthesia included electrocardiography, blood gas analysis, capnography and pulse oximetry. Horses were positioned in dorsal recumbency and prepared for an aseptic ventral midline celiotomy. The large colon was exteriorized and placed on a plastic drape on t he ventral abdomen. To induce ischemia, a 40cm segment of colon at the pelvic flexure was subjected to transmural compression by intestinal clamps at each end of the selected segment, and combined venous and arterial occlusion was achieved with umbilical tape ligatures. After induction of ischemia, the colon, colonic vasculature and associated mesentery were surgically divided at the pelvic flexure so that two 20cm segments of colon (dorsal and ventral) did not communicate. To accomplish this, the colon w as transected and sutured at each end over an intestinal clamp in Parker Kerr fashion with 20 polydioxanone, and this layer was oversewn in a continuous Cushing fashion to completely close the transected bowel. The blind ends so created were lavaged with warm sterile saline and placed in the abdomen during periods between biopsies. After the colon was replaced in the abdomen, the abdominal incision was closed temporarily with towel clamps. After 1hI the colon was reexteriorized and 1 of

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74 the two 20cm ische mic segments and a 5 to 7 cm of control (nonischemic) colon were resected for histological evaluations and in vitro experiments, alternating ventral and dorsal segments between horses (Graham et al. 2011). The transected end created by removal of these segments was closed by Parker Kerr technique as described above. At the same time, the clamps and ligatures were removed from the remaining segment of colon and it was replaced in the abdomen to allow resumption of blood flow for 4hR under general anesthes ia. Small mucosal biopsies (1 to 2 cm2) were taken before (control), and after 1hI, 1hR, 2hR and 4hR. After the reperfused tissues were sampled, the horses were humanely subjected to euthan asia with an overdose of sodium pentobarbital (88 mg/kg, IV) while under anesthesia. The same investigator performed all surgeries and sampling (DEF). Sample Preparation In each horse, four 2mmpunched mucosal biopsies of control tissues, and tissues after 1hI, 1hR, 2hR and 4hR were fixed in 2.5% glutaraldehyde and paraf ormaldehyde at 4C overnight, washed in 0.1M cacodylate buffer, and placed in 1% aqueous osmium tetroxide for 1 h our at room temperature. After washing in 0.1M cacodylate buffer and deionized water, fixed samples were dehydrated in graded concentrations of acetone, and embedded in epon. Tissues were cut into semithin sections (500 nm) with an ultramicrotome equipped with a glass knife. Sections were mounted on glass slides, stained with TB and examined by LM. Tissue blocks were further trimmed to a size of 0.5 x 0.5 mm at the area of interest for thin sectioning. Thin sections (70 nm) were cut on the same microtome equipped with a diamond knife, and mounted on Formvar coated copper mesh gri ds. The grids were stained with 2% uranyl

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75 acetate and lead citrate, and examined with the Hitachi H 7000 or Zeiss EM10A TEM at magnifications varying between 2,500 and 80,000. Additionally, samples were fixed in 10% neutral buffered formalin for 36 h ours subsequently embedded in paraffin, and cut into 5m sections. After deparaffini sing and rehydration, slides were stained with PAS in a routine manner to characterize basement membranes. A descriptive evaluation of morphological changes assessed by LM was performed, and compared with ultrastructural alterations of epithelial cells evaluated by TEM. Furthermore the reaction of subepithelial immune cells during I/R was characterized by TEM. Results Morphological Changes In TB stained semithin sections, mi nor pathohistological alterations of the colonic epithelium were evident after 1hI (Fig ure 3 1B) compared to controls (Fig ure s 3 1A D). Epithelial injury was characterized by cell edema, microvilli disintegration, apoptosis, subepithelial fluid accumulation and detachment of epithelial cells from the vacuolated basement membrane (Fig ure s 3 1B,E). Lamina propria edema, accumulation of necrotic debris, and swollen or necrotic immune cells (lymphocytes, eosinophils, mast cells) were detectable in the subepithelial space after 1hI (Fig ure 3 1B). Cytoplasmic granules in subepithelial mast cells and eosinophils were also reduced. Further deterioration of epithelial injury was not evident after reperfusion (Fig ures 3 1C, F). The colonic epithelium responded to reper fusion with dilation of the paracellular space, and infiltration of neutrophils, lymphocytes and eosinophils (Fig ure 3 1C). After 1hR, repair of the epithelial layer could be detected as a covering of small

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76 epithelial defects by interconnections between de tached epithelial cells or between membrane ex t ensions from intact adjacent neighboring cells (Fig ure 3 1C). Numerous macrophages with large phagocytic vacuoles, neutrophils and mast cells were located in the subepithelial lamina propria after 4hR. Ultrastructural Changes Ischemia After ischemia, most of the epithelial cells were shorter and dilated (Fig ure 31H) compared to controls (Fig ure 31G). Microvilli were reduced in size and number, and their core appeared less electrondense (Fig ure 3 2A). The surface coat was diminished or disappeared partially, and goblet cells were rarefied and their granules reduced. The apical part of terminal TJ was partly disrupted or dilated (Fig ure s 3 2A B). After ischemia, the less electrondense cytoplasm of epithelial cells appeared vacuolated and contained a reduced number of mitochondria, rER and golgi complexes, all of which were swollen (Fig ure s 3 1F ; 3 2A,C,D). The mitochondrial matrix was lucent, and the christae were dilated and disrupted (Fig ure 3 2D). Most of the nuclei appeared rounded and enlarged. Their less electrondense plasma, and the condensation and margination of nuclear chromatin indicated early apo p totic features (Fig ure s 3 1H ; 3 2F). Large numbers of autophagosomes evident in the cytoplasm after 1hI contained damaged cell organelles, lytic cytoplasm and lysosomes (Fig ure s 3 2C,E). Only single epithelial cells appeared necrotic, as characterized by cytoplasmic lucency and vacuolization, and severely dilated and lytic nuclei and cell organelles. A fter ischemia, large subepithelial vacuoles separated epithelial cells from the basement membrane (Fig ure 3 2F). Consequently, small groups of epithelial cells detached from the distorted basement membrane to form subepithelial clefts. Detached

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77 epithelial cells however remained connected to each other by their apical cellular junctions. Numerous lymphocytes, phagocytic active neutrophils and eosinophils infiltrated the subepithelial clefts and migrated through the paracellular space towards the intestinal lumen. The subepithelial lamina propria contained large vacuoles. Enlarged macrophages containing phagocytic vacuoles and granules were located in the subepithelium. Subepithelial mast cells, lymphocytes and eosinophils were swollen and necrotic (Fig ure 3 2F). Their ultrastructure was characterized by cytoplasmic vacuolization, decreased intracellular granules and damage to the plasma membrane. Numerous neutrophils were attached to subepithelial venules, and they migrated into the lamina propria (Fig ure 3 2F). Reperfusion Within 4 h ours of reperfusion, the ultrastructural damage to epithelial cells did not progress. A prominent feature during reperfusion was an enlargement of the paracellular and subepithelial space over time that further sepa rated detached epithelial cells from the basement membrane (Fig ure s 3 1I ; 3 3A). Within 4hR, there was no evidence that these detached epithelial cells reattached. Instead these cells became shorter and appeared to adhere to each other at the luminal surface by membrane extensions and intact apical cell junctions, thus preserving coverage of large underlying clefts (Fig ure s 3 1I ; 3 3A,D). Subepithelial clefts and paracellular spaces were infiltrated with intact and apoptotic neutrophils (Fig ure 3 3A), lymphocytes and eos inophils. Numerous neutrophils migrated into the intestinal lumen. They were located in close proximity to the epithelial surface and contained vacuoles with phagocyt iz ed bacteria

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78 and necrotic debris. Some apoptotic cells and apoptotic bodies were evident within the epithelium (Fig ures 3 3B,E). Epithelial cell nuclei appeared shrunken, irregularly lobulated and partly py knotic, and contained large nucleoli and electrondense chromatin (Figure 3 3E). Although reduced in their number, mitochondria, rER and G o lgi complexes looked normal (Fig ure 3 3E). In addition to autophagosomes, large membranebound vacuoles containing necrotic debris, bacteria, and apoptotic bodies were observed in the cytoplasm of epithelial cells, possible evidence that they became phagoc ytic during reperfusion (Fig ure s 3 3B,C). Numerous apoptotic cells, phagocytic active macrophages and neutrophils, and mast cells and lymphocytes were located in the subepithelial lamina propria (Fig ure 3 3F). Discussion Although 1hI of the equine colon resulted in minor mucosal injury on LM, examination of the mucosa by TEM demonstrated ultrastructural alterations in individual epithelial cells in response to the short ischemic period. Examination of TB stained semithin sections by LM revealed distinct mor phological alterations of the epithelium. Damage to microvilli, dilated paracellular spaces, subepithelial cleft formation and single cell necrosis evident on TEM after ischemia could cause cellular dysfunction and disruption of the intestinal barrier. Reperfusion of the ischemic injured mucosa for 4 h ours was sufficient to allow the damaged epithelium to recover epithelial barrier function (Graham et al. 2011). Whereas the naturally occurring lesion in clinical cases is an ischemia of variable duration and intensity, the lesion induced in this study may represent a milder disease because of the short duration of ischemia and because the subjects were on 100% oxygen. However, the type of injury inflicted was designed to be

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79 reversible and capture the gross and microscopic elements that are typical of colonic ischemia (Snyder et al. 1989a ). Morphological Changes Semithin sections stained with TB can demonstrate specific morphological changes of the colonic epithelium by LM after I/R. Histomorphometric and morph ological studies on paraffinembedded colonic mucosal biopsies have shown that short periods of ischemia cause minor but significant changes characterized by detachment of epithelial cells from adjacent cells and basement membranes, edema formation, hemorr hage, and accumulation of necrotic debris in the lamina propria (Meschter et al. 1991; Darien et al. 1995, Graham et al. 2011). These changes could be demonstrated in the results of the present study. Additional changes demonstrated by semithin sections included disrupted microvill i integrity, dilated paracellular spaces, vacuol iz ation of the basement membrane, single cell necrosis, early apoptosis, and epithelial repair. Epithelial alterations did not progress during reperfusion, and epithelial repair started at 1hR in the present study. Although epithelial alterations after 1hI were minor in the present study, metabolic and ultrastructural changes of single epithelial cel ls can cause epithelial barrier dysfunction (McAnulty et al. 1997; Graham et al. 2011). Sun et al. (1998) calculated a strong positive correlation between short ischemic times of 20 and 40 min utes and disruption of epithelial barrier permeability in rats, consistent with our recent findings of epithelial barrier failure after 1hI (Graham et al. 2011). However, 4hR resulted in fully recovery of the barrier function in the equine colon (Graham et al. 2011).

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80 Ultrastructural Changes Ischemia A cascade of cellu lar enzymatic and metabolic changes in the epithelium during hypoxia leads to reversible ultrastructural alterations (Snyder et al. 1992; McAnulty et al. 1997) characterized by swelling and vacuol iz ation of epithelial cells, dilation of cell organelles, and structural changes of the nucleus consistent with features of early apoptosis (Labat Moleur et al. 1998). In addition to hypoxia, activation or necrosis of subepithelial mast cells, neutrophils and eosinophils, as demonstrated in our study, could also pl ay a potential role in ischemic mucosal injury by releasing toxic and inflammatory mediators (Wardlaw 1996; Boros et al. 1999 a ; Gayle et al. 2000). One of the key findings in the present study was the disintegration or dilation of TJ between epithelial cel ls after ischemia. T ight junctions are principally responsible for regulating paracellular permeability and, therefore they play a major role in maintaining the epithelial barrier. Additionally, the lateral intercellular space is also thought to mechanical ly contribute to the TER (Madara 1998; Blikslager et al. 2007). Thus, separation of epithelial cells from their neighboring cells by intercellular fluid accumulation and expanded TJ could explain epithelial barrier failure after ischemia (Graham et al. 2011). A prominent change in colonocytes after ischemia in the present study was autophagy, a homeostatic process that removes damaged or surplus organelles, supplies nutrients and energy, eliminates intracellular pathogens and toxic proteins, and delivers endogenous antigens for presentation (Levine and Deretic 2007; Levine and Kroemer 2008). Amino acids or fatty acids recovered through autophagy may be used for ATP production, and misfolded proteins and damaged mitochondria may be

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81 removed under hypoxic condi tions (Sadoshima 2008). Alternatively, a marked upregulation of autophagy and accompanying upregulation of lysosomal enzymes can cause self digestion and eventual cell death (Sadoshima 2008). Although autophagy in epithelial cells might have caused single cell death after ischemia in the present study, it might also favor epithelial cell survival during hypoxia (Sadoshima 2008), as evident by epithelial repair and functional recovery within 4hR (Graham et al. 2011). Reperfusion Reperfusion of colonic tissues did not exacerbate epithelial cell damage in the present study, and ischemic injured epithelial cells appeared to restore the epithelial lining during reperfusion by reattaching to adjacent cells (Fig ure 3 4). Rapid self sealing by epithelial cells usual ly begins within 15 minutes after injury, and allows neighboring cells to reestablish cell to cell contacts and restore epithelial integrity (Wilson and Gibson 1997; Mammen and Matthews 2003; Blikslager et al. 2007; Figure 3 4). However, epithelial morphol ogy and ultrastructure did not appear completely normal after 4hR. Although the final fate of damaged epithelial cells cannot be established conclusively from t his study, our findings are consistent with previous descriptions of a rapid recovery process and are within timeframes previously determined for restitution (Wilson and Gibson 1997; Mammen and Matthews 2003; Blikslager et al. 2007). Final repair of the ischemic injured epithelium starts later and involves proliferation and reepithelialization (Blik slager et al. 2007). Our observation of closure of TJ or sealing of membrane extensions between surviving neighboring cells could explain functional recovery in the same tissues in Ussing chambers after 4hR (Graham et al. 2011). Although a larger epithelial defect requires restitution by migration of surviving cells in the periphery of the injury, the injury induced in our model appeared predominantly to

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82 involve recovery of the epithelial lining by reattachment between remaining cells in the zone of epithelial damage (Fig ure 3 4). Many factors that control epithelial repair are released by epithelial cells themselves, or they are produced by mucosal immune cells. Among these immune cells, neutrophils are thought to play a key role in tissue injury and repair (Serhan and Savill 2005; Nathan 2006). Despite the presence of neutrophils within the intercellular space and TJ after 4hR, the colon had improved barrier function at this time, as determined by TER and transmucosal mannitol flux (Graham et al. 201 1). Accumulation and transepithelial migration of neutrophils persist in ischemic injured colonic mucosa for at least 18 hours after ischemic injury without impairment of epithelial barrier integrity ( Grosche et al. 2008; Matyjaszek et al. 2009). This is c ontrary to what has been demonstrated in porcine ileum (Gayle et al. 2002). It is p ossibly that activated neutrophils that are recruited to the site of colonic injury (Grosche et al. 2008) secrete anti inflammatory and proresolution factors that also prom ote repair (Nathan 2006; Serhan et al. 2008). Apoptosis of neutrophils, and their clearance by inflammatory macrophages, is also an essential step in inflammation reduction and initiation of repair (Savill et al. 2002). Thus, neutrophils could play a potential role in resolution of inflammation and promoting tissue repair during reperfusion of the ischemic injured colonic mucosa in horses. The results of the current study also indicated that epithelial cells displayed phagocytic activity as demonstrated by intracellular phagocytic vacuoles. The role of this process is not clear. Phagocytosis of foreign material could provide more nutrients and energy during reperfusion or sample the microenvironment for regulation of innate

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83 and adaptive immune responses, and possibly initiate repair (Artis 2008). Because epithelial cells can phagocytiz e adjacent cells, apoptotic cells and bacteria (Monks et al. 2005; Neal et al. 2006), they could control the inflammatory response and minimize injury after I/R in horses. Resu lts of the present study indicate that 1hI causes structural alterations of the equine colonic epithelium. Initially, mucosal injury occurs at the cellular level, and leads to epithelial barrier failure. However epithelial cells can survive short term hypoxia and recover during 4hR. It was evident that the cells remaining in the zone of injury had reestablished connections with adjacent cells, despite their abnormal appearance. This is also consistent with our finding that the same tissues had reestablished functional integrity within this timeframe, based on their performance in Ussing chambers. (Graham et al. 2011). Phagocytosis of apoptotic and necrotic cells could minimize inflammation and assist epithelial repair during reperfusion. The results also indicate that repair can proceed in the presence of mucosal neutrophil activity during I/R in the equine colon.

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84 Figure 3 1. Epithelium and subepithelial lamina propria of the equine colon after I/R : A ,D, G ) control; B,E,H ) 1hI ; C,E,I) 4hR ; TB (A C, x400), PAS (D F, x400), TEM (G H).

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85 Figure 3 2. Epithelial cells and subepithelial structures after 1hI; TEM; A) Apical part of 2 epithelial cells: shortened degenerated microvilli, cytoplasmic lucency and decreased cytoplasmic granules; B) Apical junction com plex between 2 epithelial cells with disrupted TJ (arrowhead; arrow: adherens junction) ; C) Vacuolated cytoplasm, degenerated cell organelles ( rER golgi apparatus, lysosomes, mitochondria), and autophagosomes; D) Degenerated mitochondria (lower epithelial cell); E) Autophagosome; F) Apoptotic epithelial cells (nuclear chromatin margination), subepithelial vacuoles, disrupted basement membrane, vacuolated subepithelial lamina propria, degranulated mast cells.

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86 Figure 3 3. Epithelial cells and subepithelial structures after reperfusion; TEM; A) Increased intercellular and subepithelial spaces with infiltrated neutrophil (2hR). Apical part of epithelial cells remains connected to each other B) Apical part of epithelial cells containing numerous phagocytic vacuoles with necrotic cell organelles, lytic plasma, lysosomes and apoptotic bodies (4hR); C) Phagocytic vacuoles (4hR); D) Apical junction complex between 2 epithelial cells with intact TJ ( arrowhead; arrow: adherens junction; 2hR); E) Pyknoti c, lobulated epithelial cell nuclei, apoptotic nucleus, and intact mitochondria (2hR); F) Subepithelial lamina propria with phagocytic active macrophages and lymphocytes and mast cells (4hR).

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87 Figure 3 4. Schematic model of epithelial cell injury after 1hI and epithelial recovery after 4hR : Ischemia causes some single cell necrosis (middle cell) but the majority of cells (remainder in ischemia panel) undergo some degree of degeneration, nuclear chromatin margination, detachment from the basement membra ne, and disruption of terminal TJ Paracellular and subepithelial clefts formed, accompanied by infiltration of neutrophils and lymphocytes during reperfusion. Enterocytes and apical junction complexes recover during reperfusion, and singlecell defects (c aused by loss of the middle cell in this example) are closed by development of apical cell to cell connections between intact neighboring cells.

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1Reprinted with permission from Grosche et al Equine Vet. J. 2008, pages 393399. 89 CHAPTER 4 DETECTION OF CALPROTECTIN AND ITS CORRELATION TO THE ACCUMULATION OF NEUTROPHILS WITHIN EQUINE LARGE COLON AFTER ISCHEMIA AND REPERFUSION1 Neutrophils appear to play an important role in endothelial injury (Granger et al. 1986; Kurose et al. 1994; Cooper et al. 2004) and mucosal damage caused by intestinal I /R (Grisham and Granger 1988; Grisham et al. 1990a/b; Schoenberg et al. 1991; Kubes et al. 1992; Moore et al. 1994a 1995a; Gayle et al. 2000, 2002). In equine colon, the role of neutrophils during I/R is unclear (Moore et al. 1995a; Blikslager et al. 1997a ), and resident eosinophils could complicate interpretation of inflammatory responses (Meschter et al. 1986; Moore et al. 1994a; McConnico et al. 1999; Rtting et al. 2003). The infiltration of neutrophils is a time dependent process, and their activation results in degranulation and release of inflammatory mediators, such as cytokines, ROS, proteases, and other regulatory proteins (McMichael and Moore 2004). To assess activation, endothelial adherence, accumulation and migration of neutrophils into tissue s, several direct and indirect methods have been used in man, different animal models, and horses (Bochsler et al. 1992; Brandtzaeg et al. 1992; Simpson et al. 1993; Seahorn et al. 1994; McConnico et al. 1999; Sorensen and Borregaard 1999; Weiss and Evanson 2002, 2003; Little et al. 2005; Franck et al. 2006), such as intravital microscopy, flow cytometry, histological, IHC and biochemical procedures. Direct counting of neutrophils within large tissue areas, such as full thickness mucosa, is difficult and cumbersome using standard histological techniques (H&E), because separation from other cells with shared staining characteristics is highly subjective (Moore et al. 1994a). Furthermore, it is difficult to define single neutrophils

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89 within mucosal tissues after I/R due to the tremendo us accumulation of erythrocytes and other cells in the inflammatory tissue reaction. Moore et al. (1994a) measured the number of neutrophils accumulated within a small area (0.01 mm2), at the base of the colonic mucosa adjacent to the muscularis mucosae, and also scored the neutrophil accumulation from grade 0 to 4 during I/R. Both, the nu mber of neutrophils in that small mucosal area and the scored neutrophil accumulation within the mucosa revealed an increased neutrophil accumulation during I/R. The scoring system was found to be useful in Moore et al. (1994a). Other methods for measuring neutrophil infiltration, such as tissue MPO activity and leukocyte scintigraphy, do not help to localize the cells as they migrate through the tissue (Moore et al. 1994a). Gayle et al. (2002) counted neutrophils within the subepithelial and epithelial area of the small intestinal mucosa (104 m) using a calibrated grid, but, as in Moore et al. (1994a), a small and well defined area was chosen. To our knowledge, neutrophil infiltration within the full height of the lamina propria has not been determined in horses, and for that purpose, we used a scoring technique similar to that used by others in equine colon (Moore et al. 1994a; McConnico et al. 1999). The inflammatory marker, calprotectin, a calcium and zinc binding complex also described as L1 antigen, c ystic fibrosis antigen, calgranulin, MRP8MRP14, and S100A8 S100A9 (Johne et al. 1997), was originally discovered as an antimicrobial protein in the cytoplasm of neutrophil granulocytes (Dale et al. 1983). It is also found in macrophages and other cells (F lavell et al. 1987), and is emerging as a valuable marker for diagnosis, monitoring, and prognosis of gastrointestinal tract diseases (Johne et al. 1997; Poullis et al. 2003; Striz and Trebichavsky 2004). Calprotectin is predominantly

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90 located in the cytoso l, regardless of neutrophil activity, and constitutes approximately 40 to 60% of cytosolic neutrophilic proteins. Some is released in response to various agonists as an expression of cytotoxicity or to modify the local inflammatory response (Johne et al. 1 997). Although the exact biological role of calprotectin is unknown, available evidence would suggest that it can modulate inflammatory reactions through activities that inhibit growth and induce apoptosis in fibroblasts or other cell types (Yui et al. 200 3); and it can defend neutrophils against microbial infections (Johne et al. 1997). If present in an excess amount for a long period, it also seems to cause local tissue destruction (Yui et al. 2003). Immunoassays (RIA and ELISA) and IHC have been establis hed for calprotectin measurement in f eces, body fluids and tissues, based on a reaction between the murinederived monoclonal antibody, MAC 387, with the specific formalin resistant antigen L1 (Fagerhol et al. 1980b ; Brandtzaeg et al. 1988, 1992; Roseth et al. 1992; Guignard et al. 1996; Johne et al. 1997; Sorensen and Borregaard 1999). Because calprotectin can be found in large amounts within the cytoplasm of neutrophils, the goal of this study was to establish the relationship between neutrophil numbers i n H&E stained tissues with the numbers of calprotectincontaining cells within submucosal venules and the colonic mucosa during I/R in horses. The purpose was to establish a method to detect neutrophils within equine intestinal tissues using IHC identifica tion of calprotectin as a marker of acute inflammatory processes. Because neutrophils are thought to play a pivotal role in reperfusion injury in equine colonic mucosa (Moore et al. 1994a 1995a), the course of influx and migration of these cells in a model of I/R, with short and long periods of reperfusion, was followed.

