Thermal tolerance of skeletal muscle and small intestine: role of metabolism and oxidative stress

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Scott Ryan Oliver, B. S.

Biophysics Graduate Program

The Ohio State University

2009

Dissertation Committee:

Mark Wewers, Advisor

Mark Angelos

Gunjan Agarwal

Govindasamy Ilangovan

Copyright by

Scott Ryan Oliver

2009

Abstract

This research investigated the thermal tolerance (i.e. the highest temperature

which does not cause dysfunction) of skeletal muscle and the small intestine and

evaluated the effects of inhibiting eicosanoid metabolism and oxidative stress. Skeletal

muscle is a primary source of heat in exertional hyperthermia and loss of intestinal barrier

function is considered a critical event in the pathogenesis of heat stroke. A mechanism of

hyperthermia-induced dysfunction in these organ systems is poorly understood.

Contractile function was measured in rat diaphragm during and after 30 min

exposure to 40 to 43° C. Only at 43° C, a significant loss of active force was observed.

Lipoxygenase inhibitors ( and eicosatetreanoic acid) as well as cyclooxygenase

inhibitors (ibuprofen and indomethacin) further depressed the loss of function at 43° C and induced a loss of thermal tolerance at 42° C. Treatment of diaphragm muscle with reactive oxygen species (ROS) scavengers, tiron and Trolox, did not inhibit the loss of contractile force at 43° C, suggesting that the ROS that are affected by these scavengers are not involved. Blockage of phospholipase A2, causing a decrease in the enzymatic

substrate for eicosanoid metabolism, was unable to prevent the loss of contractile force at

43° C.

ii Mouse small intestine permeability was monitored using a modified everted gut protocol. Tissues were exposed to 37° C to 42.5° C for 90 minutes and permeability was

assessed using the diffusion of a fluorescent molecule (4 kDa simulating the permeability

of molecules the size of endotoxin) into the everted gut sacs. At 40.5° C, intestinal

permeability remained unchanged, but increased significantly when exposed to higher

temperatures. Treatment with baicalein, indomethacin or the antioxidant N-acetyl

(NAC) significantly protected permeability at 41.5° C. Protein carbonyl levels

were used as a marker of cellular oxidative stress and increased after 90 minutes of 41.5°

C exposure. Hyperthermia also significantly increased structural damage as measured by

microscopy. Baicalein and NAC both reduced protein oxidation, but only NAC reduced

structural damage. This suggests that ROS could play a major role in hyperthermia-

induced barrier dysfunction.

The mechanism for the increase in intestinal permeability was studied using

compounds known to induce opening of tight junctions to compare to permeability

observed during hyperthermia. Cytochalasin D was the only compound that increased

intestinal permeability at 37° C as measured by a 0.48 kDa probe. However, it resulted in

severe structural damage to the epithelial cells. Despite the observed structural damage,

opening of tight junctions did not cause enough permeability to explain all observations

during hyperthermia. Lactate dehydrogenase, a marker for cell cytotoxicity, was found to

increase during hyperthermia at 30 and 60 min. The results are consistent with cell

damage being the predominant cause of increased intestinal permeability during

hyperthermia, most likely mediated by ROS. However, in skeletal muscle a completely

iii different mechanism appears actively involved. Both antioxidants and/or inhibitors such as baicalein show potential for ameliorating morbidity of heat stroke.

iv

Dedication

Dedicated to my parents, Scott and Janice Oliver.

v

Acknowledgments

I would like to acknowledge Dr. Thomas Clanton for his helpful advice,

mentoring, support and encouragement throughout my graduate career. I would also like

to thank both my general examination committee (Dr. Wewers, Dr. Angelos, Dr.

Parinandi and Dr. Khramtsov) and my final examination committee (Dr. Wewers, Dr.

Angelos, Dr. Agarwal and Dr. Ilangovan) for their constant encouragement and helpful

insight. Finally, I would like to thank the members of the Clanton lab, past and present,

Val Wright, Jose Oca, Tony Payne, Steve Welc, Neil Phillips, and Monsour Al-Rajhi.

vi

Vita

2005...... B.S. Biology Minor in Chemistry Shawnee State University Portsmouth, Ohio, United States

2005 – 2007...... Graduate Research Associate The Ohio State University

2007-2009……………………………………..Laboratory Technician University of Florida

2009- 2009…………………………………….Graduate Research Associate The Ohio State University

Publication

1. Oliver SR, Wright VP, Parinandi N, and Clanton TL. Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid inhibition. Am J Physiol Regul Integr Comp Physiol 295: R1695-1705, 2008.

Fields of Study

Major Field: Biophysics Area of Emphasis: Physiology

vii

Table of Contents

Abstract………………………………………………………………………….. ii

Dedication……………………………………………………………………….. v

Acknowledgments……………………………………………………………… vi

Vita………………………………………………………………………………. vii

List of Tables…………………………………………………………………….. xii

List of Figures……………………………………………………………………. xiii

Chapter 1: Introduction…………………………………………………………... 1 1.1. Scope and Rationale………………….………...……………. 1 1.2. Heat Related Illnesses………………….…………………….. 2 1.2.1. Intestinal Permeability during Hyperthermia………. 5 1.2.2. Skeletal Muscle Function during Hyperthermia…… 8 1.2.3. Acute Phase of Heat Stroke………………………… 10 1.2.4. Heat Shock Response………………………………. 11 1.3. Eicosanoid Metabolism and Implications during Hyperthermia ……………………………………………….. 12 1.3.1. Cyclooxygenases…...………………………...…….. 13 1.3.2. ……………………………………… 18 1.3.3. Cytochrome P450………………………………….. 21 1.4. Research Objectives..……………………………………….... 23 1.5. Figures………………………………………………………... 27 1.6 References…………………………………………………….. 29

Chapter 2: Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition …………………………………………………………...…………….. 38 2.1. Introduction…………………………………………………... 38 2.2. Methods……………...……………………………………….. 41 2.2.1. Animal Treatment Protocols and isolated muscle Preparation.……………………….……………… 41 2.2.2. Protocols.……………..…………………………… 42 viii 2.2.3. Membrane Permeability Assessment…………… 43 2.2.4. Statistical Analysis....……………………………... 43 2.3. Results……………………………………………………….. 44 2.3.1. Thermal Tolerance of Isolated Diaphragm……... 44 2.3.2. Effects of Antioxidants and Reducing Agents on Contractile Function with Heat Exposure...……….. 44 2.3.3. Effects of Lipoxygenase Inhibition on the Loss of Contractile Function at 43º C…………………….... 46 2.3.4. Effects of Lipoxygenase and Cyclooxygenase Inhibition on Heat Tolerance to 42ºC ………..…… 46 2.3.5. Effects of Inhibition of Upstream PLA2…………… 48 2.3.6. Tests for Membrane Damage in Heat Stress ……… 49 2.4. Discussion………………………………………………….… 51 2.4.1. Critique of Approach …………………….…….….. 51 2.4.2. Mechanisms for the Loss of Contractile Function..... 52 2.4.3. Mechanisms for Changes in passive force during heat exposure...... 55 2.4.4. LOX, COX and PLA2 involvement during heat stress...... 56 2.4.5. Conclusions...... 60 2.4.6. Perspectives and Significance...... 61 2.5. Tables………………………………………………………... 63 2.6. Figures……………………………………………………….. 64 2.7. References…………………………………………………… 74

Chapter 3: Protection of hyperthermia-induced intestinal permeability in the Mouse………………………………………………………………… 81 3.1. Introduction………………………………………………….. 81 3.2. Methods………………………...……………………………. 85 3.2.1. Chemicals and drugs used…………………………. 85 3.2.2. Animal treatment protocol and gut sac preparation... 85 3.2.3. Histology…………………………………………… 86 3.2.4. Data analysis and Statistics...………………………. 86 3.3. Results………………………………………………………... 88 3.3.1. Thermal tolerance of mouse small intestine Permeability…...…………………………………… 88 3.3.2. Effects of cyclooxygenase inhibition on thermal tolerance of intestinal gut sacs ………………….… 88 3.3.3. Effects of lipoxygenase inhibition on hyperthermia induced intestinal permeability..…………………… 89 3.3.4. Effects of cytochrome P450 inhibition on hyperthermia induced intestinal permeability.…..… 89 3.3.5. Effects of antioxidants on hyperthermia induced intestinal permeability …...………………...……… 90 ix 3.4. Discussion……………………………………………………. 91 3.4.1. Critique of Approach...... ….…...………...……… 91 3.4.2. Possible mechanisms for the increase in permeability during hyperthermia...... ….…....………...……… 92 3.4.3. LOX, COX and CYP450 inhibition during heat stress...... ……………………..…...……...……… 95 3.4.4. Conclusions...... ………………..…...……...……… 98 3.5. Figures……………………………………………………….. 100 3.6. References……………………………………………………. 107

Chapter 4: Mechanism for the loss of intestinal permeability and pharmacological protection during hyperthermia in mouse...……………………………………….. 113 4.1. Introduction…………………………………………………... 113 4.2. Methods……………………...……………………………….. 117 4.2.1. Chemicals and Drugs Used…………..…………….. 117 4.2.2. Animal treatment protocol and gut sac preparation.... 117 4.2.3. Effects of Pharmacological Tight Junction Openers.. 118 4.2.4. Histology……………..…………………………….. 119 4.2.5. EM analysis…………………………………………. 119 4.2.6. LDH assay………………………………………….. 120 4.2.7. Determination of Oxidative Stress…………………. 120 4.2.8. Data analysis and Statistics……………...…………. 121 4.3. Results………………………………………………………… 123 4.3.1. Time and temp controls: LDH and permeability….... 123 4.3.2. Time and temp controls: Oxidative stress measurements………………………..……………... 123 4.3.3. Tight junction regulation during hyperthermia..…… 124 4.3.4. Histological assessment of heated and control tissues…………………………………………….... 125 4.3.5. Calcium chelation during hyperthermia…………… 125 4.3.6. LDH release of tissues treated with baicalein or NAC……………………………………………….. 126 4.3.7. Damage index of tissues treated with baicalein or NAC……………………………………………….. 126 4.3.8. Protein carbonyl and 4-HNE formation in tissues treated with baicalein or NAC…………………….. 127 4.4. Discussion……………………………………………………. 128 4.4.1. Critique of Approach………………………………. 128 4.4.2. Mechanism for increased permeability during Hyperthermia………………………………………. 129 4.4.3. Baicalein and NAC treatment during hyperthermia... 134 4.4.4. Conclusions………………………………………… 135 4.5. Figures………………………………………………………... 137 4.6. References………..……………………………………………. 150 x

Chapter 5: Conclusions and Future Directions……………………………………. 156 5.1. References…………………………………………………….. 162

Bibliography……………………………………………………………………….. 164

xi

List of Tables

Table 2.1. Drugs used in study with corresponding targets.……………………… 63

xii

List of Figures

Figure 1.1. Schematic of the various pathways heat stress affects skeletal muscle and the intestine……………………………………………… 27

Figure 1.2. Main pathways of metabolism...... 28

Figure 2.1. Muscle mounting and tissue bath setup……………………………… 64

Figure 2.2. Force frequency relationship during hyperthermia…….…………….. 65

Figure 2.3. Change in %Max Force (150Hz) of diaphragm strips exposed to 43ºC with antioxidants ………………….…………………………… 66

Figure 2.4. Change in %Max Force (150Hz) of diaphragm strips exposed to 43ºC with dithiothreitol (DTT)………………...…………………….. 67

Figure 2.5. Change in %Max Force (150Hz) of tissues exposed to specific LOX inhibitors ………………………………………….…………………. 68

Figure 2.6. Change in %Max Force (150Hz) of tissues exposed to 42ºC with LOX or COX inhibitor treatment …………………...………………. 69

Figure 2.7. Change in %Max Force (150Hz) of tissues exposed to a combination of LOX and COX inhibitors.………,,,,,,,,,,,,,,.……….……………… 70

Figure 2.8. Change in %max Force (150Hz) of tissues exposed to 43ºC and PLA2 inhibition ……………………………………….……………... 71

Figure 2.9. Change in %Max Force (150Hz) of tissues exposed to BPB...…….. 72

Figure 2.10. Membrane permeability evaluated using procion orange...……… 73

Figure 3.1. Comparison of intestinal permeability when exposed to temperatures up to 42.5°C……………………………….…………………………. 100

Figure 3.2. Gut sections exposed to 37°C or 41.5°C……………………………... 101

xiii Figure 3.3. Intestinal permeability assessed at different time points (15, 30, 60 and 90min.) when exposed to 41.5°C ……….……….. 102

Figure 3.4. Cyclooxygenase inhibition effects on intestinal permeability after 90 min. of 41.5°C exposure…………..………………..…………….. 103

Figure 3.5. Lipoxygenase inhibition effects on intestinal permeability after 90 min. of 41.5°C exposure………………...………………………... 104

Figure 3.6. CYP450 inhibition effects on intestinal permeability after 90 min. of 41.5°C exposure………………………………………… 105

Figure 3.7. Permeability assessment of gut sacs treated with antioxidants exposed to 41.5°C for 90 min………………………………………… 106

Figure 4.1. LDH release and permeability of tissues exposed to either 37°C or 41.5°C………………...…………………………………………… 137

Figure 4.2. Protein oxidation measurements of tissues exposed to 37°C or 41.5°C ……..…………………………………………………….... 138

Figure 4.3. 4-HNE measurements of tissues exposed to 37°C or 41.5°C.....…….. 139

Figure 4.4. Chemicals to induce tight junction opening …………………………. 140

Figure 4.5. EMs of 37°C control and CyD treated tissues…………………….…. 141

Figure 4.6. Tissue damage during 90 minutes of hyperthermia……..……………. 142

Figure 4.7. Calcium chelation by BAPTA-AM during hyperthermia…………….. 143

Figure 4.8. LDH release of tissues treated with NAC and exposed to 41.5°C……. 144

Figure 4.9. Damage index of tissues treated with baicalein or NAC…………….. 145

Figure 4.10. Histological sections of tissues treated with baicalein or NAC…….. 146

Figure 4.11. Electron micrographs of tissues treated with baicalein or NAC……. 147

Figure 4.12. Protein carbonyl measurements of tissues treated with baicalein or NAC………………………………………………………………. 148

Figure 4.13. 4-HNE measurements of tissues treated with baicalein or NAC…… 149

xiv

Chapter 1: Introduction

1.1. Scope and Rationale

Heat related illnesses, such as heat stroke, are becoming more prevalent in our society, possibly due to the rise in global temperatures (Pachauri, 2007). Children, the elderly and athletes are the most susceptible populations to these illnesses, but the precise causes are still not clearly understood. Treatment of these illnesses is still limited, due to the lack of knowledge and understanding of the cellular events leading up to heat stroke fatality. Currently, the most common course of action is to lower the core body temperature and to combat septic shock symptoms. These methods can be difficult to execute even when patients are diagnosed and treated in a timely manner. Unfortunately, even with treatment, heat stroke is often fatal. From 1979 to 1997, 7,000 deaths occurred in the United States alone due to heat stroke (2000). In 2003, an abnormally long lasting summer heat wave in Paris killed 15,000 people from heat related causes (Misset et al.,

2006). With a worldwide trend of increasing temperatures and predictions for increased extreme weather events across the globe (including heat waves) (Pachauri, 2007), the risks of heat related illnesses may become even more prevalent. Because of these reasons, the physiology and cellular responses to heat stress need to be studied in greater detail, in order to find better ways of treating and preventing heat stroke as well as other heat related illnesses.

1

Several areas of the body are affected during the progression of heat related illnesses

(Bouchama & Knochel, 2002). Two organs of particular interest which have been found

to be critical during hyperthermia are skeletal muscle and the intestine (Figure 1.1).

Skeletal muscle function may break down during heat stress, which could cause a number

of physiological problems. Intestinal permeability is also affected by hyperthermia

(Lambert et al., 2002a), which can be a critical health issue because a lack of the

intestine’s ability to provide the proper barrier can lead to increased susceptibility to

septic shock (Bouchama & Knochel, 2002). Eicosanoid metabolism has been linked to heat stress in a few studies (Zuo et al., 2004; Lambert et al., 2007), but what role if any

that it plays in skeletal muscle or gut permeability function is unclear. Eicosanoid

metabolism is important in regulating the temperature of the body as well as increasing

the core temperature during situations such as (Funk, 2001). This research looks at

the effects of heat stress on skeletal muscle as well as intestinal function, and the

mechanistic physiological links into how and why decreased function occurs at elevated

temperatures.

1.2. Heat Related Illnesses

Most heat illnesses begin with a heat stress, which is defined as discomfort and

strain (such as during physical work) during exposure to a hot environment (Bouchama &

Knochel, 2002). Heat stress can then lead to heat exhaustion, which can cause thirst,

discomfort, anxiety, fainting, dizziness and headache (Bouchama & Knochel, 2002).

However, changes in core body temperature may not necessarily be a symptom, and can

remain normal, increase or even decrease (Bouchama & Knochel, 2002). If the core

temperature is allowed to increase, then the body will enter a state of hyperthermia,

2

which is defined by the rise in body temperature, possibly due to an impairment of heat

dissipating mechanisms (Bouchama & Knochel, 2002). Heat stroke occurs when the core

temperature elevates enough above normal (exact temperature range varies), followed by

a decrease in sweating and an increase in central nervous system abnormalities (e.g.

convulsions, delirium, or coma) (Bouchama & Knochel, 2002). The rise in body

temperature during heat stroke is usually attributed to high environmental temperatures or

during exhaustive exercise (Bouchama & Knochel, 2002).

The pathogenesis of heat related illnesses is comprised of a complex set of events that are not yet completely understood. One of the biggest difficulties in studying heat stroke is because of the variations in observed symptoms when diagnosing a patient.

Such variability often results in a lack of correlative data (Leon, 2006). It is still

unknown why one individual with a body temperature of 39° C will suffer from heat

stroke, yet another with a body temperature of 41° C will not. Interestingly, anesthetized

rats undergoing heat stroke have been found to become susceptible (decreased survival

rate) at temperatures of approximately 41.5° C (Lim et al., 2007), but this response could

be largely different from what is seen in humans. Differences in heat shock protein

expression (Moseley, 1997; Yang & Lin, 1999; Wang et al., 2001), altered cytokine gene

expression (Lin et al., 1997; Liu et al., 2000) and coagulation protein expression (al-

Mashhadani et al., 1994; Bouchama et al., 1996) have all been considered candidates for

reasons why heat stroke temperatures can vary so widely. Despite the large variability in

the data, an overall pathogenesis for heat stroke has been able to be described in the

literature (Bouchama & Knochel, 2002).

3

One of the first impairments to occur during the onset of heat stroke is a

decreased ability to maintain thermoregulation. Normally, if body and blood temperature

increases, the body senses this and activates signaling processes to increase cardiac

output. The goal of this response is to divert a greater amount of blood (~8 liters per

minute, (Rowell, 1983)) to the skin where it can be cooled. Heat can then be dissipated through mechanisms such as sweating. However, sweating is only effective if the ambient conditions allow for a sufficient vapor pressure to permit water evaporation (i.e., the relative humidity is low enough). Six hundred kcal of heat per hour can be dissipated

in a dry environment under perfect conditions (Nelson et al., 1947; Adams et al., 1975;

Buono & Sjoholm, 1988). However, excess sweating can lead to dehydration and loss of salts, which has the potential to exacerbate heat stress symptoms (Deschamps et al.,

1989).

When blood is shunted toward the skin, the superior mesenteric artery is

constricted causing a decrease in blood flow to the intestine in order to increase the total

volume of blood flowing toward the skin (Rowell, 1974; Kregel et al., 1988; Sakurada &

Hales, 1998). This decrease in blood flow from the intestine causes ischemia (low

oxygen), and when combined with hyperthermia can lead to an increase in intestinal permeability (Rowell, 1983; Sakurada & Hales, 1998; Hall et al., 1999). After ischemia,

a secondary response causing can occur, leading to bouts of ischemia-

reperfusion (I-R), which has also been shown to cause an increase in intestinal

permeability (Kregel et al., 1988).

4

1.2.1. Intestinal Permeability during Hyperthermia

The combination of ischemia and hyperthermia could have a synergistically

deleterious effect compared to either stress alone. Studies have shown that during intense

exercise in humans (Pals et al., 1997; Smetanka et al., 1999) as well as in animals

(Shapiro et al., 1986; Prosser et al., 2004; Davis et al., 2005; Singleton & Wischmeyer,

2006), intestinal permeability increases resulting in an elevation of plasma endotoxin levels (Brock-Utne et al., 1988). Increased endotoxin levels in the blood can be detrimental and lead to multiple organ failure, shock and death (Bouchama & Knochel,

2002). Administering antibodies against endotoxin to subjects before heat stress has been shown to attenuate the symptoms seen during heat stroke and increase survival rates

(Gathiram et al., 1987). Interestingly, athletes undergoing strenuous exercise have increased levels of endotoxin (Bosenberg et al., 1988; Brock-Utne et al., 1988; Camus et al., 1998; Ng et al., 2008) and inflammatory cytokines in the blood (Bosenberg et al.,

1988; Pals et al., 1997; Camus et al., 1998; Pedersen & Hoffman-Goetz, 2000; Ng et al.,

2008). This could suggest that a certain level of tolerance exists. However, when this

tolerance is surpassed or the threshold level is lowered by a hindrance of one of the

thermoregulatory mechanisms, heat stroke is allowed to progress.

The exact cellular events in the intestine that occur during the onset of

hyperthermia are unknown. Because of this lack of knowledge, the mechanism for how

endotoxin travels into the blood stream during heat stroke is difficult to ascertain. The

main role of the intestine is to absorb nutrients from diet to supply the body. The

intestine accomplishes this by creating a selectively permeable membrane of cells which

block unwanted materials but allow nutrients to pass through (Hollander, 1992). This

5 selectively permeable membrane consists of three main parts: epithelial cells connected adjacently through protein-protein interactions called tight junctions, mucus covering the luminal side of the cells, and a variety of immune cells (Hollander, 1992; Lambert, 2009).

