THE ANATOMICAL RECORD 237:345-357 (1993) Structure, Innervation, and Age-Associated Changes of Mouse Forearm Muscles MICHAEL R. CARRY, STEVEN E. HORAN, SHELLEY M. REED, AND ROBERT V. FARRELL Departments of Cellular and Structural Biology (M.R.C.) and Neurology (S.E.H., S.M.R., R.V.F.), University of Colorado Health Sciences Center, Denver, Colorado 80262 ABSTRACT In spite of a decline in muscle strength with age, the cause of the overall decrease in motor performance in aged mammals, including rodents, is incompletely understood. To add clarity, the gross organization, innervation, histochemical fiber types, and age-associated changes are described for mouse forearm muscles used in a variety of motor functions. The anterior (flexor) and posterior (extensor)forearm compartments have the same arrangement of muscles and gross pattern of innervation as the rat. Two primary histochemical fiber types, fast/oxidative/glycolytic(FOG) and fast/glycolytic(FG),with characteristic hitsochemical staining patterns were observed in all forearm muscles. Additionally, there was a small population of slow/oxidative(SO) fibers confined to the deep region of a single muscle, the flexor carpi ulnaris (FCU). Between 18 and 26 months the FCU muscle displayed fibers with morphological features distinct from earlier ages. Fibers displayed a greater variation in size, a loss of their uniform polygonal shape, and a dramatic increase in clumps of subsarcolemmal mitochondria, lysosomes, and lipofuscin granules. Many of the fibers had a distinctly atrophic, angular shape consistent with recent denervation. Morphometric analyses of the FCU’s source of innervation, the ulnar nerve and one of its ventral roots (CS), were consistent with the denervation-like changes in the muscle fibers. Although, there was no net loss of myelinated axons between 4 and 26 months of age, there was a significant increase in the density of degenerating cells in both the ulnar nerve and ventral root C8. 0 1993 Wiley-Liss, Inc. Key words: Muscles, Peripheral nerves, Spinal nerve roots, Aging, Histochemistry, Mice It is well established that performance on motor tasks, dependent in part on muscle strength, decreases with increasing age for all mammals including humans (Larsson, 1982; Moore, 1975; Mortimer et al., 1982; Schaumberg et al., 1983; Welford, 1982). Although a variety of mechanisms may contribute to the decrease in muscle strength, it appears to be associated with a loss of muscle mass with increasing age (Faulkner et al., 1990). This senile muscle atrophy may be due either to a primary atrophy of muscle fibers or secondary to a primary loss of motor innervation to the muscle. Observations on muscle from rodents and humans support the thesis that a number of denervation-related mechanisms may cause the loss of functional limb muscle fibers with increasing age. Senile limb muscles from humans (Grimby et al., 1982) and rats (Bass et al., 1975; Caccia et al., 1979; Fujisawa, 1974; Van Steenis and Kroes, 1971) display the histopathological changes (angular fibers, type grouping, and group atrophy) consistent with cycles of denervation and re-innervation. Although not always demonstrating group atrophy, 0 1993 WILEY-LISS, INC. both rat (Basset al., 1975; Caccia et al., 1979; Eddinger et al., 1984,1985; Kugelberg, 1976; Marlin et al., 1985) and hamster (Goldspink and Ward, 1979) limb muscles commonly display a shift in the relative percentages of fiber types with increasing age. These changes may be limited to specific muscle groups (Holloszy et al., 1991) and related to long-term cycles of denervation and re-innervation, or to the loss of specific fiber types (Larsson and Ansved, 1988). Although it is technically difficult to demonstrate that fiber loss following denervation is responsible for senile muscle atrophy, the loss of muscle fibers with age has been directly demonstrated for small rodent muscles (Arabadjis e t al., 1990) and implicated in large human muscles (Grimby et al., 1982). Received December 29, 1992; accepted June 9, 1993. Address reprint requests to Michael R. Carry, Ph.D., Department of Cellular and Structural Biology, B - I l l , University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. 