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Morphological changes in skeletal muscle after transplantation
Journal of the neurological Sciences, 1975, 25:227-247 ~?~ Elsevier Scientific Publishing Company, Amsterdam
227 Printed in The Netherlands
Morphological Changes in Skeletal Muscle after Transplantation A L i g h t - a n d E l e c t r o n - m i c r o s c o p i c S t u d y o f the Initial P h a s e s o f Degeneration and Regeneration* F. L. M A S T A G L I A , R. L. DAWKINS AND J. M. PAPADIMITRIOU Departments of Medicine and Pathology, University of Western Australia, Perth Medical Centre, Verdun Street, Shenton Park, Western Australia 6008 (Australia) (Received 26 November, 1974)
INTRODUCTION
It has been found in previous muscle transplantation studies that there is an initial phase both in autografts and in homografts during which the mature muscle fibres in the graft degenerate and that this phase is followed by one of active regeneration (Elson 1929 ; Studitsky 1964; Carlson 1968, 1970; Mastaglia, Dawkins and Papadimitriou 1973a). So effective is this regenerative phase that it may lead to reconstitution of an entire muscle when minced muscle fragments are replaced in the original muscle bed (Studitsky 1964; Carlson 1968, 1970; Salafsky 1971 ; Batalin and Allbrook 1973). The mechanisms of regeneration in muscle grafts and in particular the source of myogenic cells from which the new population of muscle fibres is derived have not been fully elucidated (Bradley 1973). Previous studies have been concerned with the question of survival of muscle grafts in normal and dystrophic hosts (Laird and Timmer 1965; Cosmos and Butler 1970), the biochemical and histochemical correlates of the degenerative and regenerative phases (Gallucci, Novello, Margreth and Aloisi 1966; Snow 1973), the reinnervation of grafts (Hsu 1974) and the contractile properties of the reconstituted graft in normal and dystrophic hosts (Salafsky 1971 ; Carlson and Gutmann 1972). The morphological changes in muscle grafts have been studied at the light microscope level (Elson 1929; Studitsky 1964; Laird and Timmer 1965; Carlson 1968, 1970; Snow 1973 ; Hsu 1974) but few observations have been made on the ultrastructural *Presented in part at the 3rd Intemational Congress of Muscle Diseases, Newcastle upon Tyne, September 1974. This study was supported by research grants from the University of Western Australia, the National Health and Medical Research Council of Australia and the Muscular Dystrophy Association of Western Australia.
BALBtC
AKR
P.H.
IRRAD. P.H.
P.H
1
2
3
4
5
26 21 23 21 5
BALB/C (32) ~
BALB/C (23)
BALB/C (21)
I R R A D . BALB/C (5)
rectus abdominis
BALB/C (30)
Recipient ( Nos. o f animals)
0
0
0
11
1[b
diaphraom
2
I*. 2", 3", 4, 5, 7, 8, 9, 10, 11, 13
I *, 2", 3", 4", 5, 7, 8. 9", 10, 11, 13, 15
2, 3*, 4", 6*, 7, 8, 9", 14, 18, 21
I, 2". 3, 4", 6*, 7, 8, 9*, 13, 14", 18, 21
Days to sacrifice
I
4
0
3
16 5
4
22
5
7
E.M.
32
L.M.
Number o f 9rafts examined
17
6
10
5
Number of grafts not recovered
0/1
5/8
8/11 1/1
6/8
5/5
0/22
Number o f yrafts with histological evidence o f r~jection" > 6 day.~
3tll
0/18
0/28
Number of 9raf ts with no r egeneration > 72 hr
Infiltration with mononuclear inflammatory cells, damage to regenerate muscle fibres, depletion of muscle fibres. b In 7 ammals a graft from the rectus abdominis was implanted on one side a n d one from the d i a p h r a g m on the other. Five animals in this group received an isogeneic graft on one side and an allogenei'c graft on the other and are also included in G r o u p 1. * The asterisks indicate animals in which grafts were examined with the electron microscope.
Donor
Grolqs
Source o f graft
D E T A I L S O F A N I M A L S U S E D IN T R A N S P L A N T A T I O N E X P E R I M E N T S A N D O F M U S C L E G R A F T S S T U D I E D
TABLE 1
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MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
229
changes in muscle after transplantation (Trupin 1970; Allbrook and James 1973). The present series of experiments was undertaken to study in greater detail the fine structural changes which occur in muscle fibres in the early stages after transplantation and in particular to throw further light upon the origin of myoblasts in the initial stages of graft regeneration. Observations on the revascularization of grafts are also presented. The morphological changes relating to the rejection of allografts are the subject of another report (Mastaglia, Papadimitriou and Dawkins 1975).
MATERIALS AND METHODS
One hundred and six 6-8 week inbred BALB/C mice were transplanted with isogeneic muscle from BALB/C littermates or with allogeneic muscle from AKR or Prince Henry (PH) mice of similar age. The numbers of animals in each group and other details are summarized in Table 1. Twenty-one animals received grafts from PH mice which had been exposed to 1500 rads of total body irradiation (Siemens Stabilapan, 300 kV, 12 mA, 1 mm Cu filter, dose rate 88 rad/min) 1-2 hr beforehand. A further experiment in which muscle from non-irradiated PH mice was transplanted into BALB/C mice which had received 1000 rads of total body irradiation 1 hr beforehand was unsuccessful as the irradiated animals all died within 72 hr. The donor animals were sacrificed immediately prior to the transplantation procedure. The rectus abdominis muscle and diaphragm were chosen for the preparation of grafts, both being thin flat muscles which could be easily divided into small fragments. These muscles were removed, placed in Petri dishes containing medium 199 and divided into fragments approximately 2 x 2 mm, using a sharp scalpel blade. The recipient animals were anesthetized with ether or intraperitoneal pentobarbitone and a small vertical mid-line incision was made in the skin of the anterior abdomen. In most animals a single fragment of the donor muscle was inserted between the skin and the fascia overlying the abdominal muscles to one side of the mid-line and the incision was closed with cotton sutures. In 7 animals a graft from the rectus abdominis was placed on one side and one from the diaphragm of the same animal on the other side. In 5 animals an isogeneic rectus abdominis graft was placed on one side and an allogeneic graft on the other. The recipient animals were sacrificed at intervals of 1 to 23 days (Table 1). In 20 animals 1 ml of India ink was injected into a tail vein immediately prior to sacrifice to study the revascularization of grafts. The graft was identified in 88 instances and was usually somewhat reduced in size, particularly in the case of allografts (both irradiated and non-irradiated) after the first week. The graft and the adherent host abdominal muscles were removed en bloc and pinned out on cardboard. The graft site was then divided into two approximately equal portions using a sharp scalpel blade. One portion was fixed in Heidenhain's Susa and embedded in paraffin. Several serial sections were taken from one or more levels of each block and were stained with haematoxylin and eosin. The remainder of the grafts were carefully separated from the host tissues, fixed in cold 3 }/o glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 hr, washed in buffer, post-fixed in cold 1 ~o osmium tetroxide in 0.1 M cacodylate buffer for 1 hr and then embedded in araldite after dehydration in
230
v . L . MASTAGLIA, R. L. DAWKINS, J. M. PAPADIM1TRIOIJ
graded solutions of ethanol. Fragments of rectus abdominis muscle from some donor animals were also prepared for electron microscopy in the same way. Thin sections from selected blocks (Table 1) were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Philips 201 (80 kV) or JEM T6 (60 kV) electron microscope.
OBSERVATIONS
Non-Irradiated Grafts Light microscopy The changes in isografts and non-irradiated allografts were essentially the same during the first week after transplantation. At 24 hr all the muscle fibres in the graft showed acute hyaline change and from 48 hr onwards there was progressive fragmentation and dissolution of necrotic myoplasm with the appearance of phagocytic cells particularly in muscle fibres in the peripheral portions of the graft (Fig. la). Fibres with some residual necrotic myoplasm were still present in the central portions of some grafts at day 10 (Fig. lb). Mononucleated cells with the characteristics of myoblasts (Mastaglia and Kakulas 1970) were present at the periphery of many necrotic muscle fibres by 48-72 hr and syncytial plaques were in evidence by 72-96 hr. Early myotubes were present in some grafts by day 4 and were numerous in most grafts by day 6-7 particularly at the periphery of the graft where regenerative changes were always more advanced (Fig. lb). In isografts, muscle fibres of increasing maturity were present throughout the graft during the second week and by the end of the third week many fibres with closely-packed myofibrils and peripheral or central vesicular nuclei were present within a connective tissue capsule of varying thickness (Fig. lc). The degree of regeneration in allografts was comparable to that in isografts up to day 7 but there was a progressive reduction in the number of muscle fibres as rejection supervened over the ensuing week (Fig. ld). Allografts examined after 10 days no longer contained muscle elements but were composed solely of connective tissue with a light infiltrate of mononuclear inflammatory cells. Evidence of revascularization from adjacent host tissues was found in all grafts showing regenerative changes after day 4. Many carbon-filled vascular channels were present throughout allografts undergoing rejection (see Mastaglia et al. 1975) and were less numerous in allografts after day 10. Electron microscopy The earliest changes seen in myofibrils during the first 48-96 hr consisted of fading or disappearance of Z-bands and loss of filaments from the I-bands (Fig. 2a). A-bands. H-zones and M-lines were often well-preserved in myofibrils with absent Z-bands and marked loss of filaments from the I-bands. At a later stage, individual fdaments inthe A-bands were no longer sharply defined and in many fibres a myofibriUar pattern was no longer recognizable, the contractile elements having been reduced to a mass of lightly-staining granular or fibrillar material (Fig. 2b). Many electron-dense filamentous or rod-shaped structures of presumed Z-band origin were scattered throughout
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
231
Fig. 1. a: necrosis of muscle fibres in a 72 hr allograft; myophagia is more advanced in the peripheral portion of the graft (lower right). HE, x 160. b: 7-day isograft. A few residual necrotic muscle fibres are still present in the central portion of the graft while many myotubes are present at the periphery. HM=host abdominal muscle. HE, x 160. c: closely-packed regenerate muscle fibres in a 14-day isograft (IG). HM=host abdominal muscle. HE, x 160. d: a few residual myotubes in a 9-day allograft which is infiltrated with mononuclear inflammatory cells. HM=host abdominal muscle; N=degenerating nerve. HE, x 160. the n e c r o t i c m y o p l a s m a n d in fibres with a sufficiently p r e s e r v e d s a r c o m e r e p a t t e r n were seen to e x t e n d from one A - b a n d to the next, b r i d g i n g the space n o r m a l l y o c c u p i e d by t h e Z - a n d I - b a n d s (Fig. 2a). A transverse p e r i o d i c i t y was a p p a r e n t in some o f these s t r u c t u r e s (Fig. 2, inset). The m i t o c h o n d r i a in d e g e n e r a t i n g fibres were often p r e s e n t in large aggregates, p a r t i c u l a r l y at the p e r i p h e r y o f the fibre, a n d s h o w e d a variety o f c h a n g e s (Fig. 3). M a n y were e n l a r g e d a n d r o u n d e d with thickening o f the i n n e r a n d o u t e r m e m b r a n e s while some were u n i f o r m l y d e n s e l y - s t a i n e d with no r e c o g n i z a b l e cristae m i t o c h o n d r i a l e s (Fig. 2a). M a n y m i t o c h o n d r i a con-
232
F . L . M A S I A G L I A . R. L. I ) A W K I N S , J. M. PAI~AI)IMITRIOI.
