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Regionally Graded Injury from the Duodenum to Ileum in Heat and Simulated Ischemia

Permanent Link: http://ufdc.ufl.edu/UFE0043011/00001

Material Information

Title: Regionally Graded Injury from the Duodenum to Ileum in Heat and Simulated Ischemia
Physical Description: 1 online resource (53 p.)
Language: english
Creator: NOVOSAD,VERONICA L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: DUODENUM -- HEAT -- HYPERTHERMIA -- ILEUM -- INTESTINAL -- INTESTINE -- ISCHEMIA -- JEJUNUM -- PERMEABILITY -- REGIONAL
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: REGIONALLY GRADED INJURY FROM THE DUODENUM TO ILEUM IN HEAT AND SIMULATED ISCHEMIA By Veronica L. Novosad May 2011 Chair: Thomas L. Clanton Major: Applied Physiology and Kinesiology Heat exposure is believed to contribute to loss of intestinal barrier function due to thermal injury as well as ischemic insult. We have previously reported that regions proximal to the stomach develop greater permeability during in vitro exposure to heat. In this study we tested if the same trend was seen in an in vitro model model of simulated ischemia. Intestinal segments of adult mice were isolated and everted to create ~8 x 2 cm sacs. All tissues were first incubated at 37+ 0.5 degrees C for 30 min in fresh cell culture media. Then sacs were transferred to preheated media at 42+ 0.5 degrees C for 90 min. Permeability was measured by accumulation of fluorescent labeled dextran (4 kDa) that diffused into the sacs during heat treatment. Consistent with our previous results, multi way ANOVA showed that both treatment and region of intestine where significant determinants of permeability, with the greatest permeability seen at the duodenum, the region of the intestine closest to the stomach. As a follow up, we also examined if an in vivo heat stroke model would result in more prominent morphological injury to the duodenum. Anesthetized mice were exposed to control (37 degrees C for 2 hours) (N = 6) or heat stress (HS) treatment (39.5 degrees C with 0.5 degrees C increases every 30 min until core temperature reached 42.4 degrees C) (N = 6). After 30 min of recovery, multiple regional samples were removed, fixed, sectioned and stained with H&E for light microscopy evaluation of intestinal morphology and tissue damage. Multi way ANOVA showed that HS mice had significantly lower villi height/ crypt depth ratios (VH/CD) (p < 0.01). VH/CD was also highly dependent on region, with duodenum showing the greatest decrease and ileum showing the least (p < 0.01). HS mice also showed greater decreases in VH/CD across regions compared to control (p < 0.01). Histological analysis of intestines from in vivo experiments in HS adds additional information to our previous findings in in vitro experiments. In both cases, the results show that regions of the small intestine proximal to the stomach are more susceptible to damage. This has important implications regarding the origins of immunological responses to heat stroke and treatments for intestinal damage suffered in extreme conditions.
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 VERONICA L NOVOSAD.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Clanton, Thomas.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043011:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043011/00001

Material Information

Title: Regionally Graded Injury from the Duodenum to Ileum in Heat and Simulated Ischemia
Physical Description: 1 online resource (53 p.)
Language: english
Creator: NOVOSAD,VERONICA L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: DUODENUM -- HEAT -- HYPERTHERMIA -- ILEUM -- INTESTINAL -- INTESTINE -- ISCHEMIA -- JEJUNUM -- PERMEABILITY -- REGIONAL
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: REGIONALLY GRADED INJURY FROM THE DUODENUM TO ILEUM IN HEAT AND SIMULATED ISCHEMIA By Veronica L. Novosad May 2011 Chair: Thomas L. Clanton Major: Applied Physiology and Kinesiology Heat exposure is believed to contribute to loss of intestinal barrier function due to thermal injury as well as ischemic insult. We have previously reported that regions proximal to the stomach develop greater permeability during in vitro exposure to heat. In this study we tested if the same trend was seen in an in vitro model model of simulated ischemia. Intestinal segments of adult mice were isolated and everted to create ~8 x 2 cm sacs. All tissues were first incubated at 37+ 0.5 degrees C for 30 min in fresh cell culture media. Then sacs were transferred to preheated media at 42+ 0.5 degrees C for 90 min. Permeability was measured by accumulation of fluorescent labeled dextran (4 kDa) that diffused into the sacs during heat treatment. Consistent with our previous results, multi way ANOVA showed that both treatment and region of intestine where significant determinants of permeability, with the greatest permeability seen at the duodenum, the region of the intestine closest to the stomach. As a follow up, we also examined if an in vivo heat stroke model would result in more prominent morphological injury to the duodenum. Anesthetized mice were exposed to control (37 degrees C for 2 hours) (N = 6) or heat stress (HS) treatment (39.5 degrees C with 0.5 degrees C increases every 30 min until core temperature reached 42.4 degrees C) (N = 6). After 30 min of recovery, multiple regional samples were removed, fixed, sectioned and stained with H&E for light microscopy evaluation of intestinal morphology and tissue damage. Multi way ANOVA showed that HS mice had significantly lower villi height/ crypt depth ratios (VH/CD) (p < 0.01). VH/CD was also highly dependent on region, with duodenum showing the greatest decrease and ileum showing the least (p < 0.01). HS mice also showed greater decreases in VH/CD across regions compared to control (p < 0.01). Histological analysis of intestines from in vivo experiments in HS adds additional information to our previous findings in in vitro experiments. In both cases, the results show that regions of the small intestine proximal to the stomach are more susceptible to damage. This has important implications regarding the origins of immunological responses to heat stroke and treatments for intestinal damage suffered in extreme conditions.
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 VERONICA L NOVOSAD.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Clanton, Thomas.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043011:00001


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1 REGIONALLY GRADED INJURY FROM THE DUODENUM TO ILEUM IN HEAT AND SIMULATED ISCHEMIA By VERONICA LE A NOVOSAD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Veronica Lea Novosad

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3 To my family; by birth, choice or just plain luck

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4 ACKNOWLEDGEMENTS I would like to think Dr. Thomas Clanton for h is mentorship and the opportunity to members of the Clanton lab for their guidance, collaboration, and high spirits throughout my time spent there. Finally, I would like to thank the other members of my thesis committee, Dr. Randy Braith, and Dr. James Collins, for their suggestions and expertise.

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5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Specific Aim 1 ................................ ................................ ................................ ......... 13 Hypothesis for Aim 1 ................................ ................................ ............................... 14 Specific Aim 2 ................................ ................................ ................................ ......... 14 Hypothesis for Aim 2 ................................ ................................ ............................... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 16 Healthy Intestinal Barrier Function ................................ ................................ .......... 16 Mucosal Layer ................................ ................................ ................................ .. 16 Epithelial Layer ................................ ................................ ................................ 16 Immune Cells ................................ ................................ ................................ ... 18 Overview of Heat Stroke ................................ ................................ ......................... 18 Thermal Damage ................................ ................................ .............................. 18 Splanchnic Ischemia ................................ ................................ ........................ 19 Free Radical Production ................................ ................................ ................... 20 Intestinal Barrier Dysfunction ................................ ................................ .................. 20 Step 1: Initial Onset of Injury ................................ ................................ ............ 21 Step 2: Intestinal Permeability and Endotoxin Translocation ............................ 21 Step 3: LPS Induced Inflammation ................................ ................................ ... 21 Step 4: Continued Cytokine Production, and Intestinal Damage ...................... 21 Previous Work Examining Regional P ermeability ................................ ................... 22 3 METHODS AND MATERIALS ................................ ................................ ................ 24 Chemicals Used ................................ ................................ ................................ ...... 24 In Vitr o Simulated Ischemia Model ................................ ................................ ......... 24 Preparation of Treatment Chambers ................................ ................................ 24 Animal Treatment and Gut Sac Preparation ................................ ..................... 25 Data Analysis and Statistics for In Vitro Experiments ................................ ....... 26 In Vivo Heat Stroke Model ................................ ................................ ...................... 27 Experimental Procedure ................................ ................................ ................... 27

