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,Departments of 1 Exercise Science, 2 Internal Medicine, and 3 Radiation Oncology, The University of Iowa, Iowa City, Iowa 52242-1111 and 4 Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico 87131-5271
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of this study was to characterize intestinal permeability changes over a range of physiologically relevant body temperatures in vivo and in vitro. Initially, FITC-dextran (4,000 Da), a large fluorescent molecule, was loaded into the small intestine of anesthetized rats. The rats were then maintained at ~37°C or heated over 90 min to a core body temperature of ~41, ~41.5, or ~42.5°C. Permeability was greater in the 42.5°C group compared with the 37, 41, or 41.5°C groups. Histological analysis revealed intestinal epithelial damage in heated groups. Everted intestinal sacs were then used to further characterize hyperthermia-induced intestinal permeability and to study the potential role of oxidative and nitrosative stress. Increased permeability to 4,000-Da FITC-dextran in both small intestinal and colonic sacs was observed at a temperature of 41.5-42°C compared with 37°C, along with widespread intestinal epithelial damage. Administration of antioxidant enzyme mimics or a nitric oxide synthase inhibitor did not reduce permeability due to heat stress, and tissue concentrations of a lipid peroxidation product were not altered by heat stress, suggesting that oxidative and nitrosative stress were not likely mediators of this phenomenon in vitro. In conclusion, hyperthermia produced increased permeability and marked intestinal epithelial damage both in vivo and in vitro, suggesting that thermal disruption of epithelial membranes contributes to the intestinal barrier dysfunction manifested with heat stress.
intestine; heat stress; free radicals; nitric oxide; FITC-dextran
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE GASTROINTESTINAL (GI) mucosa protects the internal environment of the body from bacteria and bacterial products found in the gut lumen such as endotoxin [lipopolysaccharide (LPS)] (15). This protective barrier is referred to as the "GI barrier." Dysfunction of this barrier leads to an increase in intestinal permeability, which involves the passive, nonmediated diffusion of both small and large molecules from the GI lumen to the blood (50). Increased intestinal permeability can result from numerous pathophysiological conditions, including hemorrhage and endotoxemia (47, 52), and has been observed after strenuous exercise (40) and exercise combined with nonsteroidal anti-inflammatory drug use (30, 43). LPS has also been shown to increase in the blood of human heat stroke victims (3), exhausted ultraendurance runners (5), heat-stressed primates (14), and hyperthermic rats (20).
These latter observations suggest that hyperthermia can increase
intestinal permeability in a variety of mammalian species. Moreover, it
has been hypothesized that passage of LPS from the GI lumen to the
blood, possibly because of a change in permeability in the intestine,
may be a serious consequence of severe heat stroke. For instance,
investigators have documented fatal cases of heat stroke in which
endotoxemia was believed to be a major etiologic factor (7,
16). Furthermore, Bouchama et al. (3) reported
endotoxemia in heat stroke patients with concomitant increases in tumor
necrosis factor-
and interleukin-1. These cytokines are known to
be primary mediators of endotoxic shock and tissue damage.
Interestingly, Bynum and colleagues (6) demonstrated
increased heat stroke survival in dogs after reduction of GI bacteria,
whereas the administration of anti-LPS hyperimmune plasma in primates
also improved heat stroke survival (13). The studies
suggest a definite link between GI permeability to LPS and heat stroke mortality.
However, the relationship between heat stress and GI permeability has not been systematically studied in terms of the core body temperature at which this permeability phenomenon occurs. The precise mechanisms for intestinal barrier dysfunction have also not been defined. Whole body hyperthermia causes a reduction in blood flow to the GI tract (28). This circulatory adjustment allows for a greater proportion of cardiac output to reach the periphery for heat dissipation (41) but also likely results in hypoxia (18), free radical production (19-21), ATP depletion, acidosis, and cellular dysfunction (15), which may contribute to a disruption of the GI barrier. Hall et al. (20) observed increased portal plasma LPS (indicating GI barrier dysfunction) in rats after heating to 41.5°C and related this response to increased oxidative stress. It has also been suggested that a primary mediator for intestinal permeability in some pathophysiological conditions is nitrosative stress (52), which also appears to occur during hyperthermia (19).
