THE EFFECTS OF PROLONGED TRAINING ONVTHE ' RESISTANCE TD RADIATION-INQUCED CHANGES. . RN MALE ALBLNO RATS Thesis for the 059m of Ph. D. V r MICHIGAN STATE UNIVERSITY :Rbberf KértZer .1965 THESIS This is to certify that the thesis entitled THE EFFECTS OF PROLONGED TRAINING ON THE RESISTANCE T0 RADIATION-INDUCED CHANGES IN MALE ALBINO RATS presented by Robert Kertzer has been accepted towards fulfillment of the requirements for Ph.D. degree in Physical Education / 5 ///2? M / M ajor professor Date September 17, 1965 0-169 LIBRARY Michigan Stan . University KUUM USE ONLY ABSTRACT THE EFFECTS OF PROLONGED TRAINING ON THE RESISTANCE TO RADIATION-INDUCED CHANGES IN MALE ALBINO RATS by Robert Kertzer The purpose of this investigation was to study the effects of various levels of preirradiation physical activity, from weanling age to adulthood, upon the radiation resistance of mature rats. Two-hundred and fifty 26-day old male albino rats (Sprague-Dawley strain) were randomly assigned to one of five treatments. Group 1 (sedentary control) received no special treatment, other than radiation, and was confined to indi— vidual sedentary cages both prior to and after being irra— diated. Group 2 (spontaneous control) received no special treatment, other than radiation, and was housed in individual spontaneous exercise cages until irradiated and then confined to individual sedentary cages. Group 3 (sedentary forced) was housed in individual sedentary cages both prior to and after being irradiated. During the training period prior to irradiation, these animals were forced to swim daily for one-half hour with two per cent body weight attached to the Robert Kertzer base of the tail. Group 4 (spontaneous forced) was housed in individual spontaneous exercise cages until irradiated and then confined to individual sedentary cages. Prior to being irradiated these animals also were forced to swim daily for one-half hour with two per cent body weight attached to the base of the tail. Group 5 (spontaneous effect) was housed in individual spontaneous exercise cages both prior to and after being irradiated. These animals were not sub— jected to the preirradiation forced training regimen. The preirradiation treatments were begun at 27 days of age and were carried on throughout puberty and early adult- hood. On the 83rd day of the preirradiation treatments, 20 per cent of the animals, randomly selected with stratification by groups were placed in sedentary cages for an approximate 64-hour rest period prior to irradiation. The forced swim- ming regimen was terminated for these animals at this time. On the 84th, 85th and 86th days, a similar procedure was followed with 20, 30 and 30 percent of the animals respect- ively. Following the 64-hour rest period, each animal was exposed to 650 r while confined in a three-inch by nine-inch lucite chamber. Nine days before and five days following irradiation, a blood sample was drawn from the orbital sinus of each animal Robert Kertzer to determine the WBC and eosinophil count. During the 30 days immediately following irradiation, mortality and sur- vival time in hours were recorded for all groups of animals. At the end of the 30-day period, the surviving animals in all groups were sacrificed. Upon death or sacrifice, body weight as well as the weights of the spleen and adrenals were determined. Statistical analysis of the experimental data has prompted the following conclusions: 1. Forced swimming for one—half hour daily with two per cent body weight attached significantly increases spon— taneous activity. 2. Forced swimming and/or spontaneous activity for a prolonged period reduce body weight. 3. Illumination of animal quarters 24 hours daily sig— nificantly reduces volitional activity as compared to 12- hour per day illumination. 4. Irradiation decreases spontaneous activity. 5. Prolonged training does not alter WBC or eosinophil levels either prior to or following irradiation. 6. Prolonged training has no effect on either adrenal weight per body weight or gross splenic weight following irradiation. Robert Kertzer 7. Level of preirradiation physical activity has no effect on postirradiation mortality and survival time. 8. Body weight appears to be the most significant fac- tor influencing survival time following irradiation with the heavier animals in all treatment groups having significantly increased survival times. THE EFFECTS OF PROLONGED TRAINING ON THE RESISTANCE TO RADIATION-INDUCED CHANGES IN MALE ALBINO RATS BY Robert Kertzer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health, Physical Education and Recreation 1965 Copyright by ROBERT KERTZER 1966 DEDI CATI ON To my wife Joyce, for maintaining an intact family unit. ACKNOWLEDGMENTS The author owes an untold debt of gratitude to Dr. W. D. Van Huss, for being scholar, friend and chaplain, each at the appropriate time, and to Dr. W. W. Heusner for demon— strating unequivocably that all of the skills necessary for outstanding teaching, creative research and scientific coaching can be mastered by one man. Thanks are also due Dr. D. R. Lamb for translating the Russian research. The writer wishes to thank Charlyne Ball, Carol Kowalewski, Frank Hartman, Dave Lamb, Ken Coutts and Rex Carrow for faithfully stidking to an absurd work schedule during data collection. The author wishes to thank Dr. U. V. Mostosky for supervising the radiation procedures. iv TABLE OF CONTENTS DEDICATION ACKNOWLEDGMENTS LIST OF LIST OF CHAPTER THE CHAPTER TABLES FIGURES I PROBLEM Introduction Statement of the Problem Importance of the Problem Limitations of the Study Definition of Terms II REVIEW OF RELATED LITERATURE American Investigations Postirradiation Exercise Preirradiation Exercise Preirradiation Training Pre— and Postirradiation Exercise Russian Investigations CHAPTER Postirradiation Exercise Preirradiation Exercise Preirradiation Training III EXPERIMENTAL METHOD Design of Experiment Sample Treatment Groups Preirradiation Forced Training Procedures Duration of Preirradiation Treatments Radiation Blood Measures Postirradiation Period General Procedures Page iii iv vii ix u)M|Aidta+a 20 20 20 21 23 24 25 25 26 26 Statistical Methods CHAPTER IV RESULTS AND DISCUSSION Results Preirradiation Spontaneous Activity Lighting, Irradiation and Spontaneous Activity Postirradiation Spontaneous Activity Body Weight White Blood Cells Eosinophils Spleen Weight Adrenal Weight Survival Time Mortality Discussion Spontaneous Activity Body Weight Blood Parameters Organ Weights Mortality and Survival CHAPTER.V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary COnclusions Recommendations SELECTED BIBLIOGRAPHY APPENDICES A Raw Data B Body Weights C Starting Positions for Irradiation of Animals vi 27 29 29 29 33 38 4O 46 52 56 61 64 68 7O 70 71 73 75 77 78 78 83 83 85 9O 98 107 Table 10 11 12 13 14 LIST OF TABLES Analysis of Variance for Preirradiation Spontaneous Activity Analysis of Variance for Level of Preirradi- ation Activity and Survival Time Analysis of Variance for Effects of Lighting and Radiation on Activity Tukey Test for Effects of Lighting and Radiation on Activity Analysis of Variance for Postirradiation Activity Level and Survival Time Analysis of Variance for Body Weights Prior to Irradiation Tukey Test for Body Weights Prior to Irradiation Analysis of Variance for Level of Body Weight and Survival Time Analysis of Variance for WBC Preirradiation Analysis of Variance for Levels of WBC Preirradiation and Survival Time Tukey Test for Levels of WBC and Survival Time Analysis of Variance for WBC Five Days Postirradiation Analysis of Variance for WBC Level Postir- radiation and Survival Time Analysis of Variance for Eosinophil Count Nine Days Preirradiation vii Page 29 33 35 38 4O 43 43 44 46 48 51 51 52 55 15 16 17 18 19 20 21 22 Analysis of Variance for Preirradiation Eosinophil Level and Survival Time Analysis of Variance for Treatment Effects on Spleen Weight at Death Analysis of Variance for Treatment Effects on Spleen Weight at Sacrifice Analysis of Variance for Treatment Effects on Adrenal Weight Per Body Weight at Death Analysis of Variance for Treatment Effects on Adrenal Weight Per Body Weight at Sacrifice Analysis of Variance for Treatment Effects on Survival Time of All Animals Analysis of Variance for Treatment Effects on Survival Time of Only Animals which Died Chi—square Test for Treatment Effects on Mortality viii 57 59 59 61 64 66 68 Figure 10 ll 12 13 14 15 16 LIST OF FIGURES Daily Preirradiation Spontaneous Activity Mean Daily Spontaneous Activity Preirradiation Upper and Lower Halves of Spontaneous Activity and Survival Time Lighting and Radiation Effects on Activity Lighting and Radiation Effects on Activity (Mean Values) Upper and Lower Halves of Three-Day Postirradiation Spontaneous Activity and Survival Time Treatment Effects on Body Weight Mean Body Weight Prior to Irradiation Upper and Lower Halves of Body Weight Preirradiation and Survival Time WBC Nine Days Prior to Irradiation Upper and Lower Halves of WBC Preirradiation and Survival Time WBC Five Days Postirradiation Upper and Lower Halves of WBC Postirradiation and Survival Time Eosinophils Nine Days Prior to Irradiation Upper and Lower Halves of Eosinophils Preirradiation and Survival Time Treatment Effects on Spleen Weight at Death ix Page 30 31 34 36 37 39 41 42 45 47 49 50 53 54 56 58 Figure 17 18 19 20 21 22 Treatment Effects on Spleen Weight at Sacrifice Adrenal Weight per Body Weight at Death Adrenal Weight per Body Weight at Sacrifice Survival Time of All Animals Survival Time of Animals Which Died Treatment Groups and Mortality Page 60 62 63 65 67 69 CHAPTER I THE PROBLEM Introduction In recent years, the rapid development of nuclear armaments, combined with conflicting international ideologies, has contributed to the constant possibility ' of a world cataStrophe. Furthermore, automation has reduCed the amount of physical activity necessary to carry on the tasks of daily living. It would be well to know the interrelationships and possible physiological consequences of these socially superimposed phenomena. Statement of the Problem The purpose of this investigation was to study the effects of various levels of preirradiation physical activ- ity, from weanling age to adulthood, upon the radiation resist— ance of mature rats. The information gained should aid in evaluating the influence of physical conditioning upon the mortality and selected physiological and anatomical parameters of animals exposed to lethal doses of radiation. Importance of the Problem Considerable work has been done already on (a) the effects upon mortality of fatigue produced by exhaustive exercise immediately prior to and/or immediately following irradiation (7, 8, 36, 38); (b) the effects upon mortality of daily postirradiation exercise combined with various radiation doses (19, 20); and (c) the effects of radiation upon recovery from fatigue (8), the ability to perform an exhaus- tive exercise test (6, 21, 35, 40) and volitional activity (18, 22). Data are incomplete in regard to the effects of radiation upon animals subjected to physical training over a long period of time prior to being irradiated. There is substantial evidence to indicate that extended programs of relatively vigorous daily activity result in significant alterations in a number of physiological and anatomical parameters which are also affected by irra- diation (5, 9, 10, ll, l2, 16, 25, 26, 28, 29, 32, 33, 44). Some of these changes are similar to those produced by irradiation, whereas others are distinctly opposite (2, 4, 7, 8, 19, 20, 30, 31, 35, 36, 37, 40, 41). This raises the question as to whether or not various types of pro- longed physical training will influence resistance to damage caused by lethal doses of radiation. Limitations of the Study 1. Small laboratory animals may be investigated in numbers sufficient for generalization within the chosen population; however, the physiological results of animal studies cannot be translated directly to human subjects. 