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6 Data analysis and Statistics for Histological Analysis from Intact Animals ....... 28 4 RESULTS ................................ ................................ ................................ ............... 30 In Vitro Intestinal Ischemia Model ................................ ................................ ........... 30 In Vivo Heat Stroke Model ................................ ................................ ...................... 30 Villi Morphol ogy ................................ ................................ ................................ 30 Intestinal Permeability ................................ ................................ ...................... 31 5 DISCUSSION ................................ ................................ ................................ ......... 40 Summary of Result s ................................ ................................ ................................ 40 Experimental Critique ................................ ................................ .............................. 40 Comparison to Previous Results ................................ ................................ ............. 41 Pot ential Causes of Duodenal Damage ................................ ................................ .. 42 The Auto digestion Theory ................................ ................................ ............... 43 Morphology of the Duodenum and Damage Susceptibility ............................... 44 Further Applications ................................ ................................ ................................ 45 Summary of Conclusions ................................ ................................ ........................ 45 LIST OF REFERENCES ................................ ................................ ............................... 46 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 53

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7 LIST OF TABLES Table page 3 1 Simulated Ischemia Conditions ................................ ................................ .......... 29 3 2 Intestinal Ischemic Damage Grading System. ................................ .................... 29 4 1 Average Villi Measurements across Treatment and Region ............................... 32 4 2 Frequency of Specific Grades across Region and Treatment ............................ 33

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8 LIST OF FIGURES Figure page 1 1 Regional inte stina l permeability at 37 and 42 C. ................................ .............. 15 4 1 Intestinal permeability when exposed t o simulated ischemia treatments. ........... 34 4 2 Regiona l intestinal permeability in different gradients of ischemia ...................... 35 4 3 Representative his tology by region and treatment ................................ .............. 36 4 4 Villi grade averages and frequencies vs. treatment and intestinal region. .......... 37 4 5 Villi height/Crypt dept h ratio by region and treatment ................................ ......... 38 4 6 Villi height/Villi widt h ratio by region and treatment ................................ ............. 39

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9 LIST OF ABBREVIATION S FD4 F luorescein isothiocyanate ( FITC) Dextran 4000 Da; a fluorescent protei n sized to mimic lipopolysaccharide (LPS) and is used to mea sure tissue permeability. H + E H emotoxin and eosin HS Heat Stroke KCl Potassium Chloride LPS Lipopolysaccharide, or endotoxin; bacterial components in the intestine that stimulate immune and inflammatory responses TFF3 Trefoil Factor 3 TLR Toll Like Rece ptors NOC 12 3 Ethyl 3 (ethylaminoethyl) 1 hydroxy 2 oxo 1 triazene NLR NOD Like Receptors VH/CD Villi Height to Crypt Depth Ratio VH/VW Villi Height to Villi Width Ratio

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science REGIONALLY GRADED INJURY FROM THE DUODENUM TO ILEUM IN HEAT AND SIMULATED ISCHEMIA By Veronica L. Novosad May 2011 Chair: Thomas L. Clanton Major: Applied Ph ysiology and Kinesiology Heat exposure is believed to contribute to loss of intestinal barrier function due to thermal injury as well as ischemic insult. W e have previously reported that regions proximal to the stomach develop greater permeability during in vitro exposure to heat In this study we tested if the same trend was seen in an in vitro model model of simulate d ischemi a Intestinal segments of adult mice were isolated and everted to create ~8 x 2 cm sacs. All tissues were first incubated at 3 7+ 0.5 C for 30 min in fresh cell culture media. Then sacs were transferred to preheated media at 42+ 0.5 C for 90 min. Permeability was measured by accumulation of fluorescent labeled dextran (4 kDa ) that diffused into the sacs during heat treatment. C onsistent with our previous results, m ulti way ANOVA showed that both treatment and region of intestine where significant determinants of permeability, with the greatest permeability seen at the duodenum the region of the intestine closest to the stomach As a follow up, we also examined if an in vivo heat stroke model would result in more prominent morphological injury to the duodenum. Anesthetized mice were exposed to control (37 C for 2 hours) (N = 6) or heat stress (HS) treatment (39.5 C with 0.5C increases every 30 min until core temperature reached 42.4 C) (N = 6). After 30 min of recovery, multiple regional

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11 samples were removed, fixed, sectioned and stained with H&E for light microscopy evaluation of intestinal morphology and tissue damage. Mu lti way ANOVA showed that HS mice had significantly lower villi height/ crypt depth ratios (VH/CD) ( p < 0.01). VH/CD was also highly dependent on region, with duodenum showing the greatest decrease and ileum showing the least ( p < 0.01). HS mice also show ed greater decreases in VH/CD across regions compared to control ( p < 0.01). Histological analysis of intestines from in vivo experiments in HS adds additional information to our previous findings in in vitro experiments. In both cases, the results show that regions of the small intestine proximal to the stomach are more susceptible to damage. This has important implications regarding the origins of immunological responses to heat stroke and treatments for intestinal damage suffered in extreme conditions

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12 CHAPTER 1 INTRODUCTION Between 1997 and 2006, 54,983 people were treated in United States emergency rooms for heat related illness ( 1 ) Heat stroke is the most severe category of heat illness, characterized by an elevated core body temperature above 40 C central nervous system dysfunction, intestinal barr ier dysfunction, and endotoxin translocation, resulting in an inflammatory cytokine cascade Severe cases of heat stroke result in multi organ failure and death ( 2 ) with mortatiliy ranging from 1 0 to 50% Intestinal barrier dysfunction is a critical component to the developing pathology of heat stroke. In addition to volatile temperatures t he intestine becomes particularly vulnerable to ischemia, as splanchnic blood is shunted towards the perip hery in an effort to dissipate heat ( 3 4 ) Increased sweating and dehydration result in decreased plasma volume and hypotension, furthe r increas ing ischemic injury risk Ischemic injury manifests in a three part progression : 1 ) splanchnic hypoperfusion 2 ) ischemia reperfusion mediated injury, and 3 ) the loss of gut barrier function. Together, these three pathological conditions promote intestinal bacteria and endotoxin translocation into the circul atory and lymphatic systems, leading to the production o f infl ammatory cytokines and potential multi organ failure ( 5 ) Our lab has been examining the effects of heat stroke like conditions on the intestine using an in vitro model originally developed by Lambert et al in the rat (6) While our lab observe d the expected increases in permeability in hyperthermia, we did not see significant increas es in permeability when adding tight junction openers such as : E. coli heat stable enterotoxin, palmitoyl DL carnitine, and NOC 12 at 37 C S ignificant increases we re seen with the tight junction opener, cytochalasin D but not to

