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Small Bowel Susceptibility to Hyperthermia Development of a Model

Permanent Link: http://ufdc.ufl.edu/UFE0042285/00001

Material Information

Title: Small Bowel Susceptibility to Hyperthermia Development of a Model
Physical Description: 1 online resource (43 p.)
Language: english
Creator: Phillips, Neil
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: applied, duodenum, everted, gastric, heat, hyperthermia, ileum, illness, intestine, jejunum, kinesiology, model, permeability, physiology, regional, small, stomach, stress, stroke
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: Our goal was to develop a viable model of heat-induced intestinal barrier dysfunction. Additionally, we tested the hypothesis that the small intestine displays varied changes in hyperthermia-induced permeability along its length. We isolated and everted segments from the small intestine of adult mice, creating ~8 x 2 cm sacs from each mouse, which were then separated into heated and control groups (42 + 0.5oC and 37 + 0.5oC, respectively). Permeability was measured following a 90 minute exposure. The data show a significantly increased permeability in regions of the intestine closest to the stomach (P ~ 0.009). We conclude that in the mouse, the anterior portions of the small intestine (near the stomach) have an increased susceptibility to hyperthermia compared to posterior regions (the ileum). This is of importance to our laboratory, as we have identified a key variable in evaluating permeability responses seen in our model. Our data further suggest that treatments targeted to protect the upper intestine may be useful in reducing the symptoms of heat related illnesses.
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 Neil Phillips.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Clanton, Thomas.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042285:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042285/00001

Material Information

Title: Small Bowel Susceptibility to Hyperthermia Development of a Model
Physical Description: 1 online resource (43 p.)
Language: english
Creator: Phillips, Neil
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: applied, duodenum, everted, gastric, heat, hyperthermia, ileum, illness, intestine, jejunum, kinesiology, model, permeability, physiology, regional, small, stomach, stress, stroke
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: Our goal was to develop a viable model of heat-induced intestinal barrier dysfunction. Additionally, we tested the hypothesis that the small intestine displays varied changes in hyperthermia-induced permeability along its length. We isolated and everted segments from the small intestine of adult mice, creating ~8 x 2 cm sacs from each mouse, which were then separated into heated and control groups (42 + 0.5oC and 37 + 0.5oC, respectively). Permeability was measured following a 90 minute exposure. The data show a significantly increased permeability in regions of the intestine closest to the stomach (P ~ 0.009). We conclude that in the mouse, the anterior portions of the small intestine (near the stomach) have an increased susceptibility to hyperthermia compared to posterior regions (the ileum). This is of importance to our laboratory, as we have identified a key variable in evaluating permeability responses seen in our model. Our data further suggest that treatments targeted to protect the upper intestine may be useful in reducing the symptoms of heat related illnesses.
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 Neil Phillips.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Clanton, Thomas.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042285:00001


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1 SMALL BOWEL SUSCEPTIBILITY TO HYPERTHERMIA: DEVELOPMENT OF A MODEL By NEIL ANDR PHILLIPS 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 2010

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2 2010 Neil Andr Phillips

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3 To my Mom

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4 ACKNOWLEDGMENTS Thanks, to those with the time to administer words of encouragement; t o my family and coaches.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 INTRODUCTION .................................................................................................... 10 Solute Movement .................................................................................................... 11 Mucus Layer ........................................................................................................... 12 Tight Junctions ........................................................................................................ 12 Medical Interest ...................................................................................................... 13 Heat Stress ............................................................................................................. 14 Current Methods for Investigating Intestinal Permeability ....................................... 16 Specific Aims .......................................................................................................... 21 2 METHODS .............................................................................................................. 22 Chemicals Used ...................................................................................................... 22 Animal Treatment and Gut Sac Preparation ........................................................... 22 Data Analysis and Statistics .................................................................................... 23 3 RESULTS ............................................................................................................... 25 Change in Experimental Chamber .......................................................................... 25 Area Measurements ............................................................................................... 26 Analysis of Confounding Variables ......................................................................... 27 Tissue Handling ...................................................................................................... 28 4 DISCUSSION ......................................................................................................... 35 Biological Sources of Variance ............................................................................... 35 Origins of Biological Variation ................................................................................. 36 Critique of Isolated Segment Model ........................................................................ 37 Conclusion .............................................................................................................. 38 LIST OF REFERENCES ............................................................................................... 40 BIOGRAPHICAL SKETCH ............................................................................................ 43

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6 LIST OF TABLES Table page 2 1 Conditions in cuvettes. Values compiled from 3 Mice. ........................................ 30 2 2 Experimental sources affecting variance. A mul tiple logistic regression analysis .............................................................................................................. 33

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7 LIST OF FIGURES Figure page 1 1 Variance in original protocol. N = 49 Mice .......................................................... 20 2 1 Vessel change. The change to conical tubes significantly lowered permeability (p) and variance (v). N = 41 mice, P < 0.05 .................................... 31 2 2 Mean c ompliance c ur ve. Average inflection point at 30 + 5 g. N = 8 sacs .......... 31 2 3 C hange in area measurement. S witch to tissue compression lowered permeability (p) and variance (v). N = 15 mice, P < 0.05. ................................... 32 2 4 Regional permeability. Permeability increases as a function of gastric proximity. N = 4 mice ......................................................................................... 34 2 5 Improved tissue handli ng. E liminat ion of investigator induced damage (*) compared to Figure 24 N = 4 mice ................................................................... 34

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8 LIST OF ABBREVIATIONS FD4 F luorescein isothiocyanate ( FITC ) Dextran 4000 Da; a fluorescent protein sized to mimic small endotoxin ( LPS) and is used to measure tissue permeability. LPS Lipopolysaccharide, or endotoxin; bacterial components in the intestine that stimulate immune and inflammatory responses NSAID Non Steroidal Anti Inflammatory Drug

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9 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 SMALL BOWEL SUSCEPTIBILITY TO HYPERTHERMIA: DEVELOPMENT OF A MODEL By Neil A. Phillips December 2010 Chair: Thomas L. Clanton Major: Applied Physiology and Kinesiology Our goal was to develop a viable model of heat induced intestinal barrier dysfunction. Additionally, we tested the hypothesis that the small intestine displays varied changes in hyperthermiainduced permeability along its length. We isolated and everted segments from the small intestine of adult mice, creating ~8 x 2 cm sacs from each mouse, which were then separated into heated and control groups (42 + 0.5oC and 37 + 0.5oC, respectively). Permeabilit y was measured following a 90 minute exposure. The data show a significant ly increased permeability in regions of the intestine closest to the stomach (P ~ 0.009). We conclude that in the mouse, the anterior portions of the small intestine (near the stomach) have an increased susceptibility to hyperthermia compared to posterior regions (the ileum). This is of importance to our laboratory, as we have identified a key variable in evaluating permeability responses seen in our model Our data further suggest that treatments targeted to protect the upper intestine may be useful in r educing the symptoms of heat related illnesses.

