Acta neuropath. (Berl.) 34, 329--338 (1976)
Acta Neuropathologica
9 by Springer-Verlag1976
The Target Phenomenon in Human Muscle A Comparative Light Microscopic Histochemical and Electron Microscopic Study W. De Coster, J. De Reuck, and H. Vander Eecken Department of Neurology, Akademisch Ziekenhuis, de Pintelaan 135, B-9000 Ghent and Department of Pathology, University of Ghent, Belgium
Summary. A large number of target fibres is observed in a muscle biopsy from a patient with an acute flaccid paraplegia of the lower limbs due to a polyneuroradiculopathy of unknown origin. With the electron microscope up to 4 structural zones are seen in the target fibers. The histochemical and ultrastructural observations in this case, suggest that the "target fibre" phenomenon is due to a disturbed trophic influence of the nervous system. Key words: Muscle pathology -- Target muscle fibre -- Targetoid muscle fibre -- Histochemistry -- Electron microscopy.
INTRODUCTION Central alterations of the muscle fibre are described in several muscle diseases, such as myotubular myopathy (Spiro et al., 1966), centronuclear myopathy (Sher et al., 1967), central core disease or congenital non-progressive myopathy (Shy and Magee, 1956), type I fibre hypertrophy with central nuclei (Engel et al., 1968), neurogenic muscular changes with target and targetoid fibres (Engel, 1961) and target myopathy (Schotland, 1967). The "target fibre" phenomenon is generally considered to be a specific sign of muscle denervation, as proposed by Engel (1961). It is also seen in other pathological conditions, such as familial periodic paralysis (Shafiq et al., 1967), experimental re-innervation (De Reuck et al., 1973), and muscular lesions induced by p-phenylenediamine (Mascr~s and Jasmin, 1974). Dubowitz (1967) suggests that this "target" phenomenon should be considered as a sign of re-innervation. Recently Schmitt and Volk (1975) described the light microscopic findings in a case of denervation atrophy with important structural variations in the muscle fibres, such as target and targetoid phenomena. These authors suggest a continuous transition between the different types of lesions.
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MATERIAL AND METHODS A biopsy of the left gastrocnemius muscle was taken from a woman of 48 with an acute flaccid paraplegia of the lower limbs caused by a polyneuroradiculopathy of unknown origin and which did not recover after 9 months. The largest piece of the biopsy was fixed in formalin and the sections were stained with hematoxylin-eosin, phosphotungstic acid hematoxylin, periodic acid Schiff, G6mori's trichrome, Bodian's method for nerve fibres and Heidenhain-Woelcke's method for myelin shears. An other fragment was fixed in liquid nitrogen and sections were stained with hematoxylin-eosin, Sudan black, and with methods for demonstration of dehydrogenase, diaphorase, myofibrillar ATP-ase, 5-nucleotidase, alkaline and acid phosphatases, unspecific esterase and cholinesterase enzyme activities. A third fragment was fixed in a buffered 3/1 paraformaldehyde-glutaraldehyde solution (4 ~ C, pH 7,2). The material was rinsed overnight in a 0.t M phosphate buffer and postfixed in 1 ~ osmic acid during 1 h. After Epon 812 embedding, semi-thin sections were obtained with a LKB pyramitome and ultra-thin sections with a ultrotome I, using glass knives. Semi-thin sections were stained with toluidine blue, while the ultra-thin sections were put on bare ] 50 and 300 mesh grids and treated with alcoholic uranylacetate and Reynolds' leadcitrate. A Siemens Elmiskop I was used at 80 KV.
