The visualization of myosatellite cells in normal and denervated muscleA new light microscopic staining technique.код для вставкиСкачать
THE ANATOMICAL RECORD 196~373-385(1980) The Visualization of Myosatellite Cells in Normal and Denervated Muscle: A New Light Microscopic Staining Technique ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS Departments ofdnatomy and Physiology, Boston Uniuersity School of Medicine, Boston, Massachusetts 02118 ABSTRACT A new light microscopic staining technique allows the visualization of satellite cells on the surface of myofibers. Either prior to or during fixation, whole frog sartorius muscles are bathed in an acidic buffered solution containing lead nitrate and subsequently exposed to ammonium sulfide. The staining of the satellite cells resulting from this procedure reveals their positions, and the outlines of their cell processes which occasionally branch. Electron microscopy shows that the staining is due to lead deposits localized between apposing membranes of satellite cells and associated myofibers. Prior exposure to N-ethyl-maleimide (NEM) does not alter the formation of the lead deposits on the satellite cell, but reduces the amount of Pb deposits on the muscle surface and connective tissue. This technique has been applied to determine the effects of denervation on the satellite cells of frog sartorius muscles. Four weeks after denervation, the number of satellite cells is essentially the same in both denervated muscles and the intact muscles of the contralateral side. However, denervation results in a subpopulation of satellite cells with altered shapes. They have elongated cytoplasmic processes which often branch. It is suggested that these supernumerary cytoplasmic processes represent a n intermediate phase in the transition of satellite cells to myoblasts. Mauro ('61) first noticed the satellite cell in a n electron microscopic study of frog and rat striated muscle, and Ishikawa ('66) reported the presence of the satellite cell in early postnatal and mature human muscle. Since its discovery, the satellite cell has been implicated in numerous physiological and pathological events, including muscle development and growth (Allbrook et al., '66, '71; Church, '69; Hellmuth and Allbrook, '73; Ishikawa, '66, '70; Moss and Leblond, '70, '71; Muir et al., '65; Schultz, '76; Shafiq et al., '681, response to injury and regeneration (Carlson, '73; Church et al., '66; Conen and Bell, '70; Kasper et al., '69; Mastaglia et al., '75; Price et al., '64; Fkznik, '69a,b, '73, '76; Schmalbruch, '76, '77; Shafiq, '70; Shafiq and Gorycki, '65; Shafiq et al., '67; Snow, '77a,b, '78; Teravainen, '70; Walker, '63), diseases of human skeletal muscle (Aloisi,'70; Carlson, '73; Conen and Bell, '70; Laguens, '63; Mastaglia and Kakulas, '70; Mastaglia et al., '70; Shafiq e t al., '67; Van Haelst, '70; Wakayama, '76) and response to denervation (Aloisi, '70; Aloisi et al., '73; DeRecondo et al., 000-3276X/80/1964-0373$02.60 0 1980 ALAN R. LISS, INC. '66; Hess and Rosner, '70; Lee, '65; Schultz, '74, '76, '78). The response to denervation has been described as changes in the cytoplasm of satellite cells in growing (Schultz, '78) and adult (Chou and Nonaka, '77; Hess and Rosner, '70) muscles and as a n increase in satellite cell population (Aloisi, '70; Aloisi et al., '73;Hess and Rosner, '70; Ontell, '74). All of the studies cited above had to rely on electron microscopic techniques, so that without reconstructions made from serial thin sections, they could not provide a complete image of the form of the whole cell, and especially of its extensive cytoplasmic processes. Neither was this possible through the use of a light microscopic staining technique described by Ontell ('74) that allows the identification and visualization of only the nuclei of these cells. Clearly, a method for visualizing the complete myosatellite cell in light microscopic preparations would greatly accelerate the Received November 20, 1978; accepted August 29, 1979 373 374 ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS elucidation of a number of basic questions in both muscle biology and pathology. A purpose of this present article is to report