HE NOBEL-EM: umfjfs ‘N BMW Mi”: {AL MUSCLE AF}r ER CHRU Hi? ”kit“ Thesis for the Degree of M. A. MICHIGAN STATE UNEVERSI‘E‘Y R‘ L. BOWZER 1970 TH 85"; ABSTRACT HISTOCHEMICAL CHANGES IN RAT SOLEUS MUSCLE AFTER CHRONIC EXERCISE By R. L. Bowzer The purpose of this study was to determine the histochemical effects of chronic exercise on selected en- zymes of individual muscle fibers in the soleus muscles of male albino rats. Eighteen rats were forced to swim for two two-hour periods twice a day, five days a week, for six weeks in water at thirty-two degrees centigrade. The equivalent of three per cent of each animal's body weight was attached to its tail as an exercise load and to help eliminate floating. At the end of the experimental period, each animal was lightly anesthetized with ether. The right soleus was removed and quick-frozen in isopentane. Tissue sections were cut and prepared for histochemical analysis with myosin ATPase, SDH, and mito alpha-GPD staining procedures. Four to seven hundred individual fibers of each slide were subjectively analyzed according to stain in- tensity. Fibers having high stain intensity were rated R. L. Bowzer dark. Those having moderate stain intensity were rated light. A percentage of dark fibers was calculated. The same procedure was followed for seventeen control animals. The resulting percentages then were used in separate t- tests for each of the three stains to determine if signifi- cant differences between mean percentages existed. The results showed a significant increase in all three enzyme levels due to training. HISTOCHEMICAL CHANGES IN RAT SKELEIAL MUSCLE AFTER CHRONIC EXERCISE By R. L. Bowzer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Health, Recreation, and Physical Education 1970 TABLE OF CONTENTS Chapter I. INTRODUCTION Statement of the Problem Importance of the Problem Limitations of the Study II. RELATED LITERATURE Negative Results Positive Results III. EXPERIMENTAL DESIGN Forced Exercise Regimen. Sacrifice Schedule Sacrifice Procedure Histochemical Rationale and Procedures. Method of Analysis . . . . IV. RESULTS AND DISCUSSION V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. Summary Conclusions. Recommendations LITERATURE CITED APPENDICES ii é“ é 0»? a; ‘5’. 1/51- ?0 (m Page J: MNl-J \ON 114 15 15 l6 l7 l9 27 27 29 30 32 36 CHAPTER I INTRODUCTION One of the present trends in muscle research is the investigation of intracellular adaptations that may occur with a change in performance. It is known that differ- ential biochemical responses exist for the various fiber types from "red" to "white" and that mixtures of fiber types occur within muscles. A muscle with postural func— tions contains predominantly "red" fibers whereas a muscle with power functions contains predominantly "white" fibers. Evidence suggests these differences exist by nature of the metabolic demands placed upon the individual fibers accord- ing to their function. From this, it was hypothesized that special training programs could alter the ordinary daily activity of a muscle enough to cause histochemical transformations of fiber types. To test this hypothesis, glycolytic and oxidative enzymes were employed to indicate histochemical transformations of fibers. Statement of the Problem The purpose of this study was to determine the histo— chemical effects of a continued endurance activity on selected enzymes of individual muscle fibers in the soleus muscles of male albino rats. Importance of the Problem Histochemists know that histochemical differences exist between fiber types and that histochemical changes occur within fibers following myopathy, cross-innervation, dcnervation, reinnervation, and tenotomy; however, little is known about the histochemical effects of chronic exer- cise. Exercise in and of itself is not an entity. The type of exercise, whether endurance or power, and the time of data collection are variables that need histochemical investigation before an understanding can be gained. Once an understanding has been obtained, the principles might well be applied to medicine and athletic training. This study examines the histochemical effects of one form of each of two variables: a specific program of en— durance training and data collection nine hours after exercise. It is hoped that other investigators will follow a similar approach until all forms of all needed variables have been examined. Limitations of the Study 1. The results of this study can be applied only to male Sprague-Dawley rats of similar age and spontaneous activity. The amount and intensity of exercise cannot be controlled accurately in the swimming rat; there- fore, the duration of the swim served as an activity control. Other training regimens undoubtedly will produce different enzyme characteristics; therefore, these