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91 Materials and Methods Horses Forty horses used in this study were of mixed breeds with an average age of 8 years (range 1 to 21 years), and a mean body weight of 469 kg (range 300 to 543 kg), that were donated for research purposes, and that were free of gastrointestinal diseases. These horses were involved in various overlapping research projects on I/R, performed with approval and under guidelines of the Institutional Laboratory Anim al Care and Use Committee of the University of Florida. Horses were fed grass hay (2% of their body weight per day), and water was provided ad libitum. Horses were adapted to their diet and environment for at least 1 week before the study. Procedures A 14gauge, 13.3cm Teflon catheter was inserted into the left jugular vein for administration of anesthetic drugs and isotonic fluids. Horses were placed under general anesthesia according to the following protocol: Premedication using xylazine (0.3 mg/kg IV) and butorphanol (0.02 mg/kg, IV) to provide sedation, and then general anesthesia was induced with diazepam (0.2 mg/kg IV ) given to effect and then ketamine IV at 2.0 mg/kg as a bolus injection. General anesthesia was maintained with isoflurane (1 t o 3%) in 100% oxygen per inhalation. Horses were mechanically ventilated at 6 breaths/min. Isotonic polyionic fluids were infused continuously IV at 5 to 10 mL/kg/h. Mean arterial blood pressure was monitored through a 20gauge, 5.1cm Teflon catheter in the facial artery, and was maintained at or above 70 mmHg. Other monitoring tools used during anesthesia included electrocardiography, blood gas analysis, capnography and pulse oximetry.

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92 Horses were positioned in dorsal recumbency and prepared for an asept ic ventral midline celiotomy. The large colon was exteriorized and placed on a plastic drape on the ventral abdomen. To induce ischemia, a 20cm segment of left dorsal colon was subjected to transmural compression by padded clamps at each end of the select ed segment, and combined venous and arterial occlusion was achieved with umbilical tape ligatures. After induction of ischemia, the colon was replaced in the abdomen and the abdominal incision was closed temporarily with suture. After ischemia had elapsed, clamps were removed, and the colon was replaced in the abdomen. Colon segments subjected to ischemia for 1 and 2 hours were reperfused for 30 minutes under general anesthesia. In horses with a reperfusion time of 18 hours, the abdomen was closed in routine fashion and these horses were then allowed to recover from general anesthesia. They received butorphanol (0.05 mg/kg, IM, q4h) at the end of ischemia. After recovery from anesthesia, each horse was moved to a stall and monitored for signs of pain, and a pain score was recorded every 4 hours according to a previously established behavioral scoring system (Pritchett et al. 2003). This system is based on behavioral patterns that are milder manifestations of pain than those observed typically in horses with c olic. After 30minR and 18hR all horses were subjected to euthanasia with an overdose of sodium pentobarbital (100 mg/kg, IV). Mucosal biopsies were taken before (control tissues, n = 20) and after ischemic periods (1hI [n = 25]; 2hI [24]), and full thickne ss tissues were removed after reperfusion (1hI + 30minR [n = 6]; 2hI + 30minR [8]; 2hI + 18hR [7]). The same investigator performed all surgeries and sampling (DEF).

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93 Histology All samples were fixed in 10% neutral buffered formalin for 36 hours, subsequently embedded in paraffin, and cut into 4 to 5 m thick sections. Adjacent sections were mounted on several silanecoated glass slides. After deparaffini sing 3 times with xylene for 5 minutes and rehydration 2 times with 100% ethanol, once each with 95%, 70% ethanol, and with deionised water for 5 minutes slides were stained with H&E in a routine manner to assess the adhesion and accumulation of neutrophil granulocytes within submucosal venules and infiltration into the colonic mucosa. For the evaluation of the images obtained by LM, a computer based image analy z ing program ImagePro Express Version 5.0 (Media Cybernetics, Bethesda, MD, USA) was used and three randomly defined areas from each tissue with a length of 866 m (equal to the length of one imag e using the 10x objective) were examined. Colonic mucosa was scored from 0 to 3 for presence of neutrophils using a 40x objective. Grade 0 was assigned when neutrophils were absent or single cells were observed after careful inspection, grades 1, 2 and 3 w ere assigned if neutrophil accumulation was mild, moderate, or marked, respectively. The numbers of neutrophils contained within 45 randomly identified circular or oval shaped submucosal venules were counted. Collapsed vessels were excluded from examinati ons. The cross sectional areas of these venules were measured, and the mean number of cells/mm2 crosssectional venule area was calculated. Immunohistochemistry All sections were deparaffinised and rehydrated according to the above described procedure. Im munohistochemical identification of cytosolic calprotectin was performed with 1:100 mouse anti human macrophages monoclonal antibody (MAC 387; Serotec,

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94 R a l e igh, NC, USA) which is known to have cross reactivity with equine calprotectin, and a commercially a vailable biotin free detection kit (Histar Detection System; Serotec, R a l e igh, NC, USA), using a modified staining procedure for equine jejunum (Little et al. 2005). For antigen retrieval, each tissue underwent heat pretreatment using a pressure cooker (1 25C for 30 sec, 90C for 10 sec) and a retrieval buffer with a pH of 6.0 (Deloaker RTU Buffer; Biocare Medical, Concord, CA, USA). After a cooling for 15 minutes at room temperature and washing 3 times with Dulbeccos PBS for 5 minutes, sections were proc essed according to manufacturers instructions (Serotec, R a l e igh, NC, USA). The specimens were incubated in 0.03% hydrogen peroxide for 15 minutes to quench endogenous peroxidase, and after rinsing 3 times with PBS for 5 minutes, slides were incubated with bovine serum albumin for an additional 15 minutes to block nonspecific binding. Tissues were then incubated with the primary antibody (anti human MAC 387) 1:100 diluted in PBS for 30 minutes at room temperature in a humidified chamber. For each staining procedure, equine lung specimens were subjected to the same staining protocol, and calprotectinpositive alveolar macrophages were used as positive controls (Parbhakar et al. 2004; Little et al. 2005). Instead of the primary antibody, PBS only was added to lung tissues and experimental samples as negative controls. After blocking the antigenantibody reaction with Special Block Solution (Serotec, R a l e igh, NC, USA) for 20 minutes at room temperature followed by rinsing 2 times with PBS for 5 minutes, slides were incubated with a biotinfree secondary antibody, which was polymerized directly with horseradish peroxidase, for 30 minutes. To demonstrate the antigen, the desired stain intensity was developed with

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95 2.5% DA B as peroxidase substrate for 3 to 5 minutes under microscopic control. Tissues were rinsed well and incubated with distilled water for 1 minute. Then slides were counterstained with Mayers hematoxylin for 5 minutes and, after rinsing wi th tape water, color was developed for 10 seconds with ammonia water. After dehydration with 70%, 95%, twice with 100% ethanol and 3 times with xylene for 3 minutes, respectively, sections were mounted in Permount Mounting Medium ( Fisher Scientific Pitts burgh, PA, USA) and covered with glass cover slips. Tissues were observed by LM using a 40x objective, and images were taken using a 10x objective with the image analy z ing program Image Pro Express Version 5.0 ( Media Cybernetics, Bethesda, MD, USA). Cy toplasm of positively stained cells appeared brown. The mean number of calprotectinpositive cells within 45 randomly identified circular or oval shaped submucosal venules were counted and computed as described above for H&E. Additionally, the number of c alprotectinpositive cells/mm2 mucosal area was counted within three segments of colonic mucosa of 866 m length and for the full mucosal height in each segment. Calprotectin positive cells were counted within five equal sized mucosal zones that spanned the full height of the mucosa from the muscularis mucosae (M1) to the surface of the epithelium (M5), as described previously (Rtting et al. 2003). The numbers of calprotectinpositive cells/mm2 of mucosa within these 5 zones were counted, and the means of three randomly defined mucosal areas with a length of 866 m and a height that spanned the full mucosal height were evaluated to detect the accumulation of calprotectinpositive cells within different mucosal regions over time. All counts and scorings were performed blindly by 1 investigator (AG).

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96 Statistical Analysis Data were expressed as mean SEM. The statistical software program SPSS, Version 15.0 (SPSS Inc., Chicago, IL, USA ) was used for analyses. Values of P were considered as significant. The Kruskal Wallis test was performed to compare the number of calprotectinpositive cells with the number of H&E stained neutrophils within submucosal venules or with the score for H&E stained neutrophils within colonic mucosa during the different I/R tim e periods and with controls. Whenever a significant P value for I/R was identified, appropriate Bonferroni adjusted P values were used for each comparison. To assess the accuracy of IHC visualization of calprotectin in correlation to neutrophils, the numbers of calprotectinpositive cells and H&E stained neutrophils or neutrophil scores were compared using Spearmans rank correlation coefficient R. Results No horses experienced complications during the I/R periods while under general anesthesia or after rec overy. The median pain score during the 18 hours after ischemia was approximately one third the maximum score that can be obtained with this system (Pritchett et al. 2003). No horse required any analgesics other than the scheduled doses of butorphanol. Calprotectin could be detected with a variable intensity in alveolar macrophages and in sporadic appeared neutrophils within positive control lung tissues using the above described staining procedure. There was no evidence of binding of the primary antibody t o cells other than alveolar macrophages and single neutrophils and there was no evidence of nonspecific binding of the secondary antibody in lung tissue (Figure 4 1). The stain intensity of most of the calprotectinpositive cells and their differentiation from

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97 surrounding and adjacent cells within tissues was pronounced. Most of the calprotectinstained cells within submucosal vessels were neutrophils. Within vessels, monocytes and neutrophils could be detected by their shape and size so that their absolute numbers were comparable to their populations in submucosal venules. On the other hand, differences between calprotectincontaining neutrophils and macrophages within the mucosal tissue were indistinguishable. The shape and size of both cell types varied h ighly because of their migration through and accumulation within small mucosal vessels and the interstitium of the lamina propria. The number of calprotectinpositive cells in submucosal venules increased significantly after 2hI, and peaked after 2hI and a fter 30minR (Table 4 1). After 18hR, calprotectinstained cells declined significantly in the venules. Similar results were observed with H&E stained neutrophils (Table 4 1). As demonstrated in Figures 4 2A and 4 2B, most of the neutrophils and calprotecti n positive cells within submucosal venules were located close to or attached to the vessel wall. Migration of these cells through the vessel wall was also evident. The number of calprotectinpositive cells/mm2 mucosa increased during ischemia, but not significantly (Figure 4 3). After 2hI + 30minR and 2hI + 18hR, significant changes in calprotectinpositive cells followed a pattern that suggested that cells left the venules during reperfusion to migrate towards the epithelial surface. Calprotectinpositive cells within the mucosa and mucosal neutrophil score increased significantly after 2hI + 30minR, and calprotectinpositive cells increased significantly within mucosal zones M1, M2, M4 and M5 during the same period, to peak at 18hR (Table 4 1). After 2hI + 30minR, the highest number of calprotectincontaining cells could be detected within the intestinal lumen (M5), but

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98 they decreased in that zone after 18hR. The accumulation of calprotectinpositive cells within submucosal venules (R = 0.80; P < 0.001) and equine large colonic mucosa (R = 0.67; P < 0.001) correlated significantly with the scores for H&E stained neutrophils. Discussion In this study, calprotectin was shown to be a useful means for assessing neutrophil influx into equine colonic mucosa subjec ted to an injury similar to that in previously reported models of mucosal ischemia (Snyder et al. 1988; Moore et al. 1994a; Matyjaszek et al. 2007). Based on the findings of this study, movement of neutrophils into colonic mucosa during I/R followed a pattern that could be closely correlated with the sequence of pathophysiological events proposed for reperfusion injury (Moore et al. 1995a). Moo re et al. (1994a) compared the accumulation of neutrophils by direct counting and using a scoring system. They measured the number of neutrophils accumulated within a small area (0.01 mm2) at the base of the colonic mucosa adjacent to the muscularis mucosa e, and scored the neutrophil accumulation on a scale from grade 0 to 4 during I/R. Both, the number of neutrophils in that mucosal area and the scored neutrophil accumulation within the mucosa yielded similar results, and revealed an increased neutrophil a ccumulation during I/R. In the present study, H&E stained neutrophils were counted directly in mucosal venules, and a close relationship found with the actual numbers of cells and calprotectinpositive cells. The scoring system used was similar to that des cribed by Moore et al. (1994a) and McConnico et al. (1999) for the remainder of the lamina propria, and this correlated with the distribution of calprotectinpositive cells. Moore et al. (1994a) recognized that direct counting of neutrophils is timeconsum ing, subjective and prone to bias, and that counting in a

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99 selected area would be misleading if the cell distribution were heterogeneous. By dividing the mucosa into zones (Rtting et al. 2003), such a heterogeneous distribution did arise from the pattern o f neutrophil migration. The characteristic brown staining produced by IHC identification of calprotectin within the neutrophilic cytoplasm provided the opportunity to follow the timedependent migration of neutrophils from the venules towards the surface e pithelium. Most of the calprotectinstained cells within submucosal vessels could be directly identified and counted in absolute numbers as neutrophils, distinct from monocytes. This would be expected because the proportion of neutrophils in equine blood ( mean 53 %) exceeds that of monocytes, which are less than 4 % (mean 0.5 %; Jain 1993). On the other hand, it was difficult to distinguish between calprotectincontaining neutrophils and macrophages within the cellular milieu of the lamina propria. The shape and size of both cells varied considerably as they became distorted during migration from small vessels and through the interstitium of the lamina propria. The brown staining of the cytoplasm of calprotectinpositive cells can obscure the nuclear identit y of the cell. Therefore, some of the positively stained cells were either activated resident macrophages or infiltrated monocytes, in addition to neutrophils. However, of these cell types, neutrophils would be expected to be more actively involved with ti ssue infiltration and migration during I/R (Moore et al. 1994a 1995a; Gayle et al. 2000). The mean numbers of calprotectinpositive cells tended to be fewer than the H&E stained neutrophils (Table 4 1). Presumably, not every neutrophil expressed a detectable amount of calprotectin, or some neutrophils had already exhausted cytoplasmic calprotectin after activation. Alternatively, some calprotectinstained cells

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100 could be physically detached and separated from the tissue during the many steps involved in the IHC procedure. However, the significant correlation of calprotectincontaining cells and H&E stained neutrophils demonstrates that calprotectin can be used for approximation of neutrophil activation in the equine gastrointestinal tract; and may be a valuable marker of associated inflammatory processes (Oshitani et al. 1997) and innate tissue defense mechanisms (Johne et al. 1997). The ischemic model used in this study mimics the ischemic condition in naturally occurring large colonic volvulus (Snyder et al 1988). However, neutrophil infiltration during ischemia was demonstrated, possibly because some arterial inflow persisted, especially during the period shortly after clamp application (Table 4 1; Figure 4 2A,B). As tissue damage progressed during ischemi a, release of local inflammatory mediators and transepithelial leakage of lipopolysaccharide probably triggered endothelial cell responses that led to margination, rolling, adhesion, diapedesis and migration of neutrophils (Moore et al. 1995a) already within the vasculature. The number of calprotectinpositive neutrophils then increased after restoration of blood flow, which would bring additional cells to the injured tissues. Furthermore, after a reperfusion period of 18 hours, neutrophils disappeared from submucosal venules, probably because of migration into and accumulation within the adjacent tissue. In similar fashion, Little et al. (2005) found an increased neutrophilic inflammation within all intestinal layers of equine jejunum at 18 hours after a period of ischemia. This was accompanied by an increased number of calprotectinpositive cells, indicating leukocyte activation.

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101 In the present study, both neutrophils and calprotectinpositive cells were released into the intestinal lumen after 30minR (Tabl e 4 1). This movement serves as the basis for using fecal calprotectin as a nonspecific marker of diseases of the gastrointestinal tract ( Roseth et al. 1992; Johne et al. 1997; Ton et al. 2000; Aadland and Fagerhol 2002). Increased concentrations of calpr otectin in intestinal lavage fluids of patients with inflammatory bowel disease and the significant correlation with intestinal permeability suggest that calprotectin enters the gut lumen as a consequence of transepithelial migration of neutrophil granuloc ytes (Berstad et al. 2000). The findings in the present study provide some evidence that measurement of calprotectin in intestinal contents might also be worthy for evaluating equine gastrointestinal diseases. Although the function of calprotectin has not been identified, it is known to have antimicrobial features as well as apoptosis inducing and growthinhibitory activities (Yui et al. 2003). Furthermore, the protein is involved in the recruitment of inflammatory cells by interaction with endothelial cell s (Srikrishna et al. 2001), and its zinc binding function may affect physiological homeostasis (Sampson et al. 2002). Yui et al. (2003) assumed that highly expressed calprotectin could have a local deleterious effect that can cause tissue destruction comparable to tissue damage after neutrophil infiltration. The ability of calprotectin to induce apoptosis could be responsible for this effect. Other potential harmful effects of neutrophils on intestinal epithelium could occur by release of oxygen metabolites and physical disruption of TJ (Gayle et al. 2002). Although the present study was not designed to establish the relationship between the injury and neutrophil influx, the findings are compatible with a putative role for increased neutrophil influx during reperfusion in the equine colon. Although the peak tissue accumulation was at 18

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102 hours after ischemia, when tissue damage appeared to be resolving (Matyjaszek et al. 2007), neutrophils can increase paracellular permeability of ischemic injured porcine ileu m after 18hR (Gayle et al. 2002). The results of the present study suggest that IHC detection of calprotectin by the monoclonal MAC 387 antibody is a useful marker of neutrophil accumulation in equine colon. This technique therefore has the potential to be a useful, clinically relevant method to characterize the severity of acute inflammatory processes that involve neutrophil influx, specifically after reperfusion injury in equine intestine.

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103 Table 4 1. Number of neutrophils and calprotectinpositive cells/mm2 submucosal venule, scores of neutrophil accumulation within the mucosa, and number of calprotectinpositive cells/mm2 mucosa and /mm2 mucosal zone M1M5 control 1hI 2hI 1hI+30minR 2hI+30minR 2hI+18hR Krusk al Wallis Neutrophils/mm 2 venule 375.4 57.8 792.2 109.7 1693.2 270.9 a P<0.001; b P<0.05 2423.7 158.5 a P<0.001; b P<0.01 4628.3 550.3 a,b,c,d P<0.001 1824.0 353.3 a P<0.01; e P<0.001 P<0.001 Calprotectin positive cells/mm2 venule 326.6 48.4 810.9 85.9 1455.8 228.5 a P<0.001; b P<0.05 1502.0 213.8 a P<0.01 3568.4 409.1 a,b,c,d P<0.001 1496.8 314.6 a P<0.01; e P<0.001 P<0.001 Mucosal neutrophil score 0.42 0.10 0.72 0.09 1.32 0.09 a,b P<0.001 1.61 0.13 a,b P<0.001 1.92 0.15 a,b P<0.001 2.28 0.20 a,b,c P<0.001 P<0.001 Calprotectin positive cells/ mm2 mucosa 163.8 34.8 211.8 28.6 358.4 42.0 468.5 92.3 723.2 92.9 a,b P<0.001; c P<0.05 1024.5 305.4 a,b,c P<0.001; d P<0.01 P<0.001 Calprotectin positive cells/ mm2 M1 194.5 47.2 228.7 41.6 392.4 55.8 587.1 151.4 992.9 167.5 a,b P<0.001; c P<0.01 1087.1 354.7 a,b,c P<0.001 P<0.001 Calprotectin positive cells/ mm2 M2 196.8 40.7 267.0 41.4 412.5 61.5 657.9 106.6 937.6 96.8 a,b P<0.001; c P<0.01 1189.1 338.0 a,b,c P<0.001 P<0.001 Calprotectin positive cells/ mm2 M3 167.2 36.0 214.3 30.2 341.1 52.2 356.1 68.2 435.9 77.8 1052.6 406.2 a,b,c P<0.001; d,e P<0.01 P<0.001 Calprotectin positive cells/ mm2 M4 77.1 18.2 102.6 12.9 216.2 19.3 230.6 62.5 359.0 62.7 a,b P<0.01 657.5 198.6 a,b,c,d P<0.001; e P<0.05 P<0.001 Calprotectin positive cells/ mm2 M5 19.6 3.9 34.6 7.5 71.5 10.3 42.4 12.2 167.2 83.3 a P<0.001; b P<0.01 111.8 40.4 P<0.001 Note: M ean SEM ; P values for Kruskal Wallis, and aP values vs. control, vs. b1hI, vs. c2hI, vs. d1hI+30minR, vs. e2hI+30minR for Bonferroni)

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104 Figure 4 1. Calprotectin IHC to detect equine alveolar macrophages (brown cells): Negative (A) and positive controls (B) of equine lung tissues using 1:100 MAC 387 antibody (x100).