RESULTS At the light microscopic level the muscle fibres show great variation in the diameter: most fibres are large and grouped together, near a few small areas of atrophic fibres. Target and targetoid fibres are seen in about 85~o of the muscle fibres. The alterations are present in hypertrophic as well as in atrophic muscle fibres, although the structural changes are slightly different in the latter. In most fibres a central condensed core of material is present, which stains strongly blue with P T A H and red with G6mori's trichrome (Fig. 1 A). In longitudinal section this core of condensed material does not extend over the whole length of the fibre: sometimes it is very small, and restricted to a few sarcomeres. These cores appear to have a fibrillary structure without evident cross-striation. The central cores are mostly surrounded by a clear area, which, at the light microscopic level, appears to be devoid of myofibrils. The extent of this area is not uniform and superficial as well as central parts of a fibre may be involved (Fig.2A). In some instances a sarcolemmal nucleus is seen, lying between this area and the outer region. This area of rarefaction is mostly surrounded by a peripheral zone of the muscle fibre, containing longitudinally arranged myofibrils which show cross-striations. Several fibres, however, do not show the crossstriations and the myofibrils are slightly frayed. Some atrophic fibres are composed of a central core of condensed material surrounded immediately by unaffected myofibrils. In this paraffin-embedded material nerve endings are rarely present and motor end-plates cannot be recognised. The endomysium and bloodvessels appear normal. A similar histochemical pattern is found in the hypertrophic and the atrophic fibres as welt as in the target and the targetoid ones. The glycogen and fat content is similar in all the muscle fibres. The peripheral zone stains moderately for
The Target Phenomenon in Human Muscle
331
Fig. 1 A and B. Cross-sectioned target muscle fibres. (A) Note the dense central core surrounded by a pale halo, which is devoid of myofibrils. One fibre (at the bottom) contains a peripherally located target and a sarcolemmal nucleus at the border of the alteration. (PTAH x 224.) (B) The target stains negatively for PAS. The outer layer is bordered medially by a small band of increased PAS + material. ( • 224)
dehydrogenase, diaphorase and ATP-ase activities. The cross-striations are not always clearly seen. This outer layer is bordered medially by a small, irregular zone of increased PAS positive material and increased dehydrogenase and diaphorase activities (Fig.1 B). The central part of the target fibre, corresponding to the previously described compact core and the zone of myofibrillary rarefaction, is completely devoid of enzymatic activity (Fig. 2B). The condensed core stains strongly with sudan black. Unspecific esterase and cholinesterase activities are demonstrated in several m o t o r end-plates of hypertrophic, atrophic, target and targetoid fibres (Fig. 3). The nerve sheaths and the bloodvessel walls are strongly stained for 5-nucleotidase activity. Acid phosphatase activity is absent in the muscle fibres, endomysium and peripheral nerve endings. Alkaline phosphatase activity can be demonstrated in some capillaries. About 8 5 ~ of the total number of muscle fibres, seen in 1 m~z sections of Epon embedded material, show a more or less obvious alteration, of variable size and extent. The affected muscle fibre appears as a target with 4 concentric areas, which are numbered f r o m the centre to the periphery as zone 1, 2, 3 and 4. In longitudinal sections, zones 1 and 3 are not present in the whole length of the fibre, and the extent of all zones is variable.
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Fig.2 A and B. Longitudinal section of target muscle fibres. (A) Note a peripherally located target with a small dense core. The myofibrils and the cross-striation are well preserved in the outer part of this fibre. A small atrophic fibre ( ~ ) has the same staining density as the central core. (PTAH, • 240.) (B) The central core is devoid of oxidative enzyme activity and is surrounded by a band of increased activity. The peripheral parts of the fibre show a moderate enzyme activity (succinate dehydrogenase, • 160)
The Target Phenomenon in Human Muscle
333
Fig. 3. Two motor-endplates are demonstrated. One is seen in an atrophic fibre, another in a hypertrophic one (cholinesterase, • 560)
Serial Epon sections show that the alteration in the fibre can move from a peripheral to a completely central position.
Zones 1 and 2 (Fig. 4 and 5) Zone 1 corresponds to the central core, while zone 2 corresponds to the halo seen with P T A H and trichrome staining. These two areas form the central part of the alteration and are devoid of PAS positive material and enzyme activity. At the uttrastructural level zone ] consists exclusively of electron-dense material with a fibrillary structure. The density of this material is comparable to Z-band material. There are no glycogen granules nor any other cellular components present. Zone 2 consists of a mass of completely disoriented thin myofilaments, some dispersed glycogen granules and some Z-band fragments of normal electron density. Profiles of sarcoplasmic reticulum, if present, are often grouped together and have usually a swollen appearance. Triads are rare but some pentads m a y be seen. There is no distinct border between the zones. Only a few dense mitochondria may be present at the periphery of zone 2. Zone 3 This zone is not always present (Fig. 6) and shows a great variation in breadth. It consists mainly of a relatively large accumulation of glycogen granules and small, electron-dense mitochondria with indefinite cristae. Some swollen profiles of the sarcoplasmic reticulum and a few fragments of myofilaments may be present. No Z-band fragments nor cross-striation are detected in this zone.