results are part of a total enzyme profile and cannot be extrapolated to other forms of exercise. CHAPTER II RELATED LITERATURE A correlation exists between the usual type of work performed by a muscle and its enzyme content. Breast mus— cle of non-flying chickens is poor in mitochondria and oxidative enzymes (26). Similar results are found when comparing the psoas muscle of a sedentary laboratory rab- bit with that of a wild hare (21). Whether these differ- ences are due to genetic makeup, neural regulation, or work performance, or any combination of the three has not been shown. Biochemical and histochemical studies of normal mammalian skeletal muscle give the enzyme characteristics associated with "red" and "white" fibers. "Red" fibers are rich in oxidative enzymes and low in glycolytic en- zymes and myosin ATPase. "White” fibers are rich in glycolytic enzymes and myosin ATPase and poor in oxidative enzymes (u, 5, 7, 8, 11, 12, 2o, 25, 28, 31, and 35). Histochemical analysis demonstrates the relative activity of various enzymes in the metabolic pathways of individual muscle fibers. Those fibers with considerable oxidative activity would be considered "red." Frequently, too strong a case is made for using histochemistry to type fibers as "red" and "white." It must be cautioned that one or two enzymes do not necessarily identify a fiber as either "red" or "white." Any given fiber may have histo— chemical characteristics of both. The question then might be asked, how does one identify a "white" or a "red” fiber from histochemical stains? Serial sections are needed. A fiber is first identified grossly as "red" or "white" (usually by a hematoxylin and eosin stain) and then its enzyme profiles are determined by histochemical techniques. Histologic patterns seen in cross sections of skele— tal muscle supposedly reflect physiologic characteristics of individual fibers. Slow contracting postural muscles are characterized by fibers utilizing mainly oxidative enzymes as derivatives of energy, whereas fast contracting phasic muscles utilize mainly glycolytic enzymes. Yellin (38) reasoned, if muscle fiber types reflect functional differences of innervating neurons, then transposition of muscle nerves from one type of muscle to another should convert the fibers. Cross uniting the nerves of fast and slow muscle causes fast muscle to become slow and slow muscle to be- come fast as evidenced by histochemical examination (6, 29, 33, and 36). A complete reversal of fiber character- istics does not occur but the change is definite. Denervation causes atrophy of muscle fibers. Evi- dence indicates that the muscle's enzyme systems and metabolic pathways are upset. There is a reduction in glycogen content, lipides, succinic dehydrogenases and cytochrome oxidase activity and an increase in ATPase activity (2, 32). Since these profiles are used to clas— sify a fiber as "red" or "white," it is suggested that nerves determine or at least influence the fiber types and their distribution. Is the mechanism involved one of various degrees of stimulation and impulse conduction, one of conducting a chemical substance down the neuron, or one of causing the release of a substance at the myo— neural Junction? An electrically stimulated denervated muscle will atrophy but slower than an unstimulated muscle (33). This seemingly absolves the actual stimula- tion and conduction of the impulse. At present no studies have been able to prove the existence of a chemical sub- stance that accounts for the change of muscle fiber char— acteristics. Cross—innervation and denervation studies suggest neural regulation of muscle; however, tenotomy and exer- cise studies indicate that function determines fiber characteristics. If the latter is true, then biochemical adaptations in muscle can be selected by specific exer- cises. That is, training could yield laboratory findings as to the qualities necessary for prOper muscle action. In a tenotomy study by Bach (1), conversion of "red" muscle to "white" muscle was accomplished by changing the position of the tendon of insertion of a "red" muscle to that of a "white” muscle. The innervation was not changed, and the nerves were still intact as they do not enter the tendon. In thus assuming the function of a "white" synergistic muscle, the "red" muscle took on properties of "white" muscle as evidenced by: similar types of re- flexively induced contractions, similar myoglobin and iron content, and similar enzyme profiles. In addition to the preceding, the activities of a number of mitochondrial enzymes in the metabolic chain have been shown to increase significantly in response to thyroxine (22), while electron micrOSCOpic work with thyroxine has shown changes in the morphology of mito— chondria in skeletal muscle (17). Most of the work