Dystrophy: a Study Using Serial Sections

J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

Journal ofNeurology, Neurosurgery, and Psychiatry, 1975, 38, 91-99

Abnormal intrafusal muscle fibres in myotonic dystrophy: a study using serial sections

M. SWASH AND K. P. FOX From the Section of Neurological Sciences, The London Hospital, London

SYNOPSIS Studies of the extent and complexity of intrafusal muscle fibre splitting in myotonic dystrophy have been carried out in three spindles obtained at muscle biopsy, and in nine spindles found in the lumbrical muscle of a case that came to necropsy. It is suggested that splitting, due to mechanical stresses, leads to a regenerative response in the separated fragments.

In myotonic dystrophy a characteristic abnormal- dominantly affected and myotonia was present, both ity occurs in the muscle spindles. Affected clinically and electromyographically, in the whole spindles contain large numbers of abnormally skeletal musculature. Bilateral cataracts were present, and there was frontal baldness and a mild thin intrafusal muscle fibres, usually arranged in Protected by copyright. several groups within the spindle lumen. In addi- degree of mental retardation. There were several affected relatives. Consent was given for muscle tion, changes occur in both the motor and the biopsy. sensory innervation, and the spindle capsule is The pronator teres muscle, which was clinically usually thickened. affected, was chosen for biopsy, since Cooper (1966) It has been suggested that the abnormal intra- has shown that this muscle contains a large number fusal muscle fibres are formed by longitudinal of muscle spindles. The biopsy specimen was divided splitting of parent fibres (Daniel and Strich, into three pieces. One portion was pinned onto stiff 1964; Swash, 1972), but the pathogenesis of this card and fixed in 10% formol-saline for light micro- splitting, which may occur in muscles in which scopy, and another was quenched in hexane, which the extrafusal fibres are almost normal, is not had been cooled with a freezing mixture, and was understood. The abnormal intrafusal muscle prepared for study using routine enzyme histochemical methods (Dubowitz and Brooke, 1973). A fibres are often so thin that, in serial sections of third portion was cut into small pieces and fixed for paraffin-embedded muscles, it is not usually four hours in 2.5% barbiturate-buffered glutaralde- possible to identify individual fibres. Conse- at hyde 4°C, post-fixed in 2% phosphate-buffered http://jnnp.bmj.com/ quently, the longitudinal extent of the abnormal- osmium tetroxide, dehydrated in a series of graded ity has not been studied. alcohols, and embedded in Araldite. We have examined this problem by the One spindle was found in the frozen blocks method of serial transverse section. Twelve examined by enzyme histochemical methods and two spindles have been studied; three were obtained were found in the Araldite-embedded material. The by muscle biopsy and nine were found in the latter were examined in 1 or 2 ptm thick, serial sec- paraffin-embedded lumbrical muscle of a patient tions stained with toluidine blue (more than 600 Our sections). Photomicrographs of one spindle were on September 25, 2021 by guest. reported previously (Swash, 1972). findings made at a magnification of 350 diameters, at 5 or will be discussed in relation to the pathogenesis 10 .tm intervals. These were mounted in series on of the spindle abnormality in this disease. large cards and examined with a hand lens, in order to study the longitudinal extent of any 'splitting' of METHODS intrafusal muscle fibres. A woman, aged 52 years, presented with typical, At selected levels thin transverse sections were moderately advanced myotonic dystrophy. Distal, mounted on Formvar-coated, 1.0 mm diameter, cervical, and sternomastoid muscles were pre- single aperture grids, stained with uranyl acetate and (Accepted 8 August 1974). lead citrate by standard methods, and examined with 91 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