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Fig. 4. Longitudinal section of the 4 zones of the target fibre. Zone 1 has a dense fibrillary appearance, resembling Z-band material and myosin filaments. In zone 2 disoriented actin-like filaments are present with scattered glycogen granules and some dilated profiles of the sarcoplasmic reticulum. Zone 3 shows a relative accumulation of~glycogen and small electron-dense mitochondria. The peripheral zone 4 looks normal. (• 24300) Zone 3 corresponds to the small band of PAS positive material and of increased oxidative enzyme activity. Zone 4 The sarcolemma shows irregular evaginations without thickening of the basal membrane. Mitochondria and profiles of the sarcoplasmic reticulum and tubular system are rarely present. Triads are almost completely absent. The remaining mitochondria are small, with an electron-dense matrix and cristae which are difficult to discern. No accumulation of glycogen material is seen, except in the sarcolemmal evaginations. The organization of the actin and myosin filaments is not disturbed. The cross-striation however is indefinite: I-bands are absent, H-zone and M-band are irregular or absent. The Z-bands have an irregular pattern too and are not sharply demarcated. Zone 4 corresponds to the peripheral zone of low enzyme activity. Some fibres in cross-section consist only of zones 4 and 2, depending on the level of the section.
The Target Phenomenon in Human Muscle
335
Fig.5. Details of zones 1 and 2. Note the dense fibrillar appearance of zone 1 and the presence of tubular elements in zone 2. ( • 24 300) Central nuclei are seen in the epon embedded material. Subsarcolemmal lipofuscin bodies are often present, mainly in the perinuclear area. No motor end-plates nor muscle spindles are detected. Bloodvessels do not show alterations. Sometimes parallel or concentric tubular aggregates, probably of sarcoplasmic reticular origin, and containing a single row of glycogen granules, are seen in the subsarcolemmal area around the nuclei (Fig. 7). DISCUSSION As compared with the data in the literature a high percentage of target fibres is seen in this case. Several sarcolemmal nuclei are located at the border of the central alteration, as reported by Schotland (1969). Hypertrophy of sarcoplasmic reticulum has already been observed in neurogenic atrophy (Shafiq et al., 1967; Schrodt and Walker, ~966). Longitudinal serial Epon sections show a great variety in the structure and the tridimensional localization of the "central" alteration. Zones 1 and 3 may be absent, separately or both together. So, regress composed only of zones 2 and 4, in cross section, will give the picture of a targetoid fibre. This suggests that at least in this biopsy there is no difference between target and targetoid muscle fibres. "Transitional" fibre alterations, as discribed by Schmitt and Volk (1975) are not found in this case. Our light microscopic observations are similar to these of Resnick and Engel (1966). The ultrastructural findings, however, are completely different: the alter-
336
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Fig. 6. Cross section of peripheral located target. Note absence of zone 3 at this level. ( • 2 700) ations in our case seem to be much more pronounced, especially the central part and there is no distinct "intermediate zone" as described by these authors and by others (Shafiq et al., 1967; Schotland, 1969; Kovarsky et al., 1973). Characteristic cytoplasmic bodies are absent (McDonald and Engel, 1969). It is generally accepted that the dense material, described in zone 1, is probably derived from Z-line material. However direct connections between these condensed masses and the normal Z-discs in zone 4 are not observed in this case. Such
The Target Phenomenon in Human Muscle
337
Fig.7. Perinuclear, circular profiles derived from sarcoplasmic reticulum. Note the presence of a single row of glycogen granules between the tubular profiles. (• 16000)
longitudinal electron-dense bands are considered as an unspecific alteration (Schotland, 1969; Tomonaga and Sluga, 1968). They have a fibrillary appearance and are embedded in a mass of completely desoriented thin filaments. One can postulate that zone i is composed of Z-material and myosin filaments, while zone 2 is composed of actin filaments and some small remnants of Z-discs. Engel (1961) suggested that the appearance of target fibres should point to an early stage of denervation. Nevertheless this type of alteration is known to occur also in periodic paralysis, polymyositis and after tenotomy (Shafiq et al., 1967). Kovarsky et al. (1973) described the reversibility of the target fibre phenomenon in a case of Guillain-Barr6 syndrome. This was also observed in rat muscle in early stages of re-innervation (De Reuck et al., 1973). In this case most muscle fibres are hypertrophic and some atrophic fibres show with P T A H and G6mori's trichrome the same staining density as the central core of the target fibres, while others have also the light halo surrounding the core. Unspecific esterase and cholinesterase activities are demonstrated in several motor end-plates of hypertrophic, and atrophic as well as target and targetoid fibres, clearly indicating that these fibres are innervated.