The epithelial cells possess a variety of channels that allow specific molecules or ions to transport across the cell. This pathway is called transcellular transport. A second pathway of absorption, the paracellular pathway, functions by opening tight junctions through a highly regulated system that lets molecules pass freely between epithelial cells (Hollander, 1992). Under the epithelial cell layer, vessels for the vascular as well as lymphatic systems are present to send the transported materials to the rest of the body.

The milieu of the gut consists of trillions of bacteria (normal flora), which when kept in the intestine pose little threat to the human body. However, when the structural barrier of the intestine breaks down, toxic components of these bacteria (e.g. endotoxin or

LPS) or sometimes the whole bacterial cells are allowed access to the blood stream. The possible route that endotoxin can take to gain access to the blood stream has been only speculated upon in the literature. It is believed that a dysfunction in one or both of the above pathways, transcellular or paracellular, is responsible for the increase in intestinal permeability during hyperthermia. Interestingly, it has also been speculated that damage to the epithelial layer and subsequent breakdown of the barrier could be the cause for the increase in permeability. This has been partially shown by Lambert et al. (Lambert et al.,

2002a) through histology and electron microscopy, but the full extent and importance of this finding has yet to be defined.

6

Hyperthermia has been shown to cause an increase in reactive oxygen species

(ROS) in the intestine (Hall et al., 1994; Hall et al., 2001) as well as in several other

tissues (Wallen et al., 1997; Flanagan et al., 1998; Zuo et al., 2000), which could be a possible mechanism for increased permeability. During hyperthermia, studies have shown that inhibition of xanthine oxidase (known ROS producer) protects against increases in intestinal permeability (Hall et al., 2001). Xanthine oxidase inhibition decreased the portal LPS concentration in rats whose core body temperature had reached

41.5° C (Hall et al., 2001). When plasma LPS levels are decreased or neutralized, survival time of primates experiencing heat stress increased (Gathiram et al., 1987).

Also, cell culture studies using intestinal epithelial cells show that the continuity of the epithelial monolayer is disrupted, and permeability is allowed to increase when exposed to oxidants (Rao et al., 1997). Also, oxidative damage caused by increased ROS formation has been linked to a decrease in barrier integrity and cell injury caused by increased intestinal (Keshavarzian et al., 1992; McKenzie et al., 1996).

Inflammation has been strongly linked to increased intestinal permeability through the

study of disorders such as Crohn’s disease and celiac disease (Vilela et al., 2008).

Intense exercise has been shown to increase the blood concentration of inflammatory

cytokines (Leon, 2006) as well as cause intestinal permeability (Pals et al., 1997;

Smetanka et al., 1999; Davis et al., 2005). Furthermore, animal studies have found that

when the inflammatory response is decreased, during heat stress, animal survival is

increased (Gathiram et al., 1987; Lin et al., 1997; Liu et al., 2000). On the contrary,

other studies have found that when intestine is treated with antioxidants during

hyperthermia, no protection is observed (Lambert et al., 2002a). Therefore, the roles

7

ROS and inflammation play in intestinal permeability during hyperthermia is still relatively poorly understood. Further study could shed more light on these mechanisms and thus help to suggest possible treatment during and after the onset of heat stroke.

1.2.2. Skeletal Muscle Function during Hyperthermia

Several studies have been conducted that show the human body can reach a core temperature that critically affects muscle function (Marino, 2004; Tucker et al., 2004).

Fever and strenuous exercise have been found to be the most common causes of increasing core temperature (Marino, 2004), because skeletal muscle is the primary heat- producing organ (Ali et al., 2003). Skeletal muscle produces , which can alter the body’s thermoregulatory set point. Core and muscle temperatures can increase past normal levels due to high metabolic activity (Ali et al., 2003), insufficient heat dispersal mechanisms (Roth et al., 2006), high ambient temperature (Morris et al., 2005)

and temperature related diseases (Parkin et al., 1999; Roth et al., 2006), such as malignant hyperthermia.

Malignant hyperthermia is a hereditary disorder of skeletal muscle that causes uncontrolled release of calcium into the cytosol through a defect in the sarcoplasmic reticulum (SR) calcium release channel (Ryanodine) (Ali et al., 2003). This uncontrolled release of calcium is usually triggered by strong inhaled anesthetics (Ali et al., 2003).

Muscle cells try to compensate for this rise in cytosolic calcium by pumping calcium back into the SR through the calcium ATPase which reduces cellular ATP levels quickly.

This re-uptake process is not sufficient to compensate for the increased calcium, and the calcium concentration is allowed to remain high. Such high levels of calcium can then

8

lead to hypermetabolism, sustained muscle contraction, lactic acidosis and hyperthermia

(Ali et al., 2003). Increased body temperature due to continued muscle stimulation,

contraction or hypermetabolism can cause a breakdown of muscle cells and lead to

several downstream systemic effects, including rhabdomyolysis (breakdown of muscle)

(Clarkson, 2007), with the possibility of death.

Indeed, skeletal muscle can reach high temperatures. The temperature that skeletal muscle reaches, in humans, during exercise has been reported to be as high as 41

°C in hot ambient temperatures (Saltin et al., 1968; Drust et al., 2005). Brooks et al.

reported temperatures as high as 44.1° C in rat skeletal muscle during exhaustive exercise

(Brooks et al., 1971). Furthermore, core body temperatures have been reported as high as

44°C in diseases such as malignant hyperthermia (Ali et al., 2003). Elevated core

temperatures can be detrimental because they can also cause higher skeletal muscle

temperatures. This can affect skeletal muscle contractile function, and in more extended

bouts of heat stress, result in the development of rhabdomyolysis (Clarkson, 2007). Van

der Poel et al. (van der Poel & Stephenson, 2002) found that a brief exposure to 43-47° C

causes a decrease in contractile function in isolated rat extensor digitorum longus (EDL)

muscle fibers (largely fast twitch). A similar effect was seen in peroneus longus muscle

fibers (more oxidative), which have a similar composition to the diaphragm muscle. The

observed loss in contractile function in the EDL muscle was suggested to be caused by

ROS formation (van der Poel & Stephenson, 2002). ROS formation was implicated

because treatment with Tiron (superoxide scavenger) protected against heat-induced loss

of contractile function, and dithiothreitol (DTT), a thiol (-SH) reducing agent, increased

contractile function recovery time of heated fibers (van der Poel & Stephenson, 2002).

9

Interestingly, the peroneus longus muscle did not respond the same to Tiron or DTT. No

protective effect was seen upon addition of these agents (van der Poel & Stephenson,

2002), which runs contrary to the idea that because oxidative fibers have a greater

amount of mitochondria, they are believed to be a major source of ROS production. This

suggests that the response to hyperthermia could partially be fiber type dependent. Also,

exposing skeletal muscle cells to 42° C causes an increase in both intra- and extracellular

ROS formation (Zuo et al., 2000). This increase in ROS formation during hyperthermia

has been seen in other tissues (Wallen et al., 1997; Flanagan et al., 1998) as well as in whole animal studies (Hall et al., 2001), but the significance is unclear. The above findings suggest that ROS formation during hyperthermia could play a major role in muscle dysfunction during heat stress, but further study needs to be done to fully elucidate the role ROS plays in skeletal muscle function.

1.2.3. Acute Phase of Heat Stroke

If thermoregulatory mechanisms are hindered and the body temperature is allowed to climb, the next phase in the development of heat stroke is allowed to occur.

An acute-phase response occurs during heat stress, triggering an inflammatory cascade leading to an increase in proinflammatory cytokine production mostly due to the presence of endotoxin in the blood. The range of cytokines produced during heat stress and stroke can vary from patient to patient but IL-6 and a few other cytokines have been found to be produced almost 100% of the time (Bouchama et al., 1993). However, no correlation between IL-6 production and temperature variations during heat stroke has been found.

On the other hand, IL-1β serum levels have been associated with the ability to decrease core body temperatures below 40°C (Chang, 1993). IL-1β can alter several aspects of

10 cell signaling (Leon, 2006), but more important to the studies described in this thesis, has been found to alter protein activity and expression of responsible for eicosanoid metabolism (Akarasereenont et al., 1999; Yucel-Lindberg et al., 1999), which have a major role in altering thermoregulation in the hypothalamus, discussed in more detail below.

Evidence is emerging that suggests skeletal muscle produces cytokines in response to exercise which could aid in exercise adaptation and anti-inflammation

(Pedersen & Febbraio, 2008) as well as a possible source for systemic signaling. IL-6 is of particular importance since it has been shown to be expressed in skeletal muscle

(Pedersen & Febbraio, 2008) and blood plasma (Ng et al., 2008) during exercise.

Skeletal muscle comprises a large percentage of the total body weight (~45%) and since muscle has been shown to be an endocrine organ (Pedersen & Febbraio, 2008), it could play a pivotal role during the onset of heat stroke as well as the recovery after. In addition, IL-6 exposure to intestinal epithelial cells (Caco-2) before exposure to hyperthermia increased their survival rate and thermotolerance (Hershko et al., 2003).

Furthermore, IL-6 is produced in the intestine during sepsis and endotoxemia (Ebong et al., 1999) and could possibly occur during hyperthermia. This suggests that IL-6 is important in both muscle and intestine during stress.

1.2.4. Heat-Shock Response

Most cells respond to stress by producing chaperone proteins to aid in protecting protein degradation, misfolding and exposure of hydrophobic moities, these are called heat shock proteins (HSPs) (Kim et al., 2007). At the onset of heat stress, gene

11

expression for HSP is increased due to activation of transcription through heat-shock

transcription factors binding to heat-shock elements (Welch, 1992; Polla et al., 1998).

Several studies have found that the increased production of HSPs can lead to a state of

tolerance and subsequent protection from a second bout of heat stress (Welch, 1992;

Moseley, 1997; Polla et al., 1998). However, studies have shown that temperatures of

50° C or higher need to occur for degradation of isolated normal proteins (Despa et al.,

2005), which is much higher than seen physiologically. Interestingly, degradation of

normal cellular proteins does still occur but the more logical conclusions that have been

discovered is that oxidant formation during hyperthermia increases protein modifications

leading to protein degradation (Schertzer et al., 2002). HSP production has also been

mainly attributed to tolerance of tissues to a secondary bout of stress since protein

expression needs a sufficient amount of time to occur and an acute exposure to heat stress

or heat stroke may not coincide with the time line of HSP production.

1.3. Eicosanoid Metabolism and Implications during Hyperthermia

Eicosanoid metabolism has been linked to heat stress in several studies, but what

role, if any, it plays in skeletal muscle or gut permeability is unclear. Calderwood et al.

(Calderwood et al., 1989) discovered that when cells are exposed to heat stress

arachidonic acid (AA) is released (Calderwood et al., 1989), which can be toxic to all

cells at higher than normal levels. AA is a fatty acyl chain found in cellular membranes

which is one of the main substrates for eicosanoid metabolism (Funk, 2001), see Figure

1.2. Since heat stress has been found to increase the available substrate for eicosanoid

metabolism (Calderwood et al., 1989), the enzymes responsible for taking the free AA and transforming it into products, could be very important during hyperthermia.

12

Furthermore, Zuo et al. (Zuo et al., 2004) evaluated the response of muscle cells to heat stress and the production of ROS. Not only was ROS increased during hyperthermia, but one of the main sources was from lipoxygenase (LOX), which is one of the enzymes responsible for AA metabolism (Funk, 2001). However, it was unclear if the products of

LOX had any part in the production of ROS or if this could have an effect during the dysfunction seen during heat. In addition, a few of the products of eicosanoid metabolism effect thermoregulation directly (Funk, 2001) and could play a crucial role in the development of heat related illnesses. What role, if any, arachidonic acid plays during hyperthermia in skeletal muscle or the intestine is still unknown.

Lipid oxygenases are enzymes that create hydroperoxides from fatty acyl chains associated in the lipid bilayer (e.g., arachidonic acid (AA)) (Natarajan & Nadler, 1998).

In particular, phospholipase A2 (PLA2) is an enzyme that is associated with the lipid bilayers of cells (Chen & Funk, 1993). It takes AA out of the membrane and shuttles the free AA to other enzymes, which can process them (Chen & Funk, 1993). The

metabolism of AA plays an important role in several areas of the body. Cyclooxygenase

(COX), lipoxygenase (LOX) and cytochrome P450 are three main lipid oxygenase enzymes which take AA and enzymatically change it into products that can be used by the cell and tissue from which they reside (Chen & Funk, 1993). These are considered the major lipid oxygenases because of the many roles they play in cellular growth, development and causation of disease.

1.3.1. Cyclooxygenases

Cyclooxygenases (COXs) are enzymes that are responsible for taking AA and transforming it into prostaglandins, in particular PGE2, as well as and

13

prostacyclin (PGI2) (Akarasereenont et al., 1999). COXs have two isoforms in the tissue:

COX-1, which is constitutively activated and is responsible for keeping homeostasis, and

COX-2, or inducible COX, which is not constitutively active but is upregulated and

activated when stimulated (Akarasereenont et al., 1999). Stimulation can occur through

mitogens and proinflammatory cytokines (Akarasereenont et al., 1999). COXs have also

been found to play a part in returning the immune response to its inactive state after an

inflammatory stressor has passed (Akarasereenont et al., 1999).

The structure of COX is a homodimer with each subunit containing a catalytic

domain, an epidermal growth factor domain and a membrane-binding domain (Rouzer &

Marnett, 2005). The catalytic domain has two active sites where AA is first transformed

into hydroperoxy endoperoxide, PGG2, and then PGG2 is transformed into hydroxy

endoperoxide, PGH2 by way of introduction of molecular oxygen (O2) (Rouzer &

Marnett, 2005). The active site uses heme groups to help reactions take place (Rouzer &

Marnett, 2005). The membrane-binding domain of COX is mainly made of alpha helices, which are used for insertion into membranes (Rouzer & Marnett, 2005). In comparing the two isoforms of COX, their structure is almost identical but their function seems to be quite different (Rouzer & Marnett, 2005).

Functionally, COX-1 and COX-2 perform similar tasks and have similar substrate reactivity (Rouzer & Marnett, 2005). The substrate that activates the COXs are hydroperoxides (Rouzer & Marnett, 2005). The difference between COX-2 activation from COX-1 activation is the concentration of substrate required (Rouzer & Marnett,

2005). COX-2 requires a much lower threshold concentration of hydroperoxides to cause activation, compared to COX-1 (Rouzer & Marnett, 2005). This makes sense when

14

comparing the activation states of COX-1 and COX-2. COX-1 is active most of the time in cells throughout the body making prostaglandins for maintenance of homeostasis

(Caughey et al., 2001). COX-2 expression is stimulated in response to the immune system and subsequently makes prostaglandins used in the inflammatory cascades

(Caughey et al., 2001).

COXs have been shown to be involved in many processes in several tissue types throughout the body. In particular, COX and its products have been studied in the vascular system (Flavahan, 2007). Studies have shown that endothelial cells express

COX-1 constantly but COX-2 expression levels are dependent upon activation (Flavahan,

2007). In the endothelium sheer stress can cause the upregulation of COX-2, which can lead to the increased production of PGI2 causing vasodilation (Caughey et al., 2001).

Also, in , increase in the COX product thromboxane leads to increased

aggregation (Caughey et al., 2001). The important aspect of these COX products is the

balance between the production of PGI2 and thromboxane (TXA2) (Caughey et al., 2001).

Each product exerts its response in a different way in order to help maintain homeostasis

(Caughey et al., 2001). PGI2 inhibits platelet aggregation and is a vasodilator (Caughey

et al., 2001). TXA2 is the opposite of PGI2 in that it promotes platelet aggregation and is

a vasoconstrictor (Caughey et al., 2001).

COX has also been found in other areas of the body such as skeletal muscle and the intestine. Skeletal muscle cells have been found to express both COX-1 and COX-2

and are present in several different fiber types (Testa et al., 2007). Interestingly, Testa et

al. (Testa et al., 2007) found that COX products produced in skeletal muscle have the

ability to slightly alter contractile function but the mechanism and what role COX

15

products play in normal muscle physiology let alone during stressful events, is unknown.

Also, in the intestine, COX-1 and COX-2 are present but the majority of the data in the literature focus on pharmacological effects of inhibiting COX. Studies have shown that when COX inhibitors (non-steroidal anti-inflammatory drugs, NSAIDs) are ingested intestinal permeability can increase (Bjarnason et al., 1986; Lambert et al., 2007;

Petruzzelli et al., 2007; Ham & Kaunitz, 2008). This possibly hints to an important role for COX in the mechanism of permeability regulation and dysfunction which is of importance to investigations of intestinal inflammatory disorders as well as many other diseases.

Interleukin (IL)-1β and tumor necrosis factor (TNF) α have been found to increase the expression and activity of COX-2. The overall feedback loop that has been discovered for IL-1β and TNFα stimulation of COX-2 expression is as follows. The immune response is activated following a stressful event (Yucel-Lindberg et al., 1999).

IL-1β and TNFα are produced by immune cells and released into the circulation and/or tissue in which they are present (Yucel-Lindberg et al., 1999). These cytokines elicit a stimulatory affect on expression of COX-2 through a cAMP, PKC or other protein- mediated pathway (depending on the tissue type) (Akarasereenont et al., 1999; Yucel-

Lindberg et al., 1999). Production of prostaglandins by COX helps to inactivate the effects of the immune response (Akarasereenont et al., 1999; Yucel-Lindberg et al.,

1999). At the same time, prostaglandins are reverted back to the COX pathway and inhibit further activity of COX (Akarasereenont et al., 1999; Yucel-Lindberg et al.,

1999). Also, COX is regulated by substrate availability, which means that the concentration of AA available for substrate binding controls the activity of COX (Rouzer

16

& Marnett, 2005). Studies have shown that the increase in substrate availability causes

the activity of COX-2 to increase (Rouzer & Marnett, 2005).

The use of NSAIDs is a common occurrence due to their wide range of

applications. Most people have taken NSAIDs to reduce a fever, alleviate pain or relieve

a headache. The actual mechanism used by these drugs is targeted more towards the

COX enzymes (Chiang & Serhan, 2006). Aspirin was one of the first NSAIDs to be

discovered and has been continually studied due to new findings involving COX and

LOX. COX inhibition by aspirin can be broken down into two pathways: one is the

acetylation of a serine residue in the catalytic domain of COX-1 and COX-2, second is

the difference in action that the acetylation has on the COX isoforms (Chiang & Serhan,

2006). The COX-1 isoform is inhibited by the acetylation and synthesis

stops (Chiang & Serhan, 2006). However, COX-2, when acetylated, shifts production

from prostaglandins to 15R-HETE (Chiang & Serhan, 2006). 15R-HETE is then

transformed by 5-LOX in activated leukocytes into aspirin-triggered 15-epi- A4

(ATL) (Chiang & Serhan, 2006). ATL is responsible for slowing down the immune response and suppressing the effects seen during inflammation (swelling, aching, fever, etc.) (Chiang & Serhan, 2006). NSAIDs do not seem to have an effect directly on LOXs but instead LOXs play a secondary role in the production of .

NSAIDs elicit some negative effects; however, the primary organ studied has been the intestine. Little to no information is available for skeletal muscle reactions to

NSAIDs. In the intestine, several studies (Bjarnason et al., 1986; Lambert et al., 2007;

Petruzzelli et al., 2007; Ham & Kaunitz, 2008) have found that NSAID administration during exercise as well as during normal activity cause an increase in intestinal

17

permeability. This is of great importance because of the wide use of NSAIDs to decrease

pain and fever. Further study needs to be done to fully understand the detrimental as well

as possibly helpful role NSAIDs can play in skeletal muscle function and intestinal

health.

1.3.2. Lipoxygenases

LOXs are non-heme iron-containing oxygenases that take polyenoic fatty acids

(such as AA) and enzymatically oxygenate them into hydroperoxy-polyenoic fatty acids

(Schewe, 2002). They are widespread throughout animals and plants and have been implicated in several diseases (Schewe, 2002). LOXs have several isoforms; in humans the main isoforms are 5-, 12-, 15-LOX and in some cases the 12/15-LOX (Brash, 1999).

The number defining the LOX isoform (for ex. 12-LOX) indicates the carbon on which the LOX applies the oxygen molecule and creates a hydroperoxide (Brash, 1999). The main purpose of LOXs is to produce lipid metabolites such as (Schewe,

2002). Leukotrienes have important properties that involved in asthma and most importantly in mediating the immune response (chemotaxis, immigration and activation)

(Sharma & Mohammed, 2006).

Structurally, LOX are made up of one amino acid chain that is folded into two different domains (Kuhn et al., 2005). Unlike COX, LOX is a monomeric enzyme

(Radmark & Samuelsson, 2005). The N-terminal domain is composed of β-barrels, which is most likely the area associated with membrane insertion (Kuhn et al., 2005).

The larger C-terminal domain comprises the catalytic portion of the LOX and holds most of the iron ligands used in reaction catalysis (Kuhn et al., 2005).

18

LOX products have been found to be involved in processes such as signaling,

structural changes, pathological changes and β-oxidation (Brash, 1999). The particular products involved in these processes are HETEs and HPETEs (Brash, 1999). These products form the basis for production as well as lipid peroxidation (Brash,

1999). Leukotrienes have also been shown to induce ROS formation, which has been linked to apoptosis in leukemia cells (Mahipal et al., 2007), as well as chemotaxis and cell proliferation (Woo et al., 2002). Since these products play a role in several processes they are possible focal points in studying diseases such as asthma, cancer, inflammatory disorders, Alzheimers disease and possibly more acute diseases such as heat stroke and heat related illnesses.