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13 the same levels of hyperthermia alone (7) With this finding, we extensively examined the protocol and modified it as necessary to reduce variability in environmental conditions and measur ement techniques (8) After these improvements to the protocol w ere implemented, it was repeatedly observed that under heat treatment, intestinal permeability was significantly dependent on region (8) The more proximal an intestinal sac was to the pylor ic sphincter, the greater its susceptibility to developing permeability in hyperthermia. In addition, when a best fit line was applied to permeability as a function of segment location, heated intestine had a significantly higher slope than control. (Fig u re 1 1) These results contrast previous work by Lambert in the rat who found no significant difference between permeability across intestinal regions when subjected to elevated temperatures ( 6 ) M ore work with this in vitro model was needed to determine if this phenomenon was also present in the second stressor common to heat stroke, ischemia. In addition, more testing was needed to determine if a whole animal model would display the same regional susceptibility to damage as the in vitro model. The pur pose of this invest igation was to determine if intestinal sacs were regional ly susceptible to permeability in simulated ischemia and to follow this up with additional in vivo experiments using microscopy to examine regional damage in heat stroke. Specific Aim 1 To develop an in vitro ischemi a induce d intestinal permeability model in the mouse and to determine the relationship between small intestinal region and ischemi a induced permeabi lity defects

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14 Hypothesis for Aim 1 H1: S imulated ischemic stress in v itro will result in a graded change in permeability that is dependent on intestinal region such that d uodenal regions will be more permeable than ileal regions. Specific Aim 2 T o determine if the extent of intestinal damage in a model of whole animal heat stroke is localized to specific regions of the small intestine and to characterize the histopathology of hyperthermia induced intestinal damage in the mouse. Hypothesis for Aim 2 H2: The intestines from animals exposed to heat stroke will display greate r damage in the duodenum compared to the jejunum and ileum

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15 Fig ure 1 1. Regional intestin al permeability at 37 and 42 C. Multiway ANOVA showed that region and treatment were significant f actors in determining transport ( p < 0.05) Slopes where co mpared between control and heated best fit lines, and the effects of heat treatment on slope was sig nificantly greater than control ( p < 0.03)

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16 CHAPTER 2 LITERATURE REVIEW Healthy Intestinal Barrier Function The intestine is believed to play an integral role in the development of heat stroke, and is particularly susceptible to acute heat stroke and ischemia (9 10) In addition to absorbing nutrients, the healthy intestine acts as a barrier between sterile blood and non sterile luminal contents such as p athogenic bacteria or digestive enzymes. The intestinal barrier is equipped for this task with multiple defenses, includ ing a mucosal layer, an epithelial cell layer held together by tight junctions, and a variety of vascular, lymphatic and immune cells s uch as tissue macrophages dendritic cells and resident lymphocytes ( 11 ) Mucosal Layer The first layer of the mechanical barrier is a thick mucosal layer which slows down bacterial penetration, increases villi lubrication, and generally separates the ep ithelial cells from digestive contents inside the lumen A thick mucosal layer will also prevent over stimulation of immune cells ( 12 ) Mucins, proteins secreted by goblet cells, comp ose the majority of the mucosal layer In humans, goblet cells produce approximately three liters of mucin per day, ensuring a constant renewal of mucosal protection ( 13 ) Epithelial Layer Below the mucous layer lies the epithelium, a single squamous cell layer placed directly between the sterile, internal body and the bact erial laden intestinal contents ( 11 ) The epithelium is responsible for nutrient transport and bacterial sampling while still keeping intestinal contents secluded to the lumen To accomplish this task, this

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17 sheet of epithelial cells is held tightly toget her by tight junctions ( 11 ) The effect of tight junctions is great enough to even maintain the polarization of the epithelial cell s and their membrane s into basal and luminal zones, each with unique transport proteins and phospholipid ratios. However, t ight junctions remain dynamic and are equipped with myosin light chains that act as the regulatory site for the cytoskeletal junctions that function as gate keepers. When myosin light chains are phosphorylated, they contract and the tight junctions are pu lled open, and small substance s pass paracellularly ( 14 15 ) Upo n the surface of the epithelium, there are a variety of germ line encoded pattern re cognition receptors that capture bacteria, translocate them internally and promote the production of pro in flammatory cytokines ( 16 ) Epithelial cells also have the ability to take up antigens through fluid phase endocytosis and receptor mediated endocytosis using various receptors ( 12 ) Initially, it may seem counterintuitive that the epithelium is equipped pathways serve a vital function to the immune system. Pattern recognition receptors include the families of toll like receptors (TLR ) and NOD like receptors (NLR) ( 16 ) Mice deficient in TLR and NLR exhibit delayed or diminished wound healing when confronted with mucosal injury including acute DSS colitis ( 16 ) For example, mice deficient in TLR 2, will lack the ability to produce goblet cell derived TFF3, a mediator of epithelial migration and anti apoptosis ( 17 ) However, the phenotype of mice with deficiency in TFF3 can be r eversed with the addition of a synthetic TLR2 agonist ( 16 ) Additionally, simulation of the NLR cryopyrin h as been shown to promote the production of IL 18, a

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18 cytokine w hich has been shown to preserve epithelial barrier integrity during the early phase of wound healing in acute DSS colitis ( 16 ) Immune Cells If bacteria pass the epitheli um they will encounter gut associated lymphoid tissue. Gut associated lymphoid tis sue is considered to be the largest immunological organ of the body filled with B cells, T cells, granulocytes, mast cells, macrop hages and dendritic cells ready to signal an inflammatory cascade and destroy f oreign bacteria ( 11 12 ) When LPS interac ts with the surface TLRs on these immune cells, phagocytosis and the capacity to present antigens to T cells is enhanced ( 17 ) Overview of Heat Stroke Heat stroke is a life threatening condition characterized by an elev ated core body temperature above 40 C accompanied by central nervous system dysfunction, intestinal barrier dysfunction, endotoxin translocation, an d a resulting inflammatory cytokine cascade ( 2 ) However, s urvival rates are not dependent on temperature alone, as decreased survival is also de pendent on the occurrence of preceding inflammatory episode s ( 18 ) Heat stroke has multiple mechanisms of injury that contribute to multiple organ dysfunction including thermal damage to cellular components, increased free radical production, acidosis, i schemia, and intestinal barrier dysfunction. Thermal Damage T here is no specific core temperature threshold that marks a guaranteed onset of heat stroke ; the initial onset of heat stroke has been reported to fall within a range of 40 43 C specifically in the 41 42 C range ( 19 ) Heat exposure of 40.5 C for 60 minutes results in scattered neuronal death of 6% ( 20 ) A 20 second exposure t o 42 C is the projected thermal threshold required for the initial onset of lipid bilayer

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19 denaturation and necro sis in cell culture ( 21 ) The lipid b i layer is sensitive to stress and increases in cell membrane fluidity at 42 C have been shown to promote the heat shock response well before protein denaturation ( 22 ) At 60 minutes of 42 C exposure, the blood brain barrier begins to break down ( 20 ) Exposure to 43 C for 10 60 min will result in single and double strand breaks of newly synthesized DNA ( 20 ) Prolonged heat exposure may also result in dysfunctional enzyme kinetics, leading to impaired metabolism o r DNA synthesis ( 20 ) Protein degradation is exhibited at 40 C in rodent cells and 41 42 C in human cells hyperthermic environment. When 5 10 % of cellular prot ei ns are denatured, the cell wil l undergo apoptosis ( 23 ) Splanchnic Ischemia In heat stroke, ischemia is initially induced when blood is shunted from the intestine and other visceral organs to the periphery in order to dissipate heat ( 3 4 ) This compromises tissues such as the liver and intestine, which both show increased hypoxia markers when exposed to heat stress ( 24 ) D uring hyperthermia and exercise, splanchnic blood flow can be reduced by as much as 70% ( 4 ) Dehydration and reduced blood volume due to increased sweat loads can lead to hypotension mucosal acidosis hypovolemia, as well as decreased renal function ( 5, 19 ) These tissues are eventually reperfused during recovery which contributes additional free radicals to the heat stressed environment Ischemia reperfusion in jury is well known for causing increased intestinal permeability, activating an inflammatory response through TLRs, mitochondrial damage epithelial necrosis and potential multi organ failure ( 17 )

