ARTICLE |
Correspondence to: Eduard I. Dedkov, Dept. of Cell and Developmental Biology, 4643 Medical Sciences II Building, U. of Michigan, Ann Arbor, MI 48109. E-mail: ededkov@umich.edu
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Summary |
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Satellite cells (SCs) are the main source of new fibers in regenerating skeletal muscles and the key contributor to extra nuclei in growing fibers during postnatal development. Aging results in depletion of the SC population and in the reduction of its proliferative activity. Although it has been previously determined that under conditions of massive fiber death in vivo the regenerative potential of SCs is not impaired in old muscle, no studies have yet tested whether advanced age is a factor that may restrain the response of SCs to muscle denervation. The present study is designed to answer this question, comparing the changes of SC numbers in tibialis anterior (TA) muscles from young (4 months) and old (24 months) WI/HicksCar rats after 2 months of denervation. Immunostaining with antibodies against M-cadherin and NCAM was used to detect and count the SCs. The results demonstrate that the percentages of both M-cadherin- and NCAM-positive SCs (SC/Fibers x 100) in control TA muscles from young rats (5.6 ± 0.5% and 1.4 ± 0.2%, respectively) are larger than those in old rats (2.3 ± 0.3% and 0.5 ± 0.1%, respectively). At the same time, in 2-month denervated TA muscles the percentages of M-cadherin and NCAM positive SC are increased and reach a level that is comparable between young (16.2 ± 0.9% and 7.5 ± 0.5%, respectively) and old (15.9 ± 0.7% and 10.1 ± 0.5%, respectively) rats. Based on these data, we suggest that aging does not repress the capacity of SC to become activated and grow in the response to muscle denervation. (J Histochem Cytochem 51:853863, 2003)
Key Words: aging, skeletal muscles, satellite cells, M-cadherin, NCAM
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Introduction |
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SKELETAL MUSCLE satellite cells (SCs) are heavily involved in regenerative processes after muscle fiber damage and are considered to be the main source of additional nuclei to growing fibers in postnatal muscles (
On the other hand, results obtained in cross-age transplantation experiments showed that there are sufficient cellular reserves to allow both old and extremely old muscles to regenerate as well as young ones if they are both placed in comparable environments (
Nevertheless, this conclusion is mainly based on the results of experiments in which the reparative response of SCs in aged skeletal muscle was initiated by the death of associated muscle fibers. However, in such circumstances, SCs might be stimulated by two factors: lack of suppressive influence from the plasmalemma of a viable muscle fiber (
For three decades, it has been known that muscle denervation devoid of massive fiber death is able to stimulate the activation of SCs in young and adult animals (
In the previous denervation experiments, the degree of SC activation and growth in skeletal muscles was analyzed by two attributes: the changes in absolute or relative number of SCs (
SC numbers have been routinely counted under high magnification based on both morphological and topographical identification of these cells by use of the electron microscope (
It has been more recently discovered that the SCs of skeletal muscle from adult rodents express at least two distinct cell adhesion molecules on their surface; NCAM, [neural cell adhesion molecule (
The present study was designed to compare the responses of SCs from 4-month-old (young adult) and 24-month-old (old) rats to permanent denervation of skeletal muscles. The relative number of M-cad+ and NCAM+ SCs was taken as a characteristic to demonstrate a level of SC activation in tibialis anterior muscles after 2 months of motor denervation. As a result of this study, we demonstrated that advanced age did not restrain the capacity of SCs to become activated in response to muscle denervation.
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Materials and Methods |
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Animals and Experimental Design
The study was conducted on eight 4-month-old (young) and six 24-month-old (old) rats of the WI/HicksCar strain. After ether anesthesia, the right sciatic nerve was surgically exposed high in the thigh and then ligated with silk in two places. The nerve was cut between the two sutures, and both stumps (proximal and distal) were implanted into muscle tissue as far away from each other as possible. The rats were treated with oral terramycin for 5 days after surgery. All operations and subsequent animal care were carried out in accordance with the guidelines of the Unit for Laboratory Animal Medicine at the University of Michigan. One and 2 months after the surgical procedures the tibialis anterior (TA) muscles were removed from both denervated and intact contralateral (control) legs of the rats, and all animals were sacrificed by an overdose of anesthetic.