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105 Figure 4 2. A) Neutrophils and erythrocytes within a submucosal vessel after 2hI (H&E; x400); B) Calprotectinpositive cells (brown cells) and erythrocytes within a submucosal vessel after 2hI (1:100 MAC 387 antibody; x400); C) Mucosal zones from the base (M1) to the surface of the epithelium (M5) after 2hI + 18hR (1:100 MAC 387 antibody; x100). Figure 4 3. Calprotectinpositive cells (brown c ells) within colonic mucosa: A) control; B) 2hI; C) 2hI and 18hR (1:100 MAC 387 antibody; x100).

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1Reprinted with permission from Grosche et al., Equine Vet. J. 2011, pages 1625. 107 CHAPTER 5 MUCOSAL INJURY AND INFLAMMATORY CELLS IN RESPONSE TO BRIEF ISCHEMIA AND REPERFUSION IN THE EQUINE LARGE COLON1 Ischemia causes mucosal damage in horses with large colon volvulus, resulting in translocation of bacteria and toxins, endotoxic shock, and possible death (Snyder et al. 1989a ). It also sets conditions for generation of reactive oxygen species, damage to the vasculature, and activation of granulocytes after reoxygenation of the tissue (Rowe and White 2002). Moore et al. (1994a,b) found that 3 h ours of reperfusion exacerbated colonic mucosal injury and increased neutrophil influx in a low flow ischemia model in horses. However, reperfusion did not affect production of PG s and cytokines in equine colon (Moore et al. 1995 c) and response to antioxidants was not typical of reperfusion injury (Moore et al. 1995 a ). Also, PGs produced by COX during reperfusion, could contribute to tissue damage or assist repair (Crofford 2001; Wallace and Devchand 2005; Little et al. 2007 b ) and early evidence of repair by restitution was evident in Moores reperfusion model (1994 b ). The low f low ischemia study by Moore et al. (1994a) in equine colon focused on the role of neutrophils as the major inflammatory cell, although eosinophils and other cells seemed to be involved to an undetermined extent (Moore et al. 1994a ). Therefore, the reaction of inflammatory cells and the expression of COX during colonic I/R warrant further study. Cells of the innate immune system are the main initiators of acute inflam matory reactions. At the beginning of injury, resident macrophages recognize damageassociated signals, invading bacteria and toxins. Alerted by these signals, macrophages attract large numbers of neutrophils to the site of injury to assist in recognizing, ingesting and destroying the invading agents (Smith et al. 2005). Neutrophil infiltration is

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107 a crucial step in the I/R cascade, and much of the tissue injury that occurs upon reperfusion is thought to result from neutrophilic radicals and proteolytic enzymes (Gayle et al. 2000). However, neutrophils also play a key role in controlling the infection, sterilizing the wound and generating proreparative signals (Serhan and Savill 2005; Nathan 2006). Additionally, resident eosinophils and mast cells are potent immunomodulatory cells that are activated by similar signals (Rothenberg et al. 2001; Galli et al. 2008). Once activated, mast cells release toxic metabolites, initiate inflam mation and recruit other immune cells to the site of injury (Marshall 2004). Eosinophils are frequently found in association with activated mast cells and are thought to manipulate the inflammatory response triggered by mast cell degranulation (Munitz and Levi Schaffer 2004). Although mucosal neutrophils, eosinophils, mast cells and macrophages can interact and contribute to mucosal inflammation and injury (Santos et al. 2001; Chen et al. 2004; Furuta et al. 2005), the responses of these cells in colonic I/ R in the horse are unknown. The purpose of the present study was to determine the number and tissue distribution of neutrophils, eosinophils, mast cells and macrophages, and the expression of COX 1 and 2 in response to I/R in the equine colon. The hypothe sis was that colonic I/R can induce an intense inflammatory response that causes further damage to the mucosa. Material and Methods Animals Six horses used in this study were of mixed breeds with a mean age of 16 years and a mean bodyweight of 548 kg. They were donated for research purposes and they were free of gastrointestinal diseases. The following study was performed with approval

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108 and under guidelines of the Institutional Animal Care and Use Committee of the University of Florida. Horses were fed grass hay (2% of their bodyweight per day), and water was provided ad libitum. Horses were adapted to their diet and environment for at least 1 week before the study. Experimental Procedures A 14 gauge, 13.3cm Teflon catheter was inserted into the left jugular vein for administration of anesthetic drugs and isotonic fluids. Horses were placed under general anesthesia according to the following protocol: xylazine (1.0 mg/kg IV) to provide sedation, and then general anesthesia was induced with diazepam (0.1 mg/kg IV) to effect followed by ketamine (2.2 mg/kg IV) as a bolus injection. General anesthesia was maintained with isoflurane (1 to 3%) in 100% oxygen. Horses were mechanically ventilated at 6 breaths/min. Isotonic polyionic fluids were infused IV continuously at 2.5 to 5m L /kg /h. Mean arterial blood pressure was monitored through a 20gauge, 5.1cm Teflon catheter in the facial artery, and was maintained at or above 60 mmHg. Other monitoring tools used during anesthesia included electrocardiography, blood gas analysis, capnography and pulse oximetry. Horses were positioned in dorsal recumbency and prepared for an aseptic ventral midline celiotomy. The large colon was exteriorized and placed on a plastic drape on t he ventral abdomen. To induce ischemia, a 40cm segment of colon at the pelvic flexure was subjected to transmural compression by intestinal clamps at each end of the selected segment, and combined venous and arterial occlusion was achieved with umbilical tape ligatures. After induction of ischemia, the colon, colonic vasculature and associated mesentery were surgically divided at the pelvic flexure so that 2 segments of colon comparable in size (dorsal and ventral) did not communicate. The colon was then

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109 r eplaced in the abdomen and the abdominal incision closed temporarily with towel clamps. After 1h I the colon was reexteriorized and 1 of the 2 ischemic segments was resected for histological evaluations and in vitro experiments (Graham et al. 2011). At th e same time, the clamps and ligatures were removed from the remaining segment of colon and it was replaced in the abdomen to allow resumption of blood flow for 4hR under general anesthesia. Small mucosal biopsies (1 to 2 cm2) were taken before (control), a nd after 1hI, 1hR, 2hR and 4hR. After the reperfused tissues were sampled, the horses were humanely subjected to euthan asia with an overdose of sodium pentobarbital (88 mg/kg, IV) while under anesthesia. The same investigator performed all surgeries and sampling (DEF). Sample P rep aration Samples were fixed in formalin, subsequently embedded in paraffin, and cut into 5 m sections. After deparaffini sing and rehydration, sections were stained with Lunas stain (Luna 1963) to assess eosinophils within the colonic mucosa. Paraffinembedded sections were also processed for immunohistochemistry. Additionally, four 2mm punch mucosal biopsies of control tissues and tissues after 1hI, 1hR, 2hR and 4hR were processed for TB staining (histomorphometry, mast cells). Briefly, biopsies were fixed in glutaraldehyde and paraformaldehyde at 4C overnight, washed in cacodylate buffer, and placed in osmium tetroxide for 1 h our at room temperature. After washing, samples were de hydrated in graded concentrations of acetone, and embedded in epon. Tissues were then cut into semithin sections (500 nm) with an ultramicrotome equipped with a glass knife. Sections were mounted on glass slides and stained with TB

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110 Immunohistochemistry Th e neutrophil marker calprotectin was detected using 1:100 monoclonal mouse anti human macrophages antibody MAC387 (Serotec, Raleigh, NC, USA) according to a previously published protocol (Grosche et al. 2008). Although calprotectin is also expressed in activated macrophages, the correlation between the numbers of calprotectinpositive cells and neutrophils is strong in I/R models in the horse (Grosche et al. 2008). For detection of macrophages, the macrophage surface protein CD163 was stained with 10 g/m L of a monoclonal mouse anti human macrophage surface antibody (clone AM 3K ; TransGenic Inc., Kumamoto, Japan) and a commercially available ABC detection kit with DAB as chromogen (Faleiros et al. 2010 ). Cytoplasmic COX 1 and 2 were stained with polyclonal goat anti human antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) based on a previously described protocol (Morton et al. 2009). For antigen retrieval, all tissues underwent heat pretreatment using a pressure cooker and retrieval buffer with a pH of 6.0. After cooling and washing with Dulbeccos PBS, sections were processed according to manufacturer instructions. When the desired stain intensity has developed, all tissues were counterstained with Mayers hematoxylin in a routine manner before process ing for mounting. Instead of primary antibodies, Dulbeccos PBS only was added to experimental samples as negative controls. Positive stained cells appeared brown. Histomorphometry For evaluation of the images obtained by LM a computer based image analy z i ng program Image Pro Express Version 5.0 (Media Cybernetics, Bethesda, MD, USA) was used. Routine histomorphometric measures (epithelial height, epithelial width,

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111 percentage of denuded epithelium) were determined in TB stained sections of three randoml y defined mucosal segments from each tissue with a length of 217.5 to the length of one image using the 40x objective) according to a previously described protocol (Rtting et al. 2003). Additional histomorphometric measurements included the heig ht and width of 15 epithelial cell nuclei, the width of the paracellular space between 15 epithelial cells, and the dimension of the subepithelial space on 15 locations. Swollen/necrotic epithelial cells, and apoptotic cells characterized by nuclear chromatin margination and condensation, and presence of apoptotic bodies were manually counted within the epithelium of each segment, and their number was calculated per mm mucosal length. Additionally, the length of the epithelium that displayed features of ongoing regeneration and repair (covering of denuded or injured mucosal areas by detached epithelial cells, membrane extensions of intact neighboring cells or flattened epithelial cells) was expressed as percentage of repaired epithelium. Neutrophils Eosinophils, M ast Cells and Macrophages The mean numbers of calprotectinpositive neutrophils / mm2 crosssectional venule area, mucosal area and mucosal zones of equal size (M1M5) were counted and calculated (Grosche et al. 2008). In Luna stained tissues, the numbers of eosinophils / mm2 mucosal area and mucosal zones of equal size (M1M5) were counted as described above for calprotectinpositive neutrophils. Mast cells were characterized by their redpurplestained granules after staining with TB Three ran domly defined segments of colonic mucosa with a length of 217.5 (equal to the length of one image using the 40x objective) and the full height of the mucosa were photographed. The numbers of mast cells within the mucosa were counted manually, and the me an numbers of mast /m m2 mucosa, and / mm2 upper and

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112 lower half of the mucosal lamina propria were determined. The same procedure was used to determine the number and distribution of CD163positive macrophages. COX 1/ 2 Expression For evaluation of mucosal CO X expression, the epithelium, upper and lower lamina propria, and crypts were scored from 0 3 (Morton et al. 2009). Grade 0 was assigned when stained cells were absent or single stained cells were observed after careful inspection. Grades 1, 2, and 3 were assigned if accumulation of stained cells was subjectively assessed as mild, moderate and marked, respectively. Histomorphometric examinations, quantification of eosinophils, mast cells, and calprotectinand CD163positive cells within tissues were perfor med blindly by 1 investigator (AG). Statistical Analysis Data were expressed as means SEM. Values of P < 0.05 were considered as significant. Kruskal Wallis test was used to compare nonparametric data during different I/R time periods and with controls. Whenever a significant P value for ischemia and reperfusion was identified, MannWhitney U test was used for pair wise comparison. Results Histomorphometry Ischemia for 1 hour resulted in minor but significant histomorphometric changes of the colonic epithelium (Figure 5 1B) compared to controls (Figure 5 1A). Ischemic injury was characterized by cellular edema (increased epithelial cell and nuclear width), subepithelial fluid accumulation, and chromatin condensation and margination, a characteristic of apoptosis (Figures 5 2A C ). Single epithelial cells be came necrotic, although their number was not significantly increased during reperfusion (Table 5 1,

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113 Figures 5 2A D). Epithelial injury was not exacerbated by reperfusion (Figures 5 1C,D). Dominant changes during reperfusion were enlargement of paracellular spaces, formation of large subepithelial clefts that were covered with shortened epithelial cells or membrane extensions between neighboring cells (Figures 5 3B ; 5 4A B). Although the cell structure of epithelial cells did not appear completely normal aft er 4hR, small epithelial defects were reepithelialized, and large subepithelial clefts were covered by a contin u ous layer of shortened epithelial cells with apical membrane connections that appeared tightly adhered to each other (Figures 5 3A C C ; 5 4B D). This was first evident at 1hR (Table 5 1). During reperfusion, apoptotic bodies became evident (Figures 5 3A D ; 5 4A D), although it was not possible to disti n guish between apoptotic epithelial cells and intraepithelial immune cells (lymphocytes, neutrophi ls, eosinophils). N eutrophils Eosinophils, M ast Cells, Macrophages, and COX 1/ 2 Expression After ischemia, calprotectinpositive neutrophils accumulated in submucosal venules with further prog r ession after 1hR (Table 5 2). Neutrophils migrated into the lamina propria and moved towards the epithelium at 1hR, and they moved into the intestinal lumen at 2hR (Table 5 2; Figure 5 1C). Mucosal mast cells and macrophages appeared activated during I/R, but their numbers remained unchanged (Table 5 2 ; Figures 5 2 to 5 5). With LM examination on TB stained semithin sections, subepithelial mast cells were evident and they contained a decreased number of cytoplasmic granules that were located in close proximity to the cellular membra ne after ischemia (Figures 5 2A C) compared to reperfused tissues (Figures 5 3A D ; 5 4A C). Such changes could be interpreted as degranulation and release of proinflammatory molecules into the interstitium. In addition, subepithelial macrophages displayed increased phagocytic activity during I/R, characterized by large phagocytic vacuoles in

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114 the cytoplasm containing debris and apoptotic cells (Figures 5 2B D, 5 3D, 5 4A,C, D). Significantly more COX 2 was expressed by epithelial cells, and lamina propria im mune cells, especially lymphocytes, eosinophils and neutrophils, after ischemia, and after 1hR compared with controls, 2hR and 4hR (Table 5 3; Figure 5 6). Discussion Although ischemia caused mild mucosal injury in the present study, the injury to mucosal barrier function was sufficient to decrease TER and increase transmucosal mannitol flux in the same colonic tissues (Graham et al. 2011). Ischemia increased apoptosis and COX 2 expression, a possible response of the colonic epithelium to prevent further damage and initiate early recovery after reoxygenation (Savill et al. 2002; Karrasch et al. 2006). In the present study, reperfusion did not exacerbate epithelial damage, consistent with the findings of Moore et al. (1994 b ), using a different I/R model and time schedule. However, differences between ischemia and reperfusion were seen in other histomorphometric criteria not evaluated in the present study, such as the depth of mucosal loss and mucosal cellular debris index (M oore et al. 1994 b ). Although morphology of the epithelium did not return completely to normal at the end of reperfusion in our study, 4hR was sufficient to initiate epithelial repair. In this rapid repair process, defects created by loss of individual cell s were covered by apical membrane extensions from intact neighboring cells (Figures 5 4A, B). This 2 after 2 after 4hR in the same mucosal tissues mounted i n Ussing chambers (Graham et al. 2011). Studies have identified apoptosis as a major cause of cell death after intestinal I/R in equine small and large intestines (Rowe et al. 2003, Grosche et al. 201 1c ). However,

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115 it is still unclear whether apoptosis cont ributes to epithelial injury or resolves inflammation and hastens epithelial repair (Ramachandran et al. 2000). In the present study, semithin sections embedded in epon and stained with TB clearly demonstrated nuclear chromatin condensation and margination, characteristics of apoptotic cells that we previously identified with the TUNEL method after I/R in equine colon (Grosche et al. 201 1c ). Chromatin condensation and margination, and formation of apoptotic bodies were increased by ischemia, and persisted during reperfusion in the present study. Apoptosis could be a mechanism that protects the tissue from harmful exposure to inflammatory and immunogenic cell contents during reperfusion (Maderna and Godson 2003). Prostaglandins, synthesized by COX enzymes, play a key role in regulating inflammatory reactions, and COX 2 is known to be induced rapidly in sites of inflammation in the colon of horses (Matyjaszek et al. 2009; Morton et al. 2009). Although COX enzymes regulate the production of potent proinflammat ory PG s (Crofford 2001), evidence is growing that COX 2 expression may contribute to resolution of gastrointestinal inflammation, and might be crucial in regulating mucosal healing (Blikslager et al. 1999; Wallace and Devchand 2005). COX 2 expression in the epithelium and by lamina propria immune cells was significantly upregulated after ischemia and after 1hR in the present study, a time point where epithelial repair began. Shifflett et al. (2004) found enhanced recovery of barrier function in porcine isch emia induced ileal mucosa mediated by upregulation of COX 2 and activation of neutrophils. Thus, expression of COX 2 could be involved in controlling mucosal damage and recovery of epithelial function after I/R.

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116 Studies have shown that neutrophils are crit ical elements in the cascade of intestinal I/R injury and barrier dysfunction (Gayle et al. 2002; Blikslager et al. 2007), and neutrophils are involved in the inflammatory response after colonic I/R in horses (Grosche et al. 2008). Blikslager et al. (1997 b ) found massive infiltration of neutrophils during initial stages of epithelial repair in porcine intestinal ischemia. They hypothesized that mucosal injury is more likely triggered by physical damage to the repairing epithelium by migrating neutrophils at this stage. In the present study, the peak neutrophil infiltration was seen after 2hR, when epithelial repair had started. We cannot rule out possible short term damage to the epithelium by migrating neutrophils in the present study, although mucosal TER recovered fully after 4hR (Graham et al. 2011) despite the intense influx of neutrophils. Thus, the overall effect of neutrophils after I/R could be beneficial for tissue repair after colonic I/R if the severity of ischemic damage is mild (Serhan and Savil l 2005; Nathan 2006). Although, the number of mucosal eosinophils, mast cells and macrophages remained unchanged in the present study, their role as potential effector immune cells during intestinal I/R has been demonstrated in many studies (Kanwar and Kubes 1994b ; Boros et al. 1999a ; Chen et al. 2004; Furuta et al. 2005). Intestinal mast cells are thought to contribute to mucosal permeability alterations during reperfusion in canine small intestine, but might play only a minor role in I/R induced structural changes (Szabo et al. 1997). In contrast, Boros et al. (1999a ) found that mast cell degranulation can initiate tissue injury after I/R, and release of histamine contributes largely to the severity of mucosal damage. Eosinophilic granulocytes can be seen in close proximity to mucosal mast cells, and degranulation of mast cells is thought to be triggered by

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117 eosinophilic toxic proteins (Piliponsky et al. 1999). Although mast cell granule release was observed during I/R in the present study, a general effect of eosinophils and mast cells on epithelial injury and barrier dysfunction after colonic I/R could not be identified. In addition, resident macrophages are well established effector cells with proand anti inflammatory activities that could also contribut e to the inflammatory response, and therefore affect mucosal injury. Activated macrophages inhibit formation of enterocyte gap junctions in vitro (Anand et al. 2008), but influence early mucosal damage during intestinal I/R in rats by expression of MPO Egr 1 gene and proinflammatory cytokines before neutrophil infiltration occurs (Chen et al. 2004). Macrophages are also crucial for recognition and clearance of necrotic debris and apoptotic neutrophils, an essential step in resolving inflammation (Serhan and Savill 2005). Histological evaluation of the tissues in the present study demonstrated an increased phagocytic activity of resident subepithelial macrophages, suggesting a possible role during colonic I/R in horses. In conclusion, mild ischemic injury in the equine colonic mucosa was accompanied by increased apoptosis and epithelial COX 2 expression, which could facilitate early epithelial repair after reoxygenation. Epithelial repair characterized by regeneration and reepithelialization was associated with influx of neutrophils to the site of injury during intestinal I/R. Additionally, resident mast cells and macrophages may become activated in response to I/R, but their exact role in colonic I/R in horses requires further study. Despite the intense in flammation observed during reperfusion after 1hI in the present study, the equine colonic mucosa did not incur additional injury characteristic of I/R, but actually recovered according to morphologic and functional measures of repair.

