There is a growing interest in the relationship between lifestyle factors and health. Physical activity has been linked to lower disease rates, such as reduced risk of cardiovascular disease and diabetes (15). The positive effects of physical activity on health could be due to a number of adaptations in physiology, including alterations in immunity, lipid profiles, and muscle physiology.
Many studies have suggested exercise training induces alterations in immune parameters. This work has demonstrated alterations in lymphocyte subpopulations (9,10) and leukocyte populations (23,27) after treadmill exercise in rats. Other work has shown decreased tumor retention in the lungs of both freewheel- and treadmill-trained animals (12). This decrease in tumor retention occurred in the freewheel animals despite lack of increase in training enzymes such as citrate synthase, demonstrating immune alterations may be independent of training adaptations. Although it is clear that exercise can change measures of immune cells and function, most of these effects have been both acute and transient; thus, the effect of exercise on basal immunological parameters remains unclear.
The existing human and animal literature on exercise and immunity has some limitations. First, the impact of acute, high-intensity exercise on circulating white blood cell composition may reflect only temporary changes in the migration of leukocytes, and this may have little impact on host defense. Second, the majority of animal studies thus far have focused on forced treadmill exercise or swimming exercise, which allows for experimenter control of exercise duration and intensity. Treadmill- and swim-trained rats demonstrate many hallmarks of chronic stress, such as adrenal hypertrophy (16), and this chronic stress alters immune parameters (16). In addition, treadmill training typically disrupts the natural activity pattern of rats with regard to duration and circadian timing of activity. Rats that are allowed free access to a running wheel will chose to run varying distances intermittently during the dark cycle, running in short bursts (12). This is in contrast to treadmill studies, which use steady state training during the light cycle. Freewheel running may therefore serve as an appropriate model of physical activity for those who incorporate motion, such as climbing a flight of stairs, into their lifestyle and activities of daily living, as opposed to highly trained subjects.
Altered lipid profiles are a risk factor for cardiovascular health, and exercise training is known to improve lipid profiles. Application of previous findings is limited, as most of the studies have tested the effect of exercise on the obese Zucker rat (24). Although this is advantageous because of the elevated cholesterol and triglyceride levels found in these rats, it makes comparisons with other strains more difficult. This literature also focuses on treadmill or swim exercise, with only one study examining the effects of voluntary freewheel exercise in F344 rats (28). This study did not produce a significant reduction in total cholesterol, but it did find a 53% decrease in triglycerides with a 13% increase in HDL. Although these results are exciting, the freewheel-running protocol used was 35 wk in duration, making it logistically difficult for most studies.
Exercise has been shown to increase circulating red blood cell number (19). This is a positive adaptation to exercise because an increase in red blood cell number could facilitate oxygen availability to exercising muscle. In contrast, intense exercise can decrease circulating red blood cells due to the increased oxidative stress and trauma of exercise (21). The effect of exercise on red blood cells has been primarily addressed using special circumstances, such as anemia and hypoxia. Although these conditions magnify the effect of exercise, it is again difficult to apply these conclusions to rats under normal conditions.
Exercise training affects muscle physiology. For example, increases in oxidative enzymes are a commonly reported finding in trained animals. Treadmill training induces alterations in muscle enzymes, such as an increase in citrate synthase in the medial long head of the triceps after 12 wk (22). The effects of treadmill training may not be generalized to voluntary exercise. For example, one study that included both forms of exercise found increases in citrate synthase in the treadmill-trained animals alone, without effect of voluntary activity on soleus muscle (12). Thus, the effects of voluntary exercise on muscle physiology remain to be elucidated.
As physical activity holds promise as an intervention to change physiological status, along with the limitations of the previous literature, it is of interest to establish the baseline alterations in hematological characteristics caused by freewheel exercise. The purpose of the following study was to test the effects of four weeks of voluntary freewheel running in rats on white blood cell differentials, red blood cell hemoglobin, blood lipid profiles, and muscle enzyme activity.