Immunohistochemistry and Fluorescent Microscopy
Each denervated and control TA muscle was fixed at resting length in freshly prepared 2% paraformaldehyde in 0.1 M PBS for 24 hr at 4C and was then transversely cut in the mid-belly area. The samples were washed overnight in PBS and cryoprotected in a graded sucrose series at 4C. Muscle pieces oriented for transverse and longitudinal sectioning were placed in specimen molds containing TBS/Tissue Freezing Medium (Triangle Biomedical Sciences; Durham, NC) and were then quickly frozen in isopentane that had been pre-cooled by dry ice. Serial 8.0-µm sections were cut from each sample with a cryostat and mounted on warm glass slides. Before staining, the sections were washed in double-distilled water at room temperature (RT) and fixed in 100% methanol for 10 min at -20C. The slides were allowed to air-dry. The sections were then re-hydrated in PBS at RT, treated with 1% Triton X-100 for 5 min, washed in PBS, and then incubated with 20% normal goat serum for 10 min. The adjacent serial sections were double labeled with mixtures of different primary antibodies for 3 hr at 37C. Two pairs of antibodies were routinely used: (a) rabbit anti-NCAM affinity-purified polyclonal antibody (2.5 µg/ml; Chemicon International, Temecula, CA) and mouse anti-laminin B2 monoclonal antibody, clone D18 (undiluted supernatat; Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA) and (b) rabbit anti-M-cadherin affinity-purified polyclonal antibody [1:50; produced by Dr. Anton Wernig, Department of Physiology, University Bonn. The specificity of this antibody was previously characterized in the studies of
Quantitative and Statistical Analysis
Images from randomly selected areas of cross-sectioned in mid-belly control and 2-month denervated TA muscles (three of each muscle were used for young and old rats) were captured under the same magnification with a Zeiss Axiophot-2 universal microscope using a Zeiss Axiocam digital camera. Three discrete images of each examined area included the laminin-positive basal lamina of muscle fibers visualized with FITC, M-cad+ or NCAM+ SCs visualized with Cy3 and nuclei counterstained with DAPI were recorded using the proper filter for fluorescence. Three separate electronic images of the same microscopic field were transformed into a single composite figure using Adobe Photoshop. This technique makes it possible to clearly distinguish the M-cad+, or NCAM+ SCs, located beneath the basal laminae of muscle fibers (Fig 1). The numbers of muscle fibers (both associated and not associated with SCs) and M-cad+ (or NCAM+) SCs were calculated on each prepared figure. Only SC profiles cut through the nuclear region of the cell were used in our calculations. In total, M-cad+ (or NCAM+) SCs were collected in each muscle from a section area consisting of 1000 fiber profiles that altogether provided 3000 fiber profiles for both control and denervated muscles from young and old rats. The frequencies of both NCAM+ and M-cad+ SCs per muscle were expressed as a percentage of the ratio of the number of SCs to the number of fibers (associated and not associated with SCs). Quantitative data were analyzed with a two-way analysis of variance (ANOVA). The values are expressed as means ±SEM.
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Results |
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At the beginning of our study, the appropriate antibody systems were selected to obtain the optimal result for detection of both M-cad+ and NCAM+ SCs with the fluorescent microscope. According to our tests, the use of secondary antibodies conjugated with Cy3 compared with the application of FITC-labeled secondary antibodies showed the best results for clear visualization of the sites at which a primary antibody binds to M-cadherin or NCAM proteins, which are expressed in SCs. The rabbit anti-NCAM polyclonal antibody was a more suitable marker for distinguishing a larger number of SCs in the skeletal muscle as compared with the mouse anti-NCAM monoclonal antibody (data not shown). At the same time, the use of a mouse anti-laminin monoclonal primary antibody that was visualized with a FITC-conjugated secondary antibody represented an appropriate antibody system for differentiating the muscle fiber basal lamina.