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Zone 3 shows a distinct increase of the mitochondrial enzyme activities, a relatively large accumulation of glycogen and several central nuclei. Therefore, this zone is considered to be a zone of active transformation from the complete disorganized portion of the fibre (zones 1 and 2) to the peripheral, normal structured zone 4. The "target muscle fibre" seems to be the result of a disturbed trophic influence of the nervous system on the individual muscle fibre, due, in this case, to incomplete re-innervation.
REFERENCES Dubowitz, V. : Pathology of experimentally re-innervated skeletal muscle. J. Neurol. Neurosurg. Psychiat. 30, 99-- 110 (1967) Engel, W. K. : Muscle target fibers, a newly recognized sign of denervation. Nature (Lond.) 191, 389 (1961) Engel, W. K., Gold, G. N., Karpati, G. : Type I fiber hypertrophy and central nuclei. A rare congenital muscle abnormality with a possible experimental model. Arch. Neurol. (Chic.) 18, 435--444 (1968) Kovarsky, J., Schochet, S. S., McCormick, W. F. : The significance of target fibers: a clinico-pathologic review of 100 patients with neurogenic atrophy. Amer. J. clin. Path. 59, 790--797 (1973) Mascres, Chr., Jasmin, G. : Etude pathog6nique des 16sions musculaires induites par la p-ph6nyl6ne-diamine. Un. M6d. Canada 103, 672--677 (1974) McDonald, R. D., Engel, A. G. : The cytoplasmic body: another structural anomaly of the Z disk. Acta neuropath. (Berl.) 14, 99--107 (1969) Resnik, J. S., Engel, W. K. : Target fibers--structural and cytochemical characteristics and their relationship to neurogenic muscle disease and fiber types. Excerpta Medica I.C.S. n ~ 147: Exploratory concepts in muscular dystrophy and related disorders. New York: Harriman 1966 De Reuck, J., Vander Eecken, H., Roels, H. : Biometrical and histochemical comparison between extra- and intra-fusal muscle fibers in denervated and re-innervated rat muscle. Acta neuropath. (Berl.) 25, 249--258 (1973) Schmitt, H. P., Volk, B.: Relationship between target, targetoid and targetoid/core fibers in severe neurogenic muscular atrophy. J. Neurol. 210, 167--181 (1975) Schotland, D. L. : Congenital myopathy with target fibers. Trans Amer. Neurol. Ass. 92, 107 (1967) Schotland, D. L. : An electron microscopic study of target fibers, target-like fibers and related abnormalities in human muscle. J. Neuropath. exp. Neurol. 28, 214--228 (1969) Schrodt, G. R., Walker, S. M. : Ultrastructure of membranes in denervation atrophy. Amer. J. Path. 49, 33--51 (1966) Shafiq, S.A., Milhorat, A. T., Gorycki, M. A. : Fine structure of human muscle in neurogenic atrophy. Neurology (Minneap.) 17, 934--948 (1967) Sher, J. H., Rimalovski, A. D., Athanasiades, T. S., Aronson, S. M. : Familial centronuclear myopathy: a clinical and pathological study. Neurology (Minneap.) 17, 727--742 (1967) Shy, G. M., Magee, K. R. : A new congenital non-progressive myopathy. Brain 79, 610--621 (1956) Spiro, A. J., Shy, G. M., Gonatas, N. K. : Myotubular myopathy. Persistence of fetal muscle in an adolescent boy. Arch. Neurol. (Chic.) 14, 1--13 (1966) Tomonaga, M., Sluga, E. : Zur Ultrastruktur der Targetfasern. Virchows Arch., Abt. A, Path. Anat. 348, 89--104 (1969)
Received November 20, 1975; Accepted December 30, 1975
Acta Neuropathologica
9 by Springer-Verlag1976
The Target Phenomenon in Human Muscle A Comparative Light Microscopic Histochemical and Electron Microscopic Study W. De Coster, J. De Reuck, and H. Vander Eecken Department of Neurology, Akademisch Ziekenhuis, de Pintelaan 135, B-9000 Ghent and Department of Pathology, University of Ghent, Belgium
Summary. A large number of target fibres is observed in a muscle biopsy from a patient with an acute flaccid paraplegia of the lower limbs due to a polyneuroradiculopathy of unknown origin. With the electron microscope up to 4 structural zones are seen in the target fibers. The histochemical and ultrastructural observations in this case, suggest that the "target fibre" phenomenon is due to a disturbed trophic influence of the nervous system. Key words: Muscle pathology -- Target muscle fibre -- Targetoid muscle fibre -- Histochemistry -- Electron microscopy.