On the basis of these findings, the primary aims of the present investigation were to 1) determine the degree of heat stress necessary to produce a significant increase in intestinal permeability in vivo, 2) further characterize heat stress-induced intestinal permeability in an in vitro model (rat everted intestinal sac), and 3) explore the role of oxidative and/or nitrosative stress in this phenomenon. Intestinal permeability in both the in vivo and in vitro models was assessed by movement of fluorescein isothiocyanate-dextrans (FITC-dextrans) through the GI barrier. Use of these fluorescent "probes" allows for a simple, well-controlled, and highly sensitive method of assessing GI permeability during heat stress.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. All animal procedures were approved by The University of Iowa Animal Care and Use Committee. Male Sprague-Dawley rats weighing between 300 and 400 g were used for all protocols and were maintained on a 12:12 light-dark cycle and fed standard rat chow and water ad libitum. In everted colonic sac permeability studies, rats were placed on a liquid diet (GatorPro nutritional supplement; Quaker Oats) for 2 days before experiments. The liquid diet contained a laxative (Miralax; 4 g/500 ml) to soften fecal matter in the colon.
Determination of intestinal permeability in vivo. All experiments were carried out between 0700 and 1100 h. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and, by use of sterile surgical techniques, a midline abdominal incision (6-8 cm) was made. The renal arteries and small intestine at the ileocecal valve were ligated, an 18-gauge venous catheter was then inserted 1-2 cm distal to the pyloric sphincter, and a syringe containing 5 ml of FITC-dextran (FD-4; 4,000 Da; Sigma Chemical, St. Louis, MO; 25 mg/ml) in isotonic saline was connected to the catheter. The small intestine was then gently filled with this solution and ligated just distal to the catheter insertion site, and the abdomen was sutured closed. Colonic temperature (Tc) was obtained every 5 min by using a thermistor probe (YSI Instruments, Yellow Springs, OH) placed ~7-8 cm beyond the anal sphincter. Each animal was either kept on a heating pad to maintain Tc at ~37°C (control condition) for 90 min or placed under a heat lamp for a period of 90 min. The heating protocols were designed to increase Tc to ~40°C during the first 30 min (~0.1°C/min), then continue to slowly increase Tc to different peak levels over the final 60 min. These peak Tc values were either ~41, ~41.5, or ~42.5°C and were chosen to be slightly below (41°C), at (41.5°C), and slightly above (42.5°C) the 50% level (41.5°C) for heat stress mortality in rodents (25). Maintenance of anesthesia was accomplished by additional small doses of pentobarbital sodium as necessary.
Thermal area (°C · min) for each experiment was calculated as a means to quantify the level of heat stress by determining the number of minutes an animal spent above 40.4°C and multiplying this value by the average temperature during that time period (24). After the 90-min experiment, the abdomen was reopened and a heparinized blood sample (~3 ml) was collected from the abdominal aorta. The anesthetized animals were immediately euthanized by pentobarbital sodium overdose (100 mg/kg). The blood sample was centrifuged (4,000 rpm) for 10 min, and plasma FD-4 concentrations were determined by fluorescence spectrophotometry (see Analytical procedures below). Intestinal permeability was defined as the concentration of FD-4 found in the plasma after the different thermal conditions described above (52).Determination of intestinal permeability in vitro. A 6- to 8-cm midline abdominal incision was made in anesthetized rats. The intestinal area to be studied was resected and placed into a pan of chilled cell culture medium (M199; Sigma Chemical) containing L-glutamine (0.1 g/l; 6.8 mM) and sodium bicarbonate (2.2 g/l; 26 mM). The pH of this solution was ~7.4-7.6. Rats were then euthanized as described above. Resected intestinal segments were flushed with M199 medium and everted over a smooth glass or stainless steel pipette. The distal end was tied, and the proximal end was infused with M199 and ligated. The entire "loaded" segment was then sectioned into smaller "everted sacs" by tying sutures every ~2-4 cm along the segment and separating the sacs along the segment with surgical scissors. The sacs were randomly put into 25-ml Erlenmeyer flasks that had been placed in water baths set at the appropriate temperature for the experiment. The flasks contained 15 ml of pregassed (95% oxygen, 5% carbon dioxide) M199 containing 0.25 mM FD-4 or FD-10 (10,000-Da FITC-dextran). The flask was then sealed with an air-tight rubber stopper.