Fig. 2. a : four-day allograft. Selective loss of Z - a n d I - b a n d s with relative p r e s e r v a t i o n o f A - b a n d s (A J m a d e g e n e r a t i n g muscle fibre. The ele~lron-dense s t r u c t u r e s b r i d g i n g the gap between the A - b a n d s in some myofibrils (arrows) are t h o u g h t t .... c Z - b a n d remnants, x 38,250, bar 0.5/~m. Inset: higher magnification s h o w i n g a transverse periodicity m one of these structures, x 104,00(/. b: necrotic muscle fibre in a 4-day allograft. M y o f i b r i l l a r outlines are lost a n d m i t o c h o n d r i a arc s h r u n k e n and electron-dense, x 11,900, b a r ] tim.
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE At:TER TRA/~SPLANTATION
233
Fig. 3. A subsarcolemmal mitochondrial aggregate in a degenerating muscle fibre in a 48-hr allograft. Many of the mitochondria contain amorphous electron-dense deposits and show thickening or breakdown of membranes; some contain para-crystalline inclusions which are probably situated between cristal membranes (arrow and inset), x 39,000; inset x 78,000, bar 0.5 ,urn.
234
F. L. MASTAGLIA, R. L. DAWKINS~ J. M. PAPA1)IMITRIOI
Fig. 4. Necrotic muscle fibre (N) in a 5-day aUograft. The nuclear and cytoplasmic characteristics of the mononucleated cells (Mc) situated internal to the basement lamina (arrowheads) of the fibre suggest that they are phagocytic in nature. A similar cell is present at the periphery of another necrotic fibre in the top left comer, x 7,800, bar 2 #m.
tained electron-dense para-crystalline inclusions between the cristal membranes (Fig. 3). Most mitochondria contained variable amounts of lightly-staining amorphous material and in many, localized deposits of densely-staining material were also present (Fig. 3). Other changes found in degenerating fibres included dilatation of the sarcoplasmic reticulum, occasional membrane-bound dense bodies of presumed lysosomal nature, and clusters of small vesicles. Some sarcolemmal nuclei
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
235
were densely-stained and shrunken with separation of the nuclear membrane. The plasma membrane of muscle fibres could no longer be identified by 48-72 hr but the basement lamina, although separated from the underlying necrotic myoplasm and at times folded and redundant, remained intact even after 96 hr (Figs. 4-6). Mononucleated ceils of various types were present internal to the basement lamina of necrotic muscle fibres by 48-72 hr. Many of these were clearly macrophages. These cells were irregular in outline with pleomorphic processes and their cytoplasm contained lysosomes, granular endoplasmic reticulum, lipid droplets and, in some instances, membranous whorls (Fig. 4). Some cells contained recognizable ingested material from the muscle fibre. Moderate numbers of such cells were also present external to the basement lamina of muscle fibres in the interstitium. Occasional subsarcolemmal cells had the ultrastructural features of lymphocytes and others of granulocytes. Other thin elongated sub-sarcolemmal cells with a relatively smooth contour, oval or elongated nuclei with prominent nucleoli, many free and membrane-bound ribosomes and few mitochondria were considered to be premyoblasts (Mastaglia 1974) (Fig. 5). Some cells of this type were in mitosis (Fig. 5b). Cells with similar characteristics and with thin actin-like filaments (50-60 A diameter) with or without thicker myosin-like filaments (100-120 A diameter) and microtubules in the cytoplasm were identified as myoblasts (Mastaglia 1974}. Myogenic cells could usually be distinguished from other mononuclear ceils at the periphery of the sarcolemmal tube by the presence of filaments in the cytoplasm or, when filaments were not present, on the basis of a relatively smooth cell contour (Fig. 5) and the characteristics of the nuclei (Fig. 6) which were generally larger with fewer chromatin clumps and larger nucleoli than those of monocytes and macrophages. The presence within the cytoplasm of membrane-bound areas of phagocytosed material was not a useful distinguishing criterion as such areas were also present in some myoblasts as has been noted in previous studies (Larson, Jenkison and Hudgson 1970; Trupin 1970; Mastaglia and Walton 1971). Certain observations relevant to the origin of myoblasts were made. The number of satellite cells found in the rectus abdominis muscle prior to transplantation varied from one animal to another. While in some animals no satellite cells were found (presumably due to the limitations of sampling), in others up to 8 ~ of muscle fibre nuclei belonged to these sub-sarcolemmal cells. Cells with the cytological features of resting satellite cells were not seen in 24- or 48-hr grafts and no definite conclusions could be reached as to the fate of these cells after transplantation. In 48-hr grafts some elongated mononucleated sub-sarcolemmal cells rich in ribonucleoprotein (Fig. 5c) which were considered to be premyoblasts resembled the so-called activated satellite cells referred to in some previous studies (Shafiq 1970). Whether or not such cells were present in the muscle prior to transplantation cannot, however, be established. Other observations in 48-hr grafts suggested that at least some myoblast precursors were formed by a process of sequestration of sarcolemmal nuclei and paranuclear cytoplasm at the periphery of degenerating muscle fibres. As illustrated in Fig. 7, the precursors of the new cell membranes take the form of multiple small vesicles and cisternae. In some areas interconnecting membrane-lined clefts are seen in the paranuclear region which is rich in ribosomes and granular endoplasmic reticulum
~N
F-
~j
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 6. Portion of a crescentic mononucleated cell situated internal to the basement lamina (open arrow) of a necrotic muscle fibre in a 3-day allograft. The nuclear chromatin is dispersed and the nueleolus (N) is large. The nuclear characteristics in particular suggest that the cell is myoblastic in nature. Thin filaments are present in some parts of the cell but are not seen at this magnification. E R = wide vesicles of the endoplasmic reticulum some of which appear to be formed by extensions of the nuclear membrane (solid arrow). × 9,000, bar 2 #m.
and contains mitochondria of normal appearance which contrast with the degenerate organelles in the necrotic myoplasm nearby. As shown in Fig. 7, this process of nuclear sequestration may occur at a relatively early stage of necrosis before the necrotic myoplasm in the region is invaded by phagocytic cells. In Fig. 8 the degree of breakdown of the muscle fibre is more advanced and the cell presumed to be a premyoblast has a complete limiting plasma membrane. However, apparently newlyformed flat membranous cisternae are present in the nearby necrotic myoplasm. Larger mononucleated myoblasts and syncytia (Fig. 9), whose cytoplasm contained thick and thin filaments, primitive Z-bodies and assembling myofibrils were present at the periphery of sarcolemmal tubes at 3 ~ days. Many early myotubes were also present at these times both in isografts and in allografts. These fibres contained well-aligned loosely-packed myofibrils and a variable number of large nuclei with dispersed chromatin and prominent nucleoli in the central parts of the fibre. The
Fig. 5. a: undifferentiated mononucleated cell with the characteristics of a premyoblast closely applied to the inner surface of the residual basement lamina {arrowhead) of a necrotic muscle fibre in a 3-day allograft. Note the high nuclear cytoplasmic ratio, prominent nucleoli and ribosomal aggregates in the cytoplasm. Collagen fibrils (c) are present external to the basement lamina, x 12,600, bar 1 gm. b : mitotic premyoblast internal to the basement lamina of a degenerating muscle fibre (DM) in a 3-day allograft. A centriole is seen in the premyoblast (arrow). The adjacent fibre (N) shows more advanced dissolution. Collagen fibrils separate the basement laminae of the two fibres, x 9,400, bar 1 #m. c: elongated premyoblast internal to the basement lamina of a degenerating muscle fibre {DM) in a 3-day allograft. Note the smooth contour of the cell, the prominent nucleolus and the presence of numerous free and membrane-bound ribosomes in the cytoplasm. N = an adjoining fibre whose necrotic myoplasm is separated from the basement lamina. Collagen fibrils separate the basement laminae of the two fibres. × 10,000, bar 1/~m.
