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Inherited muscular disorder in mutant Japanese quail (Coturnix coturnix japonica): Relationship between the development of muscle lesions and age
j . Comp. Path. 1995 Vol. 113, 131-143
Inherited Muscular Disorder in Mutant Japanese Quail (Coturnix coturnix japonica): Relationship Between the D e v e l o p m e n t of Muscle Lesions and Age I. S. Braga III, S. Tanaka, T. Kimura, C. Itakura and M. Mizutani* Department of Comparative Pathology, Faculty of VeterinaryMedicine, Hokkaido University, Sapporo 060 and *Laboratory of Animal Research Station, Nippon Institutefor Biological Science, Kobuchizawa, Yamanishi 409-16, Japan
Summary The progression of the pathological changes that occur in the skeletal muscle was examined in 19 Japanese quail of the LWC strain, affected with an autosomal dominant inherited muscular disorder producing electrical myotonia. The muscle samples were obtained every 10 days from 20 to 70 days of age. Muscle samples from 18 age-matched commercial quail were used as normal controls. Characteristic histological lesions found in the skeletal muscles included sarcoplasmic masses, ringed fibres, internal migration of nuclei and fibre size variation. These lesions, which mainly occurred in the proximal muscles, appeared first in the pectoral region and later in the muscles of the thoracic and pelvic limbs. The most predominant lesion observed at all ages consisted of sarcoplasmic masses. The presence of histological changes did not affect muscle fibre typing by two staining methods, for myosin ATPase at pH 4"5, and by NADH-TR stain. The histological changes were observed in type 2A and less commonly in 2B fibres, but not in type 1. The pectoralis thoracicus muscle, in which lesions were particularly common, showed abnormally large type 2B muscle fibres at 20 days of age. These fibres began to decrease in size at 30 days of age, and at 70 days had become strikingly atrophic, their diameter being only about half that observed at 20 days. The atrophic type 2B muscle fibres were eventually replaced by lipocytes. Chronological staging of the histopathological changes in muscle was impossible since no inter-relationship was observed between the age of the quail, the severity of clinical signs and the extent of muscle lesions. This variability in the severity and age of onset may have been due to the variable expression or incomplete penetrance of the defective gene. Because the disorder is hereditary and progressive in nature, it can be classified as a type of progressive muscular dystrophy. 9 1995 Academic Press Limited
Introduction A new strain of m u t a n t J a p a n e s e quail (Coturnix coturnixjaponica), L W C , with an inherited muscular disorder p r o d u c i n g electrical myotonia, was developed by the L a b o r a t o r y of A n i m a l Research Station of the N i p p o n Institute for 0021-9975/95/060131 + 13 $12.00/0
9 1995 Academic Press Limited
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Biological Science. The disorder is inherited as an autosomal dominant trait and is homolethal. Since the pattern of muscular involvement is primarily proximal in distribution, the clinical manifestations of the disorder in affected quail are characterized by loss of wing lift ability and an impaired righting reflex. Histological examination of the skeletal muscles of the affected birds shows sarcoplasmic masses, ringed fibres, central nucleation, and nuclear rows with occasional solitary necrotic fibres and, in the advanced stages, fatty infiltration (Braga et al., 1995). In addition to the mode of inheritance, the presence of electrical myotonia and characteristic histopathological changes, and the presence of bilateral cataracts and testicular atrophy in some affected quail suggest a multi-systemic disorder not unlike myotonic dystrophy of man. O u r previous report (Braga et al., 1995) strongly suggested progressive degenerative changes in the muscle, but it is not known whether these changes are due to defective maturation and retention of embryonic features posthatching or develop later as the bird ages. The present study describes the histopathological changes found in the skeletal muscle of L W C quail from 20 days of age to sexual maturity at approximately 70 days. Materials and M e t h o d s
Birds Nineteen mutant (LWC)Japanese quail and 18 normal age-matched control birds from a commercial quail farm were examined. Two to five birds from each group were killed every 10 days from day 20 to 70 (Table 1) by an overdose ofpentobarbital sodium (Somnopentyl| Pitman-Moore, USA) administered intraperitoneally. Clinical Observations Quail are normally capable of raising their wings vertically, through 180 ~ from the rest position. Mutant LWC quail manifesting the inherited muscular disorder are, however, incapable of lifting their wings vertically (wing lift), and are unable to right themselves when placed on their dorsum (flip test) (Braga et al., 1995). As will be seen later (Table 1), LWC quail with wing lifts of approximately 150~ usually have a normal flip test, whereas a 90 ~ wing lift is associated with a negative flip test. Responses to the wing lift and flip tests were checked on all birds examined (Table 1). Light Microscopy and Histochemistry Where and when possible, 18 different skeletal muscle groups were collected from each bird for histopathological examination. Samples from the right side were fixed in 10% neutral buffered formalin, dehydrated through graded alcohols, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (HE). Selected sections were stained with periodic acid-Schiff (PAS) and phosphotungstic acid haematoxylin (PTAH). The nomenclature for the muscles follows that of Vanden Berge (1979). The muscles examined included: those of the pectoral girdle--Musculus pectoralis thoracicus (cranial and medial belly), M. supracoracoideus, M. subcoracoideus, M. latissimus dorsi cranialis, M. latissimus dorsi caudalis; the thoracic limb muscles--M, deltoideus major, M. triceps brachii (pars humeralis), M. triceps brachii (pars scapularis), M. biceps brachii (pars scapularis), M. tensor propatagialis; and the pelvic limb muscles--M, iliotibialis cranialis, M. iliotibialis lateralis, M. femorotibialis medius, M. iliofibularis, M. gastrocnemius (pars externa), M. fibularis longus, M. tibialis cranialis, and M. extensor digitorum longus. Samples of muscle were quickly removed from the left side, mounted on slices of
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I n h e r i t e d M u s c u l a r D i s o r d e r in M u t a n t Q u a i l Table 1 Age, sex, body weight and fllp test and wing lift responses in quail examined
Case no.