indicating transformation of muscle fibers and enzyme differences has been with normal, diseased, or experimentally altered animals. Many of these studies indicate that changes are possible. What remains to be shown is that adaptive changes can be made to occur through selective chronic exercise programs. Negative Results Unfortunately, the few studies with exercise have been confusing. Some investigators have found enzyme changes while others have not. For example, Rawlinson and Gould (30) swam rats for one half an hour, five days a week, for six weeks. Biochemical analyses for adenosine triphosphatase (ATPase) and malate dehydrogenase activity failed to show any effect of training. In another biochemical study, Hearn and Wianio (18) could not find any change in skeletal muscle succinic dehydrogenase (SDH) activity after five to eight weeks of one half hour of daily swimming. A histological study by Mani, Ito, and Kikuchi (2A) confirms the work of Rawlinson and Gould and Hearn and Wianio. Sudan black B was used to analyze lipid storage in hind leg skeletal muscles of pre-adult and adult age rats. Each trained animal completed a treadmill program of forty mtr/minute, two hundred-fifty mtr/day, six days a week for sixty days. Cross sections of muscle fibers showed no difference in distribution between the trained and the control groups; therefore, it was concluded that a transformation of muscle fibers did not occur as a result of the training program. These negative results do not necessarily indicate that with chronic exercise enzyme changes do not occur. Since the exercise programs were well within the limits of the untrained rat, it might be that a more strenuous program would bring about adaptive changes. This hypothesis seems to be validated in the findings of Holloszy, Gordon, and Edgerton. Positive Results Holloszy (19) ran six-week—old male Wistar strain rats on a progressive treadmill program. The animals were exercised five days a week on an eight-degree incline. They ran initially for ten minutes at twenty-two mtr/ minute, twice daily, four hours apart. At the end of a twelveéweek program, the animals were running continu- ously for one hundred-twenty minutes at thirty-one mtr/ minute with twelve thirty-second sprints at forty-two mtr/minute interspersed at ten-minute intervals. Bio- chemical analysis showed that SDH, reduced diphospho- pyridine nucleotide, cytochrome 0 reductase, succinate oxidase, and cytochrome oxidase activities, expressed per gram of muscle, increased approximately two hundred per cent in hind limb muscle in response to the exercise pro- gram. Gordon and associates confirm training effects on rat hind limb fiber distribution. In their study, three groups of rats were used, two of which were put on a supposed endurance or energy liberating program consist— ing of a one to eight-kilometer daily run or a half-an- hour daily swim for eight weeks. Quadriceps and gastro- cnemius examination showed an increase in sarCOplasmic 10 protein and a decrease in myofibrillar protein (13, 14, and 15). They believe the decrease in myofibrillar pro— tein might be attributed to a relative undernutrition. A third group of rats placed on a strength or myofibrillar building program carried one hundred—gram weights up a sixteen-inch pole fifty times daily. There was an in- crease in myofibrillar protein and a decrease in sarco— plasmic protein (13, 1A, and 16). The decrease was again attributed to a relative undernutrition. In Edgerton's (9) study, histochemical analysis of the soleus indicated one or two half an hour daily swim- ming periods for fifty—two days did not alter the propor— tion of intermediate or dark fibers. However, examination of the plantaris muscles of the same animals showed an increase in the proportion of dark fibers as indicated by malate dehydrogenase, mito alpha-GPD, SDH, and a trichrome stain. Examination of the two muscles shows natural differ- ences. The soleus contains only intermediate to "red" fibers and is highly adapted to repetitive exercise. The plantaris contains a continum of "white" to "red" fibers and is not highly adapted to repetitive exercise. What appears to have been stimulus enough for biochemical adaptation to repetitive activities in the plantaris was not sufficient to elicit further adaptation in the soleus. Edgerton feels that since the soleus is naturally adapted 11 to repetitive exercise and does not contain "white" fibers, little change would be expected from such a small work load. A more strenuous program might have revealed adaptations. This is the appraoch taken in this investi— gation. CHAPTER III EXPERIMENTAL DESIGN This study was carried on in connection with other studies of lactic acid determinations, measurements of pyridine nucleotide ratios, and static and dynamic