such a method. Another purpose of this paper is to describe changes in satellite cell morphology seen after muscle denervation. MATERIALS AND METHODS The experiments were performed on the frog sartorius muscle ofRanapipiens (northern and southern species, Carolina Biological and Connecticut Valley Supply Companies). Mature animals were used to assure that all mononucleated cells underlying the external lamina were myosatellite cells and not mast cells, fibroblasts, or other unclassified cells found to be contained by the external lamina in neonatal muscle (Ishikawa, ’66; Ontell, ’77). Animals were used shortly after arrival in the laboratory; when frogs had to be kept in captivity for long periods (as in the denervation experiments), they were frequently fed live tenebrio larvae. The sartorius muscles were dissected under a low power stereo microscope while bathed in amphibian Ringer’s solution (NaC1, 118 mM; KCl, 2.8 mM; CaCl,, 2.8 mM; Tris buffer, 5.0 mM, pH 7.4).Dissectedmuscles were then pinned (minuten pins) to the bottom of transparent Sylgard (Dow Corning, Midland, Michigan) chambers, where they were incubated, fixed, stained, examined under the light microscope, and photographed. Similar chambers were used to prepare the specimens for electron microscopy. The chambers were made by pouring 3 ml of uncured Sylgard mixture on a 1007 Falcon plastic Petri dish. Care was taken to avoid muscle damage during dissection. Damage of the sarcolemma results in the entry of large quantities of Pb, which upon exposure to (NH,),S obliterates visualization of any cell surface structures. A . Light microscopy The following method gave the clearest and most consistent results. The sartorius muscle was dissected and then incubated for 30 min in Ringer’s, buffered with 5 mM Tris, pH 6.15, containing 1mM NEM. Incubation in Ringer’s a t pH 6.15 reduces the formation of insoluble lead hydroxide precipitate during Pb incubation, while the addition of NEM prevents the formation of granular Pb precipitates associated with the muscle surface and connective tissue. After incubation, the tissue was briefly washed (5 sec) in a solution of 115 mM sodium cacodylate (NaCaco) buffer, pH 6.15 (titrated with nitric acid), to decrease the concentration of free chloride ions. This wash was followed by incubation in a 60 mM solution of lead nitrate (Pb(NO,),) in 70 mM NaCaco, pH 6.15, for 30 min. (The Pb incubation medium was prepared by the addition of NaCaco to a solution of Pb(NO,), in bidistilled water. Attempts to raise the pH of this solution above pH 6.15 resulted in the formation of lead hydroxide, insoluble white precipitate.) The Sylgard chamber holding the muscle was then inverted and immersed in the incubation medium to prevent deposition of any insoluble precipitates formed in the bulk solution during incubation. Furthermore, the incubation container was covered to reduce exposure t o atmospheric CO, which might result in the formation of insoluble carbonate salts. Next, the tissue was briefly washed (5 sec) in a 115 mM NaCaco buffer solution, pH 6.15, and fixed in 2.5% glutaraldehyde and 115mMNaCaco, pH 7.4, for 2 hr a t room temperature. Following fixation, the muscle was exposed to a solution of 1.1%ammonium sulfide [(NH,),S] in 115 mM NaCaco for 10-20 sec, depending upon the density of the reaction product (lead sulfide) produced. The muscle was then washed in NaCaco buffer, pH 7.4, and viewed under the light microscope. B. Electron microscopy Muscles were dissected and incubated as described above, except t h a t the incubation period in the Pb(NO,), solution was varied between 15 to 30 minutes, and the concentration of Pb(NO,), was reduced to between 15mM and 60 mM. The purpose of these changes was to find the conditions that resulted in the best morphological preservation and that produced adequate quantities of Pb deposits between the apposing membranes. The muscles were then fixed in 2.5% glutaraldehyde in a buffer solution of 115 mM NaCaco at pH 7.4. After 2 hr of fixation a t room temperature, specimens (2 mm3)containing nerve terminals were cut from the sartorius muscle, postfixed in 29’0 OsO, in 115 mM NaCaco buffer, pH 7.4, for 2 hr, dehydrated in graded alcohol solutions, and embedded in a n Epon 812-Araldite mixture. For orientation, thick sections (0.5 pm) were mounted on glass slides and stained in 0.5% toluidine blue-0, or according to the method of Ontell (‘74),and after trimming the block face, thin sections were cut with a diamond knife. (Ontell’s method consists of staining with basic fuchsin in methanol, followed by azure 11, methylene blue, Na,CO,, and methyl alcohol.) The thin sections were mounted on bare grids and stained with uranyl acetate and lead citrate before examination in an AEI-6Belectron microscope. 