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Structure innervation and age-associated changes of mouse forearm muscles.

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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.
ACKNOWLEDGMENTS
u
Electron microscopic observations were made with
the assistance of Pam Eller from the Department of
Cellular and Structural Biology at the University of
Colorado Health Sciences Center.
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