346 M.R. CARRY ET AL. The denervation-related changes in senile skeletal muscle are suggestive of a loss of motor neurons or their axons with increasing age. Although morphometric analyses of most peripheral nerves and ventral roots do not provide conclusive evidence of a loss of myelinated axons with increasing age in rodents (Knox et al., 1989; Rao and Krinke, 1983), the presence of pathological alterations in myelinated axons and shifts in fiber size distribution in the peripheral nerves are highly suggestive of age-associated axonal degeneration (Cotard-Bartley et al., 1981; Gilmore, 1972; Grover-Johnson and Spencer, 1981; Kazui and Fujisawa, 1988; Knox et al., 1989; Krinke, 1983;Thomas et al., 1980). Where senile muscle and nerve have been examined together there is a corresponding loss of both muscle fibers and motor axons (Larsson and Ansved, 1988; Ansved and Larsson, 1990; Ishihara et al., 1987). Although a few studies have examined age-associated changes in forelimb muscles (Rowe, 1969; Fujisawa, 1974) the vast majority have focused upon the distal hindlimb muscles and nerves of rodents active in locomotion. The overall purpose of this study was to describe the age-associated neuromuscular changes in rodent forelimb muscles active not only in the gross movements of locomotion, but also the fine movements of grooming and feeding. More specifically, we chose to 1)compare the macroscopic anatomy of the mouse forearm (antebrachial) muscles to those previously described for the rat, 2) describe the histochemical fiber types present in mouse forearm muscles, 3) describe the age-associated morphological changes in one of the flexor compartment muscles, the flexor carpi ulnaris, and 4) describe the structure and age-associated changes in the flexor carpi ulnaris’s source of innervation, the ulnar nerve and ventral root C8. MATERIALS AND METHODS Processing of Tissue-Muscle Histochemistry Female mice (23 total mice [9 histochemistry, 14 dissection and photography]), C57BL/6J; Jackson Laboratories) 4 months of age were killed by a n overdose of methoxyflurane and forearm muscles (Fig. 1)removed with the aid of a dissecting microscope. Single or multiple muscles were placed in a small, bisected cube of pork muscle, mounted on a cryostat chuck with gum tragacanth, and quick frozen in liquid nitrogen-cooled isopentane. The pork muscle was utilized to orient and support the small forearm muscles. The frozen muscles were stored at -70°C in closed containers. Ten micron serial sections were cut in a cryostat and stained with a series of histochemical procedures (menadione linked alpha-glycerophosphate dehydrogenase [men-a-GP], nicotinamide adenine nucleotide dehydrogenase tetrazolium reductase [NADH-TR], non-specific esterase, modified trichrome, and myofibrillar adenosine triphosphatase [ATPasel) following a n alkaline (pH 10.3) or acid (pH 4.45) preincubation (Engel and Brooke, 1968). Additional sections were incubated with commercially available monoclonal antibodies raised against fast-twitch (Sigma, labels type I1 isomyosin molecules in skeletal muscle [Havenith et al., 19901)or slow-twitch (Amersham Laboratories, labels human type I fibers [Moore et al., 19841) myosins. The antibodies were visualized using a biotinylated second an- tibody-streptavidin-peroxidaseprocedure (Kirkegaard and Perry Laboratories). Processing of Tissue-Plastic Embedded Nerve and Muscle Mice (21 total) from 2-26 months of age (110%of this strain’s mean life span [Storer, 19671)were killed by a n overdose of methoxyf lurane and perfused through the left ventricle with a glutaraldehyde (3.5%)-formaldehyde (0.5%) fixative (0.2 M phosphate buffer, pH 7.4). The ulnar nerve (just proximal to the elbow), ventral root C8, and flexor carpi ulnaris muscle were dissected free of their surrounding tissues and immersed in the same fixative for a total of 2 h. Tissues were rinsed in the same buffer and postfixed for 2 h in chilled 1% Os04. The tissues were subsequently rinsed in buffer, dehydrated in a graded series of EtOHs and propylene oxide, and infiltrated in plastic (Medcast-Araldite 502; Ted Pella). The following day the tissue specimens were embedded in fresh plastic and polymerized for 2 nights a t 60°C. Semithin (0.5-1.0 micrometer) sections were cut on a Porter-Blum ultramicrotome and stained (toluidine blue-methylene blue-Azure 11) for the demonstration of morphological characteristics. For electron microscopy, thin sections were stained with uranyl acetate and lead citrate and viewed with a Philips electron microscope. Muscle Fiber Morphometrics Forearm muscles were analyzed for fiber type (refer to Results section for criteria for typing fibers) percentage and fiber diameter. Every fourth microscopic field (original magnification of 86 x ) of NADH-TR stained sections was photographed and printed at a total magnification of 640 x . Fiber type percentage was based upon counts of the two major fiber types (FOG, fast/ oxidative/glycolytic; FOGc, fast/oxidative/glycolyticcoarse; FG, fast/glycolytic) for all photographic fields of a given muscle section (approximately 40 fibers per field with 4 fields per muscle section). The fiber diameter was determined by measuring the maximum width of the least diameter (Dubowitz and Brooke, 1973) for every typed fiber from the same photographic fields. Nerve Morphometrics Whole nerve and myelinated axon morphometric parameters were calculated from measures made