The results of immunostaining on cross-sections of both control and denervated skeletal muscles demonstrated that polyclonal primary antibodies against either M-cadherin or NCAM protein were able to distinguish SCs located beneath the basal lamina of normal and atrophied muscle fibers. The expression of M-cadherin protein was predominantly detected in the area where a SC interfaces with a muscle fiber (Fig 1A, Fig 1C, and Fig 1E), whereas NCAM protein was expressed around the cell surface and in the cytoplasm (Fig 1B, Fig 1D, and Fig 1F).
Considering the fact that SCs undergo activation in response to muscle denervation, their shape and distribution of SCs in denervated muscle were examined prior to a quantitative analysis of their number. Immunostaining on longitudinal sections revealed that SCs labeled for either M-cadherin or NCAM were commonly irregularly situated on the surface of muscle fibers, in such a way that some part of a muscle fiber might demonstrate two or three neighboring SCs while another part of the same muscle fiber was absolutely devoid of them (Fig 2). The majority of SCs in denervated muscles displayed an enlarged nuclear region and one or two slender extensions of cytoplasm that stretched along the longitudinal axis of the muscle fiber, sometimes for a long distance (Fig 3).
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Some of the SCs in a denervated skeletal muscle co-expressed both M-cadherin and NCAM proteins. This was demonstrated by the use of double labeling with a rabbit anti-M-cadherin polyclonal antibody and a mouse anti-NCAM monoclonal antibody on the same section (Fig 4). However, the application of this co-staining technique for detection and counting of SCs has a major disadvantage because of its inability to appropriately label the muscle fiber basal lamina. The absence of laminin-positive fiber profiles on cross-sections of skeletal muscle made it extremely difficult to determine accurate topographical relationships between muscle fibers and other cells that might be stained for either M-cadherin or NCAM, or for both M-cadherin/NCAM proteins.
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The fact that for calculating SC number laminin staining was mandatory on each muscle section labeled made it impossible to simultaneously immunostain the same section for both adhesion molecules. In addition, because SCs, particularly in a denervated muscle, stretch along fibers for a long distance, only SCs cut only through the nuclear region, as verified by the presence of a DAPI-labeled nucleus, were counted. These limitations made the separate application of anti-M-cadherin and anti-NCAM antibodies on adjacent serial cross-sections necessary. This technique at least allowed the discrete counting of the two "subpopulations" of SCs, which were stained positively for either M-cadherin or NCAM proteins in the same regions of the muscles (Fig 5).
The calculation of SC numbers, which was carried out on adjacent serial cross-sections, revealed that in both young rats and old rats the numbers of M-cad+ SCs were always larger than those of NCAM+ SCs for both control and denervated TA muscles (Fig 6). The percentages of M-cad+ SCs in control TA muscles from 6-month-old and 26-month-old rats were 5.6 ± 0.5% and 2.3 ± 0.3%, respectively, whereas the percentages of NCAM+ SCs in the same muscles were 1.4 ± 0.2% and 0.5 ± 0.1%, respectively. These results confirmed the reduction of the SC population in old compared with young muscle. In addition, these showed that the antibody against M-cadherin protein was able to detect a larger number of SCs than was the antibody against NCAM protein.
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Despite the fact that the numbers of M-cad+ and NCAM+ SCs were always greater in young control muscles than those in old control muscles (Fig 6), 2 months after chronic denervation the number of SCs in both cell "subpopulations" demonstrated significant increases and reached a relatively similar level among young and old rats. Specifically, the percentages of M-cad+ and NCAM+ SCs in 2-month denervated TA muscles from 6-month-old rats were 16.2 ± 0.9% and 7.5±0.5%, respectively, compared to 15.9 ± 0.7% and 10.1 ± 0.5% of those cells, respectively, in 2-month denervated TA muscles from 26-month-old rats. Therefore, these data showed that advanced age did not restrain the capacity of SCs to become activated and to grow on living fibers in response to muscle denervation.
