Igisémsiewevsa:a*-I2I:'a_-avv~ - - uu.I.'.2...'.'. ‘m .’.‘4: -' HISTOCHEMICAL OBSERVATIONS ON RAT CARDIAC MUSCLE FOLLOWING CHRONIC EXERCISE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ROBERT OTTO RUHLINAG 1970 F'ZJBRARY I‘v’lichigan State University This is to certtfg that the thesis entitled HISTOCHEMICAL OBSERVATIONS ON RAT CARDIAC MUSCLE FOLLOWING CHRONIC EXERCISE presented by Robert Otto Ruhling has been accepted towards fulfillment of the requirements for Ph.D. degree in HealthI Physical Education & Recreation m/zflé Major professor use 8/6/70 0-169 ABSTRACT HISTOCHEMICAL OBSERVATIONS ON RAT CARDIAC MUSCLE FOLLOWING CHRONIC EXERCISE BY Robert Otto Ruhling The purpose of this study was to investigate the effects of seven different levels of chronic physical activity on the metabolic and morphologic characteristics of the left ventricular myocardium of adult male albino rats. Two hundred and fifty-two, 71-day-old, male, albino rats (Sprague-Dawley strain) were brought into the labo- ratory and were assigned to one of seven treatments at random. The treatment groups were sedentary control (CON); voluntary running (VOL); short-duration, high-intensity running (SHT); medium-duration, moderate-intensity running (MED); long-duration, low-intensity running (LON); electric stimulus control (ESC); and endurance swimming (SWM). Treatments were administered Monday through Friday. Animals were provided with food and water ad libitum. The healthiest and best trained animals were selected for sacrifice. Seven selected animals were weighed and Robert Otto Ruhling sacrificed on Mondays after 0, 2, 4, 6, 8, and 10 weeks of training. The final sample consisted of 126 animals. During sacrifice, each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital. The heart was excised and cut transversely at a level just below the atria. The apical portion was placed on a chuck which was immersed in pre-cooled 2—methylbutane. Three hundred micra from the apex a minimum of six 10 u serial sections were cut. Five histochemical procedures were utilized to evalu- ate the relative glycogen, fatty acid, and aerobic enzyme concentrations in the cardiac fibers. A histologic pro- cedure was performed to evaluate the morphology of the hearts. Each histochemical stain was measured objectively using a light transmission meter. The histological sections were rated subjectively on the bases of normalcy and of pathology. The results indicate that training for eight weeks was sufficient to produce metabolic adaptations in the rats. The eight-week LON group was observed to be significantly lighter than the eight-week CON group (p < .10). The fatty acid concentrations were greater in the VOL, LON, and SWM groups at eight weeks than in the CONS group at zero-week (p < .10). Also, in the LON group at eight weeks, sur- prisingly, the glycogen concentrations were greater than in the CONS group at zero-week (p < .10). Robert Otto Ruhling No pathological changes were observed in any of the seven treatments (p < .05). This result is limited to the single section taken approximately 300 u from the apex. HISTOCHEMICAL OBSERVATIONS ON RAT CARDIAC MUSCLE FOLLOWING CHRONIC EXERCISE BY Robert Otto Ruhling 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 1970 Q: —~ w 7A A /~~ 3 "7- ”fl DEDICATION To Holly ii ACKNOWLEDGMENTS Appreciation is extended to the chairman, Dr. W. D. Van Huss and to the individual members, Dr. W. W. Heusner, Dr. R. E. Carrow, and Dr. S. D. Sleight of my guidance committee for their individual and group help that I re- ceived over the past four years. Special thanks go to Dr. R. E. Carrow for allowing me to have unlimited use of the equipment and the supplies of the Cytology Laboratory, Department of Anatomy. A word of appreciation goes to Mr. James F. Taylor who helped provide a friendly and stimulating atmosphere in which to work. Thanks also go to Mrs. Barbara Wheaton and to Miss Patricia L. Lamb for their unending technical aid and laboratory assistance. Special thanks go to Mr. David J. Anderson for drawing the figures. A thank you is also in order to Mr. Kwok-Wai Ho, Chief Animal Caretaker, and to his assistants for the care and training of the animals in the Human Energy Re- search Laboratory, Department of Health, Physical Edu- cation and Recreation. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . Vi LIST OF FIGURES O O O O O O O O O O O O Viii Chapter I 0 THE PROBLEM. O O O O O O O O O O 1 Introduction. . . . . . . . . . 1 Statement of the Problem. . . . . . 2 Rationale. . . . . . . . . . . 2 Significance of the Problem. . . . . 3 Limitations of the Study. . . . . . 3 Definitions and Abbreviations . . . . 4 II. REVIEW OF RELATED LITERATURE . . . . . 7 Myocardial Metabolism. . . . . . . 7 Related Biochemical and Histochemical Studies. . . . . . . . . . . 13 Glycogen . . . . . . . . . . 13 Lactate. . . . . . . . . . . 18 Fatty ACidS O I O O O O I O O 21 SDH and Cy Ox. . . . . . . . . 22 Myocardial Pathology . . . . . . . 25 III. RESEARCH METHODS . . . . . . . . . 32 sample. 0 O O O O O O O O O O 32 Treatment Groups . . . . . . . . 34 CON 0 O O O O O O O O O O O 34 VOL 0 O O O O O O O O O O O 34 SHT O O O O O O O O O O O O 35 MED I O O O O O O O O O O O 35 LON I O O O O O O O O O O O 36 ESC O O O O I O O O O O O O 36 SWM I O O O O O I O O O O O 37 iv Chapter Treatment Procedures . . . . . Animal Care . . . . . . . . Sacrifice Procedures . . . . . Histological Procedure . . . . Histochemical Procedures. . . . Tissue Analysis Procedures . . . Histochemical. . . . . . . Pathological . . . . . . . Statistical Procedures . . . . IV. RESULTS AND DISCUSSION . . . . . Resu1ts O O O O O O O O 0 Effectiveness of the Training Programs. . . . . . . . Histochemical. . . . . . . Pathological . . . . . . . Discussion . . . . . . . . V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. Summary . . . . . . . . . Conclusions . . . . . . . . Recommendations. . . . . . . LIST OF REFERENCES. . . . . . . . . APPENDICES Appendix A. Training Programs and Training Data. . Sacrifice Modifications. . . . . Histochemical and Pathological Data. U P I'D . Statistical Tables . . . . . . ['11 Tissue Section Description. . . . Page 37 39 40 43 43 44 45 47 47 49 49 49 53 58 58 63 63 65 65 67 74 87 89 95 101 LIST OF TABLES Table Page 1. The Number of Animals in Each Cell by Treatment and Duration . . . . . . . 34 2. Mean Performance Values from the Last Day of Training for the Sacrificed Animals. . 41 3. Scheffé Comparisons for Body Weight Between O-Wk CONS and 8-Wk Treatment . . . . . 51 4. Scheffé Comparisons for Body Weight Between 8—Wk Treatment. . . . . . . . . . 52 5. Scheffé Comparisons for LDH, FA, SDH, and PAS Between O-WK CONS and 8-WK Treatment . 53 A—l. Standard Eight-Week, Short-Duration, High- Intensity Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels . . . . . . 74 A-2. Standard Eight-Week, Medium-Duration, Moderate-Intensity Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels . . . 75 A-3. Standard Eight-Week, Long-Duration, Low- Intensity Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels . . . . . . 76 A-4. Standard Eight-Week, Endurance, Swimming Training Program for Postpubertal and Adult Male Rats . . . . . . . . . 77 C-1. Mean Per Cent Light Absorbed and Body Weight Presented by Animal Number, Treat- ment, and Duration . . . . . . . . 89 vi Table C-ZO C-2a. C-3. 13.10. D-ll. D-lZ. Page Reliability of Histochemical Data . . . . . 91 Validity of Histochemical Data . . . . . . 92 Pathologic Analysis Presented by Animal Nunber, Treatment, and Duration . . . . . 93 Analysis of Variance for Body Weight by O-Wk CONS and 8-WK Treatment. . . . . . . . 95 Analysis of Variance for Body Weight by 8-Wk Treatment . . . . . . . . . . . . 95 Analysis of Variance for LDH by O-Wk CONS and 8’Wk Treatment. 0 o o o o o o o o o 96 Analysis of Variance for PA by O-Wk CONS and 8-Wk Treatment. 0 O O I O O O O O O 96 Analysis of Variance for SDH by O-Wk CONS and 8-Wk Treatment 0 o o o o o o o o 97 Analysis of Variance for PAS by O-Wk CONS and 8-Wk Treatment . . . . . . . . . 97 Analysis of Variance for LDH by 8-Wk Treatment 0 O O O O O O O O O O O 98 Analysis of Variance for FA by 8-Wk Treatment . . . . . . . . . . . . 98 Analysis of Variance for SDH by 8-Wk Treatment . . . . . . . . . . . . 99 Analysis of Variance for PAS by 8-Wk Treament O C I C O C O O O O O O 99 Chi-Square Contingency Table for Pathology by O-Wk CONS and 8-Wk Treatment . . . . . . 100 Chi-Square contingency Table for Pathology by 8-Wk Treatments . . . . . . . . . . 100 vii Figure 1. A-6. A-7. LIST OF FIGURES Flow Sheet of Animal Arrivals, Treatments and Sacrifices . . . . . . . . . . Mean Body Weight . . . . . . . . . . LDH Mean Values . . . . . . . . . . FA Mean Values. . . . . . . . . . . SDH Mean Values . . . . . . . . . . PAS Mean Values . . . . . . . . . . Mean Daily Body Weight . . . . . . . . Mean Daily Revolutions Run (VOL). . . . . Mean Daily Total Revolutions Run (TRR) and Per Cent Expected Revolutions (PER) for SHT Mean Daily Cumulative Duration of Shock (CD8) and Per Cent Shock Free Time (PSF) for SHT. Mean Daily Total Revolutions Run (TRR) and Per Cent Expected Revolutions (PER) for MED. O O O I I O C O O O O O 0 Mean Daily Cumulative Duration of Shock (CD8) and Per Cent Shock Free Time (PSF) for MED O O O O O C I O I O O 0 Mean Daily Total Revolutions Run (TRR) and Per Cent Expected Revolutions (PER) for LON O O O O O O O O O O O O O 0 Mean Daily Cumulative Duration of Shock (CD8) and Per Cent Shock Free Time (PSF) for CON. Sectioning of the Heart. . . . . . . . viii Page 33 50 54 55 56 57 79 80 81 82 83 84 85 86 101 CHAPTER I THE PROBLEM1 Introduction Investigations reporting general effects of exercise have shown that trained animals tend to weigh less, contain less fat, have shorter but stronger and more dense bones, have larger hearts, and that their skeletal muscle metabo- lism may be altered (65, 62, 33, 37, 40, 15, 16, 28, 29). The evidence is often conflicting and impossible to inter- pret because in most instances the training regimen used has not been defined. The recent evidence suggests that exercise consists of a continuum of specific activities each eliciting a specific response within the organism. Although little is known about specific effects, weight lifting type exercise in the rat produced hypertrophy of the right side of the heart which Krames and Van Liere (36) attributed to hypoxia, whereas endurance type exercise produced hypertrophy of the left side of the heart. Other results (22, 23, 24), from studying skeletal muscle, showed a "sarcoplasmic 1This study was supported in part by National Insti- tutes of Health Grant HD 03918. hypertrophy" or an elevation of the cytoplasmic proteins following training of a high-repetitive, low-resistance nature. The converse was indicated when the work performed was of a low-repetitive, high-resistance type. A need clearly exists for additional information concerning the Specific effects of defined exercise pro- grams. Since three exercise regimens have recently been developed for the rat which simulate specific training pro- grams in man (69), an opportunity existed for contributing to this body of knowledge. Following a search of the liter- ature, no information could be found concerning the specific effects of exercise regimens on heart metabolism and mor- phology. Thus it was decided to pursue an investigation along these lines. Statement of the Problem The purpose of this study was to determine the effects of seven different levels of chronic physical activity on the metabolic and morphologic characteristics of the left ventricular myocardium of adult male albino rats. Rationale If exercise is beneficial to the heart, what type of exercise causes the heart to function most efficiently? This study was designed to explore the possibilities of a rational answer to this question. It was hypothesized that as the type of exercise progressed from the short-duration, high-intensity (SHT) end of the physical activity continuum toward the long- duration, low-intensity (LON) end of the physical activity continuum, metabolic characteristics of the myocardium would progress accordingly from those of anaerobiosis and partial aerobiosis to those of total aerobiosis, respec- tively. Significance of the Problem Specificity of exercise is a reality that investi- gators are just beginning to comprehend. Previous studies have lacked an exercise apparatus which controls the in— tensity of various exercise regimens (42, 43). Prior to this study, duration was controllable, intensity was not controllable. It behooves physical educators to know exactly what is happening to the heart when prescriptions for exercise are given. Limitations of the Study l. The results of this study cannot be applied to man or generally to other animals. 2. The morphological analysis was only made on one section taken 300 u from the apex of the heart. 