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Morphometric analysis of neuronal soma size within the motor nucleus of a transplanted murine skeletal muscle.

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THE ANATOMICAL RECORD 217:402-406 (1987)
Morphometric Analysis of Neuronal Soma Size
Within the Motor Nucleus of a Transplanted Murine
Skeletal Muscle
KATHLEEN M. KLUEBER
Medical Sciences ProgradAnatorny Section, Indiana University School of Medicine,
Bloomington, IN 47405
ABSTRACT
Since the number of motor neurons supplying a muscle graft is
reduced, the peripheral field for the surviving motor neurons would be enlarged.
This possible change in motor unit size may result in morphologic changes in the
size of motor neurons within the motor neuron pool of the graft. The extensor
digitorum longus (EDL) muscle from 19 transplanted and 17 age-matched normal
129 ReJ female mice were injected with horseradish peroxidase in order to examine
the cell size distributions of the motor neuron pools supplying these muscles. Computer-assisted morphometric analysis of cell sizes within the motor neuron pool to
the transplants indicated a significant shift in cell size, with the largest areas
ranging between 630 and 1250 pm2. A number of the alpha neurons supplying the
grafts were twice the average cell size for the control population
= 592 pm2). The
increase in the number of large motor neurons indicates a n hypertrophy of neurons
reinnervating the grafts. The long-term graft is reinnervated by a decreased population of motor neurons
= 71, which vary in size from small to very large,
reflecting both changes in the possible source of the nerve reinnervating the graft
as well as alteration in the size of the motor unit, respectively.
(x
(x
Subsequent to whole muscle transplantation, the myofibers of the denervated, devascularized graft undergo
necrosis. The necrotic myofibers are phagocytosed, leaving only the empty basal lamina1 tubes. Finally, regenerating myofibers are formed de novo within these tubes,
and most, if not all, of the myofibers receive motor
innervation. This secondary myogenesis occurs without
the presence of nerves (Carlson et al., 1979) with reinnervation occurring at two weeks posttransplantation
in the mouse (Ontell et al., 1982). Although the reinnervating neurons of the murine extensor digitorium long u s (EDL) are located within the same spinal cord level
and Rexed's lamina as those to nontransplanted EDL
muscles, the motor neuron pool of murine grafts is significantly smaller in size than those innervating the
nonoperated muscle (Klueber et al., 1984).
Since the murine EDL regenerate contains a third less
myofibers than unoperated controls (Bourke and Ontell,
1984) and the number of motor neurons supplying the
grafts is reduced (Klueber et al., 1984), the motor units
to the graft are modified as indicated by the myofiber
type grouping in the murine model of secondary myogenesis (Thomas et al., 1984).Thus, the reinnervation pattern of the EDL graft is not the same as the innervation
pattern seen prior to transplantation. The question
arises: Does the altered peripheral field of the transplant influence the soma size of the reinnervating motor
neurons supplying the graft? It was the purpose of this
study to examine the motor neuron soma sizes within
the motor neuron pool of transplanted murine EDL
muscles.
0 1987 ALAN R. LISS. INC
MATERIALS AND METHODS
Under methoxyflurane anesthesia (Metafane, PitmanMoore, Inc., Washington Crossing, NJ) administered via
inhalation, the extensor digitorum longus (EDL) muscles of 19 four-week-old 129 ReJ female mice were surgically removed and then soaked in 0.75% bupivacaine
HCl (Marcaine, Sterling Drug Inc., New York, NY), a
known myotoxic agent (Benoit and Belt, 1970; Libelius
et al., 1970). The muscle was then reattached to the
tendonous ends (Carlson and Gutman, 19741, but no
attempt was made for neurovascular anastornosis.
At 100 days posttransplantation, the grafts as well as
EDL muscles from 17 age-matched normal 129 ReJ female mice were surgically exposed. Using a n endodontic
file technique (Klueber and Ontell, 1984), a semisolid
paste of horseradish peroxidase (HRP, Sigma Type VD
was placed intramuscularly. After a n 18-24-hour survival time, the animals were anesthetized with methoxyflurane via inhalation, intravenously injected with heparin, then transventricularly perfused with physiological saline followed by 1% paraformaldehyde and 1%
glutaraldehyde in 0.1M phosphate b d e r (pH 7.2).