Fig. 7. a: degenerating muscle fibre (MF) in a 48-hr allograft. The myonucleus (N) and surrounding cytoplasm which contains numerous ribosomes and many strands of granular endoplasmic reticulum are partially separated from the degenerating m y o p l a s m by a series of membrane-lined clefts (enclosed area lower left), vesicles (arrow) and m e m b r a n o u s folds (enclosed area upper right). Arrowheads = basement lamina, x 22,400, bar 1 #m. b: photographic enlargement of lower enclosed area in (a). x 35,400. c: photographic enlargement of upper enclosed area in (a). x 35,400.
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 8. a : a thin mononucleated cell (Mc) rich in ribosomes is present at the periphery of a necrotic muscle fibre (Mf} in a 48-hr allograft. A series of interrupted flat membranous cisternae is present in the necrotic myoplasm nearby (arrows). Arrowheads =basement lamina and endomysial collagen fibrils. × 15,000, bar 1/~m. b: higher magnification o f the area indicated by arrows in (a). x 39,700, bar 0.5 #m.
paranuclear cytoplasm contained numerous polyribosomes and prominent granular endoplasmic reticulum and Golgi apparatus (Fig. 10a). Many muscle fibres with closely-packed myofibrils were present in 9-14 day isografts. Some were at the late myotube stage of development with internal nuclei while others had peripherally-
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F. L. MASTAGLIA. R. L. I)AWKINS. J. M. t'APAI)IMITRI()/'
Fig. 9. Multinucleated muscle cell internal to the basement lamina (arrowheads) of a necrotic muscle fibrc (N) in a 4-day allograft. The nuclei show a dispersed chromatin pattern and nucleoli are not present m tile plane of section. The cytoplasm contains many ribosomal particles and a few mitochondria. Myofilaments cannot be identified at this magnification. The dissociated basement lamina of an adjacent necrotic fibre is also seen {arrow). x 7,000, bar 2 ~tm.
placed nuclei. Numerous polyribosomes and strands of granular endoplasmic reticulum were present in the paranuclear region as well as at the periphery of such fibres (Fig. 10b). Other structures included longitudinally-orientated microtubules in the subsarcolemmal region (Fig. 10b), prominent paranuclear Golgi apparatus, variable numbers of mitochondria, developing triads and budding sarcoplasmic reticulum. Myofibrils in the more mature fibres varied considerably in diameter, were often split longitudinally, and showed poor transverse sarcomere alignment. Undifferentiated mononucleated cells were present internal to the basement lamina of some fibres (Fig. l(k i Newly-formed capillaries with plump endothelial cells and a narrow lumen, and small venules were first seen at the periphery of grafts at day 4 and were subsequently found throughout the graft. The continuity of these vessels with the vascular tree of the host was demonstrated by the presence of carbon particles within the lumen in grafts removed from animals injected with India ink (Fig. 11 ). Regenerating muscle fibres were often closely applied to capillaries and venules.
Irradiated Grafts While no significant differences in the initial degenerative changes and phagocytosis of necrotic debris were noted between irradiated and non-irradiated grafts, differences were apparent in the degree and time course of regeneration. Regenerative changes were found in only 5 0 ~ of irradiated grafts (Table 1). Small numbers of premyoblasts and myoblasts were present at the periphery of necrotic fibres in some grafts at 48-72 hr but were less numerous than in non-irradiated grafts. Myotubes
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 10. a: central portion of a, myotube in a 9-day isograft showing copious polyribosomes and prominent granular endoplasmic reticulum (ER) in the paranuclear region. The nucleus (N) shows a prominent nucleolus and dispersed chromatin. G = Golgi apparatus. The myofibrils on either side of the nucleus are longitudinally aligned and sectioned somewhat tangentially. × 16,800, bar 1 /Jm. b : peripheral portion of a regenerate muscle fibre in a 9-day isograft showing polyribosomes, granular endoplasmic reticulum (ERI and longitudinally-orientatedmicrotubules (arrows). M = myofibril showing Z-band streaming. × 30,500, bar 0.5/~m. c: undifferentiated mononucleated cell (M) at the periphery of a regenerate muscle fibre in a 7-day allograft. The basement lamina of the muscle fibre is indicated by the arrowheads, z 11,200~ bar 1/~m.
were p r e s e n t in s m a l l n u m b e r s in two 3-day a n d in o n e 5-day graft b u t were a b u n d a n t in o n e 9 - d a y graft. M y o t u b e s in i r r a d i a t e d g r a f t s t e n d e d to be s m a l l e r a n d to h a v e fewer n u c l e i t h a n t h o s e in n o n - i r r a d i a t e d grafts. Six o u t o f 8 g r a f t s e x a m i n e d after 6 days were v a s c u l a r i z e d a n d 5 o u t o f 8 were i n f i l t r a t e d b y m o n o n u c l e a r i n f l a m m a t o r y
242
v. 1,. MASTAGLIA, R. 1,. I ) A W K I N S , J. M. PAI'AI)IM11"1~.1{)1
Fig. 11, a: c a r b o n particles in the lumen of a capillary m a 9-day allograft. × 8,300, bar 1 Iml. b: small venule in a 7-day i r r a d i a t e d allograft. × 8,000, bar 1 lzm.
cells. As in the case of non-irradiated allografls, each of the 4 irradiated grafts examined after 10 days were composed only of connective tissue with a few fat spaces and variable numbers of mononuclear cells.
MORPHOLOGICAL CHANGES 1N SKELETAL MUSCLE AFTER TRANSPLANTATION
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DISCUSSION
The present findings emphasize the inherent capacity of skeletal muscle to regenerate even when deprived of nerve and blood supply and transplanted to a foreign site in an immunologically unrelated host. Regeneration has been found to be as active in the present heterotopically implanted homografts as in previous experiments in which autografts were re-implanted into the original muscle bed (e.g. Carlson 1968, 1970). Moreover, regeneration is initially as active in allografts as in isografts and rejection only occurs when the reconstitutive phase is well under way, a point which is of relevance to the question of therapeutic muscle transplantation. The finding of active regeneration in grafts by 48-72 hr at a stage before evidence of vascularization could be demonstrated suggests that the initial stages of regeneration may proceed even under relatively anaerobic conditions. Histochemical observations indicating that certain glycolytic enzyme systems are active in myoblasts in regenerating autografts (Snow 1973) support this conclusion. The paucity of mitochondria found in premyoblasts and myoblasts on electron microscopy in the present and in other studies (Reznik and Hansen 1969) would suggest that aerobic metabolic pathways are of lesser importance at this early stage of regeneration. The fact that myoblasts and myotubes were first seen in the peripheral portions of grafts and that regeneration was always more advanced peripherally does, however, suggest that regeneration is initially dependent upon and enhanced by the diffusion of oxygen and metabolic substrates from surrounding host tissues and subsequently upon the establishment of vascular connections with these tissues. Our observations provide support for the view that the primitive myogenic cells found in the early stages of muscle fibre regeneration after injury arise within the fibre after the injury as a result of the sequestration of myonuclei and their immediate cytoplasm by newly-formed plasma membranes. The postulated sequence of events in myoblast formation is shown schematically in Fig. 12. Similar conclusions have been reached in electron-microscopic studies in the salamander (Hay 1970), the rabbit (Reznik 1970), the guinea pig (Hess and Rosner 1970) and the rat (Aloisi, Mussini and Schiaffino 1973). Walker (1972) has also concluded on the basis of autoradiographic studies in the rat that the nuclei of regenerate muscle fibres are derived principally from myonuclei within the original muscle fibre syncytium. Although typical subsarcolemmal satellite cells were found in the muscle from which grafts were taken in some animals in the present study, no definite conclusions could be reached as to the role of these cells in graft regeneration. No evidence was found to support the view that myoblasts are derived from perivascular cells (Freund-Molbert and Ketelsen 1973). The possibility has been raised that the myoblasts which appear in muscle grafts may be derived from cells of host rather than of graft origin (Bradley 1973; Mastaglia et al. 1973a; Salafsky 1973). While this possibility cannot be absolutely excluded on the present evidence, the finding of partial or complete inhibition of regeneration when grafts from pre-irradiated animals were transplanted to the subcutis suggests that the role of cells of host origin is at the most a minor one. The various ultrastructural changes found in muscle fibres after transplantation are non-specific and identical changes have been found in muscle explants (Mendell,
244
F.L. MASTAGL1A. R. L. I)AWKINS, J. M. PAPAI)IMI1"RIOU
,iii,,i!,i,iii! °o~O
iiij,ii!i!iiii ! ,,,? o
J
Fig. 12. Schematic representation of the postulated sequence of events leading to myoblast formation in degenerating muscle fibres. A = normal peripheral myonucleus, B = accumulation of ribonucleoprotein and new membrane formation around a myonucleus at the periphery of a degenerating fibre. C = inactive satellite cell D-F=premyoblasts prior to the appearance of myofilaments, G =early myoblast with thin actin filaments in the cytoplasm, H=more advanced myoblast with thick and thin f'flaments, primitive Z-bands and assembling myofibrils. The thick interrupted line represents the original basement lamina of the muscle fibre.
