Sepsis or endotoxemia is a severe inflammatory disorder and a serious clinical problem with high mortality (2,4,6). This condition may lead to severe shock and multiple organ failure (17,29) and is one of the leading causes of mortality in critically ill patients (2,29). Lipopolysaccharide (LPS) derived from the outer member of gram-negative bacteria is strongly associated with septic shock, and it has been used for the induction of endotoxemia in animal experimentation (17,18). Binding of LPS to the CD14 receptor on the cell membrane results in the activation of monocytes, macrophages, neutrophils, and endothelial cells (5,17). Consequently, LPS induces the release of proinflammatory mediators including tumor necrosis factorα (TNFα), interleukin (IL)-1β, and nitric oxide (NO) (17,18,29).
It is generally known that adequate and appropriate exercise is essential to maintain health and to release work pressure (3,8,15,16). On the other hand, strenuous or exhaustive exercise may cause body injury and produce oxygen radical species, resulting in organ damage (1,9). Several studies have demonstrated that regular exercise can enhance immunological and antioxidant functions (20,23). Starkie et al. (27) suggest that appropriate exercise in humans increases the plasma antiinflammatory cytokine IL-6 and suppresses the endotoxemia-induced elevation in TNFα. They conclude that exercise exerts antiinflammatory effects.
We hypothesize that exercise training may attenuate inflammatory responses to sepsis and to organ dysfunction and damage. To test this hypothesis, the present investigation was designed to evaluate the effects of regular exercise on the endotoxin-induced changes in conscious rats without anesthesia. The conscious rat model was used to minimize the possible influence of anesthetics on the sepsis-induced changes and to prolong the observation time to as long as 72 h. After endotoxemia, we observed the changes in arterial pressure, heart rate (HR), blood cells, biochemical factors, and proinflammatory cytokines. Histopathological examinations of the heart, lung, and liver were performed and assessed. These changes were compared between rats with regular and gradual increases in exercise training and those remaining sedentary to elucidate the effectiveness of exercise training.
Male Wistar-Kyoto strain rats were used. Their body weight ranged from 320 to 350 g, and they were 10 wk old. The animals were obtained from the National Animal Research Center and were housed in the university animal center with adequate air, temperature, and light control. Water and food were provided ad libitum. The care and use of experimental animals was approved by the university committee of animal research. In addition, the university and the National Science Council experimental animal committee gave permission and supported the animal experiments. We followed the guidelines of the National and University Animal Research Center for animal experimentation.
The animals were randomly divided into an exercise training group (Tr; N = 12) and a control group without exercise training (Con; N = 12). Exercise training followed the protocol from low to high speed. The rats were placed on a treadmill with an electrode on the end of the machine. Electric shock was used to encourage the animals to run on the treadmill. Some animals refused to run even with electric shock and were not used. The animals received 2-wk preexperimental exercise for familiarization to the treadmill. The exercise machine was kept at an angle of 30°. The Tr group ran at a speed of 10 m·min−1 for 10 min, four to six times a day. For regular and gradual increase in exercise training, rats received daily exercise training at 12 m·min−1 for 15 min during the first week. The intensity of daily exercise was increased to 15 m·min−1 for 30 min on the second week, 18 m·min−1 for 45 min on the third week, and 21 m·min−1 for 60 min on the fourth week. The Con group was placed on the treadmill rather than running. They rested quietly on the treadmill for the same time intervals. They also received electric shocks four to six times per day. Electric shocks were given when the animals reached and stayed at the end of the treadmill.
After the exercise training, rats entered acute experiments designed to induce endotoxemia. We used a conscious, unrestrained model recently developed in our laboratory (19). Rats were anesthetized with ether inhalation for about 10 min. During the period of anesthesia, a femoral artery was catheterized and connected to a pressure transducer (Gould Instruments, Cleveland, OH) to record the arterial pressure and HR on a polygraph recorder (Power Lab, AD Instruments, Mountain View, CA). The arterial pressure and HR were continuously monitored for 72 h.
