Distribution of different fibre types in human skeletal muscles

Journal of the Neurological Sciences, 1986, 72:211-222

211

Elsevier

JNS 2610

Distribution of Different Fibre Types in Human Skeletal Muscles Fibre Type Arrangement in m. vastus lateralis from Three Groups of Healthy Men Between 15 and 83 Years J a n Lexell 1'2, D a v i d D o w n h a m 3 and Michael SjOstrOm 2 Departments of ~Anatomy and 2Neurology, Universityof Ume&, Umed (Sweden)and 3Department of Statistics and Computational Mathematics, University of Liverpool, Liverpool (U.K.) (Received 10 May, 1985) (Revised, received 6 September, 1985) (Accepted 12 September, 1985)

SUMMARY

The effects of age on the fibre type arrangement in the human muscle m. vastus lateralis were studied. There were 10,6 and 8 healthy men in the three age-groups with means 24, 52 and 77 years, respectively. For each fascicle considered, the numbers of type 1 (ST) and type 2 (FT) fibres on the boundary and internally, and the numbers of enclosed fibres of either type, were counted. The randomness of the fibre type arrangement was considered in terms of the numbers of enclosed fibres and assessed by a Monte Carlo significance test. Fibre type grouping was shown to increase with increasing age. The proportion of type 2 fibres on the boundary of a fascicle was consistently greater than internally, but the difference was less pronounced in the old group. It is argued that the process of denervation and reinnervation of individual fibres (i) has started before the age of 50, (ii) is a major factor in a progressive reduction of fibres with increasing age and (iii)is probably caused by a continuous loss of motor neurons in the spinal cord.

This work was supported by grants from the Gun and Bertil Stohne Foundation, the Hans and Loo Osterman Foundation, the Swedish MS Foundation, the Research Council of the Swedish Sports Federation and the Swedish Medical Research Council. The authors were supported by study visit awards from the Royal Swedish Academy of Sciences and the Royal Society of Great Britain (JL), and the Swedish Institute (DD). Correspondence to: Dr. Jan Lexell, Department of Neurology, University of Ume~i, S-901 85 Ume~, Sweden, Tel. (46)90101926. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

212

Key words: H i s t o c y t o c h e m i s t r y - M u s c l e denervation - M u s c l e s - N e e d l e b i o p s y - N e r v e degeneration - N e r v e regeneration

INTRODUCTION

The fibre type arrangement in muscle cross-sections is known to change as a result of various progressive neurogenic disorders. The most obvious of these changes is the occurrence of large groups of fibres with the same histochemical properties. This finding is referred to as "fibre type grouping" (Walton 1981). Lexell et al. (1983a) have defined fibre type grouping on the basis of randomness as assessed by the number of "enclosed fibres", i.e. fibres surrounded by fibres of the same histochemical type (Jennekens et al. 1971), and have described a significance test to distinguish muscles with grouping of fibre types from those with random arrangements of fibres. In the second paper in this series, the fibre type arrangement in thin cross-sections of whole m.vastus lateralis from young, healthy males was tested (Lexell et al. 1984). It was found that the fibre type arrangement, as assessed by the number of enclosed fibres, can be regarded as random. To ascertain the range of applicability of such a finding, studies of the fibre type arrangement under various "normal" conditions are needed. A factor such as the age of the individual might be of importance for the interpretation of the results: as an illustration, the proportion of type 2 fibres on the boundary of fascicles is usually greater than internally, but this difference is less pronounced with increasing age (Lexell et al. 1984; SjOstrOm et al. 1985). It is known that increasing age affects the fibre type composition: the number of fibres is reduced, and the fibre type distribution over a whole muscle cross-section is changed (Lexell et al. 1983c). This seems to be accompanied by a reduction in the number of motor units and an increase in the size of the surviving motor units (Stfdberg and Fawcett 1982). The result of this study implies that part of the fibre population undergoes a denervation and reinnervation process with increasing age. Consequently, fibre type grouping may occur without being the result of a "pathological" neuromuscular process. By increasing the knowledge about the muscle fibre type arrangement in different age-groups, the mechanisms underlying the changes caused by normal ageing would be better understood and an improvement in the diagnosis of neurogenic muscle disorders might follow. In this study, whole muscle cross-sections of m. vastus lateralis from healthy males are used. The fibre type arrangements - more particularly, the number of enclosed fibres and the differences between proportions of fibre types on the boundary of fascicles and internally - are analysed and compared for different age-groups. Since the distribution of fibres with different histochemical properties within the muscle is more homogenous with increasing age (LexeU et al. 1983c), the fibre type arrangement is also studied in different parts of the muscle cross-sections.