directly on microscopic slides or on micrographs of semithin sections of plastic embedded ulnar nerves and ventral roots. Nerve area was estimated using the point-counting method (Weibel, 1979, p. 123). Micrographs with a n original magnification of 86 x were printed ( x 645 with a n overlying grid [0.5 cm spacing]). Myelinated axons were counted on micrographs printed with a total magnification of 1,012 x . The endoneurial area fraction (total nerve cross-sectional area minus myelinated cross-sectional area) was estimated using the point-counting method. Every fourth microscopic field of cross-sectioned nerve or ventral root was photographed ( x 500) using transparency film. The transparencies were projected on a grid (1.0 cm spacing) using a modified enlarger at a total magnification of 3,500 x . Points were counted over myelinated axons AGE AND CHANGES IN MOUSE FOREARM MUSCLES and the remaining endoneurial compartment. Myelinated axon diameters were measured (maximum least diameter) using the same transparencies. The number of capillaries, cell nuclei (all nuclei within the limits of the perineurium including Schwann cells, fibroblasts, mast cells, and pericytes), and degenerating cells (those cells, regardless of type, containing inclusions, large lipofuscin deposits, or myelin figures) were counted directly from the stained plastic sections at a n original magnification of 1,000 x . Statistical Analysis The various sample sizes utilized for calculating the muscle fiber, ventral root, and ulnar nerve morphometric parameters were determined by doing sequential calculations of means and standard deviations for the measures on sample sections (Muller, 1976). Means and standard errors were calculated for all morphometric variables. Statistical significance was determined by analysis of variance comparing the 4-month-old group to the other (2-, 18-23-, and 24-26-month) groups (Statview 11; Abacus Concepts). RESULTS Flexor and Extensor Compartment Muscles Six forearm flexor compartment and four extensor compartment muscles (Fig. 1) were studied for their innervation and histochemical fiber types. The flexor carpi radialis (FCR), flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS), palmaris longus (PL), and pronator teres (PT) muscles were all innervated by branches of the median nerve. The flexor carpi ulnaris and the ulnar head of the FDP were the only forearm muscles that received branches of the ulnar nerve. The four extensor compartment muscles (abductor pollicis [AP], extensor carpi radialis [ECR], extensor carpi ulnaris [ECU] and extensor digitorum [ED]) were all innervated by branches of the radial nerve. 347 plastic sections of the FCU muscle (Fig. 4). The FG fibers also stained intensely for glycolytic enzyme activity, but stained weakly for oxidative enzyme activity (NADH-TR and non-specific esterase), and lacked the subsarcolemmal deposits evident in the FOG fibers. There was a small population of a third fiber type, SO (slow/oxidative), confined to the deep region of the FCU muscle (Fig. 3). The SO fibers were the only forearm fibers that cross-reacted with a n antibody t h a t labels type I (slow-twitch) fibers. This fiber type also stained intensely for myofibrillar ATPase activity following a n acid (pH 4.45) preincubation. In general, all of the fibers in this region of the FCU stained more intensely for oxidative enzyme activity. The relative fiber type (FOG, FOGc, and FG) percentages and diameters were calculated for the ten forearm muscles (Table 2). The fibers were typed based upon their staining characteristics for oxidative enzyme activity (NADH-TR) a s described above. No distinct pattern was evident for the percentages of the FOG, FOGc, and FG fibers in the flexor forearm muscles. In the extensor compartment muscles, there were generally greater numbers of FOGc fibers with far fewer FG fibers. In general, the FG fibers had the largest and the FOGc fibers smallest mean diameters. Age-Associated Changes in the Flexor Carpi Ulnaris Muscle The flexor carpi ulnaris muscle was studied in greater detail using plastic embedded tissue specimens of female mice aged 2-26 months. The fiber types identified in cryostat sections were readily apparent in stained (toluidine blue-methylene blue-Azure 11) semithin (0.5-1.0 micrometer) plastic sections (Fig. 4A). The fibers were polygonal in shape and uniformly distributed throughout the muscle with scant connective tissue separating adjacent fibers. The nuclei were thin and fusiform shaped and often capped by a small deposit of subsarcolemmal material in the FOG fibers. Starting at 18 months the FCU muscle displayed fiHistochemical Fiber Types bers with morphological features distinct from the earTwo primary fiber types, FOG (fastloxidativelglyco- lier ages (2,4, and 12 months of age). By 26 months of