Functionally LOX play several roles in tissues throughout the body. Examples of these roles are in the lungs, reticulocytes, platelets, leukocytes and the vascular system.

The 5-LOX enzyme is present in cells of lung tissue and has been found to be very important in the pathways involved in asthma (Brash, 1999). This has been shown by the use of 5-LOX inhibitors to control asthmatic episodes (Brash, 1999). 15-LOX has been shown to be important in reticulocytes, most likely being used for cell signaling (Brash,

1999). 12/15-LOX is activated by apoptotic thymocytes, which cause LOX to translocate to the membrane where it can be activated (when Ca2+ is present) and produce products

such as leukotrienes (Kuhn et al., 2005). Furthermore, LOX has been found to be

important in maintaining the structural composition of the membrane since it is able to

use other membrane lipids as substrate (Brash, 1999), which could be very important

when the membrane is exposed to stress events and needs to alter membrane structure

quickly.

19

Very little is known about the function of LOX in skeletal muscle and even less is

known in the intestine. Zuo et al. (Zuo et al., 2004) discovered through immunohistochemistry that LOX is present in skeletal muscle. Also, LOX and its products have been found to be able to bind to the contractile machinery in muscle

(Brock, 2008) which could affect overall function. In several different types of cells heat stress can increase the release of free arachidonic acid (Calderwood et al., 1989). This increase in free arachidonic acid has been attributed to alterations in normal cellular function such as stimulation of oxidant formation (Esenabhalu et al., 2003), disruption of the sarcolemmal Ca2+-ATPase (Cardoso & De Meis, 1993), activation of calcium

channels (Mignen & Shuttleworth, 2000), as well as activation of the mitochondrial transition pore (Qian et al., 2004; Di Paola et al., 2006). In the intestine, inhibition of

LOX has been shown to protect against an increase in permeability caused by experimental colitis (Mazzon et al., 2006), inflammatory bowel disease (Shapiro et al.,

1986) and butyrate stimulation (Ohata et al., 2005). Also, Chang et al (Chang et al.,

2007) discovered that when rats exposed to whole animal hyperthermia were given baicalein (LOX inhibitor) survival time was increased, apparently due to a lowering of the core body temperature, which alluded to the possibility of LOX enzyme activity being important during heat stress.

Leukotrienes and prostaglandins have been shown to be important in the progression of several diseases (Akarasereenont et al., 1999; Caughey et al., 2001;

Sharma & Mohammed, 2006), but their importance during hyperthermia in skeletal muscle as well as the intestine is not known. PGE2 is produced primarily by COX in

skeletal muscle during normothermic conditions (Testa et al., 2007) but could change

20

during heat stress. However, which products are produced by LOX in skeletal muscle as

well as the intestine have yet to be fully identified. Zuo et al. have shown through

immunohistochemistry that both 5-LOX and 12-LOX are present in skeletal muscle, but

12/15-LOX were not studied (Zuo et al., 2004). Also, Testa et al. (Testa et al., 2007)

have shown that both COX isoforms (1 and 2) are present constitutively in skeletal

muscle but they mainly produce only PGE2 as a product under normothermic conditions.

It could be possible that both LOX and COX activities change as the temperature of

skeletal muscle rises. This could be possible because LOX and COX are involved in

membrane restructuring (Brash, 1999), which is a very important process, especially

during heat stress. Also, LOX and COX involvement during hyperthermia could be due to the availability of free AA because of its increased release when exposed to heat

(Calderwood et al., 1989).

1.3.3. Cytochrome P450

Cytochrome P450 (CYP450) isoforms are present in both skeletal muscle (Wang

et al., 2007) and have been found in high concentration in the intestine (Paine et al.,

2006). The main products produced are epoxyeicosatrienoic acids (EETs) and

monohydroxyeicosatetraenoic acids (monoHETEs) which have been found to be

involved in drug metabolism (Watkins, 1992), control of vasoregulation (Harder et al.,

1995) and anti-inflammation (Node et al., 1999). Most literature about cytochrome

P450’s in the intestine has focused on drug absorption and metabolism. In skeletal

21 muscle the majority of the literature pertaining to CYP450 enzyme activity and products discusses vasculature regulation. To my knowledge, no studies exist that have examined the role of CYP450’s during hyperthermia as well as any role these enzymes could have in the intestine or in skeletal muscle.

22

1.4. Research Objectives

The goal of this thesis was to determine the thermal tolerance of skeletal muscle and the small intestine (i.e. the highest temperature which does not cause dysfunction); and evaluate the effects of inhibiting eicosanoid metabolism and oxidative stress on hyperthermia-induced dysfunction. Hyperthermia is one of the most primordial environmental stresses driving evolutionary diversity; it can affect all cells and integrative systems within the body. However, the mechanisms which help protect cells and tissues during hyperthermia are not well understood.

My objectives were threefold; the first was to define the thermal tolerance of skeletal muscle and gut permeability exposed to physiologically relevant temperatures and evaluate the functional response of antioxidant treatment. In pathological conditions of heat stroke or malignant hyperthermia, core temperatures can exceed 44°C (Ali et al.,

2003). Surprisingly, we know little about the effects of hyperthermia on contractile function as well as gut permeability within this temperature range (41– 44°C). Studies have found that skinned muscle fibers (van der Poel & Stephenson, 2002) as well as rat small intestine (Lambert et al., 2002a) become susceptible to hyperthermia. However, the effect of hyperthermia on whole intact muscle and on mouse small intestinal function have yet to be elucidated. Since muscle is a primary source of heat production in the body and the intestine is a critical barrier found to be involved in the pathogenesis of heat stroke, further study of the response of these tissues to heat stress is of great importance.

Also, studies have shown a possible correlation between increased ROS formation during hyperthermia and increased gut permeability (Rao et al., 1997; Hall et al., 2001) as well as muscle (van der Poel & Stephenson, 2002) dysfunction. For these reasons, skeletal

23 muscle and the intestine will be treated with antioxidants to discover if scavenging oxidants can protect against dysfunction. I hypothesized that skeletal muscle and mouse intestine will lose function when exposed to temperatures relevant to heat stroke or exercise and treatment of these tissues with antioxidants will protect against hyperthermia-induced dysfunction.

The second objective was to elucidate the effects of inhibiting eicosanoid metabolism enzymes during hyperthermia and compare these effects between skeletal muscle and intestine which are critically important during heat stress. Eicosanoid metabolism has been found to be important in several aspects of cellular function including thermoregulation; however, little to no data is present pertaining to the possible role these enzymes play during hyperthermia. Previously, an increase in extracellular but not intracellular ROS production, in muscle cells during hyperthermia, has been shown to decrease significantly when LOX enzymes are inhibited (Zuo et al., 2004). This led me to believe that eicosanoid metabolism could be detrimental to contractile function during hyperthermia and inhibition of enzymes associated with eicosanoid metabolism could provide protection during heat stress. Furthermore, studies have shown that ingesting inhibitors of eicosanoid metabolism can cause an increase in intestinal permeability in normal as well as exercise conditions (Bjarnason et al., 1986; Smetanka et al., 1999;

Lambert et al., 2007). These findings suggest that eicosanoid metabolism in the gut and in muscle could also be important in tissue viability or damage during hyperthermia. I hypothesized that eicosanoid metabolism, an area previously not linked to thermal tolerance, may actually have a protective impact on skeletal muscle function or intestinal permeability during hyperthermia.

24

The third objective was to discover the mechanism of increased gut permeability

during hyperthermia. Is it due to tight junction opening or to injury of the epithelial

lining? Furthermore, I wish to determine the extent to which changes to gut permeability

could be attributed to oxidative damage, and to other mechanisms such as elevations in

intracellular calcium and whether the overall structure of the gut lining during

hyperthermia could give clues to the mechanisms involved. I hypothesized that cellular damage due to hyperthermia would be the primary cause for the increase in permeability and that the mechanism for protection of gut hyperthermia-induced permeability changes in response to some pharmacological treatments would be through inhibition of oxidative stress and reduction in structural damage.

Discovering links between eicosanoid metabolism, intestinal permeability, muscle contractile function and responses to hyperthermia could create new therapies and treatment paradigms for heat-related illnesses that have the potential of improving thermal tolerance in athletes, soldiers, first-responders, and other vulnerable populations, such as the elderly and very young.

Summary

Specific Aims Covered in Chapter 2. 1. To determine the functional responses to brief heat exposure in isolated, largely oxidative skeletal muscle. 2. To test the hypothesis that increased oxidant production in heat stress is responsible for the loss of contractile function in intact muscle tissue. 3. To evaluate the role of eicosanoid metabolism, specifically catalyzed by lipoxygenase and cyclooxygenase, on the loss of contractile function in hyperthermia.

25

Specific Aims Covered in Chapter 3. 1. To determine the thermal tolerance of mouse intestinal tissue using an in vitro model of gut permeability. 2. To evaluate the possible protective or detrimental effects of inhibition of various eicosanoid metabolic pathways on hyperthermia-induced intestinal permeability. 3. To test the hypothesis that quenching ROS during hyperthermia, using known and contrastingly different scavengers tiron, Trolox and N-acetyl cysteine, could protect the intestine from loss of barrier function.

Specific Aims Covered in Chapter 4.

1. To determine if tight junction opening or cell damage is the predominant pathway through which permeability is increased during hyperthermia in the mouse intestine. 2. To discover how baicalein and N-acetyl cysteine protect the mouse intestine from hyperthermia-induced permeability.

26

1.5. Figures

Figure 1.1. Schematic of the various pathways heat stress affects skeletal muscle and the intestine.

27

Figure 1.2: Main pathways of arachidonic acid metabolism

28

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Chapter 2: Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition (In Press: Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008. Nov; 295(5): R1695-705)

2.1. Introduction

The cellular mechanisms responsible for maintaining skeletal muscle function at

elevated temperature are not well understood. However, since skeletal muscles are heat

generators during exercise, their ability to resist the effects of heat must be an integral

part of their normal physiology. Human limb muscles can attain temperatures up to 41

°C during intense exercise in hot ambient environments (Parkin et al., 1999) and rat limb

muscle temperature has been measured as high as 44° C during exhaustive exercise

(Brooks et al., 1971). In pathological conditions of heat stroke or malignant

hyperthermia, core temperatures can exceed 44 °C (Ali et al., 2003). Surprisingly, we

know little about the effects of hyperthermia on contractile function within this

temperature range (41-44 ºC). Therefore, one of the objectives of this study was to

identify the temperature at which significant contractile dysfunction is evident in isolated,

intact skeletal muscle over a time period that could be relevant to exercise. A second objective was to identify possible mechanisms responsible for the loss of contractile function at this temperature.

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In previous work, our laboratory observed that exposing skeletal muscle to 42 ºC

resulted in increases in both intra- and extracellular reactive oxygen species (ROS) (Zuo

et al., 2000). Similar elevations in ROS production during heat stress have been made in

a variety of tissues (Wallen et al., 1997; Flanagan et al., 1998) and in whole animals

(Hall et al., 2001) but the functional significance of these findings is not well known.

Recently, van der Poel and Stephenson (van der Poel & Stephenson, 2002) have shown

that at elevated temperatures (43-47° C), contractile function was disrupted in isolated rat extensor digitorum longus muscle fibers, a largely fast twitch muscle. ROS were

implicated because treatment with dithiothreitol (DTT), a thiol (–SH) reducing agent, or

by Tiron, an antioxidant eliminated or greatly diminished the effects of high temperature

on contractile force. However, no such effects of antioxidants were observed in a small group of more oxidative fibers from the peroneus longus muscle (van der Poel &

Stephenson, 2002). Therefore, a second objective of this study was to test the hypothesis that ROS formed during heat are responsible for the loss of contractile function in an intact muscle with a fiber population that is largely oxidative, namely the isolated

diaphragm.

The cell membrane is believed to be particularly sensitive to the effects of

temperature and has been described as the “temperature transducer” of the cell (Balogh et

al., 2005). In a number of isolated cell systems, as temperature increases to 42 ºC or

above there is a rapid release of arachidonic acid (AA), through the action of

phosopholipase A2 (PLA2) on the membrane phospholipids (Calderwood et al., 1989).

This suggests membrane repair or remodeling activities are ongoing. Since AA is further

metabolized by lipoxygenase (LOX), cyclooxygenase (COX) and cytochrome P450

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enzymes, it is likely that these enzymes also play significant roles in normal membrane

responses to hyperthermia. Interestingly, these pathways may also be responsible for

some fraction of oxidant production at high temperatures. In previous work, inhibition of

global LOX activity almost completely inhibited the extracellular ROS formed during

heat exposure (Zuo et al., 2004). Therefore, we hypothesized that by blocking LOX and

possibly other pathways of eicosanoid metabolism we might protect skeletal muscle

function during heat exposure and possibly inhibit oxidative stress at the same time.

In summary, this study had three objectives. (i) To determine the functional

responses to brief heat exposure in isolated, relatively intact, and largely oxidative

skeletal muscle where other variables such as blood flow, central neural responses to

heat, neuromuscular activation or whole body inflammation are not present. (ii) To test

the hypothesis that increased oxidant production in heat stress is responsible for the loss

of contractile function in intact muscle tissue and (iii) to evaluate the role of eicosanoid metabolism, specifically catalyzed by lipoxygenase and cyclooxygenase, on the loss of contractile function in hyperthermia.

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2.2. Methods

2.2.1. Animal Treatment Protocols and isolated muscle preparation

Adult male Sprague-Dawley rats (350-450 g) were used for this study. Animal care and treatment was performed using protocols approved by The Ohio State University and the University of Florida Institutional Animal Care and Use Committees. Rats were anesthetized by IP injection of ketamine (100 mg/kg) and xylazine (20 mg/kg),

tracheotomized, and ventilated on room air. The diaphragm was quickly excised from

the animal and placed in pre-oxygenated (95 % O2, 5 % CO2) Ringers solution (in mM:

21 NaHCO3, 1.0 MgCl2, 0.6 Na2HPO4, 0.45 Na2SO4, 2.0 CaCl2, 5.9 KCl, 121 NaCl;

11.5 glucose, and 10 μM D-tubocurarine). Strips of muscle, 5-6 mm wide, were created

with the central tendon and rib intact. The central tendon was fixed to a plastic frame

with a small amount of cyanoacrylate gel adhesive and affixed to a force transducer

(Grass FT03) via 3-0 silk suture. The rib was attached to a glass frame using a loop of

suture around the rib. The strips were placed in 20 ml water-jacketed tissue baths,

maintained at 37 ºC (Figure 2.1). Four strips were obtained from each animal and studied simultaneously. The optimum length (Lo) and stimulation current, resulting in maximum

twitch force, were determined. Throughout the study, force frequency (FF) curves were

constructed using the twitch force and the force responses to 400 ms trains of

supramaximal stimuli of 0.2 ms duration at 20, 30, 60 and 150 Hz with 20 sec rest

between contractions (Figure 2.2).

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2.2.2. Protocols

All protocols for heat exposure and pharmaceutical treatment were similar, or are

described further in the Results section. The tissues were first equilibrated in oxygenated

Ringers solution at 37 ºC for 30 min. During this time, Lo and optimum stimulation

voltage were determined. They were then stimulated at 0.05 Hz throughout the

equilibration periods for all experiments. After initial equilibration, a baseline FF

relationship was determined. The baths were then changed to either fresh, oxygenated

Ringers buffer, pre-heated to 37 ºC, or buffer containing one of a large number of pharmaceutical preparations of antioxidants, COX, LOX or PLA2 inhibitors (Table 2.1).

Matched controls always contained the corresponding vehicles (usually DMSO) in the same concentration as the maximum dosage of the vehicle in the treated baths. After 30 min of equilibration in the treatment buffers, a second FF was obtained to determine the independent influence of the drugs. The baths were changed again with the appropriate drugs or vehicle and the strips were exposed to either 37 ºC or one of 4 elevated temperatures, 40 ºC, 41 ºC, 42 ºC, or 43 ºC. Temperatures within the baths were monitored and maintained within 0.1 ºC of the target. Once the target temperature was obtained it was maintained for a 30 min exposure, at which time another FF was performed. The baths were then replaced with fresh Ringers solution and returned to 37

ºC for another 30 min after which a final FF was obtained to monitor recovery. The

length and weight of each tissue was measured to determine specific force (average value

for control tissues: 21.8 N/cm2 ±0.5, n=43). More specific combinations of drug

treatments, their doses and the specific temperature exposures in each experiment will be

described in the Results Section.

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2.2.3. Membrane Permeability Assessment

Membrane permeability was assessed using procion orange MX2R dye (Sigma

Aldrich), which only crosses damaged membranes and has been used frequently to detect

muscle cell membrane damage (Van Gammeren et al., 2005). At the end of some

experiments 0.15 % wt/vol procion orange MX2R was added to the tissue bath for 45

min. followed by a 15 min. rinse with fresh buffer. The tissue was then fixed in 10%

formalin, mounted in a 3% agar gel and later cut in 20-60 µm sections with a vibrating tissue slicer (Precisionary Instruments, Inc.). Multiple images of each section were recorded with an epifluorescence microscope using standard filter settings for fluorescein. The numbers of cells containing procion orange MX2R were delineated as a fraction of the total number of cells in the field. Three fields were chosen at random for each sample. This ratio was used to quantify the extent of damage in each specific tissue.

2.2.4. Statistical Analysis

Statistical significance was analyzed using analysis of variance (ANOVA), designed for each experimental protocol. Treatment and temperature were used as factors of interest in most studies. Data were expressed as a percent of maximum baseline force. The effects of “animal, i.e. specific experiment on matched tissues” were treated as a random variable resulting in the equivalent of a repeated measures design.

Post hoc analyses (Dunnetts) were done to determine specific effects of treatment from control measurements. Mean contrasts after ANOVA were used in some experiments to compare sample means in complex designs (SASJMP statistical package). All results are

reported as means ± SE; P < 0.05 was considered statistically significant.

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2.3. Results

2.3.1. Thermal Tolerance of Isolated Diaphragm

As shown in Figure 2.2.A, compared to control measurements at 37ºC, exposure of

diaphragm to temperatures from 40-42 ºC for 30 min had no significant effect on force

developed at all stimulation frequencies. In contrast, at 43° C, maximum force dropped markedly from control with similar relative decreases seen at all lower frequencies, including twitch. After 30 min of recovery at 37 ºC there were no improvements in contractile function (Figure 2.2.B) demonstrating that the effects were not readily reversible. In response to elevated temperature, an increase in passive force was also observed by the end of the 43° C exposure (inset, Figure 2.2.A).

2.3.2. Effects of Antioxidants and Reducing Agents on Contractile Function with Heat

Exposure

To test the hypothesis that the loss of contractile function during heat stress is due to the effects of increased oxidant production, diaphragms were exposed to 43° C in the presence of antioxidants, Tiron (1, 10 and 20 mM) and Trolox (50 µM, 100 µM and 1 mM) (Figure 2.3). The intermediate dose of Tiron (10 mM) was shown in previous studies to be effective in preserving contractile function during hypoxia (Wright et al.,

2005) and the maximum dose (20 mM) was previously used by van der Poel and

Stephenson (van der Poel & Stephenson, 2002). The lowest dose of Trolox (50 µM)

approximated the concentration shown by Betters et al. to reduce oxidant stress in the

diaphragm in the whole animal (Betters et al., 2004), whereas the highest dose has been

44 shown to be effective in reducing oxidant production in isolated myotubes (Young et al.,

2004). Contrary to expectations, no effects of these antioxidants on the heat-induced loss of contractile force were observed, although there was a significant reduction in force at the highest concentration of Trolox. There were also no improvements in function during the recovery period in antioxidant treated tissues (data not shown).

To compare our results to observations in isolated fibers (van der Poel & Stephenson,

2002) we tested whether the heat-induced reductions in force could be reversed by treatment with 5-20 mM dithiothreitol (DTT) given immediately after the heat exposure and allowed to remain in the bath during the 30 min. recovery period (Figure 2.4). The dosage range came from several sources. The dose of 5 mM was previously shown to be effective in partially reversing the effects of fatigue in in vitro diaphragm, presumably by reducing protein –SH oxidation (Diaz et al., 1998). Control tissues were performed and found to be similar to Diaz et al.’s (Diaz et al., 1998) findings which showed that a 5 mM dose of DTT has no effect on baseline muscle function after a full time-matched exposure protocol in 37 ºC buffer. The 10 mM DTT dose has also been used in mechanically skinned rat skeletal muscle fibers with no adverse reactions on twitch responses (Lamb &

Posterino, 2003). Van der Poel and Stephenson (van der Poel & Stephenson, 2002) previously used the highest (20 mM) dose in isolated fibers. To perform this experiment, the DTT was dissolved directly into buffer in the tissue bath to reduce rapid oxidation of

DTT. No significant recovery of function was observed following DTT-treatment over the 30 min recovery period.

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2.3.3. Effects of Lipoxygenase Inhibition on the Loss of Contractile Function at 43 º C

Since we previously observed that one source of ROS in heat stress arises from

one or more lipoxygenase pathways, we tested whether blockade of LOX activity could protect contractile function at 43 ºC. Initial doses of LOX inhibitors were determined from previous studies as follows: (a 5-LOX inhibitor, 50 µM, (Carter et al.,

1991) Cayman Chemical), diethylcarbamazine (a 5-LOX inhibitor, 50 µM, Sigma

Aldrich), baicalein (a 12-LOX inhibitor and partial general LOX inhibitor (Chang et al.,

2007), 50 µΜ, Sigma Aldrich) and nordihydroguaiaretic acid (NDGA, general LOX inhibitor; 50 µM, Sigma Aldrich). Contrary to our hypothesis, NDGA treatments greatly increased the loss of force during exposure to 43 ºC (Figure 2.5). The 5-LOX inhibitors,

Zileuton and diethylcarbamazine showed no significant effects compared to control.