2. The strong psychosomatic response which may be expected in man when he is aware of the fact that he has been exposed to a lethal dose of radiation limits extra- polation from the rat to the human. 3. No attempt was made either to measure or control food intake. 4. The physiological changes produced by 650 roent— gens of radiation may be vastly different than those produced by other doses. 5. Exercise regimens, other than those employed in the present investigation, might produce dissimilar results. Definition of Terms l. Roentgen. — One roentgen is that quantity of X—or gamma radiation such that the associated corpuscular emission (electrons and ions) per 0.001293 gm. of air produces, in air, ions carrying l electrostatic unit of charge of either sign (2). 2. Mode of Irradiation. — 250 k v p x—rays; 15 M.A.; .5 mm. Cu (HVL 1.5 mm Cu) 78 inches TSD; 3.6 r/minute (air dose). 3. LD 50(30. - That dosage of radiation estimated to eliminate 50% of the population within a 30-day period. CHAPTER II . REVIEW OF RELATED LITERATURE In making a study of the effects of prolonged training on the resistance to radiation—induced changes it seems wise to have some knowledge of the effects of pre- and post— irradiation exercise and the associated changes in both mortality and related physiological and anatomical par— ameters. American Investigations Postirradiation Exercise Smith and Smith (35) forced mice to exercise in a revolving cage following irradiation. Animals receiving 325 r tolerated 12 hours of continuous activity immediately after or at any time up to 13 days postirradiation. Animals receiving 400 r tolerated eight hours of continuous activity on three consecutive days beginning immediately after or at any time up to 13 days postirradiation with little, if any, effect upon survival time. All animals receiving 600 r and then forced to exercise for eight continuous hours on three consecutive days died. When the radiation effects in exercised animals were compared with those observed in nonexercised irradiated control animals, it was concluded that there were no marked consequences either of irradiation upon exercise tolerance or of post- irradiation exercise upon mortality. Smith and Smith (36) subjected mice to various levels and durations of exercise following whole-body irradiation. Three days of moderate exercise of eight consecutive hours a day in equally spaced sessions of three hours, decreased slightly, if any, the tolerance of mice to prior irradiation. Following a radiation dose which was 100 per cent lethal, seven of 30 mice could not tolerate three successive days of exercise for eight hours a day. No differences:utsurvival time were noted between those mice surviving the exercise period and irradiated controls. The radiation tolerance of mice was not increased by phenobarbital sedation following irradiation. It was concluded that radiation lethality doses not parallel the rate of O2 utilization under the experimental conditions employed. Kimeldorf and Jones (20) studied the relationship of radiation dose to lethality in exercised animals exposed to x-rays. Male Sprague-Dawley rats were forced to swim until fatigued daily for four weeks following irradiation. As a result, these animals showed a greater mortality, with increased deaths at lower doses, than similar animals receiving only radiation. The nonexercised rats displayed a calculated median lethal dose which was 28 per cent higher than that computed for exercised animals. .It was concluded that exercise increases the level of mortality when per— formed postirradiation. Kimeldorf, Jones and Castanara (21) studied the effect of irradiation upon the performance of daily exhaustive exercise by the rat. For periods as long as nine weeks post— irradiation, rats were forced to perform a standardized exhaustive swimming test five times weekly. The ability to perform this test was decreased by exposure to x-rays in the 300—1000 r range. The extent of the decrease in per- formance was dependent upon dosage. Those rats surviving the test period recovered sufficiently to attain their preirradiation exercise levels by the ninth week. However, these animals were not able to perform as well as non- irradiated exercised control animals. The rats dying within a short period following irradiation were almost normal in performance, whereas those animals dying later displayed a lower level of performance which varied with the duration of the survival period. Kimeldorf et a1. (22) studied the effect of repeated exposures to x-rays on the volitional activity of adult male rats. Under controlled conditions of light, sound and temperature, the daily volitional activity of each animal was recOrded for 15 consecutive weeks. Dose increment effects were investigated by subjecting animals to 50 r, 100 r and 150 r at weekly intervals. The resulting cumulative doses were 600 r, 1200 r and 1350 r, respect— ively. By spacing 150 r doses at seven, 14 and 28 day intervals, the effect of changing the interval between ex- posures was studied. Irrespective of increment size or interval between exposures, a decrease in activity was noted after each irradiation. Statistically significant dif- ferences were observed between experimental and nonir- radiated control groups immediately following each 150 r exposure, after six of 12 exposures to 100 r and follow— ing three of 12 exposures to 50 r. The decrease in activ— ity was a function of the increment size rather than the cumulative dose. With the exception of weekly 150 r exposureS, recovery occurred within a week following each exposure. After six weekly exposures to 150 r, recovery in activity was less than normal. The responses to each 150 r dose were comparable regardless of the intervals be— tween exposures. Jones et a1. (18) also studied the effect of ir— radiation on voluntary activity of the adult male rats utilizing single doses of x-rays, ranging from 200—1000 r. A prompt significant reduction was noted at all doses. A period of increased activity immediately followed the initial decrease. Within five days postirradiation, all animals exposed to 200 r and 300 r were completely re- covered. No further detectable effects of irradiation upon voluntary activity at these levels were noted. When animals were exposed to x—rays of 400 r or more, some animals died. In the animals which survived, there was an initial period of depressed activity, followed by a re- covery period, and then a second depression. This sub- sequent reduction in activity reached a low value during the third week postirradiation. The amount of time required for surviving animals to completely recover from the second depression in activity seemed to be proportional to the x-ray dose. Those rats which died within the first nine days postirradiation displayed voluntary activity levels which continuously decreased from the day of ir- radiation until death. In animals dying after nine days postirradiation, the first depression was followed by some recovery. However, there was a second decrease in activity which persisted until death. Kimeldorf and Baum (19) investigated the changes in 10 organ and body growth following exhaustive exercise, x- irradiation and postirradiation exercise. Eight hundred and sixty-four male Sprague-Dawley rats were used in the in- vestigation. Animals were subjected to no stress, to repeated swims to eXhaustion, to various single doses of irradiation, or to irradiation plus various numbers of bouts of postirradiation exhaustive swimming. The animals were 55 days old when the experimental treatments were begun, and sacrifices were conducted 3, 9, 18 and 30 days follow— ing. Body weight was reduced in the experimental animals by both exhaustive exercise and exposure to irradiation. Relative to body weight: (a) either exercise or irradiation produced a decrease in thymus weight, an increase in adrenal weight, and no change in kidney weight; (b) exercise produced an increase and irradiation a decrease in the weights of the gluteus maximus, heart, spleen and testes; (c) exercise had no effect upon the pituitary weight whereas irradiation reduced the weight of this gland;.and (d) ex- ercise increased thyroid weight while irradiation failed to alter it. In general, changes following irradiation were found to be immediate and to be related to dosage, whereas exercise-induced changes were accumulative and related to the number of repetitions. When animals were subjected to 11 irradiation plus exhaustive fatigue, the responses were predominately those of irradiation and were not markedly increased by exercise. Postirradiation exercise did tend to prevent recovery from radiation damage. Smith and Smith (39) studied the effects of thyroid and radiation on sensitivity to hypoxia, basal rate of O2 consumption and tolerance to exercise. Mice treated with thyroid were found to have increased basal O2 consumption, increased sensitivity to hypoxia and a depressed ability to tolerate exercise. These three parameters followed the same course of develOpment with time. Within the limits imposed by the experiment, animals eXposed to 325 r exhibited no decrease in exercise tolerance and no ob- servable changes in basal O2 consumption. These ir— radiated mice did show a decrease in sensitivity to pro— gressive hypoxia. In mice receiving both thyroid and irradiation there were no increases in basal O2 consumption or decreases in tolerance to exercise beyond those caused by thyroid alone. However, the doubly treated animals had a greater resistance to hypoxia than animals receiv— ing only thyroid. It was concluded that early failures in an exercise test and deaths of thyroid—administered animals during exercise were indicative of impaired functional capacity 12 of the heart rather than of general muscular fatigue. PreirradiationyExercise Brown and White (7) studied preirradiation fatigue as a factor in the prevention of irradiation deaths in rats. Ten female Sprague—Dawley rats were swum to exhaustion on ten successive days. Five of these animals were irradiated on the eleventh day with 1200 r of cobalt60 immediately after an exhaustive swim. The other five animals received the same dose of radiation but without immediately prior exercise. All animals which swam before being irradiated were alive one month after irradiation, whereas all of the other animals died within ten days following the radiation exposure. It was concluded that fatigue—induced anoxia serves as'a protective mechanism against lethal doses of radiation.