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10 CHAPTER 1 INTRODUCTION The small bowel is a critical site for the bodys interaction with the external environment. The majority of nutrient absorption occurs across the epithelial wall, which serves a s an anatomical barrier to anything ingested. As with most mucosal linings, the small intestine is home to a host of secretory, immune, and inflammatory cells that regulate the organisms interactions with dietary and bacterial antigens (Baumgart, 2002). The small intestine coordinates one of the bodys largest collections of endocrine and immune cell populations while maintaining a symbiotic relationship with the resident bacterial milieu ( Merchant, 2007). This relationship, hinged on proper barrier mai ntenance, is pivotal for immunological homeostasis. The outermost layer of the intestine consists of a longitudinal and subjacent circular muscle layer. These muscles produce the peristaltic movements of the small intestine and control, downstream of neural stimulation, the motility of intestinal co ntents The submucosa that follows this outer muscular layer is separated from the inner mucosa or lamina propria by the thin muscularis mucosae. The lamina propria is home to a variety of immune and inflam matory factors and is generally convoluted into finger like villi that increase the abs orptive area of the small bowel E ach villus has its own circulatory supply and is surrounded by invaginations known as crypts The lamina propria is then separated from the lumen by a unicellular layer of epithelial and differentiated cells. The luminal surface of these cells is impermeable to most hydrophilic solutes, a property accomplished through mucus secretion and strong intercellular connections (Turner, 2009).

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11 Solute Movement Solute movement across the intestinal barrier occurs either through paracellular (between cells) or transcellular (across cell membranes) pathways. Molecules too large or hydrophilic must rely on transporters on the apical surface of epithelial cells to access the body, often taking advantage of a stringent sodium gradient maintained by sodium hydrogen co transporters and intercellular tight junctions (Baumgart, 2002). Through cytoskeletal connections and intracellular signals, inter actions at the apical surface can directly affect the assembly of the tight junction proteins that control paracellular solute diffusion (Turner, 2009). Cellular and tight junction integrity must be maintained to prevent the unregulated translocation of l uminal contents, including bacteria and bacterial products (endotoxin), into the submucosa. To assist in its immunological role, the small intestine hosts a large number of B cells, T cells, macrophages and other lymphoid tissue (Baumgart, 2002). Antigen presenting cells (APCs), like dendtritic cells, and pattern recognition cells, like the tolllike receptor (TLR) family reside in the lamina propria and link the bodys innate and adaptive immune responses These components interact with specific stimuli and guide the bodys adaptive responses by activating resident B and T Cells. Toll like receptor 4 ( TLR 4 ) a member of the toll like receptor family that is sensitive to LPS in the intestine can induce inflammation by stimulating the local production of cytokines by lymphocytes Intraepithelial lymphocytes are joined at the mucosal surface by differentiated epithelial cells including the absorptive brush border cells and mucus secreting goblet cells that are constantly generated from crypt stem cells and develop along the length of the villus (Turner, 2009).

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12 Mucus Layer The first line of defense for the epithelium against fully active digestive enzymes and bacterial antigens is a continuous, secreted mucus bicarbonate layer (Allen, 1986). There ar e actually two distinct layers, a loosely and a firmly adherent mucus layer both formed by epithelial secretions of mucin, bicarbonate, and a glycocalyx or sugary coat The loosely adherent layer plays a superficial protective role and is more suscepti ble to damage by ethanol, aspirin, and high concentrations of pepsin and bile salts (Allen, 1986). The firmly adherent layer is vital in protecting mucosal cells from luminal acids and for maintaining a relatively neutral juxtamucosal pH along the length of the intestine; its thickness varies along the gastrointestinal tract and is lowest in the small intestine (Atuma, 2001). In addition to its defensive role in protection from intestinal contents, an undisrupted mucus layer improves intestinal function. It limits diffusion, aiding nutrient absorption and immune function by controlling the rate at which nutrients and antigens come into contact with the mucosal barrier By also allowing the concentration of epithelial secretions to remain high, it increases the activity of locally produced digestive enzymes, and makes bicarbonate secretion more effective at maintaining a neutral pH at the luminal surface (Allen, 2005) Tight Junctions Interactions at the mucosal surface determine epithelial cell viability and regulate the assembly and disassembly of tight junction proteins. These intercellular proteins help regulate the flux of water into the central cavity of the small intestine, or lumen, and preserv e the continuity of the intestinal barrier while contr olling the most dynamic and permeable path of solute movement (Steed, 2010). At tight junctions, the space between cells is essentially eliminated creating a physical barrier to luminal contents.