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2 0 Free Radical Production Multiple tissues increase free ra dical production as a result of heat stress including: brain ( 25 ) intestine ( 2 6 ) muscle ( 2 7 ) and splanchnic blood ( 28 29 ) Together, hyperthermia and isc hemia promote an environment with altered pH and increased free radical production. It is theorized that l actic acidosis seen in heat stroke is brought on by the onset of local hypoxia due to poor perfusion and hypovolemia ( 19 ) The body then overcompensates for this metabolic acidosis, and respiratory alkalosis follows ( 30 ) M ucosal acidosis of the i ntestine leads to increased free radical production Increased free radicals contribute to intestinal permeability and anti oxidant therapy has been shown to reduce symptoms of intestinal barrier dysfunction. A x anthine oxidase inhibitor has been shown to reduce heat stress induced intestinal permeability ( 27 ) and supplementation of the antioxidant vitamin C decreases the elevated plasma endotoxin associated with exertional hyperthermia ( 31 ) Intestinal Barrier Dysfunction There are numerous reports that increased thermal load ( 6, 32 34 ) and mucosal ischemia reperfusion ( 17 3 5 ) each separately lead to increased intestinal permeability as well as endotoxin translocation into the blood stream. As previously stated heat stroke combines these stressors. W hen e xcessive endotoxin due to increased intestinal permeability cannot be cleared by the gut associated lymphoid tissue, liver or kidneys the intestinal endothelium is activated and neutrophils are recruited ( 3 6 ) This is the start of a four step downw ard spiral towards multi organ failure where a leaky gut leads to inflammation, and in return, inflammatory cytokines further promote a leaky gut through tight junction opening and endothelial apoptosis.

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21 Step 1: Initial Onset of Injury Heat stroke has m ultiple mechanisms of intestinal injury: thermal damage to cellular components, enzymatic dysfunction, acidosis, increased reactive oxygen species, and ischemia. Cells are compromised; reduced blood flow leads to reduced waste removal and reduced oxygen d elivery. Step 2: Intestinal Permeability and Endotoxin Translocation Poo r environmental conditions and/or activation of pro inflammatory pathways induce the opening of tight junctions. When tight junctions open, the gut becomes permeable to endotoxin, t he lipopolysaccharide (LPS) components of gram negative bacteria. LPS is translocated across the epithelium and into the underlying lymphatics of the intestine as well as the blood stream ( 11 ) Step 3: LPS I nduced Inflammation Ischemia reduces renal bloo d flow and glomerular filtration rate which reduces the rate that LPS can be cleared. LPS circulates and docks with the toll like receptors on inflammatory cells and other cells such as muscle cells LPS activates TLR 4 receptors on a variety of cell mem branes and this leads to stimulat ion of the NF B pathway ( 12 ) and other stress signaling pathways In response, inflammatory cells produce pro inflammatory cytokines In particular, heat stroke creates significant increases in circulating IL 1 IL 1 IL 6, IL 10, and TNF ( 19 ) Ste p 4: Continued Cytokine Production and Intestinal D amage The same pro inflammatory cytokines that are pr oduced by inflammatory cells in response to endotoxin exposure will also promote additional endotoxin translocation. For example, TNF ( 14 37 ) IFN ( 37 38 ) IL ( 39 ) have been shown to promote the open ing of tight junctions through myosin light chain phosphorylation In particular,

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22 TNF via nF B signaling ( 37, 40 ) The presence of TNF chain kinase protein and gene activity as well ( 37, 40 ) Phosporylation of the myosin light chain kinase opens the tight junction via myosin actin interactions on the tight junction cytoskeleton proteins. With l e ngthy exposures to IFN TNF or IFN epithelial cells also undergo apoptosis promoting more intestinal barrier dysfunction ( 41 ) Pro inflammatory cytokines promote a leakier gut, which promotes endotoxin translocation, which promotes pro inflammato ry cytokine production from immune cells. Previous Work Examining Regional Permeability Previous work examining regional permeability at 37 C is conflicting Nejdfors et al. examined permeability in human, pig, and rat intestines using Ussing chambers. In man and pig, no significant differences in intestinal permeability to F luorescein isothiocyanate ( FITC) Dextran 4000 Da (FD4) between jejunum, ileum, proximal, or distal colon were seen. In rat, distal colon permeability was significantly lower than th e jejunum ( 42 ) Nejdors et al. conflicts with Jezyk et al. who showed higher permeability in the colon than jejunum in rabbit, dog, and monkey at 37 C ( 43 ) Pantzar et al. also observed greater permeability in the distal small intestine (20 to 5 cm prox imal to the caecum) compared to th e proximal small intestine (5 to 20 cm distal to the pylorus) ( 44 ) These studies primarily examined the regional differences between the small intestine and the large intestine, and no duodenal measurements were taken. I t has been repeatedly shown that heat stress increases permeability in cellular ( 32 33 ) in vitro ( 6 ) and whole animal in vivo models ( 34 ) The scientific community has repeatedly shown that heat stroke both exertional ( 4 5 47 ) and non exertional ( 34 ) will

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23 result in increased plasma LPS concentrations. Although measuring plasma LPS can signify endotoxin translocation, plasma LPS measurements alone cannot specify where the LPS came from There is a distinct lack of studies examining regional intestina l permeability in hyperthermia or heat stroke. Lambert et al. reported that there were no significant differences between duodenum, jejunum, ileum, or colon in the rat under hyperthermic or normal conditions ( 6 ) Jejunal histology sections however exhibi ted jejunal endot h eli al damage, but no duodenal or ileal histology measurements were reported. Our lab was not able to reproduce these results in the mouse and found that proximal regions were significantly more susceptible to hyperthermia than distal reg ions in in vitro hyperthermia conditions

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24 CHAPTER 3 METHODS AND MATERIAL S Chemicals U sed Medium 199 (Cellgro), L Glutamine (Lonza), sodium bicarbonate (Acros Organics), Potassium Cloride (Fisher Scientific), DL Lactic Acid Lithium Salt (Sigma), fluores cein isothiocyanate (FITC) dextran 4 kDa (FD4, Sigma Aldrich) pentobarbitra l ( Sigma ) In Vitro Simulated Ischemia Model Preparation of Treatment Chambers Ischemic treatments were modeled to mimic porcine conditions of reduced intestinal blood flow where c lear relationships between reductions in blood flow and tissue O 2 CO 2, pH and K + have been established ( 48 ) We defined m oderate ischemia as equivalent to a 50% reduction in blood flow which was found to result in 20 mm Hg P O 2 80 mm Hg PCO 2 5 mM KCl, a nd 2.5 mM lactic acid in pigs S evere ischemia treatment was defined as a an 80 % reduction in blood flow and defined to be 35 mm Hg P O 2 60 mm Hg P CO 2 8 mM KCl, and 6 mM lactic acid ( 48 ) In treatment chambers, medium 199 a common cell culture media th at includes amino acid substrates, was adjusted by the addition of KCl and lactic acid. Appropriate o xygen, c arbon d ioxide, and n itrogen mixtures were obtained in tanks and concentrations were tested via an oxygen electrode system (Oxygraph) and a fyrite CO 2 detector system, a chemical system used to calibrate tissue culture hood CO 2 levels From this, we determined that the tank gas concentrations needed to reach our target P O 2 and P CO 2 levels were 2.9 % O 2 11.2 % CO 2 for severe ischemia and 4 .9 % O 2 and 8.5 % CO 2 for moderate ischemia. All tan ks were within +/ 0.7% of the targeted gas concentrations. pH was analyzed with an

