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Ultrastructural morphometric evaluation of muscles in pigeons kept under conditions of long-lasting hypodynamic state
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
37, 26-36 (1982)
Ultrastructural Morphometric Evaluation of Muscles Pigeons Kept under Conditions of Long-Lasting Hypodynamic State MARIANNA Laboratory
MARCINIAK, of Electron
Received
WANDA
BARA~SKA,
Microscopy, Institate Warsaw, Chalubiriskiego
January
AND WOJCIECH
of Biostructure, 5, Poland
28. 1981. and in rernised form
November
Medical
in
BARAN
Academy,
20, 1981
The purpose of this research was to evaluate morphometrically the structure of muscles of pigeons in hypodynamic state, i.e., when they are deprived of flying. The pigeons were allowed to move freely in the cages, thus avoiding a stress reaction. The results of these investigations revealed, that in the 6 months following initiation of the experiment, marked changes appeared in the enzymatic reactions in the mitochondria of the muscle fibers. These changes were reflected in the accumulation of glycogen and fatty droplets. After 12 to 18 months there appeared a limitation of mobility of the muscles followed by an extensive atrophy of the myofibrils with a decreased volume of sarcomeres. The above changes may be interpreted as a consequence of lack of tissue oxygen, aberration in the catabolic pathways of carbohydrates, and diminished content of ATP necessary for proper metabolic functions.
INTRODUCTION Long-lasting immobilization is one of the new problems in contemporary medicine. A number of changes occur in various organs as the consequence of complete or partial limitation of mobility (Rokhlin and Levites, 1975; Shvets and Portugalov, 1976). Some of these modifications concern the muscular system (Eisenhauer and Key, 1945; Summers, 1951; Tomanek and Lund, 1974). Numerous publications devoted to this problem have appeared of late (Portugalov and Petrova, 1976; Hayat et al., 1978). Hypokinesis greatly reduces mobilization and a hypodynamic state (limited mobility) produces stress reactions and leads to degeneration in the muscular system (Goldspink, 1977). The extent of these changes depends on the duration of hypokinesis or the hypodynamic state and on the experimental model. Thus, the changes in the muscular system may be considered as the outcome of both immobilization and stress. It is known that corticosteroids excreted in excess during stress have a destructive effect on the muscle proteins, actin and myosin (Wawrzynska-Pagowska, 1972). No reports were found in the available literature on quantitation at the ultrastructural level of muscles in animals subjected to a hypodynamic state without the added stress factor. Therefore, it seemed desirable to use an experimental model of a hypodynamic state that probably eliminates the stress reaction. The present investigations concern the ultrastructural morphometric evaluation of pigeon muscles playing an active part in flying. The birds were placed in cages where they could move freely without being able to fly. Such conditions in our opinion should not elicit a stress reaction. MATERIALS
AND METHODS
The experiments were performed with 35 domestic female pigeons weighing approximately 350 g, aged 4 months, kept in special cages 0.13 m high, 4.0 m long, 26 0014-4800/82/040026-11$02.00/0 Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
HYPODYNAMICS
AND
MORPHOMETRIC
EVALUATION
OF
MUSCLES
27
and 2.0 m wide which allowed free movement without the possibility of flying. The birds remained under these conditions for 6 (6-H), 12 (12-H), and 18 months (18-H)) and were fed a standard diet. Control experiments were run in parallel (c). The control animals were kept under conditions where they could freely fly. Each group, both experimental and control, consisted of five birds. The control pigeons were of the same age as the experimental ones. The objects in point were the muscles taking an active part in flying: musculus supracoracoideus and musculus pectoralis. Tissue for ultrastructural examination (10 blocks from each muscle) was obtained intra &am. The pigeons were anesthetized by intraperitoneal injection of sodium pentabarbiturate (Nembutal). The muscle tissue was fixed in 3% glutaraldehyde, postfixed in 1% osmic acid buffered with 0.1 M phosphate buffer, pH 7.4. After dehydration in graded ethanols the tissue was embedded in Epon 812 (Luft, 1961). The sections were cut on a Reichert Urn3 microtome and stained with uranyl acetate (Watson, 1958) and lead citrate (Vanable and Coggeshall, 1965). The sections were inspected in a JEM 100 C Jeol electron microscope. Morphometric evaluation was done on electronograms at final magnifications of x29 000, according to the principles of microstereology (Weibel and Elias, 1967). White and red fibers were separately analyzed in reference to their ultrastructural properties described by Hess (1970). The absolute sarcomere volume (V,, pm”) was determined from their length and width. Width was measured at the level of the H band. The relative mitochondrial volume (V,, %) and the number of profiles (N,) of these structures in one surface area unit-l pm2-of the section were analyzed, and the relative volumes of the sarcoplasmic reticulum (V,,, %) and sarcoplasm (V,,, %) were also determined. In all experimental groups the number of glycogen granules (NG) was estimated per 10 pm2 of the section. The results were statistically evaluated by Student’s t test at the 95% confidence level. RESULTS Morphometric analysis of the supracoracoideus and pectoralis muscles revealed that, owing to limitation of mobility, the sarcomere volume decreases. After 6 months in a hypodynamic state, a statistically significant reduction of sarcomere volume was observed as compared with the controls, an exception being the red fibers of musculus (m) pectoralis. From the data represented (Table I) it may be seen that changes in the m. supracoracoideus were greater than those in the m. pectoralis. Reduction in sarcomere volume was most pronounced when compared with that in the controls in the white and red fibers of both of the muscles studied after 18 months of hypodynamic state. Statistically significant differences in the sarcomere volume were noted in the red fibers of m. pectoralis of experimental animals after 6, 12, and 18 months. It should be stressed that in the control group of pigeons serving as reference for the experimental groups (6-H, 12-H, 18-H) statistically significant differences were not noted as regards the studied parameters of muscle fibers, either in m. supracoracoideus or m. pectoralis. Atrophy of single myofibrils was observed after 6 months of experiments (Figs.