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118 Table 5 1. Histomorphometric measurements during I/R Control 1hI 1hI+1hR 1hI+2hR 1hI+4hR Kruskal Wallis Epithelial height (m) 27.9 1.6 25.6 2.0 23.3 1.8 24.7 1.4 23.9 1.4 0.363 Epithelial width (m) 3.8 0.1 a 5.5 0.3 b 5.1 0.6 abc 4.5 0.2 c 4.6 0.3 ) c 0.007 Nuclear height (m) 6.5 0.4 6.8 0.3 6.5 0.3 6.8 0.2 6.2 0.2 0.615 Nuclear width (m) 2.7 0.2 a 4.2 0.2 b 3.4 0.3 abc 3.4 0.2 c 3.3 0.2 c 0.003 Intercellular space (m) 0.9 0.1 ab 0.6 0.2 ac 1.6 0.6 ab 1.5 0.2 b 1.6 0.2 b 0.027 Subepithelial edema (m) 0.2 0.1 a 3.2 0.2 b 2.2 0.8 bc 1.2 0.4 c 1.1 0.5 c 0.002 Apoptotic cells/mm mucosa 14.3 4.1 a 60.4 14.0 b 80.1 11.4 b 78.4 23.8 b 83.7 23.7 b 0.010 Necrotic cells/mm mucosa 2.6 0.8 34.3 7.3 30.2 13.9 12.8 5.7 20.2 11.0 0.066 Denuded epithelium (%) 0 0 ) 2.3 1.4 2.2 2.2 0 0 0.7 0.5 0.172 Repaired epithelium (%) 0 0 a 0 8 a 29.3 8.5 b 10.1 3.6 ac 19.9 7.1 bc 0.001 Note: M ean SEM ; MannWhitney U test; P<0.05 (different letters represent significant differences between conditions)

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119 Table 5 2. Calprotectin positive neutrophils, and eosinophils per mm2 mucosal and submucosal venule (neutrophils only) area, and within mucosal zones M1M5 during I/R Control 1hI 1hI+1hR 1hI+2hR 1hI+4hR Kruskal Wallis Calprotectin pos. cells/ mm 2 mucosa 72.3 8.4 a 215.0 73.4 ab 484.1 177.6 bc 1149.7 220.6 c 857.0 179.4 c 0.000 Calprotectin pos. cells/ mm 2 M1 12.8 3.7 a 58.7 19.6 b 134.8 57.3 bc 359.8 83.9 c 196.5 47.0 c 0.000 Calprotectin pos. cells/ mm 2 M2 30.3 8.8 a 88.4 39.39 ab 179.1 85.4 bc 303.0 49.0 c 211.9 58.6 c 0.002 Calprotectin pos. cells/ mm 2 M3 22.1 7.8 a 45.5 16.5 ab 94.4 27.6 bc 220.8 48.8 d 144.4 27.3 cd 0.001 Calprotectin pos. cells/ mm 2 M4 4.6 1.4 a 16.9 7.1 ab 51.5 19.2 bc 143.6 41.8 c 72.9 11.6 c 0.000 Calprotectin pos. cells/ mm 2 M5 2.6 1.7 a 5.4 2.0 ab 24.4 8.7 b 122.7 26.5 c 231.3 78.3 c 0.000 Calprotectin pos. cells/ mm 2 venule 469.7 193.2 a 1209.9 377.6 b 4068.0 644.0 c 3861.0 779.4 cd 1757.8 384.0 bd 0.001 Eosinophils/ mm 2 mucosa 709.9 132.0 530.5 111.9 652.5 166.2 684.8 145.6 581.2 119.6 0.731 Eosinophils/mm2 M1 355.1 66.5 279.1 63.9 284.5 68.2 269.2 71.2 295.3 69.6 0.880 Eosinophils/mm2 M2 227.1 39.2 169.2 35.3 223.7 55.2 244.4 71.6 202.0 42.4 0.737 Eosinophils/mm2 M3 110.6 28.8 65.8 20.6 100.4 39.0 109.1 38.6 68.2 17.8 0.759 Eosinophils/mm2 M4 15.8 4.8 14.3 5.7 32.6 15.4 36.5 10.5 9.0 2.4 0.255 Eosinophils/mm2 M5 1.3 0.5 a 2.2 1.1 ab 11.2 4.5 c 25.6 10.3 c 6.6 2.3 bc 0.011 Note: M ean SEM ; MannWhitney U test; P<0.05 (different letters represent significant differences between conditions)

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120 Table 5 3. Scores for COX positive cells in the mucosa, epithelium, upper lamina propria (ULP), lower lamina propria (LLP), and crypts during I/ R Control 1hI 1hI+1hR 1hI+2hR 1hI+4hR Kruskal Wallis COX 1 mucosa (012) 3.8 0.4 4.9 0.6 5.4 0.7 4.2 0.7 3.5 0.6 0.259 COX 2 mucosa (012) 5.4 0.6 a 8.2 0.6 b 7.1 1.1 ab 4.7 0.4 a 5.6 0.5 a 0.018 COX 1 epithelium (03) 0.3 0.1 1.0 0.2 1.0 0.2 0.7 0.3 0.7 0.2 0.127 COX 1 ULP (03) 1.1 0.1 1.3 0.2 1.5 0.2 1.3 0.1 1.1 0.2 0.485 COX 1 LLP (03) 1.6 0.2 1.8 0.2 1.8 0.2 1.3 0.2 1.2 0.1 0.067 COX 1 crypt (03) 0.8 0.2 0.8 0.2 1.1 0.2 0.8 0.2 0.6 0.2 0.565 COX 2 epithelium (03) 0.8 0.2 a 2.0 0.1 b 1.7 0.3 b 0.8 0.2 a 1.5 0.2 b 0.002 COX 2 ULP (03) 1.6 0.2 2.0 0.1 1.9 0.3 1.4 0.2 1.6 0.1 0.169 COX 2 LLP (03) 1.7 0.2 a 2.2 0.2 ab 1.8 0.2 ac 1.2 0.1 c 1.3 0.2 ac 0.014 COX 2 crypt (03) 1.4 0.2 2.0 0.3 1.7 0.4 1.3 1.1 1.2 0.1 0.307 Note: Mean SEM; MannWhitney U test; P<0.05 (different letters represent significant differences between conditions)

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121 Figure 5 1. Colonic mucosal tissues during I/R ( TB ; x400). A ) control; B ) 1 hI ; C ) 1h I + 2hR ; D ) 1 hI + 4 hR Ischemia was characterized by epithelial cell swelling, subepithelial vacuolization and fluid accumulation, detachment from the basement membrane, small epithelial defects and single cell necrosis (B). No further exacerbation of epithelial damage was present after reper fusion (C, D). Neutrophils, lymphocytes and eosinophils infiltrated the damaged epithelium and moved into the intestinal lumen (C). Epithelial defects were covered by a continuous layer of shortened epithelial cells that appeared tightly adhered to each other (D).

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122 Figure 5 2. Characteristic cellular features of the colonic epithelium and subepithelium after 1hI ( TB ; x1000, oil immersion). One hour of ischemia resulted in epithelial swelling, subepithelial vacuolization and fluid accumulation, detachment from the basement membrane, single cell necrosis ( ) and small epithelial defects (A D). Epithelial cell nuclei were rounded and characterized by chromatin margination ( ; A C). Some apoptotic bodies were present in the epithelium and subepithelium ( ; A C). Subepithelial mast cells appeared degranulated characterized by decrease of cytoplasmic granules which were located near the cellular plasma membrane ( ; A C). Resi dent macrophages displayed phagocytic activity characterized by large cytoplasmic vacuoles containing cell debris and apoptotic bodies ( D). apoptotic body; chromatin margination; degranulated mast cells; ophage; necrotic cell; E eosinophil; L lymphocyte.

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123 Figure 5 3. Characteristic cellular features of the colonic epithelium and subepithelium after 1hI + 2 hR ( TB ; x1000, oil immersion). After 2hR epithelial cell swelling declined and epithelial cells appeared shorter resulting in the enlargement of paracellular spaces and subepithelial cleft formation (B D). Neutrophils, eosinophils and lymphocytes infiltrated subepithelial clefts and moved into the intestinal lumen (A D). Small epithelial defects were s ealed by adjacent neighboring cells ( ; A), or they were covered by a continuous layer of shortened detached epithelial cells ( ; C) or membrane extensions between neighboring cells ( ; B). Chromatin margination was only seen in single epithelial cells ( ; B), and more apoptotic bodies were present in the epithelium ( ; A D). The number and distribution of subepithelial mast cell granules appeared normal ( D), but subepithelial macrophages were phagocytic active ( apoptotic body; chromatin margination; mast cell; necrotic cell; coverage of subepithelial clefts and increased paracellular spaces by membrane extensions from neighboring cells; apical closure of epithelial defects by detached epithelial cells or intact neighboring cells; E eosinophil; L lymphocyte; N neutrophil.

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124 Figure 5 4. Characteristic cellular features of the colonic epithelium and subepithelium after 1hI + 4 hR ( TB ; x1000, oil immersion). After 4hR detached epithelial cells remained adhere to each other at the luminal surface by membrane extensions preserving coverage of large paracellular spaces and subepithelial clefts ( ; A, B). Apoptotic epithelial cells characterized by formation of apoptotic bodies ( ; C), and single necrotic c ells appeared to be replaced by migration of neighboring cells after extrusion into the intestinal lumen ( ; C), and further covering of epithelial defects by reepithelialization proceeded ( ; C, D). More neutrophils, eosinophils and lymphocytes infiltrate d the injured epithelium (A C). Cell debris and apoptotic bodies were phagocytized by subepithelial macrophages ( apoptotic body; chromatin margination; necrotic cell; coverage of subepithelial clefts and increased paracellular spaces by membrane extensions from neighboring cells; apical closure of epithelial defects by detached epithelial cells and intact neighboring cells; E eosinophil; L lymphocyte; N neutrophil.

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125 Figure 5 5. Number of macrophages (top left) and mast cells (bottom left) / mm2 lower lamina propria (LLP) and upper lamina propria (ULP) during I/R; mean; Kruskal Wallis; not significant. Top right: CD163 positive subepithelial macrophages (large brown cells; 10g/mL mouse anti human macrophage surface antibody; x400); Bottom right: subepithelial mast cells (cells with numerous redpurple granules; TB ; x400).

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126 Figure 5 6. COX e x pression in the colonic epithelium and upper lamina propria during I/R (positive cells appear brown); A C ) COX 1 (polyclonal goat anti human antibody, x400); D F ) COX 2 (polyclonal goat anti human antibody, x400); A, D ) control; B, E ) 1 hI; C, F ) 1 hI + 4 hR COX 1 expression was mainly evident in lamina propria cells in control ti ssues, and did not change during I/R. COX 2 expression increased during ischemia, especially in the epithelium and lamina propria, but declined after reperfusion.

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Reprinted with permission from Grosche et al., Am. J. Vet. Res 2011, in press. 127 CHAPTER 6 EFFECT OF ISCHEMIA AND REPERFUSION ON PRODUCTION OF NITROTYROSINE, ACTIV ATION OF EOSINOPHILS AND APOPTOSIS IN EQU INE LARGE COLONIC MUCOSA1 Ischemia/reperfusion injury is thought to play an important role in the pathophysiology and development of numerous gastrointestinal diseases (Moore et al. 1995a; Mallic k et al. 2004; McMichael and Moore 2004) During reperfusion, generation of ROS production of proinflammatory enzymes, and expression of adhesion molecules initiate an inflammatory response. Peroxynitrite generated by activated leukocytes (Gagnon et al. 1998; Takemoto et al. 2007) and endothelial cells (Beckman et al. 1990; Kooy and Royall 1994) and during I/R ( Kono and Rock 2008) is a short lived oxidant species and potent inducer of cell death that can cause tissue damage by oxidation and nitration of lipids, proteins and DNA. It may also induce apoptosis or necrosis due to acute and severe cellular energetic derangements (Szabo et al. 2007) Formation of nitrotyrosine as a result of nitration of the aromatic amino acid tyrosine could be a potential marker of the generation of RNS (Halliwell 1997; Muijsers et al. 1997; Eiserich et al. 1998) The adherence of activated neutrophils to postcapillary venules after release of pro inflammatory molecules by endothelial cells is thought to be one of the key steps in initiating I/R injury (Kurose et al 1994; Cooper et al. 2004) However, the role of neutrophils during I/R in the colon of horses is unclear (Moore et al. 1995a) and eosinophil presence also complicates interpretation of the inflammatory response (Meschter et al. 1986; Rtting et al. 2003; Rtting et al. 2008a ) Eosinophils have received little attention as proinflammatory leukocytes, although they are abundant in

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128 the colon of healthy horses (Rtting et al. 2008 a ) can generate an array of destructive mediators and toxic proteins, and contribute to pathological changes in patients with asthma and other hypereosinophilic diseases (Wardlaw 1996; Walsh 1997) Equipped with enzymes that may cause oxidative damage to biological targets activated eosinophils can also produce the respiratory burst that generates ROS and peroxynitrite (Van Dalen et al. 2006; Takemoto et al. 2007) Increased intestinal accumulation of eosinophils in horses with experimentally induced acute colitis, I/R injury, eosinophilic enteritides and parasitism (Moore et al. 1994a; Edwards et al. 2000; Archer et al. 2006; Rtting et al. 2008b) has been described. Cell death from necrosis and apoptosis is initiated by mediators released during I/R (Cummings et al. 1997; Ramachandran et al. 2000) Cells that die as a result of n ecrosis release their intracellular contents and generate an intense inflammatory reaction (Frangogiannis 2007; Kono and Rock 2008) In contrast, apoptotic cell death is a controlled, energy dependent event that regulates the physiological cell turnover i n tissues undergoing cell replication without stimulation of the immune system (Hall et al. 1994; Kono and Rock 2008) Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is associated with several pathological conditions in the gastrointestinal tract (Ramachandran et al. 2000; Tarnawski and Szabo 2001) Apoptosis could disrupt the intestinal barrier function (Abreu et al. 2000; Gitter et al. 2000) or it might control tissue damage, maintain a defen sive barrier, reduce inflammation, and initiate repair (Sun and Shi 2001) Results of p revious studies have indicated that reperfusion cause s neutrophil infiltration into equine colonic mucosa, and that the respiratory burst produced by

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129 neutrophils might be responsible for exacerbation of tissue damage after reoxygenation (Grisham et al. 1986; Moore et al. 1994a; Gayle et al. 2000) However, we are not aware of studies to assess activated eosinophils, oxidative stress and cell death in the colon of horses o ver a range of periods of I/R time. The purpose of the study reported here was to assess the effect s of ischemia and reperfusion on indicators of oxidative stress, activation of eosinophils, and apoptosis in the large colonic mucosa of horses To investigate the potential role of eosinophils nitrotyrosine generation by those cells in equine colon after ischemia and reperfusion was evaluated. Our hypothesis was that reperfusion would induce inflammation, as indicated by the activity of eosinophils and concurrently cause change s in oxidative stress and apoptosis patterns over the various periods of ischemia and reperfusion evaluated. Materials and M ethods Animals Forty horses of mixed breeds with a mean age of 9.2 years and a mean body weight o f 469 kg were used in the study. The horses were donated for research purposes and were free of gastrointestinal tract diseases. These horses were involved in several overlapping research projects (Grosche et al. 2008; Matyjaszek et al. 2009; Morton et al. 2009; Morton et al. 2011) on ischemia and reperfusion that were performed with approval and under guidelines of the Institutional Animal Care and Use Committee of the University of Florida. Horses were fed grass hay (2% of body weight /d), and water was provided ad libitum Horses were adapted to their diet and environment for at least 1 week before commencement of the study.

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130 Study Design Horses were assigned randomly to undergo 1 of 5 conditions of colonic ischemia alone or colonic isc hemia and reperfusion (Morton et al. 2009) In 13 horses, ischemia was induced in a 20cm long segment of pelvic flexure for 1 hour; tissue samples were collected immediately after the period of ischemia. In 6 horses, ischemia was induced in a 20cm long s egment of pelvic flexure for 2 hours; tissue samples were collected before ischemia (control samples) and immediately after 2hI In 6 horses, ischemia was induced for 2 hours in a 20cm long segment of pelvic flexure; tissue samples were collected before i schemia, immediately after 1hI and immediately after a second 1hour period of ischemia. In 6 horses, ischemia was induced for 1 or 2 hours in each of two 20cm long segments of pelvic flexure, after which reperfusion was allowed for 30 minutes; tissue samples were collected from both segments before ischemia, from 1 segment immediately after 1hI and again after the following 30minR and from the other segment immediately after 2hI and again after the subsequent 30minR In 2 horses, ischemia was induced in a 20 cm long segment of pelvic flexure for 2 hours after which reperfusion was allowed for 30 minutes; tissue samples were collected before ischemia, immediately after 2hI and again after the subsequent 30minR In 4 horses, ischemia was induced in a 20cm long segment of pelvic flexure for 2 hours after which reperfusion was allowed for 18 hours; tissue samples were collected immediately after 2hI and again after the subsequent 18hR In 3 horses, ischemia was induced in a 20cm long segment of pelvic flexu re for 2 hours after which reperfusion was allowed for 18 hours; tissue samples were collected immediately after the 18hour period of postischemia reperfusion.

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131 Anesthesia and Monitoring A 14 gauge, 13.3cm long polytetrafluoroethylene catheter was inserted into the left jugular vein of each horse for administration of anesthetic drugs and isotonic fluids. Xylazine hydrochloride (0.3 mg/kg), butorphanol tartrate (0.02 mg/kg), or a combination of those drugs was administered IV to provide sedation, then anes thesia was induced via IV administration of diazepam (0.02 mg/kg) to effect followed by ketamine hydrochloride (2.0 mg/kg) as a bolus IV injection. Anesthesia was maintained with isoflurane (1 to 2%) in 100% oxygen. Each horse was mechanically ventilated at a rate of 6 breaths/min. Isotonic polyionic fluids were infused continuously (5 to 10 mL/kg/h, IV). Mean arterial blood pressure was monitored through a 20gauge, 5.1cm long polytetrafluoroethylene catheter placed in a facial artery and was maintained a t 70 mm Hg. Other monitoring tools used during anesthesia included electrocardiography blood gas analysis, capnography, and pulse oximetry. Surgical Procedures Each horse was positioned in dorsal recumbency and aseptically prepared for a ventral midline celiotomy. The large colon was exteriorized and placed on a plastic drape on the ventral aspect of the abdomen. To induce ischemia, a 20cm long segment of the medial part of the pelvic flexure was selected. Transmural compression was achieved by placemen t of intestinal clamps at each end of the selected segment, and combined venous and arterial occlusion was achieved with umbilical tape ligatures. In 6 horses, 2 pelvic flexure segments approximately 20 cm apart were selected to each undergo ischemia for 1 or 2 hours, after which reperfusion was allowed for 30 minutes. In these horses, a 2hour period of ischemia was commenced in 1 of the 2 pelvic flexure segments One hour after ischemia was started in the first segment, a 1hour period of

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132 ischemia was com menced in the other pelvic flexure segment. In this manner, reperfusion (30 minutes) of both segments was instituted at the same time and full thickness samples of tissues could be obtained from both segments simultaneously. After induction of ischemia, the colon was replaced in the abdomen and the abdominal incision was closed temporarily with suture. At the end of the predetermined period of ischemia in horses for which tissue reperfusion was not planned, the colon was exteriorized, and full thickness col onic tissue from the ischemic injured, and adjacent nonischemic colon were harvested. At the end of the predetermined period of ischemia in horses for which tissue reperfusion was planned, the intestinal clamps were removed and the colon was replaced in th e abdomen. During reperfusion of pelvic flexure segments for 30 minutes, each horse remained anesthetized. To facilitate reperfusion of pelvic flexure segments for 18 hours, the horses abdomen was closed in routine fashion and it was allowed to recover fr om anesthesia. Each horse undergoing reperfusion for 18 hours received butorphanol (0.05 mg/kg, IV, q4h) at the end of the period of ischemia. After recovery from anesthesia, each of these horses was moved to a stall and monitored for signs of pain every 4 hours according to a previously established behavioral scoring system for low grade pain (Pritchett et al. 2003; Matyjaszek et al. 2009). At the end of the 18hour reperfusion period, horses were anesthetized again as described for the first surgery, the colon was exteriorized, and full thickness colonic tissues were harvested from the ischemic injured and adjacent nonischemic colon.

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133 At the end of the 1hour ischemia period, and after reperfusion (30 minutes or 18 hours), all horses were euthanized with an overdose of pentobarbital sodium (100 mg/kg, IV) while under anesthesia. Sample Collection One mucosal biopsy specimen was collected before (control samples; n = 20 horses) and after periods of ischemia ( 1hI 25 horses; 2hI 24 horses), respectively. One fullthickness tissue sample was removed after each period of reperfusion ( 1hI+30minR 6 horses; 2hI+30minR 8 horses; 2hI+18hR 7 horses). Because most of the injured colon was also used for an in vitro experiment, the size (2.5 X 2.5 cm) and number of each randomly chosen sample was limited to minimize mucosal and secondary inflammatory reactions in adjacent mucosa. The same investigator per formed all surgeries and sample collection (DEF). Histological Examinations All mucosal biopsy specimens and full thickness tissue samples were fixed in 10% neutral buffered formalin for 36 hours, subsequently embedded in paraffin, and cut into 4 to 5 m thick sections. Adjacent sections were mounted on several silanecoated glass slides. After deparaffinization and rehydration, slides were stained with H&E and Luna (Luna 1992) stains in a routine manner for histomorphometric evaluation and assessment of t he accumulation of eosinophils within the colonic mucosa. For evaluation of the images obtained by LM a computer based image analysis program (Image Pro Express Version 5.0; Medi a Cybernetics, Bethesda, MD, USA) was used. Histomorphometric examinations included determination of the ICR and the evaluation of the severity of mucosal hemorrhage. By use of the computer based analyzing program, the widths of 10 randomly selected crypts and the distances of the

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134 lamina propria between these crypts were measured by use of a 10X objective lens. The ICR was calculated as the ratio of the lamina propria occupied by the interstitium, compared with the width of the crypts. The ICR for healthy equine colonic mucosa (ie, the value in colons without I/R injury) is define d as (Snyder et al. 1988) The degree of erythrocyte accumulation within the mucosa was classified on the basis of the distribution and severity of interstitial hemorrhage by use of a scoring system from 0 to 3. A score of 0 was assigned when no erythr ocytes were observed within the lamina propria. Scores from 1 to 3 indicated focal and mild, multifocal and moderate, or diffuse and extensive hemorrhage within the tissue, respectively. The number of eosinophils was determined during examination of Lunastained tissues. Three fields of view within one section from each tissue sample were randomly use of the 10X objective lens]; full height of the mucosa) was photographed. The number of eosinophils within each region of the mucosa was counted with the aid of the computer based analyzing program, and the mean number of eosinophils per mm2 of mucosal area was calculated. Based on previous evidence that eosinophils can mig rate from the lower lamina propria to the lumen of the colon, independent of vessels, a system was used to detect such migration (R tting et al. 2003) Specifically, in each photographed region, mucosal eosinophils were counted in each of 5 mucosal zones t hat cumulatively spanned the full height of the mucosa from the muscularis mucosa to the surface epithelium to detect population changes in zones that could indicate migration (R tting et al. 2003) Four lines were drawn at intervals of onefourth of the mean distance from the muscularis mucosa to the luminal surface (delineating zones

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135 M1 up through M4); the surface of the epithelial cells was designated as zone M5. The number of eosinophils within each mucosal zone was counted, and the mean number of eosinophils per mm2 of mucosal area of 3 fields of view was used for further statistical analysis. Immunohistochemical Analysis and TUNEL Staining After tissue sections were deparaffinized and rehydrated, nitrotyrosine was detected by use of 1:50 monoclonal mouse anti human nitrotyrosine antibody ( Cayman Chemical, Ann Arbor, MN, USA) and a commercially available ABC detection kit (R&D Systems, Minneapolis, MN, USA) with 3 amino 9 ethylcarbazole as the ch romogen. The detection of apoptotic cells was determined on the basis of a TUNEL method using a commercially available apoptosis detection kit and DAB as the chromogen (Chemicon/Millipore, Billerica, M A USA) according to a protocol of Rowe et al. (2003). Apoptosis detected by staining was verified in conjunction with morphological features (cell shrinkage, chromatin condensation and margination, and apoptotic bodies) identified in H&E stained tissue sections with a 40X objective lens. For antigen retrieval all tissue samples underwent heat pretreatment by use of a pressure cooker ( Biocare Medical, Concord, CA, USA; 125C for 30 seconds and 90C for 10 seconds) and a retrieval buffer ( Biocare Medical, Concord, CA, USA; pH, 6.0). After cooling and washing wi th Dulbecco PBS solution, sections were processed according to manufacturer instructions. When the desired stain intensity developed with DAB (apoptotic cells) or 3amino 9 ethylcarbazole (nitrotyrosine), tissue sections were counterstained with Mayers hematoxylin stain in a routine manner before processing for mounting. Instead of primary antibodies (nitrotyrosine) or TdT enzyme (apoptosis), Dulbecco PBS solution only was added to experimental samples as negative controls.