Quail
Age (days)
Sex
Body weight (g)
Clinical signs 14/~nglift
Flip test
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
LWC o " ~ ~ ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
2O 20 2O 30 30 30 30 30 40 40 40 50 50 50 60 60 60 70 70
F M F F F M M F M F F F M F F M F M F
57 63 56 100 105 88 73 86 101 123 141 134 116 139 162 115 158 104 137
+ + + + + + + + + 120 ~ + 150 ~ + 90 ~ 90 ~ + 150 ~ 90 ~ 90 ~
+ + + + + + + + + + + + --+ + --
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Control ~ " ~ " ~ ~ ~ ~ ~ ~ ~ ~ " ~ ~ ~ "
20 20 3O 30 30 4O 40 40 50 50 50 60 60 60 70 70 70 70
M F F M F M F M M M F M M F F M M M
46 48 71 81 75 98 89 93 98 95 107 94 106 92 121 104 103 98
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
M, male; F, female; + , positive; --, negative. cork; fixed in place by a small amount of gum tragacanth, and frozen rapidly by immersing the mounted specimen in isopentane pre-cooled in liquid nitrogen to -160~ (DeGirolami and Smith, 1982). The frozen blocks were stored at -80~ u n t i l t h e y w e r e s e c t i o n e d . S e r i a l t r a n s v e r s e s e c t i o n s ( 6 - 1 0 ~tm) w e r e c u t w i t h a cryotome at --25 to --30~ and then mounted on glass slides coated with neoprene 0"02% in toluene. Sectioned specimens were air-dried and stored in the freezer at - 30~ until they were stained. The sections were stained for myofibrillar adenosine triphosphatase (ATPase) with the metachromatic d y e , t o l u i d i n e b l u e ( D o r i g u z z i et al., 1 9 8 3 ) , a f t e r p r e i n c u b a t i o n a t p H 4"5, f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e , r e d u c e d f o r m - t e t r a z o l i u m reductase (NADH-TR) as described by Dubowitz (1985), and for glycogen by the PAS technique (Dubowitz, 1985). Sections were also routinely stained with modified Gomori trichrome (Engel and Cunningham, 1 9 6 3 ) a n d H E ( D u b o w i t z , 1985).
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Muscle Fibre Morphomet~y Photomicrographs of the superficial and deep parts of the M. pectoralisthoracicusstained for ATPase were taken from 29 (13 control and 16 LWC) quail. The muscle fibres were classified into three major fibre types (types 1, 2A and 2B) and the minimum fibre diameters of at least 100 fibres per quail were measured. Results
Clinical Evaluation Control quail. All the control birds examined had normal (180 ~) wing lifts and positive flip tests (Table 1). LWC quail T h e quail started to show negative (< 180 ~ wing lifts, and negative flip tests, which are diagnostic (Braga et al., 1995), at 40 days of age (Table 1). Quail killed for necropsy when 20 and 30 days old were chosen at r a n d o m because identification of affected birds was not possible at this age. No macroscopical wasting of muscle was noted in any of the birds examined. Microscopical Findings Control quail. Differentiation of the skeletal muscle into the three major fibre types (types 1, 2A and 2B) was observed at 20 days of age with both the ATPase and the N A D H - T R staining methods. Apart from an occasional isolated solitary necrotic fibre, no lesions were observed in the muscles of these birds. LWC quail. As in the control birds, muscle fibre differentiation into the three major fibre types was observed at 20 days of age. Characteristic histopathological changes such as ringed fibres, sarcoplasmic masses, internal migration of nuclei, nuclear chains, fibre size variation and occasional solitary necrotic fibres were observed at all ages in all the LWC birds examined, except in nos 5, 6, 9 and 13, in which clinical signs were absent and the muscles were histologically normal. Sarcoplasmic masses (Fig. 1) were found mostly in the periphery of the affected muscle fibres. Multiple centrally located masses (Fig. 2) were also observed, particularly in mildly affected muscle groups or in muscle fibres that did not have peripheral sarcoplasmic masses. Regardless of whether they were found in the periphery or in the centre of the muscle fibre, the sarcoplasmic masses frequently contained enlarged vesicular nuclei. Histochemically, peripherally located sarcoplasmic masses retained their ATPase activity; they stained intensely with N A D H - T R (Fig. 3) and sometimes with PAS. In frozen HE sections, the sarcoplasmic masses acquired a slightly basophilic tinge. Careful inspection of sections stained with Gomori's trichrome revealed that these sarcoplasmic masses were stained a shade of green slightly darker than that of the normal periphery. Multiple masses, resembling mini-cores, found in the centre of affected muscle fibres, were negative for N A D H - T R but
135
Inherited Muscular Disorder in Mutant Quail
7 L;
Fig. 1.
M. triceps brachiipars scapularis o f a myotonic LWC quail (No. 18), 70 days old. Peripherally located
Fig. 2.
M. supracoracoideusofa myotonic LWC quail (No. 3), 21 days old. Peripheral and central sarcoplasmic
sarcoplasmic masses. Paraffin wax transverse section. PTAH. x 568. masses. Paraffin wax transverse section. PTAH. x 360,
5 84 i~
Fig. 3.
M. pectoralis thoracicus of a myotonic LWC quail (No. 2), 21 days old. Peripherally located sarcoplasmic masses stain intensely (asterisk), whereas centrally located masses appear "motheaten" (arrowheads). Frozen transverse section. N A D H - T R . x 333.
136
Fig. 4. Fig. 5.
I.S.
Braga e t
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M. pectoralis thoracicus of a myotonic LWC quail (No. 14), 50 days old. Ringed fibres with distinct sarcomeric structures. Paraffin wax transverse section. PTAH. x 475. ,~. supracoracoideusof a myotonic LWC quail (No. 12), 50 days old. Internal migration of nuclei in muscle fibres with sarcoplasmic masses and rings. Paraffin wax transverse section. HE. x 333.
appeared to coalesce with each other, giving a "moth-eaten" appearance (Fig. 3). Because type 2B fibres remained unstained with ATPase, both peripherally and centrally located masses were light blue against an unstained background; this indicated an increase in enzyme activity. Both types of sarcoplasmic masses were occasionally found in the same myofibre, but regardless of location or n u m b e r they remained unstained by P T A H , indicating the absence of myofibrils. Although occurring less frequently than sarcoplasmic masses, ringed fibres (striated annulets or "Ringbinden") were also observed and were generally associated with peripheral sarcoplasmic masses. T h e y were readily seen in PAS-stained frozen sections, or in PTAH-stained paraffin wax sections. Regular bundles of myofibrils encircled the entire periphery of the affected muscle fibre (Type A ring fibres) (Sawicka, 1991), usually between the remaining normal fibre and an outer sarcoplasmic mass. P T A H staining revealed distinct sarcomeric structures (Fig. 4). Internal migration of nuclei was seen mainly in muscle groups with minimal changes, such as those of the thoracic limb in young affected quail and in the pelvic limb of older birds. Increased numbers of internal nuclei were seen in m a n y fibres with sarcoplasmic masses (Fig. 5). Lymphorrhages and solitary necrotic fibres were observed in both LWC and control birds, and were therefore considered to be non-specific. Although a decrease in ATPase and N A D H - T R staining intensity was noted, fibre type differentiation remained distinct.
I n h e r i t e d M u s c u l a r D i s o r d e r in M u t a n t Q u a i l
Fig. 6.
137
3/1. pectoralis thoracicus of a myotonic LWC quail (No. 19), 70 days old. Inter- and intra-fascicular fatty infiltration with replacemeat of atrophic ~ e 2B (arrowheads) muscle fibres. Characteristic histological changes are seen primarily in type 2A (A) muscle fibres. Paraffin wax transverse section. HE. x 180.