lytic) and FG (fast/glycolytic), with characteristic his- age the FCU muscles (Fig. 4B) displayed a far greater tochemical staining patterns were observed in all variation in fiber size and a loss of the uniform polygmouse forearm muscles (Figs. 2, 3; Table 1). The FOG onal shape. Many muscle fibers were atrophic, includfibers were further subdivided into two populations ing some with a distinctly angular appearance. The (FOG and FOGc [fast/oxidative/glycolytic-coarse]) most consistent age-associated feature was a dramatic based upon the appearance of the intermyofibrillar net- increase in the presence of the dark staining subsarcolemmal material which often distorted the fiber cirwork in NADH-TR stained sections. All FOG and FG fibers cross-reacted with a n anti- cumference. Many of the deposits contained spherical body that labels type I1 (fast-twitch) isomyosin mole- inclusions, some of which were metachromatic in apcules in other mammalian muscles, and displayed low pearance. These deposits, comprised of aggregates of activity for myosin ATPase activity following an acid mitochondria, lysosomes, and lipofuscin granules (Fig. (pH 4.45) preincubation. The FOG fibers displayed 51, often surrounded enlarged, oval-shaped nuclei. moderately intense reactions with the NADH-TR, nonVentral Root C8 and the Ulnar Nerve specific esterase, and men-a-GP stains. The FOGc fiVentral root C8 was surrounded by a distinct peribers were distinguished by their coarse, clumped intermyofibrillar network and numerous NADH-TR neural sheath a s i t traversed its associated dorsal root reactive subsarcolemmal deposits. The remaining FOG ganglion (Fig. 6A). Myelinated axons, Schwann cells, fibers had similar subsarcolemmal deposits and fibroblasts, and capillaries were readily distinguished stained more intensely for glycolytic enzyme activity in the stained semithin plastic sections. The ventral (men-a-GPD) (Goldspink and Ward, 1979; Lojda e t al., roots increased in size with increasing age but there 1979) than the FOGc fibers. The same pattern of sub- was no significant change in the number of myelinated sarcolemmal deposits and intermyofibrillar network in axons (Table 3). There was a significant increase with FOG and FOGc fibers was evident in stained semithin age in the percentage ofmyelinated axons in two of the Fig. 1. Photographs of dissected mouse forearms displaying superficial anterioriflexor (A), intermediate anterioriflexor (B),deep anterioriflexor (0,and superficial posterioriextensor (D) muscles and nerves. AP, abductor pollicis; ECRb, extensor carpi radialis brevis. ECR1, extensor carpi radialis longus; ECU, extensor carpi ulnaris; ED, extensor digitorum; FCR, flexor carpi radialis; FCU, flexor carpi ulnaris; FDPh, flexor digitorum profundus (humeral head); FDPr, flexor digitorum profundus (radial head); FDPu, flexor digitorum profundus (ulnar head); FDS, flexor digitorurn superficialis; Mn, median pronator teres; Tr, triceps brachii; nerve; PL, palrnaris longus; IT, Un, ulnar nerve. Bar, 1 mrn. AGE A N D C H A N G E S I N MOUSE FOREARM MUSCLES 349 Fig. 2. Photomicrographs of serial cryostat sections of the flexor carpi ulnaris muscle displaying the histochemical staining properties of the FOG (fast/oxidative/glycolytic),FOGc (fastloxidativeiglycolyticcoarse), and FG (fastiglycolytic)fibers. A Nicotinamide adenine nu- cleotide dehydrogenase tetrazolium reductase (NADH-TR). B: Myofibrillar adenosine triphosphatase (ATPase) with a n acid (pH 4.45) preincubation. C Non-specific esterase. D. Menadinone linked alpha glycerophosphate dehydrogenase (men-a-GP). Bar, 50 micrometers. larger diameter ranges (7-8 and 11-12 micrometers) with an associated loss of the smallest myelinated axons (0-2 micrometers). In the oldest animals (18-23 and 24-26 months) there was a significant increase in the density of degenerating cells displaying inclusions, lipofuscin deposits, and/or myelin figures (Table 3). The ulnar nerve was unifascicular and surrounded by a distinct perineural sheath just proximal to the elbow (Fig. 6B). A wide diameter range of myelinated axons was present with the largest axons clustered near the perimeter of the nerve. The myelinated axons were separated by endoneurial Schwann cells, fibroblasts, capillaries, pericytes, and mast cells. Macrophages may have been present (particularly in the older animals) but were not readily apparent. There was a significant decrease in both myelinated axon density and endoneurial cell density with increasing age, but this was most likely due to the significant increase in nerve cross-sectional area (Table 4). Likewise the capillary density decreased with increasing age and nerve area despite a moderate increase in the number of capillaries between 4 and 24-26 months of age. There were no significant shifts in the distribution of the various