Acta Phyriol Scand 1990, 139, 297-304
Effects of denervation on enzyme- histochemical and morphometrical properties of the rat soleus muscle in relation t o age T. A N S V E D and L. L A R S S O N Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Stockholm, Sweden
ANSVED,T. & LARSSON, L. 1990. Effects of denervation on enzyme-histochemical and morphometrical properties of the rat soleus muscle in relation to age. Acta Physiol Scand 139,297-304. Received ZI December 1989, accepted 18January 1990. ISSN 00014772. Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Stockholm, Sweden. The soleus muscle of young adult (5 months), adult (1-1 I months) and old (23 months) male Wistar rats was unilaterally denervated for a period of 3 weeks and studied with regard to enzyme-histochemical and morphometrical properties. Denervation caused a marked atrophy of all fibres, irrespective of age and enzyme-histochemical type. Fibres having myofibrillar ATPase staining characteristics intermediate to type I and type IIA fibres increased in number in all age groups and a reduction in the number/proportion of type I fibres was found in adult and old animals. These results indicate that a slowto-fast shift in myofibrillar properties as a consequence of denervation, shown to occur in the soleus muscle of young animals, also takes place in old age. This supports the view that atrophic fibres with intermediate myofibrillar ATPase staining characteristics, and possibly also atrophic IIA fibres, seen in old soleus muscle could be type I fibres which have undergone a transformation in response to the age-related denervation process. Key words : ageing, denervation, muscle fibre atrophy, muscle fibre transformation, soleus.
During the period of maturation and growth, there is a decrease in the number of type I1 fibres in the rat soleus muscle due to a transformation of these fibres into type I (Kugelberg 1976, Boreham et af. 1988, Ansved & Larsson 1989). At older ages the total number of fibres decreases and the incidence of angular and atrophic fibres increases, indicating a denervation process (Ansved & Larsson 1989). T h i s is secondary to a loss of whole a-motoneurons (Ishihara et al. 1987, Hasizume et al. 1988, Ansved & Larsson 1990) and incomplete reinnervation of previously denervated muscle fibres, resulting in a decreased motor unit number and an increased innervation ratio (Edstrom & Larsson 1987, Pettigrew & Gardiner 1987). T h e few type I1 muscle fibres Correspondence : Tor Ansved, Department of Clinical Neurophysiology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
left in the old soleus often have an angular and atrophic appearance, and it has been suggested that some of these fibres are type I fibres which, through loss of neuronal contact, have been transformed into type I1 (Edstrom & Larsson 1987, Larsson & Ansved 1988). T h e reversal of the age-related fast-to-slow transformation in very old rats (Caccia et al. 1979, Boreham et al. 1988) lends some support to this hypothesis. I t is unclear, however, what effects a denervation process has on the relative occurrence of different fibre types in old age. I n young and adult animals, denervation is known to increase the number of fast-twitch type I1 fibres, the myosin ATPase activity and the occurrence of fast-type myosin light and heavy chains in slow-twitch muscle (Tomanek & Lund 1973, Jaweed et al. 1975, Syrovf 1976, Nwoye et al. 1982, HallCraggs et al. 1983, Spector 1985, Midrio et at.
1988).
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The purpose of the present study was therefore to elucidate whether a shift i n the proportion of fibre types i n response to denervation occurs i n old age as well, and, if so, whether the age-
(Brooke & Kaiser 1969). Depending on the pH sensitivity of the myofibrillar ATPase, the muscle fibres were classified into types I and 11, type I fibres being those with acid-stable ATPase and alkali-labile ATPase and type I1 fibres those showing the reverse related denervation process could be responsible pH sensitivity. Type I1 fibres were subdivided into for a transformation of type I fibres into type I1 type IIA and IIC, the former being completely as has previously been suggested. The soleus inhibited at pH 4.35.The fibres showing intermediate muscle of young adult ( 5 months), adult ( I ~ I I reactions at pH 4.35 (slightly greyish to dark grey) and months) and old (23 months) rats was denervated also after formaldehyde fixation (grey to black) were for a period of 3 weeks a n d then studied with assigned to the type IIC group (see Kugelberg 1976). respect to enzyme-histochemical a n d morpho- The type I fibres were further subdivided according to their ATPase staining characteristics (see Ansved & metrical properties. Larsson 1989). Those fibres showing strong ATPase activity at pH 4.3 (black) but intermediate activity MATERIALS AND METHODS after formaldehyde fixation (greyish) were classified as Young adult (5 months), adult (10-11 months) and type IC fibres, with staining intensities intermediate old (23 months) male Wistar rats (M~llegaard to those of type I and type IIC. The type IC fibres Breeding Centre) were studied and compared with were further divided into a lighter (type IC,,,,,) and a age-matched controls. The controls were randomly darker form (type IC,,,,), according to the ATPase selected from a group of animals which were part of activity after formaldehyde fixation. Quantitative trchnique. The total number of fibres another study concerning the effects of normal ageing and the absolute number of different fibre types and on the soleus muscle (Ansved & Larsson 1989). All subtypes were counted fiom magnified photorats were kept in conventional plastic cages and fed ad libztum with standard laboratory food and tap water. micrographs of whole muscle cross-sections. The Animals that were sick or moribund or showed gross relative number of each type and subtype was pathological organ changes were excluded from the calculated. Sizes of individual muscle fibres were study. Prior to the experiments the animals were obtained by tracing their outlines on magnified anaesthetized