375 MYOSATELLITE CELLS C.l. Concentrations of PbCNOJ, in the incubation medium for light microscopy To determine the Pb(NO,), concentration needed for optimal staining during incubation, the Pb(NO,), was varied between 30 mM and 90 mM. Dissection, incubation, fixation, and exposure to (NH,),S were performed as in method A. C.2. Concentrations of PbWO,), in the fixative medium for light microscopy Concentrations between 30 mM and 90 mM Pb(NO,), in 2.5% glutaraldehyde buffered with 70 mM NaCaco at pH 6.15 were tested for optimal staining. Muscles were dissected in normal Ringer’s, pH 7.4, rinsed briefly in a solution of 115 mM NaCaco buffer, pH 6.15, and fixed for 2 h r without prior incubation. disturb the vasculature of the hindlimb. The skin incision was sutured with 5.0 silk. Four weeks post denervation, the animals were sacrificed and the sartorius muscles were dissected and stained accordingto method A. Microscopic examination of each denervated muscle showed degeneration of the distal segment of the sartorius nerve and absence of reinnervation. The intact sartorius muscles of the contralateral limbs served as controls. RESULTS C.5. Incubation and fixation with Pb(NOJ, buffered by Bis Tris To obtain a stable Pb buffered solution a t pH 7 . 2 , i t was necessary to dissolve 15 mM Pb(NO,), and 100 mM Bis Tris in two separate volumes of bidistilled water with the addition of the Bis Tris solution to the Pb(NO,), solution immediately before use. For incubation with Pb and Bis Tris prior to fixation, method A was followed with a Bis Tris buffered wash, pH 7.2. Fixation with Pb and Bis Tris plus 2.5% glutaraldehyde was performed as in method C.2., with a Bis Tris buffered wash, pH 7.2. Light microscopic observations The spindle shaped entities (Figs. 1-4) revealed by Pb salts lie on the surface of the muscle fibers. As shown below, by means of the electron microscope, these spindle shaped entities are Pb-stained satellite cells. These cells usually occur individually, so that few of them are found in close proximity to each other (Fig. 4). The majority of satellite cells lie parallel to the lengths of the muscle fibers (Figs. 3, 4; Table 1).They are mostly fusiform in shape with a n elongated, central nuclear region and one cytoplasmic process (Figs. 1,2,4)projecting from each pole of the cell body. These cytoplasmic processes are usually of similar length. Figure 3 shows a cell that is unusual, being asymmetric in shape and having one large cytoplasmic process. In the frog sartorius muscle, the satellite cells are approximately 40 to 90 p m in length and 6 to 10 pm in their widest transverse diameter. The number of cells that can be seen on the femoral surface of Pb-stained sartorius muscles using a 40 x water immersion objective is remarkably constant, with counts of satellite cells on four control muscles ranging between 75 and 100 cells. It should be noted that after exposure to (NH4),S,the muscle fibers vary in color from yellowish to light brown. These slight variations in color are noted within individual fibers and from one fiber to another. Muscles exposed to (NH,),S without prior exposure to Pb result in the fiber acquiring a yellowish color. D . Deneruation The effect of muscle denervation on the morphology of satellite cells was studied in frog sartorius muscles. Frogs were anesthesized by immersion in a 0.1% solution of tricaine methanesulfonate (Finquel, Ayerst Laboratories). Denervation was accomplished under the dissection microscope by removal of a 10-20 mm section of the sartorius nerve, taking care not to Electron microscopic identification of lead