myelinated axon diameter ranges with increasing age (Table 5). All of the oldest (24-26 months) mice displayed ageassociated changes in their ulnar nerves (Fig. 7). Degenerating cells containing various inclusions, lipofuscin deposits, and myelin figures occurred in all nerve sections and on occasion in the intramuscular branches Fig. 3. Photomicrographs of cryostat sections of the flexor carpi ulnaris muscle (FCU). A An ATPase (pH 4.45) stained section displaying a small population of dark staining fibers (arrowheads) located in the deep (ulnar head) region of the muscle. Bar, 1 mm. B-F Serial sections displaying the histochemical staining properties of this population of SO (slowioxidative)fibers which cross-react with an antibody directed against a slow-twitch myosin. B: Slow-twitch myosin antibody. C: Fast-twitch myosin antibody. D Non-specific esterase. E: ATPase (pH 4.45). F NADH-TR. Bar, 50 micrometers. Fig. 4. Photomicrographs of toluidine blue-methylene blue-Azure I1 stained semithin plastic sections of the flexor carpi ulnaris muscle. Bar, 25 micrometers. A A cross-section of a muscle from a 4-monthold female mouse displaying uniform sized and shaped FOG (fast/ oxidative/glycolytic),FOGc (fastioxidativeiglycolytic-coarse), and FG (fastiglycolytic)fibers. Note the thin rim of dark stained sarcolemmal material (arrowheads) in the FOG and FOGc fibers. B: A representative cross-section of a muscle from a 26-month-old female mouse displaying a dramatic variation in fiber size and shape. Thin angular (arrows) fibers are interspersed between fibers with large clumps of dark stained subsarcolemmal material (small arrowheads) and spherical inclusions (large arrowheads). 352 M.R. CARRY ET AL. TABLE 1. Histochemical properties of adult (4 month) forearm muscle fibers' Fiber type SO Histochemical Drocedure ATPase-alkaline (pH 10.3) preincubation ATPase-acid (pH 4.45) preincubation Anti-fast-twitch myosin Anti-slow-twitch myosin NADH-TR Non-specific esterase Men-alpha-GP FOG FOGc FG + ++ ++ ++ +++ + + + + + + + +++ ++ +++ + +++ ++ +++ + + + + + +/++ + + + 'SO, slowioxidative; FOG, fastioxidativeiglycolytic;FOGc, fastloxidative/glycolytic-coarse; FG, fast/glycolytic. -/ + i + + i + + + indicates increasing stain intensity. ATPase, myofibrillar adenosine triphosphatase; NADH-TR, nicotinamide adenine nucleotide dehydrogenasetetrazolium reductase; Men-alpha-GP, menadione-mediated alphaglycerophosphate dehydrogenase. within the FCU. There were occasional large diameter axons devoid of their myelin sheath and clusters of thinly myelinated, small diameter axons. As with ventral root C8, the ulnar nerve displayed a significant increase in the density of the degenerating cells between 4 and 24-26 months. DISCUSSION The vast majority of aging studies on nerve and muscle have focused on the hindlimb muscles of rats utilized in locomotion. These muscles are well characterized for their gross structure and fiber types. We chose to investigate age-associated changes in a group of incompletely characterized forelimb muscles utilized not only for locomotion, but also for additional behaviors including feeding and grooming. The arrangement and innervation of flexor and extensor compartment forearm muscles of the mouse were studied by gross dissection. The mouse had the same arrangement and innervation of forearm muscles as previously described for the laboratory rat (Hebel and Stromberg, 1976). The extensor compartment muscles were all innervated by branches of the radial nerve. The bulk of the flexor compartment innervation was by the median nerve with the ulnar nerve innervation restricted to the flexor carpi ulnaris and a portion of the ulnar head of the flexor digitorum profundus. Since the development of muscle histochemistry for human diagnostic purposes (Dubowitz and Brooke, 19731, muscle fibers have traditionally been classified a s type I (slow/oxidative) or I1 (subtypes A [fast/oxidative/glycolyticl and B [fast/glycolyticl). The two types can usually be distinguished based upon their differential staining for myofibrillar adenosine triphosphatase following a n alkaline or acid preincubation, oxidative and/or glycolytic enzyme activity (Dubowitz and Brooke, 1973), and their physiological properties (Burke e t al., 1971). More recently, antibodies specific for the various myosins associated with the specific f i ber types have been developed (Schmalbruch, 1985). We identified two major and one minor fiber type in the mouse forearm muscles which did not fit well into the traditional ATPase classification scheme used for human limb muscle, but were similar to the SO/ FOGiFG fibers originally described in rabbit and guinea pig skeletal muscle (Peter et al., 1972). The FOG and FG fibers all cross-reacted with a n antibody that labels the type I1 (fast-twitch) isomyosin molecules