Follow up experiments with NDGA (data not shown) demonstrated that it caused significant reduction of contractile function in the absence of heat exposure, making it unsuitable for these kinds of experiments.

2.3.4. Effects of Lipoxygenase and Cyclooxygenase Inhibition on Heat Tolerance to 42ºC

Based on the results of the previous experiment we hypothesized that lipoxygenase and possibly other eicosanoid metabolic pathways are important components of the normal heat tolerance mechanisms of skeletal muscle. To test this hypothesis, we exposed diaphragm strips to the highest temperature that does not cause significant loss of contractile function (42 ºC, Figure 2.2A) and evaluated the effects of lipoxygenase and cyclooxygenase inhibition (Figure 2.6).

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Both treatment with baicalein (12-LOX inhibitor, doses of 50, 100 and 200 µM) and

ETYA (general LOX inhibitor, 50, 100 and 200 µM) resulted in significant decreases in

contractile function during exposure to 42° C (Figure 2.6A). In time controls at 37 ºC,

the middle dose of either of these drugs had no significant effect on contractile function

compared to time controls (baicelein, –15 ∆%Max Force ±1.4, ETYA, -15 ∆%Max Force

±4.0), indicating no measurable pharmacological effects. The decrease in function

occurred in a dose dependent manner and resulted in the greatest loss of function at 200

μM during 42 ºC heat exposure. Like the influence of heat exposure alone, recovery of

function was not observed in these tissues (data not shown). These results suggest that

functional LOX activity may be an important factor for heat tolerance in skeletal muscle

at 42 ºC.

A parallel experiment was done using COX inhibition. Since COX also metabolizes

AA, it was logical to think that this pathway may also affect heat tolerance at 42 ºC.

Addition of indomethacin at (50,100 and 200 µM) resulted in a dose dependent reduction in maximum contractile force during exposure to 42 ºC. Likewise, ibuprofen (Bond et al., 1992), given at 200, 400 and 800 µM resulted in a dose-dependent loss in heat tolerance at 42 ºC (Figure 2.6B), whereas the time controls showed no effect on contractile function (indomethacin, -2.1 ∆%Max Force ±2.3, ibuprofen, -6.9 ∆%Max

Force ±1.9). Both drugs had qualitatively similar effects but at these doses they did not decrease force to the extent that LOX inhibition did. Interestingly, recovery data showed that loss of function at 42 ºC in the presence of COX inhibition resulted in partial but modest recovery of force over 30 min (data not shown).

47

To determine whether the influences of LOX and COX during heat exposure had

overlapping or additive effects, we inhibited both enzymes simultaneously (Figure 2.7),

using Baicalein (100 µM) and indomethacin (100 µM) in the same muscle tissue at 42°

C. A small loss in function was observed in pharmacological controls done at 37° C (-

25.1 ∆%Max Force ±4.6, Figure 2.7) but a much larger effect was seen at 42° C.

Compared to all other drugs the combination of the two drugs resulted in the greatest loss

in function and suggests that the two enzyme systems do not have overlapping effects but

rather that their influence is likely additive.

Treatment with LOX and COX inhibitors resulted in an increase in baseline passive

force during 42 ºC exposure as well, (Figure 2.6C and Figure 2.6D). ETYA, baicalein,

indomethacin and ibuprofen showed a dose dependent increase in baseline passive force

with baicalein having the greatest impact.

2.3.5. Effects of Inhibition of Upstream PLA2

Based on the preceding studies, we hypothesized that the role of LOX and COX in

heat exposure might be simply to metabolize and therefore reduce the concentration of

free AA and other lipid products created at elevated temperatures that could be

detrimental to function. If so, then by blocking upstream PLA2 alone, we might cause a

+2 reduction of heat-induced contractile function at 43 ºC. To block iPLA2, a Ca –

independent PLA2 (iPLA2) inhibitor, bromoenol lactone (BEL) was used. Previous work

has demonstrated that BEL targets functional iPLA2, which has been shown to be present in skeletal muscle (iPLA2) (Gong et al., 2006). Control experiments showed a pharmacological effect of 20 µM BEL at 37° C. At 1 µM and 10 μM, however, there

48 were no significant effects at 43 ºC, but at 20 μM a significant decrease in function was observed (Figure 2.8A). We accredited this decrease at the highest dose to be, in part, a nonspecific pharmacological effect.

To block the cytosolic PLA2 (cPLA2) and iPLA2 together, arachidonyltrifluoromethyl ketone (AACOCF3) was used at a dose (40 µM) previously shown to be effective in isolated skeletal muscle (Nethery et al., 1999). AACOCF3 showed no significant effect on contractile function at 43 ºC (Figure 2.8B). Finally, 4-bromophenacyl bromide (BPB) was used to inhibit secretory PLA2 (sPLA2) at 30 µM shown to be effective in smooth muscle (Vanheel et al., 1999) (Figure 2.9). Control experiments showed a pharmacological effect of BPB during 37 ºC, which was significantly higher than 37 ºC control tissues (P < 0.01). Also, tissues exposed to BPB at 43 ºC showed a significantly larger decrease in force compared to untreated 43 ºC controls (P < 0.01). However, these effects can likely be attributed to the pharmacological effects of the drug, as shown in the

37 ºC controls.

2.3.6. Tests for Membrane Damage in Heat Stress

Based on the sensitivity of contractile function to elevated temperatures and particularly to enzymes known to be involved with maintenance of membrane function, we hypothesized that the loss of contractile function in heat stress may reflect a subpopulation of fibers within the intact muscle that have lost membrane integrity. To test this hypothesis, we evaluated the integrity of the sarcolemmal membrane using procion orange leakage into diaphragm cells (Hayot et al., 2001). Examples of the kinds of results from this experiment are shown in Figure 2.10. No additional leakage was

49

observed in response to heat stress alone (43 ºC). However, significant leakage was

observed when muscles were incubated with both indomethacin and baicalein at 43 ºC

(17.35 %positive cells ±1.37, n=6) compared to control (0.45 %positive cells ±0.21,

n=6). Muscles incubated with indomethacin and baicalein at 37° C showed no significant

increase in permeability (2.37% positive cells ±0.71, n=6) compared to both 43° C (2.32

% positive cells ±0.91, n=6) and 37° C (0.45 % positive cells ±0.21, n=6) controls.

50

2.4. Discussion

The results demonstrate that oxidative skeletal muscle, as represented by isolated rat diaphragm, is resistant to 30 min of hyperthermia between 40-42 ºC. However, exposure to 43 ºC causes a marked loss of tetanic force and an elevation in passive force.

Though the mechanisms for the loss of normal contractile function at this threshold temperature are not known, we demonstrate that they are not easily reversible in 30 min and do not appear to result from a response to oxidative stress. In addition, the response does not primarily reflect loss of cell membrane integrity, though when combined with

LOX and COX inhibition, loss of the sarcolemmal barrier was widely present. Our data also suggest that a critical aspect of the muscle’s ability to sustain normal function in hyperthermia is an intact network of membrane eicosanoid metabolic machinery, requiring functioning COX and LOX enzyme systems. Pharmacological inhibition by many contrasting pharmacological mediators, causes increased sensitivity to hyperthermia and must directly impact contractile function in some, as yet, unknown way.

2.4.1. Critique of Approach

Though it is unlikely that the diaphragm would ever attain temperatures much above body core temperature in hyperthermia, this model is useful as an intact, oxidative skeletal muscle preparation for the purposes of this study. We have little reason to believe that results would differ substantially from oxidative limb muscle, and in essence our work substantiates results seen in isolated single oxidative limb muscle fibers exposed to heat (van der Poel & Stephenson, 2002). O2 delivery is always a concern in

51

non-perfused, intact preparations, but since the muscles were not fatigued or exercised

intensely during the protocol it is unlikely that the results can be attributed to differences

in oxygen delivery. A 9 % reduction in O2 solubility occurs between 37 ºC and 43 ºC, and equivalent reductions in PO2 have little effect on rat diaphragm contractile function

(unpublished observation) or on NADH autofluorescence (Zuo & Clanton, 2005), an

indicator of tissue hypoxia. Though pharmacological approaches were used to evaluate

the influence of enzymes involved with eicosanoid metabolism as well as ROS

scavenging, we attempted to account for possible non-specific pharmacological effects of

these drugs by (i) studying their influence on time controls at 37 ºC, (ii) by studying dose

response curves, and (iii) by using multiple drugs that often worked by chemically

divergent methods. This approach revealed a significant adverse pharmacological

reaction to NDGA, a commonly used LOX inhibitor; confirming recent studies in vitro

and in vivo (Lambert et al., 2002b; Huang et al., 2004).

2.4.2. Mechanisms for the Loss of Contractile Function

The results show that there is a critical temperature, between 42-43° C, where there is a marked loss of contractile function within 30 min. This is also the range of temperatures generally required for induction of heat shock proteins (Oishi et al., 2002), rapid increases in membrane permeability for small molecular weight molecules (Bischof et al., 1995) and reduced survival in many mammalian in vitro cell types (Cress et al.,

1982). The absolute value of the critical temperature is also, no doubt, a reflection of the duration of heat exposure, as loss of contractile function (van der Poel & Stephenson,

2002) as well as tissue injury, reviewed in Despa et al. (Despa et al., 2005), are both time

52

and temperature dependent. The underlying biochemical mechanisms responsible for this

critical temperature phenomenon are unknown but most research points to accumulating

alterations in protein structure, membrane integrity or oxidative stress, as discussed

below.

Although the activity of most enzymes increases as a function of their

temperature coefficients (Q10) it is generally held that protein configuration changes at

some critical and protein-specific temperature, thereby decreasing activity or interfering

with protein-protein interactions. Temperature-induced protein misfolding, exposure of hydrophobic moities or degradation have traditionally been the primary mechanisms attributed to HSP induction in hyperthermia, as reviewed in (Kim et al., 2007). Although degradation is possible at these temperatures there is little evidence for it in isolated proteins, as studies have shown that temperatures >50°C are generally needed for denaturation (Despa et al., 2005). Many investigations have attributed protein degradation to a secondary effect of increased oxidant formation during hyperthermia, e.g. (Schertzer et al., 2002), which may be a common mechanism.

Acute hyperthermia also increases membrane fluidity (Balogh et al., 2005) and is known to disrupt membrane microdomains such as lipid rafts and the interaction of the membrane with associated channels, proteins and cytoskeleton (Nagy et al., 2007). Heat induced changes in the biophysical properties of membranes can also result in alterations in water (Prasad et al., 2007) or solute (Bischof et al., 1995) permeability that could have secondary effects on membrane potential, action potential propagation or contractile function. For example, swelling of muscle fibers, which has been shown to occur in cells

53

at high temperature, could potentially have influences on the lattice structure of the

contractile machinery and thus reduce force development (Rapp et al., 1998).

Skeletal muscle hyperthermia has also been shown to cause elevations in ROS production (Wallen et al., 1997; Zuo et al., 2000), but their complex role is not completely understood. Van der Poel et al. (van der Poel & Stephenson, 2002) reported that in rat extensor digitorum longus (EDL) muscle, a fast non-oxidative fiber, loss of maximum Ca2+-activated force during heat could be blocked by co-treatment with the

antioxidant Tiron (20 mM). In addition, it could be reversed after heat exposure with 20

mM DTT. Interestingly, these investigators saw no such effects of Tiron in more

oxidative fibers from the limb muscle (peroneus longus). Our results for diaphragm are

essentially in agreement with the studies of the peroneus (van der Poel & Stephenson,

2002) presumably because the rat diaphragm is only 3.5 % type IIb fast fibers (Farkas et

al., 1994). In addition, we tested for the influence of multiple doses of DTT and Trolox.

Trolox is a soluble vitamin E analog and a general antioxidant and has previously been

shown to be effective in intact diaphragm, protecting it from oxidative damage induced

by mechanical ventilation (Betters et al., 2004) or from secondary oxidant effects of TNF

(Hardin et al., 2008). None of these agents were effective at concentrations equal to or

much higher than those previously shown to be effective in diaphragm preparations (Diaz

et al., 1998; Betters et al., 2004; Wright et al., 2005; Hardin et al., 2008). Therefore, our

results, taken together with those of van der Poel et al. (van der Poel & Stephenson,

2002) are most consistent with the hypothesis that oxidant production is not the main

causative factor for the loss of contractile function in oxidative fibers. Since oxidative

54 fibers are rich in mitochondria, which are believed to be a primary source of ROS in pathological conditions, these conclusions seem indeed paradoxical.

It is of physiological interest that the FF relationship was relatively unchanged between 37 ºC and 42 ºC. Since large shifts of FF to the right occur as temperature increases from 23 ºC to 37 ºC, we expected to see a continuation of this behavior above

37 ºC. The fact that tetanic force at all frequencies remains relatively constant suggests that skeletal muscle has an inherent mechanism for control of muscle force for a given neural activation within a temperature range that corresponds to the range seen in heavy exercise. How it could do this with varying rates of biochemical reactions (Q10) is a mystery but would function to ensure continuity of motor control during exercise in hyperthermia.

2.4.3. Mechanisms for Changes in passive force during heat exposure

Another mechanical effect of heat exposure was an increase in passive tension

(contracture) during 43° C exposure or at 42° C when drugs affecting eicosanoid metabolism were present. In all cases, the elevation in passive force was rapidly reversed during 37 ºC recovery, during which time the loss of contractile function did not reverse.

There are at least two possible reasons for this phenomenon: first, resting cytosolic calcium concentration could increase during heat stress, thus activating the contractile machinery at low levels. To our knowledge intracellular Ca+2 has not been measured in hyperthermic skeletal muscle. However, an increase in passive tension has been attributed to elevations in [Ca+2] based on indirect observations that diaphragms with

55

RyR1 mutations associated with malignant hyperthermia have greater heat-induced contracture than wild type diaphragms (Chelu et al., 2006). Also, elevated temperature has been shown to increase Ca+2 leakage from the SR, via an oxidant-dependent

mechanism (van der Poel & Stephenson, 2007) though this would not necessarily result

in elevated cytosolic calcium if the SR Ca+2 ATPase continued to function appropriately.

A second possibility is a mechanism that has been demonstrated in glycerinated fibers

at high temperature by Ranatunga et al. (Ranatunga, 1994) that is due to non-Ca+2

activated cross bridge cycling. This is believed to reflect the heat-induced inactivation of

the steric hindrance normally blocking cross bridge interaction in the absence of Ca+2

(Fuchs et al., 1975). Interestingly, in all experiments, the observations of significant

elevations in passive force were accompanied by proportional losses of maximum

contractile function. This suggests that understanding the mechanism for elevation in

passive force may provide future insights into the mechanisms for loss of maximum

stimulated force. However, at this time, the mechanism is not clear in this muscle fiber

population.

2.4.4. LOX, COX and PLA2 involvement during heat stress

Very little is known about the functional roles of eicosanoid metabolism in

skeletal muscles, even during normothermic conditions, though literature is emerging.

With regard to cyclooxygenase, skeletal muscle cells contain both COX-1 and COX-2

and both of these enzymes are present in oxidative and non-oxidative muscle fibers as

well as in surrounding vessels (Testa et al., 2007). The primary products produced are

prostaglandin (PG)E2 and thromboxane (Tx)B2, with considerably more PGE2 being

56

formed (Testa et al., 2007). Though there is some evidence that products formed by

COX can modestly affect contractile function (Testa et al., 2007), their primary roles in physiology are not known. COX products are able to cause vasodilation or vasoconstriction, depending on which product is produced in higher amounts (Caughey et

al., 2001) and it is possible that this balance between products may be altered during heat

stress. Thus, it is also possible that production of COX products in heat stress could

affect heat dispersal mechanisms by influencing conductance of surrounding vessels.

This assumes they are capable of paracrine signaling in this tissue. COX has also been

found to be potentially important in the regulation of heat shock proteins (HSPs) (Batulan

et al., 2005) as PGA2, an enzymatic dehydration product of PGE2, appears important for

HSP70 expression over 4-8 h and for activation of c-fos and Egr-1 stress genes within 30

minutes. This process occurs by a mechanism involving increases in intracellular Ca+2

(Choi et al., 1994).

Even less is known about lipoxygenases in skeletal muscle. LOXs are non-heme

iron-containing oxygenases that take polyenoic fatty acids (such as AA) and

enzymatically oxygenate them into hydroperoxy-polyenoic fatty acids (Schewe, 2002).

Zuo et al. (Zuo et al., 2004) found immunohistochemical evidence of both 5-LOX and

12-LOX in diaphragm cells, staining predominantly along the sarcolemma of smaller, presumably oxidative fibers. There are a variety of products made from lipoxygenases and associated downstream enzymes, including the leukotrienes and HETEs, and these

are known to have a multitude of functions in immune responses, inflammation and

control of smooth muscle contraction. However, the roles of LOX in normal membrane

remodeling, breakdown of subcellular organelles and cell differentiation may be of equal

57

or greater importance in many cells (Kuhn & Borchert, 2002). Blocking global LOX

activity, even at normal temperatures, greatly decreases extracellular ROS production and

has no effect on intracellular superoxide formation in diaphragm muscle in heat exposure

(Zuo et al., 2004). Recent evidence suggests that HETEs, leukotrienes, AA and other

eicosanoid products can directly bind and interact with numerous intracellular proteins,

including cytoskeleton components associated with the contractile machinery (Brock,

2008). Such lipid-protein interactions are believed to be important in normal regulatory

processes of the cell such as water balance across the cell membrane (Ortenblad et al.,

2003) and GLUT4 translocation to the muscle cell membrane (Vahsen et al., 2006).

No mechanisms have been described that could fully account for the effects of

LOX and/or COX inhibition on contractile function during hyperthermia. However

several mechanisms are of potential significance. One working hypothesis is that

inhibition of any important AA metabolic pathway could allow AA to accumulate in the

cell to toxic levels during hyperthermia. Temperatures of 42-45° C are known to cause

rapid increases in AA release and prostaglandin synthesis in cell systems (Calderwood et

al., 1989) and AA alone has been shown to stimulate oxidant formation (Esenabhalu et

al., 2003), to activate calcium channels (Mignen & Shuttleworth, 2000), to disrupt the

sarcolemmal Ca2+-ATPase (Cardoso & De Meis, 1993) and to activate the mitochondrial

transition pore (Qian et al., 2004; Di Paola et al., 2006). The sarcolemmal Ca2+-ATPase denaturation temperature has been found to shift according to the concentration of free fatty acids in the cytosol (Mignen & Shuttleworth, 2000). Therefore, by these mechanisms blockage of any pathway that allows AA to accumulate in the cell could potentially result in cell dysfunction. A second, related idea is that LOX and COX

58

inhibitors can sometimes act as pro-oxidants. This is a controversial area because most of the pharmacological agents used in this study have been shown to be chemical antioxidants by this laboratory (Zuo et al., 2004) and others, e.g. baicalein (Piao et al.,

2008), ETYA (Takami et al., 2000), indomethacin (Petersen et al., 2008) and ibuprophen

(Costa et al., 2006). Nevertheless, there is abundant evidence, particularly in intact tissues or cells, that any of these agents can also act as pro-oxidants under certain conditions, e.g. baicalein (Woo et al., 2005), ETYA (Anderson et al., 1994), indomethacin (Basivireddy et al., 2005) and ibuprophen (McAnulty et al., 2007). Since many in vitro chemical studies have ascribed these agents as having antioxidant activities, it could be that frequent observations of oxidant reactions in more intact preparations is secondary to the oxidative effects of accumulating AA. With this in mind, it is possible that application of any eicosanoid could induce an AA- mediated oxidant stress superimposed over any damage caused by hyperthermia alone.

To test the possibility that AA accumulation could be responsible, we

attempted to reduce AA formation during 43 ºC heat stress by inhibiting PLA2 with the calcium independent PLA2 (specific iPLA2) inhibitor, BEL, as well as by a general

cPLA2 and iPLA2 inhibitor, AACOCF3. Though not studied extensively, isoforms of iPLA2 are highly expressed in skeletal muscle cell lines (Gong et al., 2006) with

+2 negligible Ca dependent PLA2 being expressed. The specificity of BEL has been found

to be 1,000-fold greater for iPLA2 compared to the cPLA2 (Hazen & Gross, 1992) and

has been used extensively in muscle (Nethery et al., 1999; Guo et al., 2003). AACOCF3

has been reported to inhibit both cPLA2 and iPLA2 at the concentrations used

(Ackermann et al., 1995). However, contrary to our hypothesis, neither BEL nor

59

AACOCF3 administration protected muscle function in 43 ºC hyperthermia. The highest

concentration of BEL contributed to a further decline in function at 43 ºC, though this

could have been due, in part, to a pharmacological effect that showed up even at 37 ºC

control experiments (Fig 2.8). The results for PLA2 inhibition by BEL and AACOCF3 do

not necessarily disprove the hypothesis that AA accumulation is responsible for the toxic

effects of LOX and COX inhibition. However, it lends some credence to an alternative

hypothesis that LOX and COX activity or products from their activity are somehow

necessary for normal responses to hyperthermia.

Finally, it is possible that the effects of each of the eicosanoid enzyme inhibitors

may represent non-specific toxic effects that are minimally evident at normal

temperatures but are accentuated at≥ 42 ºC. The extreme effects at 43 ºC and the apparent added insult on muscle membrane integrity (Figure 2.10) could represent an additive effect from the toxicity of the drugs at high temperature and direct effects of extreme heat. They may therefore be unrelated phenomena. The only circumstantial evidence that would counter this argument is that the same basic observation was seen with a variety of different drugs at a variety of doses, which in some cases are within the therapeutic range in humans. It would seem unlikely that we would observe such a uniform toxicity unless it was directly related to the normal physiology of hyperthermia.