stained cells Satellite cells of mature muscle are mononucleated and contained beneath the external lamina of the myofiber (Figs. 5-7). The external lamina does not intrude between the apposing cell membranes (sarcolemma and satellite cell membrane). Associated with both membranes are numerous caveolae with lucent contents opening to the intercellular space (Figs.5, C.3. Duration of Incubation To determine the optimum duration of incubation, muscles were incubated in 60 mM Pb(NO,,),for 5-45 min. Dissection, incubation, fixation, and exposure to (NH,),S were performed as in method A. C.4. Buffers Various buffers were tested with the purpose of creating a more physiologically compatible incubation medium (increasing the pH and removing the arsenic based NaCaco). PIPES, ADA, ACES, MES, BES, MOPS, and Bis Tris were tested for their ability to buffer a 15 mM concentration of Pb(NO,), a t pH 7.2 376 ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS MYOSATELLITE CELLS TABLE I . Percentages of 357 cells showing varying degrees of deviation from the longitudinal axes of myofibers measured in the region of the cell body (Measurements done on photographs using a protractor) Control Denervated 0"-3" 4"-lo" 11"-30" 91.5 91.4 4.5 4.0 2.7 5.9 7, 8). The plasmalemma of the satellite cell is separated from the sarcolemma by intercellular space, 15 to 20 nm wide. Lead deposits are located between the apposing membranes of the satellite cell and myofiber, and within numerous caveolae extending from these membranes (Figs. 7,8).The cleft deposits do not form a continuous layer between the apposing membranes but appear as sporadic dense patches. All satellite cells, including cytoplasmic processes examined with the electron microscope after Pb(NO,), exposure, exhibited these electron-dense deposits. Other mononucleated cells present in muscle, such as fibroblasts, pericytes, and endothelial cells described by Ontell ('74) do not exhibit Pb deposits around them or in association with their cell surfaces. It should be noted that after exposure to NEM, regions of the muscle fiber continue to exhibit Pb deposits associated with the external lamina and associated collagen matrix; however, their quantity is reduced. Occasional membranous whorls are present between apposing membranes of satellite cells and myofibers (Fig. 6, arrow). These whorls decrease in size and number with decreasing periods of Pb(NO,), incubation. Effects of p H , buffer, and P b concentration To avoid the precipitation of Pb in the incubating solution or in the fixing solution, it is necessary to use buffers that do not signifi- 377 cantly react with Pb t o form insoluble complexes. The best light microscope visualizations are obtained by incubating the muscles for 15to 30 minutes before fixation (method A) in 60 mM Pb(NO,,),in a buffer of 70 mM NaCaco a t pH 6.15 (Figs. 1-3). Higher pH values result in precipitation of Pb, while greater concentrations of lead salts (90 mM) result in intense Pb infiltration of fibers, so that it is difficult to identify satellite cells after the addition of ammonium sulfide. Concentrations lower than 60 mM Pb produce only partial staining of the cells. Adequate light microscopic visualization is also obtained with 60 mM Pb(NO,J, in a fixative solution buffered with 70 mM NaCaco, pH 6.15, with no prior incubation (method C.2.; Fig. 4). However, incubation in Pb(NO,), prior to fixation has been found t o be the method of choice because of the homogeneous lead staining of muscle fibers and the clarity of the satellite cell demarcation. It should be noted that for electron microscopy, incubation in media containing 15 to 60 mM Pb(NOJ, either before or during fixation resulted in visualization of Pb deposits (Figs. 6-8). In an effort to create a more physiologically compatible incubation medium, numerous buffers were tested (method C.4.). Empirically, it was found that with Bis Tris, i t was possible to obtain a solution of 15 mM Pb(NO,), and 100 mM Bis Tris at pH 7.2. However, no Pb deposits could be visualized with light or electron microscopy after incubation in this medium, followed by fixation and (NH,),S treatment (method C.5.). However, 30 minute incubation in