strength measurements. Since activity may influence pyridine nucleotide levels, it was decided to delete all animals having innately high or low levels of voluntary activity from the study. To do this, one hundred-five Sprague— Dawley strain, twenty-three—day old male rats were put into individual voluntary cages twenty—four centimeters long, eighteen centimeters high, and eighteen centimeters wide with an attached exercise wheel thirteen centimeters wide and thirty—five centimeters in diameter. An auto— matic counter recorded daily revolutions run for five days. Data from the third, fourth, and fifth days were made into frequency distributions, with the middle half being designated for each of the three days. Of the forty-eight animals used in the study, thirty fell within the middle half on all three days. The other eighteen animals were chosen from those in the middle half two of the three days and not more than one hundred twenty 12 13 revolutions off the mean on the third day (see Appendix A for the daily revolutions run by each of the forty-eight animals). These animals then were ranked according to their three-day total revolution count. The most active animal was assigned to group A, the second and third rank- ing animals to group B, the fourth and fifth to group A, the sixth and seventh to group B, etc. A flip of a coin designated group A as experimental and group B as seden- tary. All forty—eight eight animals then were housed in a single rack of sedentary cages, twenty-four to a side. The animals were placed on each side of the rack and in each of the four rows so that animals from groups A and B were alternated. Group A animals were put into standard sedentary cages twenty—four by eighteen by eighteen centi— meters. The cages of the animals in group B were specially adapted to restrict their activity even further. A sheet metal plate disected these cages from the upper left to the lower right side. Environmental conditions in the animal room were .held constant throughout the experimental period. Room temperature was maintained at twenty—five degrees centi- grade. Lighting consisted of twelve hours of light alternated with twelve hours of darkness. I“ No attempt was made to control food or water in- take. All animals were fed ad libitum with Wayne Labora- tory Blocks and had constant access to water. Body weights were recorded every Thursday at the same time throughout the experimental period. Group A's weights were then used to determine the swimming load of each animal until the next Thursday's weigh in (see Appendix B). Forced Exercise Regimen Each animal swam individually in a cyclinder twenty— five centimeters in diameter and 1.2 meters deep in water at thirty- to thirty-two degrees centigrade. The exercise program consisted of a 3:00 to 5:00 P.M. and 9:00 to 11:00 P.M. swim five days a week for six weeks. No weights were attached to the animal's tails during the first week of swimming although on the fourth and fifth days tiny Clothespins were attaChed. For the second week of swim— ming, two per cent of each animal's weight was attached to its tail. During weeks three, four, five, and six, three per cent of each animal's body weight was attached to its tail. All swimming loads were accurate to plus or minus ten milligrams. With the completion of each swim, the animals were dried with a towel, their weights were removed, and they were returned to their cages. 15 Sacrifice Schedule Eighteen animals were randomly chosen from each group for sacrifice. On day one of the sixth week, the first three animals from each group were sacrificed. On day two, the next three animals from each group were sacrificed. This procedure was continued through the sixth day. All animals were kept on their normal after— noon schedule until sacrifice. Sacrifice Procedure Each animal was lightly anesthetized in an anes- thetic chamber with ether. A nose cone maintained this state while (a) blood samples were taken for lactic acid determination, (b) the right soleus muscle was removed for histochemical analysis, and (c) static and dynamic contractions of the left gastrocnemius-plantaris muscle group were recorded. At the end of the ten-minute con— traction period, the gastrocnemius and plantaris muscles were quick-frozen between two liquid nitrogen cooled aluminum plates. Heart and liver samples were also re- moved and quick—frozen for biochemical assay of pyridine nucleotide levels. To prepare the soleus for histochemical analysis, a section one centimeter thick was removed from the belly of the muscle and placed on a freezing block in five per cent gum tragacanth. The tissue was then immersed for 16 approximately ten seconds in isopentane previously cooled until viscous by liquid nitrogen. Tissue sections ten-microns thick were cut at