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25 AB15 pH meter (Fisher Scientific), accurate to two de cimals All treatment chambers are bubbled for at least 30 minutes befor e experiments to ensure pH equilibration and gas saturation. Animal T reatment and G ut S ac P reparation Adult C57 B l6 mice (25 35 g) were treated according to protocols approved by the University of Florida Institutional Animal Care and Use Committee. Animal s were given food and water ad libitum an d housed at Shands Hospital. After euthanasia by carbon dioxide asphyxiation, the entire intestine was immediately removed and placed in Medium 199 prepared with L Glutamine and bubbled with 95% O 2 and 5% CO 2 Gross mesentery, cecum, and large intestine were removed. Remaining mesentery was carefully trimmed from the small intestine. Once freed from the mesentery, luminal contents were removed by perfusion of 5 ml of oxygenated medium 199. The small intestine was then inverted over a 20 l glass capillary tube. 2 3 cm intestinal sacs were filled with oxygenated media 199, tied with 2 0 sutures, and labeled. Care was taken to ensure the same hydrostatic pressure was applied to each sac during preparation ( 2 3 cm H 2 O) L ength measurements were taken at each suture site to determine the position of the tissue sample. When all sacs were prepared, they were collectively transferred to continually oxygenated chambers of 7.5 ml medium 199 in a 37 C water bath for 30 minutes. After the intestinal sacs were acclimated to 37 C the sacs were randomly assigned to one of four treatment chambers, control, moderate ischemia, severe ischemia, and hypoxia (T able 3 1) ; each contain ed 7.5 ml of medium 199 and a high molecu lar weight fluorescent marker, 4 kDa fluorescein isothiocyanate dextran (0.3 mM ;

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26 FD4) While well below the average molecular weight of endotoxin (15 20 kDa), t his molecular weight is in range the lipid component of endotoxin ( 2 4 k Da ) ( 49 ) Intestinal s acs then were subjected to 37 C treatment for 90 minutes. Data Analysis and Statistics for I n V itro Experiments The sacs were removed, their cont ents emptied into preweigh ed tubes, and the surface area of the intestinal sac measured. Permeability was d efined as transport of nmoles of F D4 per cm 2 using the following formula: (Concentration serosal fluid X Volume serosal fluid ) Mucosal surface area This method was described by Lambert et al for the rat ( 6 ) but was adapted for mouse and modified to impr ove reproducibility Volume wa s determined by the weight of intestina l contents collected, assuming the density of buffer = 1g/ml. Mucosal surface area was determined by compressing the sample under a plastic sheet with 30 g of weight, and measuring the area i n cm 2 Concentration was determined by measuring the fluorescence of the sample and comparing it to a standard curve. Changes in for unequal variance (GraphPad Pri sm). Differences in variance between groups were evaluated using (ANCOVA) was used to stratify potential sources of variance due to both categorical and continous variables (SASJMP). The slopes of l inear regressions in control and ischemic treatments were statistically compared using t tests for differences in slope and intercept (GraphPad Prism). All result s are reported as means SEM; p <0.05 was considered to be statistically significant.

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27 In Viv o H eat S troke M odel Experimental Procedure Animals were weighed and gavaged with 10 l/g of body we i ght of 20.8 mmol FD4 Then the animal was injected with 10 l/g of 10% pentobarbitrol. Supplemental anesthesia was gi ven throughout the experiment as 0.1 cc of 10% pentobarbitol per injection as needed When the mouse was fully anesthetized, a thermistor was inserted anally and two electrodes were placed subcutaneously to monitor heart rate. The mouse was placed into a heated chamber where it under went a control or a heat treatment. The core temperature of c ontrol mice was a servo controlled to keep core temperature at 36.5 C (resting day time temperature of mice which are active only at night time ) Mimicking temperatures of previous work by Leon et a l. (50) in unanesthetized mice, h eat ed mice were subjected to an initial temperature of 39.5 C for 30 minutes and for each subsequent 30 minutes, the temperature was raised 0.5 C until the internal temperature of the mouse reached 42.4 C at which poin t the mouse was taken out of the environmental chamber and allowed to recover or a 30 minute recovery period Animals that expired within the procedure due to respiratory failure were ( 30%) excluded from the final analysis because of the need for matchin g times in conditions between groups At the end of recovery, one last supplement of 0.1 0.33 cc 10% pentobarbitrol was administered before harvesting blood, intestine, soleus, and hind limb samples. Heparnized or Ca +2 chelated b lood was centrifuged at 50 00 rpm for 10 minutes and the supernatant was collected to examine the concentration FD4 marker in the blood (an indication of barrier dysfunction) Intestines were stored in 4% formalin at 4 C and transverse sections were cut from the duodenum, jejunum, and ileum for later

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28 hematoxylin and eosin staining. To en sure consistency in samples chosen, mesentery was removed from the small in testine to a point where the intestine could be straightened without strain Then the duodenum was cut 1 cm below the pylo ric sphincter; the jejunum was cut half way between the pyloric sphincter and the ileocecal junction and the ileum sample was cut 2 cm above the ileocecal junction. Data analysis and Statist ics for H istological A nalysis from I ntact A nimals Histology sli des of the small intestine were graded according to the method by Chiu et al, modified to examine individual villi (T able 3 2) ( 5 1 ) Two trained raters separately graded each slide (the author and Neil Phillips) All grading was blind; n either rater knew which samples corresponded to wh ich treatments or intestinal section. All slides that raters differed in more than one average grade were re blinded and graded a second time over one month after the initial ratings. Each slide had multiple cuts of the sa me sample, thus raters chose one representative cut based on the following factors: the least number of staining artifacts, the least number of cutting artifacts, and the most complete section of intestine. For the chosen cut, one villus without artifacts was chosen at random as a starting point. This villus and e very fo u rth villus thereafter was graded and measured until a total of 10 measurements were made or in the case of a large number of cutting artifacts, until no more villi without artifacts were available to measure. Villus blunting and swelling were graded by the use of a crypt depth/villus height ratio and a villus width/villus height ratio two measurements commonly used to determine brush border integrity ( 5 2 5 5 )

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29 Table 3 1 Simulated Isch emia C onditions Treatment % O 2 % CO 2 % N 2 Lactic Acid (mM) KCl (mM) Average pH Control 95 5 0 0.0 5 7.30 0.029 Moderate Ischemia 5 8 87 2.5 5 7.07 0.006 Severe Ischemia 3 11 86 8.0 8 6.94 0.009 Hypoxia 0 5 95 0.0 5 7.41 0.042 Gases were prem ixed in tanks that would raise the buffer to corresponding gas levels when bubbled through the media All partial pressure oxygen levels listed w ere tested in gas equilibrated buffer with an Clark oxygen electrode at 37 C and carbon dioxide gas levels whe re checked with fyrite at room temperature Average pH values were experimental values measured in individual baths. Table 3 2 Intestinal Ischemic Damage Grading System Grade Villus Appearance 0 Normal mucosal villus 1 Development of subepith elial space, us ually at the apex of the villus. 2 Extension of the sub epithelial space with moderate lifting of epithelial layer from the lamina propria. 3 Massive epithelial lifting down the sides of the villus. Tip of villus is ulcerated. 4 Den uded villus with lamina propria and dilated capillaries exposed and digested. 5 Complete digestion and disintegration of lamina propria; hemorrhage and ulceration. (51) was modified to grade one villus a t a time out of a random sample of villi from the slide. Previously, this grading system was taken as a subjective measurement of the entire slide at on c e. No modifications were made to the categories of damage specifications.