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13 Moreover, the structural and regulatory proteins of the t ight junction help establish the charge and size selectivity needed for the tightly controlled flow of solutes ac ro ss the intestinal wall. Claudins are one such family of proteins known to help govern the cation specificity epithelial linings throughout t he body (Niessen, 2007) The dynamics of the t ight junction can be regulated by the direct modulation of morphological structures or by the activity of effect or proteins and molecules (Steed, 2010). Other junctional proteins include the intercellular pr oteins cadherin and occludin, and the scaffolding protein, zonula occludens 1 (ZO 1) Extracellular activation of cadherins occur s via calcium signals through the par acellular space; this increas e s junctional tightness and trigger s intracellular signals t o increase the transcription of junctional strands (Turner, 2009). Tight junctions are normally regulated by perijunctional rings of actin and myosin that are under the direct influence of myosin light chain kinase (MLCK). MLCK phosphorylates myosin II r egulatory light chain (MLC) within the actomyosin ring, activating myosin ATPase and stimulating interactions between actin and myosin. The resulting condensation of the perijunctional ring pulls on junctional proteins, controlling the strength of the ti ght junction (Turner, 2009). MLCK is a common endpoint of physiological and pathological mechanisms regulating intestinal permeability (Turner 2009). Medical Interest Myriad disease states and disorders trace their effects to intestinal dysfunction, including ulcerative colitis, inflammatory bowel disease, and heat stroke (Cario, 2000) Barrier maintenance is regularly tested as any number of pharmacological, psychological, physiological, or pathological factors can affect mucus secretion or increase int estinal permeability (Lambert, 2009). Proper barrier function involves the

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14 establishment and maintenance of a sodium gradient across the mucosal membrane. Tight junctions help prevent the dissipation of this gradient by guarding solute movement between e pithelial cells; alterations in these mechanisms can result in diarrhea from increased water flux into the lumen or deficient absorption (Turner, 2009). Damage to the cell membrane, a direct effect of increased core temperature, challenges the bodys abili ty to maintain transmucosal gradients. Patients with C ro h ns disease and ulcerative colitis both possess upregulated TLR 4, which is primarily activated by endotoxin in the intestine and result s in the overstimulation of T Cells (Cario, 2000). The decreas ed barrier function in colitis patients has also been attributed to an overexpression of the tight junction protein claudin (Steed, 2010). The expression of this protein varies between organs and throughout development and may be modified by cytokines li ke interferon( ) and tumor necrosis factor ( TNF ) (Turner, 2009). Increased mucosal TNF production has been linked to a variety of intestinal disease states including colitis, and has been shown to regulate tight junctions in vivo and in vitro by manipulating MLC Ks enzymatic activity and by increasing MLCK transcription and translation. Because of its ability to alter mucus production, increase mucosal permeability, and cause malabsorption, TNF is often used as an experimental stimulus for intestinal dysfunction (Turner, 2009). Heat Stress Increases in core temperature, from the environment or exercise, can cause as much as a 40% reduction in intestinal blood flow (Hall, 2001) as the body attempts to simultaneously power active muscles and disperse heat by div ertin g blood to the skin. As a result, the intestine may become deficient in blood flow. This, when combined with direct thermal damage to membrane cells, disrupts tight junction proteins, allowing

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15 bacterial components like endotoxin (Hall, 2001) and di gestive enzymes to penetrate the mucosal barrier (SchmidSchonbein, 2008). The subsequent rise in bacterial translocation activates both local and systemic inflammatory responses by accessing the circulation and infiltrating the liver and other organs. W ith increasing severity, the resulting endotoxemia and inflammatory cascades can result in sepsis or death (Hall, 2001). A five hour heat exposure for three days notably decreased the integrity of tight junctions in vivo and increased in the number of dam aged mitochondria in the epithelium (Liu, 2009). The i nternal cristae of mitochondria appeared swollen and shortened and there was an increase in secondary lysososomes ; both are thought to occur in response to intestinal lesions caused by hyperthermia (Li u, 2009). Villus heights and crypt depths were significantly decreased in response to heat treatment and the desquamation of epithelial cells at villus tips and exposed lamina propria offered clear signs of mucosal damage (Yu, 2010). Lambert et al, (Lambert, 2002) also report ed thermal disruption of the epithelial membrane both in vivo and in vitro with physiological increases in temperature. The elevation of t umor necrosis factor TNF, in heat stroke patients suggest s a link between heat stress and entotoxemia (Lambert, 2002). Circulating levels of endotoxin (LPS) are also significantly increased in heat stroke victims and heat treated animals ( Hall, 2001). Dokladny et al (Dokladny, 2005) found similar effects in caco2 monolayers, noting that increases in temperature decreased transepithelial electrical resistance (TEER) and increased the perm eation of inunlin ( a paracellular permeability probe) indicating a breakdown of tight junction stability

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16 Current Methods for Invest igating Intestinal Permeability There are various in vivo ex vivo and in vitro animal models that have been verified for the investigation of intestinal function, allowing inv estigators the freedom to manipulate specific physiological parameters. Recently, the effects of long term heat exposure was investigated by subjecting experimental mini pigs to a heat stress for five hours daily for 10 consecutive days. Animals were sac rificed at different time points and epithelial tissues were excised for morphological analysis using hematoxylin and eosin (H&E) staining and transmission electron microscopy to visualize nuclei and cellular components. The relative lengths of villi and c rypts were recorded and used in conjunction with photomicrographs to quantify damage to the epithelium (Yu, 2010). After three days of treatment, thermal damage was noted in jejunal epithelium by shorter villus heights and crypt depths ; alterations in tig ht junction morphology were determined by localized changes in electron density (Liu, 2009). A common method of assessing i ntest i nal permeability both in vivo and in vitro uses the ratiometric passage of an ingested pair of hydrophilic permeability probes into the urine or blood. Probe pairs typically consist of a large and a small saccharide that are selected based on established regions of permeation along the digestive tract; the sugars lactulose and mannitol are often used to assess the permeability of the small bowel (Fink, 2003). These probes have been used to assess the in vivo human permeability of gastric or intestinal mucosa, but their use relies on several critical assumptions. First, that the larger sugar (lactulose) is limited to paracellul ar diffusion while the smaller (mannitol) can diffuse both between cells and across the apical cell membrane. This method does no t account for the possibility of active transport, and the rate of absorption of the smaller probe is presumed to be cons tant. Moreover, both