minus eighteen to a minus twenty degrees centigrade by an Ames Lab-Teck cryostat. Each section was placed on a cover slip and briefly fan dried before incubation. Histochemical Rationale and Procedures Histochemistry of fresh frozen sections involves interruption of the metabolic pathway at a particular site or place of chemical reaction. As a result of the reac— tion, electrons and/or hydrogen are freed. These react with a substance in the incubating medium to form a precipitate. The more electrons and hydrogen freed, the more precipitate formed. The more precipitate formed the darker the stain. Relative intensitives of the stains give an indication of the metabolic activities of a given fiber. Padykula and Herman's (27) technique was used to investigate myosin ATPase activity. Histochemistry of the ATPase reaction is not adequately understood. Earlier investigators employed it to help differentiate muscle fibers. Since ATPase activity has been used commonly, it was incorporated in this study as a means of compari— SOD. l7 Barka and Anderson's (3) technique using NBT [2,2'-di—p-nitrophenyl-5, 5'-diphenyl 3,3'-(3,3'- dimethoxy-A, A'—diphenylene) ditetrazolium chloride] as an electron acceptor was employed to study SDH activity. SDH is a Kreb's cycle enzyme that acts as a carrier system for hydrogen removed in the aerobic oxida- tion of carbohydrates; therefore, it can be used as an indicator of oxidative metabolism. Wattenberg and Leong's (37) method was employed to study "menadione linked" mito alpha-CPD activity. Mito alpha-CPD takes part with phospho-glyceraldehyde and DPN in the metabolism of triose phosphate. Histochemically, its activity can be regarded as an indication of glycerol fermentation. As part of the overall picture, it is con- sidered an indicator of glycolysis (see Appendix C). All histochemical procedures were incubated for thirty minutes at thirty-seven degrees centigrade. A glycerin Jelly mount was used with the SDH and mito alpha-GPD procedures. Permount was used with myosin ATPase. Freezing artifacts prevented the analysis of tis- sues from one of the control animals. Method of Analysis All slides with the same histochemical stain were put into a pile. Each slide was randomly picked and 18 analyzed without knowing the treatment group of the animal until the pile was depleted. An image of the central portion of each slide was projected on a piece of paper. A subjective evaluation of each fiber was made. If the fiber appeared dark, a number "1" was written over its image; if a fiber appeared light, a number "2" was written over its image. All marked fibers were counted and a percentage of dark fibers was calcu— lated. The resulting percentages were then used in a t-test to determine whether or not a significant differ— ence between mean percentages existed. The probability of making a Type I statistical error was set at .10. CHAPTER IV RESULTS AND DISCUSSION Stain intensity was used to subjectively evaluate each slide. Numerous cells, generally four to seven hundred, were rated as either light or dark. A ratio of light to dark determined the per cent of each fiber type. The animals were grouped as controls or exercised. Seventeen control animals and eighteen exercised animals served as subjects. Three separate t—tests were used to analyze the data. In each case, the t value was signifi- cant at the 0.10 level-~for myosin ATPase, t = 4.65; for mito alpha-GPD, t = 5.53; and for SDH, t = 3.39. (The t-values proved to be significant at the 0.005 level.) The mean percentages are given in Table I. See Appendix D for the raw data. Examination of the data reveals information con— trary to that previously reported. No other investigators have shown a corresponding increase in both oxidative and glycolytic indicating enzymes with exercise. The explana- tion has been that a relative undernutrition has led to an increase of one at the expense of the other. The results of this study suggest that a striated muscle fiber might l9 20 TABLE l.--The mean percentages and deviations for each treatment. Enzymes Treatment Group ATPase SDH -GPD i i s i i s i i 3 Control 2A.98 i 3.58 27.97 i 3.58 27.11 i 3.20 Experimental 31.18 i 4.05 31.13 i 3.56 32.88 i 2.79 have sufficient glycolytic capacity to handle a given task anaerobically but not sufficient odative capacity to handle the task aerobically. Proper training could then relieve the system of its anaerobic load and allow for a more efficient aerobic handling without increasing gly- colytic metabolism. The opposite situation would also exist. A muscle fiber might have sufficient oxidative capacity to handle a given task aerobically but not suffi- cient glycolytic capacity to handle the task anaerobically. Proper training could relieve the system of its aerobic load and allow for a more