~ Preirradiation Training Kimeldorf and Jones (20) demonstrated that a short training program prior to irradiation (for a period of ten daYS) has no significant effect upon mortality. Pre- and Postirradiation Exercise Brown and White (8) forced sixteen adult Sprague-Dawley rats to swim to exhaustion on ten successive days. The animals then were matched on mean swimming time and assigned 12 of the heart rather than of general muscular fatigue. PreirradiationyExercise Brown and White (7) studied preirradiation fatigue as a factor in the prevention of irradiation deaths in rats. Ten female Sprague-Dawley rats were swum to exhaustion on ten successive days. Five of these animals were irradiated on the eleventh day with 1200 r of cobalt60 immediately after an exhaustive swim. The other five animals received the same dose of radiation but without immediately prior exercise. All animals which swam before being irradiated were alive one month after irradiation, whereas all of the other animals died within ten days following the radiation exposure. It was concluded that fatigue-induced anoxia serves as'a protective mechanism against lethal doses of radiation.~ Preirradiation Training Kimeldorf and Jones (20) demonstrated that a short training program prior to irradiation (for a period of ten days) has no significant effect upon mortality. Pre- and Postirradiation Exercise Brown and White (8) forced sixteen adult Sprague—Dawley rats to swim to exhaustion on ten successive days. The animals then were matched on mean swimming time and assigned 13 to two treatments. On the eleventh day, the animals in Group I were swum to exhaustion, immediately irradiated with 1200 r of cobalt60, and two hours later again swum to exhaustion. Group II was irradiated first, immediately swum to exhaustion, and two hours later again fatigued by a swim to exhaustion. The groups had similar swimming times during the first swim on the day of irradiation, but Group II swam significantly longer during the second session. It was concluded that irradiation immediately following exhaustive fatigue retards the biological processes involved in recovery from fatigue more than irradiation immediately prior to fatigue. Subsequently, both groups were forced to swim to exhaustion once a day until death or for a period of seven days after irradiation. The animals in Group II were able to do significantly more work, as measured by total swimming time, during this postirradiation period but died significantly earlier. All animals in Group II died within six days after irradiation; only two animals in Group I died within six days and two lived more than 30 days. Sta— tistical analysis revealed that the difference in post— irradiation work was not responsible for the difference observed in mortality rate. Both differences were shown to be the result of the relative states of fatigue of the two 14 groups at the time of irradiation. It was concluded that (a) anoxia associated with fatigue protected the animals in Group I from radiation injury, (b) preirradiation anoxia could not explain the lack of recovery from postirradiation fatigue in Group I, and (c) a condition which increased radiation injury in Group II also increased their relative ability to do work postirradiation. Kimeldorf, Jones and Fishler (23) studied male Sprague- Dawley rats in an effort to ascertain the role of metabolic level in determining radiosensitivity. The experimental animals performed a standardized exhaustive exercise, con- sisting of swimming in individual tanks. These animals were exercised once a day for ten days prior to irradiation. Following irradiation they were exercised daily, five times per week. After irradiation with 600 r, 50% of the ex- ercised rats died and all nonexercised irradiated controls survived. At a dose (700 r) that was lethal to 44% of the nonexercised animals, mortality was 92% among exercised rats. At a highly lethal dose (860 r), exercised animals displayed symptoms of roughened coat, diarrhea and crusted nares much sooner following irradiation than did the nonexercised controls. The exercised rats had a much shorter post- irradiation survival time in addition to a higher mortality 15 rate. The authors conclude that the results provide evidence of the relation between radiosensitivity and metabolic level. Russian Investigations Zimkin and Korobkov, two Russian investigators, have reviewed the studies of their co—workers in the area of exercise and radiation (50). Postirradiation*Exercise Sergeyev, working with rabbits, demonstrated a rise in mortality in animals exercised following a radiation dose of 1000 r. Intensity of radiation sickness was increased in the exercised animals. In the investigations of Popov, all dogs died Who were forced to execute daily hourly runs on a treadmill at a speed of 6 km./hr. following irradiation. Markelov also demonstrated that following an intravenous injection of l millicure/kg. of radioactive Sr physical 89' exercise intensified radiation sickness in rats. On the other hand, Trifonov showed that daily moderate exercise following irradiation both reduces and postpones the onset of mortality. Mortality of 24 control rats sub— jected to irradiation of 1150 r was 54 per cent after 25 days with the average length of life of those dying being 3.77 days. Animals which were swum without weights for 15 minutes daily over a ten—day period following irradiation 16 with the same dose had a 35 per cent mortality with the average life span being 6.57 days. More severe exercise after irradiation (1 hour and 20 minutes daily for ten days) produced1x>further decrease in mortality but did prolong the life of those who eventually perished. PinChook and Scherban irradiated mice with either 800 or 1200 r be— fore forcing them to swim for 30 minutes in water at 25- 300 c, After a dose of 1200 r all mice died. However, mice who were only irradiated lived, on the average, less (4—12 days) than those irradiated and swum (4-75 days). Of 80 mice exposed only to irradiation with 800 r 15 (18.7 per cent) survived, but 20 (25 per cent) of those irradiated and SWum survived. For the mice who died after being sub- jected only to irradiation the average length of life was less (8.87 days) than for those subjected to irradiation and subsequent swimming (10.35 days). Preirradiation Exercise Oogodskaya and Yondin forced rats to swim for 8—10 minutes with a ten—gram weight on their tails at 15—20 minutes before or immediately before irradiation. Mortal- ity for 40 control rats was 35 per cent, while mortality for 40 exercised rats was only 22 per cent. In one exper- iment of Trifonov, 25 rats were swum to exhaustion 17 immediately prior to irradiation with 1150 r. Mortality in the experimental group was approximately the same (56 per cent) as in a group of nonexercised control rats (54 per cent). However, average length of life in the exercised rats was greater (7.36 days) than in the controls (3.77 daysL Preirradiation Training The experiments of Sergeyev demonstrated that prelim- inary physical training decreases the mortality of ir- radiated animals. Using rabbits, he showed that systematic physical exertion for 10-25 days prior to irradiation with 1000 r reduces radiation sickness. In this study, radia- tion sickness was slight in the trained rabbits, even When they were subjected to daily physical exertion following irradiation. Tovbin, using three groups of 12 mice each, found that after a dose of 726—800 r control group mortal- ity was 75 per cent; in one group trained for 50 days in dynamic work, mortality was 42 per cent; and, in a group trained during the same time in static efforts (hanging on a slender pole), mortality was 25 per cent. Trifonov also investigated the effects of pretraining on mortal- ity following irradiation. Rats, which were trained by swimming and hanging from a pole for 1 1/2 months (35- 38 training periods), died from radiation sickness in a l8 smatller percentage and on later dates than control animals. Trifonov emphasizes the importance of the exercise intensity. Longer sessions of training in swimming (up to 3 1/2 hours per day) showed a smaller prophylactic effect than shorter periods (30-60 minutes). Zimkin conducted experiments on 44 male rats of which 22 were subjected to training. Seven rats were exercised by running in a wheel which revolved at a rate of 13 revolu- tions per minute, and 14 rats were trained by hanging from a vertically suspended rOpe. The duration of ex— ercise was increased by two minutes daily for 15 days. In subsequent exercise bouts for 30 days (with interruptions for vacations) the duration was held to 30 minutes. Animals were irradiated with 600 r. All rats surviving on the 28th day after irradiation were sacrificed. Four control and four experimental animals were sacrificed immediately following irradiation in order to study histological changes in various tissues. Therefore, the analyses of mortality, body weight changes, food consumption for one hour, number of leucocytes and hemoglobin content were carried out on data obtained from 18 control and 18 ex- perimental animals. The results indicate that the ex- perimental rats were more resistant to radiation than the I (“.Jl ((4.11! .D»( all]. t :l s _ 19 controls. In the group of trained animals 15 survived (81.1%) while only 10 (55.5%) of the untrained controls lived. Six of the control animals died in the period from the 6th to the 10th day following irradiation. One con- trol animal died on the 13th day and one on the 19th day after irradiation. The average length of life for the con— trol rats who died postirradiation was 10 days while the experimental animals lived, on the average, 15 days. Food consumption and body weight were decreased in all rats, but more so in the control group. Histological changes in the heart muscle of trained and untrained animals indicated a marked protein "dystrophy”; however, Zimkin and Korobkov report it was less widespread in the trained animals. CHAPTER III EXPERIMENTAL METHOD Design of Experiment Sample Two hundred and fifty weanling (26-day-old) male Sprague- Dawley rats were randomly assigned to one of five treatment groups. A sample size of 50 for each group was chosen be- cause previous experience with animal training studies in this laboratory (15, 25, 27) indicated that the variability of the parameters studied is such that it would be desirable to be able to detect, as significant, differences at least as small as two—thirds of one standard deviation, while holding the probability of making a type I statistical error to the .05 level and that of making a type II error to the .20 level. According to these specifications a sample size of 36 in each group was calculated as sufficient to distin- guish between the effects of the various training programs whiCh were used. Assuming, however, that concurrent irradia- tion would increase the within group variances to some un- known extent and that a few animals might be lost by drowning in the forced training period, by confinement during the radiation procedure or by the anesthetic during blood sampling, a sample size of 50 animals in each group was chosen arbitrarily. 