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136 Tissue sections were examined vi a LM by use of a 40X objective lens and the image analysis program. A positive reaction was evidenced by cells that appeared red (nitrotyrosine) or had brown nuclei (apoptotic cells). The number of nitrotyrosinecontaining eosinophils (characterized by their prominent intracytoplasmic granules) and the number of other stained leukocytes (neutrophils, macrophages, and lymphocytes) were counted manually. These cells were identified by their shape, size, and location but could not be distinguished from each ot her because the staining pattern obscured identifying features. Nitrotyrosinepositive cells were counted in 3 randomly defined fields of view of 2 mucosal areas (217.5 X 162.5 m [equal to the size of 1 image by use of the 40X objective lens]). One such a rea was adjacent to the muscularis mucosa (lower lamina propria), and another was adjacent to the epithelium (upper lamina propria). Nitrotyrosinecontaining eosinophils and other leukocytes in 1 submucosal area (217.5 X 162.5 m) adjacent to muscularis mu cosa were also counted in triplicate. The mean number of nitrotyrosinepositive cells per mucosal area of 3 fields of view was used for further statistical analysis. The severity of apoptosis was determined (n umber of apoptotic cells/mm2 of mucosa) as desc ribed for assessment of eosinophil accumulation and as the apoptotic index. The apoptotic index was defined as the number of apoptotic cells per 1,000 epithelial cells, which were counted manually with the 40X objective lens. Histomorphometric examinations quantification of eosinophils, and detection of nitrotyrosineand TUNELpositive cells within the tissues were performed in a blinded fashion by 1 investigator (AG).

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137 Statistical Analysis Data are expressed as means SEM. A statistical software program (SPSS, Version 15.0; SPSS Inc., Chicago, IL, USA) was used for analyses. Values of P < 0.05 were considered significant. The Kruskal Wallis test was performed to compare the nonparametric data during the ischemia and reperfusion periods and between experi mentally treated tissues and controls. Whenever a significant P value for ischemia and reperfusion was identified, the MannWhitney U test was used for pairwise comparison. To assess the correlation among eosinophils, nitrotyrosine generation, and apoptosi s on tissue damage, the Spearman rank correlation coefficient (R) was calculated. Results In 13 horses, ischemia was induced in a pelvic flexure segment for 1 hour, after which tissue samples were collected. Mean age of these horses was 8.2 years (age range, 1 to 19 years) and mean weight was 467.7 kg (weight range, 350 to 550 kg); there was 1 stallion, 6 mares, and 6 geldings. In 6 horses, ischemia was induced in a pelvic flexure segment for 2 hours, and tissue samples were collected before and immediately after the 2 hour period of ischemia. Mean age of these horses was 8.3 years (age range, 2 to 20 years) and mean weight was 479.2 kg (weight range, 390 to 605 kg); there was 1 stallion, 3 mares, and 2 geldings. In 6 horses, ischemia was induced for 2 hours in a pelvic flexure segment, and tissue samples were collected before ischemia, immediately after 1hI and immediately after a second 1hour period of ischemia. Mean age of these horses was 9.2 years (age range, 1 to 20 years) and mean weight was 486.7 kg (weight range, 445 to 520 kg); there was 1 stallion, 3 mares, and 2 geldings. In 6 horses ischemia was induced for 1 or 2 hours in each of two 20-

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138 cm long segments of pelvic flexure, after which reperfusion was allowed for 30 minutes; tissue samples were collected from both segments before ischemia, from 1 segment immediately after 1hI and again after the following 30minR and from the other segment immediately after 2hI and again after the subsequent 30minR Mean age of these horses was 6.3 years (age range, 2 to 18 years) and mean weight was 438.3 kg (weight range, 300 to 545 kg); there were 2 stallions, 2 mares, and 2 geldings. In 2 horses, ischemia was induced in a pelvic flexure segment for 2 hours after which reperfusion was allowed for 30 minutes; tissue samples were collected before ischemia, immediately after 2hI and again after the subsequent 30minR These horses were a 21year old 500kg mare and a 32year old, 500 kg gelding. In 4 horses, ischemia was induced in a pelvic flexure segment for 2 hours after which reperfusion was allowed for 18 hours; tissue samples were collected immediately after 2hI and again after the subsequent 18R Mean age of these horses was 8.0 years (age range, 2 to 15 years) and mean weight was 478.8 kg (weight range, 420 to 515 kg); there was 1 stallion, 1 mare, and 3 geldings. In 3 horses, ischemia was induced in a pelvic flexure segment for 2 hours after which reperfusion was allowed for 18 hours; tissue samples were collected immediately after the 18hour period of postischemia reperfusion. Mean age of these horses was 11.0 years (age range, 6 to 20 years) and mean weight was 476.7 kg (weight range, 390 to 530 kg); there was 1 mare and 2 geldings. For all horses, mean age was 9.2 years and mean weight was 468.75 kg. Compared with control findings, the ICR and the mucosal hemorrhage score increased gradually an d significantly as the period of ischemia increased (Figure 6 1). There was no further significant change in either variable after reperfusion. The overall

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139 number of mucosal eosinophils per mm2 of mucosa appeared to generally decrease as a result of I/R c ompared with the control tissue value, but the changes were not significant (Figure 6 2). After 1hI and 2hI nitrotyrosine production by eosinophils had significantly increased from the control value; however, the longer period of ischemia did not result i n a further increase production. After either period of ischemia, production was maintained through 30minR although the value declined significantly after 18hR in samples that had previously undergone 2hI (Figure 6 3). In contrast, I/R resulted in increas ed nitrotyrosine production by mucosal and submucosal leukocytes, compared with the control value. A substantial increase in production was evident after 1hI which decreased somewhat as the duration of ischemia increased. The 30minute period of reperfusi on after 1hI or 2hI had little effect on nitrotyrosine production by mucosal and submucosal leukocytes but there was a significant peak in production after 18hR in samples that had previously undergone 2hI Changes in the extent of apoptosis among mucosal epithelial cells were detected as a result of I/R injury (Figure 6 4). Apoptosis (as indicated by the number of apoptotic cells/mm2 of mucosa) increased significantly from the control findings during ischemia, although the value after 2hI did not differ s ignificantly from that after 1hI (Table 6 1). A peak value was detected in samples that underwent 1hI+30minR With regard to the apoptotic index, ischemia resulted in significantly more apoptotic epithelial cells, compared with the control value; the index increased as the duration of ischemia increased. Following either period of ischemia, the index remained unchanged at the end of 30minR ; similarly, following the 2 hour period of ischemia, the index remained unchanged at the end of 18hR

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140 D iscussion Result s of the present study demonstrated that oxidative stress is involved in the inflammatory process initiated in equine colon during I/R as evidenced by increased nitrotyrosine generation. This response was localized in resident eosinophils and other mucosal leukocytes. Nitrotyrosine is considered to be a potential marker of peroxynitrite generation in tissues, although it is also generated by other molecules like nitrate, NO nitrogen dioxide or nitryl chloride (Wallace and Ma 2001) Therefore, detection of nitrotyrosine should be regarded as evidence of generated RNS rather than as a specific marker of peroxynitrite. In the colonic tissue samples asse ssed in the present study, eosinophils and other leukocytes (neutrophils, macrophages, lymphocytes) that accumulated within the mucosa and submucosa represented the main sources of nitrotyrosine, and therefore they could have a role in the pathogenesis of I/R injury in equine colon. Eosinophils are multifunctional proinflammatory leukocytes involved in numerous inflammatory processes (Gleich and Adolphson 1986) Armed with a number of toxic mediators, including granule proteins, oxygen metabolites, lipid m ediators, and proteases, eosinophils can contribute to intestinal epithelial dysfunction (Rothenberg et al. 2001; Furuta et al. 2005) Eosinophils are involved in tyrosine nitration, as well as formation of peroxynitrite and peroxidase catalyzed RNS which are capable of inflicting cell damage (Furuta et al. 2005; Van Dalen et al. 2006) However, t he biological role of eosinophils within the equine colonic mucosa during I/R is unknown. Whereas eosinophils accumulate within the human gastrointestinal tract i n gastrointestinal disorders (Rothenberg et al. 2001) eosinophilic granulocytes are resident in the gastrointestinal lamina propria in healthy horses. Most of the eosinophils

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141 in equine colon are found near the muscularis mucosae and are rarely located close to the surface of the mucosa (Rtting et al. 2008a) consistent with results of the present study. The number of eosinophils per mm2 mucosa did not change during 18hR suggesting that eosinophil adhesion molecules might not be activated during a period o f reperfusion of that duration, and that resident eosinophils in the equine colonic mucosa only respond to the ischemic insult. These results indicate that resident eosinophils in the colon could contribute to the inflammatory reaction initiated in the ear ly stage of I/R injury It has been reported that nitrotyrosine staining in mucosal leukocytes of horses with naturally acquired small intestinal strangulation obstructions is increased, compared with findings in horses without gastrointestinal tract diseases, which could reflect the presence of peroxynitrite subsequent to increased NO and superoxide production (Mirza et al. 1999) A possible cytotoxic role of NO in small intestinal strangulation obstructions was proposed (Mirza et al. 1999) Administration of peroxynitrite into the colon of rats produced widespread injury and inflammation similar to that recorded in inflammatory bowel disease (Rachmilewitz et al. 1993) which provides evidence that it could initiate intestinal inflammation and tissue damage (Singer et al. 1996; S hah et al. 2004) Although peroxynitrite has limited extracellular stability and diffusion range, it could be cytotoxic towards invading pathogens and act as a potent microbicidal compound (Muijsers et al. 1997) The second peak production of nitrotyrosine by submucosal leukocytes after 18hR in the present study could be a defen se mechanism against transepithelial passage of bacteria or bacterial products during this stage of reperfusion.

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142 In a previous study (Grosche et al. 2008) performed by our group, the number of neutrophils in equine colonic mucosa increased during I/R in a time dependent manner starting at 2h I and progressing after reperfusion. A ccumulation of neutrophils within the mucosa was significantly correlated with mucosal damage, consistent wi th the role of the neutrophil influx in reperfusion injury in the colon of horses The peak tissue accumulation of neutrophils in that study ( Grosche et al. 2008) was at 18h after ischemia, when damaged tissue started to repair (Matyjaszek et al. 2009) Th us, neutrophil influx into the tissue and production of RNS at that time could be a possible reaction of the innate immune system in defense against invading pathogens crossing the damaged epithelial barrier. Although other studies (Banda and Granger 1996) have revealed that cellular necrosis is the principal effect of ischemia on epithelial cells, the results of the present study support the hypothesis that apoptosis could contribute to cell death during I/R in equine colonic mucosa. Recently, it has been shown that ischemic injured intestine responds by undergoing programmed cell death rather than necrosis, and the severity of apoptosis depends on the differentiation stage of the enterocytes (Kerr et al. 1972; Ikeda et al. 1998; Hinnebusch et al. 2002) In the present study, apoptotic cell death started during 1hI in the epithelium, in the lamina propria and crypt cells, with further progression after 2hI in the epithelium, and after 1hI+30minR in the mucosa. Although studies (Shah et al. 1997 b ; Ikeda et al. 1998; Rowe et al. 2003) have revealed an activation of apoptosis during intestinal ischemia with further exacerbation during reperfusion, the role of programmed cell death on tissue damage and dysfunction remains controversial (Toth et al. 2007) Failu re to eleminate unnecessary and

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143 damaged cells by apoptosis will prolong inflammation through continued release of toxic agents, and repair after tissue damage requires the elimination of proliferative mesenchymal and inflammatory cells from the sites of injury (Haanen and Vermes 1995; Baylor et al. 2003; Serhan and Savill 2005; Maniati et al. 2008) On the other hand, pronounced apoptosis can contribute to leaks in the epithelial barrier and to permeability defects of the bowel (Shah et al. 1997b ; Abreu et al. 2000; Gitter et al. 2000; Schulzke et al. 2006) that could be accompanied by proteolytic cleavage of TJ proteins (Bojarski et al. 2004) In the present study, I/R in the colon of horses cause d activation of eosinophils and other leukocytes and product ion of nitrogen radicals by these cells might be involved in the inflammatory process. Additionally, apoptosis in the injured colonic mucosa appeared to be a prominent cause of cell death during IR Although the role of eosinophils or apoptosis in injury or repair remain s to be elucidated in future studies, results of the present study indicate d that resident eosinophils in equine colonic tissues are subjected to oxidative stress in the early stage of I/R thereby favoring their contribut ion to this and oth er disease processes in the colon of horses

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144 Table 61. Mean SEM apoptotic index (apoptotic epithelial cells) and number of apoptotic cells per mm2 of mucosa in colonic tissue samples obtained from 40 anesthetized horses that underwent 1 of 5 conditions of colonic ischemia alone or colonic I/R Experimental condition No. of apoptotic cells/mm 2 of mucosa Apoptotic index Control 545.4 68.8 a 98.2 13.1 a 1hI 905.4 97.3 b 199.9 18.5 b 2hI 1,138.9 135.3 bc 286.4 23.4 c 1hI+30minR 2,084.2 480.1 cd 192.9 19.1 bd 2hI+30minR 1,719.1 331.0 cd 339.4 58.3 cd 2hI+18hR 953.8 102.6 bcd 270.0 33.7 bcd Note: In 1 or 2 pelvic flexure segments in each horse, ischemia was induced for 1 or 2 hours followed by no reperfusion (1hI [n = 25 horses] and 2hI [24], respectively) or 30 minutes or 18 hours of reperfusion (1hI+30minR [6], 2hI+30minR [8], and 2hI+18hR [7], respectively). One mucosal specimen was collected before (controls; n = 20 horses only) and after each period of ischemia, respectively and one full thickness tissue sample was collected after each period of reperfusion. Sections of the tissue samples were examined microscopically. The detection of apoptotic cells was determined on the basis of a TUNEL method using a commercially available apoptosis detection kit and DAB as the chromogen. The distribution of apoptotic cells was determined in 5 mucosal zones that cumulatively spanned the full height of the mucosa from the muscularis mucosa to the surface epithelium. Four lines were drawn at intervals of onefourth of the m ean distance from the muscularis mucosa to the luminal surface (delineating zones M1 up through M4); the surface of the epithelial cells was designated as zone M5. Apoptotic cells were counted in each zone in 3 regions (each 866 m in length) of each tissu e sample, and the mean number of apoptotic cells per square millimeter of mucosa was calculated. The apoptotic index was defined as the number of apoptotic cells per 1,000 epithelial cells, which were counted manually with the 40X objective lens. a dWithin a variable, different superscripted letters represent significant (P < 0.05) differences between conditions.

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145 Figure 6 1. Mean SEM ICR (A) and mucosal hemorrhage score (B) in colonic tissue samples obtained from 40 anesthetized horses that underwent 1 of 5 conditions of colonic ischemia alone or colonic I/R In 1 or 2 pelvic flexure segments in each horse, i schemia was induced for 1 or 2 hours followed by no reperfusion (1hI [n = 25 horses] and 2hI [24], respectively) or 30 minutes or 18 hours of reperfusion (1hI+30minR [6], 2hI+30minR [8], and 2hI+18hR [7], respectively ). One mucosal specimen was collected before (controls; n = 20 horses only) and after each period of ischemia, respectively and one full thickness tissue sample was collected after eac h period of reperfusion. Sections of the tissue samples were examined microscopically in 3 randomly chosen fields of view. To calculate the ICR, t he widths of 10 random ly selected crypts and the distance s of the lamina propria between these crypts were measured by use of a 10X objective lens; t he ICR was calculated as the ratio of the lamina propria occupied by the interstitium compared with the width of the crypts. The severity of mucosal interstitial hemorrhage was scored on a scale f rom 0 to 3 represent ing no hemorrhage, focal and mild, multifocal and moderate, or diffuse and extensive hemorrhage w ithin the tissue, respectively. Different letters above the bars represent significant ( P < 0.05) differences between conditions.

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146 Figure 6 2. Mean SEM number of eosinophils per mm2 of colonic mucosa and the number of eosinophils in each of 5 mucosal zones in tissue samples collected from the horses in Figure 1 that underwent 1 of 5 conditions of colonic ischemia alone or colonic I/R As illustrated in the photomicrograph, the distribution of eosinophils was determined in 5 mucosal zones that cumulatively spanned the full height of the mucosa from the muscularis mucosa to the surface epithelium. Four lines were drawn at intervals of onefourth of the mean distance from the muscularis mucosa to the luminal surface (delineating zones M1 up through M4); the surface of the epithelial cells was designated as zone M5. Eosinophils were counted in each zone in 3 fields of view (each 866 m in length) of each t issue sample, and the mean number of eosinophils per square millimeter of mucosa was calculated. Luna

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147 Figure 6 3. M ean number of nitrotyrosinepositive eosinophils (A) and other leukocytes (C) within the submucosa (SM), lower lamina propria (LLP) and upper lamina propria (ULP) of sections of in tissue samples collected from the horses in Figure 1 that underwent 1 of 5 conditions of colonic ischemia alone or colonic I/R and representative photomicrographs of a negative control section (B) and a section in which nitrotyrosine positive mucosal eosinophils are present (D). Sections were processed by use of an ABC detection system with 3 amino 9 ethylcarbazole. Mayers hematoxylin counterstain; Figure 6 4. Representative photomicrographs of results of apoptotic cell detection in tissue samples collected from the horses in Figure 1 that underwent 1 of 5 conditions of colonic ischemia alone or colonic I/R The detection of apoptotic cells was determined on the basis of a TUNEL method using a commercially available apoptosis detection kit and DAB as the chromogen. A poptotic epithelial cells have brown nuclei A N egative control section. B C ontrol section obtained from a horse prior to induction of colonic ischem ia. C Section obtained from a horse after 2hI D Section obtained from a horse after 2hI+30minR ; TUNEL stain with DAB, and Mayers hematoxylin counterstain; bar

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148 CHAPTER 7 CALPROTECTIN, AND HE MATOLOGIC AND BIOCHEMICAL CHANGES IN SYSTEMIC AND COLONIC VENOUS BLOOD AFTER C OLONIC ISCHEMIA AND REPERFUSION IN HORSE S Strangulation obstructions of the large colon are one of the most devastating forms of colic in horses and are usually associated with high mortality from progressive ischemic damage to the intestinal wall, failure of the epithelial barrier and septic shock ( Harrison 1988; Snyder et al. 1989a; Gibson and Steel 1999; Mair and Smith 2005). Intestinal barrier dysfunction during ischemia is caused by a variety of biochemical, metabolic and ultrastructural changes of epithelial cells resulting in cell death over time (McAnulty et al. 1997 ; Logue et al. 2005; Grosche et al. 2011a,b,c ). In addition, reperfusion generates reactive metabolites and induces a pro found inflammatory response that can exacerbate mucosal damage (Moore et al. 1995a; McMichael and Moore 2004). I ntestinal i nflammation after I /R is often associated with an acute phase reaction. This prominent response of the immune system is characterized by systemic activation of neutrophils and other leukocytes, and generation and release of a large array of inflammatory, regulatory and metabolic proteins and enzymes into the circulation ( Moore et al. 1995c; Gruys et al. 2005; Vandenplas et al. 2005; Cray et al. 2009). Released cell contents and stimulation of a variety of target cells and organs cause a series of hematological and biochemical alterations in blood These includ e changes in leukocyte numbers, activation of the complement and coag ulation system s, and changes in plasma electrolytes, proteins and enzymes (Petersen et al. 2004; Gruys et al. 2005). Biochemical and hematological changes are widely used to assess disease severity after intestinal I/R in humans and other species (Parry 1987; Harrison 1988; Snyder et al. 1989 a ; Thompson et al. 1990; Fried et al. 1991; Niels e n et al. 2000;

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149 Caglayan et al. 2002; Saulez et al. 2004, 2005; Latson et al. 2005; Grosche et al. 2006; Johnston et al. 2007; Block et al. 2008; Delgado et al. 2009) Fo r example, c hanges in hydration, oxygenation, coagulation, fibrinolysis, and generation or release of metabolites and intracellular enzymes such as Llactate, D dimer and ALP have been used for diagnosis and prognosis of intestinal I/R in the horse (Parry et al. 1983; Gibson and Steel 1999; Monreal et al. 2000; Saulez et al. 2004 ; Latson et al. 2005; Delesalle et al. 2007; Delgado et al. 2009). Few studies have focused on local hematological and biochemical alterations in CB of horses with I/R (Moore et al. 1994c; Kawcak et al. 1995; Moore et al. 1995b; Moore et al. 1998a ). Moore et al. (1994c) found c han ges in blood oxygenation, lactate and pyruvate in CB but no alterations in systemic venous blood in horses with colonic I/R. Lactate, oxygen saturation and pO2 in CB were significantly correlated with the severity of mucosal damage after 3 hours of ischemia and 1hR in another study in horses (Kawcak et al. 1995). In recent years, n eutrophilic proinflammatory molecules and enzymes such as lactoferrin, neutrophil elastase, lysozyme, MPO and calprotectin were used to evalutate the activation status of neutrophils in intestinal I/R and inflammation (Kurose et al. 1994; Martins et al. 1995; Grulke et al. 1999, 2008 ; Weiss et al. 2003; DInca et al. 2007; De La Re biere De Pouyade et al. 2010). Calprotectin is a proinflammatory protein that serves as a sensitive marker of acute and chronic inflammatory conditions (Foell et al. 2004; Striz and Trebichavsky 2004; Little et al. 2005; Grosche et al. 2008). It is an ant imicrobial cytoplasmic protein mainly produced in granulocytes, monocytes and macrophages It is more likely to be released after cell disruption and cell death (Voganatsi et al 2001).