MARCINIAK,
28
BARANSKA,
AND BARAN
TABLE I Effect of Hypodynamic State on the Sarcomere Volume (V, 2 SE, pm3) of Pigeon Muscles
M. supracoracoideus Group C
6-H 12-H 18-H
White fibers 2.63 1.28 1.22 1.14
+ 2 k +
0.03 0.14* 0.18* 0.18*
M. pectoralis
Red fibers 2.18 1.70 1.52 1.32
White fibers
IT 0.20 + 0.21* 2 0.11* 2 0.13*
2.52 2.01 1.82 1.36
f 2 2 2
0.03 0.13* 0.09* 0.02*
Red fibers 2.01 1.97 1.42 1.08
2 k 2 k
0.22 0.05 0.14** O.ll**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between the experimental groups (P < 0.05).
1 and 2), whereas after 12 and 18 months of immobilization many myofibrils disappeared (Fig. 3). Degenerative changes were also observed in the sarcomeres, manifested by obliteration of H bands and deformation of Z bands (Fig. 4). Immobilization, notwithstanding its duration (6, 12, or 18 months), resulted in an increase of the relative volume of the sarcoplasm (V,,, %) as compared with that in the controls. After 6 months of hypodynamic state (Table II) the sarcoplasmic volume increased in the white fibers of both muscles and after 12 and 18 months of restricted mobility the volume was several times higher in both white and red fibers than it was in the control group.
FIG. 1. M. supracoracoideus
red fibers. Control. ~30,000.
HYPODYNAMICS
AND MORPHOMETRIC
EVALUATION
OF MUSCLES
29
FIG. 2. M. supracoracoideus red fibers after 6 months in a hypodynamic state. Atrophy of single myotibrils and accumulation of glycogen granules. ~34,000.
FIG. 3. M. pectoralis red fibers after 18 months in a hypodynamic state. Atrophy of many myofibrils. x30.000.
30
MARCINIAK,
BARANSKA,
AND
BARAN
FIG. 4. M. supracoracoideus red fibers 12 and 18 months of immobilization. in the sarcomeres manifested by deformation of Z bands. ~30,000.
Degenerative changes
The electron micrographs of muscle often showed areas devoid of myofibrils, or containing a few organelles, randomly scattered fragments of the T system, and mitochondria oriented at right angles to the longitudinal axis of the sarcomere (Fig. 5). It should be emphasized that mitochondria, appearing in the empty spaces in the degenerated fibers, had an irregular shape but preserved a normal internal structure. The sarcoplasmic reticulum did not show in any of the experimental periods statistically significant differences in the red muscles (Table III) as compared with those of the controls. Only in the white fibers of the pectoralis muscle was a significant reduction of volume observed after 18 months in a hypodynamic state. TABLE II Effect of Hypodynamic State on the Sarcoplasmic Volume (V,,, of White and Red Fibers of Pigeon Muscles V,,,
M. supracoracoideus Group C
White fibers 5.99 2 0.85
6-H
13.43 k 0.82**
12-H
19.19
18-H
21.52 k 0.74*
k 1.06**
Red fibers 6.56 t 0.5 8.92 ? 0.98 12.91 f 0.76*
15.87 f 0.94*
+
f
SE, %)
SE (%) M. pectoralis White fibers
Red fibers
1.49 k 0.85
4.2 r 1.01 6.66 25 0.49 10.52 ? 0.67* 8.08 -+ 0.72*
11.54 + 0.72* 14.4 k 0.71* 24.44 + 1.75**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between the experimental groups (P < 0.05).
HYPODYNAMICS
AND MORPHOMETRIC
EVALUATION
OF MUSCLES
31
FIG. 5. M. supracoracoideus white fibers after 18 months in a hypodynamic state. Sarcoplasmic reticulum with fragments of the T system scattered randomly and mitochondria oriented at right angles to the long axis of the sarcomeres. x 18.000.
The number of glycogen granules was considerably increased after 6 months in experimental animals in both types of fibers. The granules were accumulated between the sarcomeres and in close proximity to the cell membrane of the myolibrils (Fig. 6). A longer period of restriction of mobility (12 months) caused a further accumulation of glycogen (N,) in the white fibers of the m. pectoralis and a significant diminution of their number in the supracoracoideus muscle as compared with the state after 6 months of experimentation (Table IV). TABLE III Effect of Hypodynamic State on the Smooth Sarcoplasmic Reticulum W,,,, 5 SE, %) of White and Red Fibers of Pigeon Muscles I’,,,
+
SE (%I) M. pectoralis
M. supracoracoideus Group C 6-H 12-H 18-H
White fibers 4.55 5.13 4.69 4.47
k 0.31 + 0.56*” ” 0.83* k 0.47
Red fibers
White fibers
Red fibers
3.3 5.41 3.2 4.69
4.33 4.34 4.44 2.83
2.75 L 0.52
2 t + t
0.41 1.39 0.52 0.36
k 0.19 k 0.39 k 0.77
k 0.23
* Statistical differences as compared with a control (P < 0.05). +* Statistical differences between the experimental groups (P < 0.05).
