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The present study provides experimental data which indicate that the neutrophil is ideal for studying programmed cell death or apoptosis in vitro. Neutrophils can be obtained from human peripheral blood in large numbers with minimal experimental manipulation and are easily separated from other leukocytes, providing nearly pure cell suspensions. The neutrophil life span in vitro is sufficiently short to allow observations to be made within eight hours after experimental manipulation. Neutrophils can also be easily maintained in serum-free, chemically defined media which can be systematically altered, thereby defining specific variables that influence the apoptotic process. Since the neutrophils do not need an exogenous trigger to undergo programmed cell death, it is also an excellent model to study senescence. It was determined from this study that neutrophils undergo apoptosis most efficiently at 37 degrees C, a temperature requirement for physiologic cell death. Neutrophils undergo apoptosis at a slightly faster rate and maintain membrane integrity better when incubated in a tissue culture medium (e.g., RPMI 1640) compared with a balanced salt solution (e.g., HBBB). Cycloheximide, an inhibitor of protein synthesis, was shown to accelerate apoptosis in a dose-dependent manner. The presence of Zn++ significantly decreased the rate of apoptosis, whereas the presence of Ca++ and Mg++ had no apparent effect. These studies indicate that the process of senescence, culminating in cell death, is subject to modulation by a variety of agents and experimental conditions. In addition, the ultrastructural features of neutrophils undergoing programmed cell death in vitro were compared in detail to those occurring in vivo and were found to be comparable.

Atrophy and growth failure of muscle in a tail-cast suspension model were evaluated in hindlimbs of female Sprague-Dawley rats. Based on measurements of food consumption, animal growth rate, urinary excretion of urea and ammonia, and muscle size, 6 days seemed to be the optimum duration of suspension for studying muscle unloading. After 6 days, the soleus, plantaris, and gastrocnemius muscles from suspended animals were 27, 10, and 11% smaller (P less than 0.05), respectively, than those from tail-casted weight-bearing animals. The extensor digitorum longus and tibialis anterior muscles were unaffected by suspension (less than or equal to 6 days) while the triceps brachii hypertrophied (8%, P less than 0.05). Wet weight-to-dry weight ratios were smaller in the plantaris (-0.19, P less than 0.05) and gastrocnemius (-0.19, P less than 0.05) muscles from suspended rats. In the plantaris, this difference coincided with a higher protein concentration (+12 mg/g, P less than 0.001). In vitro measurements of protein metabolism in the soleus muscles of suspended rats showed both slower protein synthesis (P less than 0.05) and faster protein degradation (P less than 0.05), whereas these processes were unaltered in the extensor digitorum longus muscles.

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