EXERCISE-INDUCED LYMPHOCYTE APOPTOSIS ATTRIBUTABLE TO CYCLE ERGOMETER EXERCISE

J.W. Navalta¹, B.K. McFarlin², T.S. Lyons¹, J.C. Faircloth³, N.T. Bacon⁴ and Z.J. Callahan¹.

¹Department of Physical Education and Recreation, Western Kentucky University; ²Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston; ³Department of Chemistry, University of South Florida, ⁴Department of Kinesiology, University of Alabama

Figure 1. Apoptotic index before and immediately following cycle exercise. * indicates significant diffeence, P < 0.01.

Abstract

Exercise induces significant apoptosis in lymphocytes. Studies on humans have only utilized the treadmill as a mode of exercise. Two purposes of this study were, (1) to assess if cycle ergometer exercise induces changes in apoptosis, and (2) to characterize the nature of the morphological method of assessing cell death. Endurance athletes (N=10) completed a 60-min ride on a cycle ergometer at ~80%VO2 peak. Blood samples taken before (PRE) and after exercise (POST) were used to make blood films for apoptoticanalysis. A significant increase was observed in the apoptotic index following cycle exercise (PRE=7.3±2, POST=12.9±2; P<0.01). On average, it took 42±9 min to evaluate PRE sample slides. This was significantly longer than the 27±4 min needed to evaluate slides from the POST condition (P<0.01). In comparison, 2.5±0.4 cells/min wereevaluated PRE, which was significantly less than POST(3.7±0.5cells/min,P<0.01). Blood smears taken at baseline required more time to evaluate microscopically than smears following exercise. This is likely due to lymphocytosis that occurs with exercise. Characterization of the time needed to morphologically assess exercise-induced lymphocyteapoptosis should aid investigators in this area. We have shown for the first time that an exercise other than treadmill running can induce apoptosis in lymphocytes. However, these values are lower than what has been reported following treadmill exercise (~20%). While cause(s) for the diminished value remains to be elucidated, the nonweight-bearing nature of cycle exercise may play a part in these observations.

Introduction

Cells are eliminated from the body through necrosis or apoptosis. Necrosis occurs from trauma and involves swelling and emptying of contents outside of the cell, resulting in an inflammatory response. Apoptosis on the other hand is carried out by the activation of endogenous endonucleases which allows the process to proceed without the occurrence of inflammation.

Exercise is a physiological stress that has a marked effect on the cells of the immune system, and lymphocytes in particular. An increase in cell numbers is observed with the performance of high intensity exercise, followed by a reduction in numbers that is significantly lower than resting levels. A portion of this post-exercise decrease may due to programmed cell death.

Exercise-induced lymphocyte apoptosis has been evaluated using two main techniques: biochemical makers, and morphological identification. While the biochemical methodology is well described, characteristics of the morphological remain to be reported in the literature.

Investigations assessing lymphocyte apoptosis in humans have utilized mainly treadmill exercise to induce a response. Cycle ergometer exercise as an initiating stimulus for programmed cell death has been reported in a single study. In this study, only a biochemical technique was used to assess lymphocyte cell death induced by cycle exercise.

The purposes of this study were two-fold:

1.) Since no investigations to date have assessed the effect of cycle ergometry on exercise-induced lymphocyte apoptosis utilizing the morphological technique, this study was designed to determine the apoptotic response as evaluated using this classic method.

2.) Since certain aspects of the morphological technique remain undescribed in the literature, a second purpose was to further characterize the nature of this method used in the evaluation of exercise-induced lymphocyte cell death.

Methods

Protocol
Blood samples were obtained from endurance-trained subjects (N = 10) before (PRE) and after (POST) completion of cycling exercise for 60 minutes at 75-80% of VO2max.

Blood films were made for the morphological analysis of lymphocyte apoptosis.

Whole blood (5ml each) was pipetted onto glass microscope slides (VWR International) in triplicate and air dried for subsequent staining with May-Grünwald Giemsa (Sigma Aldrich) and determination of apoptosis. Slides were placed in May-Grünwald stain for 3-5 min, and subsequently placed in phosphate buffer for 3 min. Slides were then placed in modified Giemsa (Sigma Aldrich) stain for 2 min, rinsed briefly in deionized water, and allowed to air dry before evaluation.

Images of both normal and apoptotic lymphocytes were captured sequentially using a digital camera mounted to a microscope. At least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics. The amount of time required to capture the requisite number of cells per slide was noted in order to further describe this morphological method.

Lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane. Lymphocytes that displayed membrane blebbing, or apoptotic bodies were considered apoptotic as has been previously reported in the literature.

Statistical Analysis
Data were analyzed using paired t-Tests with significance set at P<0.05 (SPSS, Inc.).

Results

Cycle ergometry induced a significant increase in the apoptotic index as assessed by the morphological technique (P<0.01, see figure 1)

The mean time to evaluate PRE sample slides was 42±9 min. This was significantly longer than the 27±4 min needed to evaluate slides from the POST condition (P<0.01). In comparison, 2.5±0.4 cells/min were evaluated PRE, which was significantly less than POST (3.7±0.5 cells/min, P<0.01).

Discussion & Conclusions

Blood smears taken at baseline required more time to evaluate microscopically than those following exercise. Since post-exercise smears generally contain an increased total number of lymphocytes due to lymphocytosis, this phenomenon likely allowed digitization to occur at a more rapid rate. Characterization of the time needed to morphologically assess exercise-induced lymphocyte apoptosis should aid investigators in assessing the cost/benefit ratio for using this technique. Although the method may be time-intensive, it has been suggested to capture the apoptotic process with greater sensitivity compared to biochemical techniques alone.

We have shown that cycle ergometer exercise can indeed induce apoptosis in lymphocytes. However, these values are lower than what has been reported following treadmill exercise (~20%). While cause(s) for the diminished value remains to be elucidated, the non weight-bearing nature of cycle exercise may play a part in these observations.