213 MATERIAL AND METHODS M. vastus lateralis from the right leg of 24 men were extirpated less than three days post-mortem (storage + 6 ° C). There were 10, 6 and 8 men in the three age-groups, mean age 24years (age range 15-35 years), 52 years (49-56 years) and 77 years (71-83 years), respectively. The three age-groups will be referred to as "Young", "Middle" and "Old". Each man had suffered a sudden accidental death. None of the 24 had a history of neuromuscular disease and there was no evidence of pathological abnormalities at the post-mortem examination nor after examination of the extirpated muscles. A slice about 10 mm thick was cut from each muscle approximately 200 mm from the origin of the muscle (half way between the origin and insertion) and then was halved. Cross-sections were prepared from each slice and then stained for myofibrillar adenosine tri-phosphatase (mATPase) at pH 10.4 to visualize type 1 and type 2 fibres. (For further details about the preparative procedure, see Lexell et al. 1983b).

Sampling procedure In every 48th square millimetre of each muscle cross-section, the numbers of type 1 and type 2 fibres were counted and then used to estimate the total number of fibres and the proportion of type 1 fibres in the whole cross-section. In each cross-section, 5 areas were selected where the quality of the cross-sectioning permitted reliable identification and counting of fibres. Every part of each selected area was photographed in the microscope and an image of the whole area was formed from which all the fibre counts were obtained. Each area was classified as lying superficially (S), centrally (C) or deeply (D) within its cross-section: 19 cross-sections contained at least one area from each of superficial, central and deep parts of the muscle, but two cross-sections lacked any area from the central parts and three cross-sections lacked any area from the deep parts. Each area usually comprised one or more larger fascicles, which themselves comprised smaller fascicles. Larger fascicles were identified by clearly seen separating spaces. Narrower spaces separated the smaller fascicles and clearly there is more subjectivity in their identification: identification is facilitated, however, because outward sides of fibres on fascicle borders tend to be flatter than other sides. The fascicles in this study were the smallest identifiable. The mean number of fibres per fascicle per individual ranged from 69 to 418.

Fascicle counts The fibres on the border of a fascicle are called "boundary fibres" and all the remaining fibres are called "internal fibres". For each fascicle the number of type 1 and type 2 internal and boundary fibres were counted. An "enclosed fibre" is a fibre that is surrounded entirely by fibres with the same histochemical properties (Jennekens et al. 1971 ; Lexell et al. 1983a). The number of type 1 and type 2 enclosed fibres of each type was counted within each fascicle. The following notation is adopted:

214 n~ = m~ = n2 = m2 = n = m = o1 = o2 =

number of type 1 internal fibres, number of type 1 boundary fibres, number of type 2 internal fibres, number of type 2 boundary fibres, total number of internal fibres = n~ + n2, total number of boundary fibres = m~ + mz, number of type 1 enclosed fibres, number of type 2 enclosed fibres.

Data For each cross-section, the age of the man was known, and estimates of the number of fibres and the proportions of the two fibre types were calculated. The part of the cross-section - S, C or D - and the number of fascicles were known for each of the selected areas. There were 6 data specific to each fascicle: nl, n2, ml, m2, o I and o 2. The Young, Middle and Old age-groups consisted of totals of 254, 173 and 307 fascicles, respectively.