20 21 In fact, three animals in one of the forced activity groups and two in the other did drown in training. One animal in one of the other groups died while under anes— thesia during preirradiation blood sampling. Consequently, animals were eliminated randomly, and 47 rats in each group were irradiated. No animals were lost as a result of con- finement during irradiation. Postirradiation blood sampling resulted in the death of one additional animal. Once again, equal groups were maintained by random elimination. There— fore, 46 animals per group comprised the final sample. This yielded a minimum statistical power of .90 for the several analysis of variance calculations made. Treatment Groups The following five treatment groups of animals were utilized in this investigation: Group 1: A sedentary control group which received no special treatment, other than radiation. These animals were confined to individual sedentary cages both prior to and after being irradiated. (Each sedentary cage is 24 cm. long by 18 cm. wide by 18 cm. tall.) Group 2: A spontaneous control group which received no special treatment, other than radiation. They were housed in individual spontaneous exercise cages until irradiated 22 and then confined to individual sedentary cages. (Each spontaneous exercise cage consists of an individual seden- tary cage plus a freely revolving drum of 35 cm. diameter and 13 cm. width. These cages permit the animals to rest or exercise at will.) Group 3: A sedentary forced exercise group which was housed in individual sedentary cages both prior to and after being irradiated. During the training period prior to irradiation, these animals were forced to swim daily for one—half hour with two per cent body weight attached to the base of the tail. Group 4: A spontaneous forced exercise group which was housed in individual spontaneous exercise cages until ir- radiated and then confined to individual sedentary cages. Prior to being irradiated these animals also were forced to swim daily for one-half hour with two per cent body weight attached to the base of the tail. Group 5: A spontaneous effect group which was housed in individual spontaneous exercise cages both prior to and after being irradiated. These animals were not subjected to the preirradiation forced training regimen. From the five treatment groups, the following were determined: 23 l. The effects of sedentary cage existence upon resis- tance to radiation—induced changes. 2. The effects of spontaneous activity upon resistance to radiation—induced changes. 3. The effects of prolonged forced exercise upon resis- tance to radiation-induced changes. ‘4. The interaction between the effects produced by spontaneous and forced exercise. 5. The effects of radiation upon spontaneous activity. Preirradiation Forced Training Procedures Those animals which were subjected to the swimming regimen (Groups 3 and 4) were swum in individual cylindri— cal tanks measuring 11 inches in diameter and 30 inches in depth. Animals were always swum between 8:00 A.M. and 11:30 A.M. Beginning on the 50th day of the preirradiation treatments, a few animals were unable to complete the re- quired one-half hour swim with two per cent body weight attached to the base of the tail. On the 53rd day two rats died while swimming. On the 56th day another died. Swim- ming was omitted on the 61st day as a one—day rest period seemed advisable. A fourth animal died on the 68th day. Consequently, a second rest period was inserted on the 69th and 70th days. Beginning with the 7lst day, whenever an 24 animal was unable to complete the one-half hour swim with weight attached, he was pulled from the tank, the weight was removed, the animal was given a one-minute rest, and he was replaced in the tank without the weight attached for the remainder of the one-half hour swim. Approximately five animals required this procedure daily with only one additional rat drowning during the remainder of the pre- irradiation period. Water temperature was maintained be— tween 360 and 380 C. during swimming. After each swim period the animals were dried with a towel and replaced in their respective cages. Duration of Preirradiation Treatments The preirradiation treatments were begun at 27 days of age and were carried on throughout puberty and early adult- hood. On the 83rd day of the preirradiation treatments, 20 per cent of the animals, randomly selected with stratifica- tion by groups were placed in sedentary cages for an approxi- mate 64—hour rest period prior to irradiation. The forced swimming regimen was terminated for these animals at this time. On the 84th, 85th, and 86th days, a similar procedure was followed with 20, 30 and 30 per cent of the animals respectively. This practice was employed since Brown and White (7) found that irradiation with 1200 r of cobalt60 25 immediately following exhaustive fatigue produced no deaths within one month while all animals not exhausted by swimming died within ten days. It was assumed that exhaustive fatigue functioned as a protective mechanism. The present investi- gation was concerned with the long-term effects of physical training upon resistance to radiation rather than the im- mediate effects of fatigue. Therefore, the 64-hour rest period between the end of training and irradiation was in- corporated into the experimental design. Radiation Following the 64-hour rest period, each animal was ex- posed to 650 r while confined in a three—inch by nine-inch lucite chamber. Twenty-four animals were irradiated simul- taneously. The total time required for irradiation was 180.5 minutes. Animal positions were rotated every 36.1 minutes to provide equal exposures for all. The radiation dose was set at 650 r because Andrews (2) has reported that the LD 50/30 for the rat is between 600 and 700 r. Blood Measures Nine days before and five days following irradiation a blood sample was drawn from the orbital sinus of each ani- mal to determine the WBC and eosinophil count. A 24-hour period of confinement in sedentary cages was provided for 26 all animals, regularly in spontaneous cages, prior to the drawing of each blood sample. Postirradiation Period During the 30 days immediately following irradiation, a laboratory technician was with the animals twenty—four hours a day. Mortality and survival time in hours for all groups of animals were recorded during this period. At the end of the thirty-day period, the surviving animals in all groups were sacrificed. Upon death or sacrifice, body weight as well as the weights of the spleen and adrenals were determined. The thymus was observed subjectively, but not weighed, since its postirradiation condition prohibited proper extraction and trimming. These organs were selected for study since Andrews (2) has reported that lymphoid tissue is highly sen- sitive to irradiation with sufficient consistency to warrant the use of such tissues as biological radiation dosimeters. General Procedures Throughout the entire investigation, all animals re— ceived water and a commercial ground animal diet, ad libi- tum. The temperature in the housing quarters was maintained between 700 and 780 F. Body weight for each animal was de- termined once a week throughout the preirradiation period, 27 and the weight to be attached to the base of the tail dur- ing swimming was changed on the following day. Daily volun- tary exercise data for each animal housed in a spontaneous exercise cage was recorded at 7:45 A.M. daily. For the first 71 days of the preirradiation period, an automatic timer illuminated the animal quarters 12 hours each day. Since it was necessary for the technician to ob- serve each animal hourly following irradiation, lights Were kept on 24 hours a day beginning on the 72nd day. The 24- hour illumination was started 12 days prior to irradiation in order to provide an adjustment period for the animals. Statistical Methods The differences in mortality between the five treat— ment groups were analyzed using the chi—square test for multiple independent samples (34). A Pearson product—moment coefficient of correlation was calculated between the pre— and postirradiation spontaneous activity values for the spontaneous effect group to deter- mine the consistency of the effect of radiation on voluntary activity of rats. The difference between the pre- and post- irradiation spontaneous activity levels in group 5 was evalu- ated by the standard one-way analysis of variance tech- nique (l4) . 28 Standard one- and two—way fixed effects analysis of variance techniques were employed where appropriate to analyze the data on preirradiation spontaneous activity levels, body weights, eosinophil and White blood cell counts, organ weights and survival times (14). The Tukey method for multiple comparisons between means was used whenever significant F-values were obtained (14). CHAPTER IV RESULTS AND DISCUSSION Results Daily spontaneous activity was evaluated in order to determine the effects of (a) forced swimming on voluntary exercise (b) voluntary activity on resistance to mortality following irradiation and (c) irradiation on level of spontaneous activity. Preirradiation Spontaneous Activity Figure 1 compares the daily voluntary activity of the spontaneous control animals (Group 2) with the spontaneous forced (Group 4) for the 84-day preirradiation training period. A one-way fixed effects analysis of variance was calculated where Groups 2 and 4 were factor A and the cri- terion variable was 84-day spontaneous activity. This anal- ysis enabledcmmato determine the effects of forced swimming on spontaneous activity. Table 1 shows the results. Figure 2 indicates mean spontaneous activity values preirradiation. Table 1 Analysis of Variance for Preirradiation Spontaneous Activity Source of Variance SS DF MS F Among Groups 9342244 1 9342244 3.89 Within Groups 216031102 90 2400345 Total 225373346 91 29 >§>So< mooocwucomw cowuewpdefiuinm 1:th A whomfm 20382.11 2:23;: gm Nu (N no Go 54 u? fiw _ _ _ a 4‘ A 6:60 msomcscoam . 636“. 302283 30 rm Lu _N d _ '3' 0"! :::.