Bis Tris and PB(NO,J, produces an increase in membrane clarity and flocculent appearance of the dense chromatin seen in the electron microscope. In contrast, after fixation (without prior incubation) in the presence of Pb and Bis Tris at pH 7.2 followed by ammonium sulfide (method C.5.1, numerous satellite cells and Fig. 1. Satellite cell exhibiting two cytoplasmic processes. Incubated in 1mM N-ethylmaleimide and 60 mM lead prior to fixation, followed by exposure to ammonium sulfide. Water immersion objective. x 1,000, Frog sartorius. Fig. 2. Satellite cell exhibiting two cytoplasmic processes. Incubated in 1mM N-ethylmaleimide and 60 mM lead prior to fixation, followed by a 24 hour wash in sodium cacodylate buffer before exposure to ammonium sulfide. Water immersion objective. x 1,000,Frog sartorius. Fig. 3. Satellite cell exhibiting two cytoplasmic processes. Note the variation in size and shape of the cytoplasmic processes. Incubated in 1 mM N-ethylmaleimide and 60 mM lead prior to fixation, followed by exposure to ammonium sulfide. Water immersion objective. x 1,000. Frog sartorius. Fig. 4. Two satellite cells located on one muscle fiber. Incubated in 1mM N-ethylmaleimide prior to fixation with 60 mM lead, followed by exposure to ammonium sulfide. Water immersion objective. x 1,000.Frog sartorius. 378 ALAN A. LAROCQm, AL,BERTO L. POLITOFF, AND ALAN PETERS Fig. 5. Electronmicrograph of a satellite cell fmm normal muscle. Dllated rough endoplasmic reticulum can be seen in the satellite cell cytoplasm (arrow). The cell lies in a groove beneath the muscle basal lamina. x 26,000. Frog sartorius. Fig. 6. A satellite cell from normal muscle incubated i n 1 mM NEM and 15mM lead nitrate prior to fixation. Note the presence of lead deposits within caveolae of the satellite cell and myofiber (arrowheads), and the presence of a membrane whorl between apposing membranes of the satellite cell and myofiber (arrow). X 36,000. Frog sartorius. Fig. 7. A .iatellite cell from normal muscle. The fixative contained 15 mM lead nitrate and wad buffered at pH 7.1 with lOOmM RisTris. x 36.000. Frogsartorius. Nowthe pre;ienceofPt,depositsassociatedwith thecollagenmatrix. Fig. 8 A high mapification view of the opposing membranes illustrated in Fig. 7. Note the It~alizationof lead between the apposing mernhranes 0 1 the satellite cell and parent myufiher (arrows,. A 69,UOU. Frog iurtorius MYOSATELLITE CELLS 379 380 ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS myonuclei became visible under the light microscope. Figures 7 and 8 are without ammonium sulfide. Effect of denervation on the cytoplasmic processes of the satellite cell Four weeks after denervation, the number of satellite cells counted on the femoral surface of the muscle is remarkably similar to the number of cells counted on the femoral surface of the intact contralateral muscle. Thus in two frogs, the denervated versus control counts are 105, 82, and 99,79. No mitatic activity or cellto-cell contacts are observed following denervation. For a subpopulation of satellite cells, denervation has a clear effect on the size and number of their cytoplasmicprocesses. This is shown by the observation that in denervated muscles, 22 of 187 satellite cells have more than two branches (Figs. 9, 10, 12) with a maximum of seven cytoplasmic processes observed, while the intact contralateral muscles had 3 of 178 cells with more than two branches, with a maximum of three. Statistical analysis using Fisher's exact test gives a Fisher exact probability of 0.00008. Therefore, the increase in branch formation seen following denervation is unlikely to be a chance occurrence, but rather a result of denervation. The majority of the elongated cytoplasmic processes extend parallel to the longitudinal axis of the denervated muscle fibers (Figs. 9-11) with occasional spiraling around the myofiber. For the population of satellite cells which proliferate their cytoplasmic processes as a result of denervation, a high cytoplasmichuclear ratio develops (Figs. 9-12). Supernumerary cytoplasmic processes are either attached to the cell body (Fig. 9) or to another process (Figs. 10,111. Occasional processes extend 200 pm from the cell