Analysis of data The data were analysed by considering firstly the number of enclosed fibres and secondly the differences in the fibre type proportions on the boundary of the fascicles and internally. Although the calculations and simulations could have been done on a microcomputer as in previous studies (Lexell et al. 1983a, 1984), the IBM 4341 at Liverpool University was used on this occasion; because of the large number of fascicles and of our time constraints, it was more convenient to use a mainframe computer. To test whether or not the observed number of type 1 and type 2 enclosed fibres are non-random, the model formulated by Johnson et al. (1973) and the simulation method of Lexell et al. (1983a) were used. For this model, the probability, p say, that a fibre in a given fascicle is type 1 is given by (n I + m 0 / ( n + m) and the expected number of type 1 enclosed fibres is (n~ + nz)p v = el, say; the expected number of type 2 enclosed fibres, e 2, is similarly defined. For each area of each cross-section, the number of fascicles for which the proportion of type 1 fibres on the boundary is less than internally, and the number for which the inequality sign is reversed, were counted. The hypothesis that the proportions of type 1 fibres on the boundary and internally are the same was tested with the (simple) sign test. A measure of the difference between the fibre type proportions internally and on the boundary could be useful when comparing groups or individuals. For each fascicle we calculated the ratio of the proportions of type 1 fibres internally and on the boundary. As ratios have notorious distributional properties - in particular, they can have large variances - the natural logarithm of the ratios are considered: h = log(n I × m2)/(n 2 × m~) is calculated for each fascicle with the exception of the three for which one of n~, n 2, m 1 or m 2 is zero.

215 RESULTS

General morphology Muscle fibres were usually tightly packed in well-preserved fascicles. The technical quality of the large sections thereby permitted random sampling and reliable analysis of the fibre type arrangement. In sections from all individuals in the Old group, a variation in both shape and size of the fibres, e.g. angulated and atrophic fibres, were occasionally seen. Data on the estimated number of fibres and the proportion and range of type 1 fibres in each whole muscle cross-section, are presented in Table 1. The results show that the muscle size as well as the total number of fibres is considerably smaller in the Old group than in the Middle and Young group. There is no difference between the three age-groups in the proportion of fibres with "type 1" properties. Data on fascicle counts for each individual are given in Table 2. The mean number of fibres per analysed area ranged from 703 to 2117. The number of analysed fascicles per individual varied between 18 and 60. The mean number of fibres per fascicle ranged from 69 to 418, with a mean for each of the three age-groups of 279, 188 and 129, respectively. There were no noticable differences in fascicle sizes between the three parts of the muscles (Table 3).

Fibre type arrangements (a) Enclosed fibres The expected number of enclosed fibres depends on the assumption that each fibre is a regular hexagon in an hexagonal lattice. For each fascicle, the expected number of enclosed fibres of each type was calculated and the significance levels estimated from 100 simulated configurations (Lexell et al. 1983a). Where this rough significance level of the observed number of enclosed fibres of either type was 10yo, or less, a further 500 simulations permitted ref'mement of the limit. As fibre type grouping causes more enclosed fibres than might be expected in a random fibre type arrangement, one tailed significance tests are used here. The number of fascicles for which the number of enclosed fibres is non-random at the 5 Yo level is given for each fibre type for each individual (Table 2) and for the three parts of a muscle of each age-group (Table 3). Table 2 also includes the figures for each of the three age-groups. Both tables include the proportion of the fascicles for which the assumption of randomness is rejected. For the Old group the proportion of fascicles with a significant number of enclosed fibres (21 ~o) is greater than the proportion for the Middle (6 ~ ) and the Young (6Yo) groups. Moreover, for the Middle and Young groups almost all the 6~o belong to one individual in each group, whereas most of the older individuals show some significant fibre type grouping. There is a clear tendency in all three age-groups towards more fascicles with significant enclosed fibres in the superficial areas (Table 3). Irrespective of age group and part of the muscle, there is a tendency towards a small predominance of type 1 enclosed fibres. We also noted that only a few fascicles contained groups (as defmed by Lexell et al. 1983a) with more than five enclosed fibres.

