TISSUE AND CELL, 1992 24 (4) 499-510 0 1992 Longman Group UK Ltd.
CHUKUKA S. ENWEMEKA*, LEO C. MAXWELLS and GABRIEL FERNANDESSS
ULTRASTRUCTURAL MORPHOMETRY OF MATRICAL CHANGES INDUCED BY EXERCISE AND FOOD RESTRICTION IN THE RAT CALCANEAL TENDON Keywords: Tendon, collagen, morphometry.
exercise, food restriction
ABSTRACT. The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then. starting from 6-7 months of age, the rats in the two exercise groups performed 4&50 min of treadmill running at 1.2-1.6 miles h-’ every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libifum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of smallsized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P < 0.05). but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P < 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.
Introduction World-wide, there is a growing trend toward combining regular exercise with reduction of food intake as a means of staying well and fit. This practice is strongly supported by the literature, because just as regular exercise is well-known to promote health and fitness, so prolonged restriction of caloric intake is a *Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine and Research Service, Veterans Affairs Medical Center, Miami, FL, USA. *Division of Physical Therapy, and Departments of §Medicine and @Physiology, University of Texas Health Science Center at San Antonio. San Antonio, TX, USA. Correspondence to: Dr. Chukuka S. Enwemeka, Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine, 5915 Ponce de Leon Blvd., 5th Floor, Coral Gables, FL 33146, USA. Received 21 January 1992.
well-documented effective mean5 of extending the life span in experimental animals (Fernandes et al., 1976a; 1976b; 1984; Yu et al., 1982; 1985; Masoro, 1985; 1988; Masoro et al., 1982; 1989a, b). Exercise training modulates both the physical and chemical properties of connective tissues (Anderson et al., 1971; Heikkinen and Vuori, 1972; Tipton et al., 1975; Booth and Gould, 197.5; Kiiskinen, 1976; Suominen et al., 1980; Michna, 1984). For example, regular treadmill running strengthens the attachment of knee ligaments to bone in male dogs (Tipton et al., 1970). hypophysectomized rats (Barnard et al., 1968), and growing male rats (Zuckerman and Stull, 1969; 1973; Tipton et al., 1975), and augments the ultimate tensile strength of rabbit tendons as well (Viidik, 1967; 1969). The increased strength is associated with a rise in 3H-hydroxyproline incorporation in 499
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long bones and Achilles tendons of mice after 1 month of training (Heikkinen and Vuori, 1972). Furthermore, electron microscopic evidence indicates that the number and mean sizes of collagen fibrils in the mouse flexor digitorum longus tendon also changes in response to treadmill exercise (Michna, 1984). Treadmill running of 10-30 min duration every day produces a 30% increase in mean collagen fibril diameter and 15% increases in fibril number and cross-sectional area after 1 week. The increase in the number of collagen fibrils rises to 29% after 10 weeks of training (Michna, 1984). Life-long treadmill running has been shown to ameliorate the age-related decrease in the biosynthesis of collagen in predominantly type I muscles (Kavanen and Suominen, 1989). Similarly, 4 weeks of exercise training results in an increase in glycosaminoglycan concentration in the mouse Achilles tendon (Heikkinen and Vuori, 1972). These findings indicate that physical exercise, in particular endurance training, changes the structure and biochemical composition of connective tissues, resulting in an increase in their biomechanical strength. Despite extensive studies on the influence of exercise training on connective tissue structure and function (Tipton et al., 1970; 1975; 1986; Heikkinen and Vuori, 1972; Suominen et al., 1980; Vailas et al., 1981; 1985; Michna, 1984; Curwin et al., 1988), the possible effects of food restriction, exercise or a combination of food restriction and exercise on dense connective tissues remains virtually unknown. Yet it is well-known that a variety of food restriction programs alter the structure and function of muscles (el Haj et al., 1986; Goldspink et al., 1987; McCarter and McGee, 1987; Boreham et al., 1988; Daw et al., 1988). Because tendons transmit the forces generated by muscles to bone, it is possible that the structure and function of tendons are influenced by food restriction as well. Thus, the purpose of this study was to determine the effects of food restriction and exercise on the rat calcaneal tendon. Specifically, our aims were to study the effects of (1) food restriction (2) endurance training and (3) a combination of food restriction and endurance training, on the ultrastructural morphometry of collagen fibril populations in the calcaneal tendons of rats.
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Methods Subjects 12, 6-7-week-old specific pathogen-free (SPF) male Fischer 344 rats obtained from Charles River Laboratory (Wilmington, MA) were used for this study. Three animals each were randomly assigned to ad libitum fed sedentary control (AC), ad libitum fed endurance trained (AE), food restricted sedentary control (RC) and food restricted endurance trained (RE) groups. To maintain their SPF status, the rats were housed one per standard plastic cage in temperature and light controlled limited access rooms as previously described (Maxwell et al., see pp 491498). Food restriction
Until 6 weeks of age, all the animals were fed a standard commercially prepared pelleted food (Teklad Co, Madison, WI) ad libitum. Thereafter, the animals were fed a semipurified diet as detailed in previous papers (Laganiere and Fernandes, 1991; Maxwell, Enwemeka and Fernandes, 1992). Beginning from 6 weeks of age, rats in the AC and AE groups were fed ad libitum. Based on ad libitum food consumption determined in previous experiments, each food restricted rat received 60% of ad libitum food intake as described previously (Yu et al., 1982; Maxwell et al., see pp 491-498). Endurance
training
At 6-7 months of age, rats in the two endurance training groups were trained to run on a treadmill (Quinton Instruments, Seattle, WA) containing 10 running compartments as previously described (Fernandes et al., 1986). After 2 weeks of gradual adaptation to the exercise protocol, each exercise-trained rat performed daily 40-50 min treadmill running at 1.2-1.6 miles h-’ for another 8 weeks. The treadmill was inclined to approximately 15” throughout the study. Tendon excision
After the training program, rats were weighed and then anaesthetized. The right calcaneal tendon of each rat was approached through a skin incision medial to its visible outline. By blunt dissection, the tendons were freed from their sheaths and sur-
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rounding tissues, Subthen excised. sequently, each excised tendon was immediately immersed in a petri-dish of 2% paraformaldehyde/2.5% glutaraldehyde (pH 7.4) and sliced several times to yield minute filamentous specimens.
fibrils per group were thus measured. Parry and Craig (1977) and Parry et al. (1978) have noted that at least 2500 collagen fibrils per group are necessary to obtain statistically relevant data.
