Aging has been associated with a progressive decline in cellular function, resulting in community health problems . It is well known that aging causes a major decline in skeletal muscle mass and contractile function. These age-related morphological changes are known as sarcopenia . Previous authors have studied the age-related decrease in muscular mass in animal models and have found many ultrastructural abnormalities . These changes were later attributed to a significant reduction in sarcomeres . Several studies have indicated that the functional properties of satellite cells (the skeletal muscle progenitor cells) are altered in old age  and could influence muscle size. However, the impact of aging on satellite cell population is still controversial.
It is well known that with increasing advanced, the accumulation of various macromolecules promotes the formation of reactive oxygen species . This condition, which affects the physical functioning of elderly individuals, indicates the significance of reversing this phenomenon. In order to reduce morbidities in the elderly, a recent trend of the use of herbal medicines derived from plant extracts has emerged. Therefore, natural antioxidants are increasingly being utilized to enhance the antioxidant defense of the body and protect against several diseases . Green tea, which has been gaining popularity, contains polyphenols called catechins. These catechins, epicatechin, epicatechin-3-gallate, and epigallocatechin-3-gallate (EGCG), are well known for their antioxidant properties and have been shown to be beneficial in the protection and treatment of many diseases [8,9]. The long-term intake of tea catechins with regular exercise was shown to suppress age-related declines in physical performance and energy metabolism in senescence-accelerated mice .
On the basis of the previous data, in the present work, we aimed to determine whether administration of green tea in adulthood could combat age-related changes in rat quadriceps muscle and enhance its regenerative capacity.
Materials and methods
A total of 30 female albino rats were used in this study. The animals were maintained in the animal house of Assiut University under normal conditions at an appropriate temperature and with food and water available ad libitum. The experiment was conducted according to the ethical norms approved by Animal Ethics Committee Guidelines in the Faculty of Medicine, Assiut University, Assiut, Egypt.
Each group included 10 animals weighing ~150–200g.
Group I: (control adult) included 6-month-old rats.
Group II: (aged) included 18-month-old rats.
Group III: (green tea treated) included 12-month-old rats that received green tea at a dose of 300mg/kg body weight in 1.5ml distilled water  for 6 months till the age of 18 months. Green tea was administered in the form of 1000mg tablets purchased from Mepaco-Medifood Company (Egypt). Green tea tablets were dissolved in tap water and administered daily orally by a gastric tube.
At the end of the experiment, after the last dose of green tea was administered, the animals were anesthetized with ether, their hearts were exposed, and perfusion with the fixatives was carried out.
For light microscopy: six rats from each animal group were used. Three of these rats were perfused intracardially with bouin's fluid, whereas the others were perfused with 10% formalin. The quadriceps muscle was removed bilaterally, immersed in the fixatives for 24–48 h, and then processed for the preparation of paraffin blocks. Paraffin sections were cut into 5–7 μm thickness and stained with the following stains :
H&E for assessment of the general histological structure.
Masson's trichrome stain for the study of collagen fibers.
Silver stain (Gomori's reticulin method) for the study of reticular fibers.
For electron microscopy: four rats from each animal group were perfused intracardially with 4% gluteraldehyde in cocodylate buffer and specimens were immersed in the same fixative for 24h and then postfixed in osmium tetraoxide in phosphate buffer for 2h. Semithin sections (1μm) were cut, stained with toluidine blue, and examined using a light microscope. Ultrathin sections (500–800A) were prepared from selected areas in semithin sections, mounted on copper grids, and contrasted with uranyl acetate and lead citrate. They were subsequently examined and photographed using a JEOL100 CX Japan transmission electron microscope at 80 kV at the Assiut University Electron Microscopic Unit .
Morphometrical and statistical study
Fiber cross-sectional areas (CSA) were measured using an image-analyzing system software (Leica Q 500 MCO, Gremany) at the Histology Department, Faculty of Medicine, Assiut University. The CSA was determined in H&E-stained sections at ×40 magnification in nonoverlapping fields of the entire specimen so that at least 100 fibers were measured. Five randomly chosen sections from three animals for each group were used. Fibers were not included if they were damaged or located at the edge of the muscle.