A femoral vein was cannulated for i.v. administration of fluid or drugs and for collection of blood samples. The operation was performed under an aseptic condition, and the incision was less than 0.5 cm2. In addition, it was completed in 15 min. Local antiseptic agent was applied to the incision wound, which was closed by suture. After the operation, the rats were placed in a metabolic cage and awakened soon thereafter. Each rat's tail was fixed by a piece of tape. This simple procedure allowed experimentation in a conscious state without anesthesia. The rats were free to move their heads and had access to food and water, but they could not escape from their cages. The animals seemed comfortable and were only minimally restrained (6,17-19).
Induction of endotoxemia.
Endotoxin (LPS, E. coli, Sigma Chemical, St. Louis, MO) at a dose of 10 mg·kg−1 was infused into the femoral vein for a period of 20 min. After LPS administration, the animals were continuously observed for 72 h.
Blood samples (0.5 mL) were obtained from the femoral vein 1 h before and 0.5, 1, 3, 6, 9, 12, 18, 24, 36, 48, and 72 h after LPS administration. The same volume of blood was given to supplement blood loss via the femoral vein. The donor blood was obtained from other rats of the same strain. The supplement of blood loss from donors did not cause discernible adverse effects in 18 out of 20 pilot tests. If signs of discomfort and/or rapid decline in arterial pressure were observed, the data were not used. White blood cell (WBC), lymphocyte, neutrophil, and red blood cell (RBC) counts were determined by a blood cell analyzer (Sysmex K-100, New York, NY).
Blood samples were immediately centrifuged at 3000g for 10 min. Plasma was stored at 4°C for biochemical examination 30 min after collection. Plasma samples were diluted 1:100 with distilled water before measurements. Blood urea nitrogen (BUN), creatinine (Cr), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), amylase, and lipase were measured with an autoanalyzer (Vitros 750, Johnson and Johnson, Rochester, NY) for the evaluation of various organ functions, that is, BUN and Cr for the kidney, AST and ALT for the liver, CPK and LDH for the heart, and amylase and lipase for the pancreas and other organs. The plasma nitrite/nitrate as NO metabolites were measured with high-performance liquid chromatography (HPLC) as described previously (18). In brief, about 20 μL of plasma supernatant was mixed with 20 μL of methanol. The mixture was immediately centrifuged at 15,000g for 10 min at 4°C. The supernatant was used for nitrite/nitrate measurement with an NOx analyzer (ENO 20, AD Instruments, Mountain View, CA). The formation of methyl guanidine (MG) is an index of hydroxyl radical production in the blood (6,18). It was measured with HPLC using a guanidino pack column (Jasco 821-FP, Spectroscopic Co., Tokyo, Japan) packed with a strong acid cation-exchange resin. The emission maximum was set at 500 nm, and the excitation maximum was set at 395 nm. The assay was calibrated with authentic MG (Sigma Chemical., St. Louis, MO).
TNFα and IL-1β.
TNFα and IL-1β were measured using antibody enzyme-linked immunosorbent assays (ELISAs) with commercial antibody pair, recombinant standards, and a biotin streptavidin-peroxidase detection system (Endogen, Rockfrod, IL) (6,7). All reagents, samples, and working standards were prepared at room temperature according to the manufacturer's directions. The optical density was measured at 450- and 540-nm wavelengths by an automated ELISA reader.
At the end of the experiment, the rats were euthanized by an overdose of sodium pentobarbital (100 mg·kg−1, i.v.). The heart, liver, and lung were removed. The organs were cut into small pieces and embedded in paraffin. For microscopic examinations, the tissue was sectioned at 5 μm and stained with hematoxylin and eosin. For quantification of the organ injury score, the pathological micrographs were evaluated for the degree of inflammatory changes and necrotic lesions in a blind fashion by several laboratory assistants. The assessment of organ injury score was as follows: degree 0-4 for no, mild, moderate, and severe inflammation; and similar grading for necrotic lesions: degree 0-4 for no, mild, moderate, and severe necrosis. Each one gave a score from 0 to 8. The average total score was taken as an indicator of the extent of organ damage.
All data were expressed as mean ± SEM. Comparisons within and among groups were made using two-way analysis of variance with repeated measures, followed by a post hoc comparison with Newman-Keul test. Differences were considered to be statistically significant at P < 0.05.
Mean arterial pressure (MAP) and HR.