3.2 2 0.48 2.33 f 0.78 3.74 + 0.77
32
MARCINIAK,
BARAhKA,
AND
BARAN
FIG. 6. M. pectoralis white fibers after 12 months of hypodynamics. Deformation of the mitochondria profiles by the lipid droplets. Accumulation of glycogen granules between the sarcomeres. x 30,000.
After 18 months in a hypodynamic state a decreased number of glycogen granules was observed in the white and red fibers of both muscles studied as compared with that after 6 months. Long-lasting immobilization also causes changes in the mitochondria. After as little as 6 months of experimentation the relative volume of mitochondria decreased both in the white and red fibers of both muscles (Fig. 7), whereas the number of profiles diminished only in the red fibers of the pectoralis muscle. After 12 and 18 months statistically significant changes were found also in the relative volume of mitochondria in the white fibers of both the muscles as compared with the values in the controls. TABLE IV Effect of Hypodynamic State on the Number of Glycogen Granules (N, k SE) in the Muscle Fibers N, + SE M. pectoralis
M. supracoracoideus Group C
6-H 12-H 18-H
White fibers 1264.6 1608.0 1220.4 937.4
2 136.5 2 117.9 2 122.1** -+ 21.70*
Red fibers 1058.6 1656.4 728.0 832.6
4 153.0 ” 154.7* -c 46.1** r 152.4
White fibers 1240.2 1698.8 2062.6 940.0
+ k k c
172.9 212.6* 49.7** 194.8**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between experimental groups (P < 0.05).
Red fibers 1239.8 1842.0 1168.2 1055.0
+ + ‘k
62.0 218.9 77.6** 92.9
HYPODYNAMICS VOLUME
FRACTION
AND MORPHOMETRIC
OF THE
MITOCHONDRIA
EVALUATION
NUMBER
OF THE
OF MUSCLES
MITOCHONDRIAL
33
PROFILES
hd/lPm21 WHITE
WHITE
FIBRE
M Supracoracoideus
M Supracaracoideus
C H-6 H-12 H-18
C H-6 H-12 H-18 RED
FIBRE
C
H-6 H-12 H-18 RED
[NM hm21
C
C
H-6 H-12 H-18
C H-6 H-12 H-18
H-6 H42
H-18
C = CONTROL a H=HYPODYNAMICSO + q p < 0.05
FIBRE
M Supracaracoideus
Supracaracoideus
C H-6 H-12 H-16
FIBRE
M PeciorallS
C
H-6 H-12 H-16
FIG. 7. Effect of hypodynamic state on the mitochondria.
Such differences were noted in the pectoralis muscle in the volume fraction of the mitochondrial structures between the groups immobilized for 6 and 12 months. The number of mitochondrial profiles in the white fibers in both muscles decreased gradually with the duration of experimentation. Differences were noted both as compared with the controls and between the periods of limited mobility of 6, 12, and 18 months. In the red fibers of both muscles studied a distinct decrease of the mitochondrial fraction and in the number of profiles of these structures was found as compared with the control after 12 and 18 months of restricted mobility. In the early period, that is after 6 months of reduced mobility, only slight changes were observed in the mitochondria. Single or numerous mitochondria could be seen in close vicinity to lipid droplets which deformed their profiles. The mitochondria were of irregular shape and had distinct cristae and dark matrix. Most lipid droplets retained some homogeneous material. After longer periods of immobility, that is, after 12 and 18 months, distinct evidence of degeneration of mitochondria was noted. They were swollen and at times of gigantic dimensions and contained vacuoles and a small number of cristae and clear matrix. Occasionally, mitochondria with partly discontinuous membranes were seen. Sometimes there were disintegrated fragments of these structures (Fig. 8). DISCUSSION The objects of this investigation were the supracoracoideus and pectoralis muscles in pigeons which were prevented from flying for periods of 6, 12, and 18 months. Whereas both muscles contain similar proportions of white and red fibers (Hodges, 1974), the degenerative changes were more pronounced in the supracoracoideus than in the pectoralis muscle. This fact may be due to the greater
34
MARCINIAK,
BARAE(ISKA,
AND
BARAN
FIG. 8. M. pectoralis white fibers after 12 months in a hypodynamic damaged continuity of their internal membranes. ~20,000.
state. Mitochondria
with
participation of the former in the process of flying. This agrees with the opinion of George and Berger (1966), who consider that this muscle plays a more important role in the lifting of birds’ wings. In the experimental model applied by us the pigeons had no possibility of raising their wings, they could only straighten them in the horizontal plane. We, therefore, consider that the analyzed muscles were under conditions of a hypodynamic state. Disturbances of enzymatic metabolism were observed as the consequence of the hypodynamic state, mainly in the mitochondria of muscle fibers, manifested in accumulation of glycogen and lipid droplets in these structures. After 12 and 18 months of limited mobility considerable degenerative changes in the mitochondria and extensive atrophy of myofibrils appeared, leading to a reduction of the sarcomere volume which decreased with the duration of the hypodynamic state. The empty spaces left by the atrophied myofilaments were filled with sarcoplasm containing fragments of T systems and mitochondria of irregular shape with well-outlined cristae. These mitochondria lay perpendicular to the long axis of the sarcomere. It would seem that the presence of these unchanged structures is a manifestation of reconstruction of the muscle fibers previously damaged as the consequence of the hypodynamic state. Similar changes in the muscle fibers were observed in animals, especially rats and mice, as the consequence of hypokinesis (Baratiski et al., 1973, 1979). It should be stressed that the extent of changes was dependent both on the duration of mobility restriction and on the kind of muscle tested.
