The Effects of Ad Libitum Feeding and Marked Dietary Restriction on Spontaneous Skeletal Muscle Pathology in Sprague–Dawley Rats

Abstract

The effects of ad libitum (AL) feeding and marked dietary restriction (DR) on spontaneous age-related skeletal muscle changes in male Sprague–Dawley (SD) rats were evaluated at 1 and 2 years. SD rats were fed Certified UAR A04C Rodent Chow ad libitum (AL), or DR at 50% of AL for (106 weeks). Body weights and organ weights were measured at the 1-year interim and 2-year final necropsies. In addition to the routine histopathologic examination, determination of 5 stereologic parameters was done in the vastus lateralis muscle after histochemistry of ATPase activity at 1 and 2 years. Body and skeletal muscle weights were proportional to the food intake. In AL-fed rats, muscle weights decreased between 1 and 2 years, in correlation with decreased type 2 myofiber numbers. In this group, fibrovascular index markedly increased with aging and muscle degeneration occurred at 2 years. In DR rats, there were no significant changes in muscle weights between 1 and 2 years. No histopathological changes were observed and the fibrovascular index was unchanged. These results demonstrated a protective effect of DR on the age-related skeletal muscle pathology in SD rats.

Introduction

In mammals, muscle weakness and impaired physical performance occur with aging. These clinical changes are related, at least partly, to a loss of muscle mass, referred to as sarcopenia, resulting from muscle cell structural and functional alterations (Larsson, 1982, 1982, 1995; Ansved and Larsson, 1989; Brooks and Faulkner, 1994; Evans, 1995). Progressive atrophy of muscle fibers and replacement by fat and connective tissue with aging have been reported in laboratory animals and men (Tauchi et al., 1971; Cacchia et al., 1979; Larsson, 1982; Schoder, 1994). In Sprague–Dawley (SD) rats, ad libitum (AL) feeding has also been associated with an age-related increase in body weights resulting from an increase in body fat and a decrease in lean body mass (Keenan et al., 1992, 1994).

Age-related modifications in muscle innervation and blood supply, accumulation of oxidative damage and decreases in protein synthetic activity resulting from endocrine imbalance have been evoked as causative factors of sarcopenia (Sonntag et al., 1980, 1985, 1997, 1999; Larson, 1982; Cook et al., 1992; Nair, 1995; Ferrington et al., 1998). In old rats, decreased muscle mass is associated with low serum growth hormone (GH) and insulin growth factor 1 (IGF1) levels (Florini et al., 1981). The decrease in trophic hormone secretion by the pituitary gland correlates with a decrease in the protein synthetic activity (Mietes et al., 1987; Mietes, 1990).

Diet restriction (DR) extends maximum life span and retards the development of a broad spectrum of pathophysiological changes in rodents (Maeda et al., 1985, Keenan et al., 1994; Yu, 1995; Weindruch, 1996; Weindruch and Sohal, 1997). In SD rats, DR significantly improves the 2-year survival, controls adult body weight and delays the onset of diet-and age-related spontaneous diseases and tumors (Keenan et al., 1992, 1994, 1995, 1996, 1999, 2000; Gumprecht et al., 1993; Masoro, 1995, 1996; Laroque et al., 1997; Kemi et al., 1998; Hubert et al., 2000; Molon-Noblot et al., 2000, 2003). Although a protective effect of DR on myopathy has been reported in aged rats (Boreham et al., 1988; Daw et al., 1988; Aspes, 1997; Kemi et al., 1998), its role in the pathogenesis of skeletal muscle changes and muscle fiber composition has not been fully investigated. The effect of DR on the degenerative changes involving the skeletal muscle in rats is of particular interest since the aging rat has been shown to be a good animal model of sarcopenia (Larsson, 1995). In this species, caloric restriction has been found to reduce the rate of age-related skeletal mass loss in soleus, anterior tibialis, extensor digitorum longus, and vastus lateralis muscles (El Haj et al., 1986; Boreham et al., 1988; Aspes, 1997).

This paper describes the qualitative and quantitative morphologic features of the effects of marked DR (50% of AL feeding) on the age-related vastus lateralis muscle pathology in SD rats.

Materials and Methods

Animals

Two-hundred and forty male and 240 female Sprague–Dawley Crl:CD (SD) BR (International Standard) rats were obtained from Charles River France (Saint-Aubin-lès-Elbeuf, France). The animals were 35 days old at study initiation, with males weighing 104–195 grams and females (104–180 grams). The rats were individually housed in suspended stainless steel cages, in air conditioned rooms with a temperature of approximately 22°C, humidity between 30 and 70%, and a 12-hour light cycle. They were individually identified by tail tattoos and allocated to cages by a randomized columnar allocation scheme. The animals were assigned to 2 different treatment groups using a balanced random allocation scheme based on body weight. Each group consisted of 120 males and 120 females with 20 animals/sex/group allocated for the 14-, 27-, and 53-week interim necropsies, and with 60 animals/sex/group allocated to the 106-week final necropsy. The Institutional Animal Care and Use Committee at Merck Research Laboratories, West Point, Pennsylvania approved all procedures in this study.