92 M. Swash and K. P. Fox an AE1 6B electron microscope at 60 kV. The fine fibres. In the subsequent serial sections this pro- structure of these spindles was compared with that of cess became more complex until the abnormality normal human and baboon muscle spindles (un- was similar to that found in the other spindle. published observations) and with that of spindles of In both spindles the abnormality became less other mammals (Landon, 1966; Banker and Girvin, complex as the equator was approached and, in 1971; Barker et al., 1972; Smith and Ovalle, 1972). the equatorial sensory region itself, longitudinal In addition, serial 6 Fm transverse sections were cut through the paraffin-embedded first lumbrical splitting of the intrafusal fibres was far less muscle of one, previously studied case, that came to prominent (Fig. Id). Many of the fibres re- necropsy, aged 18 years (see Swash, 1972). In these mained smaller than normal but several clusters sections, stained with haematoxylin and eosin, nine of fibres formed bag or chain aggregations of spindles were examined through most of their length. nuclei in single, undivided, fibres of normal diameter (Fig. 2). The nuclear aggregations were not as prominent and the nuclei were often not as RESULTS tightly packed, however, as in normal spindles SERIAL SEMI-THIN SECTIONS Two muscle spindles sectioned in this region. In these abnormal were studied in serial 2 ,um thick sections stained spindles the primary sensory region was unusu- with toluidine blue. Serial sections of one spindle ally short (20-50 [Lm), but the sensory endings extended from the mid-polar region of one pole, themselves were normal (Fig. 2). The region through the equatorial region, to the juxta- between the pole and the equatorial sensory equatorial region of the opposite pole. In the region, in which fusion of abnormal fibres mid-polar sections of this spindle there were occurred, was approximately 50 tm in length. more than 100 abnormally thin intrafusal There were only slight changes in the extra- Protected by copyright. muscle fibres: the number found varied greatly fusal muscle fibres. Some contained central even in sections 10 ,um apart. The diameter of nuclei. There were several hypertrophied fibres these abnormal fibres ranged from less than 1 Vm and, rarely, part of the edge of a fibre was split to 7 Fm, but most were narrower than 2 ,um. into several smaller fragments (see Schroder They were usually arranged in clusters ofthree to and Adams, 1968). A few small, degenerate 30 fibres. fibres were present. Study of the serial sections showed that this abnormality was the result both of longitudinal ENZYME HISTOCHEMISTRY In polar sections of splitting and of fusion of individual fibres within the single spindle found in the blocks of frozen a given cluster. This process occurred along the tissue there were five normal intrafusal muscle length of the pole of the spindle (Fig. la-d). It fibres. One abnormal fibre appeared split into 11 was so complex that it was often difficult to tiny fragments which were all of the same histo- identify a single abnormal fibre in sections chemical type (Fig. 3). All the intrafusal fibres separated by only a few micra. Each abnormal showed prominent NADH tetrazolium reductase http://jnnp.bmj.com/ fibre therefore represented a fragment separated activity and relative unreactivity for phosphoryl- from its adjacent fibres for only a short part of ase. The reaction for myofibrillar ATPase after its length. Nuclei were found in some, but not in preincubation at pH 9.5 demonstrated strongly all, of these fragments. There was great variation reactive, intermediate, and weakly reactive in the staining intensity of individual fragments fibres. This variation in the ATPase reaction of intrafusal fibres even within a single cluster occurs in normal human intrafusal muscle (Fig. 1). Several ring fibres were observed. Each fibres (Spiro and Beilin, 1969). on September 25, 2021 by guest. surrounded a cluster of abnormal fibres, or passed diagonally between the fibres of a single ELECTRON MICROSCOPY Each cluster of intra- cluster toward its circumference. fusal muscle fibres was surrounded by a layer of The sections of the second spindle included basement membrane and, usually, by a thin the tip of its pole. In this region there were four cytoplasmic layer derived from intraluminal normal intrafusal muscle fibres. In sections cut invaginations of capsular cells or fibrocytes. Mid 50 ,um deeper into the block three of these fibres polar sections contained the largest number of had become split into three or more abnormal clusters. At all sectional levels there were J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

Abnormal intrafusal muscle fibres in myotonic dystrophy: a study using serial sections 93

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(c) ( ]) on September 25, 2021 by guest. FIG. 1 2 um thick, transverse sections of an Araldite-embedded muscle spindle obtained at biopsy. Toluidine blue, x 350. (a) This mid-polar section shows the typical abnormality. The intrafusal muscle fibres are all abnormal. There is marked variation in size and staining intensity of individual fibres. Several myelinated nerve fibres are present. (b) The same spindle sectioned 53 pum deeper in the block. Mid-polar section. (c) The same spindle sectioned 162 pum from section (a). Two myelinated nerve fibres encircle isolated groups of abnormal intrafusal muscle fibres. The spindle capsule is more prominent and the lumen is larger than in section (b). The6section w ..-.is juxta-equatorial. (d) The same spindle sectioned in its equatorial sensory region, 325 pum from section (a). The spindle lumen is larger and there are fewer intrafusal muscle fibres. Severalfibres, each containing several nuclei (nuclear bags), have been sectioned through their equatorial sensory regions. J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

