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ová,
Soukup
Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Zachaová, Gisela, Helena
Knotková-Urbancová, Pavel Hník, and
Tomá Soukup. Nociceptive atrophy of the rat soleus muscle induced by bone fracture: a morphometric study.
J. Appl. Physiol. 82(2): 552-557, 1997.
Reflex atrophy of the soleus muscle induced by ipsilateral
metatarsal bone fracture in Sagatal-anesthetized adult male rats was
studied by using two-dimensional stereological methods 7 days after the
operation. When compared with contralateral solei, the wet weight of
the experimental soleus muscles was decreased by ~24% and the area
of the entire muscle section by ~29%. In atrophied solei, the number
of type 1 fibers was lower by ~8%, resulting in lower total number
of fibers (by ~6%). This indicates that slow motor units might be
more sensitive to nociceptive stimulation. However, with respect to the
fiber area, the reflex atrophy induced by metatarsal bone fracture in
the rat soleus muscle resembles simple atrophy after 7 days, as the
mean muscle fiber area was decreased by ~26% with no significant
difference between atrophy in type 1 and type 2a fibers (by 27.3 and
23.0%, respectively).
muscle atrophy; muscle histochemistry; muscle two-dimensional
stereology; muscle fiber types; pain
INTRODUCTION PAINFUL AFFLICTIONS OF THE JOINTS in patients (12, 18,
23) as well as in experimental animals (11, 30) have been reported to
lead to muscle wasting. This type of atrophy was hence termed
"arthrogenic muscle atrophy." On the basis of their
results, Gutmann and Vodi Despite extensive experimentation, the mechanism of this type of
atrophy is still not clear. The development of reflex atrophy was not
influenced either by restricting the blood supply to the affected leg
or by adrenalectomy (or administration of corticosteroid hormones)
(14). Neither did incorporation studies of labeled lysine into spinal
motoneurons supplying the lower leg of rats provide any indication of
alteration in neuronal protein metabolism during reflex atrophy (16).
Disuse itself does not seem to be the primary cause of muscle atrophy
induced by chronic nociceptive stimulation. The study of Harding (11)
on cats already indicated that the muscle wasting caused by arthritis
cannot be explained simply by disuse, as elimination of sensory inflow
into the spinal cord did not influence the disuse atrophy evoked by
immobilization, whereas it reduced the arthrogenic muscle atrophy in
cats. In our previous study, electromyographic (EMG) activity in the
soleus muscle was diminished only during the first 1 or 2 h after paw fracture but returned to normal after this period (13). Furthermore, it
was shown that dorsal root section on the side of paw fracture prevented the development of muscle atrophy if performed before the
fracture; however, if deafferentation was carried out at short time
intervals (2 min to 2 h) after the application of the nociceptive stimulus, muscle wasting still occurred after 7 days (32).
Various models of muscle atrophy (e.g., denervation, transection of the
spinal cord, tenotomy, limb immobilization, or hindlimb suspension)
result either in comparable wasting of both type 1 and type 2 extrafusal fibers, so-called simple atrophy (17, 28) or in
preferential atrophy of one fiber type (2-4, 24). The present
study describes in detail the morphological changes occurring in reflex
atrophy in the rat soleus muscle, which is a muscle most afflicted by
fracture of the ipsilateral paw (32), with the aim to ascertain the
extent to which the two types of muscle fibers are affected during
reflex atrophy. Muscles were examined 7 days after the fracture,
since this type of atrophy is only transient in character and
gradually recedes after this period (13, 14).
Our study was based on two-dimensional stereological
methods recently adjusted for measurements in muscle cross sections
stained by routine histological methods (35). It provides basic
morphological data that should be of value for further investigations
of reflex atrophy evoked by nociceptive stimulation and thus enables a
more complex understanding of this type of muscle atrophy.