216 TABLE 1 ESTIMATES OF THE TOTAL NUMBER OF FIBRES AND PROPORTION OF TYPE 1 FIBRES IN CROSS-SECTIONS OF WHOLE M. VASTUS LATERALIS OF 24 MEN FROM THE AGE OF 15 YEARS TO 83 YEARS

Subject and age (yr)

No. of mm 2 a

1 2 3 4 5 6 7 8 9 10

77 78 72 80 99 66 74 91 86 81

158 135 195 163 190 124 158 167 150 141

Mean (SD): 24 ± 7

80 _+ l0

11 49 12 51 13 55 14 56 15 49 16 49

Mean No. of fibres/mm2

Total No. of fibres b

Proportion of type 1 fibres % -+ SD

Range (~o)

584000 505000 673000 626000 903000 393000 561000 729000 619000 548000

58 48 48 48 53 42 45 52 46 53

38-76 30-67 29-68 29-68 34-74 27-70 23-84 21-84 27-69 33-90

158 _+ 22

614000 _+ 137000

49 ± 5

84 68 63 49 65 67

219 232 118 179 159 178

883000 757000 357000 425000 496000 572000

66 34 54 72 47 42

Mean (SD): 52 ± 3

66 _+ 11

181 _+41

582000 ± 202000

52 ± 14

17 18 19 20 21 22 23 24

49 62 62 62 41 54 49 33

144 147 132 174 212 133 171 154

339000 437000 393000 518000 417000 345000 402000 244000

43 _+ 8 55 _+ 9 60 _+ 9 49 + 11 43_+ 7 64 + 14 40 + 9 51 _+ 11

52_+11

158+27

387000_+80000

51_+ 9

15 20 18 19 21 21 35 34 27 26

73 71 73 75 80 83 80 80

Mean (SD) : 77+4

_+ 8 ± 8 _+ 10 ± 9 ± 10 ± 8 ± 11 _+ 13 _+ 9 _+ 9

± 6 _+ 6 _+ 12 -+ 9 _+ 10 ± 8

38-86 20-53 23-80 49-88 29-75 24-59

26-61 31-75 33-76 29-67 29-60 31-100 18-59 32-78

Corresponds to approximately ~8 of the muscle cross-sectional area. Calculated by multiplying a with the mean number of fibres/mm 2 and by a factor of 48.

Subjects 19 and 22 contained two fascicles with just one fibre type. In addition, grouping of type 2 fibres tended to occur near the boundaries of fascicles and grouping of type 1 fibres tended to occur in the more central parts. Although not included in the argument, we observed the number of fascicles for which there were "too few" enclosed fibres. We noted that the fascicles with "too few" enclosed fibres were almost always from the younger men.

217

o~ O Z o

0

0

0

0

0

0

~

0

~

0

0

0

.,.~

~

0

0

0

0

~

0

O~

~ O O

I"" O l ~r5 ~

t¢~ I ' ~ ~''~ ~

~

O 0., O O

Z <

.

~

~

Z Z

i~i~*

< ZN m

1 * ~Z *~ ~Z*Z Z g r~o

v

Z ¢.

"6

O

~O

d

Z

A

~1

<

z [.-, Zm Z~)
z~

z~ O

o

i | l l l l i l l i

~O~,~

O ~

ol

m ,1 <

r..)~ m ,...1

Zg

~

l

l

l

Ig

I

I

I

I

,

l

i

l

o

,..i

~O ~

~

0 0 oo

~

1081 1076 1096

1049 1005 853

S 17 C 14 D 9

2. 2. 2. 2.