94 M. Svash and K. P. Fox

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FIG. 3 Myosin ATPase; preincubation at pH 9.5 x 900. This spindle, sectioned in its polar region, contains five normal intrafusal muscle fibres. A sixth fibre, consisting of 11 fragments, occupies an area equivalent to that ofan adjacent normalfibre. Several Protected by copyright. FIG. 2 Toluidine blue, x 1 400. This equatorial sec- nerve fibres are present in the spindle lumen tion was cut 18 fsm from section I (d). The large fibre sectioned through its nuclear bag region contains There was marked variation in electron fewer nuclei than expected, but normal sensory endings the (S) can be seen indenting the periphery of the fibre. density of adjacent abnormal fibres. This corre- There are several, additional, small fibres of varying sponded with the variability of staining intensity staining intensity. observed in the toluidine blue preparations, and was due to degeneration and loss of myofibrils, and to the presence of sarcoplasmic masses. In scattered, single, tiny fibres which sometimes re- such fibres myofilaments were misaligned, or mained discrete for several micra before fusing present in groups of 5-20 filaments separated with a nearby fibre or cluster of fibres. The base- from each other by clear sarcoplasm. Some ment membrane surrounding these isolated fibres, in which these changes were usually par- single fibres often projected in loose folds into ticularly prominent, contained electron-dense the spindle lumen. Within a cluster, individual rodlets. These rodlets showed the characteristic http://jnnp.bmj.com/ abnormal intrafusal muscle fibres, defined by periodicity of Z-band material (Fig. 5). Clear or their plasma membranes, were separated by a dark membrane-bound, lipid vacuoles and lipo- clear space 130-300 A wide. Regions of density fuscin granules were common. Several fibres and intimate membrane contact (zonula ad- contained myelin bodies, autophagic vacuoles, haerens) between apposing plasma membranes and other degenerative figures (Fig. 4). The mito- were common. In some instances a narrow fissure, chondria and sarcoplasmic reticulum were bounded by partially formed plasma membranes, usually normal, but in the atrophic fragments on September 25, 2021 by guest. passing through a region of disrupted myo- the few remaining mitochondria were often filaments and swollen, rounded mitochondria rounded and swollen; and dilated, short seg- seemed to indicate incomplete separation of two ments of the tubular system were found, usually fragments of a fibre. Pinocytotic vesicles were in close relation to isolated disarrayed myo- prominent beneath the plasma membranes of filaments. individual fibres, but very abnormal fibres, for Other fibres contained small groups of well example those containing few myofilaments, formed myofilaments in a granular cytoplasm, contained fewer vesicles (see Fig. 4). in which polysomes, free ribosomes, glycogen J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

Abnormal intrafusal muscle fibres in myotonic dystrophy: a study using serial sections 95

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FIG. 4 Electron micrograph, x 18 000. One fibrefragment contains densely packed myofibrils. An adjacent fibre contains a lamellated lipid body. Another contains a nucleus, tubules of the Golgi apparatus and rough endoplasmic reticulum. One fibre fragment (F), immediately adjacent to the basement membrane contains granular and vacuolated cytoplasm, and degenerate mito- chondrial profiles similar to those described by Hall-Craggs and Lawrence (1970) (see text). Mitochondria in adjacent fibre fragments are normal. Protected by copyright.