with an intramuscular injection of photomicrographs of muscle cross-sections with the aid of a digitizing unit connected to a microcomputer fentanyl-fluanisone (0.2 mg ml-' fentanyl [base] (Videoplan, Kontron Bildanalyse GmbH, Munich, 10 mg mi-' fluanisone), 0.2-0.3 ml kg ' body wt, followed by intraperitoneally administered pento- FRG). The maximum diameter across the lesser barbitone sodium (25-50 mg kg-' body wt). The aspect of each fibre, approximated as an elliptical-ty pe sciatic nerve was cut unilaterally in the upper hind structure, was calculated and used in order to avoid errors arising from obliquely cut fibres (see Dubowitz limb and an approximately I 5-20 mm long section of the nerve was removed. The overlying muscles and 1985). Measurements were always made in the central region of each cross-section. Measurements were skin were then sutured. After 3 weeks of denervation, the animals were again anaesthetized and the dener- made on 200 fibres of type I and 50 of each of types vated soleus muscle was carefully freed from sur- IIA, IIC, ICdarkand IC,,,,,. If the total number of fibres of the respective type was lower than these rounding tissue, as was the soleus muscle on the values, then all the fibres of this specific type were contralateral side. T h e muscles were removed and measured. clamped, using an adjustable metal clamp, at a Statzstics. Means and standard deviations were standardized length by measuring the distance becalculated from individual values by standard protween fine sutures (Ethicon, 5-0) placed in the distal tendon and in the fascia overlying the proximal part of cedures. Statistical inter-group comparisons were the muscles with the knee and ankle joint held at an made by means of the Mann-Whitney test. Since the angle of yo". The muscle was then weighed, frozen in same control groups were used for comparison with freon chilled with liquid nitrogen and stored at both denervated and contralateral muscles, the P values were accordingly adjusted to avoid the problem - 80 "C until processed further. of mass significance. Differences were considered At the motor point, the Hastologzcal techntque. muscle was cut perpendicular to its longitudinal axis significant at P < 0.05. into serial Io-pm thin cross-sections in a cryostat (- 20 "C). RESULTS The cross-sections were stained with haematoxylin-eosin (Dubowitz ry85), and for myofibrillar The weight of the denervated soleus decreased ATPase (Padykula & Herman 1955) ( I ) after 55 min ( P < 0.05-0.01)in all age groups by 4-1 % as of formaldehyde fixation at 4 "C (Hayashi & Freiman compared with control muscles (Table I ) . After 1966) and (2) after acid preincubation at pH4.35 denervation, body weight decreased (P < 0.05)
+
Body weight, muscle weight and diameters of fibres of different types and subtypes in the soleus muscle of normal and denervate alateral side) rats. Values are expressed as means SD
ed
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ed
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ed
eral
0.05,* * P
Age group (months)
n
Body weight (9)
Muscle weight (mg)
Type IIA (F)
6
414t-21
21.5
Type IIC (LLm)
Type IC dark (pm)
Tqpe IC light (pm)
Tj (pm
25.4*4.6**
25.3 +3.3*
28.5 f 3 . P
31
42.1 f 9 . 6
46.7f6.5 ( n = 4) 42.4 f 4.9
42.6k8.9
53
45.7 5 5.4 (n = 5)
54
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27.652.6" 39.5 f 4 . 2 (n = 5) 44.8k 4.9
30 56
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5
5
6
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104f40* (n = 4) 193f 18
5
6
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(n = 5) 5 0 . 1 f 13.0 (n = 4) 33.' (n =
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6 6
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88 f20"" 226 k 29
23.2f6.4"
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6
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41.6f6.6*
2I.ofr.z"
18.9f r.8"
24.0
26.8k3.7"
29
33.ok3.7
38.2 If: 6.6 (n = 2) 45.0f2.2 (n = 4)
35.9 k 4 . I
47
46.3 f 6 . 5
53
I*1
10-1
I
23
5
53'538"
1oof 18" (n = 4)
23
6
652k54
23
5
-
18jf6 ( n = 5) 182f46 (n = 4)
< 0.01in comparison with controls.
34.8 f2.9
24.2& 3.j " 31.8 f4.4
35.2 f8.2
33.6 (n = r ) 42.3 & 6.6 (n = 5)
k 3.4
52
(n = 4)
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T. Ansved and L . Larsson
Fig. I . Cryostat cross-sections of rat soleus muscle stained for myofibrillar ATPase after formaldehyde fixation. The left column represents control muscles, and the right column denervated muscles, of young adult (a, b), adult (c, d) and old (e, f) animals. A type I and a type IIA fibre are indicated with an asterisk and a circle respectively. Note the occurrence of fibres with intermediate staining characteristics and the non-specific atrophy of different fibre types in denervated muscle. Bar = 50 pm.
by 20% in the adult (1-1 I months) and 19% in the old (23 months) animals as compared with the controls, while no difference was seen in the young adult (5 months) group (Table I).
Fibre size characteristics A significant decrease ( P < 0.05-0.01) in muscle fibre size was seen in all fibre types and age
groups after denervation, although it was not possible to make a reliable statistical evaluation of the denervation atrophy in IC,,,, fibres since only a few of the adult and old control animals had fibres of this type (Table I ; Fig. I). Denervation affected all the age groups and the different fibre types _ - almost equally - except for a slightly more pronounced decrease in fibre size among type IIA fibres in the young adult group.
Age-related efeects of denervation
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Fibre type composition based on A T P a s e stainability
e m N e m 0 m N
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The atrophy was not always uniform throughout the muscle. Instead, some muscles in each age group had certain regions which were severely affected, while other parts of the muscles only showed slight atrophy. In contralateral muscles, a significant difference in fibre size was only noted in type IIC of adult and type I of old animals, where the fibres were larger compared with controls (Table I).