Roelofs and Engel 1972), after a variety of forms of focal muscle injury (Price, Howes and Blumberg 1964; Shafiq and Gorycki 1965; Reznik 1970) and in various forms of necrobiotic myopathy in man (Milhorat, Shafiq and Goldstone 1966; Mastaglia, Papadimitriou and Kakulas 1970; Mastaglia and Walton 1971) and in animals (Bray and Banker 1970; Oksanen and Poukka 1972; Mastaglia, Papadimitriou, Kakulas and Allbrook 1973b). The selective loss of Z-bands and I-band filaments in the early stages of necrosis has been noted previously in ischaemic muscle (Stenger~ Spiro, Scully and Shannon 1962; Karpati, Carpenter, Melmed and Eisen 1974) and in plasmocid intoxication (Price, Pease and Pearson 1962). The filamentous and rodshaped electron-dense structures seen in necrotic muscle fibres have been observed previously in vitamin E deficiency myopathy (Mastaglia et al. t973b). Their transverse periodicity is reminiscent of that seen in rod-bodies and they are presumably derived from Z-bands. The mitochondrial changes in necrotic muscle fibres are similar to those found by Reznik and Hansen (1969) after cold injury in the mouse, and by Karpati et al. (1974) in experimental ischaemic myopathy in the rat. The paracrystalline inclusions in mitochondria have also been found in a variety of other situations (see Chou 1969; Hudgson, Bradley and Jenkison 1972) and are probably formed as a
MORPHOLOGICALCHANGES IN SKELETALMUSCLE AFTER TRANSPLANTATION 245 result of crystallization of a protein c o m p o n e n t of the mitochondrion, possibly one of the enzymes normally located on the inner mitochondrial membrane.
ACKNOWLEDGEMENTS The authors are grateful to Professor B. A. Kakulas for his encouragement and support during this study and to Professor M. N. I. Walters for providing facilities. They are also indebted to Professor J. C, Sloper for his very constructive criticisms, Dr. T. Keenan provided surgical assistance in some of the early experiments. Dr. J. Holt, Director of the Institute of Radiotherapy, provided facilities for animal irradiation. Miss H. Dew, Mr. T. Robertson and Mr. M. Archer provided technical assistance. Mr. H. Upenieks prepared the photomicrographs and Mr. R. T. Tarrant the diagrams. Mrs. Kay Cross prepared the manuscript.
SUMMARY The morphological changes in subcutaneously implanted muscle homografts in mice were studied by light and electron microscopy 1-23 days after transplantation. The initial degenerative changes were identical in isografts and allografts and were essentially the same as those found in muscle explants in tissue culture and after various forms of muscle injury. Regenerative changes were prominent at the periphery of grafts by 48-72 hr before evidence of graft revascularization could be demonstrated by India ink perfusion. Active regeneration occurred in isografts and in allografts during the first week resulting in the formation of a new population of muscle fibres. Rejection subsequently occurred in allografts between days 7-14. Regeneration was retarded or completely inhibited by exposure o f the donor animal to 1,500 rad o f X-irradiation 1-2 hr prior to transplantation suggesting that regeneration is brought about by cells derived from the graft. Electron-microscopic observations in 48-72 hr grafts suggested that primitive mononucleated myogenic cells m a y form within degenerating muscle fibres by a process of myonuclear sequestration. Inactive satellite cells were present in the muscle from which grafts were taken in some donor animals but no definite conclusions could be reached as to the role of these cells in graft regeneration. REFERENCES ALLBROOK,D. B. ANDD. L. JAMES(1973) Electron microscopy of the regeneration of neonatal striated muscle segments within millipore chambers. In: B. A. KAKULAS(Ed,), Basic Reseach in Myoloyy (International Congress Series, No. 294), Excerpta Medica, Amsterdam. pp. 384-390. ALOlSI,M., I. MUSSINIANDS. SCHIAFFINO(1973) Activation of muscle nuclei in denervation and hypertrophy. In B. A. KAKULAS(Ed.), Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 338 342. BATALIN,N. J. ANDD. B. ALLBROOK(1973)Skeletalmuscleregeneration from fragmentedmuscleauto- and homografts in white mice. In: B. A. KAKULAS(Ed.), Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 39~404. BRADLEY,W. G. (1973) Discussion-Myogenesis. In: B. A. KAKULAS(Ed.), Basic Research hi Myoloqy (International Congress Series, No. 294), Excerpta Medica, Amsterdam, p. 451. BRAY,G. M. ANDB. W. BANKER(1970) An ultrastructural study of degeneration and necrosis of muscle
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in the dystrophic mouse, Acta neuropath. (Berl.), 15: 34~44. CARLSON, B. M. (1968) Regeneration of the completely excised gastrocnemius muscle in the frog and n~t from minced fragments, J. Morphol., 125: 447-472. CARLSON, B. M. (1970) Regeneration of the rat gastrocnemius muscle from sibling and non-sibling muscle fragments, Amer. J. Anat. 128:21 32. CARLSON, B. M. AND E. GUI'MANN (1972) Development of contractile properties orminced muscle regent. rates in the rat, Exp. Neurol., 36:239 249. Cnou, S. M. {1969)"'Megaconial" mitochondria observed in a case of chronic potymyositis, Acta new'opath. (Berl.), 12:68 89. CosMos, E. AND J. BUTLER (1970) Differentiation of muscle transplanted between normal and dystrophic chickens. In: B. Q. BANKERet al.. Research in Musch" Development and the Muscle Slfindle (International Congress Series, No. 240), Excerpta Medica, Amsterdam, pp. 149 162. ELSON, J. (1929) Auto- and homoio-transplantation of cross-striated muscle in the rat, Amer. J. PaUl.. 5: 425-438. FREUND-MOLBERT, E. AND U. P. KETELS~ (1973) The regeneration of the human striated muscle cell, Beitr. path. Anat., 148: 35-54. GALLUCCI, V., F. NOVELLO, A. MARGRETHAND M. At,otst (1966) Biochemical correlates of discontinuous muscle regeneration in the rat, Brit. J. exp. Path., 47:215 227. HAY, E. D. (1970) Regeneration of muscle in the amputated amphibian limb, In: A. MAURO. S. A. SHAtlU AND A. T. MILHORAT (Eds.), Regeneration of Striated Muscle and Myogenesis (International Congress Series, No. 218), Excerpta Medica, Amsterdam, pp. 3 2 4 . HESS, A. AND S. ROSNER (1970) The satellite cell bud and myoblast and denervated mammalian muscle fibres, Amer. J. Anat., 129: 21~,0. Hsu, L. (1974) The role of nerves in the regeneration of minced skeletal muscle in adult Anurans, Anat. Rec., 179: 119-136. HUDGSON, P., W. G. BRADLEYAND M. JENKISON(1972) Familial "mitochondrial" myopathy A myopathy associated with disordered oxidative metabolism in muscle fibres, J. neurol. Sci., 16: 343-370. KARPATL G., S. CARPENTER, C. MELMEO ANt) A. A. EISEN (1974) Experimental ischaemic myopathy. J. neurol. Sci., 23:129 161. LAIRD, J. L. AND R. F. TIMMER (1965) Homotransplantation of dystrophic and normal muscle. Arch. Path., 80: 442~46. LARSON, P. F., M. JENKISON AND P. HUDGSON (1970) The morphological development of chick embryo skeletal muscle grown in tissue culture as studied by electron microscopy, J. neurol. Sci., 10: 385405, MASTAt~UA, F. L. (1974) The development and growth of the skeletal muscles and connective tissues. In: J. A. DAvts AND J. DOBmNG (Eds.), Scientific Foundations O[ Paediatrics, William Heinemann, London, pp. 348 375. MASTAGUA, F. L. AND B. A. KAKULAS(1970) A histological and histochemical study of skeletal muscle in polymyositis, J. neurol. Sci., 10:471-487. MASTAGUA, F. L. AND J. N. WALTON (1971) An ul~rastructural study of skeletal muscle in polymyositis, J. neurol. Sci., 12: 473-504. MASTAGLIA, F. L., R. L. DAWKINS AND J. M. PAPADIMITRIOU(1973a) Morphological changes in isogeneic and allogeneic grafts of murine rectus abdominis muscle. In: B. A. KAKULAS(Ed.), Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 410-419. MASTAG11A. F. L., J. M. PAPADJMrrRIOt; AND R. L. DAWKlNS 0975) Mechanisms of cell-mediated myotoxicity Morphological observations in muscle grafts and in nmscle exposed to sensitized spleen cells in civo, J, neurol. Sci., 25: 269-282. MASTAGLIA, F. L., J. M. PAPADIMITRIOUAND B. A. KAKULAS(1970) Regeneration of muscle in Duchennc muscular dystrophy, J. neurol. Sei., 11 : 425 444. MASTAGLIA, F. L., J. M. PAPADIMITRIOU,B. A. KAKULASAND D. B. At, LBROOK (1973b) Skeletal muscle changes in the vitamin E deficient quokka An ultrastructural study. In: B. A. KAKULAS(Ed.), Bm'ic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 622~531. MENDELL, J. R., R. I. ROELOFSAND W. K. ENGEL (1972) Ultrastructural development of explanted hUman skeletal muscle in tissue culture, J. Neuropath. exp. Neurol., 3l : 433-446. MILHORAT, A. T., S. A. SHAHQ AND L. GOLDSTOYZ (1966) Changes in muscle structure in dystrophic patients, carriers and normal siblings seen by electron microscopy- Correlation with levels of serum creatine phosphokinase (CPK), Ann. N. Y. Acad. Sci., 138:246 292. OKSANEN, A. AND R. POUKKA(1972) An electron microscopical study of nutritional muscular degeneration (NMD) of myocardium and skeletal muscle in calves, Actapath. microbiol, stand. Section A, 80:440 448, PRXCE,H. M., E. L HOWLSAND J. M. BLUMBERG(1964)Ultrastructural alterations in skeletal muscle fibres
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injured by cold, Part 1 (The acute degenerative changes), Lab. Invest., 13: 1264-1278. PRICE, H. M., D. C. PEASEAND C. M. PEARSON(1962) Selective actin filament and Z-band degeneration induced by plasmocid--An electron microscopic study, Lab. Invest., 11 : 549-562. REZ~K, M. (1970) Satellite cells, myoblasts and skeletal muscle regeneration. In: A. MAURO,S. A. SnAFIQ AND A. T. MILHORAT(Eds.), Regeneration o f Striated Muscle and Myooenesis (International Congress Series, No. 218), Excerpta Medica, Amsterdam, pp. 133-156. REZNIK, M. AND J. L. HANSEN (1969) Mitochondria in degenerating and regenerating skeletal muscle, Arch. Path., 87: 601~08. SALAVSKY,B. (1971) Functional studies of regenerated muscles from normal and dystrophic mice, Nature (Lond.), 229: 270-272. SALAFSKY, B. (1973) Discussion-Myogenesis. In: B. A. KAKULAS(Ed.). Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 452 453 SHAFIQ,S. A. (1970) Satellite cells and fibre nuclei in muscle regeneration. In: A. MAURO,S. A. SHAFIQAND A. Z. MILHORAT(Eds.), Regeneration o f Striated Muscle and Myogenesis (International Congress Series. No. 218), Excerpta Medica, Amsterdam, pp. 122-132. SHAFIQ, S. A. AND M. A. GORYCKI (1965) Regeneration of skeletal muscle of mouse--Some electron microscope observations, J. Path. Bact., 90: 123-127. SNOW, M. H. (1973) Metabolic activity during the degenerative and early regenerative stages of minced skeletal muscle, Anat. Rec., 176:185 204. STENGER,R. J., D. SPIRO,R. E. SCULLYANDJ. M. SHANNON(1962) Ultrastructural and physiologic alterations in ischaemic skeletal muscle, Amer. J. Path., 40: 1-20. STUDITSKY, A. N. (1964) Free auto- and homografts of muscle tissue in experiments on animals, Ann. N. Y. Acad. Sci., 120/2: 789-801. TRUPIN, G. L. (1970) An ultrastructural study of the regeneration of the minced gastrocnemius muscle in the frog, Anat. Rec, 166:391 (Abstract). WALKER, I. E. (1972) Skeletal muscle regeneration in young rats, Amer. J. Anat., 133: 369-378.