INTRODUCTION Central alterations of the muscle fibre are described in several muscle diseases, such as myotubular myopathy (Spiro et al., 1966), centronuclear myopathy (Sher et al., 1967), central core disease or congenital non-progressive myopathy (Shy and Magee, 1956), type I fibre hypertrophy with central nuclei (Engel et al., 1968), neurogenic muscular changes with target and targetoid fibres (Engel, 1961) and target myopathy (Schotland, 1967). The "target fibre" phenomenon is generally considered to be a specific sign of muscle denervation, as proposed by Engel (1961). It is also seen in other pathological conditions, such as familial periodic paralysis (Shafiq et al., 1967), experimental re-innervation (De Reuck et al., 1973), and muscular lesions induced by p-phenylenediamine (Mascr~s and Jasmin, 1974). Dubowitz (1967) suggests that this "target" phenomenon should be considered as a sign of re-innervation. Recently Schmitt and Volk (1975) described the light microscopic findings in a case of denervation atrophy with important structural variations in the muscle fibres, such as target and targetoid phenomena. These authors suggest a continuous transition between the different types of lesions.
330
W. De Coster et al.
MATERIAL AND METHODS A biopsy of the left gastrocnemius muscle was taken from a woman of 48 with an acute flaccid paraplegia of the lower limbs caused by a polyneuroradiculopathy of unknown origin and which did not recover after 9 months. The largest piece of the biopsy was fixed in formalin and the sections were stained with hematoxylin-eosin, phosphotungstic acid hematoxylin, periodic acid Schiff, G6mori's trichrome, Bodian's method for nerve fibres and Heidenhain-Woelcke's method for myelin shears. An other fragment was fixed in liquid nitrogen and sections were stained with hematoxylin-eosin, Sudan black, and with methods for demonstration of dehydrogenase, diaphorase, myofibrillar ATP-ase, 5-nucleotidase, alkaline and acid phosphatases, unspecific esterase and cholinesterase enzyme activities. A third fragment was fixed in a buffered 3/1 paraformaldehyde-glutaraldehyde solution (4 ~ C, pH 7,2). The material was rinsed overnight in a 0.t M phosphate buffer and postfixed in 1 ~ osmic acid during 1 h. After Epon 812 embedding, semi-thin sections were obtained with a LKB pyramitome and ultra-thin sections with a ultrotome I, using glass knives. Semi-thin sections were stained with toluidine blue, while the ultra-thin sections were put on bare ] 50 and 300 mesh grids and treated with alcoholic uranylacetate and Reynolds' leadcitrate. A Siemens Elmiskop I was used at 80 KV.