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150 Calprotectin is resistant to bacterial degradation in the gut, stable in feces for up to 1 week at room temperature, and can be measured by ELISA or RIA (Fagerhol et al. 1980b; Roseth et al. 1992; Ivanov et al. 1996; Ton et al 2000; Heilmann et al. 2008b). P lasma concentrations of calprotectin ca n increase from a normal of < 3 g/mL to 11, 15 and 46 g/mL or more in patients with systemic bacterial infections, major surgeries and rheumatoid arthritis ( Johne et al. 1997). M ucosal calprotectin expression was recently associated with neutrophil infilt ration and increased inflammatory response in colonic tissues during I/R of the equine large colon (Grosche et al. 2008; Grosche et al. 2011b,c). Thus, quantification of neutrophil activity by evaluation of local and systemic serum calprotectin, in combination with routine hematological and biochemical variables could be helpful to assess the severity of inflammation during colonic I/R in horses. The aim of this study was to develop an ELISA for equine calprotectin, and to compare routine hematological and biochemical variables with calprotectin concentrations in JB and CB after colonic I/R in horses. We hypothesize that alterations caused by colonic I/R in the horse are more profound in CB than in JB, and that activation of a local inflammatory response in the colonic mucosa during I/R is associated with increased cal protectin concentrations in CB. Material and Methods Animals Six horses used in this study were of mixed breeds with a mean age of 16 years and a mean bodyweight of 548 kg. They were donated for research purposes and they were free of gastrointestinal diseases. The following study was performed with approval and under guidelines of the Institutional Animal Care and Use Committee of the University of Florida. Horses were fed grass hay (2% of their body weight /day), and

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151 water was provided ad libitum Horses were adapted to their diet and environment for at least 1 week before the study. Experimental Procedures A 14gauge, 13.3cm Teflon catheter w as inserted into the left and right jugular vein for administration of anesthetic drugs and isotonic fluids and for sampling of venous blood. Hor ses were placed under general anesthesia according to the following protocol: xylazine (1.0 mg/kg IV) to provide sedation, and then general anesthesia was induced with diazepam (0. 1 mg/kg IV ) to effect followed by ketamine (2.2 mg/kg IV) as a bolus injection. General anesthesia was maintained with isoflurane (1 to 3 %) in 100% oxygen. Horses were mechanically ventilated at 6 breaths/min. Isotonic polyionic fluids were infused IV continuously at 2.5 to 5 m L /kg/h. Mean arterial blood pressure was mo nitored through a 20gauge, 5.1 cm Teflon catheter in the facial artery, and was maintained at or above 60 mm Hg. Other monitoring tools used during anesthesia included electrocardiography, capnography and pulse oximetry. Horses were positioned in dorsal recumbency and prepared for an aseptic ventral midline celiotomy. The large colon was exteriorized and placed on a plastic drape on the ventral abdomen. To induce ischemia, a 40cm segment of colon at the pelvic flexure was subjected to transmural compression by intestinal clamps at each end of the selected segment, and combined venous and arterial occlusion was achieved with umbilical tape ligatures After induction of ischemia, the colon, colonic vasculature and associated mesentery were surgically divided at the pelvic flexure so that 2 segments of colon comparable in size (dorsal and ventral) and associated vasculature did not communicate. The colon was then replaced in the abdomen and the abdominal incision closed tempor arily with towel clamps. After 1hI the colon was reexteriorized and 1 of

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152 the 2 ischemic segments was resected for histological evaluations and in vitro experiments for another study ( Graham et al. 2011). At the same time, the clamps and ligatures were rem oved from the remaining segment of colon and it was replaced in the abdomen to allow resumption of blood flow for 4hR under general anesthesia. Blood was sampled from the jugular vein before induction of anesthesia (pre anesthesia control) before ischemi a ( post anesthesia control), and after 1hI 1h R 2h R and 4hR C olonic blood was collected before ischemia, and after 1hI, 1hR, 2hR and 4hR alternately from the left dorsal or ventral colonic vein that drained the ischemic injured part of the pelvic flexure. Heparinised blood was used for blood gas analysis, and analysis of hemoglobin, PCV, L lactate, glucose, Na+, Ca2+ and K+ which was performed immediately after sampling. In addition, a blood smear for leukocyte differenti ation was prepared. After they had been k ept on ice for less than 1 hour, blood samples were centrifuged for 10 minutes at 10, 000 rpm, and serum samples were stored at 80C for further analyses. After tissue and blood sampling, horses were humanely euthanized with an overdose of sodium pentobarbital ( 88 mg/kg, IV) while under anesthesia. The same investigator performed all surgeries and CB sampling (DEF). Calprotectin Analysis ELISA For quantification of calprotectin in equine blood samples, a noncompetitive sandwich ELISA was developed based on a modified procedure that has been used for analysis of calprotectin in human serum, plasma and urine (Immundiagnostik AG, Bensheim, Germany). The assay was designed using 2 commercially available monoclonal ant i human antibodies with cross reaction to equine calprotectin subunits

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153 S100A8 and A9 serving as capture and detection antibodies. Human recombinant calprotectin subunits S100A 8 and A9 were used as ELISA standards. Development and a nalytic v alidation of th e ELISA The ELISA was conducted at room temperature (23 to 24C) in 96well high proteinbinding capacity polystyrene ELISA plates (NuncMaxiSorp; eBioscience, San Diego, CA USA ). The plates were analyzed by the microplate reader BenchmarkTM Plus Microplate Spectrophotometer (BioRad, Hercules, CA USA ) at 650 nm and a microplate reader software program ( Microplate ManagerTM, V ersion 5.2 ; BioRad, Hercules, CA USA ). A mouse anti human monoclonal IgG1 (clone 3H2617; US Biological, Swampscott MA USA ) that has been tested to cross react with calprotectin in equine serum in a preliminary study was used as coating antibody. The detection antibody was a customized biotinylated mouse anti human monoclonal IgG1 (clone MAC387; AbD Serotec, Raleigh, NC, USA ) that has been used before to detect mucosal calprotectin in equine large colonic tissues with IHC (Grosche et al. 2008). StreptavidinHRP (Thermo Scientific, Waltham, MA USA ) and T MB (US Biological, Swampscott, MA USA) served as detection system s to produce a blue colored product that was read with the microplate reader at 650 nm. The human recombinant large (13.2k Da; S100A9; ProtEra) and small calprotectin subunits (10.8 kDa; S100A8; Pr otEra, Sesto Fiorentino, Firenze, Italy ) served as calprot ectin standards. Optimal coating and detection antibody, and standard concentrations were determined by performing antibody titration grid experiments with signal to noise ratios > 5 at background values of < 0.2 O.D. An optimal standard curve was determin ed at 250, 125, 62.5, 31. 2, 15.6, 7.8, 3.9 ng/mL of equal aliquots of S100A8 and S100A9 subunits that were preincubated in 1mM CaCl2 solution at room temperature before

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154 further dilution in 1:15 blocking buffer. Incubation of both calprotectin subunits wit h CaCl2 has been shown to increase the signal intensity in a preliminary study. The curve height was > 2.0 O.D. at 250ng/mL, and the lowest standard was > 0.2 O.D. Human venous blood of one healthy person (AG) sampled from the dorsal venous arch of the left hand was used as positive control, and background values were determined with 1:15 blocking buffer only. Positive control and equine serum samples were centrifuged for 10 minutes at 10, 000 rpm and 4C, and the supernatant was diluted in blocking buffer (positive controls: 1:100 and 1:150; samples: 1:20, 1:150, 1:200 and 1:300 depending on calprotectin concentrations) immediately before adding to wells. Precision and reproducibility (intraand inter assay experiments; CV < 10%), sensitivity (minimum dete ctable concentration 6.5 ng/mL), and linearity were tested (CV < 10%). ELISA p rotocol 10 mM PBS (pH 7.2) overnight at 4C. After coating, plates were adjusted to room temperature for 15 minutes, emptied and tap p ed out to remove residual liquid. Plates containing bovine serum albumin ( KPL, Gaithersburg, M D USA ) for 1 hour on a shaker. Plate controls diluted in blocking solution were added in triplicates to the appropriate wells, and incubated for 1 hour on a shaker. After washing 4 times with a commercially availa Gaithersburg, M D USA detection Ab (0.1 g/mL) diluted in 1:15 blocking solution was added to each well and incubated for another hour on a shaker. Unbound detection antibody was removed by a

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155 secon HRP diluted in blocking solution (1:10,000) for 1 hour on a shaker. After washing, TMB Color d evelopment was allowed for 40 minutes in t he dark on a shaker, and then plates were read with the microplate reader at 650 nm The assay results were expressed as O.D., and the concentration of positive control and unknown samples were analyzed from the standard curve by the microplate reader soft ware program. Calprotectin concentrations were expressed as ng/mL serum. Positive control samples contained 3 328 4 464 ng calprotectin/mL serum (CI 95%). Due to the high variability of serum calprotectin concentrations in horses, calprotectin values wer e expressed as relative concentrations (%) in comparison to control samples (= 100%) in each horse. Blood Gas Hematological and Biochemical Analyses Values for pH and pCO2 (mm Hg), and hematological (PCV [%] and haemoglobin [mmol/L]) and biochemical variables (Na+ [mmol/L], K+ [mmol/L], Ca2+ [mmol/L], glucose [mmol/L] and lactate [mmol/L]) were analyzed in heparinised SB and CB blood samples immediately after sampling ( GEM PremierTM 3000 Blood Gas Analyzer ; Instrumentation Laboratory, Bedford, MA USA ). The variables for HCO3 [mmol/L] and BE [mmol/L]) were calculated from plasma pH and pCO2 using the HendersonHasselbalch equation ( http://medicineworld.org/calculators/bicarbonate/bicarbonatebase excesscalculator.html ). Because pCO2 concentrations in C B exceeded the analyzers upper measurable value of 115 mm Hg after 1hI, values of 1 15 mmHg were used for calculation. Differentiation of J B and CB leukocytes (lymphocytes, neutrophils, band and necrotic neutrophils, eosinophils, basophils, monocytes [%]) was performed on Wright Giemsa stained blood smears by LM using the 40x objective. Additional

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156 biochemical variables (TP [mg/dl], albumin [mg/dl], creatinine [mol/L], ALP [IU/L] and CPK [IU/L]) were determined in serum of frozen JB and CB samples ( Siemens Dimension Expand C hemistry A nalyzer ; Siemens, Deerfield, IL, USA ). Statistical Analysis Statistical tests were performed with SPSS, Version 17.0; SPSS Inc., Chicago, IL, USA Normality was tested with Kolmogorov Smirnov test. Because values in CB and JB were not normally distributed within treatment groups (controls, 1hI, 1hR, 2hR and 4hR), nonparametric tests were used for further statistical analyses. Pre and post anesthesia control samples in JB were compared with related samples Wilcoxon test. Trea tment groups from JB and CB samples were compared with Kruskal Wallis test. Whenever a significant P value was identified, MannWhitney U test was used for pair wise treatment group comparison. JB and CB samples of each treatment group were compared with r elated samples Wilcoxon test. Data were expressed as means SEM Values of P < 0.05 were considered significant. Results After induction of anesthesia ( post anesthesia control ) h orses had significantly (P < 0.05) increased JB lactate (preanesthesia cont rol: 0.47 0.07 vs. post anesthesia control: 1.20 0.06 mmol/L) and glucose (preanesthesia control: 6.06 0.31 vs. post anesthesia control: 7.67 0.69 mmol/L), and decreased Ca2+ (pre anesthesia control: 1.54 0.02 vs. post anesthesia control: 1.40 0.02 mmol/L) and BE concentrations (pre anesthesia control: 8.56 0.70 vs. post anesthesia control: 7.32 0.73 mmol/L) compared to preanesthesia control samples All other hematological and biochemical variables did not differ between preand post anesthesia sampling.

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157 One hour of ischemia caused metabolic acidosis in CB characterized by decreased pH, bicarbonate and BE, and increased pCO2 and lactate compared to same variables in post anesthesia control samples (Figure 7 1). The glucose concentration w as decreased, and K+ and CPK were increased in CB after 1hI (Figure 7 2). Changes in JB after 1hI were characterized by increased lactate and decreased TP concentrations (Figures 7 1, 7 2). Values for pH, lactate, glucose, and K+ in CB were significantly different from same values in JB (Figures 7 1, 7 2). Values for pH, HCO3 -, glucose, K+, and CPK in CB returned to normal after 1hR whereas lactate decreased significantly but remained higher throughout reperfusion compared to cont rol values (Figures 7 1, 7 2). L actate and TP concentrations in JB remained increased during reperfusion in comparison to control samples (Figures 7 1, 7 2). After ischemia, the percentages of band neutrophils were increased in JB and CB, with further prog ression during reperfusion. This was accompanied by a relative increase of necrotic neutrophils during reperfusion (Table 7 1). The relative numbers of remaining neutrophils, lymphocytes, eosinophils, basophils and monocytes in JB and CB did not change dur ing I/R ; however lymphocytes were higher, and neutrophils were lower in CB compared to JB during I/R (Table 7 1 ). Mean c alprotectin concentrations in positive control serum samples of a healthy human (3896.3 221.0 ng/mL) in the present study were higher compared to plasma reference values of < 3000 ng/mL determined elsewhere in healthy humans (Table 7 2; Calprotectin ELISA kit ; Hycult Biotech, Plymouth Meeting, PA USA; PhiCal calprotectin; Immundiagnostik, Bensheim, Germany). E quine calprotectin in JB of post anesthesia

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158 control samples were higher than values of positive controls. There was a large individual variability in calprotectin concentrations among the horses of the present study (Table 7 2) and absolute calprotectin c oncentrations did not differ between JB and CB, and between treatment groups during I/R (Table 7 2) Howev er, relative percentages of calprotectin after 1hI, 1hR and 4hR in relation to control (defined as 100%) were significantly higher in CB compared to v alues in JB (Figure 7 3). Relative calprotectin concentrations in JB and CB did not change between treatment groups, but calprotectin trended to increase in CB, and decrease in JB after I/R (Figure 7 3). Discussion In the present study ischemia caused a p rominent but reversible metabolic acidosis within the affected part of the large colon, accompanied by disturbances in energy homeostasis (increased lactate, decreased glucose) and signs of cellular damage (increased K+ and CPK). Reoxygenation of tissues r esulted in normalization of these metabolic changes to baseline values ; however a mild elevation of lactate persisted that was only significant at 4hR Similar findings have been reported in several studies ( Moore et al. 1994c ; Kawcak et al. 1995). Moore e t al. (1994c) d emonstrated a d ecreased pH and increased lactate in CB after 30 min utes of ischemia that returned to normal within 5 min utes after reoxygenation. Experimentally induced 3 hours of ischemia caused significantly decreased pH, pCO2 and glucose, and increased lactate concentrations in CB of horses, and CB blood gas and lactate correlated with severity of mucosal damage in the equine colon (Kawcak et al. 1995). Additionally, ischemia induced a notable inflammatory response in the pres ent study characterized by activation of neutrophils (release of calprotectin) and increased neutrophil turnover (increased band neutrophils) with further progression after reperfusion.

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159 Our findings are consistent with the classic pathophysiological mechanisms of ischemia (Moore et al. 1995a, McMichael and Moore 2004). Cellular h ypoxia is associated with a decrease d pH and increased pCO2, and cause s local accumulation of lactic acid which is generated by anaerobic glucolysis (Moore et al. 1995a; Rowe and W hite 2002; McMichael and Moore 2004). Thus, decreased glucose concentration in CB after 1hI is probably caused by an increased demand and utilization of glucose as an energy substrate during ischemia (Moore et al. 1998 a ). Local changes of serum K+ and CPK as d etermined in the present study are consistent with the results published by Moore et al. (1998 a ). Increased K+ is generally seen after ischemic acidosis, and could be a result of release of intracellular K+ in response to accumulation of intracellular H+ ions and/or HCO3 to maintain electroneutrality and acid base homeostasis (Burnell et al. 1956; Fraley and Adler 1977; Moore et al. 1994c; Hoskote et al. 2008). However, increased K+ concentrations could also be secondary to ischemic cell necrosis (Graeber et al 1981; Fried et al. 1991; Mukai et al. 1995; Caglayan et al. 2002; Hoskote et al. 2008). In addition, necrosis of colonic mucosal cells and/or local intravascular cells might be responsible for increased CPK activities in CB after 1hI in the present study. Although the majority of CPK is found in the seromuscular layer of the gastrointestinal tract (Graeber et al. 1984), it is also diffusely distributed throughout the cytoplasm of inte stinal epithelial and endotheli a l cells (Wallimann and Hemmer 1994) This enzyme has been found in normal small and large intestines of dogs (Graeber et al. 1984) and release of CPK into circulation was determined within the first 3 hours after colonic ischemia in dogs and rabbits (Graeber et al. 1981; Caglayan et al. 2002). Some studies documented increased serum colonic venous blood and peritoneal fluid CPK activities

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160 associated with intestinal ischemia in horses ( Moore et al. 1998 a ; Nieto et al. 2005; Grosche et al. 2006) probably released by leakage from damaged cells (Boyd 1983) The explanantion that K+ and CPK are released by damaged intestinal cells after ischemia can be supported by the presence of single cell necrosis and small epithel ial defects present in the same colonic tissues after 1hI (Graham et al 2011; Grosche et al. 2011a,b). In agreement with previous studies ( Moore et al. 1998a ), colonic ischemia in the present study did not affect systemic metabolic homeostasis H owever co ntinuous generation of lactate and activation of neutrophils throughout reperfusion could be reflected systemically following colonic I/R (Klebanoff et al. 1986; Souza et al. 2000, 2001, 2002, 2005; Kaminski et al. 2002; Kalia et al. 2003). The decreased T P concentration in JB after I/R could be attributed to volume expansion after IV fluids throughout the study, or by protein loss through the damaged vascular endothelium during IR (Moore et al 1998a ). Possible explanations for the absence of systemic metabolic alterations in the present study could be the short ischemia time and small segments of bowel affected. Although 1hI was sufficient to cause structural changes and functional impairment of the colonic epithelium (Graham et al. 2011 ; Grosche et a l. 2011a,b), this short period of ischemia did not cause the degree of colonic injury that could affect systemic metabolic responses (Park et al. 1990; Moore et al. 1994c; Guan et al. 2009; Grootjans et al. 2010). Most of the modified biochemical variables in CB had normalized to baseline after reperfusion in the present study probably due to rapid restitution of the mucosal epithelium and improvement of barrier integrity after 4hR (Graham et al. 2011; Grosche

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161 et al. 2011a,b). However elevated lactate levels, activation of neutrophils and increased neutrophil turnover throughout reperfusion could indicate the persistence of a mild systemic inflammatory response, as documented in intestinal I/R in humans and other species (Murthy et al 1997; Souza et al. 2 000, 2001; Kaminski et al. 2002; Weiss and Evanson 2003; Franklin and Peloso 2006; Grootjans et al. 2010). In addition to increased band neutrophils in JB and CB, the response of the local immune system to colonic I/R was characterized by a decrease of th e percentages of neutrophils and accumulation of lymphocytes in CB compared with JB in the present study. However we did not evaluate absolute leukocyte numbers in JB and CB. As we previously reported, neutrophils accumulate in submucosal venules after 1hI 2hI, 30minR, 1hR and 2hR (Grosche et a l 2008; 2011b) Neutrophils migrate through the endothelium into the colonic lamina propria during reperfusion, and they move towards the damaged epithelium which was accompanied by a decrease d neutrophil number in submucosal venules after I/R over time (Grosche et al. 2008; Grosche et al 2011b). This could explain the decreased percentages of neutrophil s in CB, thus indicating neutrophil infiltration and activation of inflammation in the colonic mucosa after I/R (Granger and Korthuis 1995; Rowe and White 2002). Recruitment and activation of neutrophils is induced by microbial moieties that enter the dam a ged epithelial barrier, and/or chemotactic mediators, such as IL8, IL 17, TNF, IFN CSF released by mu cosal immune and nonimmune cells (Nathan 2006; Chin and Parkos 2007; Mantovani et al. 2011) In addition, cell necrosis is commonly seen after I/R (Guan et al. 2009) as demonstrated by the increase of necrotic neutrophil s in the differential in CB in the present study, and necrosis is thought to be responsible for activation of a

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162 systemic inflammatory response by release of cellular danger molecules and signals (Bianchi 2007; Kono and Rock 2008; Rock and Kono 2008). One of these danger molecul es released during inflammation is calprotectin, a cytoplasmic proinflammatory protein generated in activated neutrophils and monocytes (Johne et al. 1997; Kerkhoff et al. 1998; Kono and Rock 2008). In the present study calprotectin concentrations did not change significantly during I/R because of the high variability of calprotectin among the horses. There was a trend toward increased median calprotectin concentrations in CB after I/R compared to control values and to values in JB. However the percentages of calprotectin in comparison to the corresponding control values demonstrate d significant differences between CB and JB characterized by an increase of calprotectin in CB compared to the values in JB after I/R which supports the fact that attracted neut rophils became activated within the ischemic damaged colonic tissue during I/R (Moore et al. 1994a; Weiss and Evanson 2003; Grosche et al 2008, 2011b). C alprotectin release is a sensitive indicator of cytotoxicity (Johne et al. 1997). Activated neutrophi ls release this protein into the interstitium or blood by active secretion or passively during necrosis (Dale et al. 1985; Hetland et al. 1998; Voganatsi et al. 2001) The latter is consistent with the presence of necrotic neutrophils after I/R in the pres ent study. On the other hand, c alprotectin concentrations trend to decrease in JB during reperfusion probably due to leukocyte margination and compartmentalization within the inj ured segment of the colon. Alternatively systemic inflammatory stimuli might not be potent enough to cause calprotectin release by circulating neutrophils (Moore et al. 1994a; Gibson and Steel 1999; Striz and Trebichavsky 2004; Southwood

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163 2006). Longer ischemic and reperfusion times would more likely result in more prominent alterations of serum calprotectin in affected horses. In recent years, many studies documented activation of neutrophils after colonic I/R in horses based on increased mucosal and plasma MPO activities (Moore et al. 1994c; Grulke et al. 1999; Vatistas et al 1996; McConnico et al. 1999). Myeloperoxidase is a granulocytespecific enzyme that generates hypochlorous acid from hydrogen peroxide and chloride anion. It is used by granulocytes and monocytes to kill microorganisms with polysaccharidic capsules that are normally protected from granulocytederived proteolytic and hydrolytic enzymes (Klebanoff et al. 1984; Deby Dupont et al. 1999). However, available assays do not distinguish MPO activit ies in the different types of cells (Grisham et al. 1990b; Grulke e t al. 1999, 2008; McConnico et al. 1999). Because the equine colonic mucosa is repleted with eosinophils under healthy conditions (R tting et al. 2008a) calprotectin is superior to MPO to evaluate neutrophil activity in this organ in horses. With the development of an ELISA for equine calprotectin, we were able to quantify this proinflammatory neutrophilic protein for the first time in horses to the best of our knowledge. T he ELISA protocol, we developed would be suitable to measure calprotectin in serum and other body fluids of horses. The median calprotectin concentration in post anesthesia control blood samples of horses in the present study was higher than the positive human control samples. This could be a species related characteristic, or it might b e caused by activation of neutrophils during induction of anesthesia, and with beginning of the surgical procedure (Kalia et al. 2003). As in humans and dogs, equine calprotectin concentrations in JB control samples were

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164 variable among animals. S erum calprotectin concentrations in samples from healthy pet dogs ranged from 76 to 1292 ng/mL, with reference values stated to be 92 to 1221 ng/mL (Heilmann et al. 2008b). Due to the high var iability of equine serum calprotectin, single measurements of calprot ectin without appropriate control values may not allow characterization of the severity of an inflammatory response. Further studies are required to establish reference values for equine serum calprotectin, and to determine the importance of calprotectin i n horses with local or systemic inflammation. In the study reported here, local metabolic ac idosis within the large colon of horses caused by ischemia was associated with an increase in lactate, K+ and CPK and decrease in glucose in CB but not in JB Colonic metabolic changes returned to normal after reperfusion, and colonic mucosa recovered histologically and functionally, despite persistence of inflammation. Serum calprotectin can be used as a marker of neutrophil activation and inflammatory activity after colonic I/R in horses.