granules, and small mitochondria were abundant. whether these endings were normal or not. There Rough endoplasmic reticulum and a prominent seemed, however, to be fewer motor endings Golgi apparatus were frequent features of these than in normal spindles. fibres (Fig. 4). In the primary sensory region normal bag (Fig. 6) and chain nuclear aggrega- STUDIES OF NECROPSIED MATERIAL Nine spindles tions were found. There were prominent clear were studied in serial transverse section in a lipid vacuoles, bound by single membranes. single lumbrical muscle. In polar sections each of http://jnnp.bmj.com/ Myelinated and unmyelinated nerve fibres these spindles contained more than 50 abnormal were found both in the spindle capsule and in thin intrafusal muscle fibres but, in their equator- close relation to the intrafusal muscle fibres. ial regions, many of these abnormal fibres fused Sensory nerve fibres were identified by their to form fewer fibres, several of which were of diameter, and by their termination in typical normal diameter, although others remained sensory endings (Smith and Ovalle, 1972), which abnormally small. The larger fibres contained were invariably normal (see Fig. 6). Motor end- bag or chain aggregations of nuclei, but these on September 25, 2021 by guest. ings were more difficult to identify since they nuclei were pyknotic and less numerous than in were small and, apparently, simplified. Plate normal fibres. Basophilia was observed in some endings were not identified. These small motor of the abnormal intrafusal fibres studied in polar endings were similar to the gamma-trail endings sections. studied in normal cat muscle spindles by Barker et al. (1970), but the fine structure of fusimotor nerve endings has not yet been studied DISCUSSION in man and it is not possible, therefore, to state The striking feature ofthe spindle abnormality in J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

96 M. Swash and K. P. Fox

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in the myotonic dystrophy is the presence, in many Longitudinal splitting was uncommon very thin intrafusal extrafusal fibres in our Araldite-embedded spindles, of a large number of in fibres. In the present work splitting of material, although it has been recognized muscle of very intrafusal muscle fibres was prominent only in extrafusal fibres in previous studies The appearance of splitting was advanced cases of myotonic dystrophy (SchrtSder polar sections. http://jnnp.bmj.com/ found to be the result of a complex process of and Adams, 1968). recombination, each subdivided In a previous study (Swash, 1972) it was sug- subdivision and muscle fibres being of very short length. In the gested that splitting of intrafusal fragment in the of the equatorial sensory region, and at the extreme was preceded by changes pattern tip of the polar region, the intrafusal muscle fusimotor innervation. However, the small frag- normal. In the two Araldite- ments observed with the electron microscope in fibres were almost would not embedded spindles an attempt was made to the biopsy specimen, described above, number of abnormal intrafusal muscle have been apparent in the teased preparations on September 25, 2021 by guest. count the and we fibres at polar, midpolar, and equatorial levels upon which our earlier work was based that it is more that these but this was abandoned when it was found that now consider likely fusimotor innervation were due to many fragments were too small to allow resolu- changes in the were of sprouting, as part of an attempt at innervation of tion with the light microscope and others fibres. intensity that it was diffi- these abnormal,, fragmented muscle such a size and staining from studies cult to distinguish them from unmyelinated Furthermore, there is no evidence on muscle was therefore, that counting was of the effect of denervation spindle axons. It felt, that fusi- too unreliable to be useful. morphology (Swash and Fox, 1974), J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

Abnormal intrafusal muscle fibres in myotonic dystrophy: a study using serial sectionts 97 Protected by copyright. http://jnnp.bmj.com/

FIG. 6 Electron micrograph, x 5 400. This fibre, sectioned through its nuclear bag, is surrounded by several sensory endings, containing closely packed mitochondria. The fibre itself is not longitudinally split and its nuclei appear normal. There are numerous single-membrane bounded, clear lipid vacuoles of varying size. on September 25, 2021 by guest. motor denervation would account for the changes of the abnormality of the intrafusal muscle fibres found in the intrafusal fibres in myotonic dys- in relation to the minor changes seen in a few trophy. extrafusal fibres is difficult to explain on such a Isaacs et al. (1973) have studied longitudinal simple hypothesis, although it is possible that splitting of extrafusal muscle fibres in the dys- myotonia and myotonic after-discharges might trophic mouse. They thought that splitting, in induce abnormal stresses in the intrafusal fibres. these fibres, was due to mechanical stresses. In These fibres contract in a localized and graded myotonic dystrophy the severity and complexity manner in response to neural stimuli, rather than J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