227 Printed in The Netherlands
Morphological Changes in Skeletal Muscle after Transplantation A L i g h t - a n d E l e c t r o n - m i c r o s c o p i c S t u d y o f the Initial P h a s e s o f Degeneration and Regeneration* F. L. M A S T A G L I A , R. L. DAWKINS AND J. M. PAPADIMITRIOU Departments of Medicine and Pathology, University of Western Australia, Perth Medical Centre, Verdun Street, Shenton Park, Western Australia 6008 (Australia) (Received 26 November, 1974)
INTRODUCTION
It has been found in previous muscle transplantation studies that there is an initial phase both in autografts and in homografts during which the mature muscle fibres in the graft degenerate and that this phase is followed by one of active regeneration (Elson 1929 ; Studitsky 1964; Carlson 1968, 1970; Mastaglia, Dawkins and Papadimitriou 1973a). So effective is this regenerative phase that it may lead to reconstitution of an entire muscle when minced muscle fragments are replaced in the original muscle bed (Studitsky 1964; Carlson 1968, 1970; Salafsky 1971 ; Batalin and Allbrook 1973). The mechanisms of regeneration in muscle grafts and in particular the source of myogenic cells from which the new population of muscle fibres is derived have not been fully elucidated (Bradley 1973). Previous studies have been concerned with the question of survival of muscle grafts in normal and dystrophic hosts (Laird and Timmer 1965; Cosmos and Butler 1970), the biochemical and histochemical correlates of the degenerative and regenerative phases (Gallucci, Novello, Margreth and Aloisi 1966; Snow 1973), the reinnervation of grafts (Hsu 1974) and the contractile properties of the reconstituted graft in normal and dystrophic hosts (Salafsky 1971 ; Carlson and Gutmann 1972). The morphological changes in muscle grafts have been studied at the light microscope level (Elson 1929; Studitsky 1964; Laird and Timmer 1965; Carlson 1968, 1970; Snow 1973 ; Hsu 1974) but few observations have been made on the ultrastructural *Presented in part at the 3rd Intemational Congress of Muscle Diseases, Newcastle upon Tyne, September 1974. This study was supported by research grants from the University of Western Australia, the National Health and Medical Research Council of Australia and the Muscular Dystrophy Association of Western Australia.
BALBtC
AKR
P.H.
IRRAD. P.H.
P.H
1
2
3
4
5
26 21 23 21 5
BALB/C (32) ~
BALB/C (23)
BALB/C (21)
I R R A D . BALB/C (5)
rectus abdominis
BALB/C (30)
Recipient ( Nos. o f animals)
0
0
0
11
1[b
diaphraom
2
I*. 2", 3", 4, 5, 7, 8, 9, 10, 11, 13
I *, 2", 3", 4", 5, 7, 8. 9", 10, 11, 13, 15
2, 3*, 4", 6*, 7, 8, 9", 14, 18, 21
I, 2". 3, 4", 6*, 7, 8, 9*, 13, 14", 18, 21
Days to sacrifice
I
4
0
3
16 5
4
22
5
7
E.M.
32
L.M.
Number o f 9rafts examined
17
6
10
5
Number of grafts not recovered
0/1
5/8
8/11 1/1
6/8
5/5
0/22
Number o f yrafts with histological evidence o f r~jection" > 6 day.~
3tll
0/18
0/28
Number of 9raf ts with no r egeneration > 72 hr
Infiltration with mononuclear inflammatory cells, damage to regenerate muscle fibres, depletion of muscle fibres. b In 7 ammals a graft from the rectus abdominis was implanted on one side a n d one from the d i a p h r a g m on the other. Five animals in this group received an isogeneic graft on one side and an allogenei'c graft on the other and are also included in G r o u p 1. * The asterisks indicate animals in which grafts were examined with the electron microscope.
Donor
Grolqs
Source o f graft
D E T A I L S O F A N I M A L S U S E D IN T R A N S P L A N T A T I O N E X P E R I M E N T S A N D O F M U S C L E G R A F T S S T U D I E D
TABLE 1
,.q
~7
r-
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
229
changes in muscle after transplantation (Trupin 1970; Allbrook and James 1973). The present series of experiments was undertaken to study in greater detail the fine structural changes which occur in muscle fibres in the early stages after transplantation and in particular to throw further light upon the origin of myoblasts in the initial stages of graft regeneration. Observations on the revascularization of grafts are also presented. The morphological changes relating to the rejection of allografts are the subject of another report (Mastaglia, Papadimitriou and Dawkins 1975).
MATERIALS AND METHODS
One hundred and six 6-8 week inbred BALB/C mice were transplanted with isogeneic muscle from BALB/C littermates or with allogeneic muscle from AKR or Prince Henry (PH) mice of similar age. The numbers of animals in each group and other details are summarized in Table 1. Twenty-one animals received grafts from PH mice which had been exposed to 1500 rads of total body irradiation (Siemens Stabilapan, 300 kV, 12 mA, 1 mm Cu filter, dose rate 88 rad/min) 1-2 hr beforehand. A further experiment in which muscle from non-irradiated PH mice was transplanted into BALB/C mice which had received 1000 rads of total body irradiation 1 hr beforehand was unsuccessful as the irradiated animals all died within 72 hr. The donor animals were sacrificed immediately prior to the transplantation procedure. The rectus abdominis muscle and diaphragm were chosen for the preparation of grafts, both being thin flat muscles which could be easily divided into small fragments. These muscles were removed, placed in Petri dishes containing medium 199 and divided into fragments approximately 2 x 2 mm, using a sharp scalpel blade. The recipient animals were anesthetized with ether or intraperitoneal pentobarbitone and a small vertical mid-line incision was made in the skin of the anterior abdomen. In most animals a single fragment of the donor muscle was inserted between the skin and the fascia overlying the abdominal muscles to one side of the mid-line and the incision was closed with cotton sutures. In 7 animals a graft from the rectus abdominis was placed on one side and one from the diaphragm of the same animal on the other side. In 5 animals an isogeneic rectus abdominis graft was placed on one side and an allogeneic graft on the other. The recipient animals were sacrificed at intervals of 1 to 23 days (Table 1). In 20 animals 1 ml of India ink was injected into a tail vein immediately prior to sacrifice to study the revascularization of grafts. The graft was identified in 88 instances and was usually somewhat reduced in size, particularly in the case of allografts (both irradiated and non-irradiated) after the first week. The graft and the adherent host abdominal muscles were removed en bloc and pinned out on cardboard. The graft site was then divided into two approximately equal portions using a sharp scalpel blade. One portion was fixed in Heidenhain's Susa and embedded in paraffin. Several serial sections were taken from one or more levels of each block and were stained with haematoxylin and eosin. The remainder of the grafts were carefully separated from the host tissues, fixed in cold 3 }/o glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 hr, washed in buffer, post-fixed in cold 1 ~o osmium tetroxide in 0.1 M cacodylate buffer for 1 hr and then embedded in araldite after dehydration in
230
v . L . MASTAGLIA, R. L. DAWKINS, J. M. PAPADIM1TRIOIJ
graded solutions of ethanol. Fragments of rectus abdominis muscle from some donor animals were also prepared for electron microscopy in the same way. Thin sections from selected blocks (Table 1) were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Philips 201 (80 kV) or JEM T6 (60 kV) electron microscope.