The spinal cords were removed and fixed a n additional
2 hours in fresh fixative (4"C), processed through a
graded series of sucrose solutions to be stored in 30%
sucrose (4°C) overnight. Spinal cord segments L2
through L4 were mounted and cut in serial transverse
Received July 28, 1986; accepted November 19, 1986
MOTOR NEURON SOMA SIZE INNERVATING MUSCLE GRAFTS
section (42 pm) on a cryostat (Damon/IEC Division,
model CTD) and subsequently processed for HRP reaction product with the tetramethylbenzidine method of
Mesulam (1978). Selection of spinal cord segments L2 to
L4 was made based on prior work in this laboratory as
well as the description of the location of the motor neurons to the deep and superficial peroneal nerves, which
supply the muscles of the shank in the mouse (McHanwell and Biscoe, 1981a).
The number of cells within the motor neuron pool for
both the control and grafted EDL was evaluated using
a A 0 microstar microscope equipped with dark-field and
phase optics. Composite camera lucida drawings from
each spinal cord were made to record the position and
size of each motor neuron within the motor nucleus. No
correction factor was necessary since the motor neuron
pool was small and dispersed, and the individual cells
could be followed by visual inspection within the serial
sections. However, only those cells exhibiting a nucleus
or nucleolus (Strick et al., 1976), determined with phase
optics, were considered as cell bodies for counting and
morphometric analysis.
Morphometric analysis of soma size for all neurons
within each of the motor neuron pools was accomplished
by tracing the outline of soma perimeter, including the
roots of dendritic and axonal processes within the plane
of focus (Fig. 1). The resulting camera lucida drawings
(50x1 were used for morphometric analysis using the
Bioquant I1 analysis programs (R&M Biometrics), which
stores perimeter and area measurements in individual
403
files for control and experimental groups. The files for
each group were combined to produce normalized histograms of cell areas of the transplants and controls. Statistical analysis of motor neuron sizes for the control
and grafted muscles was completed using a two-tailed
Student's T-test, and the level of significance was set at
P < .05.
RESULTS
The average size of the motor neuron pool for the
control EDL was 16 f 1.03 SEM cells (N = 17),whereas
the average size of the motor neuron pool to the EDL
grafts was 7 & 0.90 SEM cells (N = 19, range 2-14).
Morphometric analysis of the motor neuron soma size
of the normal EDL indicates that the motor nucleus
consists of a trimodal population of cells (Fig. 2) whose
soma area ranges from 250 to 1250 pm2. Within the
motor nucleus for the normal EDL, there is a population
of small motor neurons ranging in area from 250 to 420
pm2 representing 18.3% of the cells. The midsized group
of neurons consisted of areas ranging from 420 to 630
pm2, while the large group of cells had soma areas from
630 to 1250 pm2. The latter groups represent 44.5% and
34.9% of the total cell population for the EDL motor
nucleus, respectively (Table 1).
The soma size distribution of the motor neurons of the
transplanted EDL was not trimodal in nature but rather
widely dispersed. When the ranges of soma size found in
the control group are superimposed on the motor neuron
Fig. 1. A phase micrograph of 6 HRP-filled neurons from the motor nucleus of the EDL. The black
dashes enclose the area used for morphometric analysis of the soma cell size. In this plane of focus, the
nuclei (n) of 4 cells was noted; thus, only those cells were measured. x 100.
404
K.M. KLUEBER
CONTROL
L
[7 GRAFTS
l
0
20
10
30
40
CELL AREA (pm’)
Bin width = 4 2 p ’
Fig. 2. Normalized histogram of soma size distribution from the motor neuron pools of control and
transplanted EDL muscles. Note that the control group has a distinct trimodal population of cell size. For
the grafts, however, the soma size distribution has a wider range with no definitive pattern.