body. DISCUSSION The present results show that incubation or fixation of striated muscle in the presence of Pb(NO&, under well defined ionic conditions, leads to the light microscopic visualization of satellite cells located on the surfaces of the muscle fibers. Electron microscopic examination of muscle treated with Pb(NOJ, under the same ionic conditions reveals that the myosatellite cells are delineated by Pb deposits contained in the cleft between the apposing cell membranes. Light microscopy This new method of demonstrating satellite cells reveals their shape, the outline of their cell processes, and their occasional branching pattern. Such features have escaped detection because previous studies on satellite cells have depended upon the use of tissue sections,rather than upon complete muscle preparations. Though our evidence is restricted to the frog sartorius muscle, it is reasonable to expect that this technique can be applied successfully to muscles of other species. The consistency in satellite cell counts per muscle suggests a precise quantitative relationship between the number of satellite cells and muscle mass. Their apparent homogeneous distribution over the muscle surface suggests that their function is not restricted to one specific portion of the muscle. Kelly ('78) has reported an increased incidence of satellite cells a t the neurojunctional region in comparison with the nonjunctional area in the rat soleus, and has suggested that the cells near the motor end plate may contribute to the repair of the synapse after injury. The difference in satellite cell dimensions reported by Muir ('70) and Muir et al. ('65) (25 pm x 4 pm) for mammalian satellite cells and the dimensions reported in this study (40 to 90 pm x 6 to 10 pm) for frog satellite cells may be due to differences in muscles, species, age, and stretch a t time of fixation. Previously, the cytoplasmic volume of the satellite cell has been reported as scanty, because the observations were made on sections through the nuclear region. These observations have resulted in the cytoplasmic nuclear volume ratio being described as low (Carlson, '73; Ishikawa, '66; Reznik, '76); however, the existence of elongated processes indicates that this ratio is higher. Numerous investigators have described the satellite cell as being situated approximately parallel t o the longitudinal axis of the muscle fiber (Carlson, '73; Reznik, '761, but the Pb staining reveals (Table 1)that a subpopulation of cells clearly deviates from this orientation. Possibly, cells that belong t o this minority do not lie in longitudinal grooves on the myofiber surface (Carlson, '73), but extend diagonally over the surface without concomitant indentation of myofibrils. The majority of satellite cells in normal frog muscle possess two elongated cytoplasmic processes. These processes project from the opposite poles of the central region and their main MYOSATELLITE CELLS Fig. 9. A satellite cell after four weeks denervation. Note the enlarged cytoplasmic processes (arrows).Water immersion objective. x 1,000.Frog sartorius. Fig. 10. A satellite cell after four weeks denervation. Note the enlarged bifurcated, cytoplasmic processes (arrows). Water immersion objective. x 1,000. Frog sartorius. 38 I 382 ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS Fig. 11. A satellite cell cytoplasmic process extends downward from the top of the field (arrows) and bifurcates at its end (arrowheads) after four weeks denervation. Water immersion objective. x 1,000. Frog sartorius. Fig. 12. A satellite cell after four weeks denervation. Note the enlarged cytoplasmic processes (arrows)marked by obvious lead precipitate. Water immersion objective. x 1,000. Frog sartorius. 