FORCED EXERCISE AND CARDIAC PATHOLOGY IN RATS Thesis for the Degree of M. A. MICHIGAN STATE UNIVERSITY JAMES L. WILSON 1968 1i LIB RA R Y '1'“ ESIS ! MiChigan State University ABSTRACT FORCED EXERCISE AND CARDIAC PATHOLOGY IN RATS by James L. Wilson This study was designed to determine the effects of an intense exercise program on cardiac muscle in adult rats and to determine if pre-pubertal forced exercise had a pro— tective effect on the heart when the animals were subjected to forced exercise later in life. One hundred and fifty male albino rats were brought into the laboratory as weanlings and divided randomly into three equal groups. These groups were randomly assigned to the forced, voluntary, and sedentary exercise groups. At thirty days of age, the pre-pubertal training period began. For thirty-five days, the forced group animals were exercised for one-half hour per day by swimming with 2% of their body weight fastened to their tails. The voluntary group could exercise at will in running wheels attached to their cages. The sedentary group remained relatively immobile within standard cages. Following the initial training period all animals were placed in voluntary cages for one hundred and eighty-two days. A second training period of thirty-five days duration followed the voluntary stage and this James L. Wilson post-pubertal or adult training program involved all but ten control animals from each group. In the second training period the animals were forced to swim one—half hour per day with 1-2% of their body weight tied to their tails. The control animals for the groups were returned to their original experimental condition with the exception of the controls in the forced group which were placed in seden- tary conditions. The design described above was duplicated at a later date on forty-two female albino rats. The heart tissues of all animals were studied histologically. Myocarditis was induced in 29.57% of the male ani- mals forced to exercise later in life. The pathological changes were independent of the pre-pubertal condition. Cardiac lesions were found in 10% of the male control ani- mals. This pathology ranged from a very mild perivascular lymphocytic infiltration to a severe myocardial necrosis and-polymorphonuclear leucocytic infiltration. No patho- logical changes were found in the female animals. On the basis of the evidence presented,pre-pubertal forced exercise does not appear to play a role in the prevention of myocardial damage later in life. The det- rimental effects of heavy exercise later in life in the male animals which was unexpectedly independent of the pre-pubertal experimental conditions warrants further investigation. FORCED EXERCISE AND CARDIAC PATHOLOGY IN RATS By [\Ij James L? Wilson A THESIS Submitted to Michigan State University in partial fulfillment of requirements for degree of MASTER OF ARTS Department of Health, Physical Education and Recreation College of Education 1968 PREFACE In the preparation of this thesis, many people devoted much time and effort.1 I would especially like to thank Drs. R. E. Carrow, w. D. Van Huss, and W. w. Heusner of the Michigan State University faculty. I would also like to thank the staff of the Human Energy Research Laboratory at Michigan State University for their assistance in this study. In addition to the above people, I would like to thank the National Institute of Health, for without its financial assistance this thesis would not have been possible. ii TABLE OF CONTENTS PREFACE. LIST OF TABLES LIST OF FIGURES Chapter I. INTRODUCTION II. METHODS. III. RESULTS. IV. SUMMARY AND CONCLUSIONS BIBLIOGRAPHY APPENDIX iii Page ii iv 10 13 Ill LIST OF TABLES Table Page 1. Pathology by Groups. . . . . . . . . 