efficient anaerobic handling without increasing oxidative metabolism. Four hours of swimming could exhaust the stored glycolytic capacity such that any additional increase in oxidative capacity would necessitate a different energy source. The most logical source would be the glycolytic pathway. This would mean that the additional increase in 21 oxidative capacity would be dependent upon a correspond- ing increase in glycolytic metabolism; thus, an increase of both oxidative and glycolytic indicating enzymes. As an interesting note, the eyeball technique indi- cates that the cells of the endurance-trained rat might be smaller than those of the sedentary rat. The following photographs are enclosed to help the reader understand the technique of analysis. 22 xosm coapmoaeaemmz msmHom pmm amoumnaam Qua: HmEHcm HORQCOQ xozm :OHSROHLstmz mSCHOm pom mam Hmeficm Hoppcoo Xena coapmofigficmmz msmHom pom ommme< cfimozz Hmeficm Homecoo Noam soapBOHmacmmz Xena coameHMflsmmz xoqm soamefihfismmz msmaom umm msmaom pmm mzofiom pmm mmolwnofim ova: mow mmmmeq camomz Hmeflcm ommfiosmxm Hmefism oomflosoxm Hmeficw ommfiosoxm ad 2A xo:m_cofipm0Hochmz msoaom pmm mmmlmsaam ova: Hmeficw Hoppcoo xoam cofipmoaeacwmz mamaom pmm mam Hmefism Hoppcoo Xena goameflMchmz mamaom pmm mmmmea memosz Hwafism Homecoo 25 xozm cofipmoaoasmmz mamaom umm omuumgaam Qua: Hmeflcm pomfiohoxm xosm :OAPROALchmz mzoaom pom mam Hmeficm comfiohmxm xosm cowpmoacfismmz mamHOm pmm mmmme< Camozz Hmafizm Ummfiohmxm 26 xozm soameHLacwmz mamaom umm omelwzaaw ova: Hmeficm Homecoo xozm soapQOHmfiswmz mamaom pmm mam HmEHcm HoppCOQ xosm coapmoaeacwmz msoaom pmm owmme¢ :fimomz Hmeficm Hospcoo CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of this study was to determine the histochemical effects of chronic exercise on male rat soleus muscle nine hours after exercise. Forty—eight Sprague-Dawley strain twenty-three—day— old male rats were randomly assigned to treatments of sedentary housing or sedentary housing with two two-hour forced swim sessions per day, five days a week, for six weeks, with three per cent body weight attached to their tails. Each control animal's movement was further re- stricted by a piece of sheet metal diagonally bisecting its cage. Normal twenty-four by eighteen by eighteen centimeter sedentary cages were used to house the swim- ming animals. Body weights were recorded once a week. Each animal's load was then determined for the coming week. All animals were sacrificed after a six-week train- ing period. Six randomly selected rats per day, three from each group, were lightly anesthetized. This was done to allow for: (a) pre-exercise and post-exercise 27 28 right femoral vein blood samples to be used in blood lactate determinations; (b) static and dynamic work re- cordings of the plantaris-gastrocnemius muscle group; and (c) pyridine nucleotide extraction after ten minutes of exercise in heart and liver tissue. To prepare the soleus for histochemical analysis, a section one-centimeter thick was removed from the belly of the muscle and placed on a freezing block. The tissue was then quick frozen for approximately ten seconds in is0pentane previously cooled with liquid nitrogen. Tissue sections ten microns thick were cut at minus eighteen to minus twenty degrees centigrade. Each section was placed on a cover slip to be fan dried before incuba— tion with myosin ATPase, SDH, and mito alpha-CPD. All tissues Were incubated for thirty minutes at thirty-seven degrees centigrade. Glycerin jelly was used to mount SDH and mito alpha-CPD slides. Permount was used with myosin ATPase. A central portion of each slide was projected on a piece of paper. Numerous fibers were subjectively eval— uated. If the fiber appeared dark, a number ”1” was written over its image; if a fiber appeared light, a number "2" was written over its image. All marked fibers were counted and a percentage of light to dark calculated. The resulting percentages were used in a t-test to show that a significant difference between means existed. 29 Conclusions Chronic physical stress can account for changes in enzyme activity; therefore, physical stress might well be used as a tool in researching the biosynthesis of various enzymes. Other implications for enzyme changes involve medicine, therapeutics, and athletic training and condi— tioning. The following conclusions were reached in accompany- ing studies: 1. The exercised animals had lower body weights upon completion of the six-week training pro— gram (10). The resting blood lactate concentrations were similar in trained and untrained animals (34). After ten minutes of direct muscle stimulation, the trained animals had higher blood lactate concentrations (3A). No difference in mean work performance between the two groups was exhibited after the ten- minute