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30 CHAPTER 4 RESULTS In Vitr o Intestinal Ischemia Model Intestinal segments treated under hypoxic, severe ischemic, or moderate ischemic conditions had significantly higher permeability than control segments when the responses of all of the segments from each animal were averaged (Fi gure 4 1) Moderate ischemic, severe ischemic, and hypoxic conditions had no significant differences between each other. Multi way ANOVA showed that region, treatment, and their crossed effect s were significant. (Region and treatment each had p value < 0 .0001, crossed effect p = 0.02). segments had statistically lower permeability than all other treatments ( p < 0.05). As shown in Figure 4 2 (inset) when the permeability data is expressed as a f unction of location along the intestine, b est fit lines across treatments showed a significant difference in slopes between oxygenated treatment and moderate or severe ischemia treatment ( p < 0.05), and a significant difference in intercepts between oxygen ated treatment and hypoxic treatment ( p < 0.0001). In Vivo H eat S troke M odel Villi Morphology Averages of control and heated villi heights, crypt depths, and villi widths can be seen in Table 4 1 and histology examples of region and treatment can be see n in F ig ure 4 3 Heated mice had significantly lower villi height/crypt depth ratios ( p < 0.0001), lower villi height/villi width ratios ( p = 0.0047), and higher average damage grades across all regions ( p = 0.0007) (Fig ure 4 4) From duodenal to ileal re gions, each region had a distinct graded response; duodenal regions exhibited higher average damage

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31 grades than regions distal to the stomach ( p < 0.0001) (Table 4 2 and F ig ure 4 4 ). Villi height/crypt depth ratios (Fig ure 4 5) were highly dependent on r egion ( p < 0.0001), with the duodenum having the highest and the ileum having the lowest. However, a crossed effect between villi height/crypt depth ratios and heat treatment was also statistically significant ( p = 0.0007), showing that heat treatment re sulted in a greater reduction in villi height/crypth depth ratios in the duodenum than in all other regions. In addition there was a significant crossed effect between region and treatment; duodenal regions were more susceptible to heat damage than ileal or jejunal regions ( p = 0.0328). Villi height/villi width ratios also were highly dependent on region ( p = 0.0133), but no crossed effect between region and heat was exhibited. Intestinal Permeability Student t tests showed that heated mice had signific antly higher blood FD4 concentration ( p < 0.05); however an outlying point in the control group had to be removed prior because this point was greater than 15 standard deviations from the mean. Heat and control treatment groups were determined to have une qual variances of blood FD4 concentration with an unequal variances test. Therefore, a nonparametric Wilcoxon/Kruskal Wallis test was used and heated mice had significantly higher concentrations of FD4 in their blood samples ( p = 0.0344).

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32 T able 4 1 A verage Villi Measurements across Treatment and Region Treatment Villi Height (M) Crypt Depth (M) Villi Width (M) VH / CD VH/VW Duodenum Control 555 21 103 4 138 14 5.46 0.32 4.39 0.72 Jejunum Control 330 26 118 5 83.5 6.0 2.80 0.16 4.15 0.62 Ileum Control 194 17 93.9 6.0 94.4 2.8 2.05 0.10 2.04 0.13 Duodenum Heat 318 31 102 10 161 10 3.23 0.36 1.97 0.12 Jejunum Heat 249 35 101 8 91.0 10.2 2.42 0.19 3.05 0.76 Ileum Heat 168 19 93.9 6.6 10 9 8 1.78 0.18 1.60 0.25 ** Graphical representation of values can be seen in Figure 4 5 and Figure 4 6. Control duodenum exhibited higher VH/CD ( villi height/crypt depth ) ratios over all other combinations of region and treatment ( p < 0.0001) an d higher VH/VW ( villi height/ villi width ) ratios in comparison to heated duodenum ( p < 0.05) when compared to control duodenum. ** ( p < 0.01) when compared to p < 0.0001) wh en compared to control duodenum

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33 T able 4 2 Frequency of Specific Grades of I njury across Region and Treatment Treatment Percentage of villi at specific grades 0 1 2 3 4 Average Grade Duodenum Control 32.9 % 10.5 30.8% 8.6 18.1% 4.5 15.6% 9.2 2.5% 2.5 1.24 0.37 Jejunum Control 42.7% 9.3 41.3% 6.5 16.0% 9.5 0% 0 0% 0 0.73 0.18 Ileum Control 58.3% 9.8 35.4% 8.1 4.4% 2.4 1.9% 1.2 0% 0 0.50 0.12 Duodenum Heat 1.9% 1.2 1.7% 1.1 39.0% 10.6 50.8% 9.3 6.7% 3.3 2.59 0.10 ** Jejunum Heat 27.7% 10.5 34.8% 5.6 23.3% 6.9 14.2% 6.0 0% 0 1.24 0.28 Ileum Heat 60.0% 17.3 25.8% 9.8 13.3% 7.7 0.8% 0.8 0% 0 0.55 0.26 Graphical representation of these values can be seen in Figure 4 4. H eated duodenum exhibited higher dama ge scores over all other combinations of region and treatment. p p <0.0001) when compared to heated duodenum.

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34 Fig ure 4 1 Intestinal permeability when exposed to simulated ischemia treatments Control t reatments had significantly lower FD4 transport than all other treatments There was no statistical difference between moderate, severe, or h ypoxic treatments. ( p < 0.05) significant increase in intestinal transport of FD4 compared to control treatment ** ( p < 0.01) significant increase in intestinal transport of FD4 compared to control treatment

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35 Fig ure 4 2 Regional intestinal permeability in different gradients of ischemia Region was defined by measuring the midpoint of the intestinal segment fr om the ileocecal junction and dividing by the total length of intestine. In this graph, data were grouped within the three segment areas. In the inserted small graph, raw data were expressed from individual locations of the intestine. All treatments exh ibited a trend of increased intestinal permeability in intestinal segments proximal to the Pyloric Spincter *M oderat e and severe ischemic treatments exhibited in the significantly higher linear slopes (inserted graph) compared to control treatment ( p < 0. 05). ** Hypoxic treatment exhibited a significantly higher intercept compared to control treatment ( p < 0.01).

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36 Representative histology by region and treatment Fig ure 4 3 Representative histology by region and treat ment Note the epithelial lifting in HS duodenum and HS jejunum and increased swelling in HS duodenum.

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37 Figure 4 4. Villi grade averages and frequencies vs. treatment and intestinal region. A ) Average villi grade vs. trea tment and intestinal region: Heated duodenum showed significantly greater levels of damage compared to all treatments ** ( p < 0.0 1 ) significant decrease in average damage score compared to heated duodenum ( p < 0.001) significant decrease in average da mage score compared to heated duodenum. ( p < 0.001) significant decrease in average damage score compared to heated duodenum. B ) Villi Grade Ratios by Region and Treatment: The same data is broken down to display the frequency of specific grades.