Data were expressed as mean ± SD. Comparison between groups was carried out using analysis of variance analysis, followed by a post-hoc (least significance difference) test for pairwise comparison between groups using SPSS program version 16 (SPSS Inc., Chicago, Illinois, USA). P value less than 0.05 was considered significant.
Light microscopic results
Group I: control adult group
Microscopical examination of H&E-stained longitudinal sections and semithin sections showed the typical histological appearances of skeletal muscle fibers in the form of parallel arranged, cylindrical-shaped myofibers with regular transverse striations and oval vesicular nuclei just beneath the sarcolemma (Figs 1 and 13a). Satellite cells were distinguished from myonuclei in semithin sections by their darker nuclei lying above the sarcolemma (Fig. 13a, inset). In cross sections, the myofibers were polygonal with oval peripheral nuclei (Fig. 2). In Masson's trichrome-stained sections, collagen distribution was mainly observed in the thick epimysium and thin fibrous septa forming the perimysium in between muscle bundles (Fig. 3). Silver-stained sections showed the distribution of reticular fibers, seen as irregular black lines representing thin endomysium around each muscle fiber, blood vessels, and nerves (Fig. 4).
Group II: aged group
In the elderly group, longitudinal and cross sections stained with H&E showed atrophy of some muscle fibers, focal degeneration (Fig. 5), and even myofiber loss (Figs 5 and 6). Multiple centrally placed nuclei were observed near areas of degeneration (Fig. 5). A few muscle fibers appeared rounded in cross section (Fig. 6). The increased appearance of lipid droplets in the spaces between myofibers was characteristic of semithin sections (Fig. 13b). Thickening of the epimysium and fibrous septa around muscle bundles was observed in Masson's trichrome-stained sections. In addition, dense collagen deposition appeared around blood vessels and nerve bundles (Fig. 7). Reticular fibers of the endomysium detected by sliver stain were interrupted or deficient (Fig. 8).
Group III: green tea-treated group
Examination of H&E-stained sections of rats treated with green tea showed preservation of myofiber continuity, variable diameter and staining intensity, and well-defined transverse striations. Myonuclei were peripherally located, although some were central (Fig. 9). An apparent increase in the diameter of some muscle fibers was observed in cross sections (Fig. 10). In semithin sections, satellite cells appeared with vesicular nuclei and prominent nucleoli (Fig. 13c). Epimysial and perimysial fibrous septa were observed in Masson's trichrome-stained sections more or less similar to the control group (Fig. 11). Reticular fibers were continuous around muscle fibers compared with the aged group (Fig. 12).
Electron microscopic results
Group I: control adult group
Electron microscopic examination of ultrathin sections showed muscle fibrils with alternating dark and light bands. The Z line was clearly seen as a dark line bisecting the light band. The euchromatic nucleus was under the sarcolemma. Small mitochondria were seen paired around Z lines and the larger ones were perinuclear. The sarcoplasmic reticulum appeared in the form of small vesicles surrounding the myofibrils (Fig. 14). A satellite cell was located between the basal lamina and the sarcolemma of a muscle fiber. It had an oval nucleus with peripheral heterochromatin clumps and a few organelles (Fig. 15).
Group II: aged group
Ultrastructurally, areas of myofibrillar loss and widening of the intermyofibrillar space were observed (Fig. 16a, b). The mitochondria were vacuolated with destroyed cristae (Fig. 16a). The satellite cell nucleus was shrunken with marked chromatin condensation (Fig. 17).
Group III: green tea-treated group
Treatment with green tea for 6 months revealed euchromatic myonuclei with prominent nucleoli and decreased heterochromatin clumps (Fig. 18). Numerous variable-sized mitochondria with well-defined cristae were accumulated in between myofibrils and beneath the sarcolemma (Fig. 18). They were even fused in some areas (Fig. 19). Focal irregularities of Z were scarcely observed (Fig. 18). Also, there was a minimal widening of the intermyofibrillar spaces (Figs 18 and 19), except for the wide perinuclear space in a few myofibers (Fig. 18). Satellite cells were frequently seen in mitosis (Fig. 20).