At the end of an experiment, the sacs were removed from the flasks and rinsed with M199, and the contents were released into a small tube by clipping one end with scissors. The volume of the sac contents was determined by the change in weight of the tube. A sample of this solution was subsequently analyzed for FD-4 or FD-10 concentration (see Analytical procedures). Flattened intestinal sacs were measured for length and width, and the surface area was calculated. Relative permeability (nmol/cm2 mucosal surface area) of everted sacs was calculated as transport of the FD-4 or FD-10 into the serosal fluid on the basis of the following equation
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70°C) for subsequent homogenization (Tekmar Tissumizer, Tekmar, Cincinnati, OH) and determination of total protein (see below). A sample of the
M199 bathing solution was also obtained for determination of cell
viability [lactate dehydrogenase (LDH) activity; see Analytical procedures].
Antioxidant enzyme mimic experiments. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; a superoxide dismutase mimic) and Ebselen [2-phenyl-1,2-benzoisoselenazol-3(2H)-one; a glutathione peroxidase mimic] were purchased from Calbiochem-Novabiochem (La Jolla, CA). A 100 mM stock solution of Tempol was initially made by dissolving the solid in M199. This solution was then added in appropriate volumes to 15 ml of M199 (in each incubation flask) to give final concentrations of 1, 5, and 10 mM. This concentration range has been used successfully to attenuate reactive oxygen species (ROS)-mediated DNA damage cell killing (35). Ebselen was initially dissolved in 100% ethanol, then diluted in 15 ml M199 to final concentrations of 10, 25, and 50 µM. These concentrations also bracket what has been found to be effective in reducing oxidative damage (i.e., lipid peroxidation) in isolated hepatocytes (37). After addition of these compounds to the incubation flasks, everted intestinal sacs were placed in the flasks and the same protocol described above was conducted.
Iron chelation/ascorbate experiments. In this set of experiments, Chelex 100 (iron-chelating resin; Sigma Chemical) was added (10 ml/l) to the M199 and gently stirred overnight in a refrigerator. The following day, a second iron chelator, Desferal (desferrioxamine mesylate; 75 µM; Sigma Chemical) and a low concentration of the antioxidant ascorbate (100 µM) were added to the M199 to further increase its antioxidant capacity. This solution was then used as the medium for intestinal permeability experiments conducted in the same manner as described above.
Lipid peroxidation experiments. In some experiments, malondialdehyde (MDA; lipid peroxidation product) concentration was determined in everted gut samples. These samples were homogenized in 20 mM Tris buffer (pH 7.4), with butylated hydroxytoluene in acetonitrile (10 µl; 0.5 M) added before homogenization to prevent oxidation during processing of the sample. A commercially available kit was used (Bioxytech LPO-586 Colorimetric assay for lipid peroxidation, OXIS International, Portland, OR) to determine MDA concentration. Tissue homogenates were also measured for protein content (see Analytical procedures), with MDA concentration expressed as nanomoles per minute per milligram protein.
L-NAME experiments. In these experiments, NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, was dissolved in M199 to a concentration of 100 mM. This stock solution was then diluted by adding appropriate volumes to incubation flasks containing 15 ml of M199. The final concentrations of L-NAME were 100, 500, and 1,000 µM (45), and experiments were conducted as described above.
Analytical procedures. In vivo plasma samples and everted intestinal sac serosal fluid samples were diluted in either phosphate-buffered saline (plasma) or M199 (everted sac fluid), and FD-4 or FD-10 concentration was determined by fluorescence spectrophotometry (Hitachi F-2000 or F-2500 fluorescence spectrophotometer, Hitachi Instruments, San Jose, CA). The same excitation wavelength (492 nm), excitation slit width (10 nm), emission wavelength (517 nm), and emission slit width (10 nm) were maintained for all measurements. Fluorescence intensity of a sample was converted to a FD-4 or FD-10 concentration by using standard curves generated for each experiment. Total protein content (Bradford method) and LDH activity were measured spectrophotometrically by use of commercially available kits (Sigma Chemical).
Histological analysis. At the conclusion of both in vivo and in vitro experiments, intestinal tissue was obtained for morphological assessment of epithelial damage as previously described (19-21). The tissue was initially placed in either 10% neutral-buffered formalin for subsequent processing and staining (hematoxylin and eosin) for LM or placed in 2.5% glutaraldehyde (in sodium cacodylate buffer, pH 7.2) for processing and staining (lead citrate and uranyl acetate) for TEM.