in human and other mammalian muscles. The FOG fibers were further subtyped based upon the appearance of their intermyofibrillar network in NADH-TR stained sections. The small FOGc, fibers with a coarse intermyofibrillar network and extensive subsarcolemmal deposits, had a n appearance similar to the coarse fibers found in human (Ringel et al., 1978) and mouse (Carry et al., 1982) extraocular muscles. TABLE 2. Fiber type characteristics of adult (4 month) forearm muscles' Flexor comnartment F T (5) Fiber diameter (micrometers) FOGc FOG FG 38 (2.1) 33 (1.8) 27 (1.5) 34 (1.5) 34 (1.3) 29 (1.2) 31 (1.8) 30 (1.7) 25 (1.5) 34 (1.3) 35 (1.6) 27 (0.9) 32 (1.6) 31 (1.4) 26 (1.5) 34 (1.9) 27 (2.6) 32 (2.4) 28 (2.1) 39 (1.9) 41 (3.9) 32 (4.7) 20 (5.1) Fiber t y p e (%) FOG 32 (1.4) 46 (2.2) 27 (2.9) 36 (1.0) 31 (2.4) 33 (2.8) 30 (1.0) 27 (4.1) 34 (3.4) 23 (4.1) 37 (3.6) 47 (5.4) Muscle (#I AP (5) ECR (5) ECU (7) ED (6) Extensor compartment Fiber d i a m e t e r (micrometers) FOGc FOG FG 19 (1.2) 27 (1.5) 30 (1.7) 34 (1.6) 41 (1.8) 43 (2.2) 30 (2.4) 23 (1.9) 29 (2.4) 26 (1.5) 35 (2.0) 36 (2.2) Fiber t y p e (%) FOGc FOG 60 (3.3) 29 (1.9) 42 (5.8) 47 (3.4) 44 (4.8) 30 (3.9) 49 (3.1) 34 (4.0) 12 (2.1) 11 (4.4) 25 (3.5) 17 (3.4) Muscle (#) FCR (5) FCU (5) FDP (5) FDS (5) PL (5) FOGc 38 (1.9) FG FG 'Data reported as mean ( 2 SEM). FG, fastiglycolytic; FOG, fastioxidativeiglycolytic; FOGc, fastioxidativei glycolytic-coarse, FCR, flexor carpi radialis; FCU, flexor carpi ulnaris; FDP, flexor digitorum profundus; FDS, flexor digitorum superficialis; PL, palmaris longus; PT, pronator teres; AP, adductor pollicus; ECR, extensor carpi radialis; ECU, extensor carpi ulnaris; ED, extensor digitorum. AGE AND CHANGES IN MOUSE FOREARM MUSCLES 353 Fig. 5. An electron micrograph of a dark staining subsarcolemmal deposit (Fig. 4)from the flexor carpi ulnaris muscle of a 26-month-old mouse. Numerous mitochondria (m), separated by sparse cytoplasm (asterisk), are clumped between the plasma membrane and myofibrils (mf) adjacent to the nucleus (Nu). A degenerating mitochondrion (large arrowhead) is associated with a cluster of lipofuscin deposits and autophagic vacuoles (small arrowheads). Bar, 1 micrometer. The remaining FOG fibers stained less intensely for non-specific esterase activity (oxidative fibers stain intensely [Brumback and Leech, 198411, but more intensely for glycolytic enzyme activity than the FOGc fibers. There was a small population of SO fibers confined to the deep region of a single forearm muscle, the FCU, which displayed a staining profile (ATPase and myosin antibody) more consistent with traditional type I fibers. Based upon their histochemical staining patterns, the mouse forearm SO, FOG, and FG fibers were, respectively, most similar to traditional types I, IIA, and IIB fibers. Numerous fiber types have previously been described for the mouse hindlimb muscles which do not always correspond with equivalent human fiber types. The forearm SO and FOG fibers had histochemical staining properties similar to the SO and FOG fibers described for the soleus (Goldspink and Ward, 1979) except that the forearm SO fibers stained more intensively for oxidative enzyme activity. The forearm FOGc and FG fibers were most similar to the I1 A-W (Suzuki, 1990) and I1 A2 (Matoba et al., 1985) fibers of the gastrocnemius. In general, all mouse fibers seem to have a more coarse intermyofibrillar network and greater number of subsarcolemmal deposits than the most similar human limb fibers. Although the FCU muscle was not studied using a comprehensive morphometric analysis, two striking morphological changes in the muscle fibers were evident with increasing age. First, there was a marked increase in variation in fiber size and shape. Although many uniformly polygonal shaped fibers remained, they were often separated by distinctly atrophic, angular fibers. Second, the slight subsarcolemmal deposits present in fibers a t 4 months of age were replaced by large aggregates of mitochondria which often surrounded the myofiber nuclei. Many of these deposits included spherical inclusions, lysosomes, and lipofuscin pigment. There is clearly a decline in muscle strength with increasing age (Grimby and Saltin, 19831, and this loss of strength appears to be directly related to a corresponding loss in muscle mass (Faulkner e t al., 1990). This decrease in muscle mass may be related to a number of mechanisms, including disuse atrophy (inactivity) and denervation atrophy following a n age associated loss of motor neurons or their axons. Where human and rodent muscles have been examined, there is clear histological evidence for denervation-related changes with increasing age (Bass et al., 1975; Caccia et al., 1979; Fujisawa, 1974; Grimby et al., 1982; Van Steenis and Kroes, 1971). The histopathological 354 M.R. CARRY