RESULTS At the light microscopic level the muscle fibres show great variation in the diameter: most fibres are large and grouped together, near a few small areas of atrophic fibres. Target and targetoid fibres are seen in about 85~o of the muscle fibres. The alterations are present in hypertrophic as well as in atrophic muscle fibres, although the structural changes are slightly different in the latter. In most fibres a central condensed core of material is present, which stains strongly blue with P T A H and red with G6mori's trichrome (Fig. 1 A). In longitudinal section this core of condensed material does not extend over the whole length of the fibre: sometimes it is very small, and restricted to a few sarcomeres. These cores appear to have a fibrillary structure without evident cross-striation. The central cores are mostly surrounded by a clear area, which, at the light microscopic level, appears to be devoid of myofibrils. The extent of this area is not uniform and superficial as well as central parts of a fibre may be involved (Fig.2A). In some instances a sarcolemmal nucleus is seen, lying between this area and the outer region. This area of rarefaction is mostly surrounded by a peripheral zone of the muscle fibre, containing longitudinally arranged myofibrils which show cross-striations. Several fibres, however, do not show the crossstriations and the myofibrils are slightly frayed. Some atrophic fibres are composed of a central core of condensed material surrounded immediately by unaffected myofibrils. In this paraffin-embedded material nerve endings are rarely present and motor end-plates cannot be recognised. The endomysium and bloodvessels appear normal. A similar histochemical pattern is found in the hypertrophic and the atrophic fibres as welt as in the target and the targetoid ones. The glycogen and fat content is similar in all the muscle fibres. The peripheral zone stains moderately for
The Target Phenomenon in Human Muscle
331
Fig. 1 A and B. Cross-sectioned target muscle fibres. (A) Note the dense central core surrounded by a pale halo, which is devoid of myofibrils. One fibre (at the bottom) contains a peripherally located target and a sarcolemmal nucleus at the border of the alteration. (PTAH x 224.) (B) The target stains negatively for PAS. The outer layer is bordered medially by a small band of increased PAS + material. ( • 224)
dehydrogenase, diaphorase and ATP-ase activities. The cross-striations are not always clearly seen. This outer layer is bordered medially by a small, irregular zone of increased PAS positive material and increased dehydrogenase and diaphorase activities (Fig.1 B). The central part of the target fibre, corresponding to the previously described compact core and the zone of myofibrillary rarefaction, is completely devoid of enzymatic activity (Fig. 2B). The condensed core stains strongly with sudan black. Unspecific esterase and cholinesterase activities are demonstrated in several m o t o r end-plates of hypertrophic, atrophic, target and targetoid fibres (Fig. 3). The nerve sheaths and the bloodvessel walls are strongly stained for 5-nucleotidase activity. Acid phosphatase activity is absent in the muscle fibres, endomysium and peripheral nerve endings. Alkaline phosphatase activity can be demonstrated in some capillaries. About 8 5 ~ of the total number of muscle fibres, seen in 1 m~z sections of Epon embedded material, show a more or less obvious alteration, of variable size and extent. The affected muscle fibre appears as a target with 4 concentric areas, which are numbered f r o m the centre to the periphery as zone 1, 2, 3 and 4. In longitudinal sections, zones 1 and 3 are not present in the whole length of the fibre, and the extent of all zones is variable.
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Fig.2 A and B. Longitudinal section of target muscle fibres. (A) Note a peripherally located target with a small dense core. The myofibrils and the cross-striation are well preserved in the outer part of this fibre. A small atrophic fibre ( ~ ) has the same staining density as the central core. (PTAH, • 240.) (B) The central core is devoid of oxidative enzyme activity and is surrounded by a band of increased activity. The peripheral parts of the fibre show a moderate enzyme activity (succinate dehydrogenase, • 160)
The Target Phenomenon in Human Muscle
333
Fig. 3. Two motor-endplates are demonstrated. One is seen in an atrophic fibre, another in a hypertrophic one (cholinesterase, • 560)
Serial Epon sections show that the alteration in the fibre can move from a peripheral to a completely central position.
Zones 1 and 2 (Fig. 4 and 5) Zone 1 corresponds to the central core, while zone 2 corresponds to the halo seen with P T A H and trichrome staining. These two areas form the central part of the alteration and are devoid of PAS positive material and enzyme activity. At the uttrastructural level zone ] consists exclusively of electron-dense material with a fibrillary structure. The density of this material is comparable to Z-band material. There are no glycogen granules nor any other cellular components present. Zone 2 consists of a mass of completely disoriented thin myofilaments, some dispersed glycogen granules and some Z-band fragments of normal electron density. Profiles of sarcoplasmic reticulum, if present, are often grouped together and have usually a swollen appearance. Triads are rare but some pentads m a y be seen. There is no distinct border between the zones. Only a few dense mitochondria may be present at the periphery of zone 2. Zone 3 This zone is not always present (Fig. 6) and shows a great variation in breadth. It consists mainly of a relatively large accumulation of glycogen granules and small, electron-dense mitochondria with indefinite cristae. Some swollen profiles of the sarcoplasmic reticulum and a few fragments of myofilaments may be present. No Z-band fragments nor cross-striation are detected in this zone.