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17 sugars are assumed to be equally affected by changes in intestinal permeability or alterations in preand post intestinal factors like transit time or renal function. The assumptions of this model have c ome under question with the temperaturedependent active transport of a variety of hydrophilic compounds, including lactulose, being verified in the small bowel (Tomita, 2000). Barrier function is investigated by analyzing the status and function of the pr otective mucus layer. As it may be easily disrupted during in vitro and staining procedures, changes in the structure of the adherent mucus layers are optimally studied in vivo Intravital microscopy is used to measure mucus thickness and production in t he exteriorized and mounted gastrointestinal tissue of anesthetized rats (Atuma, 2001). Szabo et al (Szabo, 2005) used an intravital microscopic method to measur e the accumulation of small and medium sized fluorescent markers in the intestinal interstiti um in vivo following an ischemic event. This method provides a relatively easy and applicable means of assessing epithelial permeability in vivo and of evaluat ing the eff icacy of attempts to preserve barrier function. However, it does not lend itself t o quantitative assessment between treatments. The use of cell culture systems is another option. Though their use diverges from normal physiology, it has the advantage of being highly controllable. In addition, the method sometimes yield s results that are variable and sometimes of questionable physiological significance Caco 2 cells are a line of colonic cancer cells that are often grown into monolayers and used in intestinal permeability studies The effects of temper ature on Caco2 permeability can be determined using established paracellular markers like inulin (Ma, 1991). Electrodes placed on either side of the monolayer

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18 measure are often used to measure transepithelial electrical resistance (TEER) as an additional assessment of solute movement between cells (Dokladny, 2005). Properly functioning tight junctions must establish and maintain electrical resistance across the epithelium and changes in TEER may represent early signs of cellular damage. However, there are normal openings in tight junctions that are responsible for ionic and water movements that do not represent the same cytoskelelal motions responsible for barrier dysfunction. The measurements of electrical resistance are therefore not suitable for determin ing the mechanisms responsible for diffusion of large molecular weight solutes like endotoxin. Combining the techniques of time lapse multidimensional fluorescence microscopy and measuring (TE ER), Turner et al, (Turner, 2009) visualized the organizati on of fluorescent tagged tight junction proteins. This methodology is used to examine the assembly and dissociation of these proteins before, during, and following experimental conditions. Similarly, the immunostaining of C aco2 monolayers has also been used to visualize the behavior of junction proteins and their cytoskeletal connections in response to increases in temperature (Dokladny, 2005) The use of Ussing diffusion chambers allows tissue from different regions or species to be treated similarly for the comparison of directionand conditionspecific solute movement and analysis (Nejdfors, 2000). Simultaneously, TEER can be measured across the tissue as an indication of cellular viability. Specifically size d molecular weight markers are used to examine the different pathways of solute permeation, and proper selection can offer a complete picture of barrier integrity. lactalbumin and ovalbumin are used to show mucosal

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19 permeability to macromolecules Medium sized molecules, like the 4 kDa fluorescein isothiocyanate ( FITC ) dextran (FD4) or radiolabeled ethylenediaminetetraacetic ac i d ( EDTA; an organic compound), are known to permeate the barrier using mainly the paracellular route in the absence of overt cellular damage (Nejdfors, 2000). In one such experiment, biopsies of the gastrointestinal tract were obtained from patients undergoing colonic resections and malignancy removals in the stomach, esophagus, or colon (Ungell, 1997) A fter the external muscle layers are stripped, the movement of radiolabeled m annitol, ovalbumin, alphalactalbmin, FD4, and FD70 (FITC dextran, 70 kDa) across the epithelial barrier was measured in an Ussing chamber while electrodes measured TEER By employing compounds known to use each avenue of solute permeability, investigators overc ame the limitations of ratiometric sugar and protein calcul ations. Lambert et al, (Lambert, 2002) investigated the role of oxidative and nitrosative stress in hyperthermiainduced intestinal permeability, using an in vitro segmental model in rats. After excision, the small intestine was cleaned, everted, and separated into individual buffer filled segments that were then exposed to temperature or compound specific treatments in a solution containing a medium or large fluorescent marker (0.25 mM FD 4 or FD 10). The movement of the fluorescent markers, in conjunction with volume and area measurements, was used to calculate tissue permeability in response to heat treatment Lambert et als heat induced permeability studies (Lambert, 2002) in rats utilized both in vivo and in vitro models and compared the results, finding that the segmental in vitro model used mimicked in vivo results (Lambert, 2002). This method permit s the study of multiple conditions separately using

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20 tissue from a single animal, while presumably obtaining physiologically relevant results. In addition, the method allows evaluation of the intestine, ex vivo after a treatment is given to the intact animal. For this reason our laboratory chose to develop this model in the mouse in order to study the pathophysiology of heat stroke. A previous student in our laborat o r y, S Ryan Oliver, adapted Lamberts protocol for the development of our laboratorys own murine (mouse) model of heat induced barrier dysfunction. This model was used to evaluate different treatments to the tissue that could attenuat e the hyperthermiainduced permeability, but the level of variance amongst similarly treated tissues made it difficult to measure significant effects (Figure 1 1). Changes were made in tissue and buffer preparations in attempts to reduce this variability before the decision was made to average duplicate treatments within each animal. This compensated for the variance, but failed to sufficiently address the sources of variance. Their identification and elimination became the focus of my graduate work. Figure 11. Variance in original prot ocol. N = 49 Mice

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21 Specific Aims The specific aims of the present investigation were to 1) identify and address the sources of variability in our in vitro segmental model of hyperthermiainduced intestinal permeability, 2) to determine the relationship bet ween intestinal region and heat induced permeability, and 3) to test the hypothesis that intestinal permeability varies as function of proximity to the stomach.