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165 Table 7 1 Differentiation of leukocytes (% of total leukocytes ) in JB and CB of post anesthesia controls and after I/R Treatment Post anesthesia control 1hI 1hI +1hR 1hI+2hR 1hI+4hR Kruskal Wallis Blood JB CB JB CB JB CB JB CB JB CB JB CB Lymphocytes 40.00 (2.67) 46.33 (5.43) 35.00* (3.45) 49.60* (5.05) 30.00 (2.10) 45.50 (6.14) 31.50* (3.51) 51.00* (5.22) 31.67* (3.91) 43.17* (4.73) Neutrophils 54.50 (2.46) 47.67 (5.12) 53.00* (4.78) 37.60* (6.34) 53.17 (2.48) 42.25 (7.54) 50.50 (4.39) 34.20 (4.36) 48.17* (2.71) 36.00* (3.49) Band Neutrophils 3.17a (0.70) 2.67a (0.88) 8.67b (1.73) 6.80b (1.16) 11.83abc (1.96) 6.00ab (0.41) 14.33cd (1.52) 8.40abc (2.64) 17.00d (1.61) 16.50c (1.73) 0.001 0.003 Necrotic Neutrophils 0 (0) 0.83 (0.48) 1.00 (0.63) 3.40 (0.81) 1.50 (0.62) 3.75 (2.25) 0.83* (0.54) 4.40* (0.98) 0.67* (0.33) 3.50* (1.38) Eosinophils 0.33 (0.21) 0.67a (0.21) 0.67 (0.33) 1.00a (0.32) 1.17 (0.48) 0.25ab (0.25) 1.33 (0.62) 0.20ab (0.20) 0.50 (0.22) 0b (0) 0.034 Basophils 0 (0) 0.50 (0.50) 0 (0) 0 (0) 0.33 (0.21) 0 (0) 0.17 (0.17) 0 (0) 0 (0) 0 (0) Monocytes 2.17 (0.65) 1.33 (0.49) 1.50 (0.62) 1.20 (0.20) 2.17 (0.6) 2.25 (0.25) 1.33 (0.62) 2.00 (0.55) 2.00 (0.58) 0.83 (0.54) Note: Mean (SEM); Kruskal Wallis test for sample comparison between treatment groups; MannWhitney U test for pair wise comparison (different letters represent significant differences between treatment groups); related samples Wilcoxon test for comparison between JB and CB in each treatment group ( asterisk represents significant differences between JB and CB in this treatment group, *P<0.05).

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166 Table 7 2 Calprotectin concentrations (ng/mL) in JB and CB of post anesthesia controls and after I/R Treatment Post anesthesia control 1hI 1hI+ 1hR 1hI+ 2hR 1hI+ 4hR Positive control Blood JB CB JB CB JB CB JB CB JB CB VB Mean 6343.38 4909.55 4961.13 5512.57 4552.80 5657.50 4762.10 4655.65 3921.06 5142.44 3896.30 SEM 2204.25 1365.81 2016.66 1825.15 1919.88 2065.13 1906.16 1239.17 1488.11 1603.79 221.02 Median 4946.58 4699.23 2599.81 6093.55 3477.86 6886.41 3577.89 4964.88 2563.12 4581.68 3754.86 Minimum 80.25 104.93 46.82 211.56 55.08 252.4 89.04 114.77 111.14 139.40 3387.81 Maximum 14825.11 8581.57 11735.85 9864.95 13071.16 11095.55 12743.50 8560.11 10160.21 11012.08 4799.76 CI 95% 12009.59 677.18 8420.47 1398.64 10145.13 222.87 10580 445.15 9487.99 382.40 11391.22 76.22 966.05 137.84 7841.03 1470.27 7746.36 95.75 9265.12 1019.76 4464.45 3328.15 Note: M ean SEM, median, minimum and maximum values, and confidence interval ( CI ) at 95% ; Kruskal Wallis test for sample comparison between treatment groups not significant; related samples Wilcoxon test for comparison between JB and CB in each treatment group not significant; Positive control blood serum samples obtained from the dorsal venous arch of one healthy human; VB venous blood

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167 Figure 71 Values for pH, pCO2, HCO3 -, BE, and PCV in JB and CB of post anesthesia controls and after I/R; mean (SEM); Kruskal Wallis test for sample comparison between treatment groups, JB: lactate P = 0.04; CB: pH P = 0.011, pCO2 P = 0.004, HCO3 P = 0.011, BE P = 0.009, lactate P = 0.001; MannWhitney U test for pair wise comparison (different letters represent significant differences between treatment groups); related samples Wilcoxon test for comparison between JB and CB in each treatment group ( above JB CB pairs represents significant differences between JB and CB in this treatment group, P<0.05)

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168 Figure 72 Values for TP, albumin, glucose, Na+, K+, Ca2+, creatinine, CPK, and ALP in JB and CB of post anesthesia controls and after I/R; mean (SEM); Kruskal Wallis test for sample comparison between treatment groups, JB: TP P = 0.012, ALP P = 0.024; CB: glucose P = 0.028, K+ P = 0.007, CPK P = 0.02; MannWhitney U test for pair wise comparison (different letters represent significant differences between treatme nt groups); related samples Wilcoxon test for comparison between JB and CB in each treatment group ( above JB CB pairs represents significant differences between JB and CB in this treatment group, P<0.05).

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169 Figure 73 C oncentrations (%) of serum calprotectin after I/R compared to control samples (= 100%) in JB and CB; mean (SEM); dashed line median; Kruskal Wallis test not significant; related samples Wilcoxon test for comparison between JB and CB in each treatment group ( above JB CB pairs represents significant differences between JB and CB in this treatment group, P<0.05).

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170 CHAPTER 8 CONCLUSIONS Mucosal S tructur e, Function, and Repair A fter Colonic I / R Morphological Changes Ischemic injury from large colon volvulus is an important cause of mucosal damage in horses. Structural changes of the colonic mucosa after I/R in the present study are similar to findings in pathological situations with hemorrhagic strangulation obstructi ons or low flow ischemia (Reeves et al. 1990). At 1hI minor histological alterations of the colonic mucosa were evident with further progression after 2hI (Morton et al. 2009; Graham et al. 2011; Grosche et al. 2011a,b,c). Moderate hemorrhage into the lam ina propria proceeding over time during ischemia was accompanied by profound interstitial and epithelial cell edema and increased ICR. Tissue injury usually begins in the superficial part of the mucosa within 20 minutes of total ischemia, and within 60 min utes if partial ischemia is severe enough to cause damage (Moore et al. 1995a). Lifting of epithelial cells due to fluid sequestration in the subepithelial space and loosening from the basement membrane were characteristic features at the beginning of muco sal injury in the present study ( Snyder et al. 1988; Moore et al. 1995a; Kong et al. 1998; Haglund and Bergqvist 1999). With prolonged ischemia further damage to the surface epithelium occurs as demonstrated by loss of 45% of the surface epithelium after 2hI (Morton et al. 2009). The separation progresses downward to the crypts over time, and epithelial cells are irreversibl y lost. The colonic epithelium is completely denuded after 4 hours of low flow ischemia, and complete necrosis of the mucosal epitheli um extending to the base of the crypt s occurs by 4 to 6 hours (Snyder et al. 1988; Moore et al. 1995a).

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171 Although necessary for recovery reperfusion can paradoxically create more injury than ischemia. It is generally assumed that increased release of ROS generated by XO is responsible for progression of tissue damage after reperfusion ( Schoenberg and Beger 1993; Darien et al. 1995). The reperfusion component is more pronounced after partial than total intestinal ischemia, and only the superficial mucosa is subjected to detectable reperfusion injury (Haglund et al. 1987; Park et al. 1990). If the ischemic injury is severe enoug h to cause injury to deeper mucosal layers, further damage can probably not be identified at reperfusion (Haglund 1994). Based on reperfusion time periods chosen in the present study there was no notable exacerbation of mucosal damage after 1hI and 2hI followed by reperfusion times of up to 18 hours in the equine colon (Morton et al. 2009; Graham et al. 2011; Grosche et al. 2011a,b). Our findings are consistent with results of Reeves et al. (1990), who could not show reperfusion injury in the equine colon following 1 hour of low flow ischemia. They assumed that 1hI might be too short to induce enough pathological changes to detect a reperfusion effect. However, a longer period of ischemia as chosen in previous studies ( Grosche et al. 2008; Matjyaszek et al. 2009; Morton et al. 2009) might cause injury that is already too severe to generate further deterioration of tissue damage. T he depth of mucosal loss and the mucosal cellular debris index progressed after 3 hours of experimental low flow ischemia followed by 3 hours of reperfusion in a study by Moore et al. (1994 b ) thus indicating some reperfusion injury in the equine large colon. However, different experimental designs of I/R, different I/R times, and different segments of the colon introduced to ischemia make it difficult to compare the findings with other studies (Reeves et al. 1990; Mesc h ter et al. 1991; Moore et al. 1994b; Darien

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172 et al. 1995; Moore 1997). Thus the question remains whether reperfusion exacerbated mucosal damage in the colon, or whether much of the injury observed during reperfusion was a continuation of injury during ischemia. Although short term ischemia causes only minor gross epithelial alterations as demonstrated in the present study, it can result in epithelial barrier dysfunction possible due to metabolic and ultrastructural changes of single epithelial cells not seen by routine LM (McAnulty et al. 1997; Sun et al. 1998; Graham et al. 2011). Sun et al. (1998) calculated a strong correlation between short ischemic times of 20 and 40 minutes and epithelial barrier disruption in rats. In the present study, the injury to mucosal barrier function caused by 1hI was sufficient to decrease TER and increas e transmucosal mannitol flux in the same colonic tissues (Graham et al. 2011). Although morphology of the epithelium did not return completely to normal at the end of reperfusion, 4hR was sufficient to initiate epithelial repair in the current study (Graha m et al. 2011; Grosche et al. 2011a b). In small intestine, r estor ation of epithelial continuity is initiated by villus contraction, coverage of the exposed basement membrane by epithelial cells and closure of leaky intercellular spaces and TJ within minutes after injury ( Florian et al. 2002; Derikx et al. 2008 ) This rapid process of epithelial restitution is regulated by lamina propria mediators arising from nerves, immune cells, fibroblasts, endothelial cells, and extracellular matrix ( Florian et al. 2 002; Playford and Ghosh 2005; Blikslager et al. 2007). The acute events are followed 18 to 24 hours later by increased crypt cell proliferation resulting in replacement of lost cells and reconstruction of villus architecture (Blikslager et al. 2007). Epith elial lifting of the small intestine returned to normal within 6

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173 hours after I/R injury, and severely damaged intestinal epithelium healed rapidly (Park and Haglund 1992; Haglund 1994). Small intestine that had lost surface epithelial cells were covered wi thin 18 hours and mucosal repair after 18hR in a previous study in equine colon followed the same pattern of repair (Morton et al. 2009). The rapid repair process initiated in the present study was characterized by coverage of single cell defects by apica l membrane extensions from neighboring cells (Grosche et al. 2011a,b) Th e morphological expression of repair in the present study could explain recovery of 2 2 after 4hR in the same mucosal tissues in Ussing chambers (Graham et al. 2011). In the current study we used TB stained semithin sections to improve the evaluation of colonic tissues by LM This special tissue processing and staining method enhanced assessment of epitheli al changes with 1000x magnification. We were able to characterize particular structural changes of single epithelial cells such as impaired microvillus integrity, dilated paracellular spaces, subepithelial cleft formation and single cell necrosis after ischemia These could explain cellular dysfunction and intestinal barrier failure, and would not have been detected in routine H&E sections (Grosche et al. 2011b). However, electron microscopic examination of the mucosa was superior in demonstrating typical ultrastructural alterations of individual epithelial cells in response to the short hypoxic conditions (Grosche et al. 2011a). Ultrastructural Changes Based on TEM findings in the present study, ultrastructural changes in epithelial cells after 1hI were characterized by swelling and vacuolation of cell organelles, and alterations of nuclei displaying features of early apoptosis (Labat Moleur et al. 1998) These changes are comparable with the classic cellular pathology caused by hypoxia

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174 (Meschter et al. 1991; Snyder et al. 1992; McAnulty et al. 1997; Labat Moleur et al. 1998). Disintegration or dilation of TJ between epithelial cells could explain epithelial barrier failure after ischemia (Graham et al. 2011). Autophagy was a prominent feature in epithelial cells after ischemia in TEM sections in the present study Autophagy is a homeostatic process that removes damaged or surplus organelles, supplies nutrients and energy, eliminates intracellular pathogens and toxic proteins, and deli vers endogenous antigens for presentation to immune cells (Levine and Deretic 2007; Levine and Kroemer 2008). Cellular conditions such as energy starvation, oxidative stress, mitochondrial damage and inflammation are thought to stimulate autophagy. Amino acids or fatty acids recovered through autophagy may be used for ATP production, and misfolded proteins and damaged mitochondria m ay be removed under hypoxic conditions (Sadoshima 2008). S evere I/R injury can induce marked upregulation of autophagy resulting in uncontrolled self digestion by lysosomal enzymes and eventual cell death (Sadoshima 2008). Although increased autophagy in epithelial cells might be responsible for single cell death after ischemia in the present study (Grosche et al. 2011a), it might have improved cell survival during hypoxia, as evident by restitution and barrier recovery within 4hR (Graham et al. 2011). Cove ring epithelial clefts with membrane extensions of intact neighboring cells and closure of TJ between detached epithelial cells could explain early recovery of intestinal barrier function after 4hR (Graham et al. 2011; Grosche et al. 2011a). This rapid sel f sealing response by epithelial cells usually begins within 15 minutes after injury, and allows neighboring cells to reestablish cell to cell contacts and restore epithelial integrity by regulated cell migration, independent of cell proliferation (Wilson and Gibson 1997;

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175 Mammen and Matthews 2003; Blikslager et al. 2007). Moreover, studies by McNeil and Ito (1989) demonstrated that wounded epithelial cells seem to participate in restitution of the injured mucosa by extending membrane pseudopodia over denuded lamina propria. This process could play an important role in epithelial restitution after colonic I/R, because most of the epithelial cells seen after 1hI in the present study exhibited features of structur al changes and injury. Additionally epithelial cells displayed phagocytic activity as demonstrated by detection of intracellular phagocytic vacuoles during reperfusion in the present study (Grosche et al. 2011a). However it is not clear whether phagocytosis of foreign material is used to provide more nutrients and energy during reperfusion, or to sample the microenvironment for regulation of innate and adaptive immune responses (Artis 2008). It has been shown that epithelial cells can phagocytize adjacent cells, apoptotic cells and bacteria (Morris and Harding 1979; Sexton et al. 2001; Monks et al. 2005; Neal et al. 2006) thus they could control the inflammatory response and minimize injury after I/R in horses. Cell Death Previous studies hypothesized cellular necrosis as a morphological manifestation of mucosal damage and therefore the principal effect of ischemia on epithelial cells (Shah et al. 1997a). During the last two decades however, apoptosis has been recognized as a major form of cell death distinct from necrosis. R ecent studies have shown that ischemiainduced injury to the gut responded by undergoing programmed cell death rather than necrosis, and the degree of apoptosis depended on the differentiation stage of enterocytes ( Kerr et al. 1972; Robinson et al. 1981; Mitsudo and Brandt 1992; S hah et al. 1997b ; Ikeda et al. 1998; Noda et al. 1998; Hinnebusch et al. 2002). Apoptotic cell death can be found under physiological and pathological

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176 conditions and it occur r s when cells receive deathinducing stimuli such as lack of survival or growth factors, metabolic defects, oxygen deficiency, chemical and physical damage and/or binding of ligands to death receptors (Haanen and Vermes 1995). However, it is still unclear whether apoptosis contributes to epithelial injury or resolves inflammation and hastens epithelial repair (Ramachandran et al. 2000). The results of the present study support the hypothesis that apoptosis h as an important role in cell death during I/R in the equine colonic mucosa N ecrosis contribut e d less than one third to epithelial c ell deaths in the present study (Grosche et al. 2011b) There are several standard techniques to determine the apoptotic rate in tissues such as electron microscopy, the TUNEL method, and flow cytometry (Gavrieli et al. 1992; Martinez et al. 2010). In addi tion to TEM and the classic TUNEL method used in the present study (Grosche et al. 2011a,c) we could clearly identify and count apoptotic cells in the colonic epithelium i n semithin sections stained with TB (Grosche et al. 2011b). The TUNEL method is a technique that can be used to identify apoptotic cells in various tissues by using TdT to transfer biotinylated nucleotides to free DNA termini of cleaved DNA which is generated upon DNA fragmentation in nuclei of apoptotic cells and in apoptotic bodies (Gavrieli et al. 1992; Otsuki et al. 2003). This method can detect early stages of apoptosis in cells where chromatin condensation has begun and strand breaks are fewer, even before the nucleus undergoes major morphological ch anges (Labat Moleur et al. 1998; Stadelmann and Lassmann 2000). However, cells exhibiting necrotic morphology may stain lightly or DNA fragmentation can be absent or incomplete in induced apoptosis. Therefore, the staining results have to be evaluated in conjunction with morphological criteria. For example, we could clearly recognize nuclear

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177 chromatin condensation and margination with the help of TB stained semithin sections These are characteristics of apoptotic cells that have been identified with the TU NEL method on I/R injured colonic tissues ( Kroemer et al. 2009; Grosche et al. 2011b,c). We could demonstrate that apoptotic cell death started at 1hI in the epithelium, and in lamina propria cells, and persisted during reperfusion (Grosche et al. 2011b,c) Toth et al. (2007) found a maximum increase in apoptosis after 1hI followed by 1hR in rat small intestine. The lowest level of apoptosis was observed after 24 h our of reperfusion accompanied by highest levels of mitosis as evidence of regeneration. Numer ous studies verified that apoptosis can contribute to leaks in the epithelial barrier and permeability defects (Abreu et al. 2000; Gitter et al. 2000; Baylor et al. 2003; Schulzke et al. 2006), which w ere associated with proteolytic cleavage of TJ proteins and disruption of TJ (Bojarski et al. 2004). However, despite a loss of 50% of epithelial cells by apoptosis and a critical decrease in TER in vitro the remaining epithelial cells were only permeable to small molecules such as mannitol and lactulose, but not to large molecules such as dextran and polyethylene glycol probably due to unique junctional rearrangements that compensated for cell loss (Abreu et al. 2000; Bojarski et al. 2001). Although numerous studies demonstrated activation of apoptosis during intestinal ischemia with further exacerbation during reperfusion (Noda et al. 1998; Baylor et al. 2003; Rowe et al. 2003; Fujise et al. 2006), the role of programmed cell death on mucosal damage remains controvers ial (Vaux and Haecker 1994). Apoptosis plays an important role in intestinal epithelial homeostasis, and failure to clear unwanted cells by apoptosis induces inflammation and promotes tissue injury (Haanen and Vermes 1995; Serhan and Savill 2005; Green et al. 2009). Additionally, apoptosis of immune cells is

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178 essential for regulation of immune responses, and engulfment of apoptotic cells primes macrophages to produce anti inflammatory mediators including TGF 10, PGE2 (Sun and Shi 2001, Savill et al. 2002; Maderna and Godson 2003; Serhan and Savill 2005, Erwig and Henson 2007; Maniati et al. 2008; Green et al. 2009). Thus, apoptosis could be a mechanism that protects the tissue from harmful exposure to inflammatory and immunogenic cell contents during reperfusion (Maderna and Godson 2003). Inflammatory Response Nitrotyrosine The present findings emphasize that the inflammatory response associated with colonic I/R might be a result of the combination of several factors, which includes tissue dam age caused by toxic ROS and RNS (Lefer and Lefer 1993; Haanen and Vermes 1995; Carden and Granger 2000; Denning et al. 2002). Ischemia caused generation of RNS in the colonic mucosa as demonstrated by increased production of nitrotyrosine in the present study and eosinophils and other leukocytes were the main source of nitrotyrosine (Grosche et al. 2011c). Nitrotyrosine is considered to be a potential marker of peroxynitrite generation in tissues (Ischiropoulos 1998). However, nitrotyrosine is also gener ated by other molecules such as nitrate, NO nitrogen dioxide or nitryl chloride (Wallace and Ma 2001). Nitrotyrosine is formed from nitrite in the presence of hypoclorus acid in neutrophils, and 3nitrotyrosine is generated after infiltration of neutrophi ls independent of peroxynitrite (Eiserich et al. 1998; Wallace and Ma 2001). Therefore, detection of nitrotyrosine should be regarded as evidence of generated RNS rather than as a specific marker of peroxynitrite.