MATERIALS AND METHODS
Adult male Fisher 344 (Harlan (NIA)), 4-month-old pathogen-free rats were used. Animals were given standard rat chow and water ad libitum. Rats were singly housed in Nalgene plexiglass cages (45 × 25.2 × 14.68 cm) in a pathogen-free barrier facility. All experimental procedures received approval from the Institutional Animal Care and Use Committee at the University of Colorado-Boulder. In addition, such procedures were in compliance with the policies and procedures detailed in the Guide for the Care and Use of Laboratory Animals as published by the U.S. Department of Health and Humans Services and proclaimed in the Animal Welfare Act (PL89-544, PL91-979, and PL94-279.)
Rats were housed with either mobile (run, N = 10) or immobile (sedentary, N = 10) stainless steel open running wheels (46.8 × 24.9 × 34.2 cm). Daily running distances were recorded using the automated Vital View system (Mini Mitter).
Rats were weighed weekly. After 4 wk of running, blood samples, and tissues were collected from both sedentary and running rats. All dissections occurred between 0900 and 1200. The colony light cycle began at 0600. No rat was found to run after 0600. Rats were quickly removed from their cages, and a blood sample (200 μL) was taken from the tail vein into heparinized tubes. Samples were quickly tested for white blood cell number and composition, as well as red blood cell number and hemoglobin content. After collecting tail vein samples, animals were deeply anesthetized with pentobarbital (i.p., 60.0 mg·kg−1) and sacrificed via transection of the diaphragm. Cardiac blood was collected into heparinized tubes for lipid determination. The medial and long heads of the triceps were removed and flash frozen in liquid nitrogen for citrate synthase determination.
Cell blood count.
Heparinized tail vein samples were analyzed using the CellDyn 3500 Hematology analyzer. The following parameters were assessed: total white blood cell count (WBC); differentials (neutrophils percentage, lymphocytes percentage, monocytes percentage, eosinophil percentage, and basophil percentage); red blood cell count (RBC; number of red blood cells); hematocrit (percentage of blood that is red blood cells); total hemoglobin (amount of hemoglobin in the whole blood), and mean corpuscular hemoglobin (MCH, the amount of hemoglobin per red blood cell).
Lipid profiles (cholesterol, HDL, triglycerides, LDL) were determined using Abbott Vision assay kits according to manufacturer’s instructions from heparinized whole blood cardiac samples within 30 min of cardiac puncture.
Citrate synthase assay.
Spectrophotometric analysis of citrate synthase activity was conducted according to Srere (26).
Data are reported as means ± SE. Paired t-tests were used for comparison of differences between sedentary and physically active animals. Statistical significance was established at P < 0.05.
Animal and exercise characteristics.
Twenty young male 344 Fischer rats were used in this study. Group body weights were equivalent at outset (379.1 ± 4.2 kg vs 373.3 ± 8.3 kg, P = 0.49, run vs sedentary), but run rats gained significantly less weight over the 4-wk study period than sedentary rats (382.6 ± 3.3 kg run vs 399.5 ± 7.3 kg, P = 0.01). Rats allowed free access to running wheels ran between 5.4249 km·wk−1 and 14.9156 km·wk−1, mean distance 9.89 ± 0.79 km·wk−1. This falls within reported distances for freewheel-trained rats (11,28).
Cell blood count: WBC differentials.
Run animals demonstrated resting or basal alterations in circulating white or red blood cell numbers (Fig. 1A) and subtype percentages (Fig. 1B) in comparison with sedentary controls. Although total white blood cell numbers were unchanged, the number (Fig. 1A) and percentage (Fig. 1B) of lymphocytes significantly increased (P < 0.001), whereas the neutrophil number (Fig. 1A) and percentage (Fig. 1B) decreased P < 0.0001) in run animals compared with sedentary controls. Blood monocyte (P < 0.01) and basophil (P < 0.001) numbers (Fig. 1A) and percentages (Fig. 1B) were also lower in run animals in comparison to sedentary controls. Eosinophils did not significantly change.
Run animals exhibited nonsignificant decreases in LDL, cholesterol, triglycerides, and increased HDL compared with sedentary animals (Table 1). The slight shifts in HDL and LDL in run animals resulted in an improved HDL/LDL ratio (P < 0.05).
Hemoglobin and RBC.