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22 CHAPTER 2 METHODS Chemicals Used M edium 199 (Cellgro), LGlutamine (Lonza), sodium bicarbonate (Acros Organics), fluorescein isothiocyanate (FITC) dextran 4 kDa (FD4, Sigma Aldrich) Animal Treatment and G ut Sac P reparation Our studies used adult C57bl6 mice (2535 g). Animals were treated according to protocols approved by the University of Florida Institutional Animal Care and Use Committee. Prior to euthanasia by carbon dioxide asphyxiation, mice were kept on a clear liquid diet for 10 hours to reduce luminal contents. The entire intesti ne was then rapidly excised and placed in pre oxygenated medium 199 (with glutamine and sodium bicarbonate). Excisions were made proximal to the cardiac sphincter in the stomach and distally in the large intestine. After gross removal of the mesentery responsible for vascularization the small intestine is freed of the cecum and large intestine with a cut above the ileocecal valve. The remaining mesentery is then trimmed, taking care throughout not to damage the gut. After the small intestine was made linear, its contents are flushed with oxygenated medium 199. Next, the length of the intestine is everted over a smooth glass pipet t e that has been gently inserted into the luminal cavity. The gut is then filled with oxygenated media and 12 cm sections are sequentially isolated using 20 suture. N ext, gut sacs were placed into cuvettes of oxygenated buffer containing the high molecular weight fluorescent marker 4 kDa fluorescein isothiocyanatedextran (0.3 mM FD4). The cuvettes were then covered and placed into water baths maintained at 37C or appropriately set aluminum heating blocks.

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23 The temperature was monitored with a highly accurate thermistor, accurate to two digits (Yellow Springs Model 4610). Heating blocks were controlled by a self regulating temperature controller (Digi Sense, Cole Parmer) and a heating plate. After experimental exposure, the solution inside the gut sacs were collected in individual tubes and the square area determined after longitudinal dissection. The FD4 concentration of the serosal fluid was calculated by comparing the fluorescence measurements made in a spectrofluorometer (SpectraMax M5, Molecular Devices) to those of a standard curve. Throughout my investigation and attempts to lessen the variance of this model, sever al changes were made to our methodology. Food was no longer withdrawn from the animals prior to euthanasia and smaller glass pipettes were used for eversion. P rior to gut sac preparation, the intestine was filled under a defined column pressure of ~6 cm of oxygenated media. S egments were then prepared to a standardized length of ~2.3 cm. Randomized pairs of segments were then incubated at 37C in conical tubes containing continually oxygenated buffer. Following this 30 minute period, segments were transferred to conical tubes containing 0.3 mM FD4 and placed into water baths maintained at 37C or 42C Finally, after experimentation, tissue area was measured by compression with a standardized weight instead of segmental dissection. Data Analysis and St atistics At the conclusion of the experiment, sac contents were emptied into individual, preweighed tubes. The serosal volume of the sac was calculated from t he change in weight and recorded in milliliters. In the original protocol, the segment was then dissected longitudinally and flattened to record the length and width. This was adapted so that the segment was compressed with a standardized weight and the square area

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24 measured. The micromolar concentration of the serosal fluid was determined by compari ng its fluorescence reading to that of a standard curve. Tissue permeability was normalized to segment surface area and serosal volume and was represented as the transport of nmoles of FITC dextran per cm2. This analytical method was taken from Lambert et al. (Lambert et al., 2002) who denoted permeability = (Concentrationserosal fluid X Volumeserosal fluid) Mucosal surface area. Decreases in permeability measurements were determined using Analysis of Variance (ANOVA) or Students t test for unequal variance (GraphPad Prism). Reductions in variance following adaptations were calculated from Fishers F test for difference of variance (SASJMP). Multiple logistic regression analysis was used to stratify potential sources of variance (SASJMP). The slopes of baseline and heated treatments were analyzed using comparative linear regression to examine linear plots of data (GraphPad Prism). All results are reported as means SEM; P <0.05 was considered to be statistically significant

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25 CHAPTER 3 RESULTS Ch ange in Experimental Chamber The original protocol that was developed in the laboratory (Oliver, 2009 Thesis) used small 3 mL sealed cuvettes for each intestinal segment. Though this may have modeled the expected ischemic conditions of hyperthermia in t he whole animal (Hall, 2001), I was concerned that variations in PO2, PCO2, pH and nutrient depletion within the cuvettes may have affected the permeability and been a source of variance. I therefore, first quantified the acidity and gas concentrations in the covered 3 mL cuvettes using the original protocol in both heated and nonheated cuvettes At experiments end, the values for pH, P C O2, and P O2 were measured using an arterial blood gas analyzer ( ABL800 FLEX ) (Table 2 1). Because the blood gas machine brings all samples back to 37 C, samples taken from different temperature cuvettes are renormalized to the same temperature. While the mean for pH was consistent between baseline and heated treatments individual values were lower than expected given the sodium bicarbonate buffer system we used. This was probably due to the lack of bubbling of the cuvettes during the treatment and the clear accumulation of PCO2. The values for PO2 were lower than expected. Combined with the acidity and hypercapnia noted in a number of treatments, this data verified our need for using a different system that control led the gas concentrations as well as temperature throughout the protocols. We addressed this need by replacing the sealed cuvettes with adapted fifty (50) ml conical tubes. These vessels allow ed control over a steady, low bubbling of 95% O2/5%CO2 into the incubation and treatment media, which can be accessed and monitored without disrupting experimental conditions. As high rates of bubbling can

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26 saturate the soluble CO2 and lower the pH of the solution the bubbling rate was kept low. Using this method, permeability was measured over 90 minutes from segments treated with only 37C or 42C in conical tubes These were compared with results from to similarly treated segments in the 3 ml cuvettes ( Figure 2 1). With the change in vessel, we noted a decrease in the permeability (P < 0.008, t test for unequal variance) and more importantly, a significant reduction in the variance between the temperature treatme nts (P < 0.05, F test). Area Measurements Following the success of our vessel change, we questioned the accuracy of our method of estimating surface area of the segments after treatment. This calculation is needed to normalize the measurements to the surf ace area for diffusion. The original technique had a great deal of uncertainty in it and we wondered if it may cause additional tissue damage and distort the area measured. The original method called for a longitudinal dissection of each segment and bef ore it was laid flat and its length and width recorded. To reduce the potential for variance, the decision was made to compress the tissue without cutting it, by laying it between two (2) small sheets of plastic with a standardized weight on top. We desi red to select a range of weight s that would sufficiently flatten the sample without causing tissue deformation and at which moderate increases in weight from water tension or hand pressure would not significantly alter the area measurement. To standardize this method, and select an ideal weight, we measured tissue compliance on eight segments following a ninety (90) minute 37C treatment The weights placed on the segment were progressively increas ed until a plateau was achieved. (Figure 2 2) A standardized weight of 30 grams was selected from the point of inflection on the compliance curve seen in plots of area