3. Methods that are employed for the determination of enzyme and substrate levels are quantitatively limited in that actual concentrations are not known. Definitions and Abbreviations Certain words and phrases are abbreviated throughout this thesis. It is important that the reader is able to understand the meaning of these symbols. An appropriate list follows. ATP DPN DPNH CDS CON C-RW Cy OX Diatex DNA ESC EST FA H & E Adenosine triphosphate Diphosphopyridine nucleotide (oxidized) Diphosphopyridine nucleotide (reduced) Cumulative duration of shock (sec) received by both the experimental (SHT, MED, LON), and the control (ESC) animals during all work periods of all bouts of a given train- ing period ' Sedentary control Controlled—Running Wheel (11 cm. wide and 38.8 cm. in diameter) Cytochrome oxidase Synthetic mounting medium Deoxyribonucleic Acid Electric stimulus control (paired with SHT) EXpected swim time (min) Fatty acid Hematoxylin and Eosin Histoclad LDH LON MED NBT PAS PER PET PSF SDH SHT STC SWM TCA TER TPN TPNH TRR Synthetic microscopic mounting medium Lactate dehydrogenase Long-duration, low-intensity running exercise (long C-RW program) Medium-duration, moderate-intensity running exercise (medium C-RW program) Nitro Blue Tetrazolium which was developed by Nachlas gt_al. (45) and produced by Sigmal Periodic Acid-Schiff (indicates presence of glycogen) Per cent expected revolutions; PER = 100 TRR/TER Per cent expected time (swimming); PET = 100 STC/EST Per cent shock free; PSF = lOO-(lOO CDS/TWT) Succinate dehydrogenase Short-duration, high-intensity running exercise (short C-RW program) Swim time completed (min) Swimming exercise Trichloroacetic acid Total expected revolutions that the animal would run, during all work periods of all bouts of a given training period, if he would run at the prescribed speed Triphosphopyridine nucleotide (oxidized) Triphosphopyridine nucleotide (reduced) Total number of revolutions run by the experimental animal, during all work periods of all bouts of a given training period 1Sigma Chemical Company, St. Louis, Missouri. TWT Total work time (sec) during all work periods of all bouts of a given training period VOL Voluntary running exercise CHAPTER II REVIEW OF RELATED LITERATURE The topical areas reviewed are: myocardial metabo- lism; related biochemical and histochemical studies in- cluding those on glycogen, lactate, fatty acids, and SDH and Cy Ox; and myocardial pathology. These areas were reviewed to give insight into the concept of specificity of exercise as it might apply to the heart. Myocardial Metabolism During metabolism of the normal heart, about 35% of the total myocardial oxygen extraction was accounted for by equal amounts of glucose and of lactate. Whereas, 60+% of the energy expenditure of the myocardium was sustained by fatty acids (7). Bing also indicated that as fat intake increased, the free fatty acid content in the myocardium approached 100%. Two mechanisms were proposed. Fatty acid metabolism might have increased or storage of fat in the heart muscle itself had occurred. It is unlikely that phospholipids or cholesterol esters are metabolized by the heart. Data were presented indicating that ketone bodies and amino acids were also metabolized by the heart. The aerobic extraction of ketone bodies accounted for <5% of the total oxygen extraction and was governed by the quantity of carbohydrates available to the myocardium. The aerobic metabolism of amino acids may account for as much as 40% of the total cardiac oxygen consumption. How- ever, a rather small increase (20%) in arterial blood amino acid content produced a startling disproportionate increase (245%) in the myocardial amino acid content. The metabolic activities that are present in the heart reflect the function that the myocardium performs (25). Because the heart requires an extraordinary amount of energy for its normal operation, the organization and function of myocardial metabolism are geared to the pro- duction of energy on a large scale when compared to other tissues of the body. By the process of oxidative phosphory- lation, energy is captured in the high energy bond of ATP. The systems of enzymes and cofactors necessary for oxi- dative phosphorylation to occur are present in the mito- chondrion. Each myocardial cell has an unusually large number of mitochondria. Therefore, the mitochondrion serves a key function in myocardial metabolism. The pro- duction of usable energy proceeds in essentially two steps. The first stage is the degradation of certain essential molecules to form acetyl CoA. Carbohydrates become de- graded through glycolysis, fats through hydrolysis and fatty acid oxidation and amino acids through transami- nation and deamination. The second stage is the funneling of acetyl CoA into the Tricarboxylic Acid Cycle through which carbon dioxide, water and energy are produced. From another point of View, Opie (48) has stated that glucose is important in the understanding of myocardial metabolism due to the possible use of glycolysis in main- taining anaerobic metabolism. However, glucose usage is limited by the rate of transport across the cell membrane. Anoxia and muscular work increase glucose phosphorylation. Data are presented indicating that glycolysis increases during periods of anoxia. Phosphofructokinase is inhibited by two end products of aerobic glycolysis (ATP and cit- rate). However, if the pH of the blood increases (by creatine phosphate breakdown), as occurs during exercise, and before lactate can accumulate, the ATP inhibition of phosphofructokinase is relieved. The hexose monophosphate shunt is not usually Operative in normal heart tissue. However, it is accelerated when synthetic processes are increased and when TPNH is required. Anoxia causes pyru- vate to form lactate rather than enter the Tricarboxylic Acid Cycle. If fatty acid oxidation is occurring the pyruvate dehydrogenase complex activity is further in- hibited with the accumulation of acetyl CoA and TPNH. Opie also indicated that glycolysis could be accelerated by a number of factors: hormones, exercise, and work. If any of these factors accelerate glycolysis to a degree which exceeds the capacity of the hydrogen shufflers 10 (DPN-DPNH), then lactate could form from pyruvate even in the absence of anaerobiosis. He stated that only one-tenth to one-third of the energy needs of the mammalian heart could be met by anaerobic glycolysis. This amount of energy could maintain the normal mechanical function of the dog heart during partial but not total deprivation of oxygen. There might be practical implications in any manipu- lations (exercise?) which could be shown to increase the maximal rate of anaerobic glycolysis in the mammalian heart. (Italics are mine). For example, the energy metabolism of the heart might be better sustained during angina pectoris or during myocardial infarction. The present failure of these anaerobic processes to maintain the anoxic heart may possibly indicate the presence of restraints on anaerobic energy production. Increased glycogen reserves (48) may be observed as a reflection of decreased glycolysis and the sparing of glyocgen associated with increased fatty acid metabolism by the myocardium. Phosphorylase activity may be influ- enced by exercise but catecholamines appear to be the most important of the known stimuli which convert phos- phorylase b to phosphorylase 3. Other results indicated that catecholamines have the ability to mobilize free fatty acids in the intact animal, thereby, increasing myocardial free fatty acid uptake with a consequent sparing of glycogen. In rats who had been swum, it was reported 11 that cardiac glycogen was unchanged (17) or decreased (10). The glycogen usage depended on the availability of alter- nate substrates and in part on the magnitude of the work load. It was also reported that only in extreme conditions (anoxia) was cardiac glycogen mobilized. Opie (48) reported that free fatty acid uptake was directly related to the circulating free fatty acid con- centration in both the intact heart and in the isolated perfused heart. Indications were such that the heart free fatty acid levels were about twenty times those of the blood. There was no evidence presented to suggest that endogenous lipid was an energy source for the normal heart in situ, except during intense exercise or during prolonged fasting. Evidence was presented indicating that ketone bodies were a minor substrate of the normal human heart. Ketone body uptake accounted for <10% of the total myo- cardial oxygen during exercise. In a continuation of his review of myocardial metabo- lism, Opie (49) indicated the myocardial uptake of phospho— lipids and cholesterol was insignificant. The nature of the substrate could alter the myocardial oxygen uptake. When fatty acids are added to the isolated, perfused heart preparation, lactate production from glucose increases and more glycogen is formed from the glucose, too. Therefore, the concurrent oxidation of free fatty acids by the myo- cardium can actually control the intracellular fate of glucose, especially at the level of pyruvate entry into 12 the Tricarboxylic Acid Cycle. He concluded that lipid (exogenous) was a major source of energy to the heart and that generally, acetate, ketone bodies, short-chain fatty acids, lactate and pyruvate are oxidized in "prefer- ence“ to glucose. From studies of skeletal muscle, myocardium could be considered an extreme type of aerobic muscle. There- fore, the heart was metabolically more dependent on the oxidative metabolism of lipids than on glycolysis for its energy requirements. There were no data presented indi- cating that increased left ventricular work, however pro- duced (exercise?) was a major factor factor in the control of competition between glucose and free fatty acids for oxidative metabolism in the heart. In the fasting condition, mean oxidation extraction ratios are 60% for free fatty acids, 28% for glucose and 11% for lactate. The increased energy demands of short periods of exercise are met by an increased coronary blood flow with an increased extraction of carbohydrate sub- strates (lactate), therefore glucose becomes the major source of energy in exercise (short bursts) (49). In the mitochondrion, the inhibition of citrate synthetase by ATP may indicate a regulating mechanism whereby decreased ATP concentrations, such as could be expected after increased myocardial work (exercise?) might accelerate the Tri- carboxylic Acid Cycle. Creatine phosphate experiences depletion in the heart prior to ATP depletion. About 70% 13 of the oxygen uptake is directed toward the contractile process while the other 30+% is used for regulating other cellular activities (ion transport across cell and mito- chondrial membranes, other non-phosphorylating cellular activities). The rate of oxidative phosphorylation is coupled to the rate of electron transport in all tissues. The impulse conducting system of the heart may depend more on glycolysis for its energy supply than does the rest of the myocardium. The oxygen uptake and SDH activity are much lower in the Purkinje fibers, but the glycogen concentration is higher and the capacity to sur- vive anoxia is greater than in other parts of the heart. The major part of myocardial norepinephrine seems to be synthesized within the heart. Epinephrine cannot be synthesized in the heart, but is taken up from the circu- lation by the heart. Furthermore, catecholamines may be released into the circulation when the left ventricle develops an increased systolic pressure (exercise?). The rate of glycolysis may either increase or decrease in hearts that have been exposed to epinephrine (49). Related Biochemical and Histochemical Studies Glycogen Evans (17), in 1934, acutely exercised rats by forcing them to swim and reported that the cardiac glycogen content remained unchanged as a result. 14 An attempt was made to relate the consumption of blood oxygen, glucose, lactic acid and the production of carbon dioxide to the amount and nature of cardiac work by Alella gt 2&- (2). They studied the performance of dogs' hearts in £153 under nine sets of conditions of heart work which included three levels of mean cardiac output (852, 1208, and 1665 cc/min.lOOg. heart weight) at each of three levels of mean aortic blood pressure (79, 97, 122 mm. Hg.). Results indicated that exogenous glu- cose consumption was not related to the cardiac oxygen consumption. Two forms of cardiac glycogen have been identified on the basis of solubility in TCA. The soluble glycogen is relatively labile, while the insoluble glycogen is more stable. Using male albino rats (Sprague-Dawley strain), that weighed between 100 and 150 grams, Weisberg and Rod- hard (68) attempted to demonstrate the glycogen distri- bution in the heart. Their results indicated that the total concentration of glycogen was highest in the atria, lowest in the left ventricle, and intermediate in the septum and right ventricle (p < 0.01). The distribution of the TCA soluble glycogen was similar to that for the total glycogen in each segment of the heart. While the concentrations of total glycogen in the atria did not differ statistically, the TCA soluble glycogen of the right atrium was significantly greater than that of the 15 left atrium (p < 0.05). The TCA soluble glycogen accounted for 55% of the total glycogen in the rat heart. Bloom and Russell (9) investigated the effects of injecting norepinephrine and epinephrine (0.2 mg./kg.) on the glycogen content of the rat heart. Using female albino rats (Sprague-Dawley strain), they reported that the glycogen content of the heart had increased at both two and four hours after both epinephrine and norepinephrine injections. These changes were reflected in the TCA solu- ble glycogen which thus appeared to be more subject to physiological change than was the insoluble glycogen fraction. In attempts to correlate mechanical events with bio- chemical events of the heart, researchers have agreed that as the work load was increased on the heart, in yitrg, the total glycogen reserve decreased. However, in Kilo, the depletion of the concentration of glycogen in cardiac tissues of swimming rats has not been demonstrated (10). Blount and Meyer (10) studied the effects of a swimming exercise on the cardiac glycogen concentration in the left