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Fig. 4. Longitudinal section of the 4 zones of the target fibre. Zone 1 has a dense fibrillary appearance, resembling Z-band material and myosin filaments. In zone 2 disoriented actin-like filaments are present with scattered glycogen granules and some dilated profiles of the sarcoplasmic reticulum. Zone 3 shows a relative accumulation of~glycogen and small electron-dense mitochondria. The peripheral zone 4 looks normal. (• 24300) Zone 3 corresponds to the small band of PAS positive material and of increased oxidative enzyme activity. Zone 4 The sarcolemma shows irregular evaginations without thickening of the basal membrane. Mitochondria and profiles of the sarcoplasmic reticulum and tubular system are rarely present. Triads are almost completely absent. The remaining mitochondria are small, with an electron-dense matrix and cristae which are difficult to discern. No accumulation of glycogen material is seen, except in the sarcolemmal evaginations. The organization of the actin and myosin filaments is not disturbed. The cross-striation however is indefinite: I-bands are absent, H-zone and M-band are irregular or absent. The Z-bands have an irregular pattern too and are not sharply demarcated. Zone 4 corresponds to the peripheral zone of low enzyme activity. Some fibres in cross-section consist only of zones 4 and 2, depending on the level of the section.
The Target Phenomenon in Human Muscle
335
Fig.5. Details of zones 1 and 2. Note the dense fibrillar appearance of zone 1 and the presence of tubular elements in zone 2. ( • 24 300) Central nuclei are seen in the epon embedded material. Subsarcolemmal lipofuscin bodies are often present, mainly in the perinuclear area. No motor end-plates nor muscle spindles are detected. Bloodvessels do not show alterations. Sometimes parallel or concentric tubular aggregates, probably of sarcoplasmic reticular origin, and containing a single row of glycogen granules, are seen in the subsarcolemmal area around the nuclei (Fig. 7). DISCUSSION As compared with the data in the literature a high percentage of target fibres is seen in this case. Several sarcolemmal nuclei are located at the border of the central alteration, as reported by Schotland (1969). Hypertrophy of sarcoplasmic reticulum has already been observed in neurogenic atrophy (Shafiq et al., 1967; Schrodt and Walker, ~966). Longitudinal serial Epon sections show a great variety in the structure and the tridimensional localization of the "central" alteration. Zones 1 and 3 may be absent, separately or both together. So, regress composed only of zones 2 and 4, in cross section, will give the picture of a targetoid fibre. This suggests that at least in this biopsy there is no difference between target and targetoid muscle fibres. "Transitional" fibre alterations, as discribed by Schmitt and Volk (1975) are not found in this case. Our light microscopic observations are similar to these of Resnick and Engel (1966). The ultrastructural findings, however, are completely different: the alter-
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Fig. 6. Cross section of peripheral located target. Note absence of zone 3 at this level. ( • 2 700) ations in our case seem to be much more pronounced, especially the central part and there is no distinct "intermediate zone" as described by these authors and by others (Shafiq et al., 1967; Schotland, 1969; Kovarsky et al., 1973). Characteristic cytoplasmic bodies are absent (McDonald and Engel, 1969). It is generally accepted that the dense material, described in zone 1, is probably derived from Z-line material. However direct connections between these condensed masses and the normal Z-discs in zone 4 are not observed in this case. Such
The Target Phenomenon in Human Muscle
337
Fig.7. Perinuclear, circular profiles derived from sarcoplasmic reticulum. Note the presence of a single row of glycogen granules between the tubular profiles. (• 16000)
longitudinal electron-dense bands are considered as an unspecific alteration (Schotland, 1969; Tomonaga and Sluga, 1968). They have a fibrillary appearance and are embedded in a mass of completely desoriented thin filaments. One can postulate that zone i is composed of Z-material and myosin filaments, while zone 2 is composed of actin filaments and some small remnants of Z-discs. Engel (1961) suggested that the appearance of target fibres should point to an early stage of denervation. Nevertheless this type of alteration is known to occur also in periodic paralysis, polymyositis and after tenotomy (Shafiq et al., 1967). Kovarsky et al. (1973) described the reversibility of the target fibre phenomenon in a case of Guillain-Barr6 syndrome. This was also observed in rat muscle in early stages of re-innervation (De Reuck et al., 1973). In this case most muscle fibres are hypertrophic and some atrophic fibres show with P T A H and G6mori's trichrome the same staining density as the central core of the target fibres, while others have also the light halo surrounding the core. Unspecific esterase and cholinesterase activities are demonstrated in several motor end-plates of hypertrophic, and atrophic as well as target and targetoid fibres, clearly indicating that these fibres are innervated.