HYPODYNAMICS
AND
MORPHOMETRIC
EVALUATION
OF
MUSCLES
35
In the present experiment extensive changes were observed in the muscle fibers of pigeons only as late as after 12 and 18 months of immobilization. In rats, however, hypokinesis of one day already caused degeneration in the soleus muscle of the muscle fibers (Portugalov et al., 1971). After 6 months of hypodynamic state in our investigation a decrease in the mitochondrial fraction was observed, the number of their profiles remaining unchanged. However, in the soleus muscle when the period of immobility was prolonged, degenerative changes became more intensive (Tabary et al., 1972). Our data showed intensive accumulation of glycogen and the presence of lipid droplets was also noted. These changes may be interpreted as due to early disturbances of the basic metabolic cycles of mitochondria, involving hydrocarbon metabolism. It is known that restriction of mobility leads to a reduced supply of oxygen to the tissues (Stenger et al., 1962; Ilyina Kakueva and Portugalov, 1977) and this results in insufficiencies in the oxidation of hydrocarbons to CO, and H,O and in the formation of ATP (high-energy compounds). Oxygen deficit causes enzymatic disturbances leading to excessive glycogen accumulation and the formation of defective products of anaerobic metabolism in the form of lipid droplets (triglycerides) in the muscle fibers. It should also be mentioned that restriction of mobility of long duration may lead to reduced acetylcholine release from the nerve endings. Owing to the lack of this transmitter, the sarcoplasmic membrane remains for prolonged periods in states of polarization which make electrolyte exchange (mainly of Na and K) difficult, thus causing changes in the structure of the muscle fiber. To sum up, it may be affirmed that reduction of mobility of long duration leads to a reduced oxygen supply to the tissues, and insufficiencies of hydrocarbon oxidation and of release of energy in the form of ATP-a compound indispensable for normal metabolism. REFERENCES BARAIQSKI, S., EDELWEJN, Z., and STODOLNIK-BARA~~SKA, W. (1973). Morphologic and electromyographic investigations on the influence of hypodynamia on the functional efficiency of muscle. Relt. Med. Aeronuut. Spat. 46, 380-382. BARA~SKI, S., STODOLNIK-BARA~~SKA, W., MARCINIAK, M., and ILYINA-KAKUEVA, E. I. (1979). Ultrastructural investigations of the soleus muscle after space flight on the Biosputnik 936. Aviaf. Space Environ. Med. 50(9), 930-934. EISENHAUER, J., and KEY, J. (1945). Studies on muscle atrophy: A method of recording power in situ and observations on effect of position of immobilization on atrophy of disuse and denervation. Arch. Surg. 51, 154-163. GEORGE, J. C., and BERGER, A. J., (1966). “Avian Myology.” Academic Press, New York/London. GOLDSPINK, D. P. (1977). The influence of immobilization and stretch on protein turn over of rat skeletal muscle. .I. Physiol. London 264, 267-283. HAYAT, A., TARDIEU, C., TABARY, J. C., and TABARY, C. (1978). Effect of denervation on the reduction of sarcomere number in cat soleus muscle immobilized in shortened position during seven days. J. Physiol. 74, 563-567. HESS, A. (1970). Vertebrate slow muscle fibre. Physiol Rev. 50, 40-62. HODGES, R. (1974). “The Histology of Fowl.” Academic Press, London. ILYINA-KAKUEVA, E. I., and PORTUGALOV, V. V. (1977). Combined effect of space flight and radiation on skeletal muscle of rats. Aviut. .Spuce Environ. Med. 48(2), 115% 119. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. PORTUGALOV, V. V., AND PETROVA, N. V. (1976). LDH isoenzymes of skeletal muscles of rats after space flight and hypokinesia. Aviaf. Space Environ. Med. 47(8), 834-838. PORTUGALOV, V. V., ILYINA-KAKUEVA, E. I. STAROSTIN, V. I., ROKHLENKO, K. D.. and SAVIK,
MARCINIAK,
36
BARANSKA,
AND BARAN
Z. F. (1971). Morphological and cytochemical studies of hypokinetic effects. Aerospace Med. 42(10), 1041- 1049. ROKHLIN, G. D., and LEVITES, E. P. (1975). Hypokinetic effect on the development of osteoporosis. 1, 20-22.
SHVETS, V. N., and PORTUGALOV, V. V. (1976). Space flight effects on the hemopoietic function of bone marrow of the rat. Kosmich. Biol. Avint. Space Environ. Med. 47(7), 746-749. STENGER, R. J., SPIRO, D., SCULLY, R. E.. and SHANNON, J. M. (1962). Ultrastructural and physiologic alterations in ischemic skeletal muscle. Amer. J. Pathol. 40, l-20. SUMMERS, T. B. (1951). Effect of immobilization in various positions upon the weight and strength of skeletal muscle. Arch. Phys. Med. Rehabil. 32, 142- 145. TABARY, J. C., TABARY, C., TARDIEU, G. and GOLDSPINK, G. (1972). Physiological and structural changes in the cat’s soleus muscle due to immobilization at different lengths by plaster casts. J. Physiol. London 224, 23 1- 244. TOMANEK, R. J., and LUND, D. D. (1974). Degeneration of different types of skeletal muscle fibres. II. Immobilization. J. Anat. 118, 531-541. VANABLE, J., and COGGESHALL, R. (1975). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. Biophys.
Biochem.
Cytol.
4, 475-478.
WAWRZY~SKA-PAGOWSKA, J. (1972). Leczenie farmakologiczne chor6b reumatycznych. PZWL. 1366171. WEIBEL, E. R., and ELIAS H. (1967). Introduction to stereological principles. In “Quantitative Methods in Morphology” (E. R. Weibel and H. Elias, eds), pp. 88-89. Springer, Berlin.