Tissue processing and electron microscopy The specimens obtained were then transferred into scintillation vials, fixed for 6-8 hr in 2% paraformaldehyde/2.5% glutaraldehyde (pH 7.4), buffer washed, then postfixed for another 2 hr in 1% aqueous solution of osmium tetroxide (pH 7.4). Thereafter, each specimen was washed with distilled water and dehydrated in graded alcohol, followed by final dehydration in propylene oxide. After gradual infiltration with a mixture of propylene oxide and EMBED 812 resin (Electron Microscopy Sciences, Fort Washington, PA), each specimen was embedded in 100% resin and cured at 60°C for 65-70 hr. Each resin-embedded specimen was then trimmed and sectioned with glass knives at a thickness of one micron, in order to verify the level of sectioning by light microscopy. Thereafter, a diamond knife was used to obtain representative ribbons of ultrathin (70 nm-80 nm) silver/silver grey transverse and longitudinal sections. Sections were mounted on copper grids, then stained with uranyl acetate followed by lead citrate for 10 min each. Subsequently, the sections were visualized and photographed with a JEOL IOOCX electron microscope.
Because the CSA distribution of each group of fibrils differed significantly from a normal curve, and in particular, because Cochran’s tests revealed significant heterogeneity of variances, Kruskal-Wallis H test was used to compare the CSA of the four groups of rats. Thereafter, Mann-Whitney U tests were performed to pin-point groups that differed in fibril CSA.
Data analysis
Results Body weights: Food restriction
with or without exercise, induced a remarkable decrease in the overall body weights of the rats (Fig. 1; P < 0.05). The mean body weights of rats fed ad libitum and trained on treadmill (377.00 * 26.54 g) did not differ from the mean weight of ad libitum fed sedentary control rats (385.00 rf: 21.27 g; P > 0.10). Similarly, the mean body weight of sedentary food restricted rats, 259.00 * 9.93 g, did not differ from that of food-restricted endurance trained rats (260.66 * 8.80 g; P > 0.10; Fig. 1). Collagen fibril cross-sectional area: In sed-
entary control
(AC) rats. food restriction
Morphometry
To objectively compare the ultrastructural morphometry of the collagen fibrils of the four groups of tendons, the high magnitication electron micrographs obtained were placed on a Jandel Scientific digitizing tablet (Jandel Scientific, Corte Madera, CA) interfaced to a computer that has SIGMAscan (Jandel Scientific, Corte Madera, CA) software designed to facilitate computation of morphometric measurements. After calibrating the system, the electronic pen of the digitizer was used to carefully trace the outline of each fibril. Simultaneously, the crosssectional area of each fibril was computed and stored in the computer. Only photographs containing perfectly cross-sectioned collagen fibrils were used. A total of 13,604 collagen fibrils representing 2900 to 4300
Fig. 1. Mean body weights of the four groups of animal\ Mean body weights neither differed between the two an libitum groups (P > O-10). nor between the two load restricted groups (P > 0~10). However. mean body weights differed signtficantly between the ad libitum fed ratsand the food restricted rats (I’ < 035).
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Fig. 2. Mean collagen tibril cross-sectional area. Fibril cross-sectional areas differed significantly between the two ad libirum groups (P < 0.05) and between the two food restricted groups (P < 0.05). Whereas there was no significant difference between AC and RC tibrils, AE and RE fibrils differed significantly in cross-sectional area (P < 0.05).
alone did not significantly alter the mean collagen fibril CSA (Fig. 2)) but predisposed a preponderance of small-sized collagen fibrils (Figs 3, 4). Endurance training per se induced a significant (32%) increase in mean fibril CSA (P < 0.05); consequently, there was a greater preponderance of large collagen fibrils (mean CSA > 35,000 nm’) in trained rats fed ad libitum than in any of the other three groups (Fig. 3). This adaptive response to exercise was altered by food restriction, as indicated by a 33% decline in fibril CSA (P < 0.05; Fig. 2), and the absence of large collagen fibrils (mean CSA > 35,000) in the tendons of RE rats (Figs 3, 4). 56% of the collagen fibrils in the restricted diet endurance trained group were 5000nm’ or less in cross-sectional area, compared to 22% in the ad libitum fed endurance trained group. Although there was a similar preponderance of small-sized collagen fibrils in the food restricted control group, large collagen fibrils (CSA > 40,000 nm2), which were not found in the tendons of ad libitum control and restricted diet endurance trained rats, were present in this group. This indicates the presence of a wider range of fibril CSA in the tendons of food restricted control rats than those of ad libitum controls and food restricted endurance-trained rats, but not the tendons of ad libitum fed endurancetrained rats whose CSA were as high as
Fig. 3. Frequency distribution of fibril cross-sectional area in the four groups of tendons. (a) Distribution profile of fibrils in AE tendons. Note the presence of relatively large (CSA > 40,000 nm*) lib&.. (b) Distribution of fibril CSA in AC tendons. In (c), note the preponderance of small collagen tibrils (tibril CSA < 10,000 nm2) compared to the other three groups, including the RC group (d).