Lipocytes, observed in small clusters in the interfascicular spaces of the M.
pectoralis thoracicus at 20 days of age, had increased in n u m b e r by day 60. Without any evidence of necrosis, replacement of some of the small angular (atrophied) type 2B fibres (Fig. 6) of the M. pectoralis thoracicus by lipocytes was observed at 60 days of age. Intrafascicular infiltration by lipocytes was seen in some muscle groups of older birds. Similar changes were also seen in other muscles such as the M. supracoracoideus, M. subcoracoideus, M. triceps pars humeralis, M. triceps pars scapularis, M. deltoideus major and M. femorotibialis medius (Table 2). Variation in fibre size became conspicuous in the affected fascicles when intrafascicular fatty infiltration had commenced. Even though an increase in the degree of fatty infiltration was observed at 70 days of age, fibrous connective tissue proliferation was not seen. The most frequently and most severely affected muscles were those of the pectoral girdle (Tables 2 and 3). Involvement of the muscles of the thoracic and pelvic limbs was observed only in severely affected or old birds. In quail less than 40 days old, muscle lesions were observed in the absence of clinical signs such as negative wing lift and flip test. Although the result of the flip test depends on the degree or extent of the wing lift, it showed no apparent relationship to the severity of histological changes (Table 3). Lesion distribution patterns according to fibre type were evident in the superficial fascicles of the M. pectoralis thoracicus, in which the affected muscle fibres (types 2A and 2B) could be distinguished easily. Two distinct patterns were observed, namely (1) a pattern primarily affecting type 2A fibres (Fig. 7), and (2) a pattern primarily affecting type 2B fibres (Fig. 8). T h e first pattern was more frequently observed in the quail examined (Table 3), but examples
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I . S . Braga e t al.
Table 2 The frequency of occurrence of hlstopathological changes seen in 18 different m u s c l e groups of LWC quail examined Number of samples Muscle group
Histopathological changes CN
SM
RF
sFN
FSC
FI
19 19 19 19 19 19
9 5 11 8 2 7
13 10 12 10 4 8
11 12 12 7 2 5
0 3 5 0 0 1
7 4 1 1 1 1
4 6 6 1 0 0
19 19 19 19 14
8 7 8 7 5
6 5 7 6 3
9 3 5 6 2
4 2 1 2 1
2 0 0 1 0
0 1 1 1 0
14 14 19 13 14 2 2
2 2 4 3 2 0 0
2 1 5 1 1 0 0
1 2 3 1 1 0 0
1 0 1 0 1 0 0
0 0 0 0 0 0 0
0 0 1 0 0 0 0
Pectoral Girdle M. pectoralis thoracicus (cranial belly) (PM 1) M. pectoralis thoracicus (medial belly) (PM2) M. supracoracoideus (SS) 11/1, subcoracoideus (SC) M. latissimus dorsi cranialis (LCR) M. latissimus dorsi caudalis (LCD) Thoracic Limb 3,4, biceps brachii pars scapularis (BB) M, triceps brachii pars humeratis (TH) M. triceps brachiipars scapularis (TS) M. deltoideus major (DD) M. tensorpropatagialis (PT) Pelvic Limb M. iliotibialis cranialis (IC) M. iliotibialis lateralis (IL) M. femorotibialis medius (fiT) M. gastrocnemius externus (GE) M. fibularis Iongus (FL) M. extensor digitorum longus (EDL) M. tibialis cranialis (TC)
CN, central nucleation; SM, sarcoplasmic mass; RF, ring fibre; sFN, solitary fibre necrosis; FSC, fibre size changes; FI, fatty infiltration.
of both patterns were seen in different birds of the same age (20, 30 and 40 days old). Type 1 fibres in all the muscle groups studied remained unaffected. Muscle fibre morphometry (Table 4) showed that in the normal quail, type 2B fibres gradually increased in size up to 60 days of age. LWC quail, however, had large and almost adult-sized type 2B fibres at 20 days, but from 30 days onwards the fibre diameter gradually decreased to half the normal value. By 50 days of age, a significant decrease in the size of type 2B fibres was observed. As a result, mature adult LWC quail had small atrophic type 2B fibre diameters as compared with those of normal quail of similar age. Atrophy of type 2B fibres was seen regardless of the distribution pattern of lesions between type 2 fibres. In types 1 and 2A fibres, the slight difference in size was due to an increase in the diameter associated with growth between 20 and 70 days of age (Table 4). Discussion In the young affected LWC quail, sarcoplasmic masses were the predominant lesion. Peripheral or subsarcolemmal sarcoplasmic masses observed in frozen sections, as described previously (Braga et al., 1995), had a slightly basophilic tinge with HE, and stained more intensely with N A D H - T R and sometimes with PAS. Positive ATPase staining at pH 4"5 was distinctly observed, indicating
Inherited
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I . S . B r a g a et al.
M. pectoralis thoracicus of a myotonic LWC quail (No. 1), 21 days old. Histopathological changes are seen primarily in type 2A (A) muscle fibres with normal type 2B (B) fibres in the periphery. Paraffin wax transverse section. PTAH. x 180. M. pectoralis thoracicus of a myotonic LWC quail (No. 3), 21 days old. Histopathological changes are seen primarily in type 2B (B) muscle fibres with normal type 2A (A) fibres in the centre of the fascicles. Paraffin wax transverse section. PTAH. x 213.
Table 4 Average diameters o f the three major fibre t y p e s o f the M. pectoralis thoracicus o f control and m y o t o n i c LWC quail Age of quail (days)
20 30 40 50 60 70
Average diameter (#m) of type 1 fibres
O'pe 2A fibres
type 2B fibres
Quail
n
Mean + s.e.m,
n
Mean 3- s.e.m,
n
Mean -+ s.e.m.