°. . CCO_ QCQ CCCN OCm N D.) Em CCU¢ 00mg 0000 000m AVE. / SNOIiflWOAHH 31 4500,- 2894i|519 4000- 22561I5'8 3500" 33000 *- o .9 £- a) tr >- 2500 - t 2 F— U <1 (0 2000— D O uJ Z (I +— Z ISOO - 0 CL (.0 IOOOP (A, U) 500- ;j 8 E”: E: L; "I 1’ r1 -3 L— t— V" “J I. 7 7_ U C r“ O :1: O % L. 341:) 2 4 TREATMENT GROUPS Figure 2.: Mean Daily Spontaneous Activity Preirradiation 32 The computed F value was not significant indicating that, at the 5% level of confidence, there was no difference in preirradiation spontaneous activity between the two groups. The power of the test was .90. It should be noted, however, that the analysis of variance is not a completely satis- factory technique in interpreting longitudinal data of the sort that we are dealing with here. Consequently, a sign test was employed utilizing the same data. The sign test showed that, at the 5 per cent level,the Spontaneous forced animals (Group 4) were significantly more active than the spontaneous controls (Group 2) for the preirradiation train- ing period. A two—way fixed effects analysis of variance was calcu- lated where the spontaneous control (Group 2), spontaneous -forced (Group 4) and spontaneous effect (Group 5) treat— ments were factor A; the upper and lower halves of the 84- day preirradiation spontaneous activity relative to groups was factor B; and the criterion variable was survival time in hours. This analysis permits consideration of both treat- ment and level of preirradiation spontaneous activity as fac— tors in determining survival time following irradiation. Figure 3 shows the mean values for the three groups evaluated. Table 2 shows the results of the analysis of variance test. 33 Table 2 Analysis of Variance for Level of Preirradiation Activity and Survival Time Source of Variance SS DF MS F Treatment 16168 2 8084 Level 18342 1 18342 .595 Interaction 82299 2 41149 1.334 Error 4070745 132 30838 Total 4187555 137 The computed F values indicate that, at the specified level of confidence, there were no significant differences be- tween upper and lower halves of preirradiation spontaneous activity relative to groups and survival time. For this test the probability of making a Type II statistical error was .10. Lighting, Irradiation and Spontaneous Activity For the first 71 days of the treatment period the lights in the animal room were kept on 12 hours per day. For the remainder of the experiment lights were kept on 24 hours a day in order to facilitate hourly checks on animals follow- ing irradiation. Three ll-day periods were selected: (a) the last 11 days prior to 24-hour lighting, (b) the ll-day period between the first day of 24-hour lighting and the first day of irradiation and, (c) the first 11 days follow- ing irradiation with 24-hour lighting. A two-way mixed SURVIVAL TIME (Hours) 8C0 _ seetno 6r9i|77 7OO '- 600 '- 500 +- 300 - 200 h IOU- SPONTAN EOUS CONTROL 34 " i" c 4» 639tl5i 6 3 79 5,3-2l4 5371:52 SPONTANEOUS SPONTANEOUS FORCED EFFECT l UVL 2 U L U L 4 5 UPPER (U) AND LOWER (L) HALVES OF SPONTANEOUS ACTIVITY PREIRRADIATION Figure 3: Upper and Lower Halves of Spontaneous Activity and Survival Time 35 model analysis of variance was calculated where the three ll—day periods were fixed factor A, the 46 spontaneous ef— fect (Group 5) animals were random factor B, and the cri- terion variable was mean ll—day spontaneous activity. This analysis permits one to determine the effects of both 24—hour illumination and irradiation on spontaneous activity. Figure 4 shows the effects of 24—hour lighting and irradiation on spontaneous activity from the 60th day of training until sacrifice. Table 3 indicates the results of the analysis of variance test. Figure 5 shows mean activity levels for the three periods. Table 3 Analysis of Variance for Effects of Lighting and Radiation on Activity # T Source of Variance SS DF MS F Periods 246284790 2 123142395 55.35* Animals 211791112 45 4706469 Interaction 200228515 90 ' 2224761 Total 658304418 137 * indicates that F value is significant at 5% level. Since the computed F value was significant at the Specified level of confidence a Tukey test for multiple comparisons between means was calculated. Table 4 shows the results. 36 3.. .17: 3,745.91; :2..:.._.:.Z “3:: ...._:..:Th...H E. 7:52“-.. 339 8.5a 3.7.25 m: mo_ N9 L0 05 mm E «Q. no 00 T _ _ I 4 _ 1 a j\r a l 309 I. 009 l oOON 103mm 1 000m J 00mm I 000*» A 30.3.: cm W m onflIVNW A 59 213V 8 2,33 S 3.3: 8 are... cozofiotzmon. A3 2: E AVG/SNOIlOWOAEH :34»; I .211... :.,.r.1......_._..._ 22.17.27:22.32.2711“ I .7211; ooEma u n o wN+Om [00m 1000” [009 J OOON 100mm 1 000m Notnove 1 00mm 1 ooov 1 00m? 1 88 1 000m 008 _l_ _ommm_~mn (KOO/A93) le-fillOV SflOBNVlNC‘dS 38 Table 4 Tukey Test for Effects of Lighting and Radiation on Activity Mean Comparisons Significance at 5% Period 1 > Period 2 Significant Period 1 > Period 3 Significant Period 2 > Period 3 Significant The results of the Tukey test indicate that mean activity was significantly higher during 12—hour lighting than during both 24-hour lighting preirradiation and 24-hour lighting post— irradiation. Furthermore, mean activity level for the 24-hour lights on period preirradiation was significantly higher than the 24-hour lights on period postirradiation. Postirradiation Spontaneous Activity A one—way fixed effects analysis of variance was calcu- lated where the upper and lower halves of 3—day postirradi- ation spontaneous activity for the spontaneous effect (Group 5) treatment was factor A and the criterion variable was sur- vival time in hours. This type of analysis permits one to determine the effects of level of postirradiation activity on survival time. Figure 6 shows mean survival times for the upper and lower levels of activity. Table 5 indicates the results of the analysis of variance test. The computed F value indicates that there was no difference, at the speci- fied level of confidence, between upper and lower halves of SURVIVAL TIME (Hours) 800 eostlsl I_ 583: I89 700 - 600 — '—-—1‘ 530F- 490— 200 - 100 )— U L UPPER (U) AND LOWER (L) HALVES OF SPONTANEOUS ACTIVITY ' ‘ ' I . I‘ l. r - 111% r: Luuvt‘ ant: 1.<:\- v1 .1..t.v.~ )—I)'n=.' r’l-.-%'-l'I’(:~.'..I'f.wll 5:)I)1:-.::.t ”1.5 .".'t“'i‘.1!:|15t.1" -\.:I 1'..':- .. 40 Table 5 Analysis of Variance for Postirradiation Activity Level and Survival Time Source of Variance SS DF MS F Among Levels 6362 1 6362 .175 Within Levels 1596980 44 36295 Total 1603343 45 postirradiation spontaneous activity and survival time. The power of the test was .90. A Pearson product—moment correlation coefficient was cal— culated between ll-day preirradiation spontaneous activity and 3—day postirradiation spontaneous activity for the spon— taneous effect (Group 5) treatment. The resulting r value (.344), which was significantly different from zero at the 5 per cent level, indicated that both high and low level ac— tivity animals were affected in the same way by radiation. Body Weight Figure 7 shows weekly body weight changes for the five treatment groups. Final body weights prior to irradiation are shown in Figure 8. A one—way analysis of variance was calculated where Groups 1, 2, 3 and 4 were factor A and the criterion variable was body weight during the last week prior to irradiation. This procedure enables one to determine the effects of the four treatments on body weight prior to 41 3.3.17.3 17:; 21.743221 :5 L_.;.__ .rl. ..._:.1_..r._. A282: 09sz o 2.2 _ are N. __ e a m r. m m ... _ 4 a I _ a _ _ _ _ _ a 0 [‘7 40m 00. _o:coo msomcoanw...:.::: \\M\ 365.1 30228? .||. \ 380“. 52:36,» IIIII . Germ 308283 I I I \\ 102 .8218 foEmpwm I. OON 10mm I COM I own 1 00¢ .1 Dow I 00m 1H9|3M M308 (swolg) IIIJTIII omom0m mpomzmmoom "N o l I Viwurt 43 irradiation. The results of the analysis of variance are shown in Table 6. Table 6 Analysis of Variance for Body Weights Prior to Irradiation Source of Variance SS DF MS F Among Groups 195872 3 65290 32.843* Within Groups 357836 180 1987 Total 553709 183 * indicates that the F value is significant at the 5% level. Since the computed F value was significant at the specified level of confidence the Tukey test for multiple comparisons between means was employed. Table 7 indicates the results. Table 7 Tukey Test for Body Weights Prior to Irradiation Mean Comparison Significance at 5% Group 1 > Group 2 Significant Group 1 > Group 3 Significant Group 1 > Group 4 Significant Group 2 > Group 3 Significant Group 2 > Group 4 Significant Group 3 > Group 4 Significant The results of the Tukey test show that the sedentary con- trols (Group 1) were significantly heavier than the spon— taneous controls (Group 2), the sedentary forced (Group 3) and the spontaneous forced (Group 4) during the final week prior to irradiation. Furthermore, Group 2 was significantly 44 heavier than either Group 3 or Group 4 and Group 3 was sig- nificantly heavier than Group 4. A two—way fixed effects analysis of variance was calcu— lated Where the five treatments were factor A; the upper and lower halves of body Weight relative to groups during the last week prior to irradiation were factor B; and the cri- terion variable was survival time in hours. This analysis permits consideration of both treatment and level of pre— irradiation body weight as factors in determining survival time following irradiation. Figure 9 shows the upper and lower halves of body weight relative to groups with respect to survival time. The results of the analysis of variance are shown in Table 8. Table 8 Analysis of Variance for Level of Body Weight and Survival Time Source of Variance SS DF MS F Treatment 54857 4 13714 Level 196925 1 196925 6.566* Interaction 244238 4 61059 2.036 Error 6597728 220 29989 Total 7093749 229 * indicates that the]?value is significant at the 5% level. The results of Table 8 indicate that the level effects are sig— nificant. The mean survival time of all animals who were in the upper half of their group was 636 hours. This was (Hours) SURVIVAL TIME 800 TOO 600 500 400 200 I00 Blenh- ‘r‘: Upper £12..3 I’l‘t*".1‘l'.1.I..I"_u: v r Halve :‘ '. I L“, -.-' tl.l\l \‘\LL ‘ '\ v~. .\~ I A -...k swine 555: '28 5941:93 650545 575t20| srutzoo 596374 649329 553:194 53925212 .11. _____p "—7"- __T __I ‘F_ SEDENTARY SPONTANEOUS SEDENTARY SPONTANEOUS SPONTANEOUS CONTROL CONTROL FORCED FORCED EFFECT L U L U L U l U L U I 2 3 4 5 UPPER (U) AND (L) LOWER HALVES OF BODY WEIGHT PREIRRADIATION 46 significantly greater than the mean survival time of all the animals in the lower half of their group, which was 578 hours. White Blood Cells Figure 10 shows mean WBC values 9 days prior to ir- radiation. A one-way analysis of variance was calculated where Groups 1, 2, 3 and 4 were factor A and the criterion variable was WBC 9 days prior to irradiation. This pro- cedure enables one to determine the effects of the four treatments on WBC count prior to irradiation. The results are shown in Table 9. Table 9 Analysis of Variance for WBC Preirradiation Source of Variance SS DF MS F Among Groups 36375149 3 12125049 1.919 Within Groups 1137549185 180 6319717 Total 1173924335 183 The computed F value shows that, at the specified level of confidence, there were no significant differences between groups with regard to WBC 9 days prior to irradiation. The power of the test was .90. A two-way fixed effects analysis of variance was cal— culated where the five treatments were factor A; the upper w .,. 8861 4 W 38232an m m. Quezon. 