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65,000 nm’. The abundance of small-sized fibrils in food restricted endurance-trained rats and the presence of numerous largesized fibrils in ad libitum fed endurancetrained rats were further manifested in the number of collagen fibrils per unit area (i.e. fibril density) (Fig. 5). There were 31.08 t2.28 fibrils p-’ in the tendons ad libitum fed endurance-trained rats, but 40.18 * 2.65, 48.45 2 14.92 and 55.60 ? 9.40 in ad libitum controls, restricted diet controls and food restricted endurance-trained group respectively (Fig. 5). Because of the significant differences in the sizes of the fibrils of the four groups of tendons, the number of fibrils per unit area cannot reflect the bulk of collagen fibrils per unit area. Thus, collagen fibril index (CFI), calculated as the sum of fibril GSA/unit area of tendon, was determined (Fig. 6). Without food restriction, exercise induced a significant increase in CFI (P < 0.05, Fig. 6); this change in CFI was not observed following endurance training and food restriction (P > 0.05; Fig. 6). Discussion Our findings indicate that in Fischer 344 rats, (1) 10 weeks of endurance exercise does not significantly alter body weight, but food restriction with or without exercise significantly reduces the body weight, and (2) endurance exercise of 10 weeks duration induces a significant increase in the crosssectional area and diameter of tendon collagen fibrils in 6-7 month old ad lib&urn fed rats, but food restriction prevents this response and predisposes a decrease in the cross-sectional area and diameter of collagen fibrils. Body weights: A reduction in body weight after a period of food restriction has been reported by others (Yu et al., 1985; Masoro, 1985; 1989; Daw et al., 1988). Daw et al. (1988) reported a reduction to 71% of ad libitum fed rat weights in 12-month-old rats that received 65-70% of ad libitum food
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intake. In the present study, the bodyweights of exercised and non-exercised rats declined to 70% and 67% respectively. as a result of food restriction to 60% of ad libitum food intake. This data compares very well with that of Daw et al. (1988) in spite of the minor differences in the two protocols. Whereas the animals in our study were food restricted daily, those in the Daw et al. (1988) study were food restricted by alternate day feeding. Effects of endurance exercise on fibril morphometry: The effect of endurance exercise per se on collagen fibril morphometry in our
study differs from the long-term effects reported by Michna (1984). For example. even though Michna (1984) reported differences in mean diameter and mean crosssectional area of the collagen fibrils in the flexor digitorum longus of mice after 1 week of treadmill running of lO-30min duration everyday, no significant morphometric changes were observed after 10 weeks of the same exercise program. In contrast, our study shows that endurance exercise alone induced a significant 32% increase in the mean cross-sectional area of the collagen fibrils of the rat Achilles tendon after 10 weeks of treadmill running for 40-50 min everyday. In ad libitum fed animals, an increase in collagen synthesis is often observed when additional load is imposed on connective tissues whether or not such load is in the form of an exercise program (Elliot, 1965). Thus, without food restriction, loading may be the overriding factor that triggers increased collagen synthesis in dense connective tissue. In other words, libril hypertrophy may be associated more with the amount of load imposed on the tissue than the overall amount of exercise performed by the animal. For example, a long-term running exercise program has been shown to increase the size and collagen content of swine extensor tendons (Woo et al., 1980). The same exercise protocol has no effects on swine flexor tendons (Woo et al., 1981). Thus, the differences
Fig. 4. Cross-sections of collagen fibrils in the four groups of tendons. Note the similarity of fibril sizes in AC (A) and RC (B) tendons, the significantly larger fibrils in AE tendons (C) and the significantly smaller fibrils in RE tendons (D). C = collagen. x58,CKNl.
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Fig. 5. Collagen fibril density (i.e. fibrils per square micron) in the four groups of tendons.
in our results and those of Michna (1984) may be partially accounted for by the fact that similar *exercise programs may not impose the same amount of load on each tendon or groups of tendons. Exercise-induced hypertrophy of collagen fibrils and increased collagen turnover have been reported by several investigators (Heikkinen and Vuori, 1972; Suominen, Kiiskinen and Heikkinen, 1980; Woo et al., 1980; Woo et al., 1981; Vailas et al., 1981; Michna, 1984; Vailas et al.., 1985; Tipton, Vailas and Mathes, 1986; Curwin, Vailas and Wood, 1988). Via electron microscopy and ultrastructural morphometry, Michna (1984) showed that the mean diameter and mean cross-sectional area of the collagen fib& in the flexor digitorum longus of mice increases
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Fig. 6. Collagen cross-sectional area Endurance exercise AE rats (P < 0.05). rats significantly.
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fibril index i.e. summation of fibril per square micron of tendon tissue. significantly increased fibril index in but did not alter fibril index of RE
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by 30 and 15% respectively, after 1 week of treadmill running of 10-30 min duration everyday. Similarly, 8 weeks of progressive treadmill running 5 day/week at 70-80% maximum oxygen consumption (VOZmax) induces a significant increase in tendon collagen deposition in the common tendon of the medial and lateral gastrocnemii (i.e. Achilles tendon) of 3-week-old white leghorn roosters (Curin, Vailas and Wood, 1988). The same study did not reveal any significant changes in DNA, proteoglycan, collagen concentration or tendon dry weight (Curwin, Vailas and Wood, 1988); thus implicating rapid collagen synthesis and turnover in fibril hypertrophy. Effects of endurance exercise and food restriction on jibril morphometry: Food restriction
did not alter the CSA of tendon collagen fib&, but the hypertrophying effect of 10 weeks of endurance exercise was reversed when food restricted animals were subjected to the same exercise protocol. Although there are no food restriction studies with which to compare our findings, a similar endurance exercise program has been shown to increase collagen turnover and augment the accumulation of smaller less mature collagen fibrils in the Achilles tendon of immature white leghorn roosters fed ad libitum (Curwin et al., 1988). The increased collagen turnover induced by exercise may relate to the fact that young connective tissues (including tendons) contain higher levels of reducible cross-links than mature tissues (Fujii and Tanzer, 1974; Eyre et al., 1984; Amiel et al., 1991). The literature indicates that food restriction slows aging and extends the lifespan of laboratory rats (Yu et al., 1982; 1985; Masoro, 1985; 1988; Masoro et al., 1982; 1989a, b). Therefore, it is conceivable that the tendons of food restricted animals were less mature than those of ad libitum fed animals, resulting in the differential response of food restricted and ad libitum fed rats to the same exercise regime. This line of reasoning is supported by the fact that a similar exercise protocol has been reported to produce ‘splitting’ or ‘confluence’ of individual collagen fibrils in 6-week-old mice (Michna, 1984). The mean size reported for such fibrils (Michna, 1984) is similar to that of newly synthesized collagen fibrils in rabbits (Enwemeka et al., 1990). Thus, the mixture
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Fig. 7. Longitudinal-section of collagen fibrils in RE tendons. Note the presence of relatively small newly synthesized collagen Iibrils (arrows) and older larger fibrils (C). Note the presence of a fibroblast (*) with fairly developed cytoplasmic organelks in the vicinity. x7400.
of relatively small and large collagen fibrils in Fig. 7, and the preponderance of fibrils that were 5000 nm2 or less in the tendons of endurance-trained food restricted rats, support the view that endurance exercise can provoke synthesis of new collagen fibrils in young animals. Regardless of the mechanism involved, overall, food restriction deters fibril hypertrophy. This effect is consistent with the finding that food restriction also prevents hypertrophy of the plantaris as well as the soleus muscle which generate a large proportion of the forces transmitted by the calcaneal tendon (Maxwell er al., see pp 491498).