LPS caused a biphasic systemic hypotension. The extent of the first and second hypotensive phase was greater in the Con group than in the Tr group. HR increased after LPS administration. The LPS-induced tachycardia was also greater in the Con group than in the Tr group (Fig. 1). In addition, basal levels of MAP and HR were lower in the Tr group than in the Con group.
LPS induced time-dependent decreases in WBC, lymphocytes, neutrophils, and RBC (Fig. 2). The changes were, in general, more pronounced in the Con group than in the Tr group. There was a transient increase in RBC at 1-3 h after LPS in both Tr and Con rats. Thereafter, the erythrocytes declined progressively. The late phase of erythrocytopenia was much less in Con rats than in Tr rats. The basal counts of WBC and lymphocytes were lower in Con rats than in the Tr group. The basal neutrophil count, however, was reduced after exercise training. There was no difference in basal RBC between the two groups.
BUN, Cr, amylase, and lipase.
LPS increased BUN, Cr, amylase, and lipase. The time-dependent changes in these biochemical substances were greater in Con rats than in Tr rats (Fig. 3).
AST, ALT, CPK, and LDH.
The time-dependent elevations in AST, ALT, CPK, and LDH after LPS administration were attenuated in the Tr group compared with the Con group (Fig. 4).
Nitrite/nitrate, MG, TNFα, and IL-1β.
The NO metabolites, hydroxyl radical, and proinflammatory cytokines increased to their peaks at different times after LPS. The changes were significantly reduced in rats with exercise training (Fig. 5).
LPS caused inflammatory and/or necrotic lesions in the heart, liver, and lung in the Con group. The cardiac, hepatic, and pulmonary lesions were not discernible in rats with exercise training (Fig. 6). Assessment of organ injury score revealed that the endotoxin-induced organ damage was significantly attenuated in rats with exercise training compared with rats that remained sedentary (Table 1).
Administration of endotoxin or LPS caused septicemia or septic shock characterized by systemic hypotension, tachycardia, and leukocytopenia. Biochemical changes including increases in BUN, Cr, AST, ALT, CPK, LDH, amylase, and lipase were the early markers of multiple organ injury. Furthermore, plasma nitrate/nitrite and MG were elevated, indicating enhanced production of NO and free radicals during septicemia. The septicemia-induced increases in proinflammatory cytokines such as TNFα and IL-1β were indicators of systemic inflammation. Finally, pathological changes such as inflammatory and/or necrotic lesions occurred in multiple organs including the heart, liver, lung, and others. These changes, which were attributable to septicemia or endotoxemia associated with systemic inflammatory responses, were more pronounced in conscious animals without anesthesia than in anesthetized rats (6,7,17,18).
The present study was conducted in conscious rats to minimize the possible interference of anesthetics. Our results reveal that regular exercise attenuated the septic responses and associated alterations. Exercise reduced the biphasic systemic hypotension (septic shock). It also mitigated the endotoxin-induced tachycardia and decreased the basal mean arterial pressure and HR. The endotoxin-induced leukocytopenia and lymphocytopenia and the basal count of neutrophil were reduced after exercise training. In addition, the endotoxin-induced increases in biochemical factors such as BUN, Cr, CPK, LDH, AST, ALT, amylase, and lipase were diminished after exercise training. Exercise training also attenuated the endotoxin-induced release of NO, free radicals, and proinflammatory cytokines (TNFα and Il-1β). Our results demonstrate that the early and late alterations of inflammatory responses to septicemia were attenuated in rats with exercise training. Consequently, the pathological changes in the heart, liver, and lung were abrogated by exercise training.
The nature of exercise is complex. The balance between beneficial and detrimental effects depends on many factors such as age, gender, and the extent of exercise. In general, regular exercise training tends to reduce basal arterial pressure and HR (10,26). We had similar findings in thisstudy. The effects of exercise on the immune system are still controversial (22). Mild exercise may enhance immunity, but strenuous and exhaustive exercise may be harmful to the body's defense system (3,25). It has been shown that athletes after heavy and intensive exercise training are more susceptible to infections (11). Exhaustive physical activity increases the release of cytokines that are toxic to cells (24,25). A recent study found that the plasma concentration of antiinflammatory cytokine (IL-6) was significantly elevated in humans bicycling at 75% of maximal oxygen consumption for 3 h. The physical activity also attenuated the increase in TNFα after an i.v. infusion of a low dose of LPS to induce low-grade inflammation. The study strongly suggests that a recommended dose of physical activity exerts an antiinflammatory effect (27). We did not measure the plasma IL-6 in the current study. Nevertheless, our results demonstrating that exercise attenuated the endotoxin-induced increases in proinflammatory cytokines (TNFα and IL-1β) support the contention that regular and appropriate exercise elicits antiinflammatory actions.