8 2. Location and Severity of Lesions . . . . 8 iv LIST OF FIGURES Figure Page 1. Experimental Method. . . . . . . . . 5 2. Heart Sectioning Method . . . . . . . 5 CHAPTER I INTRODUCTION It has been the contention of many research workers and educators that physical activity has beneficial effects on circulo—respiratory efficiency. In an earlier study (10) involving exercise of adult male rats (227 days), gross inspection of the hearts aroused speculation that pathology was present. The present study was designed to investigate these findings in more detail using appropriate histological techniques. Cardiac lesions have been studied in several labora- tory animals (1-6). However, none of these reports have dealt with physical exercise, and more specifically, none have attempted to Show what the early effects of exercise might have later in life. Therefore, the current study was undertaken to determine if there is any protective value of early exercise on cardiac pathology in later life, and to determine the effects of an intensive exercise pro- gram later in life. Specifically, the necrotic and fi— brotic foci, indicative of cell degeneration and scar tis- sue formation, were to be searched for. The subject of this study is currently of great pub- lic interest. With the promotion of this interest through the President's Council on Physical Fitness, the YMCA, and numerous local agencies, the effect of fitness programs on their middle-aged participants is extremely important. Generalizations from this animal study to human physiological phenomena would be difficult and are not to be implied. However, research in this area can assist in the elimination of many perplexing problems confronting the field of exer- cise physiolOgy and can provide clues for further research on man . CHAPTER II METHODS One hundred and fifty male albino rats were received in the laboratory as weanlings (25 days) and placed in indi- vidual cages (5" x 5" x 12"). No animals were removed from the study on the basis of pre-experimental activity, as had been done in the initial pre-puberty study (10). At thirty days of age, the rats were randomly assigned to three equal groups. Group A was a sedentary group and remained relatively inactive within their cages. Group B, the voluntary group, had running wheels attached to the sides of their cages, and could exercise under their own volition. Group C was the forced group. They were kept in cages of the type used for Group B, but were forced to exercise for one-half hour per day by swimming in individual tanks with 2% of their body weight tied to their tails. The rats had free access to commercial rat food (Wayne Lab Blocks) and water at all times. From thirty to Sixty-five days, the pre—pubertal stage in albino rats, the groups remained as stated above. Following the 65th day, the 182 day adolescent period was marked by the three groups being placed in voluntary- exercise-type cages. The second training program which included all animals except ten animals from each group followed. These ten animals were used as controls and returned to conditions characteristic of their pre-pubertal training period, with the exception of those from the forced group. These animals were placed under sedentary conditions. The remaining rats in each group were forced to exercise by swimming in individual tanks for one-half