3 m >mm 1 2 lower, 5 lower, 1 lower, 3 upper 3 lower > 5 lower, 1 lower, 3 upper 5 upper >.3 upper 4 upper > 3 upper 2 upper > 3 upper were factor A and the criterion variable was WBC five days postirradiation. This analysis permits one to evaluate the effects of the treatments on WBC postirradiation. The results are shown in Table 12. Table 12 Analysis of Variance for WBC Five Days Postirradiation Source of Variance SS DF MS F Among Groups 12239 4 3059 .170 Within Groups 4061250 225 lsdng Total 4073489 229 The results, as indicated in Table 12, show that there were no significant differences in WBC postirradiation between groups. The probability of making a Type II statistical error was .10. A two-way fixed effects analysis of variance was cal— culated where the five treatments were factor A; the upper and loWer halves of WBC five days postirradiation relative 52 to groups were factor B; and the criterion variable was survival time in hours. The above procedure permits con- sideration of both treatment and level of WBC postirradia- tion as factors in determining survival time following irradiation. Figure 13 shows the relationship between upper and lower halves of WBC postirradiation and survival time. The analysis of variance is shown in Table 13. Table 13 Analysis of Variance for WBC Level Postirradiation and Survival Time Source of VarianCe SS DF - MS F Treatment 54857 4 13714 Level 7123 l 7123 .227 Interaction 140011 4 35002 1.117 Error 6891757 220 31326 Total 7093749 229 The F values, as indicated in Table 13, show that there were no differences between level of WBC postirradiation relative to groups and survival time. Eosinophils Figure 14 shows mean eosinophil levels nine days prior to irradiation for Groups 1, 2, 3 and 4. A one-way fixed effects analysis of variance was calculated where Groups 1, 2, 3 and 4 were factor A and the criterion variable was eosinophil count nine days prior to irradiation. The (Hours) SURVIVAL TIME 900 r— 627 i173 637?” 800_ 624370 S'Zt'es 6l4i’I68 sootles 642114| 606tI64 ssatzol 552:198 700 r- GOOF- "'""""I I__ F———- L____1. _____I. 500*- . 400'- o 300 - 200)- IOO- SEDENTARY SPONTANEOUS SEDENTARY SPONTANEOUS SPONTANEOUS CONTROL CONTROL FORCED FORCED EFFECT . I I I I L U L U L U L U L U I 2 3 4 5 UPPER (U) AND (L) LOWER HALVES OF WBC POSTIRRADIATION Figure 15: Upper and Lower Hztlxcs (21' 1‘. NC Postirradiation and Surxixal I“. : u 47 QMJIOu 7’): 53 mzomzmmzrm g D > C> > C) C) 0: LIJ m Lu “J < 4 2‘3 4 z Z I—0 <0: I—O <0 4)— z.z: P- 'zu-I Pun F-LJ uth- Z'F' tnc> U‘L>\ u3u- ”.m, (nLu | 2 3 4 ' 5 TREATMENT GROUPS I‘Lgtlrc 18: Aulrtwial (NI-.ght IJVL'I3-HI§ \Vt-.;ht at Ih nth __ 133553 SDOENVINOdS 00012 t .00002 030803 SOOBNVINOdS .ooonstooom 03380:! AHVINBOBS oooni .ooooz 'IOtIlNOO 8003 N ‘71NOdS .ooomi ooom 'IOHlNOO VAUVINEOES 000“ tooooz 00030 (— 00020 L OOOIO '- (3w010)39|:IIHOVS 1V .LHOIEINI A008 83d LUV WVNHHGV GROUPS TREATMENT (t’ C . .‘.l\ [.1 .Il I111 I)('1' IIIII (Vt-.L-Ilt .-‘.I‘u 64 effects analysis of variance was calculated where the five treatments were factor A and the criterion variable was adrenal weight per body weight at sacrifice. The results are shown in Table 19. Table 19 Analysis of Variance for Treatment Effects on Adrenal Weight Per Body Weight at Sacrifice Source of Variance SS DF MS F Among Groups .00000004 4 .00000001 1.073 Within Groups .00000132 155 .00000001 Total .00000136 159 The results, as shown in Table 19, indicate that there were no significant treatment effects on adrenal weight per body weight at sacrifice. The probability of making a Type II statistical error was .10. Survival Time The effects of the five treatments on survival time of all animals are shown in Figure 20. It should be noted that if an animal lived until sacrifice he was assigned a survival time of 715 hours. A one—way fixed effects analysis of variance was calculated where the five treatments were factor A and the criterion variable was survival time in hours. This procedure permits one to determine the treatment 5951|88 Hummum ”Joy/3:20am 62:t|6‘.‘~ 6091' ITZ 5841186 omomOu maouzqezoau ‘F—_‘ owomou >mm_>m3m 300 - 20’);- I00- r.) TREATMENT GROUPS Shitifirtl. 15.11.001.111 .. .115 ) L. .glll‘t' 66 effects on survival time following irradiation. The results are shown in Table 20. Table 20 Analysis of Variance for Treatment Effects on Survival Time of All Animals Source of Variance SS DF MS F Among Groups 54857 4 13714 .438 Within Groups 7038892 225 31282 Total 7093749 229 The results, as shown in Table 20, indicate that there was no significant treatment effect on survival time of all animals. The power of the test was .90. Figure 21 shows treatment effects on survival time of only those animals which died during the 30-day postirradi— ation mortality period. A one-way fixed effects analysis of variance was calculated where the five treatments were factor A and the criterion variable was survival time. This procedure allows one to determine treatment effects on sur— vival time of only those animals which died. The results are shown in Table 21. The results, as shown in Table 21, indi- cate that there were no significant treatment effects on sur- vival time of those animals which died. The probability of making a Type II statistical error was .10. 3901I4O 348i'3l Belt”? hummmm msomzr3 m0 m5 m a m N _ a o 3 o 3 a a a 3 a o Swab Some Bozo... 39:58 3528 msowzatloam msouzfizoam $558.0, moomzfzoam Ear/mono 19 now tom t 9. :IC HBSWDN S'IVWINV 70 Discussion fipontaneous Activity Previous work in this laboratory by Hanson and Van Huss (15) and Van Huss, Midkelsen and Heusner (49) have shown that animals which were forced to swim for one-half hour daily for 35 days had a significantly lower voluntary activ— ity level than their spontaneous controls° Reference to Figure 1 will indicate that in the present investigation there were no differences between the two groups for the first 30 days of training. However, after 30 days and for the remainder of the preirradiation training period those animals which were forced to swim daily had consistently higher activity levels than the control animals. Apparently after 30 days the forced swimming, in some way, acted as an incentive for voluntary running. It should be noted that these differences were not significant at the specified level of confidence (Table 1) when the analysis of variance test was employed, but were highly significant when the sign test was utilized. No previous work has been done relating level of pre- irradiation spontaneous activity and survival time following irradiation. Table 2 and Figure 3 show that level of volun- tary activity does not significantly alter survival time. 71 Jones et al (18) and Kimeldorf et al (22) have shown that irradiation produces a rather dramatic decrease in voluntary activity of rats. These results have been sub- stantiated in the present investigation (Tables 3 and 4 and Figure 4). Furthermore it would be wise for future in- vestigators to carefully regulate the amount of time that the animal rooms are to be illuminated if spontaneous activ- ity is to be a measured parameter. Figure 4 clearly demon- strates that 24-hour per day illumination results in sub- stantially decreased spontaneous activity when compared with lZ—hour per day illumination. Published results on the effects of postirradiation exercise on mortality are conflicting. Trifonov (50) and Pinchook and Scherban (50) have demonstrated decreased mortality and increased survival time in animals subjected to exercise following irradiation while Kimeldorf and Jones (20), Kimeldorf and Baum (l9), Sergeyev (50), Popov (50) and Markelov (50) have shown increased mortality and decreased survival time. Smith and Smith (35), (36) report no differences between rats exercised following irradiation and control animals. It should be noted that all of the above investigations were concerned with forced exercise following irradiation while the present study involved 72 postirradiation spontaneous activity. Figure 6 and Table 5 indicate that there were no significant effects of post— irradiation voluntary activity level on survival time. Body Weight The results of the four treatments produced significant differences in body weight prior to irradiation (Figure 8 and Tables 6 and 7). The most heavily exercised group (treatment 4) had a mean body weight significantly lower than Groups 1, 2 and 3. The sedentary control group (treat— ment 1) had a mean body weight significantly higher than Groups 2, 3 and 4. The results indicate that body weight was a function of the exercise regimen. Table 8 and Figure 9 show that, regardless of treatment, the animals in the upper half of their group with respect to body weight had significantly longer survival times than animals in the lower half. Mean survival time for animals in the upper half was 636 hours as opposed to 578 hours for animals in the lower half. A plausible explanation may be that the heavier animals did not receive as high a dose as the lighter animals. Andrews states (2) "In general, de— crease in dose with depth results from attenuation by ab— sorption." An interesting question, however, does arise. Why, if the sedentary animals (Group 1) were significantly 73 heavier and, if being in the upper half of one's treatment group significantly improved survival time, didn't Group 1 have significantly better survival times? This investiga- tion cannot shed any light on the proposed question, however, if one employed similar types of treatments while attempting to maintain equal body weights between groups, conclusive evidence could be obtained. Blood Parameters Garrey and Bryan (13) and Sturgis and Bethell (46) in their reviews of literature, have concluded that a marked leucocytosis is to be expected in circulating blood immedi- ately following muscular exercise. Conclusive evidence has not been found regarding the longitudinal effects of training on the white blood cells. Thorner (47) and Ander— sen, Heusner and Pohndorf (l) have reported increases in the WBC; whereas, Hawkins (17) found no such changes. The results of the present study are in agreement with Hawkins. Analysis of the preirradiation WBC count indicates that there were no significant differences in WBC between the five treatment groups (Figure 10 and Table 9). Table 10 indicates that the interaction effects of upper and lower halves of WBC relative to groups and the five treatments were significant with respect to survival time following 74 irradiation. Table 11 indicates the existing significant differences. Although there are significant differences no pattern with regard to either treatment or level exists and interpretation is impossible. Within the next year statistical procedures will be available that will enable one to consider the combined effects of level of WBC pre- irradiation and body weight preirradiation on survival time. Table 12 and Figure 12 indicate that following irradia- tion there were dramatic decreases in WBC levels in all groups, however, no significant differences between the five treatments were detected. Bacq and Alexander (4) have reported that a large dose of radiation