+I +I +I
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z
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%
mw
#
# r?N N
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- - 0
+I +I +I
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-
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N - -
3
woo
e
r.*m
+I +I +I
o w N m e m w
+I +I +I
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After denervation, the number and proportion of type IClightfibres and the proportion of type IC,,,, fibres increased significantly (P < 0.05) in all age groups. The proportion of type I fibres decreased ( P < 0.05-0.01)in the 10- to 1 1 - and 23-month-old rats, and a similar trend, although not statistically significant, was also seen in the young adult animals. Furthermore, the proportion of type IIC fibres increased (P< 0.05-0.01)in the 5- and 10- to 11-month-old animals. The total number of muscle fibres and the number and proportion of type IIA fibres were not significantly affected by denervation in any age group (Table 2). A slight difference in fibre type profile between the control muscle and the contralateral nondenervated muscle was found in the 10- to I I month-old group, while no differences were seen in the other two age groups. However, the difference was small and the number of animals limited (Table z ) , thus allowing no speculation as to whether this difference represented an effect of altered usage, or an effect of denervation per se, on the contralateral muscle, or if the difference merely reflected a bias in the sampling of animals.
E
.-5: d
10W10
301
88 E
DISCUSSION Methodological considerations
.C
I
8 V 4 # # Lo
2
V 4 #
The classification of fibre types within the soleus muscle into types I, IIA and IIC based on their myofibrillar ATPase staining characteristics is widely accepted (Brooke &?Kaiser 1970,Kugelberg 1976). However, the staining intensities represent a continuum, and many intermediate reactions between typical type I and type IIA fibres occur. Since some of the type 1 fibres had staining characteristics intermediate to those of
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typical type I and type I I C fibres, these fibres were denoted ICriarkand IC,,,, (see Ansved & Larsson 1989) in order to facilitate the detection of a transformation process. A direct correlation between the myosin heavy chain (MHC) composition and the histochemical staining for myofibrillar ATPase has been reported in single fibres of rabbit soleus muscle (Staron & Pette 1986). The intermediate fibres, classified histochemically in a similar way as in this study, were characterized by the coexistence of fast and slow MHCs in varying proportions, i.e. type I I C had a predominance of fast MHCs while type I C had a predominance of slow MHCs (Staron & Pette 1986). Furthermore, the isoform composition of types I I C and I C with regard to myosin light chains, M- and C-proteins, a-tropomyosin and troponin I resembled that of types IIA and I respectively (Staron & Pette 1987).
Fibre size characteristics As in other studies of denervated soleus muscle (Herbison et al. 1979, Ullman et al. 1989, Wroblewski et al. 1989), fibre atrophy in this study affected the different fibre types to an approximately equal extent. This is in contrast to studies of denervated fast-twitch muscles where a preferential type I1 atrophy has been reported (Karpati & Engel 1968, Niederle & Mayr 1978, Lindboe & Presthus 1985, Ullman et al. 1989, Wroblewski et kl. 1989). There is some evidence that adult muscles are more resistant to post-denervation atrophic changes than developing muscles (Kumar & Talesara 1977). However, the present results show no difference in susceptibility with regard to denervation atrophy between young adult, adult and old animals. The atrophy was not always uniform within the muscle, suggesting that peripheral factors, such as passive tension, are different in different parts of the muscle. It has, for example, been shown that the stretch of a muscle, regardless of whether it is innervated or not, is of paramount importance for the size of a fibre (see Sola et al. 1973, Goldspink 1978).
Fibre type composition based on A T P a s e stainability In young and adult animals, experimental denervation of the soleus has been shown to increase the number of fast-twitch type I1 fibres,
myosin ATPase activity and the occurence of fast-type myosin light and heavy chains (Tomanek & Lund 1973, Jaweed et al. 1975, Syrovy 1976, Nwoye et al. 1982, Hall-Craggs et al. 1983, Spector 1985, Midrio et al. 1988). This slow-tofast shift in myofibrillar properties has been ascribed to a transformation of type I fibres into type I1 (Midrio et al. 1988). This is further substantiated by the finding that MHC isoforms of fast and slow types are expressed together in a large proportion of fibres in denervated rat soleus (see Schiaffino et al. 1990). Based on these reports and on the findings of Jenny et al. (1980) showing a synthesis of fast myosin in old, but not in young, adult rabbit soleus muscle, it has been proposed that some of the angular, atrophic type I1 fibres in old rat soleus muscle might be type I fibres which, through loss of neuronal contact, have been transformed into type I1 (Edstrom & Larsson 1987, Ansved & Larsson 1989). This assumption is partially supported by the observation by Caccia et al. (1979) of a reversal of the age-related fast-to-slow transformation, together with obvious neurogenic changes, in the soleus muscle of very old rats. Boreham et al. (1988), on the other hand, ascribed the increased number of type I1 fibres in very old age to a reduced postural load on the muscle. In fact, the reported shift in fibre-type profile at these ages might be related to both a denervation process and a reduced postural load, since both of these mechanisms are expected to reduce the level of activity of the muscle, which presumably favours a slow-to-fast transformation (see Boreham et al. 1988, Midrio et al. 1988). In the present study, the number and proportion of fibres with intermediate myofibrillar ATPase staining characteristics increased in all age groups and the number and/or proportion of type I fibres decreased in adult and old animals, in response to denervation. Since no obvious signs of fibre splitting or formation of fibres de not'o were observed, a transformation of type I fibres into intermediate types IC and I I C seems to be the most likely cause of these changes. A similar slow-to-fast shift in myofibrillar ATPase staining characteristics was not seen in the contralateral soleus muscles, thus excluding a generalized effect of inactivation. The slow-to-fast transition in the present study affected only a limited number of type I fibres. It has been speculated that in the normal rat soleus muscle there are two populations of type I fibres, with indis-
Age-related eflects q/' denervution tinguishable MHC composition and corresponding to the primary and secondary generation fibres, which respond differently to, for example, denervation (Schiaffino et ul. 1990). According to this hypothesis, those type I fibres that have been transformed from type IIA during maturation and growth constitute secondary generation fibres, and it is these fibres that may undergo transformation in response to denervation. I n conclusion, the present findings imply that a slow-to-fast shift in myofibrillar properties in response t o denervation can also take place in the soleus muscle of old animals. Atrophic fibres with intermediate ATPase staining characteristics, and possibly also atrophic IIA fibres, seen in this muscle in old age might consequently be type I fibres which have been transformed in response to the age-related denervation process, as has previously been suggested (Edstrom & 1,arsson 1987, Larsson & Ansved 1988). The authors are indebted to Lars Edstriim for placing laboratory facilities at our disposal and Birgitta Hedberg and Birgitta Lindegren for excellent laboratory assistance. This study was supported by grants from the Swedish Medical Research Council (8651,3875), the Karolinska Institute and the Swedish Society of Medicine.