Diet and Dietary Regimen

UAR A04C rodent diet (UAR, Villemoisson sur Orge) was given to all rats in the morning. In group 1, rats were fed AL. In group 2 (approximately 50% of AL daily food consumption), males and females were given daily 14 and 10 grams of food, respectively. The mean diet composition was 15.7% protein, 60.7% carbohydrate, and 3.1% fat and contained 3.34 kcal/g of physiological energy, calculated using Atwater physiologic fuel values. Drinking water was available AL. All rats were given daily 0.5 percent aqueous methylcellulose by oral gavage at a dosing volume of 5 mL/kg.

Clinical Evaluations

All rats were observed daily for clinical signs and weighed before the start of the study, once in week 1, generally twice weekly through week 14, and once a week thereafter. In study weeks 4, 8, 11, 27, 39, 51, 79, and 103, hematologic and serum biochemical examinations were conducted in 20 rats/sex/group, and urinalyses examinations were done in 10 rats/sex/group. These clinical data have been reported separately (Hubert et al., 2000).

Necropsy and Histopathology

Twenty males rats from groups 1 (AL) and 2 (50% AL) were used for the portion of the study presented in this paper. Ten rats from each group were selected for the 53-week (1-year) and 106-week (2-year) scheduled necropsies by a stratified randomization allocation procedure to optimize the probability that a truly representative sample was selected across the range of body weights in each group. At scheduled 1-year and 2-year necropsies, following an overnight fast, all rats were sacrificed by exsanguination under deep anesthesia. Terminal body weights were obtained and complete gross examination was performed on all animals. Numerous organs were weighed and tissue samples were taken for microscopic examination; these data will be reported separately. The vaste lateralis muscles were sampled, weighed, and the volume measured. Transversal sections of muscles were fixed by immersion in a 10% phosphate buffered formalin solution, and 5 μm-thick paraplast sections were stained with hematoxylin and eosin or Gomori’s trichrome for microscopic examination. Adjacent sections of muscles were snap frozen at −70°C in isopentane cooled with liquid nitrogen for histoenzymology.

Histochemistry and Stereology

Histochemical reactions for myofibrillar adenosine triphosphatases (ATPases) activities were done on 10-μm frozen sections adjacent to those used for histopathology. Incubations of the sections were done at pH 4.3 and 9.8, for the characterization of type 1 (slow twitch) and type 2 (fast twitch) myofibers, respectively.

Stereological analysis of the muscle sections was conducted using the Imagenia image analysis system (Biocom, Lyon, France). The total muscle area together with the type 1 and type 2 myofiber areas and numbers were determined on frozen sections histochemically stained for ATPases activities. For each animal, 25 randomly selected type 1 myofibers stained at pH 4.3 and 25 Type 2 myofibers stained at pH 9.8 were analyzed. The fibrovascular tissue volume fraction (%) was determined using the point counting method (Weibel, 1979) on the Gomori’s trichrome stained paraffin sections.

Statistical Analysis

Statistical analyses were performed on body weights, skeletal muscle weights, and the stereologic parameters. Data were analyzed for homogeneity of variance and normality and statistical significance at p ≤ 0.05 was based on the variance analysis using the Student’s t-test. These analyses were done in a logarithmic scale in order to satisfy the assumptions for continuous parameters that follow a normal distribution. Each parameter was analyzed separately for each necropsy.

Results

Body Weights and Muscle Weights (Table 1)

Data on body weights have been reported and discussed in a previous paper (Hubert et al., 2000). Briefly, body weights were proportional to food intake and at 1 and 2 years, the body weight values were statistically significantly lower in DR-fed than in AL-fed rats (Table 1).

Muscle weights decreased (p ≤ 0.05) from 1 year to 2 years in AL-fed rats. In DR rats, they were statistically significantly lower than in AL-fed rats, but remained unchanged between 1 and 2 years . When expressed as percentages of body weights, the muscle weights were greater in the DR rats than in AL-fed rats (Table 1).

Histopathology and Stereology (Tables 1 and 2)

Age-Related Changes

In all rats examined at 1 and 2 years, the vastus lateralis muscle was mainly composed of type-2 (fast-twitch) myofibers, which accounted for more than 90% of the muscle area. Type-1 (slow-twitch) myofibers had generally a smaller diameter and were randomly interspersed between type-2 myofibers. Myofibers were separated to each other by thin strands of fibrovascular tissue, which resulted in a low fibrovascular index in 1-year-old rats.