Aging alters the critical length necessary to maintain the appropriate tensile properties of rat tail tendons (Craig et al., 1989), facilitates the formation of larger ‘dysplastic’ collagen fibrils in the tunica media of the internal jugular vein of man (Breiteneder-Gelef, Mallinger and Brock, 1990), slows collagen synthesis in the medial collateral and anterior cruciate ligaments of rabbits (Amiel et al., 1991), promotes diminished collagen deposition (Uitto, 1989), retards the proliferation of skin fibroblasts (Uitto, 1989; Imayama and Braverman, 1989), and increases glycation of aorta1 and skin collagen in mice and rats (Deyl et al., 1990). Given the evidence that
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food restriction prolongs the life-span (Masoro, 1989), it is conceivable that these deleterious effects of aging on connective tissues can be retarded by food restriction. Already there is evidence that long term food restriction prevents pathological changes in several tissues (Maeda et al., 1985), deters irreversible glycation of cellular constituents (Cerami, 1985; Masoro et al., 1989), retards age-related loss of free radical detoxifying ability (Yu et al., 1989; Laganiere and Yu, 1987), and maintains a higher level of spontaneous exercise in experimental animals (McCarter and McGee, 1987). During maturation, the number of non-reducible crosslinks increases (Amiel et al., 1991), resulting in fibrils of larger diameter (Parry et al., 1978; Viidik et al., 1982). Therefore, the large quantity of fibrils with small CSA in the tendons of food restricted animals could be an indication that food restricted and endurance-trained food restricted rats have less mature collagen fibrils, and are perhaps more youthful than ad libitum fed controls and endurance trained ad libitum fed rats. Although a similar preponderance of smallsized fibrils has been observed in the tendons of young animals relative to those of older animals (Parry, Barnes and Craig, 1978), retardation of aging may not be the only viable explanation for this observation. For example, Michna (1984) has reported splitting of collagen fibrils in the flexor digitorum tendon of mice, after 10 weeks of endurance training. Given such phenomenon, it could be argued that the tendons of endurance trained food restricted animals do age and do hypertrophy, but split to yield small-sized fibrils. Similarly, the possibility that collagen turnover is so rapid that fibrils do not mature in food restricted and food restricted endurance-trained animals cannot be ruled out. Because these possibilities are not mutually exclusive and cannot be distinguished with our present data, future studies must include assays for collagen synthesis as well as tendon strength and tendon cross-sectional area measurements. Given that connective tissue response to food restriction and exercise i.e. the formation of a large proportion of relatively new collagen fibrils, may not be limited to the calcaneal tendon alone, it is possible that food restriction or combined food restriction and exercise can potentially minimize the
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formation of larger ‘dysplastic collagen’ fibrils induced by aging in human blood vessels (Breiteneder-Gelef, Mallinger and Brock, 1990). The preponderance of smaller collagen fibrils in food restricted endurance-trained rats but densely packed larger fibrils in ad libitum fed endurance-trained rats have obvious implications for athletes and others who perform strenuous exercise while limiting food intake at the same time. Studies have shown that there is a relationship between the sizes, number, and organization of tendon collagen fibrils and their biomechanical strength (Vogel, 1974; Parry et al., 1978; Flint et al., 1984). Given the direct relationship between biomechanical strength on one hand and fibril organization, size, and number on the other, we infer that the rate of injuries such as ligament and tendon ruptures will differ in physically active ad libitum fed individuals compared to those that limit food intake, especially in view of the leaner body mass associated with limited food intake. Further studies are necessary to further explore practical implications of these findings to the growing proportion of humans that engage in vigorous exercise while limiting dietary intake at the same time.
Conclusions
Our results mandate the conclusion that in Fischer 344 rats 10 weeks of treadmill running at 1.2-1.6 miles h-’ everyday does not significantly alter body weight, but food restriction with or without exercise results in a significant loss of body weight. This same endurance exercise program induces a significant increase in the cross-sectional area of tendon collagen fibrils in ad libitum fed 67-month-old rats, but predisposes a decrease in the cross-sectional area of collagen fibrils when combined with food restriction. Food restriction alone does not significantly alter the mean cross-sectional area of collagen fibrils even though it predisposes a preponderance of small-sized fibrils that are akin to those of younger animals.
Acknowledgements
The technical assistance of Dr Oscar Rod-
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riguez and MS Mary Ann Hart is gratefully acknowledged. We appreciate with thanks, the statistical assistance of Dr Tom Prehoda. This study was supported in part by NIH
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grants AG 03417 and AG 01188, and a Department of Veterans Affairs RR and D merit grant and a Morrison Trust Foundation grant.
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Masoro, E. J. 1985. Nutrition and aging-A current assessment. J. Nutr., 115, 842-848. Masoro, E. J., Katz, M. S. and McMahan, C. A. 1989a. Evidence for the glycation hypothesis of aging from the foodrestricted rodent model. J. Gerontol. Biol. Sci., 44,BZ&B22. Masoro, E. J. 1988. Food restriction in rodents: An Evaluation of its role in the study of aging. J. Gerontol., 43, B59B64. Masoro, E. J., Iwasaki, K., Gleiser, C. A., McMahan, C. A., Seo, E-J. and Yu, B. P. 1989b. Dietary modulation of the progression of nephropathy in aging rats: an evaluation of the importance of protein. Am. J. Clin. Nutr., 49, 1217-1227. Maxwell, L. C., Enwemeka, C. S. and Fernandes, G. 1992. Effects of exercise and food restriction on rat skeletal muscles. Tissue Cell, 24, 491498. McCarter, R., McGhee, J. 1987. Influence of nutrition and aging on the composition and function of rat skeletal muscle. J. Gerontol., 42, 432-441. Michna, H. 1984. Morphometric analysis of loading-induced changes in collagen-tibril populations in young tendons. Cell Tissue Res.,
236,465-470.
Parry, D. A. D. and Craig, A. S. 1977. Quantitative electron microscope observations of collagen fibrils in rat-tail tendon. Biopolymers, 16, 1015-1031. Parry, D. A. D., Barnes, G. R. G. and Craig, A. S. 1978. A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relationship between fibril size distribution and mechanical properties. Proc. R. Sot. Lond. [B], 203, 305-321. Suominen, H., Kiiskinen, A. and Heikkinen, E. 1980. Effects of physical training on metabolism of connective tissues in young mice. Acta Physiol. Stand., 108, 17-22. Tipton, C. M., Mathes, R. D., Maynard, J. A. and Carey, R. A. 1975. The influence of physical activity on ligaments and tendons. Med. Sci. Sports Exert., 7, 165-175. Tipton, C. M., James, S. L., Mergner, W. and Tcheng, T. 1970. Influence of exercise on the strength of the medial collateral knee ligaments of dogs. Am. J. Physiol., 218, 894-901. Tipton, C. M., Vailas, A. C. and Matthes, R. D. 1986. Experimental studies on the influences of physical activity on ligaments, tendons and joints: a brief review. Acta Med. Stand [Suppl], 711, 157-168. Uitto, J. 1989. Connective tissue biochemistry of the aging dermis. Age-associated alterations in collagen and elastin. Clin. Geriatr. Med., 5, 127-147. Vailas, A. C., Tipton, C. M., Matthes, R. D. and Gart, M. 1981. Physical activity and its influence on the repair process of medial collateral ligaments. Connect Tissue Res., 9, 24-31. Vailas, A. C., Pedrini, V. A., Pedrini-Mille, A. and Holoszy, J. 1985. Patella matrix changes associated with aging and voluntary exercise. J. Appl. Physiol., 58, 1572-1576. Viidik, A. 1967. The effect of training on the tensile strength of isolated rabbit tendons, Stand. J. P&t. Reconstr. Surg., 1, 141-147.