REFERENCES ANSVED, T. & LARSSON, L . 1989. Effects of ageing on enzyme-histochemical, morphometrical and contractile properties of the soleus muscle in the rat. 3 Neuvol Sci 93, 105-124. ANSVED, T. & LARSSON, L. 1990. Quantitative and qualitative morphological properties of the soleus motor nerve and the L5 ventral root in young and old rats. Relation to the number of soleus muscle fibres. 3 Neurol Sci (in press). C.A.G., WATT, P.W., WILLIAMS,P.E., BOREHAM, MLRRY,B.J., GOLDSPINK, G. & GOLDSPINK, D.F. 1988. Effects of ageing and chronic dietary restriction on the morphology of fast and slow muscles of the rat. 3 Anat 157, I I 1-125. BROOKE, M.H. &KAISER, K.K. 1969. Some comments on the histochemical characterization of muscle adenosine triphosphatase. 3 Histochem Cytnchem 17,431-432, BROOKE,M.H. & KAISER,K.K. 1970. Muscle fiber types: How many and what kind? Arch Neurol23, 369-379. CACCIA, M.R., HARRIS,J.B. &JOHNSON,M.A. 1979.
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Morphology and physiology of skeletal muscle in aging rodents. Muscle N e r v e 2 , 202-212. DUBOWITZ, V. 1985, Muscle Biopsy - A Practical Approach. Bailliere Tindall, London. EDSTROM,I,. & LARSSON, L. 1987. Effects of age on contractile and enzyme-histochemical properties of fast- and slow-twitch single motor units in the rat. 3 Ph.ysiol 392, 129-145. GOI.DSPINK,D.F. 1978. Changes in the size and protein turnover of the soleus muscle in response to immobilization or denervation. Biochem Soc Trans 6, 1014-1017. HALL-CRAGGS, E.C.B., WINES,M.M. & MAX,S.R. 1983. Fiber type changes in denervated soleus muscles of the hyperthyroid rat. Esp Neurol 80, 2 5 2-2 5 7. HASIIIZUME, K., KANDA,K. & BURKE,R.E. 1988. Medial gastrocnemius motor nucleus in the rat: age-related changes in the number and size of motoneurons. 3 Comp Neurol269, 425-430. HAYASHI, M. & FREIMAN, D.G. 1966. An improved mcthod of fixation for formalin-sensitive enzymes with special reference to myosin adenosine triphosphatase. 3 Histochem Cytochem 14, 577-581. HERBISON, G.J., JAWEED, M.M. & DITUNNO, J.F. 1979. Muscle atrophy in rats following denervation, casting, inflammation, and tenotomy. Arrh Ph.ys Med Rehahil60, 401-404. ISHIHARA, A,, NAITOH,H. & KATSUTA,S. 1987. Effects of ageing on the total number of muscle fibers and motoneurons of the tibialis anterior and soleus muscles in the rat. Brain Res 435, 355-358. JAWEED, M.M., HERBISON, G.J. & DITUNNO, J.F. 1975. Denervation and reinnervation of fast and slow muscles. A histochemical study in rats. 3 Ilisiochem Cyiochem 23, 808-827. JENNY,E., WEBER, H., LUTZ,H . & BILLETER, R. 1980. Fibre populations in rabbit skeletal muscles from birth to old age. In: D. Pette (ed.) Plastzcity of Muscle, pp. 97-109. de Gruyter, Berlin. KARPATI, G. & ENGEI.,W.K. 1968. IIistochemical investigation of fiber type ratios with the myofibrillar ATP-ase reaction in normal and denervated skeletal muscles of guinea pig. A m 3 Anat 122, 145-156. KUGELBERG, E. 1976. Adaptive transformation of rat soleus motor units during growth. 3Neurol Sci 27, 269-89. KUMAR,P. & TALESARA, C.1,. 1977. Influence of age upon the onset and rate of progression of denervation atrophy in skeletal muscle : relative neuronal (trophic) independence upon fiber type maturation. Ind 3 Exp Biol 15, 45-51. LARSSON, L. & ANSVED, T. 1988. Effects of age on the motor unit. A study on single motor units in the rat. I n : J.A. Joseph (ed.) Central Determinants of Agerelated Declines in Motor Function, vol. 515, pp. 303-313. New York Acadamy of Sciences.