Statistical analysis. In vivo data were analyzed by using a factorial one-way ANOVA. The Fisher protected least significant difference post hoc test was employed to identify significant differences among groups. Either a one-way or multi-way within-subjects repeated-measures ANOVA was employed for statistical analysis of the in vitro everted sac data. Tukey's honest significant difference post hoc test was utilized to identify significant differences as necessary. The level of significance was set at P < 0.05 for all comparisons. Data are presented as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In vivo study.
The highest level of heat stress (42.5°C) produced significantly
greater (P < 0.05) permeability to FD-4 than lower
levels of hyperthermia in the in vivo experiments (Fig.
1). Increasing the peak Tc
among groups also resulted in a progressive elevation in thermal loads
and heating rate (Fig. 1). Heat stress resulted in profound damage to
the epithelium of the small intestine. The intestine of rats heated to
42.5°C demonstrated vacuolization of epithelial cells and actual
sloughing of epithelium off the basement membrane at the villus tips
(Fig. 2). To further assess the effect of
hyperthermia on epithelial morphology, we performed electron microscopy
on intestinal samples (Fig. 3).
Heat-stressed enterocytes demonstrated significant damage to the
luminal membrane with loss of microvilli. Interestingly, the tight
junctions appeared intact, and the mitochondria in the heat-stressed
enterocytes were morphologically normal.
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In vitro studies.
Figure 4 depicts the viability data
obtained from the everted intestinal sac experiments. The time course
for permeability changes (FD-4 transport) and cell damage (LDH release)
over a 120-min time course in the 37°C (control) condition were very similar. The data indicate that loss of viability (i.e., increased permeability and LDH release) of the model occurred at ~90 min. In
conjunction with these results, normal intestinal epithelial morphology
was observed through 60 min of control conditions (see below). These
findings allowed further studies using this model to assess the effects
of heat stress on intestinal permeability.
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1 · mg protein1,
respectively; n = 7) in everted intestinal sac tissue
homogenates.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study is the first to characterize small intestinal permeability changes in vivo over a range of physiologically relevant body temperatures utilizing highly sensitive fluorescent permeability probes (FITC-dextrans). It is also the first to utilize the everted intestinal sac model to examine the effect of hyperthermia on intestinal permeability. The study yielded three primary findings. First, significant increases in small intestinal permeability occurred in rats heated to a Tc of ~42.5°C over 90 min (mean Tc = ~41.5°C over final 60 min). Second, the rat everted intestinal sac preparation exhibited increases in permeability similar to those observed in vivo, which permitted us to use this model to further characterize hyperthermia-induced intestinal permeability. Third, oxidative and nitrosative stress were not found to be primary mediators of hyperthermia-induced intestinal permeability in the rat everted intestinal sac.
The significance of intestinal barrier dysfunction during heat stress is related to the potential pathological conditions it may induce. It has been documented that some heat stroke victims do not necessarily succumb to the initial hyperthermic episode but rather to a sepsislike condition that follows (2, 16). This condition is thought to be mediated by leakage of LPS from the intestinal lumen into the circulation, leading to an immune (i.e., cytokine) response that can culminate in cardiovascular collapse, disseminated intravascular coagulation, and multiple-organ failure (15). Accordingly, Caridis et al. (7) and Graber et al. (16) each described cases of fatal heat stroke in which the major symptoms included GI bleeding, endotoxemia, disseminated intravascular coagulation, and renal failure. In support of the concept that LPS leakage from the GI tract is involved in the etiology of heat stroke mortality, Bynum and colleagues (6) demonstrated that reduction of bacterial contents in dogs increased survival after experimental heat stroke. Furthermore, Gathiram et al. (13) observed in heat-stressed primates that administration of anti-LPS hyperimmune plasma also significantly increased survival time.
In the present study, increased permeability occurred in vivo at a peak temperature of ~42.5°C and an average Tc of 41.5°C over the final 60 min of the experiment. These temperatures are similar to those in which permeability increased in the everted intestinal sacs (41.5-42°C). This Tc range is similar to that which has been shown to be the temperature for mortality in 50% of heat-stressed rodents (25) and which significantly increases portal LPS concentrations (20). Out of 125 cases of heat stroke studied by Malamud et al. (32), 86% had Tc values above 41.1°C on admission to the hospital. Bouchama et al. (2) reported the average Tc in heat stroke patients on admission (n = 25) to be 41.2°C, whereas O'Donnell (39) and Hart et al. (22) observed higher mean Tc values of 41.6°C (n = 15) and 42.5°C (n = 28), respectively. However, these values are likely underestimations of the actual peak Tc achieved by these patients, given that some cooling undoubtedly occurred during transport to the hospital. Thus the peak and mean Tc levels that increased GI permeability in the present study are likely representative of the core temperatures encountered both by rodents that succumb to heat stress and by many human heat stroke victims.