"O"

a Cf. Table b Cf. Table Cf. Table '~ Cf. Table

1368 1495 1381

Mean No. of fibres/ area

S 11 C 13 D 16

S 19 C 19 D 12

No. of areas

"M"

-y~,

Age group

44 (30-63) 55 (29-70) 55 (36-69)

44 (29-67) 42 (23-78) 49 (32-64)

41 (28-60) 46 (28-68) 56 (42-74)

Proportion of type 1 fibres (~o)

142 93 72

59 77 37

94 95 65

No. of fascicles

126 151 107

202 182 178

277 299 255

Mean No. of fibres/ fascicle >

88 d 60 49

56 66 c 34

85 ¢ 84 58

n

nI m

m~

52 33 23

3 10 3

8 11 7

n

n I

< m

m 1

No. of fascicles with

** ** **

*** *** ***

*** *** ***

Significance of sign test a

0.18 0.17 0.23

0.52 0.44 0.48

0.49 0.56 0.57

Arithmetic means of h

20 10 6

3 3 1

8 2 2

O~

17 (26~o) 6 (17~'o) 4 (14%)

4 (12~o) 0 (4~o) 0 (3~o)

6 (15~o) 0 (2~o) 0 (3~o)

02

No. of fascicles with significant b

THE FASCICLE COUNTS, DATA ON BOUNDARY AND INTERNAL FIBRE TYPE PROPORTIONS AND, N U M B E R A N D P R O P O R T I O N S OF FASCICLES WITH S I G N I F I C A N T NUMBERS OF ENCLOSED FIBRES FOR EACH OF THE THREE PARTS OF A MUSCLE, SUPERFICIAL (S), CENTRAL (C) AND DEEP (D), FOR EACH AGE-GROUP, Y O U N G (Y), M I D D L E (M) A N D OLD (O)

TABLE 3

tJ Oo

219

(b) Boundary versus internal parts of fascicles The number of fascicles for which the proportion of type 1 fibres internally exceeds that on the boundary - nl/n > mm/m - and that for which the inequality sign is reversed, are given for each individual in Table 2 and for each part of the muscle for each age-group in Table 3. The two-tailed sign test is used to test the (null) hypothesis that these proportions are the same against the alternative hypothesis that they are different. The results of applying this test to individuals and parts of muscles are given in Tables 2 and 3, respectively. For 8 of the 10 individuals in the Young group and 5 of the 6 in the Middle group, the hypothesis of equal proportions is rejected at the 0.1 Yo significance level, at the 5 % level for the sixth individual in the Middle group but cannot be rejected for 2 of the individuals in the Young group. For the Old group, this hypothesis is rejected at the 5 ~o level for 2 of them and at the 0.1 Yo for 1 individual (Table 2). As can be seen in Table 3, for each part of the muscle for the Old group, this hypothesis of equal proportions is rejected at the 1~o level, while for each area of the other two groups the significance level is less than 0.1Y/o. The 9 × 2 contingency table of the numbers of fascicles with n I/n > m~/m and n~/n < mm/m - two columns of Table 3 - was then tested for independence. The chi-value is significant at the 0.1 ~o level, which implies possible differences between the age-groups or between the parts of the cross-sections. By suitable partitioning the sources of the dependence can be identified. The relative frequencies for the Young and Middle group are very similar, but differ greatly when each age-group is compared with the Old: the hypothesis of homogeneity between the Old and the other two age-groups is rejected at the 0.1 ~o significance level. Within each age-group, the observed and expected numbers are very close and the homogeneity hypothesis cannot be rejected. Consequently, the rejection of the independence hypothesis at the 0.1 ~o significance level can be wholly explained by the Old differing from the Young and Middle age-groups. The arithmetic mean of h for each of the 24 individuals is given in Table 2 and for each part of the muscle for each age-group in Table 3. It can be seen in Tables 2 and 3 that there is a small reduction (15~o) in these means between the Young and the Middle groups, and a 55% reduction between the Middle and the Old groups. Thus, the proportion of type 1 fibres internally exceeds that on the boundary for all age-groups, but diminishes with increasing age. There is no discernable pattern in the ratios of proportions for the different parts of the muscles. DISCUSSION In this study, as in its predecessors (Lexell et al. 1983a, 1984), the term "fibre type grouping" is reserved for a fascicle for which the arrangement of fibre types is non-random at a given significance level. For random arrangements, roughly 5 % of the fascicles should be significant at the 5 % level, which is the case for the Young and Middle group but not for the Old group, for whom 21% of the fascicles are significant. Thus, a certain degree offibre type grouping can be considered "normal" in old muscles,