TABLE 1. Percentages of motor neuron soma sizes for control and
transplanted EDL muscles
Range of
soma area (Km2)
<250
250-420
420-630
630-1250
> 1250
Control
spinal cords (N= 17)
cells measured (N= 277)
1.4
18.3 &
44.5
34.9 &
0.8 +_
1.0
4.1
4.1*
5.1**
0.6
Transplant
spinal cords (N = 19)
cells measured (N = 135)
2.8 & 1.4
17.9 4.7
25.9 k 4.7*
51.1 f 7.3**
2.2 & 1.4
*P < ,005.
**P c . 0 5 .
size distribution of the grafts, it becomes apparent that
there is a shift in soma size for those neurons reinnervating the graft (Table 1). The percentage of neurons
falling within the control ranges decreased for the small
and medium populations, while increasing in the largest
group of neurons (17.9%, 25.9%, and 51.1%, respectively). However, the range of soma size for both the
small and large neuron populations increased for the
grafts (Table 1).For the transplant motor neurons, 2.8%
of the small neuron population were < 250 pm2 and
2.2% of the large neurons were > 1250 pm2.
Further evaluation of individual soma size distributions for both the transplants and the control group
revealed that 21.2% of the motor nuclei of the grafts did
not contain motor neurons smaller than 630 pm2, while
none of the control motor nuclei had all their motor
neurons greater than 630 pm2. Within the transplant
group, 15.8% of the motor nuclei did not contain neurons
above 630 pm2, while 11.8% of the control muscles fit
into this category. The transplant group had 63.2% of
the individual motor nuclei in which over half of the
neurons were greater than 630 pm2, while only 29.4%
of the control motor neuron pools had a similar composition. In general, there appears to be a tendency for the
small-sized motor neuron pools of the transplants to
consist of the large-sized neurons ( > 630 pm2).
Comparison of the soma size distributions for the control and transplant motor nuclei (Fig. 1; Table 1)reveal
a slight shift in the percentage of small motor neurons
for the transplant. Similarly, there is a highly significant (P < .005) decrease in the percentage of medium
motor neurons in the transplant motor nucleus, with a
significant (P < .05) increase in the percentage of very
large neurons. Overall, theaverage soma size for the
transplant’s motor neurons (X = 642.2 pm2 f 22.5 SEM)
was significantly larger than that of the control neurons
(X = 592.2 pm2 k 12.5 SEMI indicating a small yet significant (P < .05) shift in cell soma size for those neurons reinnervating the grafts.
DISCUSSION
In a n HRP study of the motor neuron pool to normal
feline muscle, Burke et al. (1982) described a bimodal
population of neuron sizes for both the medial gastrocnemius and soleus muscles. The motor nuclei of these
muscles consisted of a population of small neurons
(gamma) and a population of large (alpha) neurons. Pelligrini et al. (1977) further subdivided the large motor
neuron population of the same feline muscles into small
and large alpha neurons. The latter two types of alpha
motor neurons were correlated to the fiber type of the
muscle fibers they innervated: the small and large alpha
neurons innervate the fast twitch oxidative (Type IIa)
and the fast twitch glycolytic fibers (Type Ilk), whereas
the smallest cells (gamma) innervate the slow twitch
fibers (Type I) (Strick et al., 1976; Burke et al., 1982).
The normal EDL muscle consists of a mixed myofiber
population that contains 36% of Type IIa and 63% of
Type IIb with less than 1% Type I (Thomas et al., 1984).
In contrast, great variability of fiber type percentages
was seen in the transplanted EDL (Thomas et al., 1984);
therefore, the variation of motor neuron size to the
transplants could be the result of the myofiber types
found in the regenerated muscle. The trimodal distribu-
MOTOR NEURON SOMA SIZE INNERVATING MUSCLE GRAFTS
tion of cell size was noted in the motor nucleus of the
normal EDL muscle of this study, but not for the
transplants.