Control LWC Control LWC Control LWC Control LWC Control LWC Control LWC
109 130 127 192 138 171 135 121 135 204 198 132
12"9 -I-0"323 14"984• 17"663_+0"283 17-919__+0-399 16"603 -+ 0" 244 19"474-+0'431 18"476_+0"325 21"378_+0"362 19"902_+0"511 22"492_+0-243 18"47 _+0"268 24"641-+0"514
160 172 166 362 179 278 177 176 180 261 264 163
13"126___0'184 19'318-t-0-278 18'757___0"242 21'505-1-0"205 17"495 _ 0"26 20"263-1-0"251 20"416_+0"302 23'652_+0"310 20'8913-0.327 22'561 _+0-218 22'01 -t-0"253 20"427-+0"267
140 135 128 266 135 195 132 132 132 204 198 132
23"643-1-0"458 40"675-t-0"590 31"164-1-0"704 39"805-+0"452 40-242 -+ 0" 763 35-439-1-0"882 36"342_+0"819 24"3 _+0"617 44-958_+1'441 25-389_+0"378 43-344___0"606 23"637-+0"334
Inherited Muscular Disorder in Mutant
Quail
141
retention of ATPase activity. Type 2B fibres, which normally do not give an ATPase staining reaction at pH 4-5, showed a rim of increased staining intensity where sarcoplasmic masses were located, indicating an increase in ATPase activity. Since they did not contain myofibrils, they remained unstained with PTAH and haematoxylin. In muscle groups that were mildly affected, such as those of the trunk and thoracic limb, centrally located sarcoplasmic masses, often without accompanying subsarcolemmal masses, were observed. Since centrally located sarcoplasmic masses were not stained for NADH-TR, it was assumed that they contained very little or no mitochondria. Although sometimes resembling mini-cores, centrally located sarcoplasmic masses had irregular indistinct borders, visible in HE-stained sections, associated with nuclei, and did not contain myofibrils since they stained negatively with PTAH. Ringed fibres, which more often occurred in association with sarcoplasmic masses than otherwise, probably developed after the sarcoplasmic masses were formed. Exceptionally large type 2B fibres in the M. pectoralis thoracicus of the 20day-old LWC quail were considered to represent the most striking change noted. Despite their abnormally large size, the lack of any observable histochemical change made fibre typing possible. Hypertrophy, limited to type 2 fibres in the sartorius muscle of the dystrophic chicken, is an early manifestation of muscular dystrophy (Shafiq et al., 1971). In man, type specific hypertrophy is rarely seen (Dubowitz, 1985). Lesion distribution patterns differed among individual quail, affecting primarily either type 2A or 2B muscle fibres, and leaving type I fibres unaffected. Because the abnormality is considered to be highly non-specific (Karpati, 1979), these results support the view that type 2 fibres are especially susceptible to dystrophic processes as described in the dystrophic chicken (Shafiq et al., 1971). Type 2B fibres are more prone than type 2A to atrophy (Karpati, 1979). These findings agree with the suggestion that atrophy in type 2 fibres is largely due to sensitivity to reduction or loss of trophic factors from motor nerves (Mendell and Engel, 1971), since the atrophic fibres found in the LWC quail retained their normal enzyme reactivity and the electromyographic findings (Braga et al., 1995) showed no evidence of neuropathy. Though the primary defect in the present disorder is still unknown, it is probably related to altered metabolism of type 2 fibres. Atrophy and fatty infiltration were not observed in age-matched controls, but progressive atrophy with fatty infiltration and replacement of atrophied type 2B muscle fibres by lipocytes in the LWC quail became apparent as the birds aged. As suggested in our previous report (Braga et al., 1995), further atrophy and fatty replacement may lead to a deficiency in type 2B fibres in aged LWC quail. Lipocyte proliferation may be due to reduced intramuscular tension and inactivity resulting from the loss and atrophy of muscle fibres (Astr6m and Adams, 1979). In the LWC quail, atrophy and loss of type 2B muscle fibres may have resulted in lipocyte proliferation. Lesions tended to be more severe in the proximal than in the distal muscles, affecting the pectoral girdle and thoracic limb muscles in the early stages and later progressing to the muscles of the pelvic limb. As previously reported
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I.S. Braga et a/.
(Braga et al., 1995), regardless of observable clinical signs, histopathological changes in skeletal muscle may be seen in some quail. These changes were present to a varying degree in LWC quail, irrespective of the age and the severity of clinical signs. For this reason, chronological staging of the characteristic histopathological lesions in relation to the age of affected quail was not possible. T h e present study showed that atrophy and fatty replacement of type 2B muscle fibres in the LWC quail were due to a slow progressive deterioration of the muscle fibres and that these changes evolved with age, since they were not observed in quail younger than 20 days (unpublished data). Clinical symptoms associated with the disorder were observed from 40 days of age onwards. T h e severity and age of onset varied, possibly due to variable expression or incomplete penetrance of the defective gene. In the dystrophic chicken, there is evidence that excessive cross-linking of collagen may be associated with the wing stiffness and may play a part in the progression of the abnormality (Feit et al., 1989a,b). As already pointed out (Braga et al., 1995), the disease bears a similarity to myotonic dystrophy of man, but the results of the present study shed no light on the primary defect that causes the muscular disorder. T h e defect, which may be morphological or biochemical in origin, must be identified before LWC quail can be considered to represent an experimental model. However, heritability, progressive degeneration and loss of muscle fibres indicate that the muscular disorder in LWC Japanese quail can be classified (Astr6m and Adams, 1979) as a type of progressive muscular dystrophy exhibiting myotonia. Acknowledgments
I. S. Braga III is grateful to the Ministry of Education, Science and Culture of J a p a n for financial assistance. References
Astr6m, K. E. and Adams, R. D. (1979). The pathological reactions of the skeletal muscle fiber. In: Handbook of Clinical Neurology, Vol. 40, P.J. Vinken and G. W. Bruyn, Eds, North-Holland Publishing (3ompany, Amsterdam, pp. 197-274. Braga, I. S., Oda, K., Kikuchi, T., Tanaka, S., Sento, M., Itakura, (3. and Mizutani, M. (1995). A new inherited muscular disorder in Japanese quails (Coturnix coturnix japonica). Veterinary Pathology, 32, in press. DeGirolami, U. and Smith, T. W. (1982). Pathology of skeletal muscle diseases. American ffournal of Pathology, 107, 235-276. Doriguzzi, C., Mongini, T., Palmucci, L. and Schiffer, D. (1983). A method for myofibrillar Ca-ATPase reaction based on the use of metachromatic dyes: its advantages in muscle fibre typing. Histochemistty, 79, 289-294. Dubowitz, V. (1985). Muscle Biopsy, a Practical Approach, 2nd Edit., Bailli+re Tindall, London. Engel, W. K. and Cunningham, G. C. (1963). Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology, 13, 919-923. Feit, H., Kawai, M. and Mostafapour, A. S. (1989a). Increased resistance of the collagen in avian dystrophic muscle to collagenolytic attack: evidence for crosslinking. Muscle and Nerve, 12, 476-485.