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W. De Coster et al.
Zone 3 shows a distinct increase of the mitochondrial enzyme activities, a relatively large accumulation of glycogen and several central nuclei. Therefore, this zone is considered to be a zone of active transformation from the complete disorganized portion of the fibre (zones 1 and 2) to the peripheral, normal structured zone 4. The "target muscle fibre" seems to be the result of a disturbed trophic influence of the nervous system on the individual muscle fibre, due, in this case, to incomplete re-innervation.
REFERENCES Dubowitz, V. : Pathology of experimentally re-innervated skeletal muscle. J. Neurol. Neurosurg. Psychiat. 30, 99-- 110 (1967) Engel, W. K. : Muscle target fibers, a newly recognized sign of denervation. Nature (Lond.) 191, 389 (1961) Engel, W. K., Gold, G. N., Karpati, G. : Type I fiber hypertrophy and central nuclei. A rare congenital muscle abnormality with a possible experimental model. Arch. Neurol. (Chic.) 18, 435--444 (1968) Kovarsky, J., Schochet, S. S., McCormick, W. F. : The significance of target fibers: a clinico-pathologic review of 100 patients with neurogenic atrophy. Amer. J. clin. Path. 59, 790--797 (1973) Mascres, Chr., Jasmin, G. : Etude pathog6nique des 16sions musculaires induites par la p-ph6nyl6ne-diamine. Un. M6d. Canada 103, 672--677 (1974) McDonald, R. D., Engel, A. G. : The cytoplasmic body: another structural anomaly of the Z disk. Acta neuropath. (Berl.) 14, 99--107 (1969) Resnik, J. S., Engel, W. K. : Target fibers--structural and cytochemical characteristics and their relationship to neurogenic muscle disease and fiber types. Excerpta Medica I.C.S. n ~ 147: Exploratory concepts in muscular dystrophy and related disorders. New York: Harriman 1966 De Reuck, J., Vander Eecken, H., Roels, H. : Biometrical and histochemical comparison between extra- and intra-fusal muscle fibers in denervated and re-innervated rat muscle. Acta neuropath. (Berl.) 25, 249--258 (1973) Schmitt, H. P., Volk, B.: Relationship between target, targetoid and targetoid/core fibers in severe neurogenic muscular atrophy. J. Neurol. 210, 167--181 (1975) Schotland, D. L. : Congenital myopathy with target fibers. Trans Amer. Neurol. Ass. 92, 107 (1967) Schotland, D. L. : An electron microscopic study of target fibers, target-like fibers and related abnormalities in human muscle. J. Neuropath. exp. Neurol. 28, 214--228 (1969) Schrodt, G. R., Walker, S. M. : Ultrastructure of membranes in denervation atrophy. Amer. J. Path. 49, 33--51 (1966) Shafiq, S.A., Milhorat, A. T., Gorycki, M. A. : Fine structure of human muscle in neurogenic atrophy. Neurology (Minneap.) 17, 934--948 (1967) Sher, J. H., Rimalovski, A. D., Athanasiades, T. S., Aronson, S. M. : Familial centronuclear myopathy: a clinical and pathological study. Neurology (Minneap.) 17, 727--742 (1967) Shy, G. M., Magee, K. R. : A new congenital non-progressive myopathy. Brain 79, 610--621 (1956) Spiro, A. J., Shy, G. M., Gonatas, N. K. : Myotubular myopathy. Persistence of fetal muscle in an adolescent boy. Arch. Neurol. (Chic.) 14, 1--13 (1966) Tomonaga, M., Sluga, E. : Zur Ultrastruktur der Targetfasern. Virchows Arch., Abt. A, Path. Anat. 348, 89--104 (1969)
Received November 20, 1975; Accepted December 30, 1975