220 which has obvious implications in the examination of an unknown muscle sample. It also supports the hypothesis that muscle fibres undergo a denervation and reinnervation process with increasing age. Disorders of the peripheral nerves (i.e. polyneuropathies) and motor neuron loss (i.e. motor neuron diseases) are known to be the two principal causes of denervation and reinnervation of muscle fibres. It is generally assumed that isolated progressive motor neuron loss gives rise to small groups of fibres of the same histochemical type ("small type grouping"), while in chronic progressive neuropathies large groups of fibres of the same type are formed ("large type grouping" or "fascicular type grouping") (Swash and Schwartz 1984). In this study, all muscles showed "small type grouping" but only two "large type grouping", each in just one part of the muscle cross-section. Consequently, the ageing process seems primarily to reduce the number of motor neurons in the spinal cord and only in a minor way to affect the motor axons and/or the myelin sheath of peripheral nerves. This is supported by the anatomical findings of deaths of ventral horn cells in the spinal cord with increasing age (Tomlinson and Irving 1977). Progressive denervation and reinnervation eventually reaches a phase when fibres become permanently denervated, and finally replaced by fat, fibrous tissue etc. A continuous neurogenic process, such as ageing, thus leads to a continuous reduction of the total number of fibres. Until the age of fifty, the proportion of lost fibres is small but increases gradually thereafter reaching almost fifty per cent at the age of eighty (Table 1). This age-related process of denervation and reinnervation, caused by a gradual loss of motor neurons, must be one of the major contributors to the reduction with increasing age in fibre number and muscle volume. The results in Table 3 indicate that for all ages fibre type grouping is more common in superficial parts than elsewhere. This fmding raises several questions about the underlying mechanism. Is the fibre type grouping in superficial parts the consequence of a higher denervation rate or more favourable conditions for reinnervation ? As type 2 fibres predominate superficially (Lexell et al. 1983b), is this finding the consequence of a higher denervation rate particularly of this fibre type? Are the nerve axons, which innervate the superficial parts of the muscle, more easily damaged on the way from the main nerve? To what extent do the functional differences at various parts of the muscle influence the relationship between nerve and muscle? Is the composition of the motor units different in different parts of the muscles? Further studies are needed before any conclusions can be made. This finding, however, has an important practical implication: a more frequent occurrence of changes in the fibre type arrangement in one particular area of a muscle, as part of a normal physiological process (here: ageing), has to be taken into account when a random sample is examined. Preferably, the biopsy depth should therefore be carefully defined. This, in turn, lends support to the use of the open surgical biopsy technique, by which a well defmed sample can be obtained. The mean number of fibres per fascicle reduces linearly from the Young to the Old group, but the reduction in the total number of fibres is considerably greater between the Middle and Old group than between the Young and Middle group. Consequently, the reduction in fascicle size cannot be due merely to a loss of fibres within individual