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anterior latissimus dorsi muscles following stretch LINDBOE,C.F. & PRESTHUS,J. 1985. Effects of denervation, immobilization and cachexia on fibre with and without denervation. Exp Neurol 41, 76100. size in the anterior tibia1 muscle of the rat. Acta SPECTOR, S.A. 1985. Trophic effects on the contractile Neuropathol 66, 42-5 I . MIDRIO,M., DANIELI BETTO,D., BETTO,R., NOVENTA, and histochemical properties of rat soleus muscle. J Neurosci 5, 2189-2196. D. & ANTICO, F. 1988. Cordotomy-denervation STARON, R.S. & PETTE,D. 1986. Correlation between interactions on contractile and myofibrillar propermyofibrillar ATPase activity and myosin heavy ties of fast and slow muscles in the rat. Exp Neurol chain composition in rabbit muscle fibers. Histo100, 216-236. chemistry 86, 19-23. NIEDERLE, B. & MAYR,R. 1978. Course of denervation R.S. & PETTE,D. 1987. The multiplicity of atrophy in type I and type I1 fibres of rat extensor STARON, combinations of myosin light chains and heavy digitorum longus muscle. Anat Embryo1 153, 9-21. chains in histochemically typed single fibres. NWOYE,L., MOMMAERTS, W.F.H.M., SIMPSON, D.R., Biochem -7 243, 6 8 7 4 9 3 . SERAYDARIAN, K. & MARUSICH, M. 1982. Evidence for a direct action of thyroid hormone in specifying S Y R O VI.~ , 1976. The relation between ATPase activity and light chains of myosin in developing, muscle properties. A m 3 Physiol 242, R401-408. adult and denervated muscles of several animal PADYKULA, H.A. & HERMAN, E. 1955. The specificity species. Physiol Bohemoslov 25, 295-300. of the histochemical method of adenosine triTOMANEK, R.J. & LUND,D.D. 1973. Degeneration phosphatase. 3 Histochem Cytochem 3, 17*195. of different types of skeletal muscle fibres. I. PETTIGREW, F.P. & GARDINER, P.F. 1987. Changes in Denervation. 3 Anat 116, 395-407. rat plantaris motor unit profiles with advanced age. ULLMAN,M., ALAMEDDINE, H., SKOTTNER, A. & Mech Aging Dev 40, 243-259. SCHIAFFINO, S., GORZA, L., AUSONI,S., BOTTINELLI, OLDFORS,A. 1989. Effects of growth hormone on skeletal muscle. 11. Studies on regeneration and R., REGGIANI,C., LARSSON, L., EDSTROM,L., denervation in adult rats: Acta Physiol Scand 135, GUNDERSEN, K. & L0M0, T. 1990. Muscle fiber types expressing different myosin heavy chain 537-543. R., EDSTROM, L. & JAKOBSSON, F. 1989. isoforms. Their functional properties and adaptive WROBLEWSKI, Effect of short time denervation on intracellular capacity. In: D. Pette (ed.) The Dynamic State of elemental content and fibre atrophy pattern of slow Muscle Fibers. de Gruyter, Berlin (in press). and fast twitch rat muscle. 3 Submzcrosc Cytol D.L. & MARTIN,A.W. SOLA,O.M., CHRISTENSEN, P a t h o l z ~ 68~-600. . 1973. Hypertrophy and hyperptasia of adult chicken 1
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Effects of denervation on enzyme- histochemical and morphometrical properties of the rat soleus muscle in relation t o age T. A N S V E D and L. L A R S S O N Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Stockholm, Sweden
ANSVED,T. & LARSSON, L. 1990. Effects of denervation on enzyme-histochemical and morphometrical properties of the rat soleus muscle in relation to age. Acta Physiol Scand 139,297-304. Received ZI December 1989, accepted 18January 1990. ISSN 00014772. Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Stockholm, Sweden. The soleus muscle of young adult (5 months), adult (1-1 I months) and old (23 months) male Wistar rats was unilaterally denervated for a period of 3 weeks and studied with regard to enzyme-histochemical and morphometrical properties. Denervation caused a marked atrophy of all fibres, irrespective of age and enzyme-histochemical type. Fibres having myofibrillar ATPase staining characteristics intermediate to type I and type IIA fibres increased in number in all age groups and a reduction in the number/proportion of type I fibres was found in adult and old animals. These results indicate that a slowto-fast shift in myofibrillar properties as a consequence of denervation, shown to occur in the soleus muscle of young animals, also takes place in old age. This supports the view that atrophic fibres with intermediate myofibrillar ATPase staining characteristics, and possibly also atrophic IIA fibres, seen in old soleus muscle could be type I fibres which have undergone a transformation in response to the age-related denervation process. Key words : ageing, denervation, muscle fibre atrophy, muscle fibre transformation, soleus.
During the period of maturation and growth, there is a decrease in the number of type I1 fibres in the rat soleus muscle due to a transformation of these fibres into type I (Kugelberg 1976, Boreham et af. 1988, Ansved & Larsson 1989). At older ages the total number of fibres decreases and the incidence of angular and atrophic fibres increases, indicating a denervation process (Ansved & Larsson 1989). T h i s is secondary to a loss of whole a-motoneurons (Ishihara et al. 1987, Hasizume et al. 1988, Ansved & Larsson 1990) and incomplete reinnervation of previously denervated muscle fibres, resulting in a decreased motor unit number and an increased innervation ratio (Edstrom & Larsson 1987, Pettigrew & Gardiner 1987). T h e few type I1 muscle fibres Correspondence : Tor Ansved, Department of Clinical Neurophysiology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
left in the old soleus often have an angular and atrophic appearance, and it has been suggested that some of these fibres are type I fibres which, through loss of neuronal contact, have been transformed into type I1 (Edstrom & Larsson 1987, Larsson & Ansved 1988). T h e reversal of the age-related fast-to-slow transformation in very old rats (Caccia et al. 1979, Boreham et al. 1988) lends some support to this hypothesis. I t is unclear, however, what effects a denervation process has on the relative occurrence of different fibre types in old age. I n young and adult animals, denervation is known to increase the number of fast-twitch type I1 fibres, the myosin ATPase activity and the occurrence of fast-type myosin light and heavy chains in slow-twitch muscle (Tomanek & Lund 1973, Jaweed et al. 1975, Syrovf 1976, Nwoye et al. 1982, HallCraggs et al. 1983, Spector 1985, Midrio et at.