In AL-fed rats, muscle degeneration was observed in a few rats at histopathological examination (Figures 1 and 2) and progressed between 1 and 2 years. This change was characterized by the presence of discrete foci of vacuolated and sometimes eosinophilic muscle fibers, associated with mononuclear cell infiltrates and fibrosis in the skeletal muscle sections. Between 1 and 2 years, the number of type-2 myofibers decreased (p ≤ 0.05) while the fibrovascular index markedly increased (p ≤ 0.05). These observations correlated with the decreased (p ≤ 0.05) muscle weights observed during the same interval. Very slight to moderate sciatic nerve degeneration affected 79% of 2-year old animals.

In DR-fed rats, no histopathological changes were observed at 2 years and the fibrovascular index remained unchanged (Figure 3). The mean number of type 1 and type 2 myofibers were decreased (p ≤ 0.05), but their mean area increased (p ≤ 0.05), resulting in similar mean skeletal muscle areas. In 2-year-old animals, sciatic nerve degeneration occurred with similar incidence (83%) but lower severity (very slight to slight) than those observed in the AL-fed group.

DR-Related Changes

After 1 year, the fibrovascular index was similar in both AL and DR groups. Type-1 and type-2 myofiber areas were smaller in DR than in AL-fed rats, resulting in decreased (p ≤ 0.05) muscle areas and weights.

After 2 years, in AL rats as compared to DR rats, the fibrovascular index increased (p ≤ 0.05) while type-1 and -2 myofibers and as well as total skeletal muscle areas decreased (p ≤ 0.05).

Discussion

In the present study, a decrease in skeletal muscle weight was observed in AL-fed rats between 1 and 2 years and was associated with a decrease in the number and size of type-2 myofibers. Because the body weight increased during the period, the skeletal muscle weight expressed as percentage of body weight was lower in 2-year-old than in 1-year-old animals. These results are supported by published findings (Larson, 1983; Brown, 1987; Carlson, 1995) as well as data obtained in Wistar rats, which indicate that the marked decline in muscle strength seen with aging in this strain is associated with a reduction in muscle weight, number of myofibers (Tauchi et al., 1971) and mean fiber size (Cook et al., 1992; Shorey et al., 1992). Of note, is also the substantial decrease in the number of muscle fibers observed, in particular in the vastus lateralis muscle, in humans from birth to old age (Ansved and Larsson, 1989; Lexell, 1995). In the present study, the decreased number of type-2 myofibers, which represent the majority of myofibers in the vastus lateralis muscle, was associated with an increase in the fibrovascular tissue in 2-year-old AL rats. This observation correlated with previous findings, which demonstrated that myocyte degeneration associated with cellular infiltration and multifocal fibrosis develops similarly in the myocardium of male AL-fed SD rats between 1 and 2 years of age (Kemi et al., 1998). Sarcoplasmic degeneration and replacement of myofibers by fat and connective tissue with aging has been reported in various mammals including man (Larsson, 1982) and noted in both slow and fast skeletal muscles (Kovanen et al., 1987b).

Multiple etiologic factors have been evoked as possible causes of this degenerative process, including metabolic, neurogenic, and vascular changes. It is known that the rats fed AL are either prediabetic or diabetic as they age. In AL-fed rats, pancreatic beta cell hyperplasia and progressive fibrosis of enlarged islets develop with aging (Molon-Noblot et al., 2001). These morphological alterations, associated with obesity, hyperglycemia, hyperinsulinemia, and increased insulin resistance, result in the development of non-insulin dependent-diabetes mellitus (NIDDM) (Larsson et al., 1997; Ahuja et al., 1987). Therefore, the skeletal muscle effect could be secondary to the metabolic effect of age-related hyperinsulinemia and/or insulin resistance. Also, the skeletal muscle is the major repository of free amino acids and protein pools in the body (Nair, 1995). The turnover of proteins of the sarcoplasmic reticulum is altered in aged skeletal muscle (Ferrington et al., 1998), resulting in a generalized progressive decrease in total protein synthetic activity (Pluskal et al., 1984; Goldspink et al., 1987).

Studies in various strains of rats and mice have shown that this decline in protein synthesis capability with age is associated with a decline in anabolic hormone concentrations (Florini et al., 1991; Sonntag et al., 1999). Part of the reduction in plasma IGF-1 and protein synthesis with age results from a reduction in GH secretion from the anterior pituitary gland (Sonntag et al., 1999). GH injections to elderly patients increase muscle mass and reduced fatty tissue (Rudman et al., 1991). However, in SD rats, although IGF-1 serum levels decreased between 1 and 2 years, the pituitary GH secreting cell volume remained stable (Molon-Noblot et al., 2003). Therefore, tissue resistance to the action of GH may be a contributing factor to the low IGF-1 concentration and the decreases in growth hormone receptor signal transduction might also contribute to the decline in IGF-1 gene expression with age (Xu et al., 1995). In addition, the decline in trophic hormone concentrations and protein synthetic activity result in decreased capacity for maintenance and repair of vascular tissues (Hutchins et al., 1996).