AND
MOLECULAR
PATHOLOGY
37, 26-36 (1982)
Ultrastructural Morphometric Evaluation of Muscles Pigeons Kept under Conditions of Long-Lasting Hypodynamic State MARIANNA Laboratory
MARCINIAK, of Electron
Received
WANDA
BARA~SKA,
Microscopy, Institate Warsaw, Chalubiriskiego
January
AND WOJCIECH
of Biostructure, 5, Poland
28. 1981. and in rernised form
November
Medical
in
BARAN
Academy,
20, 1981
The purpose of this research was to evaluate morphometrically the structure of muscles of pigeons in hypodynamic state, i.e., when they are deprived of flying. The pigeons were allowed to move freely in the cages, thus avoiding a stress reaction. The results of these investigations revealed, that in the 6 months following initiation of the experiment, marked changes appeared in the enzymatic reactions in the mitochondria of the muscle fibers. These changes were reflected in the accumulation of glycogen and fatty droplets. After 12 to 18 months there appeared a limitation of mobility of the muscles followed by an extensive atrophy of the myofibrils with a decreased volume of sarcomeres. The above changes may be interpreted as a consequence of lack of tissue oxygen, aberration in the catabolic pathways of carbohydrates, and diminished content of ATP necessary for proper metabolic functions.
INTRODUCTION Long-lasting immobilization is one of the new problems in contemporary medicine. A number of changes occur in various organs as the consequence of complete or partial limitation of mobility (Rokhlin and Levites, 1975; Shvets and Portugalov, 1976). Some of these modifications concern the muscular system (Eisenhauer and Key, 1945; Summers, 1951; Tomanek and Lund, 1974). Numerous publications devoted to this problem have appeared of late (Portugalov and Petrova, 1976; Hayat et al., 1978). Hypokinesis greatly reduces mobilization and a hypodynamic state (limited mobility) produces stress reactions and leads to degeneration in the muscular system (Goldspink, 1977). The extent of these changes depends on the duration of hypokinesis or the hypodynamic state and on the experimental model. Thus, the changes in the muscular system may be considered as the outcome of both immobilization and stress. It is known that corticosteroids excreted in excess during stress have a destructive effect on the muscle proteins, actin and myosin (Wawrzynska-Pagowska, 1972). No reports were found in the available literature on quantitation at the ultrastructural level of muscles in animals subjected to a hypodynamic state without the added stress factor. Therefore, it seemed desirable to use an experimental model of a hypodynamic state that probably eliminates the stress reaction. The present investigations concern the ultrastructural morphometric evaluation of pigeon muscles playing an active part in flying. The birds were placed in cages where they could move freely without being able to fly. Such conditions in our opinion should not elicit a stress reaction. MATERIALS
AND METHODS
The experiments were performed with 35 domestic female pigeons weighing approximately 350 g, aged 4 months, kept in special cages 0.13 m high, 4.0 m long, 26 0014-4800/82/040026-11$02.00/0 Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
HYPODYNAMICS
AND
MORPHOMETRIC
EVALUATION
OF
MUSCLES
27
and 2.0 m wide which allowed free movement without the possibility of flying. The birds remained under these conditions for 6 (6-H), 12 (12-H), and 18 months (18-H)) and were fed a standard diet. Control experiments were run in parallel (c). The control animals were kept under conditions where they could freely fly. Each group, both experimental and control, consisted of five birds. The control pigeons were of the same age as the experimental ones. The objects in point were the muscles taking an active part in flying: musculus supracoracoideus and musculus pectoralis. Tissue for ultrastructural examination (10 blocks from each muscle) was obtained intra &am. The pigeons were anesthetized by intraperitoneal injection of sodium pentabarbiturate (Nembutal). The muscle tissue was fixed in 3% glutaraldehyde, postfixed in 1% osmic acid buffered with 0.1 M phosphate buffer, pH 7.4. After dehydration in graded ethanols the tissue was embedded in Epon 812 (Luft, 1961). The sections were cut on a Reichert Urn3 microtome and stained with uranyl acetate (Watson, 1958) and lead citrate (Vanable and Coggeshall, 1965). The sections were inspected in a JEM 100 C Jeol electron microscope. Morphometric evaluation was done on electronograms at final magnifications of x29 000, according to the principles of microstereology (Weibel and Elias, 1967). White and red fibers were separately analyzed in reference to their ultrastructural properties described by Hess (1970). The absolute sarcomere volume (V,, pm”) was determined from their length and width. Width was measured at the level of the H band. The relative mitochondrial volume (V,, %) and the number of profiles (N,) of these structures in one surface area unit-l pm2-of the section were analyzed, and the relative volumes of the sarcoplasmic reticulum (V,,, %) and sarcoplasm (V,,, %) were also determined. In all experimental groups the number of glycogen granules (NG) was estimated per 10 pm2 of the section. The results were statistically evaluated by Student’s t test at the 95% confidence level. RESULTS Morphometric analysis of the supracoracoideus and pectoralis muscles revealed that, owing to limitation of mobility, the sarcomere volume decreases. After 6 months in a hypodynamic state, a statistically significant reduction of sarcomere volume was observed as compared with the controls, an exception being the red fibers of musculus (m) pectoralis. From the data represented (Table I) it may be seen that changes in the m. supracoracoideus were greater than those in the m. pectoralis. Reduction in sarcomere volume was most pronounced when compared with that in the controls in the white and red fibers of both of the muscles studied after 18 months of hypodynamic state. Statistically significant differences in the sarcomere volume were noted in the red fibers of m. pectoralis of experimental animals after 6, 12, and 18 months. It should be stressed that in the control group of pigeons serving as reference for the experimental groups (6-H, 12-H, 18-H) statistically significant differences were not noted as regards the studied parameters of muscle fibers, either in m. supracoracoideus or m. pectoralis. Atrophy of single myofibrils was observed after 6 months of experiments (Figs.