1988).
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The purpose of the present study was therefore to elucidate whether a shift i n the proportion of fibre types i n response to denervation occurs i n old age as well, and, if so, whether the age-
(Brooke & Kaiser 1969). Depending on the pH sensitivity of the myofibrillar ATPase, the muscle fibres were classified into types I and 11, type I fibres being those with acid-stable ATPase and alkali-labile ATPase and type I1 fibres those showing the reverse related denervation process could be responsible pH sensitivity. Type I1 fibres were subdivided into for a transformation of type I fibres into type I1 type IIA and IIC, the former being completely as has previously been suggested. The soleus inhibited at pH 4.35.The fibres showing intermediate muscle of young adult ( 5 months), adult ( I ~ I I reactions at pH 4.35 (slightly greyish to dark grey) and months) and old (23 months) rats was denervated also after formaldehyde fixation (grey to black) were for a period of 3 weeks a n d then studied with assigned to the type IIC group (see Kugelberg 1976). respect to enzyme-histochemical a n d morpho- The type I fibres were further subdivided according to their ATPase staining characteristics (see Ansved & metrical properties. Larsson 1989). Those fibres showing strong ATPase activity at pH 4.3 (black) but intermediate activity MATERIALS AND METHODS after formaldehyde fixation (greyish) were classified as Young adult (5 months), adult (10-11 months) and type IC fibres, with staining intensities intermediate old (23 months) male Wistar rats (M~llegaard to those of type I and type IIC. The type IC fibres Breeding Centre) were studied and compared with were further divided into a lighter (type IC,,,,,) and a age-matched controls. The controls were randomly darker form (type IC,,,,), according to the ATPase selected from a group of animals which were part of activity after formaldehyde fixation. Quantitative trchnique. The total number of fibres another study concerning the effects of normal ageing and the absolute number of different fibre types and on the soleus muscle (Ansved & Larsson 1989). All subtypes were counted fiom magnified photorats were kept in conventional plastic cages and fed ad libztum with standard laboratory food and tap water. micrographs of whole muscle cross-sections. The Animals that were sick or moribund or showed gross relative number of each type and subtype was pathological organ changes were excluded from the calculated. Sizes of individual muscle fibres were study. Prior to the experiments the animals were obtained by tracing their outlines on magnified anaesthetized with an intramuscular injection of photomicrographs of muscle cross-sections with the aid of a digitizing unit connected to a microcomputer fentanyl-fluanisone (0.2 mg ml-' fentanyl [base] (Videoplan, Kontron Bildanalyse GmbH, Munich, 10 mg mi-' fluanisone), 0.2-0.3 ml kg ' body wt, followed by intraperitoneally administered pento- FRG). The maximum diameter across the lesser barbitone sodium (25-50 mg kg-' body wt). The aspect of each fibre, approximated as an elliptical-ty pe sciatic nerve was cut unilaterally in the upper hind structure, was calculated and used in order to avoid errors arising from obliquely cut fibres (see Dubowitz limb and an approximately I 5-20 mm long section of the nerve was removed. The overlying muscles and 1985). Measurements were always made in the central region of each cross-section. Measurements were skin were then sutured. After 3 weeks of denervation, the animals were again anaesthetized and the dener- made on 200 fibres of type I and 50 of each of types vated soleus muscle was carefully freed from sur- IIA, IIC, ICdarkand IC,,,,,. If the total number of fibres of the respective type was lower than these rounding tissue, as was the soleus muscle on the values, then all the fibres of this specific type were contralateral side. T h e muscles were removed and measured. clamped, using an adjustable metal clamp, at a Statzstics. Means and standard deviations were standardized length by measuring the distance becalculated from individual values by standard protween fine sutures (Ethicon, 5-0) placed in the distal tendon and in the fascia overlying the proximal part of cedures. Statistical inter-group comparisons were the muscles with the knee and ankle joint held at an made by means of the Mann-Whitney test. Since the angle of yo". The muscle was then weighed, frozen in same control groups were used for comparison with freon chilled with liquid nitrogen and stored at both denervated and contralateral muscles, the P values were accordingly adjusted to avoid the problem - 80 "C until processed further. of mass significance. Differences were considered At the motor point, the Hastologzcal techntque. muscle was cut perpendicular to its longitudinal axis significant at P < 0.05. into serial Io-pm thin cross-sections in a cryostat (- 20 "C). RESULTS The cross-sections were stained with haematoxylin-eosin (Dubowitz ry85), and for myofibrillar The weight of the denervated soleus decreased ATPase (Padykula & Herman 1955) ( I ) after 55 min ( P < 0.05-0.01)in all age groups by 4-1 % as of formaldehyde fixation at 4 "C (Hayashi & Freiman compared with control muscles (Table I ) . After 1966) and (2) after acid preincubation at pH4.35 denervation, body weight decreased (P < 0.05)
+
Body weight, muscle weight and diameters of fibres of different types and subtypes in the soleus muscle of normal and denervate alateral side) rats. Values are expressed as means SD
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Age group (months)
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Body weight (9)
Muscle weight (mg)
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21.5
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31
42.1 f 9 . 6
46.7f6.5 ( n = 4) 42.4 f 4.9
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29
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35.9 k 4 . I
47
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53
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I
23
5
53'538"
1oof 18" (n = 4)
23
6
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23
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< 0.01in comparison with controls.