hour per day with l-2% of their body weight tied to their tails. (Figure 1) All animals, having expired during the training period, or sacrificed at its completion, were subjected to the experimental method shown in Figure l. The intact body was weighed. The hearts were excised immediately by severing the blood vessels at their junction with the heart. The heart was weighed in whole, dissected in trans- versely sectioned quarters (Figure 2), and fixed in 10% formalin. The tissues were dehydrated in graded concen- trations of alcohol, embedded in paraffin, and sectioned at 7 microns. Sections from each quarter were stained with hematoxylin-eosin and Mallory's Trichrome stain (for connective tissue) and examined under the microscope. At this time, another pre-pubertal study was con- ducted in our laboratory using female albino rats. The same experimental design was used, except that the xmm< :ofluomm m coapoom n cofipomm o oonpmz wcazofipoom pnmmmll.m mhswam Am>mv mpmp OH mpmm om Am>mv mass on . mass mm A>v mass me msms mm Amv smohom A>>>v mama OH mpsm om Am>>v mpmh o: I when mm A>V mass me mzms mm A>v mhmeSHo> Am>mv mama OH mpmm om Am>mv must on u mmmo mm A>V mmms me msmu mm Amv mhmpcmoom HH .Um wcficamhe mphmpsm H .Um wcficfimme upmom mpnmnsmIOLm cospoz HapcmEHmexmnl.H mpzwflm post—pubertal voluntary exercise period was 160 days rather than 182 days. Forty-two animals were used and all were subjected to the same histological examination as the males. CHAPTER III RESULTS Myocardial lesions were observed in 3A of the 115 rats that were exposed to the second training period. This involved 12 forced, lO voluntary, and 12 sedentary animals (Tables 1 and 2). The lesions ranged from a slight perivascular lymphocytic infiltration to advanced myocardial necrosis and polymorphonuclear leucocytic infiltration (Plates 1 and 2). In certain small foci, the muscle was entirely replaced by mononuclear and poly- morphonuclear leucocytes and plasma cells. The lesions were classified by groups (Table l). The lesions were also classified as being either mild or severe and are tabulated in Table 2 by location and degree of involvement. Using a Chi Square statistical analysis, its was calculated that there was no significant difference in the frequency or severity of necrosis between the three experimental groups. The animals of all three groups were grouped together as experimental animals in the second exercise period since no difference between groups was found. In these animals 29.6% exhibited cardiac necrosis. This .OHOHmpco> pmmH map CH mums mGOHmmH Hmnuo HHH .mHOHHu:o> uanH CH mconoH :sz mHmEHc¢** .mmHOprcmsdpmmH Ocm OanL EH OcOHmmH szz mHOeHcH* O O H H O O O O O O. aHH *N O O O O O o o o o o o o *H o o o o HHco o O O H O O O H O O O N O o H O o o o *: o H m H o o H o cho D o *H H a H o o *m o o H H n w m o o o o o o H H o o o a cho m .68 .>mm .08 .>mm .UE .>om .oe .>mm .UE .>mm .UE .>mm :oHpoom m>m m>m >>> O>> m>m m>m spasm mconmH,mo zuHHm>mm cum coHmeoHII.m mHnt H NH O OH O NH m:OHmmH OOH: amassz OH Om OH Om OH Om OOOSO cH Honesz m>m m>m >>> m>> m>m m>m masomo an awOHospmmll.H OHQOB group was then compared with the controls which were found to have cardiac necrosis in 10% of the animals. The groups when compared using the Mann-Whitney U-Test were found to be different at the .06 level of probability. Since no hypothesis of this nature had been drawn up and no alpha error set, no conclusions are warranted. However, the results are highly suggestive of cardiac damage as a result of intensive exercise later in life