ET AL. Fig. 6. Photomicrographs of toluidine blue-methylene blue-Azure I1 stained semithin plastic sections displaying various sized myelinated axons. Bar, 50 micrometers. A: A cross-section of ventral root C8 from a 4-month-old female mouse. The ventral root is surrounded by a perineural sheath (arrowheads) as it passes through its associ- ated dorsal root ganglion (surrounding tissue). B: A cross-section of the ulnar nerve just proximal to the elbow (Fig. 1) from a 4-month-old female mouse. The single fascicle of this nerve is surrounded by a thick perineural sheath (arrowheads). TABLE 3. Age-associated changes in ventral root C8 morphometric parameters’ Age group (# mice) Parameter Nerve area (mm2) Axon number (#/cross-section) Axon density (#/mm2) Degenerating cell density (#/mm2) Capillary number (#/cross-section) Capillary density (#/mm2) Endoneurial area fraction (% cross-sectional area) Cell densitv (# nuclei/mm2) 2 month ( 5 ) 0.034 (0.003) 813 (28) 24,359 (1870)** 0 (0) 4.2 (0.7) 129 (29)** 23 (1) 1,520 (73) 4 month (5) 0.045 (0.004) 872 (16) 19,571 (1401) 4 (4) 3.2 (0.5) 69 (8) 19 (1) 1.409 (209) 18-23 month (3) 0.059 (0.013) 788 (56) 14,660 (3096) 67 (14)* 3.3 (0.9) 57 (7) 26 (3)* 1,311 (259) 24-26 month (7) 0.058 (0.007) 833 (69) 14,856 (894)** 87 (9)* 4.1 (0.8) 76 (16) 26 (1)* 1,404 (195) ‘Data reported as MEAN (? SEM). *Group vs. 4 month; P = 0.01. **Group vs. 4 month P = 0.05. changes associated with senile muscle have experimentally been demonstrated in rodents to be related to motor denervation of muscle fibers (angular fibers and group atrophy) or cycles of denervation and re-innervation (type grouping) (Dubowitz, 1967). The observation of angular fibers in the FCU of the mouse with increasing age is consistent with the previous studies on senile mouse muscle. The presence of the subsarcolemmal aggregates of mitochondria and lysosomes has not previously been reported for senile muscles. Similar deposits occur frequently in human extraocular muscles and give the fibers a “ragged-red appearance in trichrome stained cryostat sections (Ringel et al., 1978). Ragged-red fibers are also a distinguishing feature of human mitochondrial myopathies, particularly those involving the extraocular muscles such as Kearn-Sayre syndrome (Ringel et al., 1979). The Kearn-Sayre and related patients with ragged-red fibers have deletions in their mitochondrial genomes (Schon e t al., 1989). The proliferation of mitochondria in the fibers of these patients may be related to the overall decreased levels of respiratory chain enzymes in the muscles. The extraocular muscle fibers may be more susceptible to the formation of ragged-red fibers than other fibers due to their normally high concentrations of mitochondria (Carry et al., 1986). The mouse forearm muscle fibers, particularly the SO and FOG fibers, with their high intense staining characteristics for oxidative enzymes, and thus numerous mitochondria, may likewise be more susceptible to age-associated defects in mitochondrial function. The aggregates may be the result of a proliferation of mitochondria to compensate for overall decreased mitochondrial function. The presence of atrophic, angular shaped fibers in the older FCU muscles was suggestive of possible ongoing denervation. To clarify this possibility, we made a detailed morphometric study (Tables 3-5) of the F C U s source of innervation, the ulnar nerve and one of the ventral roots (C8) contributing axons to this nerve. The small size of both the ulnar nerve and ventral root C8 made these structures suitable for comprehensive 355 AGE AND CHANGES IN MOUSE FOREARM MUSCLES TABLE 4. Age-associated changes in ulnar nerve morphometric parameters' Age group (# mice) Parameter Nerve area (mm2) Axon number (#/cross-section) Axon density (#/mm2) Degenerating cell density (#/mm2) Capillary number (#/cross section) Capillary density (#/mm2) Endoneurial area fraction (% cross-sectional area) Cell density (# nuclei/mm2) 2 month ( 5 ) 0.033 (0.008) 1,285 (29) 39,498 (1127)* 0 (0) 4.6 (0.2) 142 (7)** 31 (1) 2,639 (126)* 4 month ( 5 ) 0.038 (0.001) 1,284 (20) 34,069 (1016) 10 (10) 3.8 (0.6) 102 (17) 29 (1) 1,982 (100) 18-23 month (4) 0.049 (0.002)* 1,255 (22) 25,957 (770)* 50 (31) 3.8 (0.4) 77 (7) 28 (1) 1,329 (108)* 24-26 month (7) 0.054 (0.002)* 1,329 (20) 24,619 (619)* 92 (19)* 4.5 (0.5) 83 (9) 32 (2) 1,528 (96)* 'Data reported as mean ( ? SEMI. *Group vs. 4 month; P = 0.01. **Group vs. 4 month; P = 0.05. TABLE 5. Age-associated changes in myelinated axon diameters' Ventral root C8 Diameter range (micrometers) 2 month ( 5 ) Diameter range 2 month ( 5 ) (micrometers) 0-2 3-4 5-6 7-8 9-10 11- (%I Age group (# mice) 18-23 month (3) 4 month ( 5 ) (%I (%I Age group ( # mice) 18-23 month (4) 4 month (5) (%I (%) 27 (1)* 46 (1) 23 (2) 3 (I)* 0 (O)** 0 (0) 17 (2) 43 (2) 27 (2) 9 (1) 