383 MYOSATELLITE CELLS axis is essentially the same as the axis of the muscle fiber (Table 1).If existing satellite cells have the potential to become myoblasts (Bischoir, '74, '75; Church et al., '66;Jirmanova and Thesleff, '72; Kahn and Simpson, '74; Moss and Leblond, '70; Schmalbruch, '77; Shafiq and Gorycki, '65; Snow, '77a,b, '78), this longitudinal orientation of the cytoplasmic processes would be consistent with the arrangement of the myofilaments. Satellite cells usually occurred individually and they were never seen making contact. This might be correlated with the fact that we have not observed mitotic figures, in agreement with Aloisi et al. ('73), Ontell ('741, Reznik ('76) and Schultz et al. ('78). In contrast, Allbrook et al. ('71) and Shdiq et al. ('68) have observed mitotic figures in normal, early postnatal muscle. If mitotic figures are present, the Pb staining used in our current studies is unable to reveal them. Electron microscopy The most remarkable feature of the Pb deposits is the consistency of their presence in the intercellular cleft between muscle and satellite cell membranes, suggesting a fairly specific phenomenon. A necessary step for the production of these deposits is the entry of Pb into the intercellular space, probably by means of simple diffusion. The entry of Pb as well as lanthanum (Chou and Nonaka, '77) into both the muscle and satellite cell caveolae suggest that they are usually open to the intercellular space, at least for some time. It is not clear why the Pb deposits form patches, instead of a continuous layer. Perhaps the extracellular space between the apposed membranes is anisotropic, due to the presence of an extracellular or membrane-bound component that has low electron-density and high affinity for Pb ions; or perhaps Pb ions are preferentially precipitated in specific areas because of a localized increased biochemical activity. Lead deposit formation Prior exposure to NEM does not detectably affect the formation of Pb deposits associated with the satellite cell. This suggests that the visualization of the satellite cell is not due to binding of Pb to SH groups. Although NEM decreases the quantity ofPb deposits associated with the collagen-matrix, some Pb deposits associated with the external lamina and the collagen-matrix persist. We assume that these deposits are responsible for the variation in color seen with the light microscope after exposure to (NH,),S. Denervation Previous studies done in mammals show an increase in the number of satellite cells after denervation. Aloisi et al. ('73) studied the effects of denervation on the number of satellite cells of the soleus and the extensor digitorum longus muscles of the adult rat. They found an increase of 85% and 250% respectively in satellite cells six days after denervation. Hess and Rosner ('70) examined limb and extraocular muscles of adult guinea pigs and found an increase after seven days of denervation. Ontell ('74) reported a n increase of 500 to 610% in adult rats 21 days after denervation of the extensor digitorum longus. Presently, two general theories exist to explain the formation of myoblasts in regenerating muscle. One, that myoblasts form from preexisting myosatellite cells (Snow, '77a,b, '78) and the other, that myoblasts form from myonuclei (Mastaglia et al., '75). Our results show that the number of satellite cells in frog sartorius muscle 28 days postdenervation is the same as in the nondenervated contralateral muscle. Provided a subpopulation of satellite cells is not produced from myonuclei in the frog sartorius muscle followingdenervation, the observed lack of change in the number of satellite cells following denervation is consistent with the absence of mitotic figures and the rare occurrence of satellite cells in close proximity to each other (Ontell, '74). To explain the lack of effect of denervation on satellite cell number, it might be hypothesized that a newly formed or latent subpopulation of satellite cells is not made visible by Pb staining: Schultz ('74, '78) has reported that in denervated growing muscle, a percent of satellite cells may become detached from the myofiber and if this detachment occurs in adult frog sartorius, the Pb deposits might not be formed. Additionally, this study examines only satellite cells on the myofiber surface. It is also possible that the time course of the denervation changes of satellite cells in frog sartorius is slower than that of mammalian muscle. Verma ('79) in a short communication reports multiplication of satellite cells 38 days post denervation in the m. rectus internus ofRana exculenta. lite cells 38 days post denervation in