MARCINIAK,
28
BARANSKA,
AND BARAN
TABLE I Effect of Hypodynamic State on the Sarcomere Volume (V, 2 SE, pm3) of Pigeon Muscles
M. supracoracoideus Group C
6-H 12-H 18-H
White fibers 2.63 1.28 1.22 1.14
+ 2 k +
0.03 0.14* 0.18* 0.18*
M. pectoralis
Red fibers 2.18 1.70 1.52 1.32
White fibers
IT 0.20 + 0.21* 2 0.11* 2 0.13*
2.52 2.01 1.82 1.36
f 2 2 2
0.03 0.13* 0.09* 0.02*
Red fibers 2.01 1.97 1.42 1.08
2 k 2 k
0.22 0.05 0.14** O.ll**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between the experimental groups (P < 0.05).
1 and 2), whereas after 12 and 18 months of immobilization many myofibrils disappeared (Fig. 3). Degenerative changes were also observed in the sarcomeres, manifested by obliteration of H bands and deformation of Z bands (Fig. 4). Immobilization, notwithstanding its duration (6, 12, or 18 months), resulted in an increase of the relative volume of the sarcoplasm (V,,, %) as compared with that in the controls. After 6 months of hypodynamic state (Table II) the sarcoplasmic volume increased in the white fibers of both muscles and after 12 and 18 months of restricted mobility the volume was several times higher in both white and red fibers than it was in the control group.
FIG. 1. M. supracoracoideus
red fibers. Control. ~30,000.
HYPODYNAMICS
AND MORPHOMETRIC
EVALUATION
OF MUSCLES
29
FIG. 2. M. supracoracoideus red fibers after 6 months in a hypodynamic state. Atrophy of single myotibrils and accumulation of glycogen granules. ~34,000.
FIG. 3. M. pectoralis red fibers after 18 months in a hypodynamic state. Atrophy of many myofibrils. x30.000.
30
MARCINIAK,
BARANSKA,
AND
BARAN
FIG. 4. M. supracoracoideus red fibers 12 and 18 months of immobilization. in the sarcomeres manifested by deformation of Z bands. ~30,000.
Degenerative changes
The electron micrographs of muscle often showed areas devoid of myofibrils, or containing a few organelles, randomly scattered fragments of the T system, and mitochondria oriented at right angles to the longitudinal axis of the sarcomere (Fig. 5). It should be emphasized that mitochondria, appearing in the empty spaces in the degenerated fibers, had an irregular shape but preserved a normal internal structure. The sarcoplasmic reticulum did not show in any of the experimental periods statistically significant differences in the red muscles (Table III) as compared with those of the controls. Only in the white fibers of the pectoralis muscle was a significant reduction of volume observed after 18 months in a hypodynamic state. TABLE II Effect of Hypodynamic State on the Sarcoplasmic Volume (V,,, of White and Red Fibers of Pigeon Muscles V,,,
M. supracoracoideus Group C
White fibers 5.99 2 0.85
6-H
13.43 k 0.82**
12-H
19.19
18-H
21.52 k 0.74*
k 1.06**
Red fibers 6.56 t 0.5 8.92 ? 0.98 12.91 f 0.76*
15.87 f 0.94*
+
f
SE, %)
SE (%) M. pectoralis White fibers
Red fibers
1.49 k 0.85
4.2 r 1.01 6.66 25 0.49 10.52 ? 0.67* 8.08 -+ 0.72*
11.54 + 0.72* 14.4 k 0.71* 24.44 + 1.75**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between the experimental groups (P < 0.05).
HYPODYNAMICS
AND MORPHOMETRIC
EVALUATION
OF MUSCLES
31
FIG. 5. M. supracoracoideus white fibers after 18 months in a hypodynamic state. Sarcoplasmic reticulum with fragments of the T system scattered randomly and mitochondria oriented at right angles to the long axis of the sarcomeres. x 18.000.
The number of glycogen granules was considerably increased after 6 months in experimental animals in both types of fibers. The granules were accumulated between the sarcomeres and in close proximity to the cell membrane of the myolibrils (Fig. 6). A longer period of restriction of mobility (12 months) caused a further accumulation of glycogen (N,) in the white fibers of the m. pectoralis and a significant diminution of their number in the supracoracoideus muscle as compared with the state after 6 months of experimentation (Table IV). TABLE III Effect of Hypodynamic State on the Smooth Sarcoplasmic Reticulum W,,,, 5 SE, %) of White and Red Fibers of Pigeon Muscles I’,,,
+
SE (%I) M. pectoralis
M. supracoracoideus Group C 6-H 12-H 18-H
White fibers 4.55 5.13 4.69 4.47
k 0.31 + 0.56*” ” 0.83* k 0.47
Red fibers
White fibers
Red fibers
3.3 5.41 3.2 4.69
4.33 4.34 4.44 2.83
2.75 L 0.52
2 t + t
0.41 1.39 0.52 0.36
k 0.19 k 0.39 k 0.77
k 0.23
* Statistical differences as compared with a control (P < 0.05). +* Statistical differences between the experimental groups (P < 0.05).