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38 Fig ure 4 5 Villi height/Crypt depth ratio by region and treatment Control duodenum had significantly higher VH:CD over all other treatments ( p < 0.0001)

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39 Figure 4 6. Villi height/Villi widt h ratio by region and treatment. *Control duodenum exhibited s ignificantly higher villi height/villi width ratios than heated duodenum ( p < 0.05).

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40 CHAPTER 5 DISCUSSION Summary of Results Our evidence shows that simulated ischemia in an in vitro model and heat stress in an intact model of heat stroke result s in a r egional gradient in intestinal damage. In each stressor tested, in vitro ischemia, in vitro heat (previous work not part of this thesis) and in vivo heat stroke, we observed that the more proximal a region is to the stomach, the more susceptible this reg ion was to damage. This gradie nt of injury appeared in both in vitro heat and ischemia as well as an in vivo model of heat stroke. More research is needed to determine if ischemia and heat have similar mechanisms of injury and/or if duodenum morphology i s more susceptible to injury regardless of the stressor. Further explored, this knowledge could lead to regionally targeted therapies that increase intestinal barrier function in certain pathological or stress conditions Experimental Critique In vitro a nd in vivo models were used to gain a more complete view of the intestinal response to heat and ischemic stress. Our in vitro model measured regional permeability and we originally tried to evaluate these in vitro segments for regional damage. However, t he procedures left tissues with extensive apparent damage, making them of questionable value for histolo pathology evaluation Previously we had adjusted our in vitro methods to reduce variability; however, we suspected that after the tissue underwent stan dardized post e xperimental measurements, it would be exposed to additional stress which we believe is responsible for making the the tissue s unsuitable for microscop ic evaluation When developing a model to test heat stroke in vivo we considered dividin g the small intestine with suture s or clamps and using

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41 multiple colored probes to examine permeability. However, additional tampering could result in additional injury and/or altered blood flow. Thus to examine damage, we modified existing in vivo model s to develop an approach minimize s invasiveness and keep s experimental conditions as physiologically relevant as possible There are multiple scoring systems to identify intestinal damage. However, many of these scoring systems fail to differentiate bet w een potentially unique types of damage Therefore, we chose to examine denudation, atrophy, edema, and intestinal permeability separately with each having its own damage scoring system. To determine denudation and ultimately ulceration, we used the inte stinal damage scoring system outlined by Chui et al. because it is widely used and its grades correlate proportionally to the exte nt and duration of ischemia ( 51 ) Our team modified the method by applying the grading system individually to a random sample of villi. By using the average grade of 10 villi, slides could be differentiated with greater accuracy and the frequencies of specific stages of denudation, could be determined. Villi height to crypt depth ratios have been previously used to objectively measure villous atrophy in intestinal barrier dysfunction in a variety of settings ( 52 58 ) and have been shown to result from cessation of blood flow ( 58 ) In the same fashion, villi height to villi width ratios were used to determine the extent of swell ing ( 52 ) Finally, intestinal permeability was tested by measuring F D4 translocation. Together, this allowed the most complete picture using the best methods available to our lab. Comparison to Previous Results As previously mentioned in Chapter 2 at ph ysiologi c temperatures regional permeability studies are conflicting with different outcomes between species ( 42 44 ) A recent study, examin in g chronic hea t exposure in pigs observed no VH:CD changes in

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42 chronic heat exposure across duodenum, jejunum, an d ileum ( 59 ) However, it should be noted that heat stroke was not induced in these animals. In acute heat stress, we observed a regional effect of permeability in the intestine while Lambert observed no significant effects ( 6 ) A weakness in work was that too few microscopy samples were taken to determine the extent of mucosal damage; only three heat stressed mice, and one control mouse was used ( 6 ) We have effectively shown regional permeability in the duodenum in vitro as well as regional damage in the duodenum in vivo. It is possible that differences observed in the rat and mouse can be attributed to species differences; however, insufficient data is available in the rat model or in other mammalian species to make a definitive statement at this time. Potential Causes of Duodenal Damage Our results showed significantly greater damage in the duodenum compared to ileum, in both heated and control treatments. The endotoxin translocation theory states that the inflammatory cascade occurrin g in heat stress is a response to endotoxin seeping out of the gut ( 36 60 ) It is hypothesized that endotoxin escapes the gut through openings in the tight junction of the epithelium, where it is transported to the rest of the body through the lymphatic system ( 36 60 ) While this experiment was not equipped to view tight junction status specifically, we have observed that in hea t stressed duodenal villi, over 50% of the villi sampled exhibited massive epithelial lifting down their sides and ulcerated, broken tips. In comparison, the jejunum exhibited less than 15% of sample villi to be ulcerated, and the ileum exhibited less than 1%. Because the majority of the damage was represented in the duodenum, one could assume that the duodenum may play a major role in the increased intestinal permeability observed in heat stroke.

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43 The endotoxin translocation theory states that the inflammatory cascade seen in multi organ failure is promoted mainly by endotoxin. However the majority of bacteria inhabit the ile um and colon (61) which in our hands are not greatly affected by hyperthermia or ischemia F ew species of bacteria can survive or thrive in the duodenum due to its hostile luminal conditions and p hasic propulsive motor activity (61) The refore, our resu lts might be more consistent to the transduction of some other inflammatory mediator than endotoxin (62). Additionally, the evidence regarding whether endotoxin is the main inflammatory mediat or in multi organ failure remains inconclusive though it is ge nerally accepted in the heat stroke literature ( 63 ). The Auto digestion Theory The Auto digestion Theory states that digestive enzymes such as proteases, lipases, nucleases, and amylases can also promote inflammation upon leakage from the intestinal lu men (62 64 ). T he pancreatic duct regularly empties digestive enzymes into the duodenum where they are activated ( 62, 64 ) Normally, these volatile digestive enz ymes are compartmentalized from the villi by a layer of mucous that covers the brush border a nd is impermeable to these enzymes ( 11 62 ) In ischemic conditions, the muc ous layer is damaged and becomes permeable to pancreatic enzymes, leaving the intestinal wall vulnerable to enzymatic penetration (62 65 ). Tissues are digested and inflammatory mediators are produced from the degraded cellular components (62 64 ). Exposure to pancreatic enzymes alone does not promote lukeocyte activation; however, the homogenate of tissues exposed to pancreatic enzymes will promote activation ( 66) In previous studies of ischemic intestinal injury, neither e ndotoxin nor TNF E xperiments were one third of the supernatant of pancreatic homogenate produced from one rat was placed