ventricle of male albino rats that weighed between 130 and 250 grams. Their analyses included the determination of the TCA soluble glycogen and the protein bound or residual glycogen. Previous experimental evidence indicated that the free form of glycogen was more readily mobilized under conditions of increased metabolic activity than was the 16 residual glycogen. Their results indicated that the exer- cised rats depleted their cardiac glycogen within the first fifteen minutes of swimming (there was no statistically significant difference between the cardiac glycogen of rats that swam for fifteen minutes and those that were forced to swim for sixty minutes). The cardiac glycogen stores appear to be adequate for only the first few minutes of exercise. The conclusion was also reached that during unusually heavy exercise, the heart is anoxic and draws upon its glycogen reserve. The TCA soluble glycogen ex- hibited greater depletion. Both the free and bound glyco- gen are rapidly restored to normal levels after extreme depletion and the TCA soluble glycogen even increased significantly above resting levels (supercompensation). Using the hearts from thirty-three infants, seventeen known or suspected diabetic adults and sixty-three non- diabetic adults, Mowry and Bangle (41) tested for the presence of glycogen using the PAS reaction. The sections were graded as containing from 0 to 5+ glycogen. The re- sults showed that the 5+ presence of glycogen does not definitely relate it to cardiac enlargement in infants. Hearts that were rich in glycogen were observed of all age groups up to eight months. Glycogen concentration was not related to sex. In the adults, they reported that glycogen storage disease was characterized by considerable cardiac enlargement without a known functional cause. Glycogen was found to be diffusely distributed throughout 17 the myocardium in concentrations exceeding those believed to occur normally. In an attempt to identify the existence of anaerobic metabolism in the heart, Neill gt_al. (46), inhibited the heart's oxidative metabolism with the intravascular adminis- tration of cyanide to six closed chest dogs (20-25 kg.). They also wanted to demonstrate that the heart could con- vert anaerobically liberated energy to mechanical work. Their results indicated that during the cyanide effect, cardiac work and heat exceeded the energy available from oxidative metabolism, therefore, the difference represented anaerobic myocardial metabolism. Also, because the energy of mechanical work output was greater than the myocardial aerobic energy source, a portion of the anaerobic energy liberated must have been converted to mechanical work. Taking twenty-two human hearts from males and nine- teen human hearts from females, Wittels and Reiner (71) investigated the relationship between myocardial ischemia and glycogen content. They looked at paraffin embedded PAS tissue sections. Glycogen was quantified in the PAS- stained sections on a 0 to 4 scale. Their results showed that in the left ventricle the glycogen content was no more than 1+ in nine control hearts. However, in hearts with myocardial infarction, 14 of the 19 hearts were found to be rich in glycogen. They concluded that although normal myocardium is apparently rich in glycogen in life, little or no glycogen was detected to be present after death. 18 Yokoyama gt 31. (72) proceeded systematically to investigate the PAS changes that occur in early myocardial infarction. Infarcts were produced experimentally by ligating a branch of the left coronary artery. Heart tissue was excised at various time intervals after ligation and samples of both affected and unaffected tissues were taken from the left ventricle. Results showed that glycogen concentration decreased in the affected portions of the hearts. Results also indicated that when healing takes place with granulation tissue, increased quantities of glycogen were observed in those myocardial fibers immedi- ately surrounding the area of the infarct. Lactate Studying dogs in situ in a variety of conditions of heart work, Alella 33 31. (2) reported that the consumption of exogenous lactic acid by the heart showed a positive relationship to its availability, to its arterial blood level, and to the myocardial oxygen consumption. It appeared to be one of the substrates readily used for the work performance of the heart. The relative consumption of exogenous glucose and lactic acid varied with their relative availabilities and are related in an inverse manner. Warbasse et 31. (67) discovered that serum LDH could not be used to differentiate myocardial injury from either skeletal muscle, lung or liver tissue damage. 19 Research by Meerson and Zayats (39) has given certain clues to the possible metabolic causes of alterations in contractile proteins of failing heart muscle. They ob- served the hearts of rabbits which had aortic stenosis experimentally induced. Their results indicated that the lactate concentration increased in the myocardium with a concomitant hypertrophy of myocardial fibers. The mito- chondrial mass also increased. Keul gt a1. (32) presented evidence that exercise altered the lactate/pyruvate ratios which can cause pyruvate output to increase by the heart. Kaplan and Goodfriend (31) showed that the M-isoenzyme of LDH was present mainly in those muscles (skeletal) that function anaerobically. While the H-isoenzyme of LDH was present in the muscle (cardiac) that functions aerobically. The H-isoenzyme is inhibited by pyruvate and it was sug- gested that this ensures the pyruvate entry into the Tri- carboxylic Acid Cycle during conditions of increased glycolytic flux. Fritz (19) presented information stating that H-LDH does not act as a regulatory enzyme as M-LDH does. The heart demands a constant supply of energy as produced by oxidation of Tricarboxylic Acid Cycle substrates, but these substrates neither act as feed back regulators nor limit their own concentration. 20 To further study the effects of physical training and hypertrophy on LDH activity in cardiac muscle, Gollnick and Hearn (20) used male albino rats (Sprague-Dawley strain) with a mean weight of 340 grams. The physical training con- sisted of forcing the rats to swim thirty minutes daily in 37°C. water for thirty-five consecutive days. Twenty-four hours after the last treatment, the experimental and the control animals (30 pairs) were sacrificed. Their results indicated that the exercised animals gained 38.7% less body weight than did the controls. On a relative basis, the heart ventricles of the exercised group were larger (p < 0.05). LDH activities of the heart ventricles of all the exercised animals were significantly increased over the controls. They concluded that these results indicated that enzyme activities could be altered as a consequence of exercise. The increase in enzyme activity for the heart, coupled with the lack of change in the skeletal muscles, would seem to indicate that the exercise, as used in this study, was a greater stress on the heart than on the skeletal muscles. Gollnick, Struck, and Bogyo (21) investigated the acute effect of exercise on myocardial and skeletal muscle LDH activity of trained and untrained rats. Forty-eight male albino rats (Sprague-Dawley strain) were assigned at random to two groups of twenty-four animals each. The trained group was exercised daily for thirty-five conse- cutive days by being forced to swim in 35°C. water. The 21 untrained group served as sedentary controls. The rats were swum in groups of six beginning with thirty minutes duration. The swim time was increased five minutes each day, until a peak of sixty minutes was attained. Both groups of animals were weighed and sacrificed the day after the last day of training. Half of the animals from each group were forced to swim for thirty minutes just before sacrifice. At sacrifice, the cardiac and the gastrocnemius muscles were removed. Their results showed that the training produced a significant increase in heart LDH activity. However, the acute exercise just before sacrifice did not produce any change in LDH activity in the trained or the untrained animals. The increased LDH activity gives the heart a greater capacity for lactate utilization from the blood and complements the oxidative pathways by supplying pyruvate and DPNH. Fatty Acids It has been shown that in the postabsorptive state, more than half of the oxygen consumption of the cardiac muscle fiber is due to the oxidation of fatty acids (5, 7, 13, 61). Bing gt 31. (8) showed that when fatty acid blood levels were increased, due to high fat intake, the myocardial extraction ratio of fatty acids approached 100%. Two mechanisms were proposed: fatty acid metabolism had increased and/or the heart had stored fatty acids. 22 Shipp (59) demonstrated that the oxidation of free fatty acids by cardiac muscle can actually control the intracellular fate of glucose, especially at the level of pyruvate entry into the Tricarboxylic Acid Cycle. Holczinger (30) reported that fatty acids may be seen histochemically utilizing a copper acetate incubating medium. The reaction was reported as being quite sensi- tive, demonstrating both saturated and unsaturated fatty acids in the same manner. The greenish-black inner com- plex afforded good localization and the sediment was reported to be extraordinarily durable. However, reliable results could only be obtained from fresh frozen unfixed tissue. SDH and Cy Ox Padykula (51) indicated that SDH was one of the few dehydrogenases that acts directly with the electron trans- port system without intervention of a coenzyme. Cardiac muscle showed a higher degree of SDH activity than skeletal muscle. Sections of myocardium presented a monotony of blue diformazan granules which were distributed throughout the tissue in great abundance. The intercalated discs and nuclei were not recognizable in these preparations. She also explained that the specificity of the histo- chemical reaction may be demonstrated by eliminating succinate from the incubating medium. 23 To study the exercise effects on SDH activity of both the heart and skeletal muscle, Hearn and Wainio (28) used male albino rats (Wistar Institute strain). The exercise consisted of forcing the animals to swim for thirty minutes daily in 32°C. water for either five weeks (15 pairs), or six, or seven, or eight weeks (10 pairs each). These ani- mals were pair-fed. Their results showed that the actual total activities of SDH were not significantly altered by the training procedure. However, the relative total activities of SDH in the heart ventricles of the exercised animals were increased (p < .02). They also reported that the weights of the heart ventricles and adrenals were sig- nificantly increased in the exercised animals (p < .01). The exercised animals gained 30-40% less body weight than the controls. They suggest that SDH appears to be present in both heart ventricles and skeletal muscle in amounts greater than that needed to c0pe with the stress offered by moderate exercise. An attempt was made to investigate the possibility of using the SDH reaction in the gross identification of early myocardial infarcts (44). This technique took ad- vantage of both substrate and enzyme loss from the infarct. Their results showed that the necrotic muscle fibers re- mained unstained or faintly stained using SDH. Using fresh frozen tissue, Nachlas gt 31. (45) demonstrated SDH activity histochemically by incorporating 24 NET as the hydrogen acceptor. NBT competes successfully with oxygen for the available electrons. The reaction gave rich blue diformazan particles as indicator sites of SDH activity. The specificity of the reaction was tested by the omission of succinate from the incubating medium. This technique permits the cytochemical visualization of the sites of SDH enzymatic activity in tissue sections. The method is further described in Barka and Anderson (6). Wachstein and Meisel (66) investigated SDH activity in several diseased states of the myocardium. They re- ported that the loss of SDH activity in those muscle fibers that were undergoing necrotic changes due to anoxia were striking. In the areas of myocardial necrosis, SDH activity decreased rapidly. In a related study, Nielson and Klitgaard (47) examined the changes in SDH activity as related to feeding. Using rats, they concluded that daily variations in the activity of SDH were related to periods of eating and fasting. These changes were of such magnitude as to possibly mask other experimental alterations in enzyme activity. Cytochrome oxidase techniques described by Burstone (ll, 12) have been frequently used by investigators inter- ested in the electron transport system. It was indicated that Cy Ox is measured in an indirect manner. Oxidized cytochrome c is reduced by p-aminodiphenylamine. This 25 reduction results in the formation of reduced cytochrome c and oxidized p-aminodiphenylamine. This latter product combines with l-hydroxy-Z-naphthoic acid and the red-brown to blue-black reaction product is produced. Cy Ox oxidizes reduced cytochrome c, thus replenishing the available oxidized cytochrome c with the formation of water. Cy Ox does not act directly on the reducing substance (64). Seligman (58) showed that Cy Ox was in the mito- chondrion and could be localized specifically by DAB (Diaminebenzadine). In rat myocardium, he used NBT and found SDH to be localized in the same place as Cy Ox. These data confirmed that the localization of SDH was similar to that of Cy Ox. These data also showed that both pigments (DAB for Cy Ox and diformazan for SDH) are appearing on one