As shown in Figure 2, MCH increased in run animals (Fig. 2A, P < 0.01). Hematocrit (Fig. 2B), total hemoglobin (Fig. 2C), and red blood cell number (Fig. 1A) were unchanged (P > 0.05).
Freewheel-running exercise did not stimulate an increase in citrate synthase activity in either the long (run mean = 12.5 ± 2.7 μmol·g−1·min−1 vs sedentary mean = 11.1 ± 1.1 μmol·g−1·min−1) or medial (run mean = 19.7 ± 2.3 μmol·g−1·min−1 vs sedentary mean = 21.2 ± 3.1 μmol·g−1·min−1) head of the triceps muscle.
This study demonstrated that 4 wk of voluntary freewheel running results in a reduction in body weight gain, alterations in WBC differentials, and improvements in HDL/LDL ratio. Voluntary freewheel running increased RBC hemoglobin content without increasing total hemoglobin, hematocrit, or triceps citrate synthase activity. This data supports that a low volume of physical activity can positively affect parameters associated with disease risk.
Freewheel running reduced body weight gain, which supports what is previously stated in the literature (4,10,28,30). This reduction in body weight gain could be related to metabolic cost of exercise or an increase in basal metabolic rate due to muscle mass. Other studies have noted this reduction in weight gain to be advantageous, as it is linked to improved insulin sensitivity and a reduction in cardiovascular disease risk (15,28).
Freewheel running also induced alterations in circulating white blood cell differentials, although it did not change the total number of WBC. We believe that the changes reported are reflective of long-term alterations in resting hematology and not acute changes associated with running. This conclusion is supported by several factors. First, all freewheel running had ceased at least 3 h before blood sampling. Given that freewheel running occurs at a moderate intensity (30), it is unlikely the acutely triggered moderate-intensity exercise effects persist this long (18). Finally, although acute moderate exercise is commonly associated with increases in circulating hormones, such as catecholamines or corticosterone, we have previously reported no elevation in basal levels of these hormones at this time of day in freewheel-run rats (8,17).
Our findings that resting or basal circulating lymphocyte number increases with voluntary exercise supports previous work in female freewheel-trained animals (1), and extends the previous finding of increased CD4+ and CD8+ number after 4 wk of treadmill exercise (6). Splenic lymphocyte proliferation is improved in physically active animals (4,27); thus, the spleen may be contributing to the basal circulating population. An increase in circulating catecholamines is associated with a rapid mobilization of lymphocytes; thus, it is possible that alterations in circulating lymphocytes are related to the repeated exposure to catecholamines during exercise (7).
This study found no change in eosinophil numbers or percentage yet a highly significant decrease in basophil numbers. To our knowledge, the literature has not examined alterations in basophils after chronic voluntary exercise in rats. One human study has demonstrated an increase in resting eosinophil in trained subjects (2), but it is impossible to determine the significance of such a change at this time.
A decrease in circulating monocyte and neutrophil percentage and number was also found in this study. Human studies have found that neutrophil adhesion increased and superoxide anion release decreased in trained subjects in comparison with untrained subjects (20). Given that excessive superoxide production can be suppressive to acquired immune responses, a reduction in resting levels of superoxide producing cells, such as monocytes and neutrophils, could be a positive adaptation. This observation needs further study to determine if this effect is advantageous or deleterious.
Voluntary exercise did improve HDL/LDL ratio. It is well established that exercise training increases HDL, and decreases LDL and triglycerides (14). HDL/LDL ratio is considered a clinical index of cardiovascular health (14), and an improvement in HDL/LDL ratio is hypothesized to protect arteries from thrombogenesis and lipid accumulation (13). Other rat studies examining lipid profiles in nonobese rats typically note improvements only when HDL is measured in proportion with total cholesterol or total triglycerides (5). Only one study using Fischer 344 has found an outright improvement in HDL, and this study used a 35-wk training protocol (28). Other studies that have demonstrated a rapid improvement in triglyceride profiles have used either obese rats, which have high baseline lipid levels, or forced exercise, including treadmill and swimming protocols that may be of higher intensity, thus allowing for more rapid alterations. It has been suggested that the decrease in plasma triglycerides after exercise training is due to decreased synthesis of triglycerides (24). A longer time course may be necessary using the lower intensity and volume of freewheel running to elucidate significant effects; whether the lipid alterations induced by freewheel running would confer health benefits will require further study.