2.4.5. Conclusions

These results support the concept that skeletal muscles have an inherent

thermotolerance within the range of temperatures they are likely to experience during

intense exercise. However, during extreme conditions, where the body cannot adequately

maintain thermal equilibrium, they are susceptible to loss of function. Furthermore, our

60

results concur with those of van der Poel and Stephenson (van der Poel & Stephenson,

2002) in that there may be a unique phenotypic response of oxidative skeletal muscles

that is not true of more fast fibers, i.e. their loss of function may not be related to oxidant

production and may not be rapidly reversible with time. Finally, we have demonstrated

that, at least in oxidative diaphragm muscles, normal thermotolerance is dependent on an

intact network of eicosanoid metabolic machinery.

2.4.6. Perspectives and Significance

We hypothesize that thermotolerance may be another important function of

eicosanoid enzyme activity that has largely gone unrecognized. Hyperthermia can affect

all cells and essentially all integrative systems within the body and comprises one of the

most primordial environmental stresses driving evolutionary diversity. Though we

studied one tissue in isolation in this study, we speculate that eicosanoid metabolism may

play a large role in thermal adaptation in many other tissues, beyond its better-known influences on local vascular regulation and as a mediator of inflammatory cascades. It is also possible that alterations in gene expression of eicosanoid enzymes, secondary to disease, chronic inflammation or drug treatment could make tissues more or less vulnerable to heat injury. Future studies are needed to discover the extent of

involvement of eicosanoid metabolism in regulation of normal skeletal muscle function,

the hyperthermic tolerance of other tissues of the body, and in the overall integrative

responses to exertional hyperthermia and environmental heat exposure. Discovering

links between eicosanoid metabolism and hyperthermia could create new therapies and

treatment paradigms for heat related illnesses that have the potential of improving

61 thermal tolerance in athletes, soldiers, first-responders and other vulnerable populations such as the elderly and very young.

62

2.5. Tables

Drugs Target Reference a BEL (1, 10 and 20µM, DMSO ) iPhospholipase A2 28

AACOCF3 (40µM, DMSO 20.4mM) cPhospholipase A2 42

BPB (30µM, 5.8mM) sPhospholipase A2 60 Diethylcarbamazine (50µM, DMSO 5.5mM) 5-Lipoxygenase 65 Zileuton (50µM, DMSO 8.3mM) 5-Lipoxygenase 14 Baicalein (50, 100 and 200µM, DMSOb) General Lipoxygenase 16 ETYA (50, 100 and 200µM, DMSOc) General Lipoxygenase 65 NDGA (50µM, DMSO 4.3mM) General Lipoxygenase 65 Indomethacin (50, 100 and 200µM, DMSOd) General Cyclooxygenase 65 Ibuprofen (200, 400 and 800µM, DMSOe) General Cyclooxygenase 9 Tiron (5, 10 and 20mM, Buffer) ROS scavenger 57,61 Trolox (50µM, 100µM and 1mM, Buffer) ROS scavenger 7,64 DTT (5 and 20mM, Buffer) Disulfide linkages 23,37,57 Table 2.1. Drugs used in study with corresponding targets

Abbreviations: Bromoenol lactone (BEL), arachidonyltrifluoromethyl ketone

(AACOCF3), 4-bromophenacyl bromide (BPB), eicosatetreanoic acid (ETYA), nordihydroguaretic acid (NDGA) and dithiothreitol (DTT).

DMSO conc. Range for drug delivery:

a. Range: 0.4-9 mM, Control = 9 mM b. Range: 9.5-38 mM, Control = 38 mM c. Range: 7-28 mM, Control = 28 mM d. Range: 5.1-20 mM, Control = 20 mM e. Range: 4.9-19 mM, Control = 19 mM

63

2.6. Figures

Figure 2.1. Muscle mounting and tissue bath setup.

64

Figure 2.2. A: Force frequency relationships obtained at the end of 30 min exposure to

37° C (n=6), 40° C (n=9), 41° C (n=9), 42° C (n=8) and 43° C (n=8). Force is expressed as a % of pre-heat, pre treatment, tetanic force at 150 Hz. Peak twitch force is represented as zero Hz. Temperature induced increases in passive tension are also shown

(inset). B: Force frequency relationships obtained after 30 min. heat exposure, followed by 30 min. of recovery at 37° C, ‡‡ P < 0.001 vs. 37° C.

65

Figure 2.3. Change in %Max Force (150Hz) of diaphragm strips exposed to 43 ºC with antioxidants. Trolox (n=6) and Tiron (n=6) data are after 30 min. at 43° C. Dose responses are shown with time-matched controls. *P < 0.05 vs. Control 43ºC.

66

Figure 2.4. Change in %Max Force (150Hz) of diaphragm strips exposed to 43 ºC with dithiothreitol (DTT). DTT (n=8) data are after 30 min. of recovery at 37° C (n=8), shown with control recovery after 43 ºC and 5 mM DTT 37 ºC control. No statistical significance was seen with DTT exposure vs. control recovery following 43 ºC.

67

Figure 2.5. Change in %Max Force (150 Hz) of tissues exposed to specific LOX inhibitors for 30 min. at 43° C (n=3). NDGA (n=4) a caused significant reduction in force but specific 5-LOX inhibition did not. *P < 0.05 vs. control.

68

Figure 2.6. Change in %Max Force (150 Hz) of tissues exposed to 42 ºC with LOX or

COX inhibitor treatment. A: General LOX inhibitors, Baicalein (n=4) and ETYA (n=4)

reduced force in a dose dependent manner. B: General COX inhibitors, indomethacin

(n=4) and ibuprofen (n=4) decreased function in a dose dependent manner. C: Passive tension increased in LOX inhibited tissues. D: Passive tension increased in COX inhibited tissues, *P < 0.05, **P < 0.01, ‡P < 0.005, ‡‡P < 0.001 vs. controls.

69

Figure 2.7. Change in %Max Force (150 Hz) of tissues exposed to a combination of

LOX (100 µM) and COX (100 µM) inhibitors. The combination of baicalein (B) and indomethacin (I) caused a reduction in force production compared to control tissues.

This reduction was greater than with individual drugs alone. **P < 0.01 ‡‡P < 0.001 vs. control 37° C (n=4).

70

Figure 2.8. Change in %max Force (150 Hz) of tissues exposed to 43 ºC and PLA2

inhibition. A: iPLA2 inhibition by BEL. BEL (20 μM) showed a significant reduction in force compared to control, whereas 1 μM and 10 μM had no effect. B: cPLA2 and iPLA2 inhibition by AACOCF3. AACOCF3 caused no change in the loss of force

production at 43 ºC ‡ P < 0.005 vs. control (n=6), @ P < 0.05 vs. 37 ºC control.

71

Figure 2.9. Change in %Max Force (150 Hz) of tissues exposed to BPB (sPLA2 inhibitor) for 30 min. at 43 ºC (n=4). BPB caused a significant reduction in contractile function at 43 ºC but it also reduced contractile function at 37 ºC, showing a possible nonspecific pharmacological response. ** P< 0.01, @ P < 0.05 vs. 37 ºC control.

72

Figure 2.10. Membrane permeability evaluated using procion orange. Baicalein and indomethacin addition at 43° C caused a significant increase in the number of procion orange positive cells. Procion orange diaphragm tissue: (A) Control 37° C, (B) Control

43° C and (C) Baicalein and indomethacin 43° C, ‡‡ P < 0.001 vs. control (n=6).

73

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Chapter 3: Protection of hyperthermia-induced intestinal permeability in the mouse

3.1. Introduction

Hyperthermia induced intestinal permeability has been shown to occur during moderate to extreme exercise (Pals et al., 1997; Smetanka et al., 1999; Davis et al., 2005;

Lambert, 2009) and heat stress (Shapiro et al., 1986; Lambert et al., 2002a; Prosser et al.,

2004; Singleton & Wischmeyer, 2006), which can allow endotoxin from the intestinal lumen to access the blood and lymph systems (Brock-Utne et al., 1988). Increased endotoxin in the blood can lead to multiple organ failure, shock and death (Bouchama &

Knochel, 2002). The amount of enhanced intestinal permeability due to exercise can vary according to duration and intensity, strenuousness, fluid consumption and ingestion of common pharmaceuticals (Lambert, 2009). The reason for the breakdown of the intestinal barrier during exercise and heat stroke is believed to be a result of a combination of both heat exposure and gut ischemia, the latter caused by diversion of blood flow to the skin for heat dissipation (Rowell, 1974; Kregel et al., 1988; Sakurada &

Hales, 1998). However, heat exposure alone can cause an increase in permeability.

Lambert et al. (Lambert et al., 2002a) showed that small intestine permeability in rats increases due to hyperthermia at 42.5° C in vivo and 41.5-42° C in vitro. This phenomenon has not previously been defined in the mouse but is important because of the recent development of reproducible mouse models of heat stroke (Leon et al., 2005)

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and the effective application of transgenic animals to unravel the molecular mechanisms

underlying heat susceptibility (Leon, 2006). Therefore, one of the objectives of this study

was to develop a relevant model to test in vitro mouse intestinal permeability during a

time period relevant to exhaustive exercise. This model will allow investigators to

rapidly evaluate alternative mechanisms underlying the response of the intestine to

hyperthermia and to test potential pharmaceutical treatments that might be useful in

future human studies designed to prevent or delay the consequences of heat shock.

There has been considerable concern regarding the facilitory role non-steroidal

anti-inflammatory drugs (NSAIDS) might have on intestinal permeability during

hyperthermia, particularly during exercise. Athletes who ingest either aspirin or

ibuprofen before vigorous prolonged exercise, have been shown to develop greater GI

permeability than in exercise alone (Smetanka et al., 1999; Lambert et al., 2007).

Interestingly, another study found that both ibuprofen and indomethacin, both

cyclooxygenase inhibitors, cause an increase in intestinal permeability in human subjects

who are not undergoing exercise or heat stress (Bjarnason et al., 1986). Inhibition of

other eicosanoid enzyme systems also has the potential for influencing tissue responses to

hyperthermia. For example, baicalein, a general lipoxygenase (LOX) inhibitor, increased

the survival time of rats undergoing whole body hyperthermia (Chang et al., 2007). In

previous studies from our laboratory, COX and LOX inhibition by a variety of

pharmacological agents reduced the thermotolerance of in vitro diaphragm muscle

exposed to 42° C (Oliver et al., 2008)(Chapter 2). However, other than the studies on

COX inhibition, there has been a paucity of information regarding the influence of other eicosanoid enzyme pathways on intestinal permeability (i.e. lipoxygenase (LOX),

82 phospholipase A2 (PLA2) and cytochrome P450 (CYP450)). Therefore, the second objective of this study was to evaluate the effects of inhibition of a variety of eicosanoid metabolic pathways on hyperthermia-induced intestinal permeability.

Both intestinal ischemia and hyperthermia have been found to cause an increase in reactive oxygen species (ROS) formation in the intestine (Hall et al., 1994; Hall et al.,

2001). It is possible that this is a general tissue response to hyperthermia, as ROS have been shown to increase during hyperthermia in skeletal muscle (Zuo et al., 2000) as well as many other tissues (Wallen et al., 1997; Flanagan et al., 1998). Whether ROS are the primary mediators of tissue dysfunction or damage is poorly understood. Previously, we were unable to reverse the harmful effects of hyperthermia in oxidative skeletal muscle by scavenging ROS with tiron and Trolox (Oliver et al., 2008)(Chapter 2), yet other investigators studying fast non-oxidative muscle could completely protect function with the same mediators (van der Poel & Stephenson, 2002). In the gut, inhibition of xanthine oxidase, a known ROS producer, by allopurinol reduces the concentration of portal LPS in rats with a core temperature of 41.5° C (Hall et al., 2001). Also, Rao et al.

(Rao et al., 1997) demonstrated that when intestinal epithelial cells (Caco-2 and T84 cell monolayers) were exposed to hydrogen peroxide, permeability was enhanced, suggesting that ROS production could lead to barrier dysfunction. Yet, others have shown that rat intestinal permeability was unaffected by treatment with antioxidants during hyperthermia (Lambert et al., 2002a). Therefore, the role ROS plays in hyperthermia- induced permeability is poorly understood and no data currently exists in the mouse intestinal model. Therefore, the third objective of this study was to test the hypothesis

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that increased reactive oxygen species production during hyperthermia is responsible for

the increase in intestinal permeability.

Though it is clear that numerous studies have concluded that hyperthermia causes

an increase in intestinal permeability, few have discovered ways of preventing or

possibly even reversing the deleterious effects. Therefore, another objective of this

research was to build the groundwork for development of nutritional or pharmaceutical

agents that could be utilized topically on the gut lumen to possibly prevent or attenuate

damage during exposure to elevated temperatures. Development of such therapeutic or

preventative agents could potentially have a major impact on the medical treatment of

athletes, soldiers and others exposed to extreme heat.

In summary, the purposes of this study were 1) to determine the thermal tolerance

of mouse intestinal tissue using an in vitro model of gut permeability, 2) to evaluate the

possible protective or detrimental effects of inhibition of various eicosanoid metabolic

pathways on hyperthermia-induced intestinal permeability and 3) to test the hypothesis

that quenching ROS during hyperthermia, using known and contrastingly different

scavengers tiron, Trolox and N-acetyl cysteine, could protect the intestine from loss of barrier function.

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3.2. Methods

3.2.1. Chemicals and drugs used. Medium 199 (Cellgro), L-Glutamine (Lonza), sodium bicarbonate (Acros Organics), formaldehyde, fluorescein isothiocyanate (FITC)-dextran

4kD [FD4, Sigma Aldrich], Eicosatetreanoic acid [ETYA, general LOX inhibitor,

Cayman Chemical], Baicalein [12-LOX inhibitor and partial general LOX inhibitor,

Sigma Aldrich], ibuprofen [cyclooxygenase inhibitor, Sigma Aldrich], indomethacin

[cyclooxygenase inhibitor, Sigma Aldrich], tiron [superoxide scavenger, Sigma Aldrich], trolox [vitamin E analogue general antioxidant, Sigma Aldrich], N-acetyl cysteine [NAC, increases the pool of free radical scavengers, Sigma Aldrich], 1-aminobenzo triazole

[ABT, general CYP450 inhibitor, Sigma Aldrich] (Strelevitz et al., 2006) and N- hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine [HET0016, selectively inhibits

CYP4A and 4F isoforms as well as CYP2C9, CYP2D6, and CYP3A4, Cayman

Chemical] (Wang et al., 2007).

3.2.2. Animal treatment protocol and gut sac preparation. Adult C57bl6 mice (25-35 g) were used for this study. Animal care and treatment were performed using protocols approved by the University of Florida Institutional Animal Care and Use Committee.

Food was withdrawn from the mice approximately 10 hours prior to sample collection to reduce intestinal contents. The mice were euthanized by carbon dioxide asphyxiation and the entire small intestine was rapidly excised and placed in pre-oxygenated medium

199 (with Glutamine and sodium bicarbonate without phenol red). The protocol for measuring permeability using everted gut sacs was modified from a previously developed procedure in the rat (Lambert et al., 2002a). Briefly, the intestinal lumen was cleaned with oxygenated medium 199 by gently pushing it through the luminal cavity until the

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intestinal contents were cleared. Following the cleaning, a glass rod was gently placed within the luminal cavity and the gut was everted over the rod, taking care to not cause damage. The gut was then filled with oxygenated medium and tied off in sections approximately 1-2 cm long with 2-0 suture. Depending on the experiment, antioxidants or inhibitors were added into the gut sac filling solution for loading of the basal side of the epithelial cells. Gut sacs were then placed into cuvettes containing oxygenated buffer, with or without antioxidants or inhibitors, and including 4kD fluorescein isothiocyanate (FITC)-dextran (0.3 mM) for 15, 30, 60 or 90 min. After gut sac preparation, cuvettes were placed in either a water bath maintained at 37° C or an aluminum heating block heated to the appropriate temperature (40.5, 41.5 or 42.5° C).

The temperature was monitored with a highly accurate thermistor, accurate to two digits

(Yellow Springs Model 4610). Heating was performed by a self regulating temperature controller (Digi Sense, Cole Parmer) and a heating plate driven by the controller. The volume of the solution inside the gut sacs (100-300 µL) and the square area were determined at the conclusion of the experiment. Fluorescence of the serosal fluid was then measured using a spectrofluorometer (SpectraMax M5, Molecular Devices) and the concentration of FITC-dextran was determined by comparison to a standard curve.

3.2.3. Histology. After the experimental protocol was finished, some tissues were

assigned to histological assessment and placed in 4 % formaldehyde for slicing and

staining with hematoxylin and eosin.

3.2.4. Data analysis and Statistics. Data were represented as the transport of nmoles of

FITC-dextran/cm2 (i.e. normalized to intestinal surface area of the gut sac). The

analytical method used for this calculation was taken from Lambert et al. (Lambert et al.,

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2002a) who denoted permeability = (Concentrationserosal fluid X Volumeserosal fluid) ÷

Mucosal surface area.

The effects of “animal, i.e. specific experiment on matched tissues” were treated as a

random variable resulting in the equivalent of a repeated measures design. Post hoc

analyses (Dunnett’s) were done to determine specific effects of treatment and temperature from control measurements. Mean contrasts after ANOVA were used in some experiments to compare sample means in complex designs (SASJMP statistical package). All results are reported as means ±SE; P <0.05 was considered to be statistically significant.

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3.3. Results

3.3.1. Thermal tolerance of mouse small intestine permeability. Compared with 37° C

control measurements (2.58 nmoles FITC/cm2 ± 0.99), intestinal permeability was significantly increased (P < 0.02) after exposure to 41.5° C (9.87 nmoles FITC/cm2 ± 1.0)

for 90 min. However, exposure to 40.5° C (2.2 nmoles FITC/cm2 ± 0.2) showed no

increase in permeability compared to 37° C (Figure 3.1). Histological assessment of

tissues exposed to 41.5° C for 90 min showed an overall loss of organization of the

epithelial lining (Figure 3.2). In a separate set of experiments, tissues were exposed to

41.5° C over a 90 min. period and sampled at 4 points, 15 min (0.52 nmoles FITC/cm2 ±

0.2), 30 min (0.92 nmoles FITC/cm2 ± 0.23), 60 min (4.51 nmoles FITC/cm2 ± 1.0) and

90 min (8.17 nmoles FITC/cm2 ± 1.2), to discover the time course of susceptibility to

hyperthermia (41.5° C). The intestine was found to become susceptible to hyperthermia between 30 and 60 minutes at 41.5° C heat exposure (P < 0.002), but 15 and 30 min of

41.5° C was insufficient to increase permeability (Figure 3.3).

3.3.2. Effects of cyclooxygenase inhibition on thermal tolerance of intestinal gut sacs.

Figure 3.4 shows intestinal permeability of tissues treated with COX inhibitors ibuprofen

(800 µM, 8.56 nmoles FITC/cm2 ± 1.13) and indomethacin (200 µM, 5.07 nmoles

FITC/cm2 ± 0.43), during hyperthermia (41.5° C, 6.83 nmoles FITC/cm2 ± 0.37).

Ibuprofen was also given at 400 µM (8.01 nmoles FITC/cm2 ± 0.79) to evaluate if dosage

was a factor. Indomethacin, but not ibuprofen, partially protected the intestine from hyperthermia (5.07 nmoles FITC/cm2 ± 0.43) compared to 41.5° C controls (P < 0.004).

Ibuprofen 800 µM showed a significant increase in permeability during hyperthermia

(P<0.02). Ibuprofen (800 µM) was found to be significantly different compared to

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indomethacin treatment (P<0.001). The doses used for ibuprofen and indomethacin were

taken from our previous study in rodent tissue (Oliver et al., 2008).

3.3.3. Effects of lipoxygenase inhibition on hyperthermia induced intestinal permeability.

LOX inhibitors baicalein (200 µM) and ETYA (500 µM) were given to tissues during

exposure to hyperthermia and intestinal permeability was measured (Figure 3.5).

Baicalein (5.14 nmoles FITC/cm2 ± 0.48) but not ETYA (9.18 nmoles FITC/cm2 ± 2.03) was found to protect the mouse intestine from an increase in permeability (P < 0.02) due to hyperthermia compared to 41.5° C control tissues (9.25 nmoles FITC/cm2 ± 1.26 and

7.5 nmoles FITC/cm2 ± 1.65, respectively). Interestingly, baicalein was also found to not

be significantly different compared to 37° C controls showing a strong protective effect.

Baicalein treated tissues were also found to be significantly different compared to 500

µM ETYA (P<0.02). ETYA was also given at 200 µM (7.16 nmoles FITC/cm2 ± 0.89) to

evaluate if dosage was a factor. Also, Baicalein, given during 42.5°C (8.24 nmoles

FITC/cm2 ± 0.78), was found to cause a significant decrease (P < 0.005) in hyperthermia

induced permeability compared to 42.5° C controls (12.45 nmoles FITC/cm2 ± 0.91),

similar to that seen at 41.5° C (data not shown), P < 0.001.

3.3.4. Effects of cytochrome P450 inhibition on hyperthermia induced intestinal

permeability. Cytochrome P450 inhibitors (ABT and HET0016) were tested as a

possible third pathway affecting eicosanoid metabolism (CYP450) (Figure 3.6). ABT

(200 µM, 7.9 nmoles FITC/cm2 ± 1.43) and HET0016 (1 µM, 9.28 nmoles FITC/cm2 ±

0.85) showed no significant difference compared to 41.5° C control (8.07 nmoles

FITC/cm2 ± 0.56).

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3.3.5. Effects of antioxidants on hyperthermia induced intestinal permeability. To test the

hypothesis that the increase in permeability during hyperthermia is not due to increased

oxidant production, tissues were treated during hyperthermia with antioxidants tiron (10

mM), Trolox (100 µM) and NAC (10 mM) (Figure 3.7). Tiron and Trolox doses were taken from our previous study (Oliver et al., 2008). In order to expose the tissue to a variety of different antioxidants NAC was used at a dose previously used by Cuzzocrea et al. (Cuzzocrea et al., 2000). Tiron (7.95 nmoles FITC/cm2 ± 0.47) and Trolox (11.82

nmoles FITC/cm2 ± 1.64) treatment showed no protection against hyperthermia induced

intestinal permeability compared to 41.5° C controls (8.38 nmoles FITC/cm2 ± 0.9).