34.8 f2.9
24.2& 3.j " 31.8 f4.4
35.2 f8.2
33.6 (n = r ) 42.3 & 6.6 (n = 5)
k 3.4
52
(n = 4)
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Fig. I . Cryostat cross-sections of rat soleus muscle stained for myofibrillar ATPase after formaldehyde fixation. The left column represents control muscles, and the right column denervated muscles, of young adult (a, b), adult (c, d) and old (e, f) animals. A type I and a type IIA fibre are indicated with an asterisk and a circle respectively. Note the occurrence of fibres with intermediate staining characteristics and the non-specific atrophy of different fibre types in denervated muscle. Bar = 50 pm.
by 20% in the adult (1-1 I months) and 19% in the old (23 months) animals as compared with the controls, while no difference was seen in the young adult (5 months) group (Table I).
Fibre size characteristics A significant decrease ( P < 0.05-0.01) in muscle fibre size was seen in all fibre types and age
groups after denervation, although it was not possible to make a reliable statistical evaluation of the denervation atrophy in IC,,,, fibres since only a few of the adult and old control animals had fibres of this type (Table I ; Fig. I). Denervation affected all the age groups and the different fibre types _ - almost equally - except for a slightly more pronounced decrease in fibre size among type IIA fibres in the young adult group.
Age-related efeects of denervation
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Fibre type composition based on A T P a s e stainability
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The atrophy was not always uniform throughout the muscle. Instead, some muscles in each age group had certain regions which were severely affected, while other parts of the muscles only showed slight atrophy. In contralateral muscles, a significant difference in fibre size was only noted in type IIC of adult and type I of old animals, where the fibres were larger compared with controls (Table I).
+I +I +I
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mw
#
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+I +I +I
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After denervation, the number and proportion of type IClightfibres and the proportion of type IC,,,, fibres increased significantly (P < 0.05) in all age groups. The proportion of type I fibres decreased ( P < 0.05-0.01)in the 10- to 1 1 - and 23-month-old rats, and a similar trend, although not statistically significant, was also seen in the young adult animals. Furthermore, the proportion of type IIC fibres increased (P< 0.05-0.01)in the 5- and 10- to 11-month-old animals. The total number of muscle fibres and the number and proportion of type IIA fibres were not significantly affected by denervation in any age group (Table 2). A slight difference in fibre type profile between the control muscle and the contralateral nondenervated muscle was found in the 10- to I I month-old group, while no differences were seen in the other two age groups. However, the difference was small and the number of animals limited (Table z ) , thus allowing no speculation as to whether this difference represented an effect of altered usage, or an effect of denervation per se, on the contralateral muscle, or if the difference merely reflected a bias in the sampling of animals.
E
.-5: d
10W10
301
88 E
DISCUSSION Methodological considerations
.C
I
8 V 4 # # Lo
2
V 4 #
The classification of fibre types within the soleus muscle into types I, IIA and IIC based on their myofibrillar ATPase staining characteristics is widely accepted (Brooke &?Kaiser 1970,Kugelberg 1976). However, the staining intensities represent a continuum, and many intermediate reactions between typical type I and type IIA fibres occur. Since some of the type 1 fibres had staining characteristics intermediate to those of
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typical type I and type I I C fibres, these fibres were denoted ICriarkand IC,,,, (see Ansved & Larsson 1989) in order to facilitate the detection of a transformation process. A direct correlation between the myosin heavy chain (MHC) composition and the histochemical staining for myofibrillar ATPase has been reported in single fibres of rabbit soleus muscle (Staron & Pette 1986). The intermediate fibres, classified histochemically in a similar way as in this study, were characterized by the coexistence of fast and slow MHCs in varying proportions, i.e. type I I C had a predominance of fast MHCs while type I C had a predominance of slow MHCs (Staron & Pette 1986). Furthermore, the isoform composition of types I I C and I C with regard to myosin light chains, M- and C-proteins, a-tropomyosin and troponin I resembled that of types IIA and I respectively (Staron & Pette 1987).
Fibre size characteristics As in other studies of denervated soleus muscle (Herbison et al. 1979, Ullman et al. 1989, Wroblewski et al. 1989), fibre atrophy in this study affected the different fibre types to an approximately equal extent. This is in contrast to studies of denervated fast-twitch muscles where a preferential type I1 atrophy has been reported (Karpati & Engel 1968, Niederle & Mayr 1978, Lindboe & Presthus 1985, Ullman et al. 1989, Wroblewski et kl. 1989). There is some evidence that adult muscles are more resistant to post-denervation atrophic changes than developing muscles (Kumar & Talesara 1977). However, the present results show no difference in susceptibility with regard to denervation atrophy between young adult, adult and old animals. The atrophy was not always uniform within the muscle, suggesting that peripheral factors, such as passive tension, are different in different parts of the muscle. It has, for example, been shown that the stretch of a muscle, regardless of whether it is innervated or not, is of paramount importance for the size of a fibre (see Sola et al. 1973, Goldspink 1978).