The improvement in mean corpuscular hemoglobin is significant because it represents an increase in oxygen-carrying capacity. Such an adaptation would suggest the rat is trained and has an increase in work capacity. This is supported by an increased run to exhaustion time in freewheel training, with freewheel-trained rats running over three times as long as sedentary rats (3). The lack of improvement in total hemoglobin and hematocrit after chronic exercise is in agreement with other studies (29).
This study failed to find an increase in citrate synthase in the triceps after freewheel running. The mean distance covered by this group of rats was only 9.89 km·wk−1, and other work has found improvements in V̇O2max occur only after the rats run greater than 11.6 km·wk−1 (11). Perhaps this training volume was insufficient for improvements in oxidative metabolism and, therefore, insufficient for stimulating an increase in citrate synthase. This is in agreement with other studies, which have found that treadmill exercise alone, not freewheel exercise, brought about an increase in CS activity (12). Sexton (22) did note an increase in triceps CS with freewheel running, but these were Sprague-Dawley rats that met the above distance criteria for V̇O2max changes. Triceps were assayed in this study because previous work in this laboratory has failed to demonstrate an increase in soleus CS activity with freewheel running, and it was thought the triceps would play a greater role in wheel running because of the vertical component.
Although speculative, it is intriguing to suggest alterations in resting immune parameters produced by voluntary freewheel running may positively affect health outcome would be via affecting formation of atherosclerotic plaques. Atherosclerotic lesions contain CD4+and CD8+ T cells, as well as monocytes, macrophages, and endothelin, which react with LDL. These reactions lead to the formation of plaques and fatty streaks that result in coronary artery disease and tissue ischemic events (25). It is, therefore, possible that the reduction in both the immune reactive cells, such as monocytes, and alterations in the LDL ratios found in this study could be indicative of improved vascular health. Further study into the interactions between these immune cells and their respective cytokine secretion and effect on atherosclerosis is needed.
This study serves to establish the baseline hematological and metabolic adaptations of voluntary freewheel running on Fischer 344 rats. Freewheel running provides adequate stimulus to alter resting white blood cell distributions, as well as to improve blood lipid profiles and increase RBC mean corpuscular hemoglobin. It failed to stimulate significant changes in CS activity. Furthermore, this study is important because it suggests the amount of exercise necessary to confer many health benefits may be lower than those required to induce hallmark muscular training adaptations.
This work was funded by NIH RO1AI48555.
1. Baldwin, D. R., Z. C. Wilcox, and G. Zheng. The effects of voluntary exercise and immobilization on humoral immunity and endocrine responses in rats. Physiol. Behav.
2. Benoni, G., P. Bellavite, A. Adami, et al. Effect of acute exercise on some haematological parameters and neutrophil functions in active and inactive subjects. Eur. J. Appl. Physiol.
3. Campisi, J., T. H. Leem, B. N. Greenwood, et al. Habitual physical activity
facilitates stress-induced HSP72 induction in brain, peripheral, and immune tissues. Am. J. Physiol. Regul. Integr. Comp. Physiol.
4. Coleman, K. J., and D. R. Rager. Effects of voluntary exercise on immune function in rats. Physiol. Behav.
5. Devi, S. A., S. Prathima, and M. V. V. Subramanyam. Dietary vitamin E and physical exercise: I. Altered endurance capacity and plasma lipid profile in ageing rats. Exp. Gerontol.
6. Ferry, A. P. R, F. Laziri, A. El Habazi, C. Le Page, and M. Rieu. Effect of moderate exercise on rat T-cells. Eur. J. Appl. Physiol.
7. Gabriel, H., L. Schwarz, P. Born, and W. Kindermann. Differential mobilization of leucocyte and lymphocyte subpopulations into the circulation during endurance exercise. Eur. J. Appl. Physiol.