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Discussion |
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In this study, we demonstrated that (a) the expression of M-cadherin protein allows one to recognize a larger number of SCs in control and denervated skeletal muscles from both young and old rats than does the expression of NCAM protein, (b) the number of M-cad+ and NCAM+ SCs in control TA muscle from young rats exceeds that in old rats, and (c) 2 months after permanent denervation of the TA, the muscle numbers of M-cad+ and, to a lesser degree, NCAM+ SCs achieve a significant increase to levels that are comparable between 6-month-old and 26-month-old rats.
Is It Proper to Use M-cadherin or NCAM Expression as a Solitary Marker of Quiescent and Activated Satellite Cells in Adult Murine Skeletal Muscle?
Since skeletal muscle SCs were discovered, the standard and most precise method for quantitative detection of these cells has been to electron microscopy (
M-cadherin and NCAM are two distinct transmembrane glycoproteins that mediate homophilic cell adhesion in skeletal myogenesis (
Although the functions of M-cadherin, and NCAM, have been methodically studied in myotube formation in vitro and during myogenesis in vivo, their role in SCs of adult muscle remains poorly elucidated. This thought raised the question of whether each of these two cell adhesion molecules would identify the same quiescent and activated SCs in mature skeletal muscle.
According to our results and those of other authors (
Ultrastructural evidence previously obtained in denervated skeletal muscles has demonstrated that some activated SCs could be deeply embedded in muscle fibers (
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Furthermore, taking into account the fact that NCAM on the surface of the cell is competent to alter cell mobility (
M-cadherin itself most likely contributes to the attachment of quiescent and activated SCs to the surface of muscle fibers, supporting their alignment along the longitudinal axis of normal and denervated fibers. This idea is supported by data of
Despite the fact that we could identify SCs that were positively stained with either anti-M-cadherin or NCAM antibodies, the question remains whether the entire SC population in normal and denervated skeletal muscles can be distinguished by this technique. It has been definitely shown by studies of
Taking into consideration our results and those of other authors, we suggest, that without further verification, immunostaining with antibodies against either M-cadherin or N-CAM cannot be assumed to be solitary markers to demonstrate the entire SC population in either normal or denervated skeletal muscles in the rat. We also propose that expression patterns of M-cadherin and NCAM proteins detected in an individual SC most likely reflect the momentary instant functional status in a cell that is dynamically able to change in response to novel local or systemic demands. Nevertheless, we suggest that the simultaneous examination of these cell adhesion proteins with the goal of SC counting would be justified if it is used to compare the number of M-cadherin and/or NCAM "cell subpopulations" calculated in different muscles under similar experimental conditions.
Does Aging Have a Profound Impact on a Population of SCs: from a Decline in Number to an Essential Alteration in Growth Potential?
According to our results and those of other authors (
These results raise the question of whether the decrease of SCs is a reflection of the influence of a deteriorating microenvironment at the tissue and organ levels or if it may be the execution of an unavoidable age-associated intercellular program. In our study we made no attempts to methodically distinguish between these two options, but we specifically designed our experiments to compare the capacity of distinct SC populations from young and old rats to respond to a similar stimulus (2 months of denervation) under different (young and old) local and systemic environments.
Previous experimental results from our (reviewed by
However, the regenerative response of SCs in these experiments has always occurred in response to the degeneration of muscle fibers that would be obligatorily associated with an enormous release of mitogens (
The question has arisen what conditions may permit SCs to become activated in the absence of death of the associated muscle fibers. It has been clearly shown that SCs situated on living muscle fibers leave their quiescent status and become activated in response to muscle denervation (
The data of
In conclusion, our data suggest that even though aging affects the number of SCs and probably their initial response to an activating stimulus, the intrinsic overall growth potential of the entire SC population after denervation remains comparable between young and old skeletal muscles, regardless of their local and systemic environment. These results on the reaction to denervation correspond to an earlier report by
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Acknowledgments |
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Supported by NIH grant PO1 AG-10821 and by a grant from the European Union.
Received for publication October 21, 2002; accepted February 19, 2003.
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