the m. rectus internus of R a m exculenta. As shown by Pb staining following denervation, a subpopulation of satellite cells change shape. This is in agreement with the observations of Hess and Rosner ('70), who noted that, following denervation, long stretches of satellite cell cytoplasm extend away from the nu- 384 ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS clear region. In electron microscopic preparations, Schultz ('74, '78) also demonstrated a n increased number of cytoplasmic processes among a specific subpopulation of satellite cells in denervated growing muscle. The processes described by Schultz ('78) appear to be limited to the nuclear region of the satellite cell and there is no report of cytoplasmic processes extending beyond the immediate vicinity of the cell body as described above. There is a great variability in the number, size, and branching patterns of the cytoplasmic processes that are induced by denervation. The majority of the processes extend parallel to the longitudinal axis of the muscle fiber with occasional spiraling around the myofiber. The functional significance of the increased size and number of cytoplasmic processes is not readily apparent. The enlarged supernumerary cytoplasmic processes, and the resulting increase in cytoplasmic volume and organelles may represent a n intermediate phase in the cytoplasmic transition from satellite cell to myoblast, and the subsequent synthesis of muscle filaments (Chou and Nonaka, '77; Hess and Rosner, '70). If this subpopulation of satellite cells with increased size and number of cytoplasmic processes induced by denervation represents an intermediate phase in the transition from satellite cell to myoblast and if the origin of this subpopulation is from preexisting myosatellite cells, then this report is in agreement with Bischoff, '74, '75; Church et al., '66 Jirmanova and Thesleff, '72; Kahn and Simpson, '74; Moss and Leblond, '70; Schmalbruch, '77; Shafiq and Gorycki, '65; and Snow, '77a,b, '78 who indicate that regenerating mononucleated myoblasts form from preexisting myosatellite cells rather than from myonuclei (Elyakova, '72; Hay, '59, '74; Hess and Rosner, '70; Lee, '65; Mastaglia et al., '75; Ontell, '74; Reznik, '69; Teravainen, '70; Yanko et al., '74). If frog myoblasts are derived from myonuclei, it is possible that the satellite cell is a n intermediate stage. Reznik ('76) suggests that, depending on the stimulus, myoblasts are formed either from preexisting satellite cells or myonuclei, and it is conceivable that both pathways might occur simultaneously. Finally, the pathway taken during regeneration may be species-dependent. We have demonstrated in this report the application of a Pb staining technique that results in the light microscopic visualization of satellite cells in normal and denervated muscles. It is reasonable to expect that this technique will be applied successfully to other questions involving satellite cells in muscle biology, development, and regeneration. ACKNOWLEDGMENTS This study was supported by NIH grants NS 11588 and CA 16778 to A.L. Politoff and Training Grant GM-01979 to the Department of Anatomy, Boston University School of Medicine. Material included in this article has been submitted by Alan Larocque in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Anatomy a t Boston University Graduate School. We thank the reviewers for their helpful comments and Ms. J.M. Henry and Ms. K.A. Larocque for their enthusiastic clerical help. LITERATURE CITED Allbrook, D.B., W.C. Baker, and W.H. Kirkaldy-Willis (1966) Muscle regeneration in experimental animals and in man. J. Bone Jt. Surg.,488:153-169. Allbrook, D.B., M.F. Han, and A.E. Hellmuth (1971)Population of muscle satellite cells in relation to age and mitotic activity. Pathology, 3r233-243. Aloisi, M. (1970)Patterns ofmuscle regeneration. In: Regeneration of StriatedMuscle and Myogenesis. A. Mauro, S.A. Shafiq, and A.T. Milhorat, eds. Excerpta Medica, Amsterdam, I.C.S., No. 218:180-193. Aloisi, M., I. Mussini, and S. Schiaffiano (1973)Activation of muscle nuclei in denervation and hypertrophy. In: Basic Research in Myology. 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