Viidik, A. 1969. Tensile strength properties of Achilles tendon systems in trained and untrained rabbits. Acta Orthop. Stand., 40, 261-272.
Viidik, A., Danielson, C. C. and Oxlund, H. 1982. On fundamental and phenomenological models, structure and mechanical properties of collagen, elastic and glycosaminoglycan complexes. Biorheology, 19, 437. Vogel, H. G. 1974. Correlation between tensile strength and collagen content in rat skin. Effect of age and cortisol treatment. Connect Tissue Res., 2, 177. Woo, S. L.-Y., Ritter, M. A., Amiel, D., Sanders, T. M., Gomez, M. A., Kuel, S. C., Garlin, S. R. and Akeson, W. H. 1980. The biomechanical and biochemical properties of swine tendons: long-term effects of exercise on the digital extensors. Connect Tissue Res., 7, 177-183, 1980. WOO, S. L.-Y., Gomez, M. A., Amiel, D. 1981. The effect of exercise on the biomechanical and biochemical properties of swine digital flexor tendons. J. Biomech. Eng. Trans. ASME, 103, 51-56. Yu, B. P., Masoro, E. J., Murata, I., Bertrand, H. A. and Lynd, F. T. 1982. Life span study for SPF Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J. Gerontol., 37, 13&141. Yu, B. P., Masoro, E. J. and McMahan, C. A. 1985. Nutritional influences on aging of Fischer 344 rats. I Physical, metabolic, and longevity characteristics. J. Gerontoi., 40, 657-670. Zuckerman, J. and Stull, G. A. 1969. Effects of exercise on knee ligament separation force in rats. J. Appl. Physiol., 26, 716-719. Zuckerman, J. and Stull, G. A. 1973. Ligamentous separation force in rats as influenced by training, detraining and cage restriction. Med. Sci. Sports Exert., 5, 4449.
CHUKUKA S. ENWEMEKA*, LEO C. MAXWELLS and GABRIEL FERNANDESSS
ULTRASTRUCTURAL MORPHOMETRY OF MATRICAL CHANGES INDUCED BY EXERCISE AND FOOD RESTRICTION IN THE RAT CALCANEAL TENDON Keywords: Tendon, collagen, morphometry.
exercise, food restriction
ABSTRACT. The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then. starting from 6-7 months of age, the rats in the two exercise groups performed 4&50 min of treadmill running at 1.2-1.6 miles h-’ every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libifum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of smallsized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P < 0.05). but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P < 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.
Introduction World-wide, there is a growing trend toward combining regular exercise with reduction of food intake as a means of staying well and fit. This practice is strongly supported by the literature, because just as regular exercise is well-known to promote health and fitness, so prolonged restriction of caloric intake is a *Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine and Research Service, Veterans Affairs Medical Center, Miami, FL, USA. *Division of Physical Therapy, and Departments of §Medicine and @Physiology, University of Texas Health Science Center at San Antonio. San Antonio, TX, USA. Correspondence to: Dr. Chukuka S. Enwemeka, Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine, 5915 Ponce de Leon Blvd., 5th Floor, Coral Gables, FL 33146, USA. Received 21 January 1992.
well-documented effective mean5 of extending the life span in experimental animals (Fernandes et al., 1976a; 1976b; 1984; Yu et al., 1982; 1985; Masoro, 1985; 1988; Masoro et al., 1982; 1989a, b). Exercise training modulates both the physical and chemical properties of connective tissues (Anderson et al., 1971; Heikkinen and Vuori, 1972; Tipton et al., 1975; Booth and Gould, 197.5; Kiiskinen, 1976; Suominen et al., 1980; Michna, 1984). For example, regular treadmill running strengthens the attachment of knee ligaments to bone in male dogs (Tipton et al., 1970). hypophysectomized rats (Barnard et al., 1968), and growing male rats (Zuckerman and Stull, 1969; 1973; Tipton et al., 1975), and augments the ultimate tensile strength of rabbit tendons as well (Viidik, 1967; 1969). The increased strength is associated with a rise in 3H-hydroxyproline incorporation in 499
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long bones and Achilles tendons of mice after 1 month of training (Heikkinen and Vuori, 1972). Furthermore, electron microscopic evidence indicates that the number and mean sizes of collagen fibrils in the mouse flexor digitorum longus tendon also changes in response to treadmill exercise (Michna, 1984). Treadmill running of 10-30 min duration every day produces a 30% increase in mean collagen fibril diameter and 15% increases in fibril number and cross-sectional area after 1 week. The increase in the number of collagen fibrils rises to 29% after 10 weeks of training (Michna, 1984). Life-long treadmill running has been shown to ameliorate the age-related decrease in the biosynthesis of collagen in predominantly type I muscles (Kavanen and Suominen, 1989). Similarly, 4 weeks of exercise training results in an increase in glycosaminoglycan concentration in the mouse Achilles tendon (Heikkinen and Vuori, 1972). These findings indicate that physical exercise, in particular endurance training, changes the structure and biochemical composition of connective tissues, resulting in an increase in their biomechanical strength. Despite extensive studies on the influence of exercise training on connective tissue structure and function (Tipton et al., 1970; 1975; 1986; Heikkinen and Vuori, 1972; Suominen et al., 1980; Vailas et al., 1981; 1985; Michna, 1984; Curwin et al., 1988), the possible effects of food restriction, exercise or a combination of food restriction and exercise on dense connective tissues remains virtually unknown. Yet it is well-known that a variety of food restriction programs alter the structure and function of muscles (el Haj et al., 1986; Goldspink et al., 1987; McCarter and McGee, 1987; Boreham et al., 1988; Daw et al., 1988). Because tendons transmit the forces generated by muscles to bone, it is possible that the structure and function of tendons are influenced by food restriction as well. Thus, the purpose of this study was to determine the effects of food restriction and exercise on the rat calcaneal tendon. Specifically, our aims were to study the effects of (1) food restriction (2) endurance training and (3) a combination of food restriction and endurance training, on the ultrastructural morphometry of collagen fibril populations in the calcaneal tendons of rats.