OBSERVATIONS
Non-Irradiated Grafts Light microscopy The changes in isografts and non-irradiated allografts were essentially the same during the first week after transplantation. At 24 hr all the muscle fibres in the graft showed acute hyaline change and from 48 hr onwards there was progressive fragmentation and dissolution of necrotic myoplasm with the appearance of phagocytic cells particularly in muscle fibres in the peripheral portions of the graft (Fig. la). Fibres with some residual necrotic myoplasm were still present in the central portions of some grafts at day 10 (Fig. lb). Mononucleated cells with the characteristics of myoblasts (Mastaglia and Kakulas 1970) were present at the periphery of many necrotic muscle fibres by 48-72 hr and syncytial plaques were in evidence by 72-96 hr. Early myotubes were present in some grafts by day 4 and were numerous in most grafts by day 6-7 particularly at the periphery of the graft where regenerative changes were always more advanced (Fig. lb). In isografts, muscle fibres of increasing maturity were present throughout the graft during the second week and by the end of the third week many fibres with closely-packed myofibrils and peripheral or central vesicular nuclei were present within a connective tissue capsule of varying thickness (Fig. lc). The degree of regeneration in allografts was comparable to that in isografts up to day 7 but there was a progressive reduction in the number of muscle fibres as rejection supervened over the ensuing week (Fig. ld). Allografts examined after 10 days no longer contained muscle elements but were composed solely of connective tissue with a light infiltrate of mononuclear inflammatory cells. Evidence of revascularization from adjacent host tissues was found in all grafts showing regenerative changes after day 4. Many carbon-filled vascular channels were present throughout allografts undergoing rejection (see Mastaglia et al. 1975) and were less numerous in allografts after day 10. Electron microscopy The earliest changes seen in myofibrils during the first 48-96 hr consisted of fading or disappearance of Z-bands and loss of filaments from the I-bands (Fig. 2a). A-bands. H-zones and M-lines were often well-preserved in myofibrils with absent Z-bands and marked loss of filaments from the I-bands. At a later stage, individual fdaments inthe A-bands were no longer sharply defined and in many fibres a myofibriUar pattern was no longer recognizable, the contractile elements having been reduced to a mass of lightly-staining granular or fibrillar material (Fig. 2b). Many electron-dense filamentous or rod-shaped structures of presumed Z-band origin were scattered throughout
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 1. a: necrosis of muscle fibres in a 72 hr allograft; myophagia is more advanced in the peripheral portion of the graft (lower right). HE, x 160. b: 7-day isograft. A few residual necrotic muscle fibres are still present in the central portion of the graft while many myotubes are present at the periphery. HM=host abdominal muscle. HE, x 160. c: closely-packed regenerate muscle fibres in a 14-day isograft (IG). HM=host abdominal muscle. HE, x 160. d: a few residual myotubes in a 9-day allograft which is infiltrated with mononuclear inflammatory cells. HM=host abdominal muscle; N=degenerating nerve. HE, x 160. the n e c r o t i c m y o p l a s m a n d in fibres with a sufficiently p r e s e r v e d s a r c o m e r e p a t t e r n were seen to e x t e n d from one A - b a n d to the next, b r i d g i n g the space n o r m a l l y o c c u p i e d by t h e Z - a n d I - b a n d s (Fig. 2a). A transverse p e r i o d i c i t y was a p p a r e n t in some o f these s t r u c t u r e s (Fig. 2, inset). The m i t o c h o n d r i a in d e g e n e r a t i n g fibres were often p r e s e n t in large aggregates, p a r t i c u l a r l y at the p e r i p h e r y o f the fibre, a n d s h o w e d a variety o f c h a n g e s (Fig. 3). M a n y were e n l a r g e d a n d r o u n d e d with thickening o f the i n n e r a n d o u t e r m e m b r a n e s while some were u n i f o r m l y d e n s e l y - s t a i n e d with no r e c o g n i z a b l e cristae m i t o c h o n d r i a l e s (Fig. 2a). M a n y m i t o c h o n d r i a con-
232
F . L . M A S I A G L I A . R. L. I ) A W K I N S , J. M. PAI~AI)IMITRIOI.
Fig. 2. a : four-day allograft. Selective loss of Z - a n d I - b a n d s with relative p r e s e r v a t i o n o f A - b a n d s (A J m a d e g e n e r a t i n g muscle fibre. The ele~lron-dense s t r u c t u r e s b r i d g i n g the gap between the A - b a n d s in some myofibrils (arrows) are t h o u g h t t .... c Z - b a n d remnants, x 38,250, bar 0.5/~m. Inset: higher magnification s h o w i n g a transverse periodicity m one of these structures, x 104,00(/. b: necrotic muscle fibre in a 4-day allograft. M y o f i b r i l l a r outlines are lost a n d m i t o c h o n d r i a arc s h r u n k e n and electron-dense, x 11,900, b a r ] tim.
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE At:TER TRA/~SPLANTATION
233
Fig. 3. A subsarcolemmal mitochondrial aggregate in a degenerating muscle fibre in a 48-hr allograft. Many of the mitochondria contain amorphous electron-dense deposits and show thickening or breakdown of membranes; some contain para-crystalline inclusions which are probably situated between cristal membranes (arrow and inset), x 39,000; inset x 78,000, bar 0.5 ,urn.
234
F. L. MASTAGLIA, R. L. DAWKINS~ J. M. PAPA1)IMITRIOI
Fig. 4. Necrotic muscle fibre (N) in a 5-day aUograft. The nuclear and cytoplasmic characteristics of the mononucleated cells (Mc) situated internal to the basement lamina (arrowheads) of the fibre suggest that they are phagocytic in nature. A similar cell is present at the periphery of another necrotic fibre in the top left comer, x 7,800, bar 2 #m.
tained electron-dense para-crystalline inclusions between the cristal membranes (Fig. 3). Most mitochondria contained variable amounts of lightly-staining amorphous material and in many, localized deposits of densely-staining material were also present (Fig. 3). Other changes found in degenerating fibres included dilatation of the sarcoplasmic reticulum, occasional membrane-bound dense bodies of presumed lysosomal nature, and clusters of small vesicles. Some sarcolemmal nuclei
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
235
were densely-stained and shrunken with separation of the nuclear membrane. The plasma membrane of muscle fibres could no longer be identified by 48-72 hr but the basement lamina, although separated from the underlying necrotic myoplasm and at times folded and redundant, remained intact even after 96 hr (Figs. 4-6). Mononucleated ceils of various types were present internal to the basement lamina of necrotic muscle fibres by 48-72 hr. Many of these were clearly macrophages. These cells were irregular in outline with pleomorphic processes and their cytoplasm contained lysosomes, granular endoplasmic reticulum, lipid droplets and, in some instances, membranous whorls (Fig. 4). Some cells contained recognizable ingested material from the muscle fibre. Moderate numbers of such cells were also present external to the basement lamina of muscle fibres in the interstitium. Occasional subsarcolemmal cells had the ultrastructural features of lymphocytes and others of granulocytes. Other thin elongated sub-sarcolemmal cells with a relatively smooth contour, oval or elongated nuclei with prominent nucleoli, many free and membrane-bound ribosomes and few mitochondria were considered to be premyoblasts (Mastaglia 1974) (Fig. 5). Some cells of this type were in mitosis (Fig. 5b). Cells with similar characteristics and with thin actin-like filaments (50-60 A diameter) with or without thicker myosin-like filaments (100-120 A diameter) and microtubules in the cytoplasm were identified as myoblasts (Mastaglia 1974}. Myogenic cells could usually be distinguished from other mononuclear ceils at the periphery of the sarcolemmal tube by the presence of filaments in the cytoplasm or, when filaments were not present, on the basis of a relatively smooth cell contour (Fig. 5) and the characteristics of the nuclei (Fig. 6) which were generally larger with fewer chromatin clumps and larger nucleoli than those of monocytes and macrophages. The presence within the cytoplasm of membrane-bound areas of phagocytosed material was not a useful distinguishing criterion as such areas were also present in some myoblasts as has been noted in previous studies (Larson, Jenkison and Hudgson 1970; Trupin 1970; Mastaglia and Walton 1971). Certain observations relevant to the origin of myoblasts were made. The number of satellite cells found in the rectus abdominis muscle prior to transplantation varied from one animal to another. While in some animals no satellite cells were found (presumably due to the limitations of sampling), in others up to 8 ~ of muscle fibre nuclei belonged to these sub-sarcolemmal cells. Cells with the cytological features of resting satellite cells were not seen in 24- or 48-hr grafts and no definite conclusions could be reached as to the fate of these cells after transplantation. In 48-hr grafts some elongated mononucleated sub-sarcolemmal cells rich in ribonucleoprotein (Fig. 5c) which were considered to be premyoblasts resembled the so-called activated satellite cells referred to in some previous studies (Shafiq 1970). Whether or not such cells were present in the muscle prior to transplantation cannot, however, be established. Other observations in 48-hr grafts suggested that at least some myoblast precursors were formed by a process of sequestration of sarcolemmal nuclei and paranuclear cytoplasm at the periphery of degenerating muscle fibres. As illustrated in Fig. 7, the precursors of the new cell membranes take the form of multiple small vesicles and cisternae. In some areas interconnecting membrane-lined clefts are seen in the paranuclear region which is rich in ribosomes and granular endoplasmic reticulum
~N
F-
~j
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
237
Fig. 6. Portion of a crescentic mononucleated cell situated internal to the basement lamina (open arrow) of a necrotic muscle fibre in a 3-day allograft. The nuclear chromatin is dispersed and the nueleolus (N) is large. The nuclear characteristics in particular suggest that the cell is myoblastic in nature. Thin filaments are present in some parts of the cell but are not seen at this magnification. E R = wide vesicles of the endoplasmic reticulum some of which appear to be formed by extensions of the nuclear membrane (solid arrow). × 9,000, bar 2 #m.