However, Trolox did cause a significant increase (P<0.005) in permeability compared to

control 41.5° C tissues. However, NAC treatment showed a significant protection (4.57

nmoles FITC/cm2 ± 0.38) during heat exposure (P < 0.005).

Dimethyl sulfoxide (DMSO) was the vehicle in several experiments and was kept to 0.33

% of the total volume or lower. Since DMSO can work as an antioxidant as well,

experiments were performed at this dose with DMSO alone and yielded no significant

increase or decrease in permeability at 37° C or 41.5° C (4.09 nmoles FITC/cm2 ± 1.1 and

8.86 nmoles FITC/cm2 ± 1.22, respectively, n=4) compared to 37° C (2.28 nmoles

FITC/cm2 ± 0.45, n=4) or 41.5° C (7.78 nmoles FITC/cm2 ± 0.69, n=4) controls (Data not

shown).

90

3.4. Discussion

The results demonstrate that mouse intestine has an inherent thermal tolerance

over 90 min to temperatures of 40.5° C, but permeability rises rapidly when exposed to

temperatures of 41.5° C and above. Exposure to 41.5° C for 30 min yielded no

significant increase in permeability, only at 60 and 90 min of 41.5° C exposure was an

increase in permeability seen. Histological assessment showed a marked increase in

damage to the overall structure of the intestine when exposed to 41.5° C for 90 min.

Though the mechanism for the increase in intestinal permeability during hyperthermia is

not completely understood, we show that the introduction of a few pharmacological

agents already given to humans in other medical conditions can protect the mouse

intestine from hyperthermia induced permeability. However, the mechanism of

protection by these pharmacological agents is still unknown.

3.4.1. Critique of Approach

The everted gut sac model has been used in several studies (Barthe et al., 1998;

Lambert et al., 2002a; Higuchi et al., 2008), including Lambert et al. (Lambert et al.,

2002a) who evaluated the stability of the preparation. The authors discovered that lactate

dehydrogenase (LDH), a marker for cytotoxicity, stayed relatively low up to 60min after

gut sac preparation but significantly increased after 90 min (Lambert et al., 2002a).

Because of this increase at 90 min. we performed control experiments at 37° C for every experimental group as well as performing time controls for 37° C and 41.5° C. Also, drugs with divergent inhibitory mechanisms were used to test if pharmacological effects

91 were present. A drawback of this preparation is that, physiologically, an increase in core body temperature seen by the intestine is accompanied by ischemia, where as in our system hyperthermia was studied alone. However, Lambert et al (Lambert et al., 2002a) found that in vivo and in vitro permeability due to hyperthermia compared closely; because of these findings I felt comfortable in only using the in vitro gut sac model to evaluate the objectives of this study.

3.4.2. Possible mechanisms for the increase in permeability during hyperthermia

Distinguishing potential underlying mechanisms for loss of intestinal barrier function is important because it could help focus on potential therapeutic targets and approaches, as well as help in understanding why some agents seem to be protective while others are not. Unfortunately, our understanding of the causes of permeability loss in hyperthermia is incomplete. Hall et al. have shown that hypoxia occurs in the intestine of heat stressed animals with a subsequent increase in ROS and reactive nitrogen species formation (Hall et al., 1994; Hall et al., 1999; Hall et al., 2001). Furthermore, these investigators have implicated ROS in the rise of portal LPS concentrations due to the protective effect of xanthine oxidase antagonists (known to lower ROS production).

How ROS are being produced and how they are eliciting their effects is still unknown but studies have suggested that ischemia/reperfusion (I-R) is occurring in the intestine during hyperthermia due to the constriction of the superior mesenteric artery and the subsequent relaxation of this artery once a core temperature of 41.5° C is achieved (Kregel et al.,

1988). I-R has been studied extensively and has been found to cause an increase in

92 permeability. However, I-R is not occurring in our current in vitro model, as the intestinal segments remain well oxygenated throughout the protocol. Antioxidants have been found to partially protect intestinal permeability after I-R in the rat (Sun et al.,

2002). However, Lambert et al. (Lambert et al., 2002a) studied the permeability response of rat small intestine to hyperthermia (Tempol and Ebselen, antioxidant treatment) and suggested that ROS and RNS do not play a role in the response of rat everted gut sacs to heat stress. Interestingly, two of the antioxidants given in this study (tiron and trolox) showed a response similar to that of Lambert et al. (Lambert et al., 2002a) and did not protect the mouse small intestine from increases in permeability, while NAC treatment did protect the intestine from increases in permeability. The antioxidants used by

Lambert et al. (Lambert et al., 2002a) and those used in this study have similar properties. Tempol and tiron both reduce superoxide levels. Tempol is a superoxide dismutase mimic (Krishna et al., 1996) and tiron directly scavenges superoxide (Ledenev et al., 1986) and may have dismutase properties (Ledenev et al., 1986). Trolox is a water soluble vitamin E analogue and a general antioxidant (Betters et al., 2004; Young et al.,

2004). Unlike some antioxidants, Trolox does not alter leukotriene production (Young et al., 2004). Ebselen and NAC both increase the availability of glutathione to scavenge oxidants; ebselen is a glutathione peroxidase mimic and NAC increases the available pool of GSH (Moldeus & Cotgreave, 1994) and may have other antioxidant properties

(Moldeus & Cotgreave, 1994). However, the mechanism of NAC’s antioxidant properties has been debated due to speculation of alternative pathways of oxidant reduction. These findings suggest that ruling out ROS as a mechanism for increased

93 intestinal permeability during hyperthermia may be premature, unless NAC is eliciting a secondary response other than ROS scavenging.

The structural barrier of the intestine is achieved by enterocyte membranes, protein-protein interactions between adjacent enterocytes forming tight junctions as well as mucus and the presence of immune cells (Lambert, 2009). Dysfunction of the intestinal barrier can lead to an increase in permeability due to a breakdown of the barrier function of the enterocyte monolayer of the intestine. Transport of molecules from the lumen to the blood stream can occur in two different ways. First, molecules can be transported through the enterocytes’ transcellular pathways i.e. by receptor mediated or transporter mechanisms (Baumgart & Dignass, 2002). Second, molecules can travel between enterocytes when tight junctions are opened; this pathway is called the

‘paracellular pathway’ (Baumgart & Dignass, 2002). Molecules that have a relatively small molecular weight are usually the only ones that are transported through the paracellular pathway. The opening of these tight junctions is a highly regulated system which requires cell signaling and stimulation of the actin-myosin ring which provides the cellular structure for the tight junctional proteins (Turner, 2000). Non-specific opening of the tight junctions is one route believed to occur during hyperthermia. However, it is still undetermined if opening of tight junctions by itself can account for the large increase in permeability during hyperthermia.

A third mechanism through which permeability can occur is via damage to the enterocytes, leading to the production of holes in the barrier. This route seems to be the more plausible, since histology studies (Figure 3.2 as well as Lambert et al’s (Lambert et al., 2002a) findings) show a marked increase in damage to the overall structure of the

94

epithelium lining the lumen. Damage to the mucosa would lead to a decrease in barrier function and direct access of the luminal contents to the blood and lymph system.

Interestingly, Lambert et al. (Lambert et al., 2002a) found that severe membrane damage to the intestine occurred during in vivo heat stress but tight junctions remained closed, possibly due to the fixing protocol of the tissues. Consequently, in the parameters of this study, we are unable to fully distinguish between the above mechanisms during hyperthermia, but our histological data combined with that of Lambert et al.’s suggests damage is the predominant mechanism.

If cell damage is the predominant phenomenon occurring to the intestinal lining then a variety of injury mechanisms could be responsible, including mitochondrial dysfunction leading to a decrease in ATP production (Christiansen & Kvamme, 1969;

Gwozdz et al., 1978), mobilization of Ca+2 stores resulting in increased protease activity

(Chakrabarti & McClane, 2005; Sergeev, 2009), or release of the epithelial contacts from

their basal lamina or basement membrane (Lambert et al., 2002a).

3.4.3. LOX, COX and CYP450 inhibition during heat stress

Previously, arachidonic acid metabolism has been found to be important for

muscle function during hyperthermia; but what role, if any, arachidonic acid plays in the

mouse intestine during heat stress is unknown. It is known, for example that in isolated

cells, hyperthermia results in massive release of arachidonic acid (Calderwood et al.,

1989), which can be toxic to all cells. Arachidonic acid is metabolized by the eicosanoid

family of enzymes which compete for substrate (Robinson et al., 1986). COX-1 and

95

COX-2 are both present in the intestine, though COX-2 can be induced whereas COX-1 is constitutively expressed. The main products produced by COX-1 and -2 are prostaglandins (PGs) (Petruzzelli et al., 2007; Ham & Kaunitz, 2008). COX inhibition in the intestine is of clinical importance because of the widespread use of COX inhibitors as categories of NSAIDs for pain and arthritis relief. Chronic use of NSAIDs has been found to cause mucosal injury (Petruzzelli et al., 2007; Ham & Kaunitz, 2008) and an increase in permeability during normothermic conditions. Ibuprofen, indomethacin and aspirin have all been found to increase intestinal permeability (Bjarnason et al., 1986;

Lambert et al., 2007). COX inhibition has been discussed in the literature, which shows a correlation between ibuprofen (and other NSAIDs) ingestion before exercise and increased intestinal permeability in athletes (Lambert et al., 2007). Most of the present studies discussing NSAIDs and the intestine have shown a deleterious effect and there is increasing concern of overuse. However, in contrast to other studies, our current results showed that administration of indomethacin caused a small but significant protection against hyperthermia induced permeability. This suggests that use of indomethacin during heat stress or exercise could lower the risk of intestinal permeability. However our findings do not show ibuprofen as having a similar response, leading us to believe that a secondary pharmacological mechanism for indomethacin protection (other than

COX inhibition) could be present.

LOXs are enzymes responsible for the transformation of arachidonic acid to leukotrienes and other eicosanoid products (such as HETEs). Leukotrienes and HETEs stimulate the inflammatory response by attracting immune cells to the site of injury as well as activating and regulating aspects of bronchoconstriction (Sharma &

96

Mohammed, 2006). Lipoxygenases also play a role in maintenance of membranes throughout the cell (Kuhn et al., 2002). Various isoforms of LOX have been found throughout the body and, although some forms are critical in normal function, many play predominant roles in disorders such as asthma (5-LOX) and inflammatory bowel disease

(12/15-LOX) (Sharma & Mohammed, 2006). Therefore, inhibiting leukotriene

production has been found to be a valuable therapeutic approach to controlling the

negative effects seen during several disorders.

A few studies have examined the effects of inhibiting LOX during hyperthermia;

in particular, Chang et al. (Chang et al., 2007) found that baicalein caused a significant

increase in animal survival time when exposed to whole body hyperthermia. This

increase in survival time was attributed to the animals having a lower core temperature

during a given environmental heat stress compared to animals without baicalein treatment

suggesting a highly integrative influence of the drug (Chang et al., 2007). In addition, we

have previously demonstrated that LOX metabolism is important during hyperthermia in

skeletal muscle to protect against loss of contractile function (Oliver et al., 2008)(Chapter

2). Also, LOX inhibition has been found to protect intestinal permeability caused by

experimental colitis (Mazzon et al., 2006), inflammatory bowel disease (Shannon et al.,

1993) and butyrate stimulation (Ohata et al., 2005). Butyrate causes an increase in LOX

mRNA and HETE production in Caco-2 cells; this finding suggests that LOX activation

is an important step in tight junction alterations (Ohata et al., 2005). Our results are

complimentary to the above studies. However, the absence of comparative responses to

another LOX inhibitor over a dose range (e.g. ETYA) leads us to believe that some other,

less specific mechanism might be responsible for the effects of baicalein. A reduction in

97

LOX products might be a logical mechanism for other responses such as inflammatory bowel disease but hyperthermia is a more acute disorder. The sequence of events leading up to an increase in permeability is more likely not fully explained through leukotriene production alone.

Cytochrome P450 enzymes are present throughout the body and have been found in high concentration in the intestine (Paine et al., 2006). Cytochrome 450s produce epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids

(monoHETEs), which have been found to be involved in drug metabolism (Watkins,

1992), control of vasoregulation (Harder et al., 1995) and anti-inflammation (Node et al.,

1999). Most literature about cytochrome P450s in the intestine has focused on drug

absorption and metabolism. To our knowledge, no studies exist that have examined the

role of CYP450s during hyperthermia in the intestine. Following the COX and LOX

inhibition data, speculation about the third pathway of eicosanoid metabolism also being

involved was considered. However, in this study no protective or detrimental effects

were seen when CYP450 were inhibited showing at least a subset of CYP450 enzymes

are not needed for alterations in permeability during hyperthermia.

3.4.4. Conclusions

Mouse intestine has an inherent thermotolerance to temperatures that resemble the

thermal tolerance seen in rat intestine. Though 41.5° C is a higher core temperature than

is generally seen in human exercise, it has been observed in extreme conditions, usually

leading to heat illness (Dematte et al., 1998). The exact relevance of mouse to humans remains unresolved but the similarities of the response across species suggest that the

98

mouse is a suitable model for use in the early exploration of mechanisms and therapeutic

approaches. More prolonged exposure and concomitant ischemia are other factors

relevant to the intact organism in heat stress but these factors are likely to reduce this

thermotolerant threshold even more. Thermotolerance, for example, has a strong time- dependent characteristic in most tissues (Despa et al., 2005). During the course of this study, we found several unexpected, potentially therapeutic reagents that could be used in humans, NAC, indomethacin and baicalein. All have been given orally in humans

(Kelly, 1998; Lai et al., 2003; Sekar & Corff, 2008) and would therefore be easily accessible to the intestinal mucosa. Furthermore, they would be expected to have only limited side effects. Therefore, our study provides some potential starting points for the design of therapeutic preventative approaches to reduce the consequences of severe heat illness in humans at risk. Future studies need to explore the effective utilization of baicalein, indomethacin and NAC in animal and human exercise in hyperthermic environments.

99

3.5. Figures

Figure 3.1. Comparison of intestinal permeability when exposed to temperatures up to

42.5° C. Gut sacs showed an increase in permeability when exposed to 41.5° C but not

40.5° C. Two-way ANOVA (N=4 in each group). Temperature (P<0.05) and animal number (P<0.03) were factors of interest. Animal number was treated as a random variable. Post comparison using least mean square contrast tested individual means.

100

A B

Figure 3.2. Gut sections exposed to 37° C (A) or 41.5° C (B). Heat exposure causes a breakdown in gut structure, e.g. structural components such as the lamina propria and the epithelial cell monolayer are indistinguishable.

101

Figure 3.3. Intestinal permeability assessed at different time points (15, 30, 60 and 90 min.) when exposed to 41.5° C. An increase in permeability was only seen after 60 and

90 min. of heat exposure. Two-way ANOVA (N=4) was performed with time and animal

(random) being the factors of interest. P < 0.005 and P < 0.001 compared to 15 min. time point using post analysis contrast of least squares means.

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Figure 3.4. Cyclooxygenase inhibition effects on intestinal permeability after 90 min. of

41.5° C exposure. Indomethacin (200 µM), but not ibuprofen (800 µM), partially protected the intestine from hyperthermia. Ibuprofen showed a significant increase in permeability during hyperthermia. Multi-way ANOVA (N=4 in each group) with temperature, drug treatment, drug treatment x temperature and animal (random) being factors of interest. Post analysis of least squares contrast of means was performed.

103

Figure 3.5. Lipoxygenase inhibition effects on intestinal permeability after 90 min. of

41.5° C exposure. Baicalein (200 µM) protected intestinal permeability after 41.5° C treatment compared to control 41.5° C. ETYA treatment (500 µM) did not significantly protect the intestine from hyperthermia. Multi-way ANOVA (N=4 in each group) with temperature, drug treatment, drug treatment x temperature and animal (random) being factors of interest. Post analysis of least squares contrast was performed.

104

Figure 3.6. CYP450 inhibition effects on intestinal permeability after 90 min. of 41.5° C exposure. HET0016 (1 µM) and ABT (200 µM) showed no protection compared to 41.5°

C controls. Multi-way ANOVA (N=4 in each group) with temperature, drug treatment, drug treatment x temperature and animal (random) being factors of interest. Post analysis of least squares contrast was performed.

105

Figure 3.7. Permeability assessment of gut sacs treated with antioxidants exposed to

41.5° C for 90 min. Tiron (10 mM) and trolox (100 µM) showed no protection compared to 41.5° C controls but were significantly different to 37° C controls. NAC treatment protected intestinal gut sacs from hyperthermia induced permeability. Multi-way

ANOVA (N=4 in each group) with temperature, drug treatment, drug treatment x temperature and animal (random) being factors of interest. Post analysis of least squares contrast was performed.

106

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Chapter 4: Mechanism for the loss of intestinal permeability and pharmacological protection during hyperthermia in mouse

The intestine performs two seemingly conflicting tasks. Firstly, it is an efficient

absorbing organ for nutrients and water. Secondly, it forms a barrier between the outside

(lumen) and the inside of the body, thus limiting the unregulated entry of foreign

substances into the circulation. Several diseases and conditions, such as Crohn’s disease

(Vilela et al., 2008), celiac disease (Vilela et al., 2008), inflammatory bowel disease

(Aiko & Grisham, 1995; Kerr et al., 1999) and heat stroke (Shapiro et al., 1986; Lambert

et al., 2002a; Prosser et al., 2004; Singleton & Wischmeyer, 2006), have been found to

alter the barrier function of the intestine and allow nonselective permeability to increase.

Even intense and prolonged exercise can result in an increase in intestinal permeability

(Pals et al., 1997; Smetanka et al., 1999; Davis et al., 2005; Lambert, 2009), allowing

substances such as endotoxin to enter the blood stream (Brock-Utne et al., 1988). An

increase in blood endotoxin levels seen during heat stroke is believed to be the major

factor contributing to multiple organ failure and death (Bouchama & Knochel, 2002).

The causes of increased intestinal permeability during hyperthermic conditions are not

fully understood. Tight junction opening and cell damage of the epithelial lining have

been the predominant theories explaining this phenomenon, but the issue remains

unresolved.

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Tight junctions in the intestine are created by protein-protein interactions which are supported internally by an actin-myosin ring skeleton (Turner, 2000). Tight junction opening is a highly regulated process requiring cell signaling and activation of the actin- myosin ring, causing contraction (Turner, 2000). This contraction then leads to structural changes, which decrease articulation with adjacent cells and can increase the amount of molecules (and possibly small particles) allowed to pass in between the epithelial cells, i.e. the so called paracellular route (Turner, 2000).

Several stimuli have been found to cause an increase in permeability due to tight junction opening, e.g. viral infections (Medigeshi et al., 2009), certain ions such as chloride, calcium and magnesium (Beyenbach, 2003), influences of the gut immune system (Shao et al., 2001), reactive oxygen species (ROS) (Rao et al., 1997; Cuzzocrea et al., 2000; Sun et al., 2002) and alterations in intracellular calcium (Suzuki & Hara, 2006).

Some stimuli are needed in normal physiology to open tight junctions for nutrient absorption; however, other stimuli may lead to unregulated opening of tight junctions and may result in permeability increases. During hyperthermia, it is unclear if tight junction opening alone can account for the resulting increase in permeability.

Cell damage in the intestinal lumen has also been shown to occur in several different cell types during hyperthermia (Kamel et al., 1988; Roti Roti, 2008), but the exact effect heat has on the breakdown of intestinal cells is unknown. In the preceding chapter it was shown that mouse small intestine exposed to hyperthermia (41.5° C) for 90 minutes can cause a severe breakdown of intestinal structure (Chapter 3). Also, Lambert et al. (Lambert et al., 2002a) discovered that rat small intestine permeability (in vitro and in vivo) becomes susceptible to hyperthermia and is associated with epithelial cell

114 damage. To address the conflicting theories pertaining to the cause of increased permeability due to hyperthermia, the first objective of this study was to evaluate the mechanism(s) for increased permeability during hyperthermia in the intestine. Is increased permeability due to tight junction opening or to cell damage of the epithelial barrier?

Previously, I discovered that two inhibitors of eicosanoid metabolism (baicalein and indomethacin) protect mouse small intestine from hyperthermia-induced permeability

(Chapter 3, “Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation). Paradoxically, these same two inhibitors caused a reduction in the thermotolerance of skeletal muscle, based on measures of contractile function (Oliver et al., 2008), suggesting completely different mechanisms of action in the two organ systems. In addition, though the muscle studies showed that analogous drugs of both bacalein and indomethacin (namely ETYA and ibuprofen, respectively) had similar effects as their respective counterparts, no such effects were seen when these analogous drugs were used in the intestine (Chapter 3, “Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation). I also found that treatment of mouse intestine with N-acetyl cysteine (NAC), a well known antioxidant and reducing agent, protected permeability during hyperthermia. These observations led me to believe that another secondary mechanism for the pharmacological agents must be occurring that is not directly related to known effects of these agents on eicosanoid metabolism.

Furthermore, these effects could be unique to the intestine exposed to hyperthermia.

Studies have shown that baicalein (Piao et al., 2008) as well as NAC (Moldeus &

Cotgreave, 1994) have antioxidant properties, which could possibly be the mechanisms

115 for their protective effects. Because of these findings, the second objective of this study was to discover the mechanism of protection for baicalein and NAC. Understanding the effects of these drugs might also provide insights in the underlying pathophysiology of loss of barrier function.