The in vivo model used in the present study was adapted from experiments described by Unno et al. (52). FD-4 has a molecular weight of 4,000 Da and a molecular radius of 1.4 nm, which is similar to inulin (molecular weight = 5,000 and molecular radius = 1.5 nm), a molecule known to have minimal permeability in the small intestine (31). FITC-dextrans have been used to determine intestinal permeability in animal models of hemorrhagic shock (42) and endotoxemia (52). Because our model most closely resembles that used by Unno et al., a comparison of our data to their results seems warranted. In the Unno et al. study, rats (same strain and similar size as the present study) were rendered endotoxemic by injection of LPS and were assessed for intestinal permeability 24 h later by loading the small intestine with FD-4 (same concentration used in the present investigation). Plasma values of FD-4 peaked at 60 min postloading, with the highest value reaching ~3.6 µg/ml in the endotoxemic group. This value is approximately three- to fourfold lower than the value obtained during the highest heat stress condition in the present study and underscores the severe intestinal barrier dysfunction that hyperthermia can produce.
The everted intestinal sac has been utilized for many years to study both absorption and permeability characteristics of the intestinal tract. It has been modified over time to permit studies to be conducted on carbohydrate absorption (11), drug absorption (9), LPS permeability (38), and permeability to other large molecules (8). Recently Barthe et al. (1) reported an improved method with greater viability of the sacs using cell culture medium (i.e., M199) rather than buffers for incubation and loading of the sacs. In the present study, viability of everted intestinal sacs was assessed by examining changes in permeability to FD-4 over time along with measurement of LDH release by the sacs into the bathing medium (Fig. 4). The sacs did not become significantly more permeable to FD-4 nor increase release of LDH until 90 min under control conditions (37°C). Histological assessment of the epithelium also served as a guide to viability over time. As highlighted in the TEM photomicrographs (Fig. 8), the sacs maintained normal epithelial morphology during the course of the experiments (i.e., no changes in the integrity of the cellular membranes or apical interjunctional complexes). These findings are in agreement with those reported by Barthe et al. However, when the sac bathing temperature was increased to 41.5-42°C, a marked increase in permeability (Figs. 5 and 6) and epithelial damage was observed (Fig. 9).
Previously, Shapiro et al. (48) examined 125I-LPS permeation in noneverted intestinal gut sacs. The sacs were incubated in Tyrode's solution at either 37 or 45°C, and, as in the present study, the authors observed increased permeability due to heat stress. However, cautious interpretation of their results should be made because of the nonphysiological level of heat stress (45°C) imposed.
The mechanism(s) responsible for increased intestinal permeability and epithelial damage with heat stress are unclear. Hall et al. (18) found evidence of hypoxia in the intestinal villi of heat-stressed rodents. Furthermore, the same group has reported increased production of ROS and reactive nitrogen species (RNS) in the portal blood of heat-stressed rats, suggesting increased production of these radicals in the intestine (19, 20). These investigators further observed that significantly increased LPS concentrations in the portal blood of heat-stressed animals could be reduced by administration of the xanthine oxidase antagonist allopurinol (implicating ROS in intestinal permeability changes during hyperthermia). The mechanism that stimulates increased ROS generation during hyperthermia, though, has not been clarified. Kregel et al. (28) noted that superior mesenteric artery blood flow declines significantly (up to 50%) with heat stress, then increases sharply at a Tc of ~41.5°C. This effectively simulates mild ischemia-reperfusion (I/R) of the gut. I/R is well known to increase intestinal epithelial damage and permeability, likely via increased ROS production (23). Heat itself has also been shown to increase ROS production in cells (12) and appears to play a role in hyperthermia-induced apoptosis (27).