METHODS Experimental Animals
ka (10) and Vodika (33) denoted
the atrophy arising from chronic nociceptive stimulation, evoked by
crushing the paw in rats and/or by injection of a small amount
of turpentine oil into the planta, as reflex atrophy.
Histology and Histochemistry
The soleus muscles of both paws of experimental rats were excised 7 days after the bone fracture under Sagatal anesthesia, with the contralateral soleus muscles serving as control. Furthermore, soleus muscles from control rats were excised to compare the right and left side of normal animals. The excised muscles were weighed immediately, then covered with talcum powder and fixed by ligatures to iron clamps, preserving their resting length and natural shape. The muscles were immediately frozen in liquid nitrogen and stored in a cryostat at
25°C for up to 2 h. Serial cross sections 10 µm thick were
cut perpendicular to the longitudinal axis through the muscle midbelly
(never containing aponeurosis or tendons), mounted on glass slides,
air-dried for ~60 min, and processed further for histological and
histochemical evaluation.
Staining with hematoxylin and eosin according to Dubowitz and Brooke (6) and staining for myofibrillar adenosinetriphosphatase (mATPase; E.C. 3.6.1.3.) by using alkaline (pH 10.3) and acid (pH 4.3 and 4.6) preincubations according to Guth and Samaha (9) and Dubowitz and Brooke (6) were carried out. The mATPase procedure stains slow type 1 fibers strongly after acid preincubations and lightly after alkaline preincubation, whereas type 2a fibers exhibit opposite staining characteristics. Type 2b fibers that, unlike 2a fibers, exhibit moderate mATPase activity after acid preincubation at pH 4.6 are not present in normal soleus muscles (1, 27). Fibers with other combinations of alkali-stable and acid-stable mATPase activity were classified as transitional fibers. The stereological measurements (see below) of the fiber number and fiber area did not allow to evaluate two different sections simultaneously and to compare single muscle fibers in both mATPase reactions. The measurements were based on the acid-preincubated mATPase reaction (pH 4.3) by which the 2a fibers were clearly distinguished from transitional and type 1 fibers, as all fibers with explicitly low activity at pH 4.3 exhibited high activity after alkaline preincubation.
Muscle Morphometry
The total number of muscle fibers or the number of fibers according to fiber type in each examined section was assessed by two methods: 1) direct counting from photographs of the sections (all fibers in entire muscle sections were counted) and 2) a statistical estimate by using a system of unbiased sampling frames (8). In the second method, a system of frames was superimposed on the muscle section (by scanning the frame across the entire section), and the number of profiles within the frames [n(fib)] was counted. The total number of fiber profiles in the whole muscle section [N(fib)] was then estimated (est) by the formula
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The total area of all profiles of a given fiber type [A(fib)] was estimated by a point-counting method (34). A point-test system was superimposed on the muscle section (by scanning the frame across the entire section), and the test points hitting the fiber profiles were counted and evaluated according to the following equation
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The mean cross-sectional fiber area was calculated as the ratio of the total area A(fib) of all profiles of a given fiber type and the number of profiles in the muscle section examined.
The area of the entire muscle section was estimated analogously to the total fiber area. Stereological measurements were performed under a light microscope with an ocular test system designed so that both the area and the number of muscle fibers could be determined simultaneously (for more details see Ref. 35).
The results are given as means ± SE of values obtained for individual animals. The latter value was obtained as the mean of 3-6 muscle cross sections. Statistical analysis was performed by the paired t-test for comparison between experimental and contralateral solei of experimental rats and between right and left solei of normal rats. The level of significance was set at P < 0.05.
RESULTS
Control Rats
Muscle wet weight and morphological parameters were assessed in soleus muscles of normal rats to compare right and left soleus muscles. The wet weight of the right and left muscles was 117.5 ± 6.9 and 117.2 ± 6.5 mg (0.042 ± 0.002% body wt), respectively (n = 6). Cross-sectional morphological parameters were measured in three controls. No statistically significant differences were found between the solei from left and right side (Table 1). The fiber type composition (91.6 ± 2.1 and 90.4 ± 3.3% of type 1 fibers in right and left soleus muscles, respectively) also did not differ.