side of the mitochondrial membrane (the outer surface of the inner mitochondrial membrane). Myocardial Pathology Reporting on several pathological conditions found in the heart, Opie (50) said the early occurrence of mitochondrial damage explained the decreased rates of oxidative phosphorylation with a shift from aerobic to anaerobic metabolism in terminal heart failure. The co- existence of the serum LDH pattern, which indicated myo- cardial damage, with electrocardiographic and radiological abnormalities was strong evidence for myxedema heart disease. In obstructive cardiomyopathy, there was a 26 tendency to increase lactate production and increase anaerobic metabolism by the heart. Within sixty seconds of infarction, the myocardial cells failed to contract. Opie stated that there was no obvious reason as to why the anoxic heart should stop beating so soon. Creatine phosphate disappears almost immediately and within fifteen minutes ATP is no longer present. The duration of anoxia which is required to produce irreversible damage appears to be greater than fifteen minutes and probably about thirty to sixty minutes. In ischemic heart disease it is not known which factors restrain anaerobic glycolysis from maximal rates which could, perhaps, contribute to the energy supply of the anoxic heart. Bing (7) indicated that a number of pathologic states may induce anoxia with a consequent shift of cardiac metabolism toward anaerobiosis. With myocardial anoxia, the heart goes into a negative balance of lactate and pyruvate which may be seen in hemorrhagic shock, ventricu- lar tachycardia and fibrillation, atrial fibrillation and in the temporary interruption of coronary flow by emboli- zation of the coronary arteries. The anoxia leads to a disappearance of glycogen with an increase of lactate and glucose-6-phosphate in the myocardium. The lactate accumu- lates due to the lack of oxygen present; therefore, there is a decreased utilization of lactate. During ventricular tachycardia and fibrillation, the cardiac glycogen decreases 27 while lactate, pyruvate, glucose-6-phosphate and phos- phorylase a increase. Shnitke and Nachlas (60) observed fresh frozen sections of dog myocardium after they had been incubated for SDH activity. The diformazan pigment, which indicated SDH activity, was restricted to the myocardial cell in the normal heart. Complete loss of SDH activity was noted one day to five days after an infarct in the area of necrosis. Five days after the infarct, DPN diaphorase activity and Cy Ox activity markedly declined, also. Reichenbach and Benditt (55) described a response of the myocardial cell to injury which was different from coagulation necrosis of ischemic infarction. The major features of the lesion included the following: segmen- tation of myocardial cell cytoplasm seen as transverse bands between areas of granularity; localization of this cytoplasmic alteration within a short time after injury; frequent localization of damage in the subendocardial cells of the ventricles; a proliferative interstitial cellular reaction; no neutrophils were seen to be present; sparing of the reticulum stroma, blood vessels and nerves; and in some cases, mineralization had occurred in the mitochondria. This lesion may be produced experimentally by several chemi- cal, metabolic and mechanical means (54). The injury affects the structural organization of the myofibrils and is at least partially reversible. 28 There is a considerable amount of disturbance to the normal metabolism occurring in the region of an infarct. According to Rubel (56), various zones with different levels of enzymatic activity are formed. In the unaffected regions of the heart, metabolism increases presumably to compensate for the losses which may lead to cardiac hyper- trophy. In an attempt to evaluate the susceptibility of domesticated white rats and of wild gray Norwegian rats to myocardial necrosis formation under sensory and emotional stresses, researchers (53) examined H & E stained sections of myocardium from these animals. Upon analysis, the lesions were rated on a scale from 0 to 3. Seventy per cent of the wild rats had myocardial lesions while about 40 per cent of the domesticated rats exhibited myocardial necroses. Letunov (37) presented data which showed a small percentage of sportsmen differing from the normal in terms of cardiovascular abnormalities. However, in a majority of the cases, the origin of the cardiovascular disease could not be traced to sports activities. He explained that these pathologic conditions usually occurred because of a previous or current infection not related to athletics. He concluded that the adaptation of the individual to muscular activity in the course (years) of athletic train- ing was accompanied by functional changes in middle-aged people that were characteristics of trained athletes. 29 Investigating the effects of hard physical training on the cardiovascular system of youth, Kitamura (33) re- ported that excessive training in the very young could be injurious to the heart. After training 200 25-day-old mice for varying periods of time, his data indicated that after six weeks of training there was hypertrophy of the myo- cardial fibers and of their nuclei. After ten weeks of training, he reported hypertrophy, interstitial fibrosis, cellular infiltration and petechial hemorrhages to be present within the myocardium. He also showed that the capillary to fiber ratio first decreased then increased after terminating the training which was concomitant with atrophy of the muscle fibers. Investigators (34) performed comparative studies on mitochondria of left hypertrophic heart ventricles of rats. Some of the rats had aortic stenosis experimentally in- duced, while others were subjected to physical training. Hypertrophy due to both types of experimental treatment showed an increased voluminal mitochondria-to-myofibrillae ratio. The density of the cristae remained approximately normal in the trained animals, whereas the stenosed rats showed a poor internal structure of mitochondria. The mitochondrial protein level of the myocardium was reduced in the stenosed rats, while being unchanged in the swim- ming rats. Enzyme assays showed a rise of malate de- hydrogenase activity amounting to 70% in the stenosed rats and to 170% in the swimming rats. 30 French (18) described myocardial lesions in potassium- deficient rats. There is a primary degeneration of muscle substance which eventually leads to necrosis and disinte- gration of myocardial fibers. The muscle debris is removed by phagocytes. Secondary to the fiber degeneration, there are changes in the connective tissue. These changes con- sist of interstitial edema, proliferation of vascular endo- thelium and fibroblasts with mobilization of macrophages. He reported no conclusive evidence of effective muscle regeneration. Healing takes place (after potassium is added to the diet) mainly by hypertrophy of the surviving muscle fibers and by condensation of the surrounding con- nective tissue. Klionsky (35) reported that the earliest lesion, in evidence of myocardial ischemia is the loss of the labile fraction of glycogen as measured by PAS. Bajusz and Homburger (3) indicated that myocardial cellular necrosis evolves along a number of different path- ways. Ischemia causes two types of necrosis. In the center of the ischemic area, the edematous cardiac cells undergo cytolysis. While in the periphery of the ischemic area, there is an initial contraction of portions of the myo- fibrils which terminate in coagulation necrosis. The areas of the myocardial cell that are most susceptible to injury include (in order): the sarcoplasmic reticulum, the nucleus, and the mitochondrion. 31 Bajusz and Raab (4) stated that some forms of human necrotizing cardiomyopathies are attributed to the reflex liberation of catecholamines which exert a contributory noxious effect on cardiac metabolism. Comparative histo- chemical studies showed that when there was an early de- cline in myocardial phosphorylase activity with a con— comitant decline in the amount of stainable glycogen, anoxic myocardial damage could be deduced. They wanted to study the types of cardiac damage that would be pro- duced with injections of epinephrine. Their results showed that a decrease of histochemically demonstrable potassium occurs within some areas of the myocardium about ten to fifteen minutes after the administration of epine- phrine. The involved foci showed a decrease of potassium with a parallel depletion of glycogen reserves, as well as a decrease in phosphorylase activity. These areas were mainly located in the subendocardium and apex where necrotic lesions usually occur. The reactions for oxi- dative enzymes remained normal or were even somewhat ele- vated within the respective areas._ They reported that these early histochemically demonstrable alterations in the myocardium following injection of epinephrine are identical to those seen in anoxic areas of myocardium following coronary artery ligature. CHAPTER III RESEARCH METHODS Sample Two hundred and fifty-two, 71-day-old, male, albino rats (Sprague-Dawley strain) were brought into the labo- ratory in eight shipments. The animals were assigned at random to one of seven treatment groups as shown in Figure 1. Prior to the treatment period, each animal was allowed thirteen days to adjust to the laboratory. The housing for the adjustment period varied according to the treatment group into which an animal was placed. The ani- mals which were placed into the CON group were housed in individual sedentary cages during the adjustment period while the animals that were placed into the remaining six groups were housed in individual voluntary activity cages during the adjustment period. One hundred and twenty-six of the healthiest and best trained animals were selected for the final sample. Table 1 depicts the numerical arrangement of the final sample by treatment and duration. 32 33 892.com RD 2:953.» .2954 .958 .o 89% 6E I _ 26¢ R. C r. o. C to 0 oi: oi: e :9 «assesses eeee_eve {I is“ - ~ 09.....va . 0 is 2.; I209: Elm. .lih . 1:28 .228 2...... Boa . omw - £28K 2.6 9.3 . col - £23... 27o .56.: . o! . I285 .56 to.» . Em . 3.05-u £9.53) . 40> . .223 32:88 . x8 . III>MK COUOUKO 34 TABLE l.--The number of animals in each cell by treatment and duration. . Treatment Duration CON VOL SHT MED LON ESC SWM O-wk 2a 2a 2a 2a 2a 2a 2a 2-wk 2 2 2 2 2 2 2 4-wk 2 2 2 2 2 2 2 6-wk 2 2 2 2 2 2 2 8-wk 8 8 8 8 8 8 8 lO-2k 2 2 2 2 2 2 2 aSince none of these animals had received any experi- mental treatment, it was deemed appropriate to group these animals together (N=l4) and call them O-wk CONS. Treatment Groups The seven treatment groups used in this study were as follows: CON These animals were housed in individual sedentary cages (24 cm. long by 18 cm. wide by 18 cm. tall) during both the adjustment and treatment periods. Egg These animals were housed in individual voluntary activity cages (sedentary cage with access to a freely revolving activity wheel) during both the adjustment and the treatment periods. The attached freely revolving 35 activity wheel had a width of 13 cm. and a diameter of 35 cm. An automatic revolution counter which was attached to the activity wheel recorded each revolution of each wheel. These animals exercised at will. sill These animals were housed in individual voluntary activity cages during the adjustment period and in indi- vidual sedentary cages during the treatment period. The exercise treatment that these animals received (short C-RW program) was such that on each of the last four days of the eight-week training program, they were required to run 49 ten-second sprints at six feet per second (69). Those animals that were trained for ten weeks followed the fortieth day SHT program during each of the last ten days (see Appendix A). @222 These animals were housed in individual voluntary activity cages during the adjustment period and in indi- vidual sedentary cages during the treatment period. The exercise treatment that these animals received (medium C-RW program) was such that on each of the last four days of the eight-week training program, they were required to run 40 thirty-second sprints at four feet per second (69). Those animals that were trained for ten weeks followed the fortieth day MED program during each of the last ten days (see Appendix A). 36 fl These animals were housed in individual voluntary activity cages during the adjustment period and in indi- vidual sedentary cages during the treatment period. The exercise treatment that these animals received (long C-RW program) was such that on each of the last four days of the eight-week training program, they were required to complete 4 twelve-minute-thirty-second runs at two feet per second (69). Those animals that were trained for ten weeks followed the fortieth day LON program during each of the last ten days (see Appendix A). $9 These animals were housed in individual voluntary activity cages during the adjustment period and in indi— vidual sedentary cages during the treatment period. These animals received no exercise treatment. However, during the treatment period, these animals were paired with ani- mals from the SHT group. When an SHT animal entered the C—Rw wheel for his daily exercise, the paired ESC animal was placed into an adjacent stimulus control cage (21.5 cm. long by 14 cm. wide by 10.5 cm. tall) which had a grid floor (69). The ESC animal was exposed to the same light stimulus to which the SHT animal was exposed. The amount of shock that the SHT animal received was, at the same time and with the same intensity, transferred to the ESC animal. 