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Feit, H., Kawai, M. and Mostafapour, A. S. (1989b). The role of collagen crosslinking in the increased stiffness of avian dystrophic muscle. Muscle and Nerve, 12, 486-492. Karpati, G. (1979). The principles of skeletal muscle histochemistry in neuromuscular diseases. In: Handbook ofClinicalNeurology, Vol. 40, P.J. Vinken and G. W. Bruyn, Eds, North-Holland Publishing Company, Amsterdam, p. 14. Mendell, J. R. and Engel, W. K. (1971). The fine structure of type II muscle fibre atrophy. Neurology, 21,358-365. Sawicka, E. (1991). Origin of the ring muscle fibers in neuromuscular diseases. Neuropatologica Polska, 29, 29-40. Shafiq, S. A., Askansas, V. and Milhorat, A. T. (1971). Fiber types and preclinical changes in chicken muscular dystrophy. Archives of Neurology, 25, 560-571. Vanden Berge, J. C. (1979). Myologia. In: Nomina Anatomica Avium, J. J. Baumel, Ed., Academic Press, London, pp. 175-219. Received, December 19th, 1994] Accepted, March 8th, 1995 J
I
Inherited Muscular Disorder in Mutant Japanese Quail (Coturnix coturnix japonica): Relationship Between the D e v e l o p m e n t of Muscle Lesions and Age I. S. Braga III, S. Tanaka, T. Kimura, C. Itakura and M. Mizutani* Department of Comparative Pathology, Faculty of VeterinaryMedicine, Hokkaido University, Sapporo 060 and *Laboratory of Animal Research Station, Nippon Institutefor Biological Science, Kobuchizawa, Yamanishi 409-16, Japan
Summary The progression of the pathological changes that occur in the skeletal muscle was examined in 19 Japanese quail of the LWC strain, affected with an autosomal dominant inherited muscular disorder producing electrical myotonia. The muscle samples were obtained every 10 days from 20 to 70 days of age. Muscle samples from 18 age-matched commercial quail were used as normal controls. Characteristic histological lesions found in the skeletal muscles included sarcoplasmic masses, ringed fibres, internal migration of nuclei and fibre size variation. These lesions, which mainly occurred in the proximal muscles, appeared first in the pectoral region and later in the muscles of the thoracic and pelvic limbs. The most predominant lesion observed at all ages consisted of sarcoplasmic masses. The presence of histological changes did not affect muscle fibre typing by two staining methods, for myosin ATPase at pH 4"5, and by NADH-TR stain. The histological changes were observed in type 2A and less commonly in 2B fibres, but not in type 1. The pectoralis thoracicus muscle, in which lesions were particularly common, showed abnormally large type 2B muscle fibres at 20 days of age. These fibres began to decrease in size at 30 days of age, and at 70 days had become strikingly atrophic, their diameter being only about half that observed at 20 days. The atrophic type 2B muscle fibres were eventually replaced by lipocytes. Chronological staging of the histopathological changes in muscle was impossible since no inter-relationship was observed between the age of the quail, the severity of clinical signs and the extent of muscle lesions. This variability in the severity and age of onset may have been due to the variable expression or incomplete penetrance of the defective gene. Because the disorder is hereditary and progressive in nature, it can be classified as a type of progressive muscular dystrophy. 9 1995 Academic Press Limited
Introduction A new strain of m u t a n t J a p a n e s e quail (Coturnix coturnixjaponica), L W C , with an inherited muscular disorder p r o d u c i n g electrical myotonia, was developed by the L a b o r a t o r y of A n i m a l Research Station of the N i p p o n Institute for 0021-9975/95/060131 + 13 $12.00/0
9 1995 Academic Press Limited
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Biological Science. The disorder is inherited as an autosomal dominant trait and is homolethal. Since the pattern of muscular involvement is primarily proximal in distribution, the clinical manifestations of the disorder in affected quail are characterized by loss of wing lift ability and an impaired righting reflex. Histological examination of the skeletal muscles of the affected birds shows sarcoplasmic masses, ringed fibres, central nucleation, and nuclear rows with occasional solitary necrotic fibres and, in the advanced stages, fatty infiltration (Braga et al., 1995). In addition to the mode of inheritance, the presence of electrical myotonia and characteristic histopathological changes, and the presence of bilateral cataracts and testicular atrophy in some affected quail suggest a multi-systemic disorder not unlike myotonic dystrophy of man. O u r previous report (Braga et al., 1995) strongly suggested progressive degenerative changes in the muscle, but it is not known whether these changes are due to defective maturation and retention of embryonic features posthatching or develop later as the bird ages. The present study describes the histopathological changes found in the skeletal muscle of L W C quail from 20 days of age to sexual maturity at approximately 70 days. Materials and M e t h o d s
Birds Nineteen mutant (LWC)Japanese quail and 18 normal age-matched control birds from a commercial quail farm were examined. Two to five birds from each group were killed every 10 days from day 20 to 70 (Table 1) by an overdose ofpentobarbital sodium (Somnopentyl| Pitman-Moore, USA) administered intraperitoneally. Clinical Observations Quail are normally capable of raising their wings vertically, through 180 ~ from the rest position. Mutant LWC quail manifesting the inherited muscular disorder are, however, incapable of lifting their wings vertically (wing lift), and are unable to right themselves when placed on their dorsum (flip test) (Braga et al., 1995). As will be seen later (Table 1), LWC quail with wing lifts of approximately 150~ usually have a normal flip test, whereas a 90 ~ wing lift is associated with a negative flip test. Responses to the wing lift and flip tests were checked on all birds examined (Table 1). Light Microscopy and Histochemistry Where and when possible, 18 different skeletal muscle groups were collected from each bird for histopathological examination. Samples from the right side were fixed in 10% neutral buffered formalin, dehydrated through graded alcohols, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (HE). Selected sections were stained with periodic acid-Schiff (PAS) and phosphotungstic acid haematoxylin (PTAH). The nomenclature for the muscles follows that of Vanden Berge (1979). The muscles examined included: those of the pectoral girdle--Musculus pectoralis thoracicus (cranial and medial belly), M. supracoracoideus, M. subcoracoideus, M. latissimus dorsi cranialis, M. latissimus dorsi caudalis; the thoracic limb muscles--M, deltoideus major, M. triceps brachii (pars humeralis), M. triceps brachii (pars scapularis), M. biceps brachii (pars scapularis), M. tensor propatagialis; and the pelvic limb muscles--M, iliotibialis cranialis, M. iliotibialis lateralis, M. femorotibialis medius, M. iliofibularis, M. gastrocnemius (pars externa), M. fibularis longus, M. tibialis cranialis, and M. extensor digitorum longus. Samples of muscle were quickly removed from the left side, mounted on slices of
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I n h e r i t e d M u s c u l a r D i s o r d e r in M u t a n t Q u a i l Table 1 Age, sex, body weight and fllp test and wing lift responses in quail examined
Case no.
Quail
Age (days)
Sex
Body weight (g)
Clinical signs 14/~nglift
Flip test
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
LWC o " ~ ~ ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
2O 20 2O 30 30 30 30 30 40 40 40 50 50 50 60 60 60 70 70
F M F F F M M F M F F F M F F M F M F
57 63 56 100 105 88 73 86 101 123 141 134 116 139 162 115 158 104 137
+ + + + + + + + + 120 ~ + 150 ~ + 90 ~ 90 ~ + 150 ~ 90 ~ 90 ~
+ + + + + + + + + + + + --+ + --
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Control ~ " ~ " ~ ~ ~ ~ ~ ~ ~ ~ " ~ ~ ~ "
20 20 3O 30 30 4O 40 40 50 50 50 60 60 60 70 70 70 70
M F F M F M F M M M F M M F F M M M
46 48 71 81 75 98 89 93 98 95 107 94 106 92 121 104 103 98
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
M, male; F, female; + , positive; --, negative. cork; fixed in place by a small amount of gum tragacanth, and frozen rapidly by immersing the mounted specimen in isopentane pre-cooled in liquid nitrogen to -160~ (DeGirolami and Smith, 1982). The frozen blocks were stored at -80~ u n t i l t h e y w e r e s e c t i o n e d . S e r i a l t r a n s v e r s e s e c t i o n s ( 6 - 1 0 ~tm) w e r e c u t w i t h a cryotome at --25 to --30~ and then mounted on glass slides coated with neoprene 0"02% in toluene. Sectioned specimens were air-dried and stored in the freezer at - 30~ until they were stained. The sections were stained for myofibrillar adenosine triphosphatase (ATPase) with the metachromatic d y e , t o l u i d i n e b l u e ( D o r i g u z z i et al., 1 9 8 3 ) , a f t e r p r e i n c u b a t i o n a t p H 4"5, f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e , r e d u c e d f o r m - t e t r a z o l i u m reductase (NADH-TR) as described by Dubowitz (1985), and for glycogen by the PAS technique (Dubowitz, 1985). Sections were also routinely stained with modified Gomori trichrome (Engel and Cunningham, 1 9 6 3 ) a n d H E ( D u b o w i t z , 1985).