In summary, the objectives of this study were to 1) determine if tight junction opening or cell damage is the predominant pathway through which permeability is increased during hyperthermia in the mouse intestine and 2) discover how baicalein and

NAC protect the mouse intestine from hyperthermia-induced permeability.

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4.2. Methods

4.2.1. Chemicals and drugs used. Medium 199 (Cellgro), L-Glutamine (Lonza), sodium bicarbonate (Acros Organics), formaldehyde, fluorescein isothiocyanate (FITC)-dextran

4 kDa (FD4, Sigma Aldrich), fluorescein-5-(6)-sulfonic acid trisodium salt (low molecular weight probe, Invitrogen), baicalein (12-LOX inhibitor and partial general

LOX inhibitor, Sigma Aldrich), N-acetyl cysteine (NAC, increases the pool of free radical scavengers, Sigma Aldrich), 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'- tetraacetic acid, tetraacetoxymethyl ester [BAPTA-AM, intracellular calcium chelator,

BIOMOL], E. coli heat labile enterotoxin b subunit (Sigma Aldrich), palmitoyl carnitine

(Sigma Aldrich), 3-Ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene [NOC-12,

Sigma Aldrich], cytochalasin D (CytD, Sigma Aldrich).

4.2.2. Animal treatment protocol and gut sac preparation. This study used C57bl6 mice

(25-35 g). Animal care and treatment were performed using protocols approved by the

University of Florida Institutional Animal Care and Use Committee. Food was withdrawn from the mice, to reduce luminal contents, 10 hours prior to excision of the intestine. The mice were euthanized by carbon dioxide asphyxiation and the entire small intestine was rapidly excised and placed in pre-oxygenated medium 199 (with glutamine and sodium bicarbonate without phenol red). The everted gut sac preparation has been modified from Lambert et al. (Lambert et al., 2002a) and has previously been described in Chapter 3 (“Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation). After gut sacs were made, they were treated at either 37° C or 41.5° C with baicalein (200 µM), NAC (10 mM), BAPTA-AM (100 µM) (Hofman et al., ;

Hoque et al.), or neither compound in order to serve as a control. In some experiments,

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permeability measurements were calculated using the permeability protocol in chapter 3

(“Protection of hyperthermia-induced intestinal permeability in the mouse” in

preparation). The volume of the solution inside the gut sacs (100-300 µL) and the tissue

area were determined at the conclusion of the experiment. Fluorescence of the serosal

fluid was then measured using a spectrofluorometer (SpectraMax M5, Molecular

Devices) and the concentration of the low molecular weight probe (0.956 kDa) or the

high molecular weight probe (4 kDa, FITC-dextran), depending on the experiment, was

determined by comparison to a standard curve. However, in other experiments tissues

were weighed and frozen for use in protein oxidation (Oxyblot) as well as 4-

hydroxynonenal (4-HNE) quantitative analysis (see Analytical Procedures). In

conjunction with the tissue collection, luminal samples were taken for lactate

dehydrogenase (LDH) activity determination, to measure cytotoxicity (see Analytical

Procedures).

4.2.3. Effects of Pharmacological Tight Junction Openers. In a separate set of

experiments, tissues were exposed to compounds previously found to cause opening of

tight junctions in intestinal epithelial cells and measurements of permeability were

determined. The compounds were E. coli heat-stable enterotoxin (Hoque et al., 2001)

(dissolved in buffer), palmitoyl-DL-carnitine (Oh et al., 1988) (dissolved in buffer),

NOC-12 (Yamamoto et al., 2001)(dissolved in sodium hydroxide, < 0.0001 % of total volume) and cytochalasin D (Madara et al., 1986) (dissolved in DMSO, < 0.003 % of total volume). The compounds were tested by measuring the permeability of isolated gut sacs to low molecular weight (0.956 kDa) fluorescein-5-(6)-sulfonic acid. This was initially modeled as a positive control to evaluate the effects of tight junction opening.

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The compounds which caused an increase in the low molecular weight probe were then analyzed using the FD4 (higher molecular weight, 4 kDa) probe, which is a probe believed to be only slightly smaller than most endotoxin molecules (endotoxin has been estimated to be between 10 and 20 kDa in size in most preparations). Permeability was then assessed and compared to 37° C and 41.5° C control tissues.

4.2.4. Histology. A few tissues were collected for histological assessment after the experimental protocol. Fixation with 4 % formaldehyde was used prior to slicing and staining with hematoxylin and eosin. Structural damage was assessed using a scaled system from 1-5. 1: low amount of damage seen with distinct structural components

(epithelial layer and lamina propria). 2: Structural components still differentiated from each other, but the epithelial layer noticeably lifting from the lamina propria, i.e. the layer of connective tissue below the epithelial layer containing vascular and lymph vessels). 3:

Disorganization of the villi beginning, and differentiation between structural components difficult. 4: Organization of the villi is chaotic. 5: Structure of villi is chaotic and many villi are completely destroyed down to the lower layers of tissue. Samples were analyzed randomly by blinding the sample conditions during collection. Each histological segment was divided up into 4 areas and each area was given a score according to the above scale.

The scores for each area were then averaged to give an overall score for the segment.

This process was repeated for each sample and data was collected for statistical analysis.

4.2.5. EM analysis. Tissues selected for EM viewing were fixed in Tyrods buffer with 4

% paraformaldehyde and 1 % glutaraldehyde for processing and staining with lead citrate and uranyl acetate. Micrographs were taken on a Hitachi 7600 electron microscope equipped with digital imaging at magnifications of x 12,000.

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4.2.6. LDH Assay. Everted gut sac luminal (outside of the gut sacs) fluid samples (50

µL) were taken after the completion of the experimental protocol, for control tissues as

well as baicalein, NAC and CytD treated tissues. These samples were then analyzed for

LDH activity as a determination of cytotoxicity. A commercially available kit (Cayman

Chemical) was used for this determination. Briefly, LDH catalyzes the reduction of

NAD+ to NADH and H+. This is accomplished by oxidation of lactic acid to pyruvate.

Next, the produced NADH and H+ are used by diaphorase which reduces a tetrazolium

salt (INT) to formazan (highly colored). The highly colored formazan strongly absorbs at

wavelengths of 490 - 520 nm. A spectrofluorometer (SpectraMax M5, Molecular

Devices) was used for absorption determination. A standard curve of known LDH concentrations was determined and used for the quantification of LDH activity in samples. LDH standard curves were calculated for baicalein, NAC and CytD to see if interactions with the assay were present. Baicalein was found to cause both a downward

shift in y-intercept and a change in the slope of the standard LDH curve. Because of this,

statistical analysis was not able to be performed on the data from the baicalein

experiments and is omitted from the results.

4.2.7. Determination of Oxidative Stress. Frozen samples taken for oxidative stress

analysis were homogenized in phosphate buffered saline (PBS) with an anti-protease cocktail to reduce protein degradation during preparation. Following the homogenization, protein concentration was determined (Bradford Assay) and samples were all diluted to the same concentration. The samples were then used for two different assays: protein carbonyl formation and 4-HNE. Protein carbonyl sample preparation was performed using a commercially available kit (Millipore) and 4-HNE samples were

120 prepared using 95 % Laemmli buffer and 5 % 2-mercaptoethanol. Following this, 4-

HNE samples were allowed to boil for 5 minutes with subsequent cooling before loading onto gels. After samples were prepared, a standard western blot protocol was followed: protein separation gels were ran at 150 V for 60 minutes followed by transfer to nitrocellulose at 275 mA for 90 minutes, ponceau (short term protein stain) was used for quick determination of equal protein loading in each lane, followed by washing and application of 1° and 2° antibodies with repeated washes in between each application.

The membranes were then developed using enhanced chemiluminescence plus (ECL plus) and exposed to film.

4.2.8. Data analysis and Statistics. Permeability data were represented as the transport of nmoles of FITC-dextran per cm2 (i.e. normalized to intestinal surface area of the gut sac and serosal volume) (Equation in Chapter 3 methods, “Protection of hyperthermia- induced intestinal permeability in the mouse” in preparation). LDH release was measured by activity and represented as µU/mg tissue. Analysis of gels was accomplished by using Image J (NIH). For normalization of samples and combined analysis of different gels, the same sample was loaded on all gels. Also, seven bands were chosen across all gels for comparison of overall protein oxidation signal. This was done to reduce background noise. For protein oxidation and 4-HNE, data were expressed as fold changes compared to either the first time point in a series (15 min.) or the heated control (41.5° C).

Student t-test, Tukey’s and mean contrast analysis were performed after ANOVA to determine specific effects of treatment and temperature compared to control measurements (SAS JMP statistical package). The effects of “animal, i.e. specific

121 experiment on matched tissues” were treated as a random variable resulting in the equivalent of a repeated measures design. All data are represented as means ± SE. P <

0.05 was considered to be statistically significant.

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4.3. Results:

4.3.1. Time and temp controls: LDH and Permeability. LDH activity measurements,

used as an indicator of cell damage, were taken over time during exposure of isolated gut

sacs to either 37° C or 41.5° C. As shown in Figure 4.1.A, 41.5° C exposure resulted in a

significant increase (P < 0.005) in LDH release after 60 minutes (N=4) and 90 minutes

(N=4) of heat exposure compared to 15 minutes (N=4) of 41.5° C exposure. This shows

that cytotoxicity is occurring within the 90 minute time course of heat exposure. LDH release was also significantly increased (P < 0.05) after 90 minutes of 37° C exposure, indicating that the viability of the everted gut sac model may be starting to decrease or that cells on the cut edges of the gut sac may be undergoing further deterioration. To determine if the increases in LDH release at 37° C caused a subsequent increase in permeability, time controls were done at 15, 30, 60 and 90 minutes (N=4 for all time

points, Figure 4.1.B). Tissues exposed to 37° C showed no significant increase in

permeability. The permeability data for 41.5° C time controls, previously presented in

Chapter 3 (Figure 3.1C, “Protection of hyperthermia-induced intestinal permeability in

the mouse” in preparation), are reproduced here for clarity and demonstrate that much larger permeability occurs in the hyperthermic tissue compared to the normothermic tissue during this time period.

4.3.2. Time and Temp controls: Oxidative stress measurements. To evaluate the extent

of oxidative stress during hyperthermia, protein carbonyl and 4-HNE formation were

measured. Using the protein carbonyl assay, tissues exposed to 90 min. at 41.5° C (N=6)

yielded a significant increase (P < 0.01) in protein oxidation compared to 15 minutes

(N=4) of 41.5° C exposure (Figure 4.2.B). Interestingly, protein oxidation was found to

123 be significantly decreased (P < 0.005) after 90 minutes of 37° C exposure compared to 15 minutes at 37° C (Figure 4.2.A). Arrows in Figure 4.2.A indicate the bands which were used in the quantification of total protein oxidation. The representative lanes for the

41.5° C controls are all from the same gel. In contrast, 4-HNE analysis yielded no significant differences between any of the groups studied at any time point or at any temperature (Figure 4.3.).

4.3.3. Tight junction regulation during hyperthermia. To test the hypothesis that tight junction opening can account for the increase in permeability during hyperthermia, compounds utilized in previous literature to induce opening of intestinal epithelial tight junctions were tested at 37° C exposure. The rationale here was that if tight junctions are responsible for increasing the permeability to substances the size of endotoxin, then it should be possible to artificially increase the permeability in the gut intestine by applying these pharmacological tight junction openers in the absence of heat exposure. To do this, a smaller molecular weight fluorescein (fluorescein-5-(6)-sulfonic acid) molecule was employed for the initial testing of permeability. This was used as a positive control because it is likely that it would penetrate the tight junction openings if they were open, whereas larger MW indicators (i.e. our 4 kDa fluorescent probe) might not. E. coli heat- stable enterotoxin (Ent, 30 µg/mL), palmitoyl carnitine (Palm, 150 µM) and NOC-12

(NOC, 100 µM) treatment did not cause an increase in permeability to the low molecular weight fluorescein (N=5, Figure 4.4.A). However, Cytochalasin D (CytD, 20 µM) caused a significant (P < 0.05) increase in permeability of the smaller molecular weight

FITC compared to 37° C control. This increase in permeability was not comparable to

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the elevation seen with 41.5° C exposure for this low molecular weight probe (dark bar in

Figure 4.4.A).

Further study utilizing the larger FITC-dextran molecule (4 kDa) that more closely resembles the MW of endotoxin also elicited a significant increase (P < 0.005) in permeability with CytD exposure at 37° C compared to 37° C control (N=6, Figure

4.4.B). However, again, CytD treatment was found to be significantly decreased compared to 41.5° C control (P < 0.05), suggesting that CytD was unable to cause an increase in permeability equal to that of hyperthermia alone. Also, to determine if CytD was eliciting a cytotoxic effect during treatment, LDH release was monitored (far right of

Figure 4.4.B). LDH release was not significantly different from 37° C control tissues or from 41.5° C control tissues. Electron micrographs (Figure 4.5.) of tissues treated with

CytD showed severe gaps between enterocytes (indicated by two ended arrows) as well as massive changes in cellular morphology.

4.3.4. Histological assessment of heated and control tissues. To assess the extent of structural damage to the intestine during hyperthermia, a scale of damage (both cellular disruption and/or cyto-structural disorder) was implemented for statistical analysis

(Figure 4.6.). Tissues exposed to 90 minutes of 41.5° C showed a significant increase (P

< 0.001) in damage compared to tissues exposed to 90 minutes at 37° C. Representative histological pictures of tissues exposed to 37° C and 41.5° C are depicted in Figure 4.6.

4.3.5. Calcium chelation during hyperthermia. To test the hypothesis that an increase in intracellular calcium during hyperthermia is causing permeability dysfunction, either through recruitment of the cytoskeleton elements controlling the tight junction opening

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(Suzuki & Hara) or prevention of injury through activation of Ca+2 sensitive protease activity (Marzocco et al., 2004), treatment of tissues undergoing hyperthermia was performed with BAPTA-AM, an intracellular calcium chelator (Figure 4.7.). BAPTA-

AM (100 µM) was unable to protect tissues from hyperthermia-induced intestinal permeability, as results were not statistically significant compared to the control at 41.5°

C (N=4).

4.3.6. LDH release of tissues treated with NAC. LDH release was measured in tissues treated with NAC to test for its protective effects on hyperthermia-induced permeability through cytotoxicity reduction (Figure 4.8). LDH release with NAC (10 mM) treatment was not found to be significantly different compared to the 37° C or 41.5° C controls.

Matched pairs analysis of these controls also showed no statistical differences from NAC treatment (data not shown). However, the variability in the LDH data made it difficult to fully determine whether NAC protected against LDH release.

4.3.7. Damage index of tissues treated with baicalein or NAC. Tissues exposed to 41.5°

C with either baicalein (200 µM) or NAC (10 mM) were analyzed for the extent of damage due to hyperthermia (Figure 4.9). NAC, but not baicalein, showed significant protection against hyperthermia-induced structural damage compared to untreated 41.5°

C exposed tissues. Representative histological sections of tissues treated with baicalein or NAC are shown in Figure 4.10. The histological sections show that NAC is protecting overall structural changes of the villi. This protection is seen with the ability to discern between epithelial cells and the lamina propria. Tissues treated with 41.5° C alone in the presence or absence of baicalein showed a complete breakdown in villus structure, indicated by the inability to discern between epithelial structures. As shown in Figure

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4.11., the electron micrographs indicate that hyperthermia causes a breakdown in the cellular structure of epithelial cells but there were no additional insights into mechanisms or specific structural abnormalities that could be discerned by EM analysis.

4.3.8. Protein carbonyl and 4-HNE formation in tissues treated with baicalein or NAC.

Protein carbonyl and 4-HNE measurements were determined in baicalein (200 µM) and

NAC (10 mM) treated tissues to discover if reducing oxidative stress is a possible mechanism for protection. Both baicalein and NAC caused a significant decrease (P <

0.001) in protein oxidation compared to 41.5° C and protein oxidation controls, shown in

Figure 4.12. Interestingly, 4-HNE formation remained unchanged for the drug treated tissues (N=6, Figure 4.13).

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4.4. Discussion

The results of this study are consistent with the hypothesis that cell damage to the intestinal epithelium is the predominant pathway by which permeability to large molecules is increased during hyperthermia. I found no data in this model that are consistent with the alternative hypothesis that changes in permeability can be attributed to epithelial tight-junction opening. The mechanism by which epithelial injury occurs appears to be related to oxidative stress. This conclusion is based on the observation that heat exposure causes an elevation in protein oxidation in the intestine, and that baicalein or NAC treatment reduced both the permeability and the oxidative stress associated with hyperthermia in the gut. Though the complete mechanism for the increase in permeability with hyperthermia is still not completely resolved, this study provides the strongest evidence to date that the mechanisms is dominated by rapidly progressing cell damage of the luminal epithelial barrier, via oxidative stress-related pathways.

4.4.1. Critique of Approach.

The everted gut sac model has been previously developed and utilized by Lambert et al. (Lambert et al., 2002a) as well as in my previous work where I adapted it to a mouse model (Chapter 3, “Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation). In the present study, LDH release was monitored during exposure to 37° C for 90 minutes to test the viability of the gut sac preparation (Figure

4.1.A). It was discovered that after 90 minutes of 37° C exposure a slight increase in

LDH release was observed. Similar data was seen by Lambert et al. (Lambert et al.,

2002a) in the rat. Also, permeability changes were determined after 90 minutes of 37° C

128 exposure and were found to not increase. Because of these findings, I felt comfortable using the everted gut sac model for the investigation of hyperthermia-induced permeability mechanisms.

4.4.2. Mechanism for increased permeability during hyperthermia.

Heat related illnesses, such as heat stroke, have been found to increase the permeability of the intestine and can result in increased levels of lipopolysaccharide

(LPS) in the blood stream (Hall et al., 2001). Both hyperthermia and ischemia/reperfusion have been found to occur during heat related illnesses, and are believed to contribute to an increase in intestinal permeability (Hall et al., 1999;

Bouchama & Knochel, 2002), possibly through ROS related pathways (Hall et al., 2001).

However, this remains controversial. Studies have speculated that this increase in permeability can occur through two possible routes, tight junction opening and epithelial cell damage. Previous studies have not resolved which of these pathways is responsible for the most permeability increase during hyperthermia.

Tight junction opening is a highly regulated cellular event which has been shown to occur in response to stress (Cuzzocrea et al., 2000; Banan et al., 2002). Most studies have evaluated tight junction regulation in cell culture, which isolates the monolayer of epithelial cells (Rao et al., 1997; Banan et al., 2000a; Cuzzocrea et al., 2000). In these preparations, tight junctions have been shown to be regulated by changes in calcium

(Suzuki & Hara) and exposure to ROS (Rao et al., 1997; Cuzzocrea et al., 2000; Banan et al., 2002) as well as induction of other downstream cellular events (Banan et al.). The importance of tight junction regulation is not only due to its role as a barrier against toxic

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materials, but also its ability to allow substances across the enterocyte monolayer (i.e. its normal function in providing nutrients to the blood stream) (Ballard et al., 1995).

However, during hyperthermia the importance of tight junction opening in the

progression of heat related illnesses has only been speculated upon. In the present study,

the use of compounds known to induce tight junction opening resulted in either no

increase or a low increase in permeability. All the compounds and dosages used for tight

junction opening have previously been evaluated in various models of epithelial barrier

function (Madara et al., 1986; Oh et al., 1988; Hoque et al., 2001; Yamamoto et al.,

2001; Deli, 2009). In my model, only cytochalasin D showed an increase in permeability. However, a frequently overlooked consequence of cytochalasin D treatment is its drastic effects on cytoskeletal structure and cell morphology (Figure 4.5.),

which has been previously observed in other cell culture studies (Ma et al., 2000).

Though cytochalasin D caused severe structural changes, the increase in permeability was

still not to the degree seen during hyperthermia. I have to conclude that even with this

most drastic of treatments, I cannot attribute permeability changes in the intact intestine

to epithelial tight junction opening alone.

Previously, calcium chelation by BAPTA-AM has been shown to protect tight junction opening and subsequent permeability in caco-2 cells (a model of intestinal cells) undergoing stimulation by sodium caprate (a known tight junction opener) (Suzuki &

Hara, 2006). Actin filaments partially comprise the internal structure of tight junctions, and when stimulated by calcium and several kinases, their contraction has been shown to cause opening of tight junctions (Hirase et al., 2001; Suzuki & Hara, 2004). Though

BAPTA-AM is a cell permeable molecule that has been shown to enter intestinal cells

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and inhibit tight junction opening (Suzuki & Hara, 2006), I did not find that it was

capable of protecting against hyperthermia-induced intestinal permeability in my model.

Though not presented here, other pilot experiments reducing extracellular Ca+2 also failed

to influence permeability changes. However, the use of only one calcium reducing

method does not completely negate the possibility that calcium could play a role in

intestinal dysfunction during hyperthermia.

Previously, Lambert et al. (Lambert et al., 2002a) showed that in rat intestine after

exposure to 41.5° C tight junctions remain closed, though the authors were unsure

whether or not this was due to the tissue fixation process. The authors have speculated

that tight junction opening could be the main pathway for increased permeability during

hyperthermia. However, severe breakdown in the overall structure of the intestine during

hyperthermia was discovered by these same authors (Lambert et al., 2002a), which seems

to negate the idea that tight junction opening is the main cause for increased permeability.

The findings in my study (increased LDH release, damage index, EMs and histology)

suggest that tissue damage is extensive, which corresponds to histological findings by

Lambert et al. in the rat (Lambert et al., 2002a). According to these results tight junction

regulation, during the time course of hyperthermic exposure, does not account for the rise

in permeability during hyperthermia.