and contains mitochondria of normal appearance which contrast with the degenerate organelles in the necrotic myoplasm nearby. As shown in Fig. 7, this process of nuclear sequestration may occur at a relatively early stage of necrosis before the necrotic myoplasm in the region is invaded by phagocytic cells. In Fig. 8 the degree of breakdown of the muscle fibre is more advanced and the cell presumed to be a premyoblast has a complete limiting plasma membrane. However, apparently newlyformed flat membranous cisternae are present in the nearby necrotic myoplasm. Larger mononucleated myoblasts and syncytia (Fig. 9), whose cytoplasm contained thick and thin filaments, primitive Z-bodies and assembling myofibrils were present at the periphery of sarcolemmal tubes at 3 ~ days. Many early myotubes were also present at these times both in isografts and in allografts. These fibres contained well-aligned loosely-packed myofibrils and a variable number of large nuclei with dispersed chromatin and prominent nucleoli in the central parts of the fibre. The
Fig. 5. a: undifferentiated mononucleated cell with the characteristics of a premyoblast closely applied to the inner surface of the residual basement lamina {arrowhead) of a necrotic muscle fibre in a 3-day allograft. Note the high nuclear cytoplasmic ratio, prominent nucleoli and ribosomal aggregates in the cytoplasm. Collagen fibrils (c) are present external to the basement lamina, x 12,600, bar 1 gm. b : mitotic premyoblast internal to the basement lamina of a degenerating muscle fibre (DM) in a 3-day allograft. A centriole is seen in the premyoblast (arrow). The adjacent fibre (N) shows more advanced dissolution. Collagen fibrils separate the basement laminae of the two fibres, x 9,400, bar 1 #m. c: elongated premyoblast internal to the basement lamina of a degenerating muscle fibre {DM) in a 3-day allograft. Note the smooth contour of the cell, the prominent nucleolus and the presence of numerous free and membrane-bound ribosomes in the cytoplasm. N = an adjoining fibre whose necrotic myoplasm is separated from the basement lamina. Collagen fibrils separate the basement laminae of the two fibres. × 10,000, bar 1/~m.
Fig. 7. a: degenerating muscle fibre (MF) in a 48-hr allograft. The myonucleus (N) and surrounding cytoplasm which contains numerous ribosomes and many strands of granular endoplasmic reticulum are partially separated from the degenerating m y o p l a s m by a series of membrane-lined clefts (enclosed area lower left), vesicles (arrow) and m e m b r a n o u s folds (enclosed area upper right). Arrowheads = basement lamina, x 22,400, bar 1 #m. b: photographic enlargement of lower enclosed area in (a). x 35,400. c: photographic enlargement of upper enclosed area in (a). x 35,400.
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 8. a : a thin mononucleated cell (Mc) rich in ribosomes is present at the periphery of a necrotic muscle fibre (Mf} in a 48-hr allograft. A series of interrupted flat membranous cisternae is present in the necrotic myoplasm nearby (arrows). Arrowheads =basement lamina and endomysial collagen fibrils. × 15,000, bar 1/~m. b: higher magnification o f the area indicated by arrows in (a). x 39,700, bar 0.5 #m.
paranuclear cytoplasm contained numerous polyribosomes and prominent granular endoplasmic reticulum and Golgi apparatus (Fig. 10a). Many muscle fibres with closely-packed myofibrils were present in 9-14 day isografts. Some were at the late myotube stage of development with internal nuclei while others had peripherally-
240
F. L. MASTAGLIA. R. L. I)AWKINS. J. M. t'APAI)IMITRI()/'
Fig. 9. Multinucleated muscle cell internal to the basement lamina (arrowheads) of a necrotic muscle fibrc (N) in a 4-day allograft. The nuclei show a dispersed chromatin pattern and nucleoli are not present m tile plane of section. The cytoplasm contains many ribosomal particles and a few mitochondria. Myofilaments cannot be identified at this magnification. The dissociated basement lamina of an adjacent necrotic fibre is also seen {arrow). x 7,000, bar 2 ~tm.
placed nuclei. Numerous polyribosomes and strands of granular endoplasmic reticulum were present in the paranuclear region as well as at the periphery of such fibres (Fig. 10b). Other structures included longitudinally-orientated microtubules in the subsarcolemmal region (Fig. 10b), prominent paranuclear Golgi apparatus, variable numbers of mitochondria, developing triads and budding sarcoplasmic reticulum. Myofibrils in the more mature fibres varied considerably in diameter, were often split longitudinally, and showed poor transverse sarcomere alignment. Undifferentiated mononucleated cells were present internal to the basement lamina of some fibres (Fig. l(k i Newly-formed capillaries with plump endothelial cells and a narrow lumen, and small venules were first seen at the periphery of grafts at day 4 and were subsequently found throughout the graft. The continuity of these vessels with the vascular tree of the host was demonstrated by the presence of carbon particles within the lumen in grafts removed from animals injected with India ink (Fig. 11 ). Regenerating muscle fibres were often closely applied to capillaries and venules.
Irradiated Grafts While no significant differences in the initial degenerative changes and phagocytosis of necrotic debris were noted between irradiated and non-irradiated grafts, differences were apparent in the degree and time course of regeneration. Regenerative changes were found in only 5 0 ~ of irradiated grafts (Table 1). Small numbers of premyoblasts and myoblasts were present at the periphery of necrotic fibres in some grafts at 48-72 hr but were less numerous than in non-irradiated grafts. Myotubes
MORPHOLOGICAL CHANGES IN SKELETAL MUSCLE AFTER TRANSPLANTATION
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Fig. 10. a: central portion of a, myotube in a 9-day isograft showing copious polyribosomes and prominent granular endoplasmic reticulum (ER) in the paranuclear region. The nucleus (N) shows a prominent nucleolus and dispersed chromatin. G = Golgi apparatus. The myofibrils on either side of the nucleus are longitudinally aligned and sectioned somewhat tangentially. × 16,800, bar 1 /Jm. b : peripheral portion of a regenerate muscle fibre in a 9-day isograft showing polyribosomes, granular endoplasmic reticulum (ERI and longitudinally-orientatedmicrotubules (arrows). M = myofibril showing Z-band streaming. × 30,500, bar 0.5/~m. c: undifferentiated mononucleated cell (M) at the periphery of a regenerate muscle fibre in a 7-day allograft. The basement lamina of the muscle fibre is indicated by the arrowheads, z 11,200~ bar 1/~m.
were p r e s e n t in s m a l l n u m b e r s in two 3-day a n d in o n e 5-day graft b u t were a b u n d a n t in o n e 9 - d a y graft. M y o t u b e s in i r r a d i a t e d g r a f t s t e n d e d to be s m a l l e r a n d to h a v e fewer n u c l e i t h a n t h o s e in n o n - i r r a d i a t e d grafts. Six o u t o f 8 g r a f t s e x a m i n e d after 6 days were v a s c u l a r i z e d a n d 5 o u t o f 8 were i n f i l t r a t e d b y m o n o n u c l e a r i n f l a m m a t o r y
242
v. 1,. MASTAGLIA, R. 1,. I ) A W K I N S , J. M. PAI'AI)IM11"1~.1{)1
Fig. 11, a: c a r b o n particles in the lumen of a capillary m a 9-day allograft. × 8,300, bar 1 Iml. b: small venule in a 7-day i r r a d i a t e d allograft. × 8,000, bar 1 lzm.
cells. As in the case of non-irradiated allografls, each of the 4 irradiated grafts examined after 10 days were composed only of connective tissue with a few fat spaces and variable numbers of mononuclear cells.