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Muscle Fibre Morphomet~y Photomicrographs of the superficial and deep parts of the M. pectoralisthoracicusstained for ATPase were taken from 29 (13 control and 16 LWC) quail. The muscle fibres were classified into three major fibre types (types 1, 2A and 2B) and the minimum fibre diameters of at least 100 fibres per quail were measured. Results
Clinical Evaluation Control quail. All the control birds examined had normal (180 ~) wing lifts and positive flip tests (Table 1). LWC quail T h e quail started to show negative (< 180 ~ wing lifts, and negative flip tests, which are diagnostic (Braga et al., 1995), at 40 days of age (Table 1). Quail killed for necropsy when 20 and 30 days old were chosen at r a n d o m because identification of affected birds was not possible at this age. No macroscopical wasting of muscle was noted in any of the birds examined. Microscopical Findings Control quail. Differentiation of the skeletal muscle into the three major fibre types (types 1, 2A and 2B) was observed at 20 days of age with both the ATPase and the N A D H - T R staining methods. Apart from an occasional isolated solitary necrotic fibre, no lesions were observed in the muscles of these birds. LWC quail. As in the control birds, muscle fibre differentiation into the three major fibre types was observed at 20 days of age. Characteristic histopathological changes such as ringed fibres, sarcoplasmic masses, internal migration of nuclei, nuclear chains, fibre size variation and occasional solitary necrotic fibres were observed at all ages in all the LWC birds examined, except in nos 5, 6, 9 and 13, in which clinical signs were absent and the muscles were histologically normal. Sarcoplasmic masses (Fig. 1) were found mostly in the periphery of the affected muscle fibres. Multiple centrally located masses (Fig. 2) were also observed, particularly in mildly affected muscle groups or in muscle fibres that did not have peripheral sarcoplasmic masses. Regardless of whether they were found in the periphery or in the centre of the muscle fibre, the sarcoplasmic masses frequently contained enlarged vesicular nuclei. Histochemically, peripherally located sarcoplasmic masses retained their ATPase activity; they stained intensely with N A D H - T R (Fig. 3) and sometimes with PAS. In frozen HE sections, the sarcoplasmic masses acquired a slightly basophilic tinge. Careful inspection of sections stained with Gomori's trichrome revealed that these sarcoplasmic masses were stained a shade of green slightly darker than that of the normal periphery. Multiple masses, resembling mini-cores, found in the centre of affected muscle fibres, were negative for N A D H - T R but
135
Inherited Muscular Disorder in Mutant Quail
7 L;
Fig. 1.
M. triceps brachiipars scapularis o f a myotonic LWC quail (No. 18), 70 days old. Peripherally located
Fig. 2.
M. supracoracoideusofa myotonic LWC quail (No. 3), 21 days old. Peripheral and central sarcoplasmic
sarcoplasmic masses. Paraffin wax transverse section. PTAH. x 568. masses. Paraffin wax transverse section. PTAH. x 360,
5 84 i~
Fig. 3.
M. pectoralis thoracicus of a myotonic LWC quail (No. 2), 21 days old. Peripherally located sarcoplasmic masses stain intensely (asterisk), whereas centrally located masses appear "motheaten" (arrowheads). Frozen transverse section. N A D H - T R . x 333.
136
Fig. 4. Fig. 5.
I.S.
Braga e t
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M. pectoralis thoracicus of a myotonic LWC quail (No. 14), 50 days old. Ringed fibres with distinct sarcomeric structures. Paraffin wax transverse section. PTAH. x 475. ,~. supracoracoideusof a myotonic LWC quail (No. 12), 50 days old. Internal migration of nuclei in muscle fibres with sarcoplasmic masses and rings. Paraffin wax transverse section. HE. x 333.
appeared to coalesce with each other, giving a "moth-eaten" appearance (Fig. 3). Because type 2B fibres remained unstained with ATPase, both peripherally and centrally located masses were light blue against an unstained background; this indicated an increase in enzyme activity. Both types of sarcoplasmic masses were occasionally found in the same myofibre, but regardless of location or n u m b e r they remained unstained by P T A H , indicating the absence of myofibrils. Although occurring less frequently than sarcoplasmic masses, ringed fibres (striated annulets or "Ringbinden") were also observed and were generally associated with peripheral sarcoplasmic masses. T h e y were readily seen in PAS-stained frozen sections, or in PTAH-stained paraffin wax sections. Regular bundles of myofibrils encircled the entire periphery of the affected muscle fibre (Type A ring fibres) (Sawicka, 1991), usually between the remaining normal fibre and an outer sarcoplasmic mass. P T A H staining revealed distinct sarcomeric structures (Fig. 4). Internal migration of nuclei was seen mainly in muscle groups with minimal changes, such as those of the thoracic limb in young affected quail and in the pelvic limb of older birds. Increased numbers of internal nuclei were seen in m a n y fibres with sarcoplasmic masses (Fig. 5). Lymphorrhages and solitary necrotic fibres were observed in both LWC and control birds, and were therefore considered to be non-specific. Although a decrease in ATPase and N A D H - T R staining intensity was noted, fibre type differentiation remained distinct.
I n h e r i t e d M u s c u l a r D i s o r d e r in M u t a n t Q u a i l
Fig. 6.
137
3/1. pectoralis thoracicus of a myotonic LWC quail (No. 19), 70 days old. Inter- and intra-fascicular fatty infiltration with replacemeat of atrophic ~ e 2B (arrowheads) muscle fibres. Characteristic histological changes are seen primarily in type 2A (A) muscle fibres. Paraffin wax transverse section. HE. x 180.