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Methods Subjects 12, 6-7-week-old specific pathogen-free (SPF) male Fischer 344 rats obtained from Charles River Laboratory (Wilmington, MA) were used for this study. Three animals each were randomly assigned to ad libitum fed sedentary control (AC), ad libitum fed endurance trained (AE), food restricted sedentary control (RC) and food restricted endurance trained (RE) groups. To maintain their SPF status, the rats were housed one per standard plastic cage in temperature and light controlled limited access rooms as previously described (Maxwell et al., see pp 491498). Food restriction
Until 6 weeks of age, all the animals were fed a standard commercially prepared pelleted food (Teklad Co, Madison, WI) ad libitum. Thereafter, the animals were fed a semipurified diet as detailed in previous papers (Laganiere and Fernandes, 1991; Maxwell, Enwemeka and Fernandes, 1992). Beginning from 6 weeks of age, rats in the AC and AE groups were fed ad libitum. Based on ad libitum food consumption determined in previous experiments, each food restricted rat received 60% of ad libitum food intake as described previously (Yu et al., 1982; Maxwell et al., see pp 491-498). Endurance
training
At 6-7 months of age, rats in the two endurance training groups were trained to run on a treadmill (Quinton Instruments, Seattle, WA) containing 10 running compartments as previously described (Fernandes et al., 1986). After 2 weeks of gradual adaptation to the exercise protocol, each exercise-trained rat performed daily 40-50 min treadmill running at 1.2-1.6 miles h-’ for another 8 weeks. The treadmill was inclined to approximately 15” throughout the study. Tendon excision
After the training program, rats were weighed and then anaesthetized. The right calcaneal tendon of each rat was approached through a skin incision medial to its visible outline. By blunt dissection, the tendons were freed from their sheaths and sur-
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rounding tissues, Subthen excised. sequently, each excised tendon was immediately immersed in a petri-dish of 2% paraformaldehyde/2.5% glutaraldehyde (pH 7.4) and sliced several times to yield minute filamentous specimens.
fibrils per group were thus measured. Parry and Craig (1977) and Parry et al. (1978) have noted that at least 2500 collagen fibrils per group are necessary to obtain statistically relevant data.
Tissue processing and electron microscopy The specimens obtained were then transferred into scintillation vials, fixed for 6-8 hr in 2% paraformaldehyde/2.5% glutaraldehyde (pH 7.4), buffer washed, then postfixed for another 2 hr in 1% aqueous solution of osmium tetroxide (pH 7.4). Thereafter, each specimen was washed with distilled water and dehydrated in graded alcohol, followed by final dehydration in propylene oxide. After gradual infiltration with a mixture of propylene oxide and EMBED 812 resin (Electron Microscopy Sciences, Fort Washington, PA), each specimen was embedded in 100% resin and cured at 60°C for 65-70 hr. Each resin-embedded specimen was then trimmed and sectioned with glass knives at a thickness of one micron, in order to verify the level of sectioning by light microscopy. Thereafter, a diamond knife was used to obtain representative ribbons of ultrathin (70 nm-80 nm) silver/silver grey transverse and longitudinal sections. Sections were mounted on copper grids, then stained with uranyl acetate followed by lead citrate for 10 min each. Subsequently, the sections were visualized and photographed with a JEOL IOOCX electron microscope.
Because the CSA distribution of each group of fibrils differed significantly from a normal curve, and in particular, because Cochran’s tests revealed significant heterogeneity of variances, Kruskal-Wallis H test was used to compare the CSA of the four groups of rats. Thereafter, Mann-Whitney U tests were performed to pin-point groups that differed in fibril CSA.
Data analysis
Results Body weights: Food restriction
with or without exercise, induced a remarkable decrease in the overall body weights of the rats (Fig. 1; P < 0.05). The mean body weights of rats fed ad libitum and trained on treadmill (377.00 * 26.54 g) did not differ from the mean weight of ad libitum fed sedentary control rats (385.00 rf: 21.27 g; P > 0.10). Similarly, the mean body weight of sedentary food restricted rats, 259.00 * 9.93 g, did not differ from that of food-restricted endurance trained rats (260.66 * 8.80 g; P > 0.10; Fig. 1). Collagen fibril cross-sectional area: In sed-
entary control
(AC) rats. food restriction
Morphometry
To objectively compare the ultrastructural morphometry of the collagen fibrils of the four groups of tendons, the high magnitication electron micrographs obtained were placed on a Jandel Scientific digitizing tablet (Jandel Scientific, Corte Madera, CA) interfaced to a computer that has SIGMAscan (Jandel Scientific, Corte Madera, CA) software designed to facilitate computation of morphometric measurements. After calibrating the system, the electronic pen of the digitizer was used to carefully trace the outline of each fibril. Simultaneously, the crosssectional area of each fibril was computed and stored in the computer. Only photographs containing perfectly cross-sectioned collagen fibrils were used. A total of 13,604 collagen fibrils representing 2900 to 4300
Fig. 1. Mean body weights of the four groups of animal\ Mean body weights neither differed between the two an libitum groups (P > O-10). nor between the two load restricted groups (P > 0~10). However. mean body weights differed signtficantly between the ad libitum fed ratsand the food restricted rats (I’ < 035).
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Fig. 2. Mean collagen tibril cross-sectional area. Fibril cross-sectional areas differed significantly between the two ad libirum groups (P < 0.05) and between the two food restricted groups (P < 0.05). Whereas there was no significant difference between AC and RC tibrils, AE and RE fibrils differed significantly in cross-sectional area (P < 0.05).
alone did not significantly alter the mean collagen fibril CSA (Fig. 2)) but predisposed a preponderance of small-sized collagen fibrils (Figs 3, 4). Endurance training per se induced a significant (32%) increase in mean fibril CSA (P < 0.05); consequently, there was a greater preponderance of large collagen fibrils (mean CSA > 35,000 nm’) in trained rats fed ad libitum than in any of the other three groups (Fig. 3). This adaptive response to exercise was altered by food restriction, as indicated by a 33% decline in fibril CSA (P < 0.05; Fig. 2), and the absence of large collagen fibrils (mean CSA > 35,000) in the tendons of RE rats (Figs 3, 4). 56% of the collagen fibrils in the restricted diet endurance trained group were 5000nm’ or less in cross-sectional area, compared to 22% in the ad libitum fed endurance trained group. Although there was a similar preponderance of small-sized collagen fibrils in the food restricted control group, large collagen fibrils (CSA > 40,000 nm2), which were not found in the tendons of ad libitum control and restricted diet endurance trained rats, were present in this group. This indicates the presence of a wider range of fibril CSA in the tendons of food restricted control rats than those of ad libitum controls and food restricted endurance-trained rats, but not the tendons of ad libitum fed endurancetrained rats whose CSA were as high as
Fig. 3. Frequency distribution of fibril cross-sectional area in the four groups of tendons. (a) Distribution profile of fibrils in AE tendons. Note the presence of relatively large (CSA > 40,000 nm*) lib&.. (b) Distribution of fibril CSA in AC tendons. In (c), note the preponderance of small collagen tibrils (tibril CSA < 10,000 nm2) compared to the other three groups, including the RC group (d).