Morphometric and statistical results
The morphometric results are summarized in Table 1, where it can be seen that the aged group showed a significant decrease in the CSA of myofibers compared with the adult group. In the green tea-treated group, there was a significant increase in the CSA compared with the aged group. However, the difference was not statistically significant compared with the adult group.
The extent of age-related changes affecting skeletal muscle varied from one muscle to another, but the weight-bearing muscles were more susceptible to the senescence process than the non-weight-bearing ones . The focus of our study was the quadriceps muscle, one of the postural muscles with more type 1 red fibers . Despite the extensive literature on the geriatric changes in skeletal muscle [1,4], there are not enough data on its regeneration. The present study aimed to determine the role of green tea in alleviating age-related changes in quadriceps muscle and whether green tea can crucially influence the process of muscle regeneration.
In the current study, histological analysis of the aged group showed atrophy, degeneration, and myofiber loss associated with widening of intermyofiber spaces. Multiple centrally located nuclei were also observed, which is indicative of tissue repair by satellite cells. This would lead to the formation of new muscle fibers or myoblasts that fuse either to themselves or to the damaged myofibers. In addition, migration of macrophages for phagocytosing cell debris and proliferation of fibroblasts resulting in the production of new temporary ECM (Extracellular matrix) components were observed. In aged muscles, the repair process will result in reduced sizes of newly formed myofibers or defective repair, which may be attributed to decreased satellite-cell myogenic potential .
Our histomorphometric analysis showed a significant decrease in the mean CSA of the individual muscle fiber. These results were in agreement with those of researchers who suggested that senescent muscle atrophy could result from a reduction in the CSA of a single fiber . Some authors [1,18] have observed a progressive age-related reduction in the diameter of type 2 fibers in quadriceps femoris, biceps, and deltoid irrespective of sex, whereas others  have suggested that aging atrophy seems to be because of a reduction in both the number and the size of muscle fibers, mainly type 2 fibers. However, there is conflicting evidence on the changes in the proportion of fiber types in age-related sarcopenia .
We assume that connective tissue appearing in the form of dense collagen deposition in Masson's trichrome-stained sections of the present study compensated for the decrease in myofiber size. Previous studies have found that amino acid supplementation decreased sarcopenia and fibrosis in aged animals because of an increase in the cellular metabolism and synthesis of myofibrils . It was found that type I collagen increased and type III collagen decreased in aged rats, suggesting a more marked contribution of type I collagen to the age-related accumulation of total muscular collagen . However, others have postulated that age-associated fibrosis in skeletal muscle was not a result of increased collagen expression but was more likely because of impairment in collagen degradation .
In silver-stained sections of the present study, there was interruption in reticular fibers forming the endomysium. Although fibroblasts are the major collagen-producing cells, myofiber-associated satellite cells have also been shown to express significant levels of interstitial collagens I and III . Recent reports  have showed that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic into a fibrogenic lineage, that is, an age-related decrease in the satellite cell number or function would affect the formation of reticular fiber. Interestingly, satellite cells were hardly identified in semithin sections of the aged group, which supported the later postulation. In semithin sections, fat droplets appeared prominently in the spaces between myofibers. This occurs because when regeneration fails, the fibrotic scar is infiltrated with adipocytes (fatty degeneration) . The cellular origin of fatty infiltration has recently been identified as a novel type of resident muscle cell called the fibro/adipogenic progenitor cell, which was believed to be a source of prodifferentiation signals for myoblasts during the process of muscle regeneration, and more importantly, they show a strong tendency to generate adipose cells .