Lipocytes, observed in small clusters in the interfascicular spaces of the M.
pectoralis thoracicus at 20 days of age, had increased in n u m b e r by day 60. Without any evidence of necrosis, replacement of some of the small angular (atrophied) type 2B fibres (Fig. 6) of the M. pectoralis thoracicus by lipocytes was observed at 60 days of age. Intrafascicular infiltration by lipocytes was seen in some muscle groups of older birds. Similar changes were also seen in other muscles such as the M. supracoracoideus, M. subcoracoideus, M. triceps pars humeralis, M. triceps pars scapularis, M. deltoideus major and M. femorotibialis medius (Table 2). Variation in fibre size became conspicuous in the affected fascicles when intrafascicular fatty infiltration had commenced. Even though an increase in the degree of fatty infiltration was observed at 70 days of age, fibrous connective tissue proliferation was not seen. The most frequently and most severely affected muscles were those of the pectoral girdle (Tables 2 and 3). Involvement of the muscles of the thoracic and pelvic limbs was observed only in severely affected or old birds. In quail less than 40 days old, muscle lesions were observed in the absence of clinical signs such as negative wing lift and flip test. Although the result of the flip test depends on the degree or extent of the wing lift, it showed no apparent relationship to the severity of histological changes (Table 3). Lesion distribution patterns according to fibre type were evident in the superficial fascicles of the M. pectoralis thoracicus, in which the affected muscle fibres (types 2A and 2B) could be distinguished easily. Two distinct patterns were observed, namely (1) a pattern primarily affecting type 2A fibres (Fig. 7), and (2) a pattern primarily affecting type 2B fibres (Fig. 8). T h e first pattern was more frequently observed in the quail examined (Table 3), but examples
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I . S . Braga e t al.
Table 2 The frequency of occurrence of hlstopathological changes seen in 18 different m u s c l e groups of LWC quail examined Number of samples Muscle group
Histopathological changes CN
SM
RF
sFN
FSC
FI
19 19 19 19 19 19
9 5 11 8 2 7
13 10 12 10 4 8
11 12 12 7 2 5
0 3 5 0 0 1
7 4 1 1 1 1
4 6 6 1 0 0
19 19 19 19 14
8 7 8 7 5
6 5 7 6 3
9 3 5 6 2
4 2 1 2 1
2 0 0 1 0
0 1 1 1 0
14 14 19 13 14 2 2
2 2 4 3 2 0 0
2 1 5 1 1 0 0
1 2 3 1 1 0 0
1 0 1 0 1 0 0
0 0 0 0 0 0 0
0 0 1 0 0 0 0
Pectoral Girdle M. pectoralis thoracicus (cranial belly) (PM 1) M. pectoralis thoracicus (medial belly) (PM2) M. supracoracoideus (SS) 11/1, subcoracoideus (SC) M. latissimus dorsi cranialis (LCR) M. latissimus dorsi caudalis (LCD) Thoracic Limb 3,4, biceps brachii pars scapularis (BB) M, triceps brachii pars humeratis (TH) M. triceps brachiipars scapularis (TS) M. deltoideus major (DD) M. tensorpropatagialis (PT) Pelvic Limb M. iliotibialis cranialis (IC) M. iliotibialis lateralis (IL) M. femorotibialis medius (fiT) M. gastrocnemius externus (GE) M. fibularis Iongus (FL) M. extensor digitorum longus (EDL) M. tibialis cranialis (TC)
CN, central nucleation; SM, sarcoplasmic mass; RF, ring fibre; sFN, solitary fibre necrosis; FSC, fibre size changes; FI, fatty infiltration.
of both patterns were seen in different birds of the same age (20, 30 and 40 days old). Type 1 fibres in all the muscle groups studied remained unaffected. Muscle fibre morphometry (Table 4) showed that in the normal quail, type 2B fibres gradually increased in size up to 60 days of age. LWC quail, however, had large and almost adult-sized type 2B fibres at 20 days, but from 30 days onwards the fibre diameter gradually decreased to half the normal value. By 50 days of age, a significant decrease in the size of type 2B fibres was observed. As a result, mature adult LWC quail had small atrophic type 2B fibre diameters as compared with those of normal quail of similar age. Atrophy of type 2B fibres was seen regardless of the distribution pattern of lesions between type 2 fibres. In types 1 and 2A fibres, the slight difference in size was due to an increase in the diameter associated with growth between 20 and 70 days of age (Table 4). Discussion In the young affected LWC quail, sarcoplasmic masses were the predominant lesion. Peripheral or subsarcolemmal sarcoplasmic masses observed in frozen sections, as described previously (Braga et al., 1995), had a slightly basophilic tinge with HE, and stained more intensely with N A D H - T R and sometimes with PAS. Positive ATPase staining at pH 4"5 was distinctly observed, indicating
Inherited
Muscular
Disorder
in Mutant
Quail
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Fig. 7.
Fig. 8.
I . S . B r a g a et al.
M. pectoralis thoracicus of a myotonic LWC quail (No. 1), 21 days old. Histopathological changes are seen primarily in type 2A (A) muscle fibres with normal type 2B (B) fibres in the periphery. Paraffin wax transverse section. PTAH. x 180. M. pectoralis thoracicus of a myotonic LWC quail (No. 3), 21 days old. Histopathological changes are seen primarily in type 2B (B) muscle fibres with normal type 2A (A) fibres in the centre of the fascicles. Paraffin wax transverse section. PTAH. x 213.
Table 4 Average diameters o f the three major fibre t y p e s o f the M. pectoralis thoracicus o f control and m y o t o n i c LWC quail Age of quail (days)
20 30 40 50 60 70
Average diameter (#m) of type 1 fibres
O'pe 2A fibres
type 2B fibres
Quail
n
Mean + s.e.m,
n
Mean 3- s.e.m,
n
Mean -+ s.e.m.