stimulation period. Since the trained animals were lighter, they did more work per unit of body weight (23). Trained animal muscle had a higher oxidized to reduced ratio of pyridine nucleotides (10). The exercised animals had more triphospho- pyridine nucleotide; therefore, their 3O diphosphopyridine nucleotide to triphospho- pyridine nucleotide ratio was lower (10). After the ten-minute exercise stress, a de— crease in muscle pyridine nucleotides was accompanied by a decrease in liver pyridine nucleotides (10). Recommendations In order to establish and understand the effects of acute and chronic physical stress, further studies should include: 1. Provisions to determine how rapidly enzyme stores are depleted and replenished. This could be done by examining enzyme activities before, during, and at intervals after exercise. Provisions to determine the effects of different exercise stresses such as running, lifting, jump- ing, etc. Provisions to determine the specific activities of each enzyme and what they mean in terms of an increase or a decrease in enzyme level. Provisions to determine the relationship of cell size in the normal and the physically stressed animal. Examination of the photographs on pages 22-26 indicate that the swimming animals might have smaller soleus muscle cells. LITERATURE C ITED 31 10. LITERATURE CITED Bach, L. M. N. Conversion of red muscle to pale muscle. Society for Experimental Biology and Medicine. 67:268, 1938. Bajusy, Eors. Red skeletal muscle fibers: Relative independence of neurol control. Science. 1A5(3635):938, August 28, 1968. Barka, T., and Anderson, P. J. Histochemistry Theory, Practice, and Biblioggaphy. New York: Harper and Row, 1963. Beatty, C. H.; Peterson, R. D.; and Bocek, R. M. Metabolism of red and white muscle fiber groups. Am. J. Physiol. 20A(5):939, 1963. Beckett, E. B. Some applications of histochemistry to the study of skeletal muscle. Rev. canad. Biol. 21(3 and A):39l, 1962. Close, R. Effects of cross—union of motor nerves to fast and slow skeletal muscles of the rat. Nature. 206(A986):831, May 22, 1965. Dawson, David M., and Romanul, F. C. A. Enzymes in muscle: II. Histochemical and quantitative studies. Arch. of Neurol. 11:369, 196A. Dubowitz, V., and Pearse, A. G. Comparative histo- chemical study of oxidative enzymes and phosphorylase activity in skeletal muscle. Histochem. 2:105, 1960. Edgerton, R. V. "Histochemical Changes in Rat Skeletal Muscle after Exercise." Unpublished Ph.D. diSsertation, Michigan State University, 1968. . Edington, D. W. "Pyridine Nucleotide Concentra— tions and Ratios in Rat Muscle, Heart, and Liver in Response to Acute Chronic Exercise.” Unpublished Ph.D. Dissertation, Michigan State University, 1968. 32 ll. l2. 13. 14. 15. l6. 17. 18. 19. 20. 21. 33 Gauthier, G. F., and Padykula, H. A. Cytological studies of fiber types in skeletal muscle. J. of Cell Biol. 28(2):333, 1966. Goldstein, D. J. Some histochemical observations of human straited muscle. Anat. Rec. 134(2):217, 1959. Gordon, E. E. Anatomical and biochemical adapta- tions of muscle to various exercises--studies in rats. JAMA. 199:103, Jan. 19, 1967. Gordon, E. E.; Kowalski, K.; and Fritts, M. Adap- tations of muscle to various exercises—- stgdies in rats. JAMA. 199:103, Jan. l9, l9 7. Changes in rat muscle fiber with forceful exercises. Arch. of Physical Medicine and Rehabilitation. Vol. A8, Nov., 1967. Protein changes in quadriceps muscle of rat with repetitive exercises. Arch. of Physical Medicine and Rehabilitation. Vol. A8, June, 1966? Gustafsson, R.; Tata, J. R.; Lindberg, D.; and Ernster, L. The relationship between the structure and activity of rat skeletal muscle mitochondria after thyroidectomy and thyroid hormone treatment. J. of Cell Biol. 26(2): 555, 1965. Hearn, G. R., and Wainio, W. W. Succinic dehydro- genase activity of the heart and skeletal muscle of exercised rats. Am. J. Physiol. 185:3u8, 1956. Holloszy, John 0. Biochemical adaptations in muscle - effects of exercise on mitochondrial oxygen uptake and recovery enzyme activity in skeletal muscle. J. Biol. Chem. 2A2(9): 2278, May 10, 1967. Lawrie, R. A. Biochemical differences between red and white muscles. Nature. 170:122, 1952. Effects of enforced exercise on myoglobin concentration in muscle. Nature. 17(A36A): 1069, 1953. 22. 23. 2A. 25. 26. 27. 28. 29. 30. 31. 34 Lee, Y. P., and Lardy, H. A. Influence of thyroid hormones on L-alpha-glycerophosphate dehydro— genases and other drhydrogenases in various organs of the rat. J. Biol. Chem. 2A0(3): 1A27, 1965. Lund, D. D. "Effects of Physical Training on Static Strength and Dynamic Work of the Gastrocnemius—plantaris Muscle Group in Male Albino Rats." Unpublished Master's thesis, Michigan State University, 1968. Mani, M.; Ito, K.; and Kikuchi, K. Histological studies of muscle training: Report I. Effect of training upon skeletal muscle fibers. Res. J. Phys. Ed. 11(3):153. Nishiyama, Akira. Histochemical studies on red, white, and intermediate muscle fibers of sksletal muscles: I. SDH activity and physiological function of intercostal muscle fibers. Acta Med. Okayama. 