ROS production has been shown in cells undergoing hyperthermic stress

(Flanagan et al., 1998; Gorman et al., 1999; Zuo et al., 2004). Epithelial cells exposed to

a very high concentration of hydrogen peroxide showed a marked increase in

permeability, suggesting a role for ROS in the development of hyperpermeability (Rao et

al., 1997; Cuzzocrea et al., 2000). However, these studies could be challenged on the

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basis of the degree of oxidative stress exogenously induced. Also, oxidative damage has

been linked to a decrease in barrier integrity and cell injury caused by increased intestinal

inflammation (Keshavarzian et al., 1992; McKenzie et al., 1996; Banan et al., 2000a;

Banan et al., 2000b). In particular, inflammatory bowel disease has been shown to cause

“leaky gut” which induces inflammation of the intestine and can lead to oxidant

production as well as hyperpermeability (Yamada et al., 1993).

ROS have also been implicated in permeability changes with whole animals. Hall

et al. (Hall et al., 2001) found that when antagonists of xanthine oxidase (a known ROS

producer) were given to heat stressed animals, portal LPS levels decreased. In addition, I

have already shown that in mouse intestine exposed to hyperthermia (41.5° C) and

treated with NAC (antioxidant), baicalein (LOX inhibitor) or indomethacin (COX

inhibitor), permeability was protected (Chapter 3, “Protection of hyperthermia-induced

intestinal permeability in the mouse” in preparation). However, the addition of two other

antioxidants (tiron and Trolox) did not protect permeability (Chapter 3, “Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation). Also,

Assimakopoulos et al. (Assimakopoulos et al., 2004) discovered that intestinal

permeability and subsequent endotoxin release into the blood, caused by obstructive

jaundice was partially due to an increase in oxidative stress. Protein oxidation, lipid

oxidation as well as redox state were altered in the intestine due to the stress of inducing

obstructive jaundice (Assimakopoulos et al., 2004). Similarly, in this study, protein

oxidation was increased due to hyperthermia. NAC, which has been found to alter the

redox state of cells by increasing free intracellular glutathione (Moldeus & Cotgreave,

1994), protected against increases in both permeability and protein oxidation. The

132 increase in protein oxidation coincides, at least in part, with the increase in permeability seen during hyperthermia. Another point of interest is that 4-HNE levels (downstream indicator of lipid peroxidation) did not increase in any of the control or treatment groups.

This finding is similar to that of Lambert et al.’s (Lambert et al., 2002a) who found that lipid peroxidation levels (indicated by MDA) remained unchanged due to hyperthermia in rat intestine (Lambert et al., 2002a). These data combined with the other known roles for ROS suggest that elevated ROS formation could be a cause for the increase in permeability during hyperthermia in the mouse.

Hyperthermia could be eliciting different cellular effects other than opening tight junctions or damage to the enterocyte monolayer, such as disrupting structural components deeper in the anatomy of the intestine. The main area of study for intestinal permeability has been to focus on the epithelial cells since they are the first line of defense against foreign materials reaching the systemic circulation. However, it could be presumptuous to think that a single layer of enterocytes is the only defense and site of dysfunction during hyperthermia. Within the structure of the intestine, lymph nodes

(Shao et al., 2001) aid in defense along with a host of immune cells (Shao et al., 2001), and could be involved in the intestinal response to hyperthermia. Lymph as well as endothelial permeability may need to be altered in conjunction with enterocyte permeability to allow for luminal contents to reach the circulation. Certainly in models of ischemia-induced barrier dysfunction, the role of endothelial cells in the vasculature is believed to be of extreme importance (Victorino et al., 2008). Hyperthermia has been shown to alter endothelial permeability (Zhao et al., 2001; Sun et al., 2002). Endothelial permeability has been shown to be of particular importance in the dysfunction of the

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blood brain barrier during heat exposure (Jeliazkova-Mecheva et al., 2006). Also, NAC protects against increased endothelial permeability (Zhao et al., 2001; Sun et al., 2002), which could be a primary or secondary mechanism in the present model. In addition to possible deep anatomy changes, the intestinal immune system which has been found to be important during several diseases (Yamada et al., 1993), could cause immune disturbances during hyperthermia and subsequent tissue damage. Though, it is doubtful that it would play a role in this acute in vitro study.

4.4.3. Baicalein and NAC treatment during hyperthermia.

In the previous chapter, baicalein and NAC were shown to have the largest protective influence compared to all other pharmacological agents tested during hyperthermia, but until now the mechanism remained elusive. Other inhibitors and antioxidants used in parallel with baicalein (ETYA) and NAC (tiron and Trolox) showed no protection to hyperthermia (Chapter 3, “Protection of hyperthermia-induced intestinal permeability in the mouse” in preparation) in the intestine, suggesting a pharmacological effect was occurring. Both baicalein and NAC have antioxidant properties, and directly inhibited protein oxidation caused by hyperthermia (Figure 4.8). Baicalein, while primarily known as a general inhibitor of lipoxygenases, has also been found to scavenge superoxide, hydrogen peroxide and hydroxyl radical (Hamada et al., 1993). Furthermore, baicalein reduces the thermal tolerance of skeletal muscle (Oliver et al., 2008), and others have shown that it increases the survival time of animals undergoing hyperthermia

(Chang et al., 2007) and attenuates ROS formation and cell death in cardiomyocytes undergoing hypoxia (Shao et al., 2002). NAC is an antioxidant which increases the pool of free glutathione (Moldeus & Cotgreave, 1994) and has other antioxidant and -SH

134

reducing properties as well (Moldeus & Cotgreave, 1994). Sun et al. have shown that

NAC is able to protect endothelial and epithelial permeability induced by ischemia/reperfusion in the rat (Sun et al., 2002). The authors speculated that free radicals play an important role in intestinal damage during ischemia/reperfusion (Sun et

al., 2002). In a separate study, intestinal damage due to ischemia/reperfusion was

attenuated by NAC treatment, which is similar to the results of this study (Cuzzocrea et

al., 2000). Altered redox state of cells to stresses such as hyperthermia (Mitchell &

Russo, 1983) and ischemia/reperfusion (Zhao et al., 2001) has been shown to occur.

Studies have monitored the glutathione levels (GSH) as an indicator of cellular or tissue redox state (Mitchell & Russo, 1983) and have found that glutathione is important in protecting intestinal cells against oxidative stress (Lash et al., 1986; Kelly, 1993; Rao et al.,

2000; Kelly et al., 2004). Since NAC has been shown to increase free glutathione in

treated cells (Moldeus & Cotgreave, 1994) and altered glutathione levels have been

shown to occur during hyperthermia (Mitchell & Russo, 1983), this strongly suggests that

NAC is protecting intestinal cells and possibly permeability. This protection could occur

through a mechanism of altering glutathione levels and subsequently maintaining less

threatening levels of oxidants. Because of the above studies and the present findings,

baicalein and NAC seem to be promising therapeutic agents to protect against

hyperthermia-induced dysfunction due to oxidant damage.

4.4.4. Conclusions

According to the results of the present study, cell damage is a more plausible

explanation than tight junction opening for hyperthermia-induced permeability, as

indicated by increased LDH release and the breakdown of intestinal structure. The

135

underlying mechanism for the increase in intestinal permeability seems to be partially due to increased ROS formation because of the rise in protein oxidation levels during heat.

Previously, I had discovered that a few pharmacological agents (baicalein and NAC, in particular) protected mouse intestine from hyperthermia-induced permeability. In the present study, NAC and baicalein caused a reduction in hyperthermia-induced protein oxidation, which could be the possible route of permeability protection. Understanding the pathway of increased permeability during hyperthermia and the mechanism for

protection by baicalein and NAC have great implications in developing treatments for

heat stroke and other heat related illnesses.

136

4.5. Figures

A

B

Figure 4.1. LDH release (A) and permeability (B) of tissues exposed to either 37° C or

41.5° C over 90 minutes. Permeability of tissues exposed to 37° C showed no increase but LDH release after 90 minutes was slightly elevated. Exposure to 41.5° C caused a significant increase in both permeability and LDH release. Multi-way ANOVA was performed with time, temperature and animal (random) being the factors of interest. Post analysis least squares contrast of means was performed. *P < 0.05 compared to 15 minutes exposure to 37° C. ‡P < 0.005 compared to 15 minutes exposure to 41.5° C.

# P < 0.05 comparing 60 and 90 minutes at 41.5° C and 37° C.

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15 30 60 90 min. 37 °C ►

► ►

► ►

► ►

15 30 60 90 min. 41.5 °C

Figure 4.2. Protein oxidation measurements of tissues exposed to 37° C or 41.5° C over

90 minutes. Tissues exposed to 90 minutes of 37° C showed a decrease in protein oxidation at 90 minutes. Tissues exposed to 90 minutes of 41.5° C significantly increased protein oxidation but only at the 90 minute time point. Protein oxidation was measured using an Oxyblot kit detected using western blot analysis. Data is depicted as fold change in band intensity. Representative gels are shown. Example lanes for 15, 30, 60 and 90 minute exposure to 41.5° C are from the same gel but intervening lanes are cut for clarity. These have exactly the same exposure and no manipulation of image. Two-way

ANOVA was performed with time and animal (random) being the factors of interest.

Post analysis least squares contrast of means was performed. **P < 0.01 compared to 15 minutes exposure to 41.5° C. ‡P < 0.005 compared to 15 minutes exposure to 37° C.

138

37°C 15 30 60 90 min.

41.5°C 15 30 60 90 min.

Figure 4.3. 4-HNE measurements of tissues exposed to 37° C or 41.5° C over 90

minutes showed no increase. Protein oxidation was measured using an Oxyblot kit detected using western blot analysis. Data is depicted as fold change in band intensity.

Representative gels are shown. Example lanes for 15, 30, 60 and 90 minute exposure to

41.5° C are from the same gel but intervening lanes are cut for clarity. These have

exactly the same exposure and no manipulation of image. Two-way ANOVA was

performed with time and animal (random) being the factors of interest. Post analysis

least squares contrast of means was performed.

139

HMW Probe A LMW Probe B

Figure 4.4. Chemicals to induce tight junction opening compared to 37° C and 41.5° C controls. A: Chemicals to induce tight junction opening with low molecular weight fluoresein, only 37° C exposure. Control 41.5° C (C41.5) is only for comparison. B:

CytD treatment on permeability of the higher molecular weight FD4 probe at 37° C

(C37) and LDH release compared to C37. Both measurements were compared to C41.5.

CytD was the only chemical which was found to significantly increase permeability but not to the same level as 41.5° C exposure. Low molecular weight (LMW) and high molecular weight (HMW) probe. LDH release caused by CytD treatment was not different from either C37 or C41.5. Multi-way ANOVA was performed with temperature, drug treatment, temperature x drug treatment and animal (random) being the factors of interest. Post analysis least squares contrast of means was performed. *P <

0.05, ‡P < 0.005 and ‡‡P < 0.001 compared to C37. @P < 0.05compared to C41.5.

140

A B

► ► ↨

Figure 4.5. Electron Micrographs of 37° C control and CyD treated tissues. Severe changes in epithelial cell morphology were seen with CytD treatment during 37° C

exposure (B) compared to 37° C control tissue (A) (12,000 x magnification). Large gaps

or vacuoles were created between epithelial cells (shown as two ended arrow). Tight junctions are designated with arrow tips.

141

37°C 41.5°C A B

► *

Figure 4.6. Tissue damage or disordered cellular structure during 90 minutes of hyperthermia. Tissues exposed to 41.5° C (B) showed a significant increase in damage compared to 37° C controls (A). Representative tissues exposed to 37° C or 41.5° C are

shown, asterisk designates lamina propria and arrow designates the epithelial cell layer.

One-way ANOVA (N=10) was performed with temperature being the factor of interest.

Post analysis least squares contrast of means was performed. ‡‡P<0.001 compared to

C37.

142

Figure 4.7. Calcium chelation by BAPTA-AM during hyperthermia. BAPTA-AM showed no protective or detrimental effects during exposure to hyperthermia. Multi-way

ANOVA (N=4) was performed with temperature, drug treatment, temperature x drug treatment and animal being the factors of interest. Post analysis least squares contrast of means was performed. *P<0.05 and ‡‡P < 0.001 compared to 37° C control (C37).

143

*

Figure 4.8. LDH release of tissues treated with NAC (NAC41.5) and exposed to 41.5°

C. NAC treatment did not show a significant difference compared to 37° C (C37) or

41.5° C (C41.5) controls. Multi-way ANOVA (N=6) was performed with temperature, drug treatment, temperature x drug treatment and animal being the factors of interest.

Post analysis least squares contrast of means was performed. *P < 0.05 compared to

C37.

144

Figure 4.9. Damage index of tissues treated with either baicalein (B41.5, N=10) or NAC

(NAC41.5, N=8) and exposed to 41.5° C. NAC treatment caused a significant reduction in the degree of damage seen during exposure to hyperthermia. Multi-way ANOVA was performed with temperature, drug treatment and temperature x drug treatment being the factors of interest. Post analysis least squares contrast of means was performed.

‡‡P<0.001 compared to C37. @P < 0.001 compared to 41.5° C (C41.5) control.

145

A B

► *

C D

► *

Figure 4.10. Histological sections of tissues treated with baicalein (C) or NAC (D) compared to 37° C (A) and 41.5° C (B) controls. The protection NAC elicits is apparent in D, which shows tissue with a distinct epithelial layer (arrow) and lamina propria

(asterisks). This is similar to the 37° C control tissues (A).

146

A B ►

►

*

C D ► ► *

Figure 4.11. Electron micrographs of tissues treated with either baicalein (C) or NAC

(D) compared to 37° C (A) and 41.5° C (B) controls. x 12,000 magnification. Arrows indicate tight junctions. Control 41.5° C (B) and baicalein treated (B) tissues showed cellular damage (indicated by asterisks); in particular, rupturing of the luminal membrane was seen.

147

Figure 4.12. Protein carbonyl measurements of tissues treated with either baicalein or

NAC. Both baicalein and NAC treatment when exposed to hyperthermia showed a

significant reduction in protein oxidation. Protein oxidation was measured using an

Oxyblot kit detected using western blot analysis. Data is depicted as fold change in band intensity. Multi-way ANOVA (N=6) was performed with temperature, drug treatment, temperature x drug treatment and animal being the factors of interest. Post analysis least squares contrast of means was performed. ‡P < 0.005 compared to C41.5.

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Figure 4.13. 4-HNE measurements of tissues treated with baicalein or NAC. 4-HNE showed no change with either baicalein or NAC treatment. Protein oxidation was measured using an Oxyblot kit detected using western blot analysis. Data is depicted as fold change in band intensity. Multi-way ANOVA (N=6) was performed with temperature, drug treatment, temperature x drug treatment and animal being the factors of interest. Post analysis least squares contrast of means was performed.

149

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Chapter 5: Conclusions and future directions

In conclusion, this thesis had three main objectives; the first was to define the thermal tolerance of skeletal muscle and gut permeability exposed to physiologically relevant temperatures and evaluate the functional response of antioxidant treatment. The second objective was to elucidate the effects of inhibiting eicosanoid metabolism enzymes during hyperthermia and compare these effects between skeletal muscle and intestine which are critically important during heat stress. Finally, the third objective was to discover the mechanism of increased gut permeability during hyperthermia. Gut permeability issues could stem from tight junction opening or injury of the epithelial lining, and the role of oxidative damage in causing such permeability issues was studied, along with a detailed structural analysis of gut lining in order to determine exactly which mechanisms were involved.

Each of these broader objectives crossed over to multiple specific studies in this research. The first study had three specific aims; the first was to determine the functional responses to brief heat exposure in isolated, largely oxidative skeletal muscle. Skeletal muscle contractile function remained intact up to 42° C but significantly dropped when exposed to 43° C. The results of this thesis provide evidence that skeletal muscle has an inherent thermal tolerance within temperature ranges likely to be experienced during intense exercise. The second aim was to test whether increased oxidant production in heat stress is responsible for the loss of contractile function in intact muscle tissue. The

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use of antioxidants during hyperthermia did not protect against the loss of contractile

function, demonstrating that oxidant damage does not seem to be a main pathway during

heat stress in muscle, which may be unique to oxidative fibers. Finally, the third aim was

to evaluate the role of eicosanoid metabolism, specifically catalyzed by lipoxygenase and

cyclooxygenase, on the loss of contractile function in hyperthermia. Lipoxygenase as

well as cyclooxygenase inhibition further depressed the loss of function at 43° C and also

caused a loss of thermal tolerance at 42° C. Blockage of phospholipase A2 was unable to

prevent the loss of contractile force at 43° C. Results showed that, at least in oxidative

diaphragm muscles, normal thermal tolerance is dependent on an intact network of eicosanoid metabolic machinery.

Chapter three had three specific aims; the first was to determine the thermal

tolerance of mouse intestinal tissue using an in vitro model of gut permeability. The data

show that the mouse small intestine has an inherent thermal tolerance within the range of

temperatures it is likely to experience during intense exercise. Interestingly, the

temperature which causes dysfunction in the intestine is lower than what was observed in

muscle, possibly due to muscle being a heat generator and the intestine seeing core

temperatures in normal conditions. The second aim was to evaluate the possible

protective or detrimental effects of inhibition of various eicosanoid metabolic pathways

on hyperthermia-induced intestinal permeability. The results of this study indicate that

the introduction of a few pharmacological agents (responsible for eicosanoid metabolism)

can protect the mouse intestine from hyperthermia-induced permeability. Interestingly, the lack of reproducible protection with multiple inhibitors of similar enzymes with chemically divergent mechanisms, leads one to believe that a more pharmacological

157 effect rather than a physiological effect is occurring in the intestine. These results show promise for developing treatment therapies, as the pharmacological agents used in this study are already approved for use in human treatments. Finally, the third aim was to test the hypothesis that ROS was responsible for loss of barrier function during hyperthermia by using the known scavengers tiron, Trolox and NAC. NAC protected the mouse intestine from hyperthermia-induced permeability, but not tiron or Trolox, which suggests that ROS able to be scavenged by free glutathione could play a role in intestinal dysfunction.

Chapter four had two aims; the first was to determine if tight junction opening or cell damage is the predominant pathway through which permeability is increased during hyperthermia in the mouse intestine. The results suggest that cell damage is the predominant cause of increased intestinal permeability during hyperthermia. The second aim was to discover how baicalein and NAC protect the mouse intestine from hyperthermia-induced permeability. Baicalein and NAC both reduce the amount of protein oxidation during hyperthermia which is a possible therapeutic mechanism. Also,

NAC reduced the extent of structural damage during hyperthermia. These results show that ROS formation is a cause for intestinal dysfunction during hyperthermia, and baicalein as well as NAC protect intestinal permeability by lowering the extent of oxidative stress and damage to the epithelial lining.

Understanding the pathways in which intestinal permeability and muscle function become susceptible to hyperthermia has great implications in developing treatments for heat stroke and other heat related illnesses. It is encouraging that some of the pharmacological agents used in this study are already approved for treatment in humans

158

in other areas. Further understanding of the mechanisms for hyperthermia-induced dysfunction goes beyond just scientific discovery, it allows for the development of new

treatment modalities.

Future directions

The main findings of this thesis open up several avenues of research which could

be pursued in greater detail. For example, the extent of eicosanoid metabolism

involvement in muscle dysfunction needs to be fully elucidated. The products of

eicosanoid metabolism are involved in several aspects of cellular function (Kuhn et al.,

2002) and could be directly connected to muscle function during hyperthermia. Treating

skeletal muscle with the products of LOX and COX during hyperthermia could increase

our understanding of how inhibiting these enzymes cause a reduction in muscle thermal

tolerance.

Pertaining to the intestine, immune system disturbances could play an important

role in permeability dysfunction because of their known ability to alter tight junction

regulation (Youakim & Ahdieh, 1999; Ceponis et al., 2000; Kinugasa et al., 2000;

Baumgart & Dignass, 2002), induce cell damage (Gitter et al., 2000; Baumgart &

Dignass, 2002) as well as alter eicosanoid metabolism (Akarasereenont et al., 1999;

Yucel-Lindberg et al., 1999). In animal models, agents which reduce the immune

response have been found to increase animal survival during heat exposure (Gathiram et

al., 1987; Lin et al., 1997; Liu et al., 2000). However, the role the immune system plays

during hyperthermia was out of the scope of this study, and could benefit from further

159

study. Inflammatory mediators such as tumor necrosis factor α and interferon-γ have been shown to cause increased intestinal permeability (Rodriguez et al., 1995; Fish et al.,

1999; Bruewer et al., 2003). Furthermore, the levels of these cytokines in the blood

stream are increased during hyperthermia (Leon, 2006). In particular, IL-6 knockout

animals have shown that mice undergoing hyperthermia have a lower survival rate than

controls, suggesting that IL-6 is partly involved in thermal regulation (Leon, 2006).

Hyperthermia-induced apoptosis has also been shown to occur with the assistance of

ROS (Katschinski et al., 2000) and could possibly be occurring in the time course studied

in this thesis. This avenue could be a very promising direction to take since dysfunctions

of the immune system are prevalent.

Furthermore, a whole body mechanism for eicosanoid metabolism could be occurring

during hyperthermia. Since muscles have been shown to produce products of eicosanoid

metabolism (Testa et al., 2007), prostaglandins and leukotrienes could enter the

circulation and elicit effects in other areas of the body which have been found to be

important during hyperthermia. Two main areas of interest are the intestine and the

blood brain barrier. The intestine is susceptible to hyperthermia, evidenced by the

findings of this thesis, but products of eicosanoid metabolism delivered from the systemic

circulation could affect the permeability of the intestine. Secondly, the blood brain

barrier has been shown to become permeable to potential toxins during hyperthermia,

most likely due to an increase in endothelial permeability (Jeliazkova-Mecheva et al.,

2006). Inhibitors of eicosanoid metabolism have been found to alter endothelial function

(Caughey et al., 2001) and could be important in the mechanism of blood brain barrier

dysfunction. The idea of eicosanoid metabolism eliciting systemic effects during

160 hyperthermia is a completely novel venture and could bring about greater understanding of how the body communicates as a whole during stressful events.

161

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