MORPHOLOGICAL CHANGES 1N SKELETAL MUSCLE AFTER TRANSPLANTATION
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DISCUSSION
The present findings emphasize the inherent capacity of skeletal muscle to regenerate even when deprived of nerve and blood supply and transplanted to a foreign site in an immunologically unrelated host. Regeneration has been found to be as active in the present heterotopically implanted homografts as in previous experiments in which autografts were re-implanted into the original muscle bed (e.g. Carlson 1968, 1970). Moreover, regeneration is initially as active in allografts as in isografts and rejection only occurs when the reconstitutive phase is well under way, a point which is of relevance to the question of therapeutic muscle transplantation. The finding of active regeneration in grafts by 48-72 hr at a stage before evidence of vascularization could be demonstrated suggests that the initial stages of regeneration may proceed even under relatively anaerobic conditions. Histochemical observations indicating that certain glycolytic enzyme systems are active in myoblasts in regenerating autografts (Snow 1973) support this conclusion. The paucity of mitochondria found in premyoblasts and myoblasts on electron microscopy in the present and in other studies (Reznik and Hansen 1969) would suggest that aerobic metabolic pathways are of lesser importance at this early stage of regeneration. The fact that myoblasts and myotubes were first seen in the peripheral portions of grafts and that regeneration was always more advanced peripherally does, however, suggest that regeneration is initially dependent upon and enhanced by the diffusion of oxygen and metabolic substrates from surrounding host tissues and subsequently upon the establishment of vascular connections with these tissues. Our observations provide support for the view that the primitive myogenic cells found in the early stages of muscle fibre regeneration after injury arise within the fibre after the injury as a result of the sequestration of myonuclei and their immediate cytoplasm by newly-formed plasma membranes. The postulated sequence of events in myoblast formation is shown schematically in Fig. 12. Similar conclusions have been reached in electron-microscopic studies in the salamander (Hay 1970), the rabbit (Reznik 1970), the guinea pig (Hess and Rosner 1970) and the rat (Aloisi, Mussini and Schiaffino 1973). Walker (1972) has also concluded on the basis of autoradiographic studies in the rat that the nuclei of regenerate muscle fibres are derived principally from myonuclei within the original muscle fibre syncytium. Although typical subsarcolemmal satellite cells were found in the muscle from which grafts were taken in some animals in the present study, no definite conclusions could be reached as to the role of these cells in graft regeneration. No evidence was found to support the view that myoblasts are derived from perivascular cells (Freund-Molbert and Ketelsen 1973). The possibility has been raised that the myoblasts which appear in muscle grafts may be derived from cells of host rather than of graft origin (Bradley 1973; Mastaglia et al. 1973a; Salafsky 1973). While this possibility cannot be absolutely excluded on the present evidence, the finding of partial or complete inhibition of regeneration when grafts from pre-irradiated animals were transplanted to the subcutis suggests that the role of cells of host origin is at the most a minor one. The various ultrastructural changes found in muscle fibres after transplantation are non-specific and identical changes have been found in muscle explants (Mendell,
244
F.L. MASTAGL1A. R. L. I)AWKINS, J. M. PAPAI)IMI1"RIOU
,iii,,i!,i,iii! °o~O
iiij,ii!i!iiii ! ,,,? o
J
Fig. 12. Schematic representation of the postulated sequence of events leading to myoblast formation in degenerating muscle fibres. A = normal peripheral myonucleus, B = accumulation of ribonucleoprotein and new membrane formation around a myonucleus at the periphery of a degenerating fibre. C = inactive satellite cell D-F=premyoblasts prior to the appearance of myofilaments, G =early myoblast with thin actin filaments in the cytoplasm, H=more advanced myoblast with thick and thin f'flaments, primitive Z-bands and assembling myofibrils. The thick interrupted line represents the original basement lamina of the muscle fibre.
Roelofs and Engel 1972), after a variety of forms of focal muscle injury (Price, Howes and Blumberg 1964; Shafiq and Gorycki 1965; Reznik 1970) and in various forms of necrobiotic myopathy in man (Milhorat, Shafiq and Goldstone 1966; Mastaglia, Papadimitriou and Kakulas 1970; Mastaglia and Walton 1971) and in animals (Bray and Banker 1970; Oksanen and Poukka 1972; Mastaglia, Papadimitriou, Kakulas and Allbrook 1973b). The selective loss of Z-bands and I-band filaments in the early stages of necrosis has been noted previously in ischaemic muscle (Stenger~ Spiro, Scully and Shannon 1962; Karpati, Carpenter, Melmed and Eisen 1974) and in plasmocid intoxication (Price, Pease and Pearson 1962). The filamentous and rodshaped electron-dense structures seen in necrotic muscle fibres have been observed previously in vitamin E deficiency myopathy (Mastaglia et al. t973b). Their transverse periodicity is reminiscent of that seen in rod-bodies and they are presumably derived from Z-bands. The mitochondrial changes in necrotic muscle fibres are similar to those found by Reznik and Hansen (1969) after cold injury in the mouse, and by Karpati et al. (1974) in experimental ischaemic myopathy in the rat. The paracrystalline inclusions in mitochondria have also been found in a variety of other situations (see Chou 1969; Hudgson, Bradley and Jenkison 1972) and are probably formed as a
MORPHOLOGICALCHANGES IN SKELETALMUSCLE AFTER TRANSPLANTATION 245 result of crystallization of a protein c o m p o n e n t of the mitochondrion, possibly one of the enzymes normally located on the inner mitochondrial membrane.
ACKNOWLEDGEMENTS The authors are grateful to Professor B. A. Kakulas for his encouragement and support during this study and to Professor M. N. I. Walters for providing facilities. They are also indebted to Professor J. C, Sloper for his very constructive criticisms, Dr. T. Keenan provided surgical assistance in some of the early experiments. Dr. J. Holt, Director of the Institute of Radiotherapy, provided facilities for animal irradiation. Miss H. Dew, Mr. T. Robertson and Mr. M. Archer provided technical assistance. Mr. H. Upenieks prepared the photomicrographs and Mr. R. T. Tarrant the diagrams. Mrs. Kay Cross prepared the manuscript.
SUMMARY The morphological changes in subcutaneously implanted muscle homografts in mice were studied by light and electron microscopy 1-23 days after transplantation. The initial degenerative changes were identical in isografts and allografts and were essentially the same as those found in muscle explants in tissue culture and after various forms of muscle injury. Regenerative changes were prominent at the periphery of grafts by 48-72 hr before evidence of graft revascularization could be demonstrated by India ink perfusion. Active regeneration occurred in isografts and in allografts during the first week resulting in the formation of a new population of muscle fibres. Rejection subsequently occurred in allografts between days 7-14. Regeneration was retarded or completely inhibited by exposure o f the donor animal to 1,500 rad o f X-irradiation 1-2 hr prior to transplantation suggesting that regeneration is brought about by cells derived from the graft. Electron-microscopic observations in 48-72 hr grafts suggested that primitive mononucleated myogenic cells m a y form within degenerating muscle fibres by a process of myonuclear sequestration. Inactive satellite cells were present in the muscle from which grafts were taken in some donor animals but no definite conclusions could be reached as to the role of these cells in graft regeneration. REFERENCES ALLBROOK,D. B. ANDD. L. JAMES(1973) Electron microscopy of the regeneration of neonatal striated muscle segments within millipore chambers. In: B. A. KAKULAS(Ed,), Basic Reseach in Myoloyy (International Congress Series, No. 294), Excerpta Medica, Amsterdam. pp. 384-390. ALOlSI,M., I. MUSSINIANDS. SCHIAFFINO(1973) Activation of muscle nuclei in denervation and hypertrophy. In B. A. KAKULAS(Ed.), Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 338 342. BATALIN,N. J. ANDD. B. ALLBROOK(1973)Skeletalmuscleregeneration from fragmentedmuscleauto- and homografts in white mice. In: B. A. KAKULAS(Ed.), Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 39~404. BRADLEY,W. G. (1973) Discussion-Myogenesis. In: B. A. KAKULAS(Ed.), Basic Research hi Myoloqy (International Congress Series, No. 294), Excerpta Medica, Amsterdam, p. 451. BRAY,G. M. ANDB. W. BANKER(1970) An ultrastructural study of degeneration and necrosis of muscle
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injured by cold, Part 1 (The acute degenerative changes), Lab. Invest., 13: 1264-1278. PRICE, H. M., D. C. PEASEAND C. M. PEARSON(1962) Selective actin filament and Z-band degeneration induced by plasmocid--An electron microscopic study, Lab. Invest., 11 : 549-562. REZ~K, M. (1970) Satellite cells, myoblasts and skeletal muscle regeneration. In: A. MAURO,S. A. SnAFIQ AND A. T. MILHORAT(Eds.), Regeneration o f Striated Muscle and Myooenesis (International Congress Series, No. 218), Excerpta Medica, Amsterdam, pp. 133-156. REZNIK, M. AND J. L. HANSEN (1969) Mitochondria in degenerating and regenerating skeletal muscle, Arch. Path., 87: 601~08. SALAVSKY,B. (1971) Functional studies of regenerated muscles from normal and dystrophic mice, Nature (Lond.), 229: 270-272. SALAFSKY, B. (1973) Discussion-Myogenesis. In: B. A. KAKULAS(Ed.). Basic Research in Myology (International Congress Series, No. 294), Excerpta Medica, Amsterdam, pp. 452 453 SHAFIQ,S. A. (1970) Satellite cells and fibre nuclei in muscle regeneration. In: A. MAURO,S. A. SHAFIQAND A. Z. MILHORAT(Eds.), Regeneration o f Striated Muscle and Myogenesis (International Congress Series. No. 218), Excerpta Medica, Amsterdam, pp. 122-132. SHAFIQ, S. A. AND M. A. GORYCKI (1965) Regeneration of skeletal muscle of mouse--Some electron microscope observations, J. Path. Bact., 90: 123-127. SNOW, M. H. (1973) Metabolic activity during the degenerative and early regenerative stages of minced skeletal muscle, Anat. Rec., 176:185 204. STENGER,R. J., D. SPIRO,R. E. SCULLYANDJ. M. SHANNON(1962) Ultrastructural and physiologic alterations in ischaemic skeletal muscle, Amer. J. Path., 40: 1-20. STUDITSKY, A. N. (1964) Free auto- and homografts of muscle tissue in experiments on animals, Ann. N. Y. Acad. Sci., 120/2: 789-801. TRUPIN, G. L. (1970) An ultrastructural study of the regeneration of the minced gastrocnemius muscle in the frog, Anat. Rec, 166:391 (Abstract). WALKER, I. E. (1972) Skeletal muscle regeneration in young rats, Amer. J. Anat., 133: 369-378.