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Experimental Rats
One experimental rat (rat 8 in Figs. 2 and 3) exhibited atypical morphometric parameters (see below). The following calculations were therefore made without this animal, but the statistics of the results did not substantially differ whether it was included or not.
Muscle weight. The wet weight of soleus muscles from the experimental legs was 95.5 ± 6.7 mg, i.e., 0.031 ± 0.002% body wt (n = 8), which was lower by 24.3 ± 2.8% (P < 0.001) relative to control contralateral muscles (128.6 ± 13.2 mg, i.e., 0.041 ± 0.003% body wt). The area of the entire muscle section in midbelly was 4.35 ± 0.28 mm2 and was smaller by 29.2 ± 3.8% when compared with control muscles (6.32 ± 0.73 mm2).
Muscle morphometry. The mATPase
reaction (Fig. 1) revealed the presence of
type 1, type 2a, and transitional fibers both in control and in
experimental solei. In all measurements described below, the
transitional fibers were included in the group of type 1 fibers. No
type 2b fibers were found either in experimental or contralateral
solei.
FIBER CROSS-SECTIONAL AREA. The mean cross-sectional fiber area (irrespective of fiber type) was significantly smaller (by 25.7 ± 1.9%) in the soleus muscles on the side of bone fracture, and this was consistent in all the rats examined, with the exception of rat 8 mentioned above (Fig. 2); no significant difference was found between type 1 and type 2a fibers (mean area on the side of bone fracture was smaller by 27.3 and 23.0%, respectively). Neither was there a difference between type 1 and type 2a mean fiber area in experimental or in control muscles. Corresponding results were obtained when the mean fiber areas were expressed relative to the body weight (Table 2).
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DISCUSSION
Our results demonstrate that the muscle atrophy caused by ipsilateral metatarsal bone fracture, when compared with contralateral solei, is characterized by 1) a substantial decrease in the muscle wet weight and a corresponding decrease in the mean fiber area of both type 1 and type 2a muscle fibers; and 2) a lower number and smaller total area of muscle fibers, both on account of type 1 fibers.
The severe extent of muscle wasting after 7 days (by 24%) found in our experiments confirmed previous findings regarding reflex atrophy (10, 13, 14, 32) and was comparable with those reported in other models of muscle atrophy analyzed after 1 wk (suspension hypokinesia by 37%, Ref. 5; immobilization in the shortened position by 27%, Ref. 15; nerve crush by 30%, Ref. 17), after 10 days (suspension hypokinesia by 26%, Ref. 19), or after 14 days (suspension hypokinesia by 33%, Ref. 20). The weight loss can mainly be explained by the marked reduction in mean fiber area of both type 1 and type 2a fibers and partly by a decrease in the total number of fibers. A comparison with normal rats makes it unlikely that compensatory hypertrophy of contralateral soleus muscles could account appreciably for the difference between ipsilateral and contralateral solei of experimental rats, since the mean fiber area relative to body weight in control soleus muscles of experimental rats did not significantly differ from that in soleus muscles of normal rats, whereas it was significantly smaller in the soleus muscles from the experimental limbs (Table 2).