In the present experiments, we studied the direct effect of heat stress (i.e., no influence of hypoxia or I/R) on the intestinal barrier in our everted intestinal sac model. However, our attempts to attenuate hyperthermia-induced permeability in this model by reducing oxidative and/or nitrosative stress (i.e., Tempol, Ebselen, L-NAME administration) were unsuccessful. As previously mentioned, GI barrier dysfunction has been attenuated during heat stress in vivo (20) by reducing superoxide production. This has also been observed after I/R (53). Furthermore, Sorrells et al. (49) and Unno and colleagues (52) have shown that NOS inhibition during endotoxemia can significantly reduce GI permeability. Nitric oxide donor administration also induces intestinal cell monolayer hyperpermeability in vitro (34, 46). Interestingly, nitric oxide appears to also be protective in the intestinal tract. For instance, Hall et al. (20) found that NOS inhibition during heat stress in vivo induced a further increase in portal blood LPS. Kubes (29) also noted that inhibition of NOS augmented GI permeability during I/R. This observation is supported by the findings of Kanwar et al. (26), who demonstrated increased gut permeability during NOS inhibition under basal conditions in vivo.
The present results, along with the finding that lipid peroxidation was not increased with heat stress, strongly suggest that ROS and RNS did not play major roles in the permeability changes observed in the everted intestinal sac. A possible reason for the lack of ROS or RNS effect in this model compared with the in vivo findings of Hall et al. (20) is that the present experiments examined the direct effect of hyperthermia on intestinal permeability. Although hyperthermia has been shown to independently increase radical flux (12, 54), it is likely that during and after heat stress in vivo even greater amounts of ROS and/or RNS are generated because of I/R of the intestine (23, 53) and release of inflammatory mediators such as cytokines (2, 3). Thus it appears that high temperature alone (for 60 min) does not induce sufficient oxidative or nitrosative stress in the everted intestinal sac to cause the observed disruption in the GI barrier.
Other possible mechanisms for hyperthermia-induced intestinal hyperpermeability include 1) acidosis due to hyperthermia (4) or hypoxia (44) and 2) ATP depletion due to mitochondrial damage (10, 17), uncoupling of oxidative phosphorylation (17), glycolytic inhibition, or hypoxia (44). Acidosis and ATP depletion have both been shown to increase intestinal permeability either in vivo or in vitro (33, 44, 51). Another possible mechanism for intestinal barrier dysfunction is direct thermal damage to the epithelial cell membranes.
In both the in vivo and in vitro studies, histological assessment revealed hyperthermia-induced epithelial damage (i.e., epithelial sloughing, subepithelial edema, membrane damage, cellular swelling, and vacuolization). This damage may be due to some of the mechanisms previously mentioned and indicates that permeability to large molecules is likely via damaged cell membranes and/or lesioned mucosa. The paracellular pathway (via "opened" tight junctions) is also a likely route on the basis of previous findings by Moseley et al. (36) that hyperthermia (41.3°C) increased permeability to mannitol in MDCK cell monolayers. Surprisingly, a defective tight junction was not detected in the TEM images obtained after heat stress in vivo despite evidence of severe membrane damage. The reason for this observation could be that the tissue was not fixed in vivo and any "opening" may have "closed" when the tissue was removed for fixation.
In summary, findings from the present study indicate that high levels of hyperthermia increase intestinal permeability to large molecules. Both in vivo and in vitro results indicate this permeability change occurs at temperatures of ~41.5-42°C (when sustained for ~60 min). This Tc corresponds to the core temperature at which increased incidence of heat stroke occurs in rodents and humans. The exact mechanisms underlying intestinal barrier dysfunction during heat stress are not known but were not found to be related to the production of ROS and/or RNS in the intestinal tissue. On the basis of histological findings in the present study, a likely mechanism is thermal disruption of intestinal epithelial membranes that results in enterocyte necrosis and increased permeability of the GI barrier.
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ACKNOWLEDGEMENTS |
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We thank Ronald Matthes for excellent technical assistance, Kathy Walters and Dr. Paul Heidger for histological expertise, Dr. William Clarke for statistical consultation, Dr. Garry Buettner for helpful advice, and Joan Seye for manuscript preparation.
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FOOTNOTES |
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Deceased 3 June 2000.
This research was supported by National Institutes of Health Grants HL-61389, AG-12350, AG-14687, and AR-40771.
Address for reprint requests and other correspondence: K. C. Kregel, Integrative Physiology Laboratory, 532 Field House, The Univ. of Iowa, Iowa City, IA 52242-1111 (E-mail: kevin-kregel{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 2, 2001;10.1152/japplphysiol.00787.2001
Received 26 July 2001; accepted in final form 31 October 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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