37 gym These animals were housed in individual voluntary activity cages during the adjustment period and in indi- vidual sedentary cages during the treatment period. The exercise treatment that these animals received (swimming program) was such that at the conclusion of the eight-week training program, each animal was required to swim one sixty-minute bout with 3 per cent of his body weight attached to his tail. Those animals that were trained for ten weeks followed the fortieth day SWM program dur- ing each of the last ten days (see Appendix A). Treatment Procedures The treatments began on the thirteenth day that the animal was in the laboratory (84 days of age). Three of the exercise treatments (SHT, MED, LON) and one of the control treatments (ESC) used the C-RW apparatus which " . . . is a unique animal-powered wheel which is capable of inducing small animals to run at a prescribed speed for one or more prescribed durations of time" (69, p. 4). A low-intensity controlled shock current provided motivation for the animal to run. However, the shock was always preceded by a light; therefore, the animal could avoid the shock by responding (running) to the light. The time during which the light was on was termed the acceleration time. This acceleration time began each running period 38 of exercise. At the end of the acceleration time, the light was turned off and the shock current was applied to the running (grid) surface of the wheel to induce the animal to run at the prescribed speed. In those cases in which the animal had responded to the light and had attained the prescribed speed during the acceleration time, the light was turned off and no shock current was applied. If the animal had attained the prescribed speed while it was being shocked, the shock was immediately discontinued. If the animal slowed down below the pre- scribed speed, the light and shock (if necessary) sequences were repeated. A typical running program consisted of alternate periods of work and rest. During the work periods (running period of exercise), the wheel was free to turn, while during the rest periods, the wheel was braked automatically to prevent spontaneous activity. A specified number of alternating work and rest periods (repetitions) constituted one bout of exercise. A single training period could include several bouts separated by time between bouts (rest periods). See Appendix A for the individual eight-week training programs. The animals in the SWM group were swum in individual cylindrical tanks which measured 27.94 cm. in diameter and 76.20 cm in depth. Tail weights, when required, were calculated, using body weight before treatment from the previous Friday. These weights were attached to the tips of the tails by means of miniature plastic Clothespins. 39 if an animal was unable to complete the EST, the animal was removed and his STC for that day was recorded. After each swim, the animals were initially dried with a towel and then put into a carrying cage which was placed under a heat lamp until the animals were completely dry. See Appendix A for the individual eight-week training program. The animals in the VOL group had revolution counter readings taken at approximately the same time (A.M.) Monday through Friday. Total voluntary activity (revolutions run) for the previous twenty-four hour period, four days per week, Tuesday through Friday, was calculated. Treatments were given Monday through Friday. The SHT, MED, LON, ESC, and SWM treatments were given in the treatment room in subdued normal room light. The animals in treatment groups SHT, MED, LON, and ESC were weighed daily before and after treatments. SWM group animals were weighed before their daily treatment. TRR and CBS were collected daily for treatment groups SHT, MED, and LON, while ESC used the values for SHT. These data facilitated the calculation of PER and PSF. For the SWM group, STC was collected daily which facilitated the calculation of PET. Animal Care Each animal was placed into a clean cage every two weeks. The animals were provided with food (Wayne Labo- ratory-Blox) and water ad libitum. Ambient air 40 temperature in both the animal quarters and the treatment room was maintained between 18°C. and 27°C. The relative humidity was maintained between 15% and 44%. The water temperature for the SWM group was maintained between 28°C. and 32°C. The animals were exposed to a sequence of twelve hours of light followed by twelve hours without light daily which was automatically controlled by an electrical timer. When possible, the paper in the animal quarters was changed daily, and usually not less fre- quently than every other day. The paper under the C-RW apparatus (wheel and stimulus control cage) was changed daily. The C-RW apparatus was washed daily. Each C-RW apparatus was cleaned thoroughly once a week. When possible all animals in all treatments were handled daily, Monday through Friday, during both the adjustment and the treat- ment period. Sacrifice Procedures Animals were selected for sacrifice on the bases of their health and their performance during the treatment programs. Good health was determined subjectively by observing each animal. Adequate performance was deter- mined objectively by observing each animal's graphed values for PER, PSF, PET, and revolutions run, where applicable. The daily mean data (TRR, PER, CDS, PSF) for the SHT, MED, and LON treatments appear graphically in Appendix A. The daily mean body weights for the SHT, MED, LON, ESC, and 41 SWM treatments also appear graphically in Appendix A as do the daily mean revolutions run by the VOL group. Animals were selected after their last treatment on Friday for sacrifice on Monday. Selection of animals, in general, was based upon their overall training curve. However, when such animals performed poorly on the last day of training, they were replaced by the next most qualified animals. Mean values for their performance on the last day of training may be seen in Table 2. TABLE 2.--Mean performance values from the last day of training for the sacrificed animals. Data Treatment . Revolutions PER PSF PET Run VOL 921 0 2 O O O O O O SHT . . 54.1 60.6 . . MED . . 63.2 67.8 . . LON . . 72.3 65.9 . . ESC . . . . 60.6 . . SWM . . . . . . 100 Body weight was taken at sacrifice. Seven animals were sacrificed every two weeks for thirty-six consecutive weeks (see Figure 1). During the course of this experiment, certain pro- cedures were either added to or deleted from the original 42 sacrifice sequence. For the sake of clarity to this pre- sentation, only those procedures that were followed the majority of the time will be reported here. A detailed account of the modifications may be read in Appendix B. Each animal was anesthetized by an intraperitoneal injection with 3.5 cc. of a 6.48% Halatall solution (sodium pentobarbital). A transverse laparotomy was made at about the level of L4 to L6' After cutting through the over- lying skin, fascia, and connective tissue, the incision was continued through the following musculature: Obliquus Externus Abdominus, Obliquus Internus Abdominus, Transversus Abdominus, and Rectus Abdominus (26). Once into the abdominal cavity, the incision was extended cranially as a parasagittal incision to the right of the xiphoid process, through the costal cartilages to the level of the second rib. The heart was located within the thoracic cavity. The parietal pericardium was removed and with the heart being held over toward the right, the major blood vessels were severed as close to the atria as possible. Once the heart was removed, it was cut transversely at a level just below the atria. The ventricular portion of the heart, with the apex pointing up, was held in place on an Ames Lab-Tek Cryostat Chuck by 5% gum tragacanth. The prepared check was held with forceps and was lowered, lFrom Jensen-Salsbery Laboratories, Division of Richardson-Merrell, Inc., Kansas City, Missouri. 43 for approximately twenty seconds, into pre-cooled 2- methylbutane (isopentane). The isopentane had been pre- viously cooled to a viscous fluid (-173°C.)l by liquid nitrogen. The chuck was placed into an empty 35 mm. film can. The can was put onto a shelf in the cryostat. Within thirty hours, the heart was sectioned at two levels approxi- mately 300 u and 600 u from the apex (see Appendix E). Serial sections of 10 u each were taken at each level at a temperature of -20°C. in the cryostat. The sections were immediately placed onto cover glasses and air-dried for a minimum of one hour and a maximum of two hours. Only the sections cut at the 300 u level are presented herein. Histological Procedure A fresh frozen H & E technique (38) was used to examine the morphology of the left ventricle. Histochemical Procedures Presence of glycogen was investigated using the PAS reaction (38). Fatty acids were depicted using Holczinger's technique (30). LDH activity was studied using NBT as the electron acceptor and DPN as the cofactor (52). SDH activity was examined using NBT as the electron acceptor as described by Barka and Anderson (6). The presence of lAs measured by T 2120-3 thermometer which was supplied by Scientific Products, Evanston, Illinois. 44 Cy Ox activity was studied using the technique of Burstone (52). Incubation times varied according to the individual procedure used. Slides stained with H & E and PAS were mounted in Histoclad while the other slides were mounted in glycerin jelly. Within three days of sacrifice, all slides were sealed with Diatex. Previous studies from this laboratory had ascertained the specificity of the enzyme reactions using control sections (16). Control sections were incubated with each of the enzyme procedures during the last three sacrifices so that specificity of the enzyme reaction could be deter- mined. The control incubating media excluded the sub- strate in some cases (SDH, LDH), and instituted a formalin fixation in the other case (Cy Ox) (64). At variable time intervals during the course of the study, the slides from the sacrifices were coded by inde- pendent members from the laboratory. Therefore, when the analysis was to be performed on the individual slide, there was no information present to indicate either animal number, treatment group, duration, or date of sacrifice. Tissue Analysis Procedures Because the heart is a rather homogeneous tissue in terms of its metabolic characteristics (13), the decision was made to choose an area of the left ventricle that could be located consistently in each tissue section. 45 The constrictor band of muscle (57) which corresponds closely to the cylindrical layer of muscle (63) in the left ventricle was chosen for study. Histochemical Each histochemical stain was evaluated objectively with the use of a light transmission meter which used a magnification of 80 X (70). The operator of the light 'transmission meter was able to isolate the photometric beam on one cardiac muscle fiber (on cross section). The meter was able to determine (on a scale from 0 to 100) the per cent of light that was transmitted through that fiber. The light transmission meter was judged to be accurate to i 0.3% (70). Sixty-six fibers were selected at random within the constrictor band of muscle in the left ventricle for light transmission readings from those tissue sections which were taken closest to the apex of the heart. Therefore, sixty-six readings were taken for each stain for each animal. It was felt that more meaning could be gleaned from the histochemical data if the per cent light transmitted values were converted to per cent light absorbed values. This change was instituted by subtracting each of the per cent light transmitted values from one hundred. Histochemical Reliability.--Reliability of the histo- chemical data was determined by selecting twenty-five slides at random. Sixty-six light transmission readings 46 were taken from each slide. The mean values were compared to the mean values from the original data using a rank order correlation coefficient (Rho). Statistical signifi- cance was determined with the use of a t-test (1). Rho was +.998 which was significant at the .05 level. The data may be seen in Appendix C. Histochemical Validity.--Validity of the histo- chemical data could only be determined by using previously recorded histochemical skeletal muscle staining intensity data from this laboratory. This was necessary because the skeletal muscle (gastrocnemius) showed adjacent cell stain- ing intensity differences, whereas, the heart showed adjacent cell staining intensity homogeneity. Therefore, to subjectively rate the hearts by eye, one would need to assess staining intensity of adjacent hearts. This would be quite difficult. Rho was calculated which compared expert subjective eye ratings (dark: 1 to light: 3) to objective per cent light absorbed values (100% to 0%). This was done on ten fibers from three stains. Calculated Rho's were +.885, +.900, and +.870, for SDH, Sudan Black B, and ATP'ase, respectively. Each was significant at the .05 level. The data may be seen in Appendix C. The Cy Ox staining intensity faded inconsistently between sacrifice and meter readings. Therefore, these data were not statistically analyzed. However, the indi- vidual mean per cent light absorbed values of Cy Ox 47 are located in Appendix C by animal number, treatment, and duration. No information important to the study was lost (58). Pathological The heart sections stained with H & E were evaluated subjectively. They were divided into two groups on the bases of normalcy and pathology exhibited. Pathological Reliability.