3 (1) 1(1) (%) 16 (3) 46 ( 5 ) 27 (2) 9 (2) 2 (1) 2 (2) 24-26 month (7) (%) 24-26 month (7) (%) 19 (2) 47 (2) 25 (2) 7 (1) 2 (0) 0 (0) 'Data expressed as mean (2SEM) of total percentage of myelinated axons. *Group vs. 4 month; P = 0.01. **Group vs. 4 month; P = 0.05. morphometric analyses. Ventral root C8 is multifascicular as i t traverses its associated dorsal root ganglion, whereas the mouse ulnar nerve, proximal to the elbow, is a typical unifascicular nerve (Mitsumoto and Bradley, 1987). Concentric layers of perineural cells surround Schwann cells, fibroblasts, capillaries, and a wide diameter range (1-12 micrometer) of myelinated axons. Two striking features of both the aged ventral root and ulnar nerve were the dramatic increase in the cross-sectional area and the increase in the density of degenerating cells or axonal processes. The cross-sectional area of ventral root C8 and the ulnar nerve increased by 30 and 42%, respectively, between 4 and 26 months. In ventral root C8 this increase was most likely due to a combined increase in the percentage of large diameter myelinated axons and the relative area fraction of the endoneurial connective tissue. The shift in percentage of large diameter axons occurred without a n overall loss of myelinated axons, suggesting a n in- crease in axonal diameter with old age. The increase in endoneurial area fraction may have been accompanied by some proliferation of Schwann cells or fibroblasts since there was no decrease in the cell (nuclear) density with increasing age. A similar increase in the area of the tibia1 nerve with increasing age was due to a distinct increase in endoneurial connective tissue (Samorajski, 1974), but the mechanisms responsible for the increase in the ulnar nerve are less clear. The nerve showed no increase in the percentage of large diameter axons, no overall loss of myelinated axons, and only a slight increase in the relative area fraction of the endoneurial connective tissue. Interestingly, the increase in the area of both ventral root and ulnar nerve was associated with a marked but non-significant increase in endoneurial capillaries. The presence of degenerating axons and myelin alterations appears to be a universal feature of the aged peripheral nerve in rodents (Ansvard and Larsson, 1990; Cotard-Bartley et al., 1981; Kazui and Fujisawa, 356 M.R. CARRY ET AL. Fig. 7. Photographs of toluidine blue-methylene blue-Azure I1 stained semithin plastic sections of the ulnar nerve from a 26-monthold female mouse displaying age-associated degenerative changes. Bar, 10 micrometers. A A myelin figure (arrow) and a cell containing pleomorphic inclusion material (large arrowheads) are found between normal appearing myelinated axons. B A cell containing pleomorphic inclusions (large arrowheads) and a n axon devoid of myelin (arrow) are found adjacent to a group of small, thinly myelinated regenerating axons (small arrowheads). 1988; Knox e t al., 1989; Samarajiski, 1974; Thomas et al., 1980). We were not able to detect any overall loss in the number of myelinated axons in ventral roots and ulnar nerves despite the dramatic increase in the density of degenerating cells between 4 and 26 months of age. Although electron microscopic observations were not made on the ventral roots and ulnar nerves, the appearance of the degenerating cells was consistent with axonal or myelin degeneration (Morris et al., 1972a; Weller and Cervos-Navarro, 1977). It was not possible to distinguish between sensory and motor axons, but the presence of the angular fibers in the FCU suggests that many of the degenerating axons were motor. Clusters of thinly myelinated axons were occasionally observed in the ventral roots and ulnar nerves from the older mice. The presence of clusters of thinly myelinated axons is consistent with active axonal regeneration (Bots and Maat-Schieman, 1987; Morris et al., 197213). The axonal regeneration may be sufficient to replace all degenerating axons, resulting in no net loss a t the oldest age (26 months) examined in the present study. The mouse forearm muscles have the same basic organization and innervation pattern as other rodent forearm muscles. These muscles involved in the finer movements of grooming and feeding show many of the same age-associated changes as other rodent hindlimb muscles active primarily in locomotion. The increased variation in fiber size, loss of uniform polygonal shape, and atrophic, angular shape are consistent with loss of motor innervation and are reflected in the increased density of degenerating cells in the peripheral nerves. Yet to be determined is the mechanism responsible for the accumulation of the subsarcolemmal mitochondria and Ivsosomes. and anv association with the degenerativechanges in the peripheral nerves. 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