Control LWC Control LWC Control LWC Control LWC Control LWC Control LWC
109 130 127 192 138 171 135 121 135 204 198 132
12"9 -I-0"323 14"984• 17"663_+0"283 17-919__+0-399 16"603 -+ 0" 244 19"474-+0'431 18"476_+0"325 21"378_+0"362 19"902_+0"511 22"492_+0-243 18"47 _+0"268 24"641-+0"514
160 172 166 362 179 278 177 176 180 261 264 163
13"126___0'184 19'318-t-0-278 18'757___0"242 21'505-1-0"205 17"495 _ 0"26 20"263-1-0"251 20"416_+0"302 23'652_+0"310 20'8913-0.327 22'561 _+0-218 22'01 -t-0"253 20"427-+0"267
140 135 128 266 135 195 132 132 132 204 198 132
23"643-1-0"458 40"675-t-0"590 31"164-1-0"704 39"805-+0"452 40-242 -+ 0" 763 35-439-1-0"882 36"342_+0"819 24"3 _+0"617 44-958_+1'441 25-389_+0"378 43-344___0"606 23"637-+0"334
Inherited Muscular Disorder in Mutant
Quail
141
retention of ATPase activity. Type 2B fibres, which normally do not give an ATPase staining reaction at pH 4-5, showed a rim of increased staining intensity where sarcoplasmic masses were located, indicating an increase in ATPase activity. Since they did not contain myofibrils, they remained unstained with PTAH and haematoxylin. In muscle groups that were mildly affected, such as those of the trunk and thoracic limb, centrally located sarcoplasmic masses, often without accompanying subsarcolemmal masses, were observed. Since centrally located sarcoplasmic masses were not stained for NADH-TR, it was assumed that they contained very little or no mitochondria. Although sometimes resembling mini-cores, centrally located sarcoplasmic masses had irregular indistinct borders, visible in HE-stained sections, associated with nuclei, and did not contain myofibrils since they stained negatively with PTAH. Ringed fibres, which more often occurred in association with sarcoplasmic masses than otherwise, probably developed after the sarcoplasmic masses were formed. Exceptionally large type 2B fibres in the M. pectoralis thoracicus of the 20day-old LWC quail were considered to represent the most striking change noted. Despite their abnormally large size, the lack of any observable histochemical change made fibre typing possible. Hypertrophy, limited to type 2 fibres in the sartorius muscle of the dystrophic chicken, is an early manifestation of muscular dystrophy (Shafiq et al., 1971). In man, type specific hypertrophy is rarely seen (Dubowitz, 1985). Lesion distribution patterns differed among individual quail, affecting primarily either type 2A or 2B muscle fibres, and leaving type I fibres unaffected. Because the abnormality is considered to be highly non-specific (Karpati, 1979), these results support the view that type 2 fibres are especially susceptible to dystrophic processes as described in the dystrophic chicken (Shafiq et al., 1971). Type 2B fibres are more prone than type 2A to atrophy (Karpati, 1979). These findings agree with the suggestion that atrophy in type 2 fibres is largely due to sensitivity to reduction or loss of trophic factors from motor nerves (Mendell and Engel, 1971), since the atrophic fibres found in the LWC quail retained their normal enzyme reactivity and the electromyographic findings (Braga et al., 1995) showed no evidence of neuropathy. Though the primary defect in the present disorder is still unknown, it is probably related to altered metabolism of type 2 fibres. Atrophy and fatty infiltration were not observed in age-matched controls, but progressive atrophy with fatty infiltration and replacement of atrophied type 2B muscle fibres by lipocytes in the LWC quail became apparent as the birds aged. As suggested in our previous report (Braga et al., 1995), further atrophy and fatty replacement may lead to a deficiency in type 2B fibres in aged LWC quail. Lipocyte proliferation may be due to reduced intramuscular tension and inactivity resulting from the loss and atrophy of muscle fibres (Astr6m and Adams, 1979). In the LWC quail, atrophy and loss of type 2B muscle fibres may have resulted in lipocyte proliferation. Lesions tended to be more severe in the proximal than in the distal muscles, affecting the pectoral girdle and thoracic limb muscles in the early stages and later progressing to the muscles of the pelvic limb. As previously reported
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I.S. Braga et a/.
(Braga et al., 1995), regardless of observable clinical signs, histopathological changes in skeletal muscle may be seen in some quail. These changes were present to a varying degree in LWC quail, irrespective of the age and the severity of clinical signs. For this reason, chronological staging of the characteristic histopathological lesions in relation to the age of affected quail was not possible. T h e present study showed that atrophy and fatty replacement of type 2B muscle fibres in the LWC quail were due to a slow progressive deterioration of the muscle fibres and that these changes evolved with age, since they were not observed in quail younger than 20 days (unpublished data). Clinical symptoms associated with the disorder were observed from 40 days of age onwards. T h e severity and age of onset varied, possibly due to variable expression or incomplete penetrance of the defective gene. In the dystrophic chicken, there is evidence that excessive cross-linking of collagen may be associated with the wing stiffness and may play a part in the progression of the abnormality (Feit et al., 1989a,b). As already pointed out (Braga et al., 1995), the disease bears a similarity to myotonic dystrophy of man, but the results of the present study shed no light on the primary defect that causes the muscular disorder. T h e defect, which may be morphological or biochemical in origin, must be identified before LWC quail can be considered to represent an experimental model. However, heritability, progressive degeneration and loss of muscle fibres indicate that the muscular disorder in LWC Japanese quail can be classified (Astr6m and Adams, 1979) as a type of progressive muscular dystrophy exhibiting myotonia. Acknowledgments
I. S. Braga III is grateful to the Ministry of Education, Science and Culture of J a p a n for financial assistance. References
Astr6m, K. E. and Adams, R. D. (1979). The pathological reactions of the skeletal muscle fiber. In: Handbook of Clinical Neurology, Vol. 40, P.J. Vinken and G. W. Bruyn, Eds, North-Holland Publishing (3ompany, Amsterdam, pp. 197-274. Braga, I. S., Oda, K., Kikuchi, T., Tanaka, S., Sento, M., Itakura, (3. and Mizutani, M. (1995). A new inherited muscular disorder in Japanese quails (Coturnix coturnix japonica). Veterinary Pathology, 32, in press. DeGirolami, U. and Smith, T. W. (1982). Pathology of skeletal muscle diseases. American ffournal of Pathology, 107, 235-276. Doriguzzi, C., Mongini, T., Palmucci, L. and Schiffer, D. (1983). A method for myofibrillar Ca-ATPase reaction based on the use of metachromatic dyes: its advantages in muscle fibre typing. Histochemistty, 79, 289-294. Dubowitz, V. (1985). Muscle Biopsy, a Practical Approach, 2nd Edit., Bailli+re Tindall, London. Engel, W. K. and Cunningham, G. C. (1963). Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology, 13, 919-923. Feit, H., Kawai, M. and Mostafapour, A. S. (1989a). Increased resistance of the collagen in avian dystrophic muscle to collagenolytic attack: evidence for crosslinking. Muscle and Nerve, 12, 476-485.
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Feit, H., Kawai, M. and Mostafapour, A. S. (1989b). The role of collagen crosslinking in the increased stiffness of avian dystrophic muscle. Muscle and Nerve, 12, 486-492. Karpati, G. (1979). The principles of skeletal muscle histochemistry in neuromuscular diseases. In: Handbook ofClinicalNeurology, Vol. 40, P.J. Vinken and G. W. Bruyn, Eds, North-Holland Publishing Company, Amsterdam, p. 14. Mendell, J. R. and Engel, W. K. (1971). The fine structure of type II muscle fibre atrophy. Neurology, 21,358-365. Sawicka, E. (1991). Origin of the ring muscle fibers in neuromuscular diseases. Neuropatologica Polska, 29, 29-40. Shafiq, S. A., Askansas, V. and Milhorat, A. T. (1971). Fiber types and preclinical changes in chicken muscular dystrophy. Archives of Neurology, 25, 560-571. Vanden Berge, J. C. (1979). Myologia. In: Nomina Anatomica Avium, J. J. Baumel, Ed., Academic Press, London, pp. 175-219. Received, December 19th, 1994] Accepted, March 8th, 1995 J
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