and certainly warrant further investigation. CHAPTER IV SUMMARY AND CONCLUSIONS The experimental production of cardiac necroses has been observed by many investigators (5, 6, 9). Foci of destruction, usually of microscopic size, appear regularly in the ventricular myocardium of corticoid-sensitized rats after application of a variety of physical stresses (restraint, exposure to cold and heat, forced exercise, surgical trauma, etc.) (6). The forced exercise program in the adult rats in the current study was considered to be an extremely stressful physical experience. Therefore, cardiac lesions were expected in the experimental animals. The pathology observed in the present study was very similar in structure to that induced in the aforemen- tioned research. The current investigation was designed to study the protective value of pre-pubertal forced exercise against cardiac necrosis induced as the result of a post- pubertal forced exercise regimen. Within the forced vol- untary and sedentary exercise groups, no significant dif- ferences were observed in either the total incidence or severity of the process of necrosis. The conclusion is 10 ll warranted that pre—pubertal forced exercise did not have a protective value on the heart for post-pubertal forced activity. The animals remaining sedentary in later life showed only 10% necrosis compared to a 29.57% involvement in exercised rats. The pathology tended to be more localized and more severe in the forced group animals as opposed to the voluntary and sedentary groups. This revelation is contrary to the above hypothesis. The great majority of animals suffered lesions in the apical region of the heart, specifically in the left ventricle. Of the A2 female animals examined, none Showed any heart pathology. Perhaps this could be related to the continued voluntary exercise period during the post- pubertal voluntary exercise period. In albino rats, the female is approximately nine times as active in voluntary activity as the male (ll). If exercise does have a protective value, it might be based on the amount of regular physical activity. Recommendations for future research in this area would be to repeat this particular study with more rigid controls and more varied staining techniques. During the voluntary exercise period, these animals only had the basic drive to exercise, then in adulthood, they were confronted with a severe exercise regimen. Addi- tional sedentary and forced exercise groups with these 12 conditions carried through the voluntary exercise period used in the current study would help clarify the situation. 10. ll. BIBLIOGRAPHY Bronson, L. H. Anatomical and chemical changes in the myocardium following short-term coronary occlusion in dogs. Yale J. Biol. and Med. 10:405, 1937- Bryant, R. E., Thomas, W. A., and O'Neal, R. M. An electron—micrOSCOpic study of myocardial ischemia in the rat. Circ. Res. 6:699, 1958. Lenke, S. E., and Loewe, L. Cardiac lesions resembling Aschoff bodies in mice. Am. J. Path. 17:857, 1941. Maruffo, C. A., and Malinow, M. R. Pathologic changes of the heart in free-ranging Howler monkeys. Am. J. Vet. Res. 28:237, 1967. Miller, C. P., Jr. Spontaneous interstitial myo- carditis in rabbits. J. Exp. Med. 40:543, 1924. Raab, W.; Chaplin, J. P., and Bafusa, E. Myocardial necrosis produced in domesticated rats and in wild rats by sensory and emotional stress. Proc. Exp. Biol. and Med. 116:665, 1964. Schmidt, E. C. H. Virus myocarditis. Pathologic and experimental studies. Am. J. Path. 24:97, 1948. Siegel, S. Non-Parametric