19:191, 1967. Paul, M. H., and Sperling, E. Correlation of cyclophorase activity and mitochondrial density in straited muscle. Proc. Soc. Exp. Biol. Med. 70(3):352, 1952. Padykula, H. A., and Herman, E. Histochemistry Theory,gPractice, and Bibliography. New York: Harper and Row, 1963. Pearse, A. G. E. Histochemistry Theoretical and Applied. 2d ed. Boston: Little, Brown and Company, Reprinted 1961. Prewitt, M. A., and Salafsky, B. Effect of cross innervation on biological characteristics of sksletal muscle muscles. Am. J. Physiol. 21A:295, 1967. Rawlinson, W. A., and Gould, M. K. Biological adaptation as a response to exercise: 1. ATP and creatin phosphokinase activity in muscles of exercised rats. Biochem. J. 73(1):AA, 1959. Romanul, F. C. A. Enzyme in muscle: I. Histo- chemical studies in individual muscle fibers. Arch. of Neurol. 11:355, 196A. 32. 33. 3“. LA‘ U: 36. 37. 38. 35 Romanul, F. C. A., and Hogan, E. L. Enzymatic changes in denervated muscle. Arch. of Neurol. 13:263, 1965. Romanul, F. C. A., and Van Der Meulen, J. P. Reversal of the enzyme profiles of muscle fibers in fast and slow muscle by cross innervation. Nature. 212(5086):1369, 1966. Sarenac, B. " H Unpublished Master's thesis, Michigan State University, 1968. Stein, J. M., and Padykula, H. A. Histochemical classification of individual skeletal muscle fibers of rat. Amer. J. Anat. 110:103, 1962. Thimann, V., and Padykula, H. A. Some histo- chemical properties of normal and denervated red and white muscle fibers in the rat. Anat. Rep. l2:A19, 1955. Wattenberg, L. W., and Leong, J. L. Histochemistry Theory, Practice, and Bibliography. New York: Harper and Row, 1963. Yellin, H. Neural regulation of enzymes in muscle fibers of red and white muscle. Exp. Neurology. 19:92, Sept, 1967. APPENDICES 36 37 APPENDIX A.—-Revolutions run by animals during pre experimental period. Animal Number First Day Second Day Third Day Total 1 1059 903 1039 2996 2 906 991 1085 2982 3 1151 1026 783 2960 9 1299 797 996 2937 5 818 871 1232 2921 6 520 997 1393 2910 7 788 902 1032 2722 8 598 691 1952 2691 9 606 986 1096 2688 10 1188 539 959 2686 11 1380 572 727 2679 12 1267 59C 781 2696 13 957 909 793 2609 19 933 920 795 2598 15 835 599 1210 2599 16 638 790 1105 2983 17 625 659 1181 2960 18 916 936 603 2955 19 831 893 761 2935 20 959 825 611 2390 21 839 707 789 2330 22 910 739 660 2309 23 795 779 709 2278 2“ . 771 898 2275 25 823 552 395 2270 26 763 626 878 2267 27 1303 505 996 2259 28 801 876 595 2222 2 781 591 779 2151 30 757 590 753 2050 31 851 779 922 2097 32 637 876 972 2035 33 962 659 893 2019 39 996 709 767 1972 35 992 582 896 1870 36 906 606 82 1839 37 610 599 656 1815 38 695 565 595 1755 39 995 539 595 1629 90 551 585 966 1602 91 816 322 960 1598 92 590 511 525 1576 93 695 533 396 1529 99 337 511 620 1968 95 393 989 633 1965 96 528 971 931 1930 97 909 925 510 1399 98 598 355 922 1329 38 sow mom mmm HNH mmfi :ma x mam oam msm mam mmfi me .M 2mm Nmm mam oma oma mma m: mmm sow com mod Hwa mmH s: smm mam mom mzfi oma mHH m: 3mm mom 22m msa moa mHH m: 0mm smm mmm Fwd oza mHH a: mmm mmm 2mm New HNH HMH mm mmm mmm mom HmH mma NHH mm Hmm cam new mom 05H mma mm mmm mam cam mofi mzfi omH mm ozm mmm mom mom mod sma mm 2mm 0mm wmm wsa boa mma mm mmm mam mam omm me mmfi Hm mmm mmm cam omfi H©H mad mm mmm mmm 0mm mmm mwa mma om wow mam wsm mad mma Hma mm :om 0mm mmm cam mma mma um :om 2mm wmm swa :ma 53H :m msm mam wwm mm Ham oma om :om mum New And oma omH om mmm mam mam msm :mfi ::H mm mzm mmm mom smfi mza mma NH mzm mam mum smm :ma oza mm :om :mm mmm omfi mmfi mmfi ma mmm mom mom msa sma :mH 0H mom mum :mm 25H msa oma ma mzm mam owm Hmm mma :ma ma mom mam :Hm omfi sma mmfi ma mmm :Hm msm mmm mmfi Hzfi ma mmm mmm 0mm mma HNH omH m mmm mom owm mmm mmfi mzfi 2H mam wsm 0mm mwa mwa omfi w mmm :om mmm mmm mmfi omfi Ha 2mm mom mmm sad omH wma m mmm mmm :mm mmm sow omH o mam mzm oom owfi mma moa H mmm mmm mam mom qua oma m w m z m m H popesz o m z m m H Lonesz xm03 xmmS xmmZ xmmB x003 xmm3 HQEHC< xmmB xmmz xmmB xm®3 xmmZ Xm®3 HmEflqu Hmucmefimooxm mHoppcoo .xooz comm Lou Hmeficm comm mo mEmmm :H pcMfimz zoomll.m xHszmm¢ APPENDIX C HISTOCHEMICAL PROCEDURES USED IN THE STUDY MYOSIN ATPase 0.10 M Na barbital 0.18 M CaC12 Distilled water ATP Adjust pH to 9.9 with 0.10 N NaOH Incubate thirty minutes at 37° C Three one minute changes of 1% CaCl2 Distilled water wash 2% cobaltous chloride for three minutes Three one minute changes of distilled water Dehydrate (Ethol of 95% - 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