--Re1iability of the patho- logical data was determined by selecting ten slides at random and subjectively re-evaluating them. These evalu- ations were compared to the original data using Rho. The calculated Rho was +.976 which was significant at the .05 level. Pathological Validity.--Validity of the pathological data was determined after consultation with a pathologist, who specializes in diseases of the cardiovascular system, and after consultation with an anatomist, who specializes in research on cardiac muscle. Each professor subjectively evaluated ten slides. These values were compared to the original observations which were made by the author. Calculated Rho's were +.964 and +.952 for the pathologist, and the anatomist, respectively. Each was significant at the .05 level. Statistical Procedures Mean values for each animal for the histochemical data were determined using the UNEQl routine by the 48 Michigan State University Control Data 3600 Computer (CDC 3600). Statistical calculations on these mean values and on body weight at sacrifice, using the LS routine for two-way analysis of variance (treatment and duration), and for adjusted cell means for the interaction were also per- formed by the CDC 3600. Subsequent statistical calcu- lations on the mean histochemical values and on the mean body weight at sacrifice were performed by the CDC 3600 using the LS routine for one-way analysis of variance (O-wk CONS and 8-2k by treatment), and for another one-way analysis of variance (8-wk by treatment). Scheffé's method for multiple comparisons was employed when the Efratio was judged to be significant (27). This work was also done by the CDC 3600. When the Efratio was judged to be not sig— nificant, the power of the test was hand calculated (27). The probability of committing a Type I error (a) was set at .05 for the one-way analyses of variance. The proba- bility of committing a Type II error (8) was set at .25 for the one-way analyses of variance. Significance levels for the Scheffé tests were set at a = .10. The pathological data were statistically analyzed using chi-square contingency tables (14). One table was designed to compare the O-wk CONS data with the 8-wk treat- ment results. A second table compared the values of the various treatment groups at 8-wk. CHAPTER IV RESULTS AND DISCUSSION Results Effectiveness of the Training Programs The initial concern in this study was the effective- ness of the training programs. The programs were rela- tively new and although considerable pilot testing had been done there was still some mild apprehension present. Since the entire study depended upon the results of these programs, meticulous records of the rats' performances were kept. The body weight results are shown in Figure 2. When analyzed by one-way analysis of variance and subsequent to obtaining a significant Efratio, Scheffé comparisons run, the LON group at 8-wk was significantly lighter than the CON group at 8-wk. However, the number of cases were small with eight rats in each group. When one utilizes all of the body weight data in Figure 2, it can be observed that all mean values for the exercise groups, except for the sixth week value for SWM, were lower than the mean body 49 WY WEUGHY (GM) 50 01% V 2 DURATIO‘J (WKS) 5-4 0-4 mwyziem N 2 (0' corn ovum d aura-Mons 2. 4. 6. if; an N= 8 (or each grow 0! Ouchon 8 ch N = L4 '0' O at 51 weights for the CONS. Therefore, using the sign test, the body weights for VOL, SHT, MED, LON, and ESC were all sig- nificantly lower than the body weights for CONS (p < .05). The programs utilized were effective in that body weights of the trained animals were lower than the body weights of the control animals. The significant differences shown in Table 3, comparing 8-wk data with 0—wk controls, were expected on the basis of normal growth. The lack of significance in the LON merely reflects the greater effect of that program on the rats in terms of body weight. TABLE 3.--Scheffé comparisons for body weight between 0-wk CONS and 8-wk treatment. Scheffé Comparison Body Weight F = 12.09 O-wk 8-wk p < 0.0005 CONS — CON sa CONS - VOL S CONS - SHT S CONS - MED Sb CONS - LON N CONS - ESC S CONS - SWM S aSignificant at .10 level. Not significant. Considerable insight into the training programs can be obtained from the mean daily TRR, PER, CDS, PSF, and revolutions run for the SHT, MED, LON, and VOL groups as shown in Appendix A. These data indicate that the MED and the LON groups increased their daily TRR from day one 52 through day forty. The SHT group's TRR tapered off after day twenty-five. Even though the LON group has higher CDS values than either the SHT or the MED group, the PSF values indicate that between day thirty and day forty the LON group ran better than 75% shock free. TABLE 4.--Scheffé comparisons for body weight between 8-wk treatment. Scheffé Comparison Body Weight F = 4.07 p 0.002 b CON - VOL N CON - SHT N CON - MED N CON - LON sa CON - ESC N CON - SWM N VOL - SHT N VOL - MED N VOL - LON N VOL - ESC N VOL - SWM N SHT - MED N SHT - LON N SHT - ESC N SHT - SWM N MED - LON N MED - ESC N MED - SWM N LON - ESC N LON - SWM N ESC - SWM N aSignificant at .10 level. Not significant. 53 Histochemical The group mean values of percentage of light absorbed may be seen by treatment and duration in Figures 3 through 6 for LDH, FA, SDH, and PAS, respectively. The individual mean values of percentage of light absorbed may be seen by animal number, treatment, and duration in Appendix C. The one-way analysis of variance tables are located in Appendix D. The Scheffé comparisons between the 0-wk CONS and the various 8-wk treatment groups are indicated in Table 5. TABLE 5.--Scheffé comparisons for LDH, FA, SDH, and PAS between O-wk CONS and 8-wk treatment. Scheffé Comparison L2H EA. §2H_ PAS F = 0.64 4.65 0.98 3.06 O-wk 8-wk p = 0.723 <0.0005 0.457 <0.008 CONS - CON . .C Nb . . N CONS - VOL . . Sa . . N CONS - SHT . . N . . N CONS - MED . . N . . N CONS - LON . . S . . S CONS - ESC . . N . . N CONS - SWM . . S . . N aSignificant at .10 level. bNot significant. cJudgment was reserved because the calculated power was <.30. None of the one-way analyses of variance on the 8-wk treatment groups for LDH, FA, SDH, or PAS was significant. Power was calculated and was <.30 in each case; therefore, judgment was reserved on all comparisons. '7. LIGHT ABSORBED 54 751 50“ \a I -1 com. .1 VOL-O SKY-A _( If)" Lou-I q [SC‘A J sun-C] 45 q I I I I 1 O 2 4 6 8 l0 DURATION (WKS) Figure 3 - LOH Moon Voluos Nn 2 foroochmoimvmz,4.6,IO-u. N: a taroochmwmrav'mem mm for 01k 55 W9??? DDIIDO. I l l I 2 4 6 0 l0 OURATIW (WKS) Figs 4 — FA Mam Values N- Zlaoochwmnofmionsz,4,6,lo as N- eiaoochmofwmionaut N-M forOIlL I O 2 4 6 DURATIW (WKS) a: 5 Rye 5 - SDH Mom Values N- Zfor oodnwcw of duration: 2.4.6.10 m. N: efaoochmofmalionans. NumtorOvvh. HE LIGHY EMU 57 DURATIM (W5) F) re 6 - PAS Mean Venues N~2 10- tech 904: o! Mamas NIB Iov now won a! Manon N-m rc- ,. n 2. ‘. 6. I0 all 8 vi! 58 Control slides of SDH, LDH, and Cy Ox for the last three sacrifices reflected the same information that was previously obtained (16). In this case, the mean percent- age of light absorbed was <5% for all slides. Pathological The hearts were diagnosed either as being normal (within the range of normality) or as having a minimal number of focal accumulations of lymphocytes present. The individual diagnoses may be seen by animal number, treat- ment, and duration in Appendix C. The chi-square con- tingency tables appear in Appendix D. Neither of the chi-squares was statistically significant at the .05 level. Essentially, the hearts were non-pathologic. Discussion A primary interest in this study was the effective- ness of the training programs. Were the endurance training programs effective? The results indicate that in terms of lowered body weight, all endurance training programs were effective. Gollnick and Hearn (20) reported that the rats they had swum thirty minutes daily for thirty-five con- secutive days gained 38.7% less body weight than the rats they had maintained as sedentary controls. Similar results were reported by Hearn and Wainio (28) in which their exercised animals gained 30-40% less body weight than their sedentary controls. Van Huss, Heusner, and 59 Mickelsen (65) indicated that at puberty the forced-exercise animals had lower body weights than the sedentary control animals. Effectiveness of a training program is indicated by a decreased body weight gain. The histochemical and pathological data take on new meaning if in fact a training effect had occurred to the rats. For the purposes of this discussion, the assumption is made that a training effect, as indicated by lowered body weight, had occurred to those rats who were in the en- durance training programs. Histochemically, the data support the original hypothesis in terms of the fatty acid concentrations at eight weeks in the VOL, LON, and SWM groups. It was hy- pothesized that the LON group would have its ventricular metabolism move more toward that of total aerobiosis after the treatment period. It was stated by Opie (49) that the heart (aerobic muscle fibers) would rather use lipid than carbohydrate as its energy source. When the physical activity becomes one of daily submaximal loads, aerobiosis is enhanced in the myocardium. Although the 8-wk CON, SHT, MED, and ESC groups did not significantly increase their fatty acid values as compared to the O-wk CONS group, the data do indicate that some type of metabolic adaptation was occurring in the heart which indicated a shift of the metabolism toward more total aerobiosis than was present prior to the treatment period. 60 It was interesting that the only significant histo- chemical differences found were in the substrate data in which fatty acid and glycogen concentrations were being measured. The enzyme concentrations of LDH and SDH were not different. These results support the earlier bio- chemical data of Hearn and Wainio (28). The fact that the LDH and SDH concentrations did not increase as a result of chronic exercise, was surprising. Since SDH and LDH are well recognized enzymes in the aerobic pathway, increased concentrations were expected. Because this did not occur, it would appear that the heart, even of sedentary control animals, contains these enzymes in amounts greater than that needed to cope with the exercise stress afforded by these programs. It is possible that in cardiac metabolism an alternate biochemical pathway exists but at present there is no evidence to support this position. The histochemical results also indicate that more glycogen is present in the ventricles of the LON group after eight weeks of training than is present in the ventricles of the CONS group at zero-week. Blount and Meyer (10) reported that all rats swimming for varying periods of time depleted their cardiac glycogen within the first fifteen minutes of exercise. Those hearts that experienced severe depletion were shown to undergo super- compensation. This latter statement might very well be the case for the LON group animals, since these animals were clearly the most highly trained as reflected by the 61 body weight and the training data. Supercompensation of cardiac glycogen might have occurred. Opie (48) stated that the glycogen stores become depleted in the early stages of exercise only to be restored at the completion of the activity. If Opie is correct it would seem that the SHT and the MED groups would also have shown super- compensation. Since this did not occur it would appear from these data that glycogen supercompensation in the heart results from severe depletion as observed in the LON group. It was not hypothesized as to what the effect of the shock stimulus might be on the SHT, MED, and LON animals. Therefore, the ESC group was paired with the SHT group to measure for those effects. From the results, it is apparent that the ESC group did not differ from either the SHT or the CON group in terms of their metabolic characteristics in the myocardium. Apparently, the shock did not have an adverse effect on those animals that were trained in the C-RW apparatus. However, these results were not statisti- cally significant. As one Observes the histochemical results on Figures 3, 4, 5, and 6, there appear, generally, to be sequential fluctuations of the mean values between zero weeks and ten weeks. LDH, FA, and PAS values generally increase at two weeks and decrease at four weeks. There is another rise at six weeks and a decrease by LDH at eight weeks. FA and 62 PAS rise at eight weeks over their six week values and all values (LDH, FA, and PAS) drop off at ten weeks. The SDH values generally show a drop at two weeks with a further drop at four weeks. An increase in the values occurs at six weeks followed by a drOp at eight weeks. The values rise and fall at ten weeks. However, there is a rhythmicity present. Whether or not this is related to the general adaptation syndrome in any way must await further investigation. The morphological analyses were included primarily to indicate normalcy in the area of the heart from which the histochemical sections were taken. It was gratifying to discover that no significant pathology was present in any Of the hearts from the various treatment groups. CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of this study was to investigate the effects of seven different levels of chronic physical activity on the metabolic and morphologic characteristics of the left ventricular myocardium of adult, male, albino rats. Two hundred and fifty-two, 7l-day-old, male, albino rats (Sprague—Dawley strain) were brought into the labo- ratory and were assigned to one of seven treatments at random. The treatment groups were sedentary control (CON); voluntary running (VOL); short-duration, high-intensity running (SHT); medium-duration, moderate-intensity running (MED); long-duration, low-intensity running (LON); electric stimulus control (ESC); and endurance swimming (SWM). Treatments were administered Monday through Friday. Ani- mals were provided with food and water 3d libitum. The healthiest and best trained animals were selected for sacrifice. Seven selected animals were weighed and sacrificed on Mondays after zero, two, four, six, eight, 63 64 and ten weeks of training. The final sample consisted of 126 animals. During sacrifice, each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital. The heart was excised and cut transversely at a level just below the atria. The apical portion was placed on a chuck which was immersed in pre-cooled 2-methy1butane. Three hundred micra from the apex a minimum of Six 10 u serial sections were cut. Five histochemical procedures were utilized to evalu- ate the relative glycogen, fatty acid, and aerobic enzyme concentrations in the cardiac fibers. A histologic pro- cedure was performed to evaluate the morphology of the hearts. Each histochemical stain was measured objectively using a light transmission meter. The histological sections were rated subjectively on the bases of normalcy and of pathology. The results indicate that training for eight weeks was sufficient to produce metabolic adaptations in the rats. The eight-week LON group was observed to be signifi- cantly lighter than the eight-week CON group (p < .10). The fatty acid concentrations were greater in the VOL, LON, and SWM groups at eight weeks than in the CONS group at zero-week (p < .10). Also, in the LON group at eight weeks, surprisingly, the glycogen concentrations were greater than in the CONS group at zero-week (p < .10). 