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65,000 nm’. The abundance of small-sized fibrils in food restricted endurance-trained rats and the presence of numerous largesized fibrils in ad libitum fed endurancetrained rats were further manifested in the number of collagen fibrils per unit area (i.e. fibril density) (Fig. 5). There were 31.08 t2.28 fibrils p-’ in the tendons ad libitum fed endurance-trained rats, but 40.18 * 2.65, 48.45 2 14.92 and 55.60 ? 9.40 in ad libitum controls, restricted diet controls and food restricted endurance-trained group respectively (Fig. 5). Because of the significant differences in the sizes of the fibrils of the four groups of tendons, the number of fibrils per unit area cannot reflect the bulk of collagen fibrils per unit area. Thus, collagen fibril index (CFI), calculated as the sum of fibril GSA/unit area of tendon, was determined (Fig. 6). Without food restriction, exercise induced a significant increase in CFI (P < 0.05, Fig. 6); this change in CFI was not observed following endurance training and food restriction (P > 0.05; Fig. 6). Discussion Our findings indicate that in Fischer 344 rats, (1) 10 weeks of endurance exercise does not significantly alter body weight, but food restriction with or without exercise significantly reduces the body weight, and (2) endurance exercise of 10 weeks duration induces a significant increase in the crosssectional area and diameter of tendon collagen fibrils in 6-7 month old ad lib&urn fed rats, but food restriction prevents this response and predisposes a decrease in the cross-sectional area and diameter of collagen fibrils. Body weights: A reduction in body weight after a period of food restriction has been reported by others (Yu et al., 1985; Masoro, 1985; 1989; Daw et al., 1988). Daw et al. (1988) reported a reduction to 71% of ad libitum fed rat weights in 12-month-old rats that received 65-70% of ad libitum food
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intake. In the present study, the bodyweights of exercised and non-exercised rats declined to 70% and 67% respectively. as a result of food restriction to 60% of ad libitum food intake. This data compares very well with that of Daw et al. (1988) in spite of the minor differences in the two protocols. Whereas the animals in our study were food restricted daily, those in the Daw et al. (1988) study were food restricted by alternate day feeding. Effects of endurance exercise on fibril morphometry: The effect of endurance exercise per se on collagen fibril morphometry in our
study differs from the long-term effects reported by Michna (1984). For example. even though Michna (1984) reported differences in mean diameter and mean crosssectional area of the collagen fibrils in the flexor digitorum longus of mice after 1 week of treadmill running of lO-30min duration everyday, no significant morphometric changes were observed after 10 weeks of the same exercise program. In contrast, our study shows that endurance exercise alone induced a significant 32% increase in the mean cross-sectional area of the collagen fibrils of the rat Achilles tendon after 10 weeks of treadmill running for 40-50 min everyday. In ad libitum fed animals, an increase in collagen synthesis is often observed when additional load is imposed on connective tissues whether or not such load is in the form of an exercise program (Elliot, 1965). Thus, without food restriction, loading may be the overriding factor that triggers increased collagen synthesis in dense connective tissue. In other words, libril hypertrophy may be associated more with the amount of load imposed on the tissue than the overall amount of exercise performed by the animal. For example, a long-term running exercise program has been shown to increase the size and collagen content of swine extensor tendons (Woo et al., 1980). The same exercise protocol has no effects on swine flexor tendons (Woo et al., 1981). Thus, the differences
Fig. 4. Cross-sections of collagen fibrils in the four groups of tendons. Note the similarity of fibril sizes in AC (A) and RC (B) tendons, the significantly larger fibrils in AE tendons (C) and the significantly smaller fibrils in RE tendons (D). C = collagen. x58,CKNl.
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Fig. 5. Collagen fibril density (i.e. fibrils per square micron) in the four groups of tendons.
in our results and those of Michna (1984) may be partially accounted for by the fact that similar *exercise programs may not impose the same amount of load on each tendon or groups of tendons. Exercise-induced hypertrophy of collagen fibrils and increased collagen turnover have been reported by several investigators (Heikkinen and Vuori, 1972; Suominen, Kiiskinen and Heikkinen, 1980; Woo et al., 1980; Woo et al., 1981; Vailas et al., 1981; Michna, 1984; Vailas et al.., 1985; Tipton, Vailas and Mathes, 1986; Curwin, Vailas and Wood, 1988). Via electron microscopy and ultrastructural morphometry, Michna (1984) showed that the mean diameter and mean cross-sectional area of the collagen fib& in the flexor digitorum longus of mice increases
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Fig. 6. Collagen cross-sectional area Endurance exercise AE rats (P < 0.05). rats significantly.