Statistics. McGraw-Hill, New York. 116, 1956. Selye, H. The Pluricausal Cardiopathies. Spring- field, Ill., 1961, Charles C. Thomas. Van Huss, w. D.; Heusner, W. W.; Weber, J.; Lamb, D.; and Carrow, R. The effects of pre—pubertal forced exercise upon post-puberty physical activity, food consumption, and selected physiological and ana- tomical parameters. First Int. Cong. of Psych. of Sport, Roma, 1965. Greenisen, M. "Environmental Effects upon Voluntary Activity in Rats." Proceedings, Research Section, AAHPER, St. Louis, Mo., 1968. 13 APPENDIX A BASIC DATA-—EXPERIMENTAL ANIMALS — L J j ‘— Body Heart Heart Wt.(lO-3) Animal No. Weight Weight Body Wt. F-4 600 gms. 2.0880 gms. 3.408 F-5 600 1.7054 2.842 F-6 450 1.5137 3.364 F-8 546 1.5542 2.828 F-11 581 2.2865 3.935 F-12 550 1.9160 3.484 p-13 573 1.6815 2.935 F-l4 430 1.4110 3.281 F-lS 599 1.9688 3.287 F-16 572 1.7692 3.093 F-17 588 1.8404 3.130 F-18 603 1.6134 2.676 F-19 572 1.9270 3.369 F-20 511 1.8830 3.685 F-21 490 1.5160 3.094 F-25 593 2.0515 3.460 F-26 597 1.8844 3.156 F-27 554 1.7570 3.171 F-29 599 1.8370 3.067 F-3o 516 1.6889 3.273 F-31 516 1.5969 3.095 F-32 558 1.6480 2.953 F—33 480 1.4698 3.062 F—34 462 1.8129 3.924 F-35 488 1.7480 3.582 F-36 519 1.4706 2.834 F-40 474 1.4792 3.121 F-ul 610 1.5994 2.622 F—y3 530 1.4812 2.795 F—44 530 2.0030 3.779 F-45 520 1.4580 2.804 F-46 632 1.7635 2.790 F-47 571 1.6577 2.903 F-49 530 1.7283 3.261 F-5o 502 1.4770 2.942 F-55 573 2.0790 3.628 F-58 600 1.5872 2.645 F-59 572 2.0436 3.573 F-60 581 2.5500 4.389 14 15 M Body Heart Heart Wt.(lO-3) Animal No. Weight Weight Body Wt. MEAN 565.6 1.9511 3.452 V-4 535 1.8570 3.471 V-5 528 1.4715 2.787 V-6 568 '3.7150 6.540 v-8 450 1.6783 3.730 V-ll 420 1.4961 3.562 V-12 532 2.0920 3.932 V-l3 610 1.9435 3.186 V—14 579 2.0937 3.616 v-15 544 1.7558 3.228 v-16 510 1.7575 3.446 V-17 584 1.4655 2.509 v-18 570 1.8421 3.232 V-19 554 1.6102 2.906 v-20 417 1.2981 3.113 V-21 528 1.4442 2.735 v-25 500 1.9020 3.804 v-26 528 1.5000 2.841 V-27 526 1.8847 3.583 v-29 500 2.0737 4.147 V-30 428 1.4938 3,490 V-31 492 1.5854 3.222 V-32 550 2.0750 3.773 V—33 546 1.8944 3.470 V-34 510 1.4325 2,809 v—35 456 1.3177 2.890 V—36 573 1.6202 2.828 v-40 452 1.4390 3.184 v—41 512 1.5490 3.025 V-43' 565 2.0103 3.558 V-44 450 1.5665 3.481 V-45 528 1.5591 2.953 V-46 570 1.8161 3.186 V-49 458 1.5306 3.342 v-50 556 1.7190 3.092 V-55 513 1.6844 3,283 v-58 488 1.3609 2.789 v-59 601 1.6851 2.804 V-60 590 1.7049 2,890 MEAN 547.2 2.0234 3.865 8-4 528 1.5282 2.894 S-5 498 1.5830 3,179 S-6 497 1.6298 3.279 3-8 512 1.6692 3.260 3-11 460 1.4829 3.224 16 Body Heart Heart Wt.(10-3) Animal No. Weight Weight Body Wt. S-12 549 1.5544 2.831 8-13 462 1.5054 3.258 8-14 558 1.8867 3.381 8-15 482 1.5310 3.176 8-16 580 1.5386 2.653 8-18 532 1.5621 2.936 S-19 460 1.4860 3.230 8-20 575 1.7404 3.027 8-21 502 1.5511 3.090 8-25 471 1.6320 3.465 8-26 617 1.6743 2.714 8-2? 594 1.6703 2.812 8—29 536 1.7248 3.218 8—30 574 1.7421 3.035 8-31 488 1.5532 3.183 8-32 546 1.6770 3.071 S-33 566 1.5546 2.747 8-34 559 1.7606 3.150 8-35 480 1.5407 3.210 8-36 538 1.5615 2.902 S-40 438 1.4219 3.246 8-41 466 1.3690 2.983 8-43 524 1.6531 3.155 8-44 469 1.4958 3.189 S-45 566 1.6939 2.993 8-46 432 1.3860 3.208 8-47 602 1.7401 2.891 8-49 570 1.7534 3.076 8-50 668 1.8147 2.717 8-55 550 1.7090 3.107 8-58 495 1.6035 3.239 8-59 634 1.8929 2.986 8-60 564 2.1040 3.730 MEAN ' 561.0 1.9954 3.556 APPENDIX B BASIC DATA--CONTROL ANIMALS m Body Heart Heart Wt.