98 M. Swash and K. P. Fox by an all-or-nothing membrane depolarization. in the electron microscopic preparations active Any theory of the pathogenesis of the abnormal- Golgi apparatus and rough endoplasmic reticu- ity must explain the patchy distribution of the lum were identified. In others the presence of spindle abnormality within a given muscle and myelin bodies, lipofuscin, and changes in the the observation that it is often severe in muscles myofibrillar pattern suggested that degenerative that are, otherwise, only moderately abnormal changes were taking place. The occurrence of rod (Swash, 1972). bodies in some of these fibres is probably not The possibility that the abnormal intrafusal specific (Cape et al., 1970), although Karpati et muscle fibres are new muscle fibres derived from al. (1971) have suggested that rod bodies may be satellite cells (Mauro, 1961), formed as part of an an indication of a long-standing abnormality of active regenerative process, must also be con- the innervation of muscle. However, in myotonic sidered. In extrafusal muscle, regeneration after dystrophy, abnormalities in the fusimotor ischaemic necrosis (Kaspar et al., 1969), and after innervation are probably secondary to frag- denervation (Drachman et al., 1969; Hess and mentation of the intrafusal fibres. Rosner, 1970), occurs from satellite cells formed from, or remaining in, the damaged fibres. In addition, it has been shown, in the rat, that during HYPOTHESIS compensatory hypertrophy satellite structures We propose, as a working hypothesis, that the are formed at the periphery of extrafusal muscle abnormalityofintrafusal muscle fibre in myotonic fibres resulting in a muscle containing an in- dystrophy may occur as a result of longitudinal creased number of fibres of greatly varying splitting, or fragmentation, due to mechanical diameter (Hall-Craggs and Lawrence, 1970; stresses, perhaps associated with the myotonia, Protected by copyright. James, 1973). Mechanical stress may, therefore, or with myotonic after-discharges. The relative be a stimulus to the formation of satellite struc- normality of the equatorial sensory region may, tures. Longitudinal splitting, similar in charac- perhaps, be explained by the viscous resistance ter, but of less complexity, to that found in the to stretch which is a feature of this region in intrafusal fibres in myotonic dystrophy, has been normal spindles. Splitting may induce the forma- observed in rat muscle fibres in the vicinity of tion of satellite structures, as an attempt at experimental crush lesions (Hall-Craggs and regeneration, and changes consistent with both Lawrence, 1970). In this work it was suggested regeneration and degeneration would then occur that the observed longitudinal division followed in the same group of abnormal intrafusal muscle damage to the fibres and that it might be a fibres. Proliferation of fusimotor nerve fibres mechanism which allowed rejection of degener- may represent the attempted innervation of the ated portions from affected fibres. Regeneration, fragmented, abnormal muscle fibres. In order to it was suggested, occurred from viable peripheral test this suggestion, further studies of the fine

portions of affected fibres by satellite cell forma- structure of the polar and equatorial regions of http://jnnp.bmj.com/ tion. In myotonic dystrophy individual frag- affected intrafusal fibres and of their innervation ments of intrafusal fibres were separated by their will be necessary in spindles with varying degrees plasma membranes from adjacent fibres, and of abnormality. were arranged in clusters encircled by a single layer of basement membrane. Only some of This work was supported by the London Hospital these abnormal fragments contained sarco- Medical College and by a grant for equipment from the These could be Wellcome Trust. lemmal nuclei. regarded as on September 25, 2021 by guest. satellite structures (Hess and Rosner, 1970). Those fibres which did not contain nuclei on the REFERENCES other hand, must have been formed by frag- Banker, B. Q., and Girvin, J. P. (1971). The ultrastructural features of the mammalian muscle spindle. Journal of mentation or splitting of parent fibres, rather Neuropathology and Experimental Neurology, 30, 155-195. than by a regenerative process. Barker, D., Harker, D., Stacey, M. J., and Smith, C. R. Some of the abnormal fibres, however, (1972). Fusimotor innervation. In Research in Muscle Development and the Muscle Spindle, pp. 227-250. Inter- appeared metabolically active. Basophilic fibres national Congress Series No. 240. Excerpta Medica: were seen in the paraffin-embedded material, and Amsterdam. J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.38.1.91 on 1 January 1975. Downloaded from

Abnormal intrafusal muscle fibres in myotonic dystrophy: a study using serial sections 99

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