In many species including humans, alterations in the morphology of arteriolar vasculature occur with aging and result in decreased blood flow and diminished skeletal muscle performance. In Fisher 344 rats, a significant reduction in skeletal muscle contractile force is associated with a decreased blow flow between 1 and 2 years (Cook et al., 1992). In the old Brown Norway rat, a reduction in the numbers of arteriolar endpoints and connections has also been demonstrated (Hutchins et al., 1996). This decrease in vascular tissue components, associated with a diminution of cardiovascular and microvascular control, results in muscle degeneration and altered performance. Neuronal dysfunction has also been evoked as a cofactor of sarcopenia. In humans, histopathological and electrophysiological findings support a denervation reinnervation process due to a loss of motoneurons as the major cause of the myofiber loss during aging (Larsson, 1982, 1995). In old rats, an incomplete reinnervation of previously denerved muscle fibers results in age-related decline in total muscle fiber numbers (Ansved and Larsson, 1989; King, 1994). In the present study, the decrease in type-2 myofiber numbers observed in 2-year old AL-fed rats correlated with nerve degeneration that affected most of the animals. This change could therefore have been a contributing factor to the muscle degeneration and fibrosis observed at 2 years in this group.

As expected, the skeletal muscle weights and myofiber sizes were smaller in DR rats than in AL rats. However, when expressed as percentages of body weights, the muscle weights and muscle areas were higher in DR rats than in AL-fed rats. This demonstrates that decreases in muscle weights in DR rats correlated with decreases in body weights and that DR had no adverse effect on the skeletal muscle. Previous studies had demonstrated that chronic DR up to 50% of the normal food intake dramatically reduces the body weight and prolongs the life span of rodents (Lewis et al., 1984, 1985). In SD rats, the diaphragm and extensor digitorum longus muscles showed a slower postnatal development in response to marked DR (Goldspink et al., 1987). Decreased growth of myofibers in DR fed animals is thought to result from a decrease in pituitary hormone secretion (Shorey et al., 1992). In a similar 2-year study, the decrease in body weight observed in DR SD rats correlated with a decrease in GH secreting cell population in the pituitary gland (Molon-Noblot et al., 2003).

Although marked DR resulted in decreased skeletal muscle weights and myofiber sizes it maintained the normal morphological organization of the skeletal muscle. These observations were in accordance with previous findings, which demonstrated that DR had a similar protective effect on the spontaneous cardiomyopathy in male SD rats (Kemi et al., 1998). The protective effect of DR on the skeletal muscle structure has been related to its effect on protein turnover as marked (50%) DR decreases protein synthetic rates (El Haj et al., 1986; Goldspink et al., 1987; Aspes, 1997). This effect was more effective in fast-twitch than in slow-twich muscles. DR also prevented age-related vascular changes: in old DR rats the arteriolar density and anastomotic connection were similar to those observed in young animals (Hutchins et al., 1996; Lynch et al., 2003).

The preventive action of DR on sarcopenia could also result from a general effect on the oxidative metabolism. During the aging process and particularly under AL feeding, oxidative damages increase while antioxidant defense systems decrease (Laganiere and Yu, 1989; Masoro, 1996). The skeletal muscle is particularly vulnerable to accumulative oxidative damage (Luhtala et al., 1994), and it is admitted that oxidative stress in this tissue may contribute to the development of sarcopenia (Weindruch, 1996). In DR rats, previous studies demonstrated that the biochemical markers of oxidative stress, including lipid peroxidation products, protein oxidation products and superoxide dismutase activity were decreased, and cytoprotective hepatic glutathione peroxidase activity was increased (Yu, 1995; Adams et al., 1996). These beneficial DR effects on cell metabolism were associated with decreased incidence and severity of pathophysiological changes in 2-year-old animals.

Conclusion

Long-term AL feeding of SD rats resulted in sarcopenia as evidenced by histopathological and histochemical changes in the vastus lateralis muscle. Marked diet (caloric) restriction had a beneficial effect by preventing or delaying the onset of skeletal muscle degeneration and fibrosis, thus contributing to the increased survival of DR animals observed in this study. The results obtained herein demonstrated that marked DR maintained the normal morphological organization of the skeletal muscle, without evidence of adverse effects significative of malnutrition.