causes rapid damage to the bone marrow and lymphoid tissue, thus greatly suppressing the mitosis of leucocytes for eight to ten days. These observations have been substantiated in this investi- gation. Figure 13 and Table 13 show that there were no signifi- cant differences between the five treatment groups regarding the level of WBC postirradiation relative to groups and survival time in hours. Figure 14 and Table 14 indicate that there were no significant differences in eosinophil level prior to irradi- ation between the five treatment groups. Irradiation 75 virtually eliminated circulating eosinophils in all animals except six. These six showed eosinophil levels of 5.5/cu.mm blood. Similarly, there were no significant differences between the five treatment groups concerning level of eosino- phils relative to groups preirradiation and survival time in hours. (Figure 15 and Table 15) Organ Weights It will be recalled from Chapter III that the thymus was not weighed, since its postirradiation condition pro— hibited proper extraction and trimming. There is conflicting evidence in regard to the effects of exercise upon organ weights. Donaldson (11, 12) and Kimeldorf and Baum (19) found increases in Splenic weight after exercise; Donaldson (9, 10), Hatai (16); and Mon- toye et a1. (25) reported decreases; and, Hanson and Van Huss (15) and Montoye et a1. (26) found no changes. Boro- vansky (5), Donaldson (9, 10, 11, 12), Hanson and Van Huss (15), Kimeldorf and Baum (l9), and Montoye et a1. (25, 27) cite increases in adrenal weights following physical train— ing; whereas Hatai (l6) and Montoye et al (26) report no effects. Asahina et a1. (3) have shown that excessive physi- cal training can produce histological deterioration and hypo— function of the thymus. 76 Kimeldorf and Baum (19) found that in the rat the weights of both the thymus and the spleen are reduced fol— lowing irradiation. They also reported increases in adrenal weights with exposure to x—rays. Patt et a1. (31) ob- served similar splenic and adrenal responses in rats. Such adrenal changes were not found in mice by Smith and Smith (39) following irradiation. In a subsequent study, however, Smith (37) found significant fluctuations in adrenal weights which apparently were dependent upon the amount of time elapsing between irradiation and sacrifice. Since splenic and adrenal weights were not made prior to irradiation, it was not possible to evaluate the effects of irradiation on the weights of these organs. Figure 16 and Table 16 show that there were no signifi- cant differences between the five treatments with respect to spleen weight at death. Similar results were found with spleen weight at sacrifice (Figure 17 and Table 17). The five treatments produced no differences in adrenal weight per body weight at death (Figure 18 and Table 18). Likewise, Figure 19 and Table 19 reveal no treatment effects on adrenal weight per body weight at sacrifice. In summary, the five treatments had no effects on splenic and adrenal weights either at death or at sacrifice. 77 Mortality and Survival Sergeyev (50), Tovbin (50), Trifonov (50) and Zimkin (50) found that pre—training increases survival time and decreased mortality following irradiation. Kimeldorf and Jones (20) found no differences in mortality between trained and untrained animals. The results of the present investigation are in direct contrast to the results of Sergeyev, Tovbin, Trifonov and Zimkin. Figure 20 and Table 20 show no significant differences between treatments with respect to survival time for all animals. Similar results were found with treatment effects on survival time of only those animals which died during the 30-day mortality period (Figure 21 and Table 21). The Chi-square test showed no treatment effects on mor— tality (Figure 22 and Table 22). It should be pointed out that the four authors who found increased survival time with pre-training did not see fit to subject their data to statistical analysis. The resultant differences may well have been chance, rather than treatment, effects. However, it would be well to keep in mind that both the intensity and duration of training employed in this investigation was dif- ferent than that employed by the authors who conclude that there were treatment differences in mortality and survival time. _ CHAPTER v SUMMARYr CONCLUSIONS AND RECOMMENDATIONS Summary The purpose of this investigation was to study the effects of various levels of preirradiation physical ac- tivity, from weanling age to adulthood, upon the radiation resistance of mature rats. Two-hundred and fifty 26—day old male albino rats (Sprague-Dawley strain) were randomly assigned to one of five treatments. Group 1 (sedentary control) received no special treatment, other than radiation, and was confined to individual sedentary cages both prior to and after being irradiated. Group 2 (spontaneous control) received no special treatment, other than radiation, and was housed in individual spontaneous exercise cages until irradiated and then confined to individual sedentary cages. Group 3 (sedentary forced) was housed in individual sedentary cages both prior to and after being irradiated. During the training period prior to irradiation, these animals were forced to swim daily for one—half hour with two per cent body weight attached to the base of the tail. Group 4 (spontaneous forced) was housed in individual spontaneous exercise cages until irradiated and then confined to 78 79 individual sedentary cages. Prior to being irradiated these animals also were forced to swim daily for one— half hour with two per cent body weight attached to the base of the tail. Group 5 (spontaneous effect) was housed in individual spontaneous exercise cages both prior to and after being irradiated. These animals were not sub— jected to the preirradiation forced training regimen. Three animals in one of the forced activity groups and two in the other drowned in training. One animal in one of the other groups died while under anesthesia during preirradiation blood sampling. Consequently, animals were eliminated randomly, and 47 rats in each group were ir- radiated. Postirradiation blood sampling resulted in the death of one additional animal. Once again, equal groups were maintained by random elimination. Therefore, 46 animals per group comprised the final sample. Those animals which were subjected to the swimming regimen (Group 3 and 4) were swum in individual cylindrical tanks measuring 11 inches in diameter and 30 inches in depth. Water temperature was maintained between 360 and 380 C. during swimming. After each swim period, the animals were dried with a towel and replaced in their respective cages. 80 The preirradiation treatments were begun at 27 days of age and were carried on thrOughout puberty and early adult- hood. On the 83rd day of the preirradiation treatments, 20 per cent of the animals, randomly selected with stratifica- tion by groups were placed in sedentary cages for an ap- proximate 64—hour rest period prior to irradiation. The forced swimming regimen was terminated for these animals at this time. On the 84th, 85th and 86th days, a similar pro- cedure was followed with 20, 30 and 30 per cent of the animals respectively. Following the 64-hour rest period, each animal was ex- posed to 650 r while confined in a three—inch by nine—inch lucite chamber. Twenty-four animals were irradiated simultaneously. The total time required for irradiation was 180.5 minutes. Animal positions were rotated every 36.1 minutes to provide equal exposures for all. Nine days before and five days following irradiation, a blood sample was drawn from the orbital sinus of each animal to determine the WBC and eosinophil counts. A 24— hour period of confinement in sedentary cages was pro— vided for all animals, regularly in spontaneous cages, prior to the drawing of each blood sample. During the 30 days immediately following irradiation, a 81 laboratory technician was with the animals twenty-four hours a day. Mortality and survival time in hours for all groups of animals were recorded during this period. At the end of the thirty—day period, the surviving animals in all groups were sacrificed. Upon death or sacrifice, body weight as well as the weights of the spleen and adrenals were deter- mined. The thymus was observed subjectively, but not weighed, since its posirradiation condition prohibited proper extraction and trimming. Throughout the entire investigation, all animals re- ceived water and a commercial ground animal diet, ad libi— tum. Body weight for each animal was determined once a week throughout the preirradiation period, and the weight to be attached to the base of the tail during swimming was changed on the following day. The temperature in the hous- ing quarters was maintained between 700 and 780 F. Daily voluntary exercise data for each animal housed in a spon- taneous exercise cage was recorded at 7:45 A.M. daily. For the first 71 days of the preirradiation period, an automatic timer illuminated the animal quarters 12 hours a day. Since it was necessary for the technician to observe each animal hourly following irradiation, lights were kept on 24 hours a day beginning on the 72nd day. The 24-hour 82 illumination was started 12 days prior to irradiation in order to provide an adjustment period for the animals. Statistical analysis (Sign test) showed that the spon— taneous forced animals (Group 4) were significantly more active than the spontaneous controls (Group 2). Both pre- irradiation and postirradiation activity level had no effect on survival time following irradiation. Twenty- four hour lighting significantly reduced activity levels when compared with 12 hour lighting. Irradiation signifi— cantly reduced activity levels. Body weight differences between groups prior to irradiation were observed with the most heavily exercised (Group 4) being lighter than Groups 1, 2 and 3. Preirradiation WBC and eosinophil (counts showed no differences between groups. Similar re- sults were found with postirradiation blood sampling. No differences between groups were noted with respect to spleen weight at death and sacrifice and adrenal weight per body weight at death and sacrifice. The comparison of both mortality and survival time in hours between the treatment groups Showed no differences. Those animals which were in the upper half of their group with respect to body Weight had longer survival times than those animals in the lower half. 83 Conclusions Analysis of the results of this investigation has led to the following conclusions: 1. Forced swimming for one—half hour daily with two per cent body weight attached significantly increases spon— taneous activity. 2. Forced swimming and/or spontaneous activity for a prolonged period reduce body weight. 3. Illumination of animal quarters 24-hours daily sig- nificantly reduces volitional activity as compared to 12— hour per day illumination. 4. Irradiation decreases spontaneous activity. 5. Prolonged training does not alter WBC or eosino— phil levels either prior to or following irradiation. 