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27 measurements against the corresponding weight applied. To examine the effect of this change on the variance and the value of the permeabili ty measurements technique, we compared data from control samples that were first compressed vs. those that were split and dissected following 37C or 42C treatment ( Figure 2 3). The means were compared with ANOVA and variances compared using Fishers F test. Compression resulted in stark reductions both in the variability within temperaturespecific groups and in permeability measurements (P < 0.05). The lowering of variance in both baseline and heated treatments indicates the elimination of another so urce of variance in our system allowing it to be more efficiently used to examine manipulations of barrier function. Analysis of Confounding Variables Despite successes improving the consistency of our model by altering our apparatus and by standardizing t he area measurement we were still faced with a level of variability that hindered our ability to assess treatment effects. A multiple regression analysis was then performed using essentially every different variable that we could measure in order to poss ibly reveal any strong experimental factors that would affect the variability of our outcomes ( Table 2 2). The factors area, fluorescence, serosal volume, and FITC dextran concentration were expected to have major effects on the variance as they are each included in the calculations of permeability However, the region of the small intestine from which the sac was made, denoted as segment number and later as a relative distance along the intestine, was found to be highly significant in affecting our var iability (P < 0.0 0 5). We then examined empirically the effect of segment number, which was previously treated as arbitrary. This was not originally thought to affect permeability as two different studies reported that it made no difference (Oliver, 2009; Lambert, 2002). To

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28 more specifically record the tissues location along the small intestine, the distances of calculated midpoints of each segment along its length were then used instead of arbitrary segment numbers. Sequential regions of the small bowels of four (4) adult mice were exposed to either 37 or 42C in one of four (4) assigned conical tubes. The midpoint for each segment was plotted against the permeability value calculated from the volume, area, and fluorescent measurements made following their specific treatment. In the plot of these points, distinct lines can be fit to similar treated tissues ( Figure 2 4). The slopes of these lines may then be statistically compared using linear regression analysis. At 37C there is a proportional relationship between the proximity of the tissue sample to the stomach and its permeability. This indicates an increased susceptibility to epithelial damage in the duodenum and jejunum, the proximal small intestine, when compared to the more distal ileum. This relationship appeared stronger in heated tissues i n the increased slope of the line for heated tissues but failed to reach statistical significance. Tissue Handling In attempts to further reduce the variability in our intestinal experiments, I mad e a number of modifications in our tissue handling procedure. This came through working closely with another student, Veronica Novosad, in the laboratory to refine a standardized methodology for isolation. Efforts were made to limit mechanical stresses on the intestine during eversion and gut sac preparation. We ensured proper oxygenation throughout segment preparation, incubation, and treatment, and standardized the amount of buffer us ed to flush intestinal contents and the hydrostatic pressure on intestinal walls during gut sac preparation. Together, these methodological adaptations further lowered the variance of our data ( Figure 2 5). This can be seen in

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29 the increased accuracy of the line s associated with both baseline and heated groups. Gastric s egments are more sensitive to increases in permeability with our improved methodology. This regional susceptibility is maintained at higher temperatures and appears to be strengthened with heating, though not sufficiently for statistical significance.

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30 T able 2 1. Conditions in cuvettes. Values compiled from 3 Mice. Treatment pH pCO2 pO2 Permeability 37C 7.1 73 91 1.86 37C 7.4 40 96 1.77 37C 7.2 53 94 5.22 37C 7.3 45 88 1.27 Mean 7.3 53 92 2.53 42C 7.2 52 80 8.33 42C 7.3 49 86 6.37 42C 7.2 53 80 6.14 42C 7.5 28 76 14.83 42C 7.2 52 72 8.15 Mean 7.3 47 79 8.76 Heat treatment increases permeability (P < 0.05)

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31 Figure 21. The Effect of Vessel on Permeability. (p) and (v) indicate that permeability and variance are lower in conical tubes than cuvettes N = 41 mice, P < 0.05 Figure 22. Mean Compliance Curve. Average inflection point at 30 + 5 g. N = 8 sacs

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32 Figure 23. Change in A rea M easurement. (p) and (v) indicate that permeability and variance are lower with compression than dis section. N = 15 mice, P < 0.05.

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33 Table 2 2. Experimental sources affecting variance. A multiple logistic regression analysis Technical Sources Significanc e (P Value) FD4 Concentration < 0.0005 Serosal Fluorescence < 0.0005 Seros al Volume ~ 0.1 Tissue Area < 0.05 Biological Sources Segment / Region < 0.0005 Temperature < 0.0 00 5 FD4 Concentration: Serosal concentration of fluorescent probe Serosal Fluorescence: Raw fluorescence reading of s erosal volume Serosal Volume: Fluid contents of intestinal segment Tissue Area: Square area of intestinal segment Segment: Anatomical region from which segment was prepared Temperature: Temperaturespecific experimental exposure Statistical Significance a t P < 0.05

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34 Figure 24. Regional permeability. Permeability increases as a function of gastric proximity. N = 4 mice Figure 25. Improved tissue handling. Specific considerations to eliminate investigator induced damage increased predictability (*) compared to Figure 24 and made more visible the influence of anatomical region. N = 4 mice

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35 CHAPTER 4 DISCUSSION At the outset, I was presented with a model having such a large degree of variance to it that the laboratory was unable to distinguish the ef fects of treatment over and above the inherent variability of the measurements. Through the systematic and rigorous deconstruction and analysis of our experimental model, I successfully resolved a number of technical issues that when understood and accounted for significantly improved the overall methodology and improved the resolution. My goal was to identify, explore, and resolve the sources of variance within our model, thus successfully increasing the predictability of control segments and allowing us to achieve full model viability E xperimental confounders may result from investigator manipulation or may be biological, result ing from the physiological and morphological nature of the intestinal mucosa. Surprisingly, based on the work of previous inv estigators, anatomical region of the small intestine, a biological variable, was paramount amongst these confounders. The susceptibility of gastric regions to heat induced permeability makes it a region of interest for future investigations and the target ing of treatment paradigms Biological Sources of Variance We began to address potential biological sources by allowing the mice ad libitum access to both food and water until euthanasia. Chief amongst biological sources of variance was the anatomical region from which the segment was made. Our data show that tissue from the gastric regions of (duodenum and jejunum) is more permeable and susceptible to heat induced barrier disruption that the distal ileum. Further investigation is required to differentially analyz e regions of the small bowel to elucidate the mechanisms controlling the susceptibility of the gastric regions.