(lO-3) Animal No. Weight» Weight Body Wt. F-2 607 gms. 1.6680 gms. 2.748 F-7 599 1.8895 3.154 F-9 581 1.9798 3.408 F-lO 516 1.3165 2.551 F-22 558 1.4441 2.588 F-37 484 1.4032 2.899 F-42 523 1.4913 2.851 F-48 574 1.9236 3.351 F-51 551 1.6331 2.964 F-52 606 1.5986 2.638 V-2 556 1.5584 2.803 V-7 622 1.5371 2.471 V-9 564 1.5401 2.731 V-10 572 1.4594 2.551 V-22 488 1.5296 3.134 V-37 560 1.5063 2.690 V-42 584 1.6703 2.860 v-48 584 1.7482 2.993 V-51 672 1.7230 2.564 V-52 562 1.6784 2.986 8-2 510 1.3950 2.735 8-7 554 1.7346 3.131 S-9 639 1.6814 2.631 8-10 638 1.7428 2.732 8-22 564 1.4016 2.485 3-37 593 1.5590 2.629 8-42 522 2.0274 3.884 8-48 568 1.5154 2.668 8-51 582 1.6631 2.858 S-52‘ 596 1.6680 2.799 1? APPENDIX C BASIC DATA--PATHOLOGY Code No. Degree & Ht. Animal Lesion of Section No. Location Severity Comments 2b V-l9 LV (Ec) S Localized - one area only 3a,b F—19 LV (Ec,Mc) S Several foci 5b F-4 LV, RV (Ec) S Local - one area in each ventricle 6b F-15 LV (Ec,Mc, S Local - one foci Epc) in each area 8a V—6 LV (MC) S Several diffuse foci 12a S-14 LV (Ec) S Local — one area b LV, RV S Local - several (Ec,Mc,Epc) area c LV (Ec) S Diffuse - large area 15b F-29 LV (Ec) S Local - one area 16b F-16 LV (EC) S Diffuse — large area 30b S-5 LV (Ec) Md Local — one small.area 32b S—27 LV (Ec) Md Local - one small area c RV (Ec) Md Local - one small area 35a S-49 LV (EC) S Local - one area b LV (Mc) S Local - one area 18 l9 Code No. Degree & Ht. Animal Lesion of Section No. Location Severity Comments 37a F—45 LV (Ec) S Diffuse - one area b RV (Mc) S Diffuse - one area 41c V-8 LV, RV (Ec) S Local - one area 42b V-52 LV, RV (Ec) Md Local - one small area 47a,b F—2l LV (Ec) Md Local - one small area 50a,b,c S—60 RV(Ec) S Local - one area 54 a,b S-43 LV (EC) Md Local - one small area 68c 8-34 LV (Ec) Md Local - one small area 74b V-34 LV (Ec) S Local - one area only 75a S-45 LV (Ec) S Local - one area 94b V-35 LV (Ec) Md Local - one small area 97a S-32 LV (Ec) S Local - one area 99b F-48 LV (Mc) S Local - one area 102a V-15 LV (Mc) Md Local - one small area 103b V-49 LV (Mc) Md Local - one small area 107b,c F-51 LV (Mc) Md Local — one small area 117b,c F-42 LV (Mc) Md Local — one small area W Code No. Degree & Ht. Animal Lesion of Section No. Location Severity Comments 122a,b F-5 LV (Ec) S Local - one area 125a,b V-50 RV (Mc) S Diffuse - one area 126b S-19 LV (Ec) Md Local - one small area 129b F-46 LV (Ec) S Local — one area 134a S-47 LV (EC) S Local - one area 135b V—36 LV (Ec) Md Local - one small area 137a S—21 LV (Ec) S Local - one area 143b V-9 LV (Ec) S Local - one area 144b F—36 LV (EC) S Local - one area l45b,c V-21 LV (Mc) Md Local - one . small area Figure Figure Figure Figure Figure 21 1.—-Rat myocardium of nine hour live-in anxiety study. Hematoxylin and eosin. X 165. 2.——Same area as Figure 1, demonstrating lack of succinic dehydrogenase activity. SDH stain. X 180. 3.--Myocardium of rat involved in injection study Showing diverse damage. Hematoxylin and eosin. X 205. 4.-—Same area as Figure 3, demonstrating lack of beta-hydroxybuterate dehydrogenase. B—OH DH stain. X 180. 5.--Same area as Figures 3 and 4 demonstrating lack of succinic dehydrogenase activity. SDH stain. X 195. Figure Figure Figure Figure 23 6.--Diverse damage in endocardium (left ventricle) of rat in Wilson‘s study. Hematoxylin and eosin. X 180. 7.--Recent heart damage in myocardium of rat from Wilson's study. Hematoxylin and eosin. X 180. 8.-—Scar tissue in myocardium of rat from Wilson's study. Hematoxylin and eosin. X 165. 9.—-Older scar tissue in endocardium of rat from Wilson's study. Hematoxylin and eosin. X 185. ”VIII II 11311711117114 VIII?!“