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fibril index i.e. summation of fibril per square micron of tendon tissue. significantly increased fibril index in but did not alter fibril index of RE
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by 30 and 15% respectively, after 1 week of treadmill running of 10-30 min duration everyday. Similarly, 8 weeks of progressive treadmill running 5 day/week at 70-80% maximum oxygen consumption (VOZmax) induces a significant increase in tendon collagen deposition in the common tendon of the medial and lateral gastrocnemii (i.e. Achilles tendon) of 3-week-old white leghorn roosters (Curin, Vailas and Wood, 1988). The same study did not reveal any significant changes in DNA, proteoglycan, collagen concentration or tendon dry weight (Curwin, Vailas and Wood, 1988); thus implicating rapid collagen synthesis and turnover in fibril hypertrophy. Effects of endurance exercise and food restriction on jibril morphometry: Food restriction
did not alter the CSA of tendon collagen fib&, but the hypertrophying effect of 10 weeks of endurance exercise was reversed when food restricted animals were subjected to the same exercise protocol. Although there are no food restriction studies with which to compare our findings, a similar endurance exercise program has been shown to increase collagen turnover and augment the accumulation of smaller less mature collagen fibrils in the Achilles tendon of immature white leghorn roosters fed ad libitum (Curwin et al., 1988). The increased collagen turnover induced by exercise may relate to the fact that young connective tissues (including tendons) contain higher levels of reducible cross-links than mature tissues (Fujii and Tanzer, 1974; Eyre et al., 1984; Amiel et al., 1991). The literature indicates that food restriction slows aging and extends the lifespan of laboratory rats (Yu et al., 1982; 1985; Masoro, 1985; 1988; Masoro et al., 1982; 1989a, b). Therefore, it is conceivable that the tendons of food restricted animals were less mature than those of ad libitum fed animals, resulting in the differential response of food restricted and ad libitum fed rats to the same exercise regime. This line of reasoning is supported by the fact that a similar exercise protocol has been reported to produce ‘splitting’ or ‘confluence’ of individual collagen fibrils in 6-week-old mice (Michna, 1984). The mean size reported for such fibrils (Michna, 1984) is similar to that of newly synthesized collagen fibrils in rabbits (Enwemeka et al., 1990). Thus, the mixture
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Fig. 7. Longitudinal-section of collagen fibrils in RE tendons. Note the presence of relatively small newly synthesized collagen Iibrils (arrows) and older larger fibrils (C). Note the presence of a fibroblast (*) with fairly developed cytoplasmic organelks in the vicinity. x7400.
of relatively small and large collagen fibrils in Fig. 7, and the preponderance of fibrils that were 5000 nm2 or less in the tendons of endurance-trained food restricted rats, support the view that endurance exercise can provoke synthesis of new collagen fibrils in young animals. Regardless of the mechanism involved, overall, food restriction deters fibril hypertrophy. This effect is consistent with the finding that food restriction also prevents hypertrophy of the plantaris as well as the soleus muscle which generate a large proportion of the forces transmitted by the calcaneal tendon (Maxwell er al., see pp 491498).
Aging alters the critical length necessary to maintain the appropriate tensile properties of rat tail tendons (Craig et al., 1989), facilitates the formation of larger ‘dysplastic’ collagen fibrils in the tunica media of the internal jugular vein of man (Breiteneder-Gelef, Mallinger and Brock, 1990), slows collagen synthesis in the medial collateral and anterior cruciate ligaments of rabbits (Amiel et al., 1991), promotes diminished collagen deposition (Uitto, 1989), retards the proliferation of skin fibroblasts (Uitto, 1989; Imayama and Braverman, 1989), and increases glycation of aorta1 and skin collagen in mice and rats (Deyl et al., 1990). Given the evidence that
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food restriction prolongs the life-span (Masoro, 1989), it is conceivable that these deleterious effects of aging on connective tissues can be retarded by food restriction. Already there is evidence that long term food restriction prevents pathological changes in several tissues (Maeda et al., 1985), deters irreversible glycation of cellular constituents (Cerami, 1985; Masoro et al., 1989), retards age-related loss of free radical detoxifying ability (Yu et al., 1989; Laganiere and Yu, 1987), and maintains a higher level of spontaneous exercise in experimental animals (McCarter and McGee, 1987). During maturation, the number of non-reducible crosslinks increases (Amiel et al., 1991), resulting in fibrils of larger diameter (Parry et al., 1978; Viidik et al., 1982). Therefore, the large quantity of fibrils with small CSA in the tendons of food restricted animals could be an indication that food restricted and endurance-trained food restricted rats have less mature collagen fibrils, and are perhaps more youthful than ad libitum fed controls and endurance trained ad libitum fed rats. Although a similar preponderance of smallsized fibrils has been observed in the tendons of young animals relative to those of older animals (Parry, Barnes and Craig, 1978), retardation of aging may not be the only viable explanation for this observation. For example, Michna (1984) has reported splitting of collagen fibrils in the flexor digitorum tendon of mice, after 10 weeks of endurance training. Given such phenomenon, it could be argued that the tendons of endurance trained food restricted animals do age and do hypertrophy, but split to yield small-sized fibrils. Similarly, the possibility that collagen turnover is so rapid that fibrils do not mature in food restricted and food restricted endurance-trained animals cannot be ruled out. Because these possibilities are not mutually exclusive and cannot be distinguished with our present data, future studies must include assays for collagen synthesis as well as tendon strength and tendon cross-sectional area measurements. Given that connective tissue response to food restriction and exercise i.e. the formation of a large proportion of relatively new collagen fibrils, may not be limited to the calcaneal tendon alone, it is possible that food restriction or combined food restriction and exercise can potentially minimize the
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formation of larger ‘dysplastic collagen’ fibrils induced by aging in human blood vessels (Breiteneder-Gelef, Mallinger and Brock, 1990). The preponderance of smaller collagen fibrils in food restricted endurance-trained rats but densely packed larger fibrils in ad libitum fed endurance-trained rats have obvious implications for athletes and others who perform strenuous exercise while limiting food intake at the same time. Studies have shown that there is a relationship between the sizes, number, and organization of tendon collagen fibrils and their biomechanical strength (Vogel, 1974; Parry et al., 1978; Flint et al., 1984). Given the direct relationship between biomechanical strength on one hand and fibril organization, size, and number on the other, we infer that the rate of injuries such as ligament and tendon ruptures will differ in physically active ad libitum fed individuals compared to those that limit food intake, especially in view of the leaner body mass associated with limited food intake. Further studies are necessary to further explore practical implications of these findings to the growing proportion of humans that engage in vigorous exercise while limiting dietary intake at the same time.
Conclusions
Our results mandate the conclusion that in Fischer 344 rats 10 weeks of treadmill running at 1.2-1.6 miles h-’ everyday does not significantly alter body weight, but food restriction with or without exercise results in a significant loss of body weight. This same endurance exercise program induces a significant increase in the cross-sectional area of tendon collagen fibrils in ad libitum fed 67-month-old rats, but predisposes a decrease in the cross-sectional area of collagen fibrils when combined with food restriction. Food restriction alone does not significantly alter the mean cross-sectional area of collagen fibrils even though it predisposes a preponderance of small-sized fibrils that are akin to those of younger animals.
Acknowledgements
The technical assistance of Dr Oscar Rod-
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riguez and MS Mary Ann Hart is gratefully acknowledged. We appreciate with thanks, the statistical assistance of Dr Tom Prehoda. This study was supported in part by NIH
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grants AG 03417 and AG 01188, and a Department of Veterans Affairs RR and D merit grant and a Morrison Trust Foundation grant.
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