6. Prolonged training has no effect on either adrenal weight per body weight or gross splenic weight following irradiation. 7. Level of preirradiation physical activity has no effect on postirradiation mortality and survival time. 8. Body weight appears to be the most significant fac- tor influencing survival time following irradiation. Recommendations 1. Unless histological or pathological investigations 84 are to be conducted, further research should diSpense with the weighing of organs. 2. If spontaneous activity is to be measured, careful attention should be given to the amount of time daily that the animal quarters are to be illuminated. 5. A study should be undertaken in which spontaneous activity is measured for 50 days prior to irradiation with 650 r. Spontaneous activity will be measured for three days postirradiation. Use 12 hours of light and 12 hours of darkness throughout investigation. This will enable one to quantitate the effect of irradiation on voluntary activ- ity. 4. It is recommended that the adrenals of all animals be subjected to histological examination (volume of three cortical zones) and plotted against survival time. This procedure will provide evidence of radiation damage and recovery trends. 5. It is recommended that the basic Study be redone utilizing a radiation dose of 700-900 r. 10 SELECTED BIBLIOGRAPHY Andersen, K. L., W. W. Heusner and R. H. Pohndorf. The progressive effects of athletic training on the red and white blood cells and the total plasma protein. Internat. g, angew Physiol. einschl. Arbeitsphysiol. 2g, 16:120- 128, 1955. Andrews, H. L. Radiation Biophysics. Prentice-Hall, Inc., Englewood Cliffs, 1961. Asahina, K., F. Kitahara, M. Yamanaka and T. Akiba. In- fluences of excessive exercise on the structure and func- tion of rat organs. Japanese g, Physiol. 9:322-326, 1959. Bacq, Z. M. and P. Alexander. Fundamentals 9§_Radiobiology. Pergamon Press, New York, 1961. Borovansky, L. Les modifications morphologiques et de croissance apres un travail musculaire de longue duree chez 1es rats. Bulletin International g§_1'Academie des Sciences Q§_Boheme, 1930. Braun, H. Uber die Steigerung der Leistungsfahigkeit der Ratten nach Ganzkorperbestrahlung, gepruft im Schwimm- versuch. Strahlentherapie 119:462-466, 1962. Brown, W. L. and R. K. White. Preirradiation fatigue as a factor in the prevention of irradiation deaths in rats. g, Genetic Psych. 93:287-290, 1958. Brown, W. L. and R. K.-White. A study of fatigue and mortality in irradiated rats. Radiation Research 13:610- 616, 1960. Donaldson, H. H. On the effects of exercise carried through seven generations on the weight of the musculature and on the composition and weight of several organs of the albino rat. Ag, Q, Anat. 50:359-396, 1932. Donaldson, H. H. On the effects of exercise beginning at different ages on the weight of the musculature and of several organs of the albino rat. Am, g, Anat. 53:403- 411, 1933. 85 11 12 l3 14 15 16 17 18 19 20 21 86 Donaldson, H. H. Effects of prolonged rest following exercise on organ weights of albino rat. Ag, g, Anat. 56:45-55, 1935. Donaldson, H. H. Summary of data for effects of exer- cise on organ weights of albino rat: comparison data from dog. Ag, g, Anat. 56:57—70, 1935. Garrey,.W. E. and W. R. Bryan. Variations in WBC Counts. Physiol. Rev. 15:597-638, 1935. Guenther, W. C. Analysis 9f_Variance. Prentice-Hall, Inc., Englewood Cliffs, 1964. Hanson, D. L. and W. D. Van Huss. The effects of forced exercise upon the amount and intensity of spontaneous activity of young male albino rats. A report to the Amer- ican College of Sports Medicine. Minneapolis, 1963. Hatai, S. On the influence of exercise on the growth of organs in the albino rat. Anat. Rec. 9:647—665, 1915. Hawkins, C. C. The Effects of Conditioning and Training Upon the Differential White-Cell Count. (unpublished Ph.D. Thesis, New YOrk University, New York, 1937). Jones, D. C., D. J. Kimeldorf, D. O. Rubadeau, G. K. Os— born and T. J. Castanera. Effects of x—radiation on per— formance of volitional activity by the adult male rat. Am, g, Physiol. 177:243-250, 1954. Kimeldorf, D. J. and S. J. Baum. Alterations in organ and body growth of rats following daily exhaustive exercise, x-irradiation and postirradiation exercise. Growth 18:79-96, 1954. Kimeldorf, D. J. and D. C. Jones. The relationship of radiation dose to lethality among exercised animals ex— posed to roentgen rays. Am, g, Physiol. 167:626—632, 1951. Kimeldorf, D. J., D. C. Jones and T. J. Castanera. Ef- fect of x-irradiation upon the performance of daily ex- haustive exercise by the rat. Am, g, Physiol. 174:331— 335, 1953. 22 23 24 25 26 27 28 29 3O 31 87 Kimeldorf, D. J., D. C. Jones, T. J. Castenera, D. O. Rubadeau and G. K. Osborn. Repeated exposure to x—rays and volitional activity performance of rats. Fed. Proc. 13:79, 1954. Kimeldorf, D. J., D. C. Jones and M. C. Fishler. The effect of exercise upon the lethality of roentgen rays for rats. Science. 112:175-176,rl950. Maksud, M., W. D. Van Huss, W. W. Heusner, E. Smith, R. Kertzer and K. Coutts. The resultant effects of elec- trical stress and physical activity on blood cholesterol levels, adrenal histology and cardiac necrosis of adult male rats. Report given to the 1st Congresso Internazion- ale Di Psicologia Dello Sport. Rome, 1965. Montoye, H. J., K. ACkerman, W. D. Van Huss and R. Nel- son. Effects of milk and training on swimming perform- ance and organ weights in rats. Res. Quart. 33:104—110, 1962. ‘ Montoye, H. H., R. Nelson, P. Johnson and R. MacNab. Effects of exercise on swimming endurance and organ weight in mature rats. Res. Quart. 31:434-439, 1960. Montoye, H. J., J. Sherburne, K. Ackerman, E. M. Jones and D. Cederquist. Effects of exercise and milk con- sumption on blood serum cholesterol in rats. Egg, Quart. 33:430-438, 1962. Morris, J. N. and M. D. Crawford. Coronary heart dis— ease and physical activity of work. Brit. Med. J, 2:1485—1496, 1958. Morris, J. N., J. A. Heady, P. A. B. Raffle, C. G. Rob- erts and J. W. Parks. Coronary heart disease and physical activity of work. -Lancet 2:1053—1057, 1953. Nims, L. F. and E. Sutton. (Weight changes and water composition of rats exposed to whole-body x~irradiation Am, g, Physiol. 171:17—21, 1952. Patt, H. M., M. N. Swift, E. B. Tyree and E. S. John. Ad— renal response to total body x—radiation. Am, Q,~Physiol. 150:480-487, 1947. 32 33 34 35 36 37 38 39 4o 41 42 43 44 88 Pedley, F. S. Coronary disease and occupation. Canad. Med. Assn. g, 46:147-151, 1942. Ryle, J. A. and W. T. Russell. The natural history of coronary disease; clinical and epidemiological study. Brit. Heart g, 11:370-389, 1949. Seigal, S. Nonparametric Statistics. McGraw—Hill Co., New York, 1956. Smith, F. and W. W. Smith. Exercise following whole- body irradiation of mice. Fed. Proc. 9:117, 1950. Smith, F. and W. W. Smith. Exercise effects on tolerance to radiation. Am, J, Physiol. 165:662—666, 1951. Smith, W. W. Acute KCl and histamine tolerance and ad- renal weight in x-irradiated mice. Am, g, Physiol. 167:321-327, 1951. Smith, W. W. Survival after radiation exposure. Nu: cleonics. 10:80—83, 1952. Smith, W. W. and F. Smith. Effect of thyroid hormone on radiation lethality. Am, g, Physiol. 165:639-650, 1951. Smith, W. W. and F. Smith. (Effects of thyroid and ra- diation on sensitivity to hypoxia, basal rate of 02 con- sumption and tolerance to exercise. Ag, Q, Physiol. 165: 651-661, 1951. Smith, D. E. and E. B. Tyree. Influence of x—irradiation upon body weight and food consumption of the rat. Am, g, Physiol. 177:251—260, 1954. Smith, W. W., L. Gonshery, I. Alderman and J. Cornfield. Effect of Granulocyte Count and Litter on Survival of Irradiated Mice. Am, g, Physiol. 178:474-476, 1954. Steel, R. G. D. and J. H. Torrie. Principles and Pro- cedures 9f_Statistics. McGraw—Hill Co., New York, 1960. Steinhaus, A. H. The chronic effects of exercise. Phys- iol. Rev. 13:103-147, 1933. 45 46 47 48 49 50 89. Stone, S. H. Method for obtaining venous blood from the orbital sinus of the rat or mouse. Science 119: 100, 1954. Sturgis, C. C. and F. H. Bethell. Quantitative and qualitative variations in normal leucocytes. Physiol. Revs. 23:279-303, 1943. ThOrner, W. Uber die Zellemente des Blutes im Train— ingszustand. Untersuchung an Olympiakampfein in Am- sterdam. Arbeitsphysiologie 2:116, 1930. Van Huss, W. D., W. W. Heusner, O. Mickelsen,J1'Weber, R. Carrow and L. Dellonsanta. The effects of forced pre pubertal exercise upon post puberty exercise pat- terns, food intake and selected physiological para— meters. Report given to the lst Congresso Internazi- onale Di Psicologia Dello Sport. Rome, 1965. Van Huss, W. D., O. Mickelsen and W. W. Heusner, The effects of pre puberty forced exercise. Unpublished data in the files of the Human Energy Research Labora- tory. Zimkin, N. V. and A. V. Korobkov. Physical exercise as a means of increasing resistance of the organism to un- favorable environmental influences. Theory and Practice 9§_Physical Culture. 23:348-355, 1960b. OFUOZEL“ X- a U) W APPENDIX A Raw Data Legend for Raw Data Animal Number Treatment Mean Daily Voluntary Activity During Total Preirradi- ation Period. Level of Mean Daily Voluntary Activity Relative to Groups During Total Preirradiation Period. Mean Daily Voluntary Activity During 11 Days Preirradi- ation Prior to Lights On. Mean Daily Voluntary Activity Last 11 Days Preirradia- tion with Lights On. Mean Daily Voluntary Activity During First 11 Days Postirradiation. Mean Daily Voluntary Activity First 3 Days Postirradi— ation. Level of Mean Daily Voluntary Activity Relative to Group During First 3 Days Postirradiation. Body Weight Last Week Prior to Irradiation. Level of Body Weight Relative to Groups Last Week Prior to Irradiation. WBC 9 Days Prior to Irradiation. 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June Fri. 25 June Sat. 26 June Starting Positions for Irradiation of Animals Time Finished 12:00 Noon 12:00 Noon 108 APPENDIX C Nos. Sed. Box II III IV II III III IV II III IV 1-50 Forced Pos. U 0 w P m 0 m > m U m > m U 0 > U U 0 w m U 0 w W m U 0 w > 11 19 10 34 14 Nos. Spont. Box I II III IV V I II III 51-100 Forced Pos. m U 0 m V U 0 w > m 0 w > M U U W U U 0 W M U 0 w W M U 0 w An. 86 98 55 73 63 100 77 61 85 84 69 9O 53 65 88 99 71 76 79 Nos. Spont. Box I II III IV V I II III IV V I II III IV V I II III IV V I II III IV V I II III IV V {PUUOW WUOW>.UOCUD>L11 00135th U133L'UUO w b m U 0 101-150 Control Pos. An. 124 112 113 123 128 142 119 107 115 127 134 141 133 138 114 139 104 150 109 140 145 130 120 129 122 135 106 105 101 Nos. Spont. Box I II III IV V I II III IV V I II III IV V I II III IV V I II III IV ' V I II III IV V 109 w P m U 0 F m U 0 m U U 0 w 5 U 0 w P m 0 w W m U 0 w W m U 151-200 Effect Pos. An. 188 153 180 190 184 156 198 163 166 200 189 181 177 169 168 173 162 155 178 161 171 170 158 197 154 183 191 151 152 Nos. Sed. Box I II III IV V I II III IV V I II III IV V I II III IV V II III IV II III IV 201-250 Control Pos. 0 wzthtJ w ptyc3(a >tntjrau1 m U