3.2 2 0.48 2.33 f 0.78 3.74 + 0.77
32
MARCINIAK,
BARAhKA,
AND
BARAN
FIG. 6. M. pectoralis white fibers after 12 months of hypodynamics. Deformation of the mitochondria profiles by the lipid droplets. Accumulation of glycogen granules between the sarcomeres. x 30,000.
After 18 months in a hypodynamic state a decreased number of glycogen granules was observed in the white and red fibers of both muscles studied as compared with that after 6 months. Long-lasting immobilization also causes changes in the mitochondria. After as little as 6 months of experimentation the relative volume of mitochondria decreased both in the white and red fibers of both muscles (Fig. 7), whereas the number of profiles diminished only in the red fibers of the pectoralis muscle. After 12 and 18 months statistically significant changes were found also in the relative volume of mitochondria in the white fibers of both the muscles as compared with the values in the controls. TABLE IV Effect of Hypodynamic State on the Number of Glycogen Granules (N, k SE) in the Muscle Fibers N, + SE M. pectoralis
M. supracoracoideus Group C
6-H 12-H 18-H
White fibers 1264.6 1608.0 1220.4 937.4
2 136.5 2 117.9 2 122.1** -+ 21.70*
Red fibers 1058.6 1656.4 728.0 832.6
4 153.0 ” 154.7* -c 46.1** r 152.4
White fibers 1240.2 1698.8 2062.6 940.0
+ k k c
172.9 212.6* 49.7** 194.8**
* Statistical differences as compared with a control (P < 0.05). ** Statistical differences between experimental groups (P < 0.05).
Red fibers 1239.8 1842.0 1168.2 1055.0
+ + ‘k
62.0 218.9 77.6** 92.9
HYPODYNAMICS VOLUME
FRACTION
AND MORPHOMETRIC
OF THE
MITOCHONDRIA
EVALUATION
NUMBER
OF THE
OF MUSCLES
MITOCHONDRIAL
33
PROFILES
hd/lPm21 WHITE
WHITE
FIBRE
M Supracoracoideus
M Supracaracoideus
C H-6 H-12 H-18
C H-6 H-12 H-18 RED
FIBRE
C
H-6 H-12 H-18 RED
[NM hm21
C
C
H-6 H-12 H-18
C H-6 H-12 H-18
H-6 H42
H-18
C = CONTROL a H=HYPODYNAMICSO + q p < 0.05
FIBRE
M Supracaracoideus
Supracaracoideus
C H-6 H-12 H-16
FIBRE
M PeciorallS
C
H-6 H-12 H-16
FIG. 7. Effect of hypodynamic state on the mitochondria.
Such differences were noted in the pectoralis muscle in the volume fraction of the mitochondrial structures between the groups immobilized for 6 and 12 months. The number of mitochondrial profiles in the white fibers in both muscles decreased gradually with the duration of experimentation. Differences were noted both as compared with the controls and between the periods of limited mobility of 6, 12, and 18 months. In the red fibers of both muscles studied a distinct decrease of the mitochondrial fraction and in the number of profiles of these structures was found as compared with the control after 12 and 18 months of restricted mobility. In the early period, that is after 6 months of reduced mobility, only slight changes were observed in the mitochondria. Single or numerous mitochondria could be seen in close vicinity to lipid droplets which deformed their profiles. The mitochondria were of irregular shape and had distinct cristae and dark matrix. Most lipid droplets retained some homogeneous material. After longer periods of immobility, that is, after 12 and 18 months, distinct evidence of degeneration of mitochondria was noted. They were swollen and at times of gigantic dimensions and contained vacuoles and a small number of cristae and clear matrix. Occasionally, mitochondria with partly discontinuous membranes were seen. Sometimes there were disintegrated fragments of these structures (Fig. 8). DISCUSSION The objects of this investigation were the supracoracoideus and pectoralis muscles in pigeons which were prevented from flying for periods of 6, 12, and 18 months. Whereas both muscles contain similar proportions of white and red fibers (Hodges, 1974), the degenerative changes were more pronounced in the supracoracoideus than in the pectoralis muscle. This fact may be due to the greater
34
MARCINIAK,
BARAE(ISKA,
AND
BARAN
FIG. 8. M. pectoralis white fibers after 12 months in a hypodynamic damaged continuity of their internal membranes. ~20,000.
state. Mitochondria
with
participation of the former in the process of flying. This agrees with the opinion of George and Berger (1966), who consider that this muscle plays a more important role in the lifting of birds’ wings. In the experimental model applied by us the pigeons had no possibility of raising their wings, they could only straighten them in the horizontal plane. We, therefore, consider that the analyzed muscles were under conditions of a hypodynamic state. Disturbances of enzymatic metabolism were observed as the consequence of the hypodynamic state, mainly in the mitochondria of muscle fibers, manifested in accumulation of glycogen and lipid droplets in these structures. After 12 and 18 months of limited mobility considerable degenerative changes in the mitochondria and extensive atrophy of myofibrils appeared, leading to a reduction of the sarcomere volume which decreased with the duration of the hypodynamic state. The empty spaces left by the atrophied myofilaments were filled with sarcoplasm containing fragments of T systems and mitochondria of irregular shape with well-outlined cristae. These mitochondria lay perpendicular to the long axis of the sarcomere. It would seem that the presence of these unchanged structures is a manifestation of reconstruction of the muscle fibers previously damaged as the consequence of the hypodynamic state. Similar changes in the muscle fibers were observed in animals, especially rats and mice, as the consequence of hypokinesis (Baratiski et al., 1973, 1979). It should be stressed that the extent of changes was dependent both on the duration of mobility restriction and on the kind of muscle tested.