221 fascicles. It has been demonstrated that the network of collagen fibres, which forms the tissue sheath around single muscle fibres and groups of fibres to make up the fascicles, increases in size as a result of denervation (Salonen et al. 1985). A plausible explanation to the reduction in fascicle size up to the age of fifty is that a denervation related increase in connective tissue subdivides larger fascicles and forms new fascicle borders. Thereafter, the reduction in fascicle size seems to be due more to a loss of fibres within individual fascicles. This rearrangement of fascicles may explain why the difference between the proportions of type 2 fibres on the boundary of fascicles and internally reduces with age. However, as there is no discernible or significant difference between the Young and Middle group, and the difference between both these age-groups and the Old group are highly significant, there must also be other explanations. Since the total number of fibres is known to be reduced with increasing age, this reduced difference between the proportions of fibre types within a fascicle could be the result of a greater reduction either in the proportion of type 1 fibres internally, or in the proportion of type 2 fibres on the boundary. The second explanation is the more plausible as the first explanation would cause a noticable increase in the proportion of type 2 fibres in the whole muscle. Possible causes of the reduction in the proportion of type 2 fibres on the fascicle boundary are, in turn (i) an elimination of type 2 fibres as a result of a higher denervation rate of this fibre type with increasing age and (ii) an increase in the number of type 1 fibres. Alternative (i) is likely as a tendency towards a reduction in the number of type 2 fibres with increasing age has been reported (Lexell et al. 1983c). Alternative (ii) would involve a functional transformation of type 2 fibres to type 1. Such a process may be explained by a slow adaptation of the fibres due to changes in the physical activity pattern of individuals; since the movements of old individuals are usually more limited than those of the young, the functional demands on the fibre population may thereby be different. In summary, the results show that increasing age affects the fibre type arrangement, leading to an increased occurrence of enclosed fibres. Consequently, ageing involves a continuous denervation and reinnervation process, most likely caused by a loss of functioning motor neurons in the spinal cord. This age-related progressive neurogenic process, probably beginning before the age of fifty, must be a major contributor to the gradual loss of fibres with increasing age. The changes with increasing age in fascicle size and the distribution of fibre types within individual fascicles imply that other processes also exist. The age of an individual must therefore be taken into account when examining an unknown sample and testing the arrangement of fibres for non-randomness as an indication of a denervation and reinnervation process. ACKNOWLEDGEMENT The authors would like to thank Miss Mona LindstrSm for assistance in preparing the muscle cross-sections.

222 REFERENCES Jennekens, F. G. I., B.E. Tomlinson and J.N. Walton (1971) Data on the distribution of fibre types in five human limb muscles - - An autopsy study, J. Neurol. Sci., 14: 259-276. Johnson, M.A., J. Polgar, D. Weightman and D. Appleton (1973) Data on the distribution of fibre types in thirty-six human m u s c l e s - An autopsy study, J. Neurol. Sci., 18:111-129. Lexell, J., D. Downham and M. Sj6str6m (1983a) Distribution of different fibre types in human skeletal muscles - - A statistical and computational model for the study of fibre type grouping and early diagnosis of skeletal muscle fibre denervation and reinnervation, J. Neurol. Sci., 61: 301-314. Lexell, J., K. Henriksson-Lars6n and M. SjOstr6m (1983b) Distribution of different fibre types in human skeletal muscles, Part 2 (A study of cross-sections of whole m. vastus lateralis), Acta PhysioL Scand., 117: 115-122. Lexell, J., K. Henriksson-Lars6n, B. Winblad and M. SjSstrOm (1983c) Distribution of different fbre types in human skeletal muscles - - Effects of aging studied in whole muscle cross sections, Muscle & Nerve, 6: 588-595. Lexell, J., D. Downham and M. Sj~strSm (1984) Distribution of different fibre types in human skeletal muscles - - A statistical and computational study of the fibre type arrangement in m. vastus lateralis of young, healthy males, J. Neurol. Sci., 65: 353-365. Salonen, V., M. Lehto, H. Kalimo, R. Penttinen and H. Aro (1985) Changes in intramuscular collagen and fibronection in denervation atrophy, Muscle & Nerve, 8: 125-131. Sj6strOm, M., D. Downham and J. Lexell (1985) Distribution of different fibre types in human skeletal muscles - - Why is there a difference within a fascicle?, Muscle & Nerve, In press. Stgdberg, E. and P.R.W. Fawcett (1982) Macro EMG in healthy subjects of different ages, J. Neurol. Neurosurg. Psychiat., 45: 870-878. Swash, M. and M.S. Schwartz (1984)Biopsy Pathology of Muscle, Chapman and Hall Medical, London. Tomlinson, B. E. and D. Irving (1977) The numbers of limb motor neurons in the human lumbosacral cord throughout life, J. Neurol. Sci., 34: 213-219. Walton, J., (1981) Disorders of Voluntary Muscle, Churchill Livingstone, London.