The decreases in blood cellular elements including WBC, neutrophils, lymphocytes, and RBC after administration of LPS have been reported in many animal experiments (7,17,18). Activation of leukocytes, lymphocytes, and macrophages has been implicated in the pathogenesis of sepsis-associated formation of NO, free radicals, and proinflammatory cytokines (5,17). However, the ultimate mechanisms of changes in blood cellular elements during sepsis remain unclear. We also observed a transient increase in RBC, followed by a gradual decline. The early increase in RBC and the late erythrocytopenia were greater in the Tr group than in the Con group. In addition, exercise reduced the basal counts of neutrophil, but it did not affect basal RBC before endotoxin administration. The interactions between exercise and sepsis on blood cells may be complicated, requiring further investigation.
Under septic or inflammatory conditions, inducible NO synthase in endothelial cells, vascular smooth-muscle cells, and fibroblasts is activated (5,17). NO is a free radical. It also leads to the formation of hydroxyl radical, superoxide anion, and peroxynitrite, which are extremely toxic to cells, tissues, and organs. Although endogenous NO is pivotal for the maintenance of homeostatic conditions, overproduction of NO may be harmful in certain pathological conditions such as sepsis, infections, and inflammation (6,14,28). Recent studies from our laboratory have found that endogenous and exogenous NO were detrimental to the lungs after endotoxemia (6,7). In this regard, the reduction of plasma nitrate/nitrite and MG may account, at least in part, for the benefits of exercise training. On the other hand, exhaustive aerobic and isometric exercise increased NO and free radical production because of increases in oxygen consumption and shear stress of the vascular walls, resulting in endothelial release of NO and reactive oxygen species (1). Whether strenuous exercise produces detrimental effects in septic humans and animals is a subject deserving investigation.
Despite extensive investigation on the prevention and treatment of endotoxemia and septic shock, the results have been not very successful at the present time (21,29). Zeni et al. (30) have reviewed clinical trials using various antiinflammatory therapies for sepsis and septic shock. The therapeutic regimen included antagonists against IL-1 receptor, bradykinin, platelet-acting factor, TNFα, prostaglandins, and endotoxin. The mortality rate in clinically ill patients has varied in different clinical reports. It seems that the overall evaluation of the antiinflammatory therapies was not very positive. Recent studies from our laboratory have reported that insulin and N-acetylcysteine exerted antiinflammatory effects on endotoxin-induced biochemical and pathophysiological abnormalities (6,12,13). The present study has provided evidence to indicate that exercise training improves the septic condition and associated responses. N-acetylcysteine has been known as an antioxidant. Previous studies have suggested that regular exercise is able to enhance the immunological and antioxidant functions (20,23). The link between the effects of exercise and insulin is a subject for further investigation.
In conclusion, exercise training attenuates endotoxin-induced systemic hypotension, tachycardia, decreases in blood cells, increases in biochemical factors, nitrite/nitrate, MG, and proinflammatory cytokines (TNFα and IL-1β). In addition, exercise training mitigates the biochemical indicators for cardiac, hepatic, and, possibly, other organ dysfunction. Consequently, the pathological changes in the heart, liver, and lung after endotoxemia were abrogated in rats with regular exercise training.
This study was supported in part by grants from the National Science Council (NSC 95-2320-B-320-004 and NSC 95-2320-B-320-008). The authors are grateful to Ms. Lucy Chen and A. Huang for the manuscript preparation.
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Keywords:©2007The American College of Sports Medicine
EXERCISE; SEPSIS; ORGAN INJURY; NITRIC OXIDE; FREE RADICALS; PROINFLAMMATORY CYTOKINES