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36 Previously, our lab analyzed the effect of the anatomical regions from which segments were created on permeability values. We did not observe a significant difference in permeability between gastric and ileal regions of the small bowel. This was supported by Lambert et als findings that permeability did not vary, at baseline or with heat, as a function of intestinal location ( Lambert, 2002). In my analyses, however, I found a significant and proportional relationship between permeability and anatomical region. Segments in the gastric region of the small intestine appear more susceptible to permeability increases, both at baseline and with heat. This is supported by Yu et als (Yu, 2010) in vivo findings of a similar relationship of increased damage in gastric regions after long term heat exposure. Morphological alterations in epithelial ultrastructure following three (3) day s of exposure were most severe in the jejunum. Microvillus height was significantly shorter in heated jejunum compared to control; no difference was found in ileal tissue. The j ejunal epithelium also had a higher number of damaged mitochondria and showed more organelle debri within lysosomes (Yu, 2010) We saw regional differences within the two ( 2 ) hours of our protocol. It is possible that this effect may have been previously indecipherable in our coveredcuvette protocol because of the extreme varianc e of the other factors Origins of Biological Variation The susceptibility of this region to damage may be explained by fact that the protective mucus layer of the digestive tract is already thinnest in the small intestine (Allen, 2005). This, when combined with larger crypt to villus ratios in the proximal small intestine, likely to increase absorptive capacity, provides a larger surface area of exposed epithelial cells for potential damage. This may explain why the regional differences in permeability appear exacerbated with the heat stress. Also of interest is

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37 the regional expression of tight junction proteins; the number of junctional strands correlates with junctional tightness (Steed, 2010) and deficient expression may increase susceptibility to permeability Regional differences in drug or sodium transporters along the intestine (Englund, 2006) provide possible explanations for the varying susceptibility These regulators of transcellular transport regulate the flux of water into the lumen have t he potential to drive paracellular solute diffusion across tight junctions (Turner, 2009). Regional susceptibility to damage, in vivo may be attributed to fully active digestive enzymes emptied directly into the duodenum from the stomach and pancreas (Al len, 2005) Potential proteolytic degradation of epithelial cells compounds the potential effects of increased heat on barrier function (SchmidSchonbein, 2008). Critique of Isolated Segment Model The development of this in vitro segmental model of intestinal susceptibility to hyperthermia is invaluable to our laboratory and to our understanding of epithelial function. It allows the efficient testing of potential treatments for hyperthermia along the length of the intestine. However, in the body, increases in core temperature are associated with intestinal ischemia, a facet of hyperthermia currently missing from our model. Furthermore, we question the sensitivity of our system, as we have previously been unable to successfully increase tissue permeabil ity with compounds known to initiate tight junction opening. These compounds have yet to be tested with our improved methodology. The method of gut sac preparation is also likely to disrupt the integrity of the adherent mucus layer s, normally responsible for mucosal protection. In our attempt to physiologically explore the mechanisms of intestinal dysfunction in hyperthermia, we have address ed these shortfalls in the development of multiple functioning models of intestinal analysis. Our laboratory has since developed

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38 functioning anesthetized and unanesthetized whole animal models of heat stres s. Throughout either of these courses of research it will be important to examine the regional status of barrier properties to more physiologically explore the regional intestinal permeability identified in our in vitro model Conclusion The gastric regions of the small intestine are more susceptible to heat damage. This results in an increased danger of unregulated solute movement and runaway inflammatory and i mmune responses. Moreover, the variation noted along intestinal length serves as a caveat for investigators seeking the universal application of findings from manipulations of only a single region. The regional differences in similar ly treated tissues i n our model highlight the importance of considering the entire intestinal tract in investigations. It provides insight into the relative importance of SchmidSchonbeins autodigestion theory, which traces the multi organ failure that is symptomatic to shock conditions, such as occurs in heat stress, to fully active digestive enzymes being emptied directly into the duodenum (Schmid Schonbein, 2008). Pancreatic enzymes have the power to degrade most biological molecules and under normal conditions, the epit helium is protected, in part, by mucus bicarbonate secretion. Under ischemic conditions however, the intestinal wall becomes permeable to these enzymes, exacerbating the mucosal injury in heat stress and stimulating the over activation of the production of inflammatory mediators. The identification and control of mechanisms regulating the preservation of tight junctions and epithelial cell viability are needed to direct efficacious environmental, nutritional, or pharmaceutical interventions Our data suggest that interventions for heat related illnesses may be most effective when targeted to gastric regions of the

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39 small intestine. Other important areas for future research include the bacterial epithelial interactions through toll like and other receptors the mechanisms f or the sensing of ischemia and those regulating mitochondrial membrane potential, and the real time assessment of cellular and subcellular structures related to mucosal defense.