In the aged group, ultrathin sections showed degenerative changes, confirming light microscopic observations as areas of myofibrillar loss and widening of the intermyofibrillar space. These observations were in agreement with previous conclusions that a decrease in the synthesis of myofibrillar proteins occurs in elderly compared with young individuals . The sarcopenia that occurred during aging can be attributed to disruption in the regulation of protein turnover, leading to an imbalance between protein synthesis and degradation. This theory is in agreement with previous studies  that have reported alterations in protein metabolism in old muscles. In the current study, mitochondrial cristae were destroyed or absent. Similar mitochondrial changes were observed . This was attributed to mitochondrial DNA mutations associated with aging and with electron transport abnormalities . Previous results have confirmed the existence of an age-associated decline in the mitochondrial function of mixed skeletal muscle, which is significantly correlated with higher levels of mitochondrial oxidative damage . Previous studies  have shown that there was a marked decrease in the volume and the number of mitochondria of old mice, leading to a decrease in the aerobic capacity of skeletal muscle.
In the present study, ultrastructural examination of satellite cells in the control adult group showed its characteristic location underneath the basal lamina surrounding each myofiber. The structural features of their nuclei are typical of quiescent cells. They contained abundant clumps of condensed chromatin, which is a marker of low nuclear activity .
Satellite cells observed in the aged group showed condensation of its nuclear chromatin; this indicates that a major role of apoptosis in earlier phases cannot be excluded. In agreement with these results, previous studies have reported decreased density of satellite cells in aged rats compared with adult rats, consistent with the impaired regenerative capacity of muscles in sarcopenia . This is why they were difficult to detect in semithin sections of the aged group in the present study.
The histological results of aged rats treated with green tea for 6 months showed improved muscle regeneration. Under the light microscope, the myofibers showed a picture that was more or less similar to that of the control group, except for the appearance of central myonuclei, which indicated regeneration . Also, morphometric analysis showed a significant increase in the CSA compared with the aged group.
In Masson's trichrome-stained sections, collagen fibers in the epimysium and perimysium were more or less similar to those of the control group. These observations were in agreement with Babu et al. , who reported that administration of green tea reduced the total collagen content in the tail tendon of diabetic rats. They reported that tea flavonoids exerted an effect on the prolyl hydroxylase enzyme (an ascorbic acid dependent enzyme), which is required to maintain the normal properties of collagen. In addition, reticular fibers present in the endomysium were continuous more or less similar to that in the control group. This observation confirmed previous postulations that satellite cells were the main contributors in collagen III secretion  and they must have been activated on treatment with green tea.
It is known that satellite cells are undifferentiated mononuclear myogenic precursor cells that have a self-renewing property and promote the generation of a population of differentiation-competent myoblasts that participate in muscle growth, repair, and regeneration . There are two populations of precursor cells: committed satellite cells, which are available for immediate differentiation without the preceding cell division, and stem satellite cells, which undergo mitosis before yielding one daughter cell for differentiation and another for future proliferation [34,36]. In the light of this concept, we suppose that active satellite cells observed in the semithin sections of the present study were dividing for future differentiation.
Examination of ultrathin sections of the green tea-treated group showed signs of improvement in age-related changes. EGCG a major polyphenol in green tea reduced the expression of a known STAT-1 proapoptotic target gene, the Fas receptor, in isolated rat heart . A recent study of Sheng et al.  reported that EGCG reduced apoptosis by inhibiting the telomere-dependent apoptotic pathway. Moreover, EGCG induced an increase in the levels of antioxidant enzymes in aged rats . The increased antioxidant activity was evidenced in our ultrathin sections by the increased size of the mitochondria and even fusion at some sites. This could be because of the division of the preexisting mitochondria, aiming to regenerate. Our postulations have been previously supported by researchers reporting that the cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria, compensating for the defects produced by various kinds of damage .
In the green tea-treated group, satellite cells nuclei were observed in different stages of mitosis, confirming our observations in semithin sections. Therefore, we suppose that one of the mechanisms of action of green tea in improving age-related changes in skeletal muscle is the activation of satellite cell proliferation.
This work complements previous studies that have shown that green tea can prevent age-related sarcopenic changes in old muscles and enhance the regenerative activity of satellite cells.
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Conflicts of interest
There are no conflicts of interest.
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