65 No pathological changes were observed in any of the seven treatments (p < .05). This result is limited to the single section taken approximately 300 u from the apex. Conclusions The results of this study have led to the following conclusions: 1. The training programs utilized, were effective in that body weights of the trained animals were lower than the body weights of the control ani- mals. 2. Fatty acid concentrations were greater in the VOL, LON, and SWM groups at eight weeks than in the CONS group at zero-week. 3. Surprisingly, the glycogen concentrations were greater in the LON group at eight weeks than in the CONS group at zero-week. 4. No pathological changes were observed in any of the seven treatments in terms of the single tissue section taken. Recommendations l. A similar study should be completed in which the trained animals have mean PER and mean PSF values of nothing less than 90% and 90%, respectively. 2. 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APPENDICES APPENDIX A TRAINING PROGRAMS AND TRAINING DATA 74 TABLE A-l.--Standard eight-week, short-duration, high-intensity endurance training program for postpubertal and adult male rats in controlled-running wheels. A M Q) 0 ill E e “-40 [-4 ~04 .K 5 g 03 5A or O B a: a :3; .- . s . :5. :r 2.2—: .3 t. 3 9 ea) EC) ‘5 v4“ 0 +iE E 8.. ch X:A 012 an v40 U3 0" V 00 9" NU 36* '44 N 0" 80 [-0 #8 H m 0.41 :3 . ° ° 2:. .2 .5 v s .3 :5 "=4 :5. :3 :1: a z; 2: as “-3 :§ §~ a .5= 2 5:: :2 2;: :2 3 Q G <8 3" I!" M3. 28 [*8 U) 0:" E-‘Da BO: 9" 1 l-M l 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 Z-T 2 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 3-W 3 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 4-T 4 2.0 00:10 10 4O 3 5.0 1.0 2.5 39:45 750 1200 S-F 5 2.0 00:10 10 40 3 5.0 1.0 2.5 39:45 750 1200 2 1'! 6 2.0 00:10 10 40 3 5.0 1.0 2.5 39:45 750 1200 Z-T 7 1.5 00:10 10 40 3 5.0 1.0 3.0 49:30 900 1200 3-W 8 1.5 00:10 10 40 3 5.0 1.0 3.0 49:30 900 1200 4-T 9 1.0 00:10 10 40 3 5.0 1.0 3.0 49:30 900 1200 SI? 10 1.0 00:10 10 40 3 5.0 1.0 3.0 49:30 900 1200 3 1-M 11 1.0 00:10 10 40 3 5.0 1.0 3.0 49:30 900 1200 2-M 12 1.5 00:10 15 40 3 5.0 1.0 3.5 59:15 1050 1200 3-W 13 1.5 00:10 15 4O 3 5.0 1.0 3.5 59:15 1050 1200 4-T 14 1.5 00:10 15 40 3 5.0 1.0 3.5 59:15 1050 1200 SI? 15 1.5 00:10 15 40 3 5.0 1.0 3.5 59:15 1050 1200 4 l-M 16 1.5 00:10 15 40 3 5.0 1.0 3.5 59:15 1050 1200 2-T 17 1.5 00:10 20 34 3 5.0 1.0 4.0 60:00 1020 1020 3-W 18 1.5 00:10 20 34 3 5.0 1.0 4.0 60:00 1020 1020 4-T 19 1.5 00:10 20 34 3 5.0 1.0 4.0 60:00 1020 1020 S-F 20 1.5 00:10 20 34 3 5.0 1.0 4.0 60:00 1020 1020 S 1-H 21 1.5 00:10 20 34 3 5.0 1.0 4.0 60:00 1020 1020 ZIT 22 1.5 00:10 25 22 4 2.5 1.0 4.5 57:10 990 880 3-W 23 1.5 00:10 25 22 4 2.5 1.0 4.5 57:10 990 880 4-T 24 1.5 00:10 25 22 4 2.5 1.0 4.5 57:10 990 880 5.? 25 1.5 00:10 25 22 4 2.5 1.0 4.5 57:10 990 880 6 1-M 26 1.5 00:10 25 22 4 2.5 1.0 4.5 57:10 990 880 2-T 27 1.5 00:10 30 14 5 2.5 0.8 5.0 54:10 875 700 3-W 28 1.5 00:10 30 14 5 2.5 0.8 5.0 54:10 875 700 4'? 29 1.5 00:10 30 14 5 2.5 0.8 5.0 54:10 875 700 5.? 30 1.5 00:10 30 14 5 2.5 0.8 5.0 54:10 875 700 7 II“ 31 1.5 00:10 30 14 5 2.5 0.8 5.0 54:10 875 700 2'? 32 2.0 00:10 35 10 6 2.5 0.8 5.5 54:00 825 600 3'" 33 2.0 00:10 35 10 6 2.5 0.8 5.5 54:00 825 600 4'? 34 2.0 00:10 35 10 6 2.5 0.8 5.5 54:00 825 600 5'? 35 2.0 00:10 35 10 6 2.5 0.8 5.5 54:00 825 600 8 l-H 36 2.0 00:10 35 10 6 2.5 0.8 5.5 54:00 825 600 2-T 37 2.0 00:10 40 7 7 2.5 0.8 6.0 51:10 735 490 3-W 38 2.0 00:10 40 7 7 2.5 0.8 6.0 51:10 735 490 4'? 39 2.0 00:10 40 7 7 2.5 0.8 6.0 51:10 735 490 SI! 40 2.0 00:10 40 7 7 2.5 0.8 6.0 51:10 735 490 This standard program was designed using male rats of the Sprague-Dawley Strain. All animals were between 70 and 170 days-of-age at the beginning of the program. The duration and intensity of the program were established so that 75 per cent of all such animals should have PS? and PER scores of 75 or higher dur- ing the final two weeks. Alterations in the rest time, repetitions per bout, number of bouts, or time between bouts can be used to affect changes in these values. Other strains or ages of animals could be expected to respond differ- ently to the program. All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the training program. Failure to provide this adjustment period will impose a double learning situation on the animals and will seriously impair the effectiveness of the training program. Standard short-duration, high-intensity endurance maintenance program for post- pubertal and adult male rats in controlled-running wheels. .8 .. .s I: I: O“ 5" 3" 2 ”‘3 o E .3 .2 .§ .- 3 °. u 5': a :3 .5-3 “.9. "E g- s: 3. ° 3. - 33 M a; 2 F48) I": “A .33 a. I 8 mm H0 04'" H“ 05 XI: 08 0 U 04) U \ GD 60 RIO 3.. '. e" §= 5= 2 5:: :2 s: 2;: .v :5 3...: .3 .3 .. .v .. .. .4 2.0 00:10 40 5 6 5.0 0.8 6.0 46:00 450 300 75 TABLE A-2.--Standard eight-week, medium-duration, moderate-intensity endurance training program for postpubertal and adult male rats in controlled-running wheels. A a: 4) o m E G. g: r: “63 E" I": 6 a a- 2 :2 - .. .2 .. ~ ~ “° .1 w 2. : a: a :- a: :2 :5 3 Ed 3 3 -§ 8 -S u a 0.. ~' 0 0 9‘“ n1» 3 'H u 0" BID [-1 ~48 u m 0.0 . :3 A 0 0 H n a- u a n x m m H r4H H x o o .x_: u 8 a: u §.u 8 :13 3 g! 3 g 3 8 8 a a 85 SE 3. 86 3 .3 n .6 on 00 on 3 o 0 4E4 3“ ¢~v mum 2:8 618 m “.5 6¢m Eu“ 8" 1 1a" 1 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 2-T 2 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 3-W 3 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 4=T 4 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 58F 5 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 2 lsM 6 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 2-T 7 1.5 00:10 10 27 4 5.0 1.0 3.0 50:20 810 1080 3=w 8 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 4-T 9 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 S-F 10 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 3 l-M 11 l.0 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 Z-T 12 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 3-W 13 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 4-T 14 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 S-F 15 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 4 l-M 16 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 2-T 17 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 3-W 18 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 4-T 19 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 S-P 20 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 5 1-M 21 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 2-T 22 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 3-W 23 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 4-T 24 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 S-F 25 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 6 l-M 26 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 Z-T 27 1.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 1100 1100 3-W 28 1.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 1100 1100 4-T 29 1.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 1100 1100 S-P 30 1.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 1100 1100 7 l-H 31 1.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 1100 1100 Z-T 32 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 3-W 33 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 4-T 34 1.5 00.25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 5a? 35 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 8 l-H 36 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 Z-T 37 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 3-W 38 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 4-T 39 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 S-F 40 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 This standard program was designed using male rats of the Sprague-Dawley strain. All animals were between 70 and 170 days-of-age at the beginning of the program. The duration and intensity of the program were established so that 75 per cent of all such animals should have PSF and PER scores of 75 or higher during the final two weeks. Alterations in the work time, rest time, repetitions per bout, number of bouts, or time between bouts can be used to affect changes in these values. Other strains or ages of animals could be expected to respond differently to the program. All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation on the animals and will seriously impair the effectiveness of the training program. Standard medium-duration. moderate-intensity endurance maintenance program for postpubertal and adult male rats in controlled-running wheels. A K 0 O m s w 0 8 - I: u g on a 9‘ ‘SA ‘3 0‘: A 0‘: o: X :3 2* ° 2. 3 :a a 3 s: :2 a; ND «48 '5 US 0" V 00 5"" [114-3 35* 0" B“ E‘ "-48 u Q 0.4) :1 a. .2 .~ : :3 .3 x ”a a: :3 :3 as 3: :§ 6: 3” 5’ § 5: :2 s: 3: <9 3" G" can. 28 ("8 U) Cl“ PD: PM [4" 1.5 00:30 30 8 3 5.0 0.8 4.0 32:30 720 720 76 TABLE A-3.--Standard eight-week, long-duration, low-intensity endurance training program for postpubertal and adult male rats in controlled-running wheels. ». m 0 U m E ' «40 B «I c C C O‘: tn B 6 ’3 .S,. 2 3‘3 .2 e c on x :’ ~ :3: :2: :3 .2. “a 5': a B- a: :2 a: B utn M e ‘H +13 0" ~' 0 U 9" hlu 36* '44 w 0" E401 £4 "-48 14 CD 0.4.) D o o H u a~ u g m m x m m H - rid H‘~ *5 00 Xi: U0 4) U 434-! 0 \ «30‘ ‘00 MU 3: :~ :~ 3.5 “a :3 a: 53 .53 .2 5:: :2 2;: a: 3 Q 0 (Ed 8‘“ m~v mix zxn 94m U) m~v 54m E u u U-A o E w m m m o -d o .c 3~u o u o e p m (as 3.. m~v m o. zcn 54m m x~v Pam E«m r~v 0.5 10:00 O 1 3 2 5 0 8 2.0 35 00 900 1800 77 TABLE A-4.--Standard eight-week, endurance, swimming train- ing program for postpubertal and adult male rats. Day of Da of Per Cent Expected Week Week Trainin Tail Swim Time 9 Weight (min) EST 1 1=M 1 O 30 2=T 2 0 4O 3=W 3 C* 50 4=T 4 C 60 5=F 5 C 60 2 1=M 6 2 40 2=T 7 2 40 3=W 8 2 40 4=T 9 2 45 5=F 10 2 50 3 1=M 11 3 3O 2=T 12 3 30 3=W 13 3 30 4=T 14 3 35 5=F 15 3 35 4 1=M l6 3 35 2=T l7 3 4O 3=W 18 3 40 4=T 19 3 40 5=F 20 3 4O 5 1=M 21 3 40 2=T 22 3 45 3=W 23 3 45 4=T 24 3 45 5=F 25 3 45 6 1=M 26 3 45 2=T 27 3 50 3=W 28 3 50 4=T 29 3 50 5=F 30 3 50 7 1=M 31 3 50 2=T 32 3 55 3=W 33 3 55 4=T 34 3 55 5=F 35 3 55 78 TABLE A-4.--Continued. Per Cent Expected Week Dageif ngzni: Tail Swim Time 9 Weight (min) EST 8 1=M 36 3 55 2=T 37 3 60 3=W 38 3 60 4=T 39 3 60 5=F 40 3 6O This standard program was designed using male rats of the Sprague-Dawley strain. All animals were between 70 and 90 days-of-age at the beginning of the program. The duration and intensity of the program were established so that 75 per cent of all such animals should have PET scores of 75 or higher during the final two weeks. Alterations in the percentage of tail weight or eXpected swim time can be used to affect changes in these values. Other strains or ages of animals could be expected to respond differently to the program. All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. Failure to provide this adjustment period will impose a severe, sudden exercise stress upon the ani- mals and will seriously impair the effectivensss of the training program. Standard endurance swimming maintenance program for post- pubertal and adult male rats Expected Eer Cent Swim Time Tail Weight (min) EST 2 40 *C = Clothespin only. 79 p, III 4 29...; 38 :60 622 u _-< 9:3 e 0v on On mm m_ p—F_L_F~_~_____p_____rPL__—r%bbbpph N lllllellllI o. r: e'lllelIl! I: . m o n a 1‘4 o _ n c . n _ N n o. _ U: 4“. 0mm 1.9% 7 own .I 0mm 1:00? [0.0 .l ON? 1.89 .l Oct 1009 T Om? I. 02.0 s 2: . ll.— anoam _. z . is 2.45 o _ m :3 .23: (swmb) 1H9HM A008 80 303 Si mcozgoim 260 :82 I m-< 939.1 r N xTelir .2 ._T 2 1.1.. a o s m n v n N O. rp—b—nbbFn—bpr_P_Lb_b__F_.L—_—_p__h A . X: d> o. _ m _ m _ s _ w _ m _ c _ n _ N _ . x; .235 cm 3 ow mm 8 mm 8 m. o. m :8 2.5: L__________r:__:__:.p:__h_____~__________:__u. 41 o. Too~ 4 1. 