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40 LIST OF REFERENCES A. Allen, D. A. Hutton, A. J. Leonar d, J. P. Pearson, & L. A. Sellers. (1986). The Role of Mucus in the Protection of the Gastroduodenal Mucosa. Scand J Gastroenterol: (2l), 71 78 Allen, A., & Flemstrom, G. (2005). Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin. Am J Physiol Cell Physiol, 288(1), C1 19. Atuma, C., Strugala, V., Allen, A., & Holm, L. (2001). The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo Am J Physiol Gastrointest Liver Physiol 280(5), G922 929. Cario E, Podolsky DK. (2000). Differential alteration in intestinal epithelial cell expression of toll like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 2000; 68:70107017. Daniel C. Baumgart and Axel U. Dignass. (n.d.). Intestinal barrier function. Current Opinion in Clinical Nutrition and Metabolic Care Issue: Volume 5(6), November 2002, 685694. Dokladny, K., Moseley, P. L., & Ma, T. Y. (2006). Physiologically relevant increase in temperature causes an increase in intestinal epithelial t ight junction permeability. Am J Physiol Gastrointest Liver Physiol 290(2), G204212. Englund, G., Rorsman, F., Rnnblom, A., Karlbom, U., Lazorova, L., Grsj, J., Kindmark, A. (2006). Regional levels of drug transporters along the human intestinal tract: Co expression of ABC and SLC transporters and comparison with Caco 2 cells. European Journal of Pharmaceutical Sciences 29(3 4), 269277. Fink, M. P. (2003). Intestinal epithelial hyperpermeability: update on the pathogenesis of gut mucosal barrier dysfunction in critical illness. Current Opinion in Critical Care (9), 143151. Hall, D. M., Buettner, G. R., Oberley, L. W., Xu, L., Matthes, R. D., & Gisolfi, C. V. (2001). Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia. Am J Physiol Heart Circ Physiol, 280(2), H509521. Lambert, G. P., Gisolfi, C. V., Berg, D. J., Moseley, P. L., Oberley, L. W., & Kregel, K. C. (2002). Molecular Biology of Thermoregulation: Selected Contribution: Hyperthermiainduced intest inal permeability and the role of oxidative and nitrosative stress. J Appl Physiol 92(4), 17501761. Lambert, G. P. (2009). Stress induced gastrointestinal barrier dysfunction and its inflammatory effects. J. Anim Sci. 87(14), E101108. Merchant, J. L. (2007). Tales from the crypts: regulatory peptides and cytokines in gastrointestinal homeostasis and disease. The Journal of Clinical Investigation, 117(1), 6 12.

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41 Mignen, O., Le Gall, C., Harvey, B. J., & Thomas, S. (1999). Volume regulation following hy potonic shock in isolated crypts of mouse distal colon. The Journal of Physiology 515(2), 501 510. Nejdfors P., Ekelund M., Jeppsson B., & Westr, B. R. (2000). Mucosal in vitro Permeability in the Intestinal Tract of the Pig, the Rat, and Man: Species and RegionRelated Differences. Scandinavian Journal of Gastroenterology 35, 501507. Niessen, C. M. (0000). Tight Junctions/Adherens Junctions: Basic Structure and Function. J Invest Dermatol 127(11), 25252532. Quigley, E. M., & Turnberg, L. A. (1987). pH of the microclimate lining human gastric and duodenal mucosa in vivo Studies in control subjects and in duodenal ulcer patients. Gastroenterology 92(6), 18761884. Robins, H. I. (1984). Whole body hyperthermia (41 42 C): A simple technique for unanesthetized mice. Medical Physics 11(6), 833. Rosario, H. S., Waldo, S. W., Becker, S. A., & Schmid Schonbein, G. W. (2004). Pancreatic Trypsin Increases Matrix Metalloproteinase9 Accumulation and Activation during Acute Intestinal IschemiaReperfusion in the Rat. Am J Pathol 164(5), 17071716. Schmid Schnbein, G. W. (2008). Biomechanical Aspects of the Autodigestion Theory. Molecular & cellular biomechanics : MCB 5 (2), 8395. Shen, L., & Turner, J. R. (2005). Actin Depolymerization Disrupts Tight Junctions via Caveolaemediated Endocytosis. Mol. Biol. Cell, 16(9), 39193936. Smecuol, E., Bai, J. C., Sugai, E., Vazquez, H., Niveloni, S., Pedreira, S., Maurio, E., et al. (2001). Acute gastrointestinal permeability responses to different nonster oidal anti inflammatory drugs. Gut 49(5), 650 655. Steed, E., Balda, M. S., & Matter, K. (2010). Dynamics and functions of tight junctions. Trends in Cell Biology 20(3), 142149. Szab, A., Vollmar, B., Boros, M., & Menger, M. D. (2008). In vivo Flu orescence Microscopic Imaging for Dynamic Quantitative Assessment of Intestinal Mucosa Permeability in Mice. Journal of Surgical Research, 145(2), 179185. Tomita, M., Menconi, M. J., Delude, R. L., & Fink, M. P. (2000). Polarized transport of hydrophili c compounds across rat colonic mucosa from serosa to mucosa is temperature dependent, Gastroenterology, 118(3), 535543.

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42 Turner, J. R. (2009). Intestinal mucosal barrier function in health and disease. Nat Rev Immunol 9 (11), 799809. Ungell, A., Nylan der, S., Bergstrand, S., Sjberg, ., & Lennerns, H. (1998). Membrane transport of drugs in different regions of the intestinal tract of the rat. Journal of Pharmaceutical Sciences 87(3), 360366. Yu, J., Yin, P., Liu, F., Cheng, G., Guo, K., Lu, A., & Zhu, X.(2010). Effect of heat stress on the porcine small intestine: A morphological and gene expression study. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 156(1), 119 128.

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43 BIOGRAPHICAL SKETCH Neil has been inv olved with Dr. Thomas Clantons laboratory for just over a year. At the commencement of his scientific career there, his work centered mainly on the development of a genetically encoded calcium indicator that will allow the detection of calcium transients and rapid action potentials in excitable cells. Since then, he has worked on the development and refinement of an in vitro mouse model of investigating the mechanisms of heat induced intestinal barrier d ysfunction. During this time, he c a me across the novel finding that the distal region of the mouse small intestine is less susceptible to hyperthermiainduced permeability increase. One possible application of this information is the directing of heat related illness treatments to the gastric regions of the small intest ine. To further explore this, he will begin testing tissue samples from unrestrained, unanesthetized mice that have been allowed to experience increased core temperatures. The development of a viable wholebody heat stress model will allow his lab to investigate regional intestinal sensitivity in an intact animal system. After the completion of his doctoral program in Applied Physiology and Kinesiology N eil plans to achieve his childhood goal of procuring a medical school education. He is certain that the skills and knowledge garnered throughout his graduate experience will solidify my competence as a scientist and his role in increasing our understandi ng of the workings of the human body.