If the reflex atrophy is evaluated according to the changes in fiber area of individual fiber types, it resembles simple atrophy 7 days after bone fracture. Similarly to our findings, Desplanches et al. (5) and Jaweed et al. (17) found that the extent of type 1 fiber atrophy 1 wk after denervation or hindlimb suspension performed in adult rats was only slightly greater than that of type 2 fibers. Our results do not exclude the possibility that differences in the extent of atrophy of type 1 and type 2a fibers might appear if the duration of the nociceptive stimulus were prolonged. For instance, the atrophy of type 1 fibers does not become clearly dominant until after 2-5 wk of hindlimb suspension (2, 5; for review see Ref. 29). The lower total number of muscle fibers (namely, type 1 fibers) in the solei from fractured limbs suggests that rapid degeneration of ~6% of the fibers had occurred. This was rather surprising, although a few observations on the degeneration of muscle fibers in some other atrophy models have been reported. About 1% of fibers were necrotic in the atrophic rat soleus muscle after 1-wk spaceflight (24). Soleus muscles reloaded for 2 days after 10-day hindlimb suspension were strongly invaded by those subpopulations of macrophages that are supposed to be associated with the necrotic stage of muscle injury (28a). As in reflex atrophy, predominant reduction in the number of type 1 fibers was reported in the following immobilization studies: a marked 24% decrease in the number of muscle fibers (namely, type 1 fibers) was observed in the rat soleus muscle immobilized for 4 wk (4), and degeneration affecting predominantly type 1 fibers was observed in soleus muscles of the guinea pig 1-3 wk after immobilization (31). The present data support observations from other experimental models that, in the soleus muscle, slow-twitch fibers with tonic antigravity function are more sensitive to various stimuli than are type 2a fibers.
Although reflex muscle atrophy has many features in common with other types of muscle atrophy, the exact mechanism by which it is induced is hitherto not known. Disuse itself does not seem to be the primary cause of reflex atrophy, since recordings of resting spontaneous EMG activity from the experimental soleus muscles returned to normal within several hours (13). Partial preservation of EMG activity and its relatively fast recovery in other atrophy models (suspension hypokinesia, immobilization) also indicate that functional inactivity is not exclusively responsible for the observed atrophic changes (7, 20; for review see Ref. 29). In reflex atrophy, the role of the nociceptive stimulus seems to be unequivocal. The afferent pathway is essential for reflex atrophy to develop, since dorsal root section performed immediately before paw fracture, but not if carried out shortly after the fracture, prevented the onset of muscle wasting (32). This implies that nociceptive information and its arrival in the spinal cord triggers the atrophy-inducing mechanism during early stages of its action and that its continuous presence throughout the experimental period is not necessary.
It seems plausible to expect that excitatory amino acids and/or neuromodulators such as substance P are released from primary afferents in the spinal cord in response to injury discharges. These substances can cause sensitization of dorsal horn neurons (21) and, perhaps, activate their second-messenger systems. Moreover, it appears that besides purely spinal mechanisms supraspinal centers may also be involved in the development of reflex atrophy, as myelotomy performed before nociceptive stimulation eliminates the difference between reflex and denervation atrophy (the first being greater in nonmyelotomized animals; see Ref. 13). Nothing is known about other aspects of motoneuronal activity, i.e., frequency of nerve discharges, end-plate morphology and function, or afferent-efferent loops in connection with reflex muscle atrophy. It should be mentioned in this context that no basic morphological or histochemical changes of muscle spindles and of intrafusal fibers were observed in reflex atrophy. In addition, changes of cellular homeostasis (e.g., intracellular calcium concentrations) may also be involved in the mechanism of reflex atrophy, similar to the way suggested for immobilization atrophy of the soleus muscle (25). Reflex muscle atrophy thus seems to be more complex than was originally anticipated. It is obvious that multiple factors are involved in the development of reflex muscle atrophy and that it, therefore, deserves further investigation.
ACKNOWLEDGEMENTS
We are grateful to T. Valentová for technical assistance, to
Dr. J. Vorlíek for the statistical evaluations, and to
Dr. J. Zelená for critical reading of the manuscript. The
collaboration of Dr. R. Vejsada during the early stages of the
experiments is gratefully acknowledged.
FOOTNOTES
Address for reprint requests: G. Zachaová, Institute of
Physiology, Academy of Sciences of the Czech Republic,
Víde
ská 1083, 142 20 Prague 4, Czech Republic
(E-mail: gzacharo{at}biomed.cas.cz).
Received 6 February 1996; accepted in final form 30 September 1996.
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