HYPODYNAMICS
AND
MORPHOMETRIC
EVALUATION
OF
MUSCLES
35
In the present experiment extensive changes were observed in the muscle fibers of pigeons only as late as after 12 and 18 months of immobilization. In rats, however, hypokinesis of one day already caused degeneration in the soleus muscle of the muscle fibers (Portugalov et al., 1971). After 6 months of hypodynamic state in our investigation a decrease in the mitochondrial fraction was observed, the number of their profiles remaining unchanged. However, in the soleus muscle when the period of immobility was prolonged, degenerative changes became more intensive (Tabary et al., 1972). Our data showed intensive accumulation of glycogen and the presence of lipid droplets was also noted. These changes may be interpreted as due to early disturbances of the basic metabolic cycles of mitochondria, involving hydrocarbon metabolism. It is known that restriction of mobility leads to a reduced supply of oxygen to the tissues (Stenger et al., 1962; Ilyina Kakueva and Portugalov, 1977) and this results in insufficiencies in the oxidation of hydrocarbons to CO, and H,O and in the formation of ATP (high-energy compounds). Oxygen deficit causes enzymatic disturbances leading to excessive glycogen accumulation and the formation of defective products of anaerobic metabolism in the form of lipid droplets (triglycerides) in the muscle fibers. It should also be mentioned that restriction of mobility of long duration may lead to reduced acetylcholine release from the nerve endings. Owing to the lack of this transmitter, the sarcoplasmic membrane remains for prolonged periods in states of polarization which make electrolyte exchange (mainly of Na and K) difficult, thus causing changes in the structure of the muscle fiber. To sum up, it may be affirmed that reduction of mobility of long duration leads to a reduced oxygen supply to the tissues, and insufficiencies of hydrocarbon oxidation and of release of energy in the form of ATP-a compound indispensable for normal metabolism. REFERENCES BARAIQSKI, S., EDELWEJN, Z., and STODOLNIK-BARA~~SKA, W. (1973). Morphologic and electromyographic investigations on the influence of hypodynamia on the functional efficiency of muscle. Relt. Med. Aeronuut. Spat. 46, 380-382. BARA~SKI, S., STODOLNIK-BARA~~SKA, W., MARCINIAK, M., and ILYINA-KAKUEVA, E. I. (1979). Ultrastructural investigations of the soleus muscle after space flight on the Biosputnik 936. Aviaf. Space Environ. Med. 50(9), 930-934. EISENHAUER, J., and KEY, J. (1945). Studies on muscle atrophy: A method of recording power in situ and observations on effect of position of immobilization on atrophy of disuse and denervation. Arch. Surg. 51, 154-163. GEORGE, J. C., and BERGER, A. J., (1966). “Avian Myology.” Academic Press, New York/London. GOLDSPINK, D. P. (1977). The influence of immobilization and stretch on protein turn over of rat skeletal muscle. .I. Physiol. London 264, 267-283. HAYAT, A., TARDIEU, C., TABARY, J. C., and TABARY, C. (1978). Effect of denervation on the reduction of sarcomere number in cat soleus muscle immobilized in shortened position during seven days. J. Physiol. 74, 563-567. HESS, A. (1970). Vertebrate slow muscle fibre. Physiol Rev. 50, 40-62. HODGES, R. (1974). “The Histology of Fowl.” Academic Press, London. ILYINA-KAKUEVA, E. I., and PORTUGALOV, V. V. (1977). Combined effect of space flight and radiation on skeletal muscle of rats. Aviut. .Spuce Environ. Med. 48(2), 115% 119. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. PORTUGALOV, V. V., AND PETROVA, N. V. (1976). LDH isoenzymes of skeletal muscles of rats after space flight and hypokinesia. Aviaf. Space Environ. Med. 47(8), 834-838. PORTUGALOV, V. V., ILYINA-KAKUEVA, E. I. STAROSTIN, V. I., ROKHLENKO, K. D.. and SAVIK,
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Z. F. (1971). Morphological and cytochemical studies of hypokinetic effects. Aerospace Med. 42(10), 1041- 1049. ROKHLIN, G. D., and LEVITES, E. P. (1975). Hypokinetic effect on the development of osteoporosis. 1, 20-22.
SHVETS, V. N., and PORTUGALOV, V. V. (1976). Space flight effects on the hemopoietic function of bone marrow of the rat. Kosmich. Biol. Avint. Space Environ. Med. 47(7), 746-749. STENGER, R. J., SPIRO, D., SCULLY, R. E.. and SHANNON, J. M. (1962). Ultrastructural and physiologic alterations in ischemic skeletal muscle. Amer. J. Pathol. 40, l-20. SUMMERS, T. B. (1951). Effect of immobilization in various positions upon the weight and strength of skeletal muscle. Arch. Phys. Med. Rehabil. 32, 142- 145. TABARY, J. C., TABARY, C., TARDIEU, G. and GOLDSPINK, G. (1972). Physiological and structural changes in the cat’s soleus muscle due to immobilization at different lengths by plaster casts. J. Physiol. London 224, 23 1- 244. TOMANEK, R. J., and LUND, D. D. (1974). Degeneration of different types of skeletal muscle fibres. II. Immobilization. J. Anat. 118, 531-541. VANABLE, J., and COGGESHALL, R. (1975). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. Biophys.
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WAWRZY~SKA-PAGOWSKA, J. (1972). Leczenie farmakologiczne chor6b reumatycznych. PZWL. 1366171. WEIBEL, E. R., and ELIAS H. (1967). Introduction to stereological principles. In “Quantitative Methods in Morphology” (E. R. Weibel and H. Elias, eds), pp. 88-89. Springer, Berlin.