Fibre type composition based on A T P a s e stainability In young and adult animals, experimental denervation of the soleus has been shown to increase the number of fast-twitch type I1 fibres,
myosin ATPase activity and the occurence of fast-type myosin light and heavy chains (Tomanek & Lund 1973, Jaweed et al. 1975, Syrovy 1976, Nwoye et al. 1982, Hall-Craggs et al. 1983, Spector 1985, Midrio et al. 1988). This slow-tofast shift in myofibrillar properties has been ascribed to a transformation of type I fibres into type I1 (Midrio et al. 1988). This is further substantiated by the finding that MHC isoforms of fast and slow types are expressed together in a large proportion of fibres in denervated rat soleus (see Schiaffino et al. 1990). Based on these reports and on the findings of Jenny et al. (1980) showing a synthesis of fast myosin in old, but not in young, adult rabbit soleus muscle, it has been proposed that some of the angular, atrophic type I1 fibres in old rat soleus muscle might be type I fibres which, through loss of neuronal contact, have been transformed into type I1 (Edstrom & Larsson 1987, Ansved & Larsson 1989). This assumption is partially supported by the observation by Caccia et al. (1979) of a reversal of the age-related fast-to-slow transformation, together with obvious neurogenic changes, in the soleus muscle of very old rats. Boreham et al. (1988), on the other hand, ascribed the increased number of type I1 fibres in very old age to a reduced postural load on the muscle. In fact, the reported shift in fibre-type profile at these ages might be related to both a denervation process and a reduced postural load, since both of these mechanisms are expected to reduce the level of activity of the muscle, which presumably favours a slow-to-fast transformation (see Boreham et al. 1988, Midrio et al. 1988). In the present study, the number and proportion of fibres with intermediate myofibrillar ATPase staining characteristics increased in all age groups and the number and/or proportion of type I fibres decreased in adult and old animals, in response to denervation. Since no obvious signs of fibre splitting or formation of fibres de not'o were observed, a transformation of type I fibres into intermediate types IC and I I C seems to be the most likely cause of these changes. A similar slow-to-fast shift in myofibrillar ATPase staining characteristics was not seen in the contralateral soleus muscles, thus excluding a generalized effect of inactivation. The slow-to-fast transition in the present study affected only a limited number of type I fibres. It has been speculated that in the normal rat soleus muscle there are two populations of type I fibres, with indis-
Age-related eflects q/' denervution tinguishable MHC composition and corresponding to the primary and secondary generation fibres, which respond differently to, for example, denervation (Schiaffino et ul. 1990). According to this hypothesis, those type I fibres that have been transformed from type IIA during maturation and growth constitute secondary generation fibres, and it is these fibres that may undergo transformation in response to denervation. I n conclusion, the present findings imply that a slow-to-fast shift in myofibrillar properties in response t o denervation can also take place in the soleus muscle of old animals. Atrophic fibres with intermediate ATPase staining characteristics, and possibly also atrophic IIA fibres, seen in this muscle in old age might consequently be type I fibres which have been transformed in response to the age-related denervation process, as has previously been suggested (Edstrom & 1,arsson 1987, Larsson & Ansved 1988). The authors are indebted to Lars Edstriim for placing laboratory facilities at our disposal and Birgitta Hedberg and Birgitta Lindegren for excellent laboratory assistance. This study was supported by grants from the Swedish Medical Research Council (8651,3875), the Karolinska Institute and the Swedish Society of Medicine.
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anterior latissimus dorsi muscles following stretch LINDBOE,C.F. & PRESTHUS,J. 1985. Effects of denervation, immobilization and cachexia on fibre with and without denervation. Exp Neurol 41, 76100. size in the anterior tibia1 muscle of the rat. Acta SPECTOR, S.A. 1985. Trophic effects on the contractile Neuropathol 66, 42-5 I . MIDRIO,M., DANIELI BETTO,D., BETTO,R., NOVENTA, and histochemical properties of rat soleus muscle. J Neurosci 5, 2189-2196. D. & ANTICO, F. 1988. Cordotomy-denervation STARON, R.S. & PETTE,D. 1986. Correlation between interactions on contractile and myofibrillar propermyofibrillar ATPase activity and myosin heavy ties of fast and slow muscles in the rat. Exp Neurol chain composition in rabbit muscle fibers. Histo100, 216-236. chemistry 86, 19-23. NIEDERLE, B. & MAYR,R. 1978. Course of denervation R.S. & PETTE,D. 1987. The multiplicity of atrophy in type I and type I1 fibres of rat extensor STARON, combinations of myosin light chains and heavy digitorum longus muscle. Anat Embryo1 153, 9-21. chains in histochemically typed single fibres. NWOYE,L., MOMMAERTS, W.F.H.M., SIMPSON, D.R., Biochem -7 243, 6 8 7 4 9 3 . SERAYDARIAN, K. & MARUSICH, M. 1982. Evidence for a direct action of thyroid hormone in specifying S Y R O VI.~ , 1976. The relation between ATPase activity and light chains of myosin in developing, muscle properties. A m 3 Physiol 242, R401-408. adult and denervated muscles of several animal PADYKULA, H.A. & HERMAN, E. 1955. The specificity species. Physiol Bohemoslov 25, 295-300. of the histochemical method of adenosine triTOMANEK, R.J. & LUND,D.D. 1973. Degeneration phosphatase. 3 Histochem Cytochem 3, 17*195. of different types of skeletal muscle fibres. I. PETTIGREW, F.P. & GARDINER, P.F. 1987. Changes in Denervation. 3 Anat 116, 395-407. rat plantaris motor unit profiles with advanced age. ULLMAN,M., ALAMEDDINE, H., SKOTTNER, A. & Mech Aging Dev 40, 243-259. SCHIAFFINO, S., GORZA, L., AUSONI,S., BOTTINELLI, OLDFORS,A. 1989. Effects of growth hormone on skeletal muscle. 11. Studies on regeneration and R., REGGIANI,C., LARSSON, L., EDSTROM,L., denervation in adult rats: Acta Physiol Scand 135, GUNDERSEN, K. & L0M0, T. 1990. Muscle fiber types expressing different myosin heavy chain 537-543. R., EDSTROM, L. & JAKOBSSON, F. 1989. isoforms. Their functional properties and adaptive WROBLEWSKI, Effect of short time denervation on intracellular capacity. In: D. Pette (ed.) The Dynamic State of elemental content and fibre atrophy pattern of slow Muscle Fibers. de Gruyter, Berlin (in press). and fast twitch rat muscle. 3 Submzcrosc Cytol D.L. & MARTIN,A.W. SOLA,O.M., CHRISTENSEN, P a t h o l z ~ 68~-600. . 1973. Hypertrophy and hyperptasia of adult chicken 1
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