1 u H 8 ON 1 . IOOn . 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I o. 1 .108 l l T a B 81 . room H on 1 Ioov J I ov 1 Icon ./ J 1., . on 1 loom ... .. om .x)... .. .. r02 . ‘ . . . r 2. 1 . . . . . loom 1. . . . . . . . f Om] . . . Y100m 1 1 om1 . 108. oo.L loo: IooN. 84 H :ISd ems. .2 Ema: NE.» 8.... x85 :80 8a 9.0 808 xoocm No 5:830 3:22:30 2.00 :85. I m-< 8:9... 7‘ N 4‘ 0. 1J1] N. J? v. J? o. cumin: o. _ m o _ N o _ m w _ n N _ x; 2.5: cc On ON 0 on no on 8 n. . m. >323”... .._r__.__.I..._______.F__.____:__..r.__.._____T1.r o.1 108 3 .. u C 3 ON... .lOOmH on!“ [00¢ owl loom om1 . loom owl I O C. O. I '00“ A I. O. 0... O 0 1 2.1 . .. . loom 81 . . .Toom 81 I80. 8.1 loo: FooN_ = 85 H BBC! On] 00.1 On] owl Obi 0m! 00. g 20... .2 €va mco_S_o>mm Ugowaxm Emu 5Q uco Sag: cam mco_3_o>mm .20... 2.00 com—2 l N-< 939... «mm; J!!! m. VII 0. J11? N. x v. 0N m _ o _ h _ m _ m _ v _ n _ N _ 0v on OM ON ON 0. o. m ___r__r__~____________________b[__~____ 33>: 35> 23 .231... >40 2.4m... b J h I oo~ IOOn H 881. T 00v IOOm I000 I 00... I 000 I com I 009 I00: I CON. 86 H 33d 20.. 5. Ema. NE... mm... 605 E8 .oa 96 808 .305 No 8.650 N>zo3Eau 360 cows. I m-< 9:9... 1 N JA[ o. if N. J? c. it o. «8.52 o. _ m o _ N o _ n v _ n N _ 23.2.45 on 3 oo mm on mN 8 m. o. m .30 .225 :....._.___.....__..__...___N_____._p____:. J. o. 1 looN O 1 . 0 S 81 loom H 1 . r oml roov . r 9.1 . loo... 1 o T oml loom .. . . ‘ .. oo1 . . . . . . 102 A o o o o .0 0.. o 21 . . ‘. looo owl . Ioom A . .. om1 . .1000. 8.1. loo: J1 APPENDIX B SACRIFICE MODIFICATIONS APPENDIX B SACRIFICE MODIFICATIONS First Sacrifice: Each animal was anesthetized with 0.35 cc. of the Halatal solution. Once into the abdominal cavity, an in vivo injection, of a 2% heparinized solution of black Pelikan ink (10 ml.) into the in- ferior vena cava between the junction of the common iliac veins and the first set of iliolumbar veins: was performed. The injection was accomplished while the heart was still beating which allowed three to five minutes continued in vivo circulation. (This was to be used later as an histologic demonstration of the capillaries of the triceps surae and plantaris muscle groups, which was another part of the study). Second Sacrifice: Each animal was anesthetized with 0.35 cc. of the Halatal solution. Once into the abdominal cavity, the left common iliac artery and vein were clamped with a taped hemostat. The modifi- cations are the same as in the first sacrifice (above), except for the fact that 5 m1. of Pelikan ink were injected. Third Sacrifice: Each animal was anesthetized with 2.50 cc. of the Halatal solution. Procedures followed those as reported in Chapter III. Fourth Sacrifice: Each animal was anesthetized with 2.50- 3.00 cc. of the Halatal solution. Procedures followed those as reported in Chapter III. Fifth Sacrifice through the Twelfth Sacrifice: No modifications from those reported in Chapter III. Thirteenth Sacrifice through the Fifteenth Sacrifice: Modifications were identical to those reported in the first sacrifice (above), except that 5 m1. of Pelikan ink were injected. 87 88 Sixteenth Sacrifice through the Eighteenth Sacrifice: Modifications were identical to those reported in the first sacrifice (above), except that 6 ml. of pelikan ink were injected. APPENDIX C HISTOCHEMICAL AND PATHOLOGICAL DATA 89 TABLE C-1.--Mean per cent light absorbed and body weight presented by anima1 number, treatuent, and duration. Dur. 8d. Wt. Mean Per Cent Light Absorbed An. No. Treat. (wk) ‘9 ’ pas LDH n son Cy Ox 4 CON 0 372 33.5 76.0 30.6 90.5 74.7 86 CON 0 401 25.8 48.7 46.1 89.7 66.2 13 VOL 0 378 17.9 73.6 34.3 90.6 80.4 87 VOL 0 400 28.5 48.0 41.6 87.5 63.8 32 SHT 0 339 22.0 74.0 33.1 92.4 75.9 88 SHT 0 393 22.1 31.2 42.2 85.0 57.3 48 M80 0 373 37.2 80.9 34.7 96.8 72.8 89 MED 0 377 19.8 38.9 38.6 85.3 52.5 57 LON 0 375 21.0 84.4 43.1 98.0 74.1 96 LON 0 370 17.2 33.1 26.0 84.0 57.8 68 ESC o 351 29.5 73.4 34.0 91.8 77.6 101 ESC o 362 17.7 42.4 34.3 84.8 50.5 74 SWM 0 332 29.3 66.5 36.1 96.6 75.5 162 SWM 0 422 19.5 23.6 36.4 86.7 56.9 8 CON 2 454 31.6 78.0 63.3 91.0 60.8 92 CON 2 432 16.8 43.6 51.3 76.5 37.9 15 VOL 2 392 31.3 78.0 53.2 86.1 58.0 103 VOL 2 390 35.4 37.2 50.3 79.0 44.0 35 SHT 2 393 26.6 81.3 61.6 86.8 70.4 117 SHT 2 371 44.6 45.6 45.7 86.6 46.2 46 MED 2 360 38.0 75.2 65.2 92.6 63.8 126 MED 2 425 13.2 50.8 34.1 71.2 34.1 55 LON 2 373 29.9 78.9 59.7 87.3 63.4 144 LON 2 340 37.1 48.7 42.5 79.3 44.8 71 ESC 2 428 28.8 80.7 42.5 90.0 48.9 153 ESC 2 413 23.8 48.2 38.6 78.6 45.7 76 SWM 2 423 16.8 79.2 44.6 93.7 68.2 164 SWM 2 447 10.6 54.9 47.1 82.1 41.8 12 CON 4 433 12.7 67.4 24.9 73.8 35.6 94 CON 4 437 16.7 49.5 28.6 75.7 11.9 20 VOL 4 422 24.4 79.1 51.5 83.3 46.5 105 VOL 4 440 22.4 48.1 44.0 75.2 8.1 31 SHT 4 388 20.1 71.2 41.0 77.6 45.6 111 SHT 4 419 25.6 41.4 47.0 79.8 18.0 39 M80 4 397 25.1 71.6 41.7 80.8 45.6 130 MED 4 395 20.1 53.4 31.8 79.8 19.9 50 LON 4 425 17.9 75.2 35.0 63.4 41.4 141 LON 4 387 20.0 46.0 36.4 83.5 11.1 67 BSC 4 432 21.7 78.0 42.4 82.0 41.2 147 ESC 4 393 23.4 42.3 36.0 81.9 14.2 84 SWM 4 400 16.9 72.4 12.0 65.5 41.5 158 SWM 4 433 27.7 50.3 50.2 86.3 18.7 2 CON 6 442 20.1 81.5 47.0 85.3 47.7 91 CON 6 440 20.4 56.4 32.9 77.2 35.7 21 VOL 6 428 13.0 79.9 35.9 79.9 41.1 97 VOL 6 423 31.8 43.4 43.8 87.4 41.3 27 SHT 6 414 36.4 83.8 45.5 80.2 48.5 114 SHT 6 408 31.5 39.1 47.4 85.1 31.6 44 MED 6 420 38.4 80.8 52.2 83.5 48.3 127 MED 6 420 32.8 51.1 43.5 85.9 53.7 49 LON 6 418 16.9 81.1 37.5 85.6 38.5 138 LON 6 418 36.6 60.7 49.3 84.9 39.6 63 ESC 6 443 14.7 81.5 50.6 84.5 51.0 150 ESC 6 408 21.1 46.7 38.1 76.1 43.8 79 SWM 6 444 25.1 84.4 54.2 84.9 51.0 165 SWM 6 460 26.5 54.0 41.0 88.1 51.8 7 CON 8 451 14.7 68.5 19.7 53.6 35.1 93 CON 8 457 28.3 64.6 47.0 86.3 10.8 169 CON 8 462 36.9 57.4 40.2 86.0 53.6 184 CON 8 472 43.0 67.8 45.4 81.1 72.2 197 CON 8 546 47.0 77.4 51.1 87.9 8.6 212 CON 8 468 47.4 74.0 41.1 77.3 40.2 TABLE C-1.--continued. 90 Mean Per Cent Light Absorbed An. No. Treat. Dur. Bd’ Wt’ ("k’ ‘9) PAS LDH PA 308 Cy Ox 225 CON 8 470 48.6 50.5 53.5 90.0 22.1 240 CON 8 455 35.8 67.3 56.5 85.9 92.2 16 vOL 8 435 14.0 78.6 40.0 71.1 67.9 106 VOL 8 454 28.2 67.9 41.2 85.9 3.6 186 VOL 8 438 50.1 68.6 57.0 89.1 78.7 188 VOL 8 446 30.1 61.6 39.9 86.9 71.8 214 VOL 8 407 44.0 55.6 48.0 71.6 51.9 215 VOL 8 427 44.0 55.0 60.2 78.1 53.1 242 VOL 8 447 28.6 56.4 56.7 81.6 92.8 243 vOL 8 428 35.5 63.9 68.8 85.4 93.2 28 SHT 8 448 14.6 88.2 34.9 42.3 63.1 116 SHT 8 434 34.0 57.6 39.2 87.9 10.2 172 SHT 8 433 43.2 52.9 47.6 89.3 65.6 173 SHT 8 464 50.0 53.6 42.7 90.3 66.0 199 SHT 8 434 44.9 73.9 53.3 84.4 10.1 202 saw 8 479 42.6 76.2 44.4 82.4 8.2 228 SHT 8 423 50.7 55.1 42.8 85.8 13.9 229 saw 8 394 28.4 49.6 50.6 81.1 9.5 43 MED 8 423 9.5 65.2 23.2 51.1 63.1 132 N80 8 461 38.0 58.8 45.6 85.1 5.8 175 N80 8 470 38.6 49.5 37.1 90.5 59.0 178 MED 8 407 36.0 57.9 41.7 92.4 76.2 203 MED 8 458 47.4 75.2 44.3 86.7 10.9 206 N80 8 398 52.1 71.1 53.9 85.7 10.3 232 MED 8 386 30.1 42.0 41.2 82.3 14.6 234 N80 8 412 46.5 50.7 59.5 89.7 18.5 52 LON 8 389 44.8 72.6 40.0 56.4 71.5 140 LON 8 464 46.7 51.2 55.8 86.7 7.8 190 LON 8 372 44.9 70.5 50.3 91.3 81.3 192 LON 8 424 47.8 64.9 54.6 94.1. 82.5 219 LON 8 405 43.9 57.2 47.7 76.0 44.5 220 LON 8 375 28.2 64.4 41.3 74.2 43.6 246 LON 8 392 31.1 61.7 60.3 87.0 91.3 248 LON 8 422 51.4 62.9 62.2 83.3 90.8 64 ESC 8 417 26.0 65.3 42.8 83.7 67.6 152 ESC 8 423 26.7 52.3 44.5 87.9 6.8 180 ESC 8 522 49.4 52.8 43.6 90.3 66.8 181 ESC 8 456 42.0 59.5 42.3 89.4 60.7 207 ESC 8 426 47.8 71.8 54.6 84.3 16.2 210 85C 8 500 39.8 75.9 41.5 82.7 7.0 236 85C 8 427 28.4 37.8 47.5 82.3 14.8 237 ESC 8 410 36.9 41.0 52.0 89.2 14.2 83 swu 8 430 8.4 78.4 25.1 36.6 36.2 160 swu 8 434 32.9 61.3 55.3 90.2 10.5 193 sun 8 460 42.5 65.1 57.0 90.2 76.4 195 sz 8 496 51.0 62.9 63.9 94.4 77.8 221 sun 8 470 40.5 60.1 53.8 82.8 46.4 224 swu 8 440 30.8 56.3 60.8 81.2 46.1 249 SNN 8 433 53.3 58.8 71.7 89.9 90.8 251 sz 8 444 44.6 63.4 64.2 86.5 94.5 3 CON 10 466 24.9 44.5 28.5 70.1 64.1 90 CON 10 533 31.9 74.7 41.2 90.3 90.5 24 VOL 10 462 33.6 47.5 26.9 82.6 65.0 100 VOL 10 409 29.4 64.4 34.9 85.8 78.4 29 SHT 10 444 19.3 52.6 32.1 85.0 70.0 109 SHT 10 457 34.6 63.6 34.8 89.3 93.8 38 MED 10 412 22.4 37.9 35.0 82.3 70.3 125 MED 10 430 44.5 71.8 44.3 91.8 94.1 51 LON 10 434 22.0 39.3 50.4 86.2 63.1 133 LON 10 530 30.5 59.2 36.0 89.0 93.9 65 ESC 10 434 23.2 40.9 18.1 78.0 68.2 145 ESC 10 460 22.8 60.1 40.8 86.6 89.3 82 swu 10 443 34.3 40.1 50.0 56.9 73.0 157 swn 10 479 24.2 58.6 35.4 85.6 93.1 91 TABLE C-2.--Reliability of histochemical data. Mean Per Cent Duration Light Ab' An. No. Treatment Stain - (Wk) 2 mos Original ' later 4 CON 0 Cy Ox 74.8 72.0 13 VOL 0 LDH 73.6 75.7 32 SHT 0 FA 33.1 36.1 57 LON 0 LDH 84.4 83.9 15 VOL 2 FA 53.2 54.7 35 SHT 2 PAS 26.6 28.3 71 ESC 2 SDH 90.0 90.0 76 SWM 2 Cy Ox 68.2 73.5 105 VOL 4 SDH 75.2 69.0 31 SHT 4 SDH 77.6 74.5 67 ESC 4 LDH 78.0 75.8 67 ESC 4 SDH 82.0 80.2 44 MED 6 LDH 80.8 79.5 127 MED 6 PAS 32.8 36.4 127 MED 6 FA 43.5 48.2 49 LON 6 Cy Ox 38.5 47.0 138 LON 6 SDH 84.9 83.1 63 ESC 6 PAS 14.7 14.4 79 SWM 6 FA 54.2 60.4 106 VOL 8 FA 41.2 41.9 214 VOL 8 PAS 44.0 44.5 228 SHT 8 LDH 55.1 58.6 229 SHT 8 Cy OX 9.5 14.3 248 LON 8 FA 62.2 55.0 38 MED 10 LDH 37.9 40.2 92 TABLE C—2a.--Validity of histochemical data. Per Cent Light Eye Stain Absorbed Rating SDH 70 56 60 74 57 53 72 59 53 66 NUJWHwWF-‘WWH Sudan Black B 16 13 16 19 15 14 18 13 15 20 I—‘WWHLONF-‘NWH ATP'ase 8O 63 52 84 63 59 82 60 60 81 l-‘LUWHDJWI-‘wwH 93 TABLE C-3.--Pathologic analysis presented by animal number, treatment, and duration. An. NO. Treat. ?::5 Diagnosis 4 CON 0 Minimal focal accumulations of lymphocytes 86 CON 0 Normal 13 VOL 0 Normal 87 VOL 0 Normal 32 SHT 0 Normal 88 SHT 0 Minimal focal accumulations of lymphocytes 48 MED 0 Normal 89 MED 0 Normal 57 LON 0 Normal 96 LON 0 Normal 68 ESC 0 Normal 101 ESC 0 Normal 74 SWM 0 Normal 162 SWM 0 Normal 8 CON 2 Normal 92 CON 2 Minimal focal accumulations of lymphocytes 15 VOL 2 Normal 103 VOL 2 Normal 35 SET 2 Normal 117 SET 2 Normal 46 MED 2 Normal 126 MED 2 Minimal focal accumulations of lymphocytes 55 LON 2 Minimal focal accumulations of lymphocytes 144 LON 2 Minimal focal accumulations of lymphocytes 71 ESC 2 Normal 153 ESC 2 Normal 76 SWM 2 Normal 1 164 SWM 2 Minimal focal accumulations of lymphocytes 12 CON 4 Normal 94 CON 4 Normal 20 VOL 4 Normal 105 VOL 4 Minimal focal accumulations of lymphocytes 31 SHT 4 Normal lll SHT 4 Normal 39 MED 4 Minimal focal accumulations of lymphocytes 130 MED 4 Normal 50 LON 4 Normal 141 LON 4 Minimal focal accumulations of lymphocytes 67 ESC 4 Normal 147 ESC 4 Minimal focal accumulations of lymphocytes 84 SWM 4 Normal 158 SWM 4 Minimal focal accumulations of lymphocytes 2 CON 6 Minimal focal accumulations of lymphocytes 91 CON 6 Minimal focal accumulations of lymphocytes 21 VOL 6 Normal 97 VOL 6 Minimal focal accumulations of lymphocytes 27 SHT 6 Normal 114 SHT 6 Minimal focal accumulations of lymphocytes 44 MED 6 Minimal focal accumulations of lymphocytes 127 MED 6 ' Minimal focal accumulations of lymphocytes 49 LON 6 Minimal focal accumulations of lymphocytes 138 LON 6 Minimal focal accumulations of lymphocytes 63 ESC 6 Minimal focal accumulations of lymphocytes 150 ESC 6 Normal 79 SWM 6 Normal 165 SWM 6 Minimal focal accumulations of lymphocytes 7 CON 8 Normal 93 CON 8 Minimal focal accumulations of lymphocytes 169 CON 8 Normal 184 CON 8 Normal 197 CON 8 Normal 212 CON 8 Minimal focal accumulations of lymphocytes 225 CON 8 Normal TABLE C-3.--Continued. 94 An. No. Treat. ?::; Diagnosis 240 CON 8 Normal 16 VOL 8 Normal 106 VOL 8 Normal 186 VOL 8 Minimal focal accumulations of lymphocytes 188 VOL 8 Minimal focal accumulations of lymphocytes 214 VOL 8 Normal 215 VOL 8 Minimal focal accumulations of lymphocytes 242 VOL 8 Normal 243 VOL 8 Normal 28 SHT 8 Normal 116 SHT 8 Normal 172 SET 8 Minimal focal accumulations of lymphocytes 173 SET 8 Normal 199 SHT 8 Minimal focal accumulations of lymphocytes 202 SET 8 Normal 228 SHT 8 Minimal focal accumulations of lymphocytes 229 SET 8 Normal 43 MED 8 Normal 132 MED 8 Normal 175 MED 8 Normal 178 MED 8 Normal 203 MED 8 Normal 206 MED 8 Normal 232 MED 8 Normal 234 MED 8 Normal 52 LON 8 Normal 140 LON 8 Minimal focal accumulations of lymphocytes 190 LON 8 Minimal focal accumulations of lymphocytes 192 LON 8 Minimal focal accumulations of lymphocytes 219 LON 8 Minimal focal accumulations of lymphocytes 220 LON 8 Normal 246 LON 8 Normal 248 LON 8 Minimal focal accumulations of lymphocytes 64 ESC 8 Normal 152 ESC 8 Normal 180 ESC 8 Minimal focal accumulations of lymphocytes 181 ESC 8 Normal 207 ESC 8 Normal 210 ESC 8 Normal 236 ESC 8 Normal 237 ESC 8 Minimal focal accumulations of lymphocytes 83 SWM 8 Normal 160 SWM 8 Normal 193 SWM 8 Normal 195 SWM 8 Normal 221 SWM 8 Normal 224 SWM 8 Minimal focal accumulations of lymphocytes 249 SWM 8 Normal 251 SWM 8 Normal 3 CON 10 Normal 90 CON 10 Minimal focal accumulations of lymphocytes 24 VOL 10 Minimal focal accumulations of lymphocytes 100 VOL 10 Minimal focal accumulations of lymphocytes 29 SET 10 Minimal focal accumulations of lymphocytes 109 SET 10 Normal 38 MED 10 Normal 125 MED 10 Normal 51 LON 10 Normal 133 LON 10 Minimal focal accumulations of lymphocytes 65 ESC 10 Normal 145 ESC 10 Normal 82 SWM lO Minimal focal accumulations of lymphocytes 157 SWM 10 Normal APPENDIX D STATISTICAL TABLES 95 TABLE D-l.--Analysis of variance for body weight by O-wk CONS and 8-wk treatment. Summary of Analysis Of Variance Source SS df MS F P Treatment 68350.62 7 9764.37 12.09a Figure E~l - Sectioning 0f the Heart