8. Greenwood, B. N., S. Kennedy, T. P. Smith, S. Campeau, H. E. Day, and M. Fleshner. Voluntary freewheel running selectively modulates catecholamine content in peripheral tissue and c-Fos expression in the central sympathetic circuit following exposure to uncontrollable stress in rats. Neuroscience
9. Jonsdottir, I. H., A. Asea, P. Hoffmann, et al. Voluntary chronic exercise augments in vivo natural immunity in rats. J. Appl. Physiol.
10. Jonsdottir, I. H., C. Johansson, A. Asea, et al. Duration and mechanisms of the increased natural cytotoxicity seen after chronic voluntary exercise in rats. Acta Physiol. Scand.
11. Lambert, M. I., and T. D. Noakes. Spontaneous running increases V̇O2
max and running performance in rats. J. Appl. Physiol.
12. MacNeil, B., and L. Hoffman-Goetz. Chronic exercise enhances in vivo and in vitro cytotoxic mechanisms of natural immunity in mice. J. Appl. Physiol.
13. Masumura, S., H. Furui, M. Hashimoto, and Y. Watanabe. The effects of season and exercise on the levels of plasma polyunsaturated fatty acids and lipoprotein cholesterol in young rats. Biochim. Biophys. Acta
14. Mazzeo, R. S., P. Cavanaugh, W. J. Evans, et al. Exercise and physical activity
for older adults. Med. Sci. Sports. Exerc.
15. Miyasaka, K., M. Ichikawa, T. Kawanami, et al. Physical activity
prevented age-related decline in energy metabolism in genetically obese and diabetic rats, but not in control rats. Mech. Ageing Dev.
16. Moraska, A., T. Deak, R. L. Spencer, D. Roth, and M. Fleshner. Treadmill running produces both positive and negative physiological adaptations in Sprague-Dawley rats. Am. J. Physiol. Regul. Integr. Comp. Physiol.
17. Moraska, A., and M. Fleshner. Voluntary physical activity
prevents stress-induced behavioral depression and anti-KLH antibody suppression. Am. J. Physiol. Regul. Integr. Comp. Physiol.
18. Natale, V. M., I. K. Brenner, A. I. Moldoveanu, P. Vasiliou, P. Shek, and R. J. Shephard. Effects of three different types of exercise on blood leukocyte count during and following exercise. Sao Paulo Med. J.
19. Navas, F. J., and A. Cordova. Iron distribution in different tissues in rats following exercise. Biol. Trace Element Res.
20. Ndon, J. A., A. C. Snyder, C. Foster, and W. B. Wehrenberg. Effects of chronic intense exercise training on the leukocyte response to acute exercise. Int. J. Sports Med.
21. Senturk, U. K., F. Gunduz, O. Kuru, et al. Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats. J. Appl. Physiol.
22. Sexton, W. L. Vascular adaptations in rat hindlimb skeletal muscle after voluntary running-wheel exercise. J. Appl. Physiol.
23. Shek, P. N., B. H. Sabiston, A. Buguet, and M. W. Radomski. Strenuous exercise and immunological changes. Int. J. Sports Med.
24. Simonelli, C., and R. P. Eaton. Reduced triglyceride secretion: a metabolic consequence of chronic exercise. Am. J. Physiol.
25. Smith, J. K., R. Dykes, J. E. Douglas, G. Krishnaswamy, and S. Berk. Long-term exercise and atherogenic activity of blood mononuclear cells in persons at risk of developing ischemic heart disease. JAMA
26. Srere, P. Citrate synthase. Methods Enzymol.
27. Sugiura, H., H. Sugiurra, H. Nishida, R. Inaba, S. M. Moirbod, and H. Iwata. Immunomodulation by 8-week voluntary exercise in mice. Acta Physiol. Scand.
28. Suzuki, K., and K. Machida. Effectiveness of lower level voluntary exercise in disease prevention of mature rats. Eur. J. Appl. Physiol.
29. Xiao, D. S., and Z. M. Qian. Plasma nitric oxide and iron concentrations in exercised rats are negatively correlated. Mol. Cell. Biochem.
30. Yancey, S. L., and J. M. Overton. Cardiovascular responses to voluntary and treadmill exercise in rats. J. Appl. Physiol.