The size range for motor neurons for the control and
transplanted muscles in the current study correspond to
those described by McHanwell and Biscoe (1981b)for the
deep peroneal nerve. The EDL motor nucleus would be
included within this motor column. The morphometric
analysis of motor neuron cell size distribution for the
transplanted EDL indicates a change in the size and
number of small motor neurons and the addition of a
population of very large motor neurons. This is the first
study evaluating cell size distribution of motor neurons
supplying a muscle graft. Similar changes in cell size
distributions have been described following nerve section (Brushart and Mesulam, 1980).The transplantation
procedure used in the current study involved the cutting
of the muscle nerve to the EDL without reanastomosis
following the repositioning of the graft. The size of the
motor neurons to the grafts is similar to the size of motor
neurons supplying the soleus muscle in murine muscular dystrophy (Parry et al., 1982). Since both the transplant and the dystrophic muscle undergo secondary
myogenesis, similar changes in reinnervation may be
expected.
The decrease in the number of small neurons (250420 pm2) reinnervating the graft may be due to the poor
reinnervation potential of gamma motor neurons following nerve section (Brushart and Mesulam, 1980).Following nerve section, motor neurons have been shown to
retract dendrites in response to the peripheral injury
(Kreutzberg, 1986). This type of response could account
for the presence of the population of small neurons
(< 250 pm2) within the motor columns of the grafts
since the peripheral nerve is cut at the time of transplantation. Another possibility is that the small neurons
found reinnervating the grafts may belong to the motor
column of a more distal muscle. McHanwell and Biscoe
(1981) describe such a relationship of neuronal size to
muscle placement in the hindlimb of the mouse with a
decrease in neuronal cell size for the more distal muscles. The possibility of nerves from surrounding muscles
sprouting to supply denervated myofibers has been described (see Brown et al., 19811, but this possibility of
muscle neurotization has not been examined in the current study.
The increase in size of some of the alpha motor neurons for the graft could be the result of hypertrophy
normally seen during the chromatolytic response of a
neuron following nerve section (Gutmann, 1961). Similar increases in the large motor neuron size were found
in a study of the motor innervation of the dystrophic
soleus muscle (Parry et al., 1982). Muscular dystrophy
is thought to be a neuropathy resulting in denervation
of dystrophic myofibers (Parry et al., 1982). Although
the neuronal swelling remains only as long as the regeneration of the damaged axon persists, it has been reported to last up to two years in the rat (Ducker, 1972).
Another explanation for the increased size of some of
the alpha motor neurons to the graft is a true hypertrophy in response to a change in motor unit size following
reinnervation. After nerve section, both the muscle and
nerve undergo changes (Brown et al., 1981). Among the
changes that can take place in the motor nucleus is a
loss of neurons capable of reinnervation due to severe
405
damage and death (Turner, 1943), or diversion of the
regenerating axons away from their target (Barron et
al., 1981). In either case there would be a reduction of
nerve supply to the graft. Thus the decrease in the
number of neurons to the graft would result in a n increase in motor unit size (Lowrie et al., 1985).
Although the number of myofibers found within the
murine graft is reduced by 32% (Bourke and Ontell,
1984) and the motor neuron pool is reduced by 53%
(Klueber et al., 1984), a change in the motor unit size is
likely a s can be inferred in this study. The grafts with
the smallest number of motor neurons exhibit the tendency to be reinnervated by the largest motor neurons.
Since motor neurons have been noted to be capable of
sustaining a much larger peripheral field when the normal number of neurons is unavailable (Betz et al., 1979),
the expansion of the graft’s motor unit could be reflected
in a n increase in motor neuron cell body size as noted
by Lowrie et al. (1985) following nerve section in the rat.
In summary, the long-term graft is reinnervated by a
decreased population of motor neurons, which vary in
size from small to very large, reflecting possible changes
in the source of the nerve supply as well as alteration in
the size of the motor unit, respectively. The source of the
motor neuron population, whether they came from the
original motor neuron pool or from those of the surrounding muscles, as well as changes in the graft’s motor unit size remain to be determined.
ACKNOWLEDGMENTS
This research was supported by a Biomedical Research
Support Grant for Indiana University School of Medicine. The competent technical assistance of Ms. Deborah
Pryor and Ms. Penelope J. Waggoner is gratefully
acknowledged.
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