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44 into a second, healthy, ane sthetized rat resulted in 100% d eath in minutes ( 67 ). Further experiments sh owed that if the intestine is flushed with saline and the protease and phospholipase inhibitor, ANGD (6 amidino 2 naphtyl p guanidinobenzoate dimethanesulfate, nafamostat mesilate ), is administered, activation of circulating leukocytes will be almost completely inhibited in ischemia reperfusion injury ( 68 ). Together this suggests that pancreatic enzymes create distinct inflammatory mediators and that these inflammatory mediators c an induce an inflammatory cascade without the presence of endotoxin. In addition, enzymatic digestion of intestinal tissues could be partially r esponsible for the villi ulceration and disintegration of the lamina propria seen in some specimens of duodenum Morphology of the Duodenum and Damage Susceptibility Despite ximity to the pancreatic duct, there are substantial levels of pancreatic enzymes throughout the small intestine. However, the duodenal barrier is less equipped to prevent these enzymes from coming in to contact with the brush border. R esearch has shown that rat duodenum has a lower percentage of mucous producing goblet cells than other regions, starting at 4% at the duodenum and gradually increasing to 16% at the distal colon ( 6 9 ) The thickness of the total rat duodenum mucous layer is 170 m, while the ileum is 476 m ( 70 ) When this mucosal layer is removed by suction the ileum replaces mucous at a faster rate compared to duodenum as well ( 71 ) With a decreased thickness an d reduced restoration rate of mucous, the duodenum faces disadvantages to buffer itself against luminal contents D uodenal villi are also typically longer than jejunal or ileal villi ( 71 73 ) The increased length may contribute to a greater oxygen dispar ity at the tip of the duodenal villi, making it more susceptible to ischemia (74) and ultimately mucosal damage ( 62 ) A longer villus will

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45 also have increased surface area that is available to pancreatic enzyme inflitration We theorize that t he increase d sus c eptiblity to damage in the duodenum may result from this unique combination of increased proximi ty to digestive enzymes a thinner mucosal barrier with reduced regenerative properties as well as an increased ischemic risk factor. Further Application s Severe cases of exer tional hyperthermia are associated with GI dysfunction and endotoxemia ( 75 77 ) particularly in extended endurance events such as marathons ( 78 ) or triathlons ( 79 ) Recent finding s suggest that 25 50% of elite athletes are afflicted with gastrointestinal symptoms that may affect their training ( 80 ) It is theorized that this exercise induced GI dysfunction is brought on by ischemia and increased intesti nal permeability as exercise further reduces splanchnic blood flow when blood is s hunted to the muscles ( 4 80 ) More research is needed to determine if to what role and to what extent duodenal damage plays in these exercise induced gastrointestinal dis turbances. Summary of C onclusions In conclusion, our results show that the duodenu m is regionally susceptible to heat stroke in mice during and after exposure to extreme hyperthermia consistent with temperatures experienced in heat stroke in humans This trend is exhibited both in an intestinal segment model and in anesthetized mice. More research is needed to determine to what extent the duodenum plays a role in non exertional and exertional heat stroke In addition, work is needed to determine if a heat stroke damaged duodenum compromises exercise performance.

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46 LIST OF REFERENCES 1. Nelson NG, Collins CL, Comstock RD, McKenzie LB Exertional heat related injuries treated in emergency departments in the U.S., 1997 2006. Am J Prev Med 40: 54 60 2011 2. Bouchama A, Knochel JP. Heat stroke. N Engl J Med 346: 1978 19 88 2002 3. Hales JR, Rowell LB, King RB. Regional distribution of blood flow in awake heat stressed baboons. Am J Physiol 237: H705 H7 12 1979 4. Rowell LB. Cardiovascular aspects of human thermoregulation. Circ Res 52: 367 3 79 1983 5. Deitch EA. Bacterial translocation or l ymphatic drainage of toxic products from the gut: what is important in human beings? Surgery 131: 241 24 4 2002 6. Lambert GP, Gisolfi CV, Berg DJ, Moseley PL, Oberley LW, Kregel KC. Selected contribution: Hyperthermia induced intestinal permeability and t he role of oxidative and nitrosative stress. J Appl Physiol 92: 1750 17 61 2002. 7. Oliver SR. Thermal tolerance of skeletal muscle and small intestine: role of eicosanoid metabolism and oxidative stress Ohio State University 2009 8. Phillips NA. Small bowe l susceptibility to hyperthermia: Development of a model. University of Florida 2010 9. Eshel GM, Safar P, Stezoski W. The role of the gut in the pathogenesis of death due to hyperthermia. Am J Forensic Med Pathol 22:100 10 4 2001 10. Dewhirst MW, Viglianti B L, Lora Michiels M, Hanson M, Hoopes PJ. Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia. Int J Hyperthermia 19:267 2 94 2003 11. Balzan S, de Almeida Quadros C, de Cleva R, Zilberstein B, Cecconello I. Bacte rial translocation: overview of mechanisms and clinical impact. J Gastroenterol Hepatol 22: 464 4 71 2007 12. Baumgart DC, Dignass AU. Intestinal barrier function. Curr Opin Clin Nutr Metab Care 5: 685 6 94 2002 13. Wiest R, Rath HC. Gastrointestinal disorders of the critically ill. Bacterial translocation in the gut. Best Pract Res Clin Gastroenterol 17: 397 425 2003

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47 14. Ye D, Ma TY. Cellular and molecular mechanisms that mediate basal and tumour necrosis factor alpha induced regulation of myosin light chain k inase gene activity. J Cell Mol Med 12: 1331 1346, 2008. 15. Ma TY, Hoa NT, Tran DD, Bui V, Pedram A, Mills S, Merryfield M. Cytochalasin B modulation of Caco 2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol 279: G875 G8 85 2000 16. Cario E. Heads up! How the intestinal epithelium safeguards mucosal barrier immunity through the inflammasome and beyond. Curr Opin Gastroenterol 26: 583 5 90 2010 17. Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of inte stinal mucosa in mice lacking intestinal trefoil factor. Science 274: 262 26 5 1996 18. Lim CL, Wilson G, Brown L, Coombes JS, Mackinnon LT. Pre existing inflammatory state compromises heat tolerance in rats exposed to heat stress. Am J Ph ysiol Regul Integr Comp Physiol 292: R186 R1 94 2007 19. Hashim IA. Clinical biochemistry of hyperthermia. Ann Clin Biochem 47: 516 5 23 2010. 20. Goldstein LS, Dewhirst MW, Repacholi M, Kheifets L. Summary, conclusions and recommendations: adverse temperature levels in the huma n body. Int J Hyperthermia 19: 373 3 84 2003 21. Despa F, Orgill DP, Neuwalder J, Lee RC. The relative thermal stability of tissue macromolecules and cellular structure in burn injury. Burns 31: 568 5 77 2005 22. Balogh G, Horvth I, Nagy E, Hoyk Z, Benk S, Bensaude O, Vgh L. The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response. FEBS J 272: 6077 60 86 2005 23. Lepock JR. Cellular effects of hyperthermia: relevance to the minimum dose for thermal damage Int J Hyperthermia 19: 252 2 66 2003 24. Hall DM, Baumgardner KR, Oberley TD, Gisolfi CV. Splanchnic tissues undergo hypoxic stress during whole body hyperthermia. Am J Physiol 276: G1195 G1 203 1999 25. Chang CK, Chang CP, Liu SY, Lin MT. Oxidative stress and ischemic injuries in heat stroke. Prog Brain Res 162: 525 5 46 2007

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53 BIOGRAPHICAL SKETCH Vero nica Le a Novosad completed her applied physiology and k inesiology undergraduate degree at the University of Florida, specializing in exercise p hysiology. After graduating with her Bachelor of Science in August 2009, she stayed at the University of Florida and imm physiology and k inesiology. She has since worked as a research assistant under Dr. Clanton. Within the Clanton lab, Veronica developed a simulated ischemia model, examined intestinal integrity in heat st roke, has contributed to four abstracts, and personally presented two of them at the international 2010 and 2011 Experimental Biology Meetings She has currently been accepted into several medical school s and plans to attend in the Fall of 2011.