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179 Macrophages, endothelial cells and neutrophils are major biological sources of RNS during inflammatory processes (Radi et al. 1991b; Bian and Murad 2001). Gagnon et al. (1998) demonstrated the ability of leukocytes to produce high amounts of peroxynitrite in response to lipopolysaccharide consist ent with the findings of the current study. Moreover, mucosal leukocytes of horses with naturally acquired small intestinal strangulation obstruction generated increased amounts of nitrotyrosine, which reflects increased NO and superoxide production (Mirza et al. 1999). The investigators hypothesized a possible cytotoxic role of NO in small intestinal strangulation obstructions in horses. Although the ischemic models used in the present study mimic ischemic conditions in naturally occurring large colonic volvulus (Snyder et al. 1988), nitrotyrosine generation during ischemia could be caused by some persistent arterial flow, especially shortly after clamp application (Grosche et al. 2008). This might explain the generation of nitrotyrosine with the start of ischemia with out further progression after reoxygenation (Grosche et al. 2011c). Peroxynitrite is a highly reactive but short lived RNS that could contribute to tissue injury during intestinal I/R in the horse ( Beckman et al. 1990; Ischiropoulos et al. 19 92; Grisham et al. 1999; Mirza et al. 1999; Kubes and McCafferty 2000; Takemoto et al. 2007; Goldstein and Merenyi 2008; Kono and Rock 2008). Peroxynitrite increases lipid peroxidation and nitration, and protein and DNA modifications that could cause acut e and severe cellular and energetic derangements, and cell death by apoptosis and necrosis (Baskin and Salem 1997; Cerqueira et al. 2005; Szabo et al. 2007). Administration of peroxynitrite into the colon produced widespread injury and inflammation as seen in inflammatory bowel disease (Rachmilewitz et al. 1993), and

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180 iNOS might be responsible for tissue injury by generation of NO dependent nitrating species (Haddad et al. 1994; Shah et al. 2004). Thus, overproduction or uncontrolled formation of RNS could be an important factor in reperfusion injury in the equine colon (Muijsers et al. 1997). Although their roles as agents of cytotoxicity is widely accepted, oxidants can also serve as impor tant mediators of specific cellular and molecular responses and expression of genes involved in inflammation (Aw 1999; Zweier and Talukder 2006). Lefer et al. (1997) demonstrated an inhibition of P selectin expression by peroxynitrite and protection agains t myocardial I/R injury in rats. B ecause of its limited extracellular stability and diffusion range, one of the major physiological actions of peroxynitrite might be the cytotoxic activity towards invading pathogens, and it is thought to be a potent microb icidal compound (Muijsers et al. 1997). This can explain the second peak production of nitrotyrosine by submucosal leukocytes after 18hR which could be an immunoregulatory reaction or it might be a defense mechanism due to transepithelial passage of bacter ia or bacterial products during reperfusion (Grosche et al 2011c). Cyclooxygenase Numerous factors that control the inflammatory response are released by epithelial cells themselves, or they are produced by a variety of mucosal immune and other cells. Pro staglandins, synthesized by COX enzymes in numerous cells of the intestinal lamina propria (Krause and DuBois 2000; Morton et al. 2009; Hilton et al. 2011) play a key role in regulating inflammatory reactions (Krause and DuBois 2000), and COX 2 is known t o be rapidly induced at sites of inflammation in the colon of horses (Matyjaszek et al. 2009; Morton et al. 2009; Grosche et al. 2011b). Although COX enzymes regulate the production of potent proinflammatory PG s ( Sakamoto 1998; Crofford 2001), evidence

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181 is growing that COX 2 expression may contribute to resolution of gastrointestinal inflammation, and it might be crucial in regulating mucosal healing (Eberhardt and DuBois 1995; Blikslager et al. 1999; Krause and DuBois 2000; Wallace and Devchand 2005; Fukut a et al. 2006 ; Little et al. 2007b ). COX 2 expression in the epithelium and by lamina propria immune cells was significantly upregulated after ischemia and after 1hR in the present study, a time point where epithelial repair began (Grosche et al. 2011b) T hese findings are similar to what we found in a previous study where 1hI caused upregulation of COX 2 mainly in epithelial cells with minimal changes in COX 1 expression. Generation of COX 2 continued, and was associated with mucosal restitution after 18hR (Morton et al. 2009). Shifflett et al. (2004) found enhanced recovery of barrier function in porcine ischemic injured ileal mucosa mediated by upregulation of COX 2 and activation of neutrophils. In most studies COX 2 expression was identified in lamina propria immune cells, such as neutrophils, macrophages, lymphocytes and mast cells, and generation of COX 2 derived PGD2 was associated with inhibition of granulocyte infiltration in a rat colitis model (Reuter et al. 1996; Wallace 2006). Moreover, it is thought, that upregulation of COX 2 in epithelial cells controls cell function, protection and signaling (Morton et al. 2009), and COX 2 dependent PGE2 production is essential for colonic epithelial proliferation, migr ation and homeostasis in response to injury (Krause and DuBois 2000; Brown et al. 2007). Thus, increased COX 2 expression after ischemia in the present study could be a possible response of the colonic epithelium to prevent further damage and initiate earl y recovery after reoxygenation (Savill et al. 2002; Karrasch et al. 2006).

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182 Inflammatory Cells Cells of the innate immune system are the main initiators of inflammation after intestinal I/R ( Medzhitov 2007, 2008; Schenk and Mueller 2008). Whether macrophages, neutrophils, eosinophils, lymphocytes, or other immune cells predominate in I/R injury is determined by the expression of specific molecular signals that control traffic of particular leukocyte classes (Jefferies et al. 2003). At the beginning of an inf lammatory response, resident macrophages and infiltrating monocytes immediately recognize damageassociated signals, invading bacteria and toxins (Duffield 2003; Bianchi 2007; Kono and Rock 2008; Smith et al. 2011). Alerted by these signals, macrophages at tract large numbers of leukocytes to the site of injury to assist in recognizing, ingesting and/or destroying the invading agents (Platt and Mowat 2008; Smith et al. 2005 2011). Neutrophils are critical elements in the cascade of intestinal I/R injury and barrier dysfunction (Gayle et al. 2002; Rowe and White 2002; Blikslager et al. 2007). However, mucosal eosinophils mast cells and macrophages have also been proven to be potential effector immune cells during intestinal I/R in numerous studies (Kanwar and Kubes 1994b; Mahida et al. 1997; Szabo et al. 1997; Kanwar et al. 1998; Kimura et al. 1998; Boros et al. 1999a,b; Torihashi et al. 2000; Chen et al. 2004; Furuta et al. 2005; Smith et al. 2005; Anand et al. 2008; Santen et al. 2008; Laskin et al. 2011). Mast Cells Although the number of mast cells did not change during colonic I/R, they displayed features of degranulation after 1hI evident by the decreased number of intracellular granules in subepithelial mast cells and their sub membranous localization in the present study (Grosche et al. 2011b) These findings support the fact that effector mast cells could also regulate inflammation after intestinal I/R by releasing proand

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183 anti inflammatory cytokines, and chemokines (Stent on et al. 1998; Boros et al. 1999b; Penissi et al. 2003; Galli et al. 2008; Santen et al. 2008). Int estinal mast cells contribute to mucosal permeability alterations during reperfusion in the canine small intestine, but they might play only a minor role in I/R induced structural changes (Szabo et al. 1997). In contrast, Boros et al. (1999a) found initiation of tissue injury after mast cell degranulation during intestinal I/R, with release of histamine contributing most to the severity of mucosal damage. Additionally, mast celldependent secretion of chemokines regulates I/R induced leukocyte recruitment in the colon (S anten et al. 2008). Although subepithelial mast cells appeared to respond to ischemia by degranulation in the present study, their role during colonic I/R in the horse remains uncertain. Eosinophils Eosinophilic granulocytes can be seen in close proximity to mucosal mast cells, and degranulation of mast cells is thought to be triggered by eosinophilic toxic proteins (Piliponsky et al. 1999). Un like in humans where eosinophil accumulation within the gastrointestinal tract is a common feature of eosinophilic gastrointestinal disorders (Rothenberg et al. 2001), the eosinophilic granulocyte is resident in the gastrointestinal lamina propria in horse s under healthy conditions with different pattern of distribution throughout the intestines (Rtting et al. 2008a). In the equine colon, most of the eosinophils were found near the muscularis mucosae and were rarely located close to the surface of the muc osa consistent with our findings (Grosche et al. 2011b,c). However with beginning of ischemia and in some extent during reperfusion the absolute number of mucosal eosinophils decreased while migrating towards the epithelium because of releasing into the l umen or by apoptosis (Grosche et al. 2011b,c). Eosinophils are important inflammatory cells of the innate immune system, and the

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184 stimulatory effect of eosinophil major basic protein on activation of neutrophils and IL8 production has been demonstrated in several studies (Haskell et al. 1995; Page et al. 1999). However Meschter et al. (1986) assumed that eosinophils may also play an essential role in suppression of inflammatory reactions as the number of eosinophils in the intestinal mucosa was significant ly greater in horses that survived naturally occurring strangulation obstructions. As one of the main sources of nitrotyrosine in the current study, eosinophils could have key functions in responding to ischemia in equine colon. It is known that eosinophils are equipped with enzymes that inflict oxidative damage upon biological targets (Van Dalen et al. 2006). They are also involved in tyrosine nitration, and form ation of peroxynitrite and peroxidase catalyzed reactive nitrogen species from eosinophils is implicated in the lung damage in asthma patients (Saleh et al. 1998; MacPherson et al. 2001; Van Dalen et al. 2006). Although we could demonstrate an increased generation of nitrotyrosine in eosinophils during ischemia and reperfusion, the reason of their accumulation and migration, and their biological role within the equine colonic mucosa during I/R remains unknown. Despite the activation of eosinophils observed during I/R in the present study, a general effect of eosinophils on epithelial injury and barrier dysfunction could not be identified under conditions of the study Macrophages M acrophages are well established effector cells with proand anti inflammator y activities that could also contribute to the inflammatory response (Duffield 2003). Activated macrophages inhibit formation of enterocyte gap junctions in vitro (Anand et al. 2008), and influence early mucosal damage during intestinal I/R in rats by expr ession of MPO Egr 1 gene and proinflammatory cytokines before neutrophil

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185 infiltration occurs (Chen et al. 2004). Macrophages are also crucial for recognition and clearance of necrotic debris and apoptotic neutrophils, an essential step in resolving infla mmation (Savill et al. 2002; Serhan and Savill 2005; Erwig and Henson 2007; Laskin et al. 2011). Although the number of mucosal macrophages did not change during I/R in the present study (Grosche et al. 2001b), histological evaluation of the tissues demons trated an increased phagocytic activity of resident macrophages during I/R thus suggesting a possible role during recovery from ischemic injury in horses (Grosche et al. 2011a,b) Neutrophils In the current study, neutrophils accounted for most of the leukocytes infiltrating the colonic mucosa, and we could demonstrate that neutrophils are involved in the inflammatory response after colonic ischemia in horses (Grosche et al. 2008; Grosche et al. 2011a,b). Ne utrophils play a dual role in inflammation, combining proinflammatory properties and effects on resolution of inflammation (WitkoSarsat et al. 2000; Serhan and Savill 2005; Nathan 2006; Blikslager et al. 2007; Chin and Parkos 2007). Based on our findings movement of neutrophils into colonic mucosa during I/R followed a pattern that could be closely correlated with the sequence of pathophysiological events proposed for reperfusion injury (Moore et al. 1994a, 1995a). The number of neutrophils within submuc osal venules increased in a timedependent manner during ischemia, and peaked after 1hR (Grosche et al. 2008, 2011b) Apparently neutrophils could reach submucosal and mucosal vessels continuously in a small amount from some persistent but much reduced blo od flow during ischemia. As tissue damage progressed after occlusion of the arterial blood flow the permeability of the venule endothelium increases over time, and cells within the vessel lumen can move across the damaged vessel wall

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186 into the surrounding tissue (Henninger et al. 1992; Darien et al. 1993; Dabareiner et al. 1995). In addition, release of local inflammatory mediators trigger s endothelial cell responses that led to margination, rolling, adhesion, diapedesis, and migration of neutrophils already in the vasculature at the time ischemia started (Moore et al. 1995a; Granger 1997; Chamoun et al. 2000; Olanders et al. 2002; Seal and Gewertz 2005; Chin and Parkos 2007). The number of neutrophils increased after restoration of blood flow in the present study (Grosche et al. 2008, 2011b), which would bring additional cells to the injured tissues. Furthermore, we could demonstrate that, after 4hR neutrophils disappear from submucosal venules because of transmigration and accumulation within the adjacent t issue (Grosche et al. 2011b). Neutrophils travel into the mucosal lamina propria during reperfusion, triggered by release of pro inflammatory chemoattractants at the side of injury ( Witko Sarsat et al. 2000; Luster et al. 2005). The peak neutrophil infilt ration seen after 2hR and 18hR was accompanied by epithelial repair and restitution of the epithelial barrier function (Grosche et al. 2008, 2011a,b ; Graham et al. 2011). Neutrophil accumulation in the colonic mucosa declined after 4hR possible due to timedependent expression of different leukocyte adhesion molecules. P Selectin plays a key role in transendothelial migration of neutrophils during I/R ( Chamoun et al. 200 0 ). It is constitutively stored in Weibel Palade bodies of endothelial cells, and can be translocated to the cell surface by activation with thrombin, histamine, hydrogen peroxide and inhibition of NOS (Chamoun et al. 2000; Lefer 2000). Expression of P sel ectin on endothelial cell surfaces occurs about 10 to 2 0 minutes following reperfusion (Weyrich et al. 1995; Lefer 2000), which promotes increased adhesion of neutrophils, and initiates rolling of neutrophils along the endothelium, the

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187 first step in leukoc yte recruitment (Butcher 1991). Adhesion of neutrophils and transendothelial migration slows within 180 minutes post reperfusion, which could explain decreased neutrophil adherence in submucosal venules and decreased number of neutrophils in the lamina propria after 4hR in the present study (Lefer 2000; Grosche et al. 2011b). New P selectin can be expressed only after upregulation of transcription selectin to the cell surface later in time (Lefer 2000). The second adhesion molecule that may also play a role in later stages of reperfusion is E selectin. E selectin is expressed on endothelial cells only after de novo synthesis following activation of endothelial cells by TNF a process that requires 48 hours and returns to baseline by 24 hours (Bevilacqua et al. 1989; Lasky 1995; Chamoun et al. 2000; Lefer 2000). Thus de novo synthesis of P selectin and expression of E selectin could explain the second peak of neutrophil acc umulation in mucosal tissues after 18hR in the present study (Grosche et al. 2008). In similar fashion, Little et al. (2005) found an increased neutrophilic inflammation within all intestinal layers of equine jejunum at 18 hours after ischemia. We did not examine reperfusion time periods between 4 hours and 18 hours, so that mucosal neutrophil traffic during that period is unknown. D espite the intense influx of neutrophils during reperfusion, and the presence of neutrophils within the intercellular space and TJ after 4hR ( Grosche et al. 2011a,b) the colon did not display any evidence of impaired barrier function at this time, as determined by TER and transmucosal mannitol flux (Matyjaszek et al. 2009; Graham et al. 2011). Our results agree with those of Bli kslager et al. (1997b) who found massive infiltration of neutrophils during initial stages of epithelial repair in a porcine ischemic model. They hypothesized that mucosal

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188 injury is more likely triggered by physical damage to the repairing epithelium at this stage (Gayle et al. 2002). Milks et al. (1986) concluded that epithelial integrity can be affected by the emigration of neutrophils, but this effect is either completely or partially reversible within 65 minutes consistent with the findings of the present study (Graham et al. 2011; Grosche et al. 2011b). Moreover, activated neutrophils that are recruited to the site of injury can secrete anti inflammatory and proresolution factors that promote repair (Cassatella 1995; Mammen and Matthews 2003; Serhan and Savill 2005; Nathan 2006; Blikslager et al. 2007; Serhan et al. 2008). In addition, apoptosis of neutrophils, and their clearance by inflammatory macrophages, is an essential step in inflammation reduction and initiation of repair (Savill et al. 2002; Simon 2003). We cannot rule out possible short term damage to the epithelium by migrating neutrophils in the present study, although mucosal TER recovered fully after 4hR and 18hR (Matyjaszek et al. 2009; Graham et al. 2011). Thus, the overall effect of neutrophils could be beneficial for tissue repair after colonic I/R in the horse if the severity of ischemic damage is mild (Serhan and Savill 2005; Nathan 2006). Calprotectin and O ther Clinic opa thological Variables The migration of a ctivated neutrophils into the intestinal lumen in patients with inflammatory conditions of the gastrointestinal tract was the basis for using calprotectin as a nonspecific marker of gastrointestinal diseases (Roseth et al. 1992; Johne et al. 1997; Ton et al. 2000; Aadland and Fagerhol 2002). Although the function of calprotectin has not been fully identified, it is present in high concentrations at sites of inflammation where activated neutrophils and monocytes belong to the most abundant cell types (Srikr ishna et al. 2001; Yang et al. 2001; Roth et al. 2003; Ryckman et al. 2003). The concentration of calprotectin depends on two characteristics: their

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189 production by injured cells and the extent of tissue injury (Rubartelli and Lotze 2007). This explains why serum calprotectin correlates specifically with disease activity in different inflammatory disorders (Roth et al. 2003; Leach et al. 2007). The findings of the present study provide some evidence that serum calprotectin could be related to the severity of inflammation induced by colonic I/R in horses. However individual calprotectin concentrations in horses of the present study were highly var iable, so that relative percentages of calprotectin related to corresponding control values were used for comparison. Although, the calprotectin concentration did not change significantly during colonic I/R in the present study, it was significantly higher in CB than in JB during I/R. These findings support the fact that neutrophils attracted to mucosal venules bec a me activated within the ischemic damaged colon (Moore et al. 1994a; Weiss and Evanson 2003; Grosche et al. 2008, 2011b). Moreover, release of ca lprotectin is a sensitive indicator of cytotoxicity (Johne et al. 1997), so that cell damage during colonic I/ R could explain the increased calprotectin values associated with the presence of necrotic neutrophils in CB (Dale et al. 1985; Hetland et al. 199 8; Voganatsi et al. 2001; Grosche et al. 2011b). Absence of changes in JB calprotectin in our study could be explained by dilution and a mild systemic inflammatory response that w as not sufficient to initiate calprotectin release from circulating neutrophi ls (Moore et al. 1994a; Gibson and Steel 1999; Striz and Trebichavsky 2004; Southwood 2006). In addition, fading of activated neutrophils from circulation due to margination and compartmentalization within the injured colon could also explain lower JB calprotectin concentrations. Although, we did not determine the absolute numbers of neutrophils in JB and CB we observed an increased local

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190 accumulation of neutrophils in submucosal venules within the first 2 hours after reperfusion by LM (Grosche et al. 2008; Grosche et al. 2011b) which could reduce circulating numbers Despite possible activation of neutrophils during colonic I/R characterized by an increased neutrophil turnover and left shift (Cornbleet 2002), 1hI followed by 4hR did not result in systemi c metabolic alterations in the present study. Ischemia caused a moderate local metabolic acidosis, disturbances of the energy metabolism and signs of cell damage within the affected colonic tissue. Most of the metabolic changes in CB were reversible, and reoxygenation resulted in normalization of the values to baseline in the presence of systemic homeostasis probably due to rapid restitution of the mucosal epithelium as demonstrated by histological and functional improvement of tissue structure and barrier integrity after 4hR (Graham et al. 2011; Grosche et al. 2011a,b). The short ischemia time followed by a mild systemic inflammatory response could be a possible reason for the absence of major systemic alterations in the present study Although 1hI was sufficient to cause notable structural changes and functional impairment of the colonic epithelium (Graham et al. 2011; Grosche et al. 2011a,b), the ischemic time was not long enough to cause irreversible damage to the mucosa (Park et al 1990; Moore et al. 1994c; Guan et al. 2009; Grootjans et al. 2010). Similar to findings of Moore et al. (1998 a ) colonic ischemia in the present study did not affect the systemic metabolic h omeostasis in general. However continuous generation of lactate and activation of neutrophils during reperfusion could indicate some systemic effects induced by colonic ischemia (Klebanoff et al. 1986; Souza et al. 2000, 2001, 2002, 2005; Kaminski et al. 2002; Kalia et al. 2003). Thus, longer ischemia and in larger col on

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191 segments could prominent alterations of metabolic variables and calprotectin co ncentration in affected horses. Changes of intestinal mucosal structure and function from hypoxic conditions are caused by multifactorial, precisely coordinated pathophysiological mechanisms that are affected by the time and severity of ischemia, and the dimension of tissue involved. Although i nterruption of arterial and venous blood flow in the equine large colon for a short period of time g enerated minor defects on LM the majority of e pithelial cells developed considerable ultrastructural changes associated with disruption of TJ Th ese alterations result ed in significant epithelial barrier failure after 1 hI accompanied by notable clinic o pathological changes Moreover, ischemia caused activation of the innate immune system characterized by degranulation of resident mast cells, increased phagocytic activity of macrophages, and oxidative burst of mucosal inflammatory cells Reoxygenation of the colonic tissue after 1hI did not result in further exacerbation of mucosal damage. Four hours of r eperfusion following 1hI was sufficient for restitution of epithelial cell structure and barrier integrity and recovery of biochemical homeostasis P rese rvation of nutrients and degradation of necrotic or toxic components by epithelial cell autophagy and phagocytosis and the anti inflammatory and repair promoting effects of COX 2 and apoptosis might be involved in early repair mechanisms initiated after r eperfusion of the ischemic injured colon. Epithelial repair was associated by accumulation of activated neutrophils and release of neutrophilic calprotectin. H owever the exact role of these effector immune cells in the equine colon during a longer period of reperfusion remains to be elucidated. Thus further studies are required to investigate

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192 the effect s of innate immune cells inflammatory mediators and regulatory enzymes on epithelial structure and function, and mucosl repair in the equine colon.

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231 BIOGRAPHICAL SKETCH Astrid Grosc he was born and raised in Magdeburg, SachsenAnhalt, Germany. In September 1986 she moved to Leipzig, Sachsen, Germany to attend the Leipzig University, where she majored in Biology, and later in Veterinary Medicine. Astrid completed her B.S. in Biology in September 1991, and received her degree in Veterinary Medicine in January 1997. Astrid worked as a scientific assistant in the Clinic of Large Animal Internal Medicine, at the Faculty of Veterinary Medicine, Leipzig University from February 1997 to August 2006. During that time, she received her doctoral degree in Veterinary Medicine in April 2001, and she specialized in Veterinary Internal Medicine and Clinical Laboratory Medicine, in April 2004 and June 2006, respectively. In September 2006, Astrid recei ved a scholarship from the Max Kade Foundation, and moved to Gainesville, Florida, where she worked as a research scholar at the Department of Large Animal Clinical Sciences, University of Florida for 16 months Astrid began her PhD studys with the UFs Large Animal Clinical Sciences program in January 2008 supported by the Deedie Wrigley Hancock Fellowship. She rec eived her Ph. D from the University of Florida in December 2011.