HISTOLOGICAL AND HISTOCHEMICAL CHANGES OF THE SKELETAL MUSCLE IN HUMAN CHRONIC CHAGAS' DISEASE ANALIA TERESA
TARATUTO FUMO
Μ. A. PAGNO OLG P. SANZ R.
E. P.
SICA
In a recent electrophysiological investigation Pagano et al. demonstrated the presence of mild denervation in the skeletal muscle of patients with chronic Chagas' disease. Later on, the Ramos Mejia Hospital's group confirmed the findings of Pagano et al. and pointed out the spinal alpha motoneurone soma as the site of the lesion within the motor unit (m.u.) . 4
e
The aim of the present study has been to obtain information concerning the eventual histological changes of the skeletal muscle of patients with chronic Chagas' disease. MATERIAL AND
METHODS
Open b i o p s i e s w e r e carried o u t u n d e r local a n a e s t h e s i a on t h e g a s t r o c n e m i u s m e d i a l h e a d m u s c l e i n 7 s u b j e c t s , 5 m a l e s a n d 2 f e m a l e s , a g e d b e t w e e n 25 and 44 y e a r s . A t h o r o u g h h i s t o r y w a s o b t a i n e d f r o m e a c h p a t i e n t and a c o m p l e t e clinical a n d laboratory examination w a s also performed; on these b a s e s the selected subjects w e r e j u d g e d to b e h e a l t h y , b e i n g t h e o n l y a b n o r m a l i t y d e t e c t e d t h e p r e s e n c e of 2 o u t of 3 p o s i t i v e s e r u m t e s t s for Chagas* d i s e a s e . N o n e of t h e m h a d b e e n p r e v i o u s l y t r e a t e d w i t h specific d r u g s . C o i n c i d e n t a l c a u s e s of m u s c l e d e n e r v a t i o n w e r e a v o i d e d b y r e j e c t i n g p a t i e n t s over t h e a g e of 60 y e a r s ι,β a n d t h o s e w i t h a h i s t o r y of t o x i c o r m e t a b o l i c d i s o r d e r s k n o w n to b e a s s o c i a t e d w i t h m u s c l e d e n e r v a t i o n . Also were eliminated those patients w i t h p e r s o n a l o r f a m i l y h i s t o r y of m u s c l e d i s e a s e . F r e s h m u s c l e s p e c i m e n s w e r e ffrozen r o z e n i n i s o p e n t a n e c o o l e d t o -150C i n liquid n i t r o g e n . C r y o s t a t s e c t i o n s w e r e c u t a t t h i c k n e s s o f 2 to 10 urn and w e r e s t a i n e d or reacted w i t h t h e f o l l o w i n g t e c h n i q u e s : h e m a e t o x i l i n - e o s i n , periodic acid Schiff, m o d i f i e d Gomori trichrome, rreduced educed nicotinamide adenine dinucleotide dehidrogenase ( N A D H ) , miofibrillar a d e n o s i n e t r i p h o s p h a t a s e ( A T P a s e ) a t p H 9.4 a n d 4.6. a l k a l i n e phosphatase and non-specific esterase. RESULTS T h e m o s t c o m m o n f i n d i n g w a s t h e p r e s e n c e of c l u s t e r s of u n i f o r m t y p e of m u s c l e fibres, e i t h e r t y p e I or t y p e I I , i n 56 of t h e 7 s t u d i e d p a t i e n t s . B o t h t y p e s of c l u s t e r s , p l a c e d s i d e b y s i d e , c o u l d o f t e n b e f o u n d i n t h e s a m e s p e c i m e n ( F i g u r e 1. Table 1).
Section Argentina.
of
Clinical
Electroneurophysiology,
Hospital
Ramos
Mejia,
Buenos
Aires.
328
ARQ-
NEURO-PSIQUIATRIA
(8Ã0
PAULO)
VOL.
36 No k, DEZEMBRO, Λ
1978
T h e c l u s t e r s w e r e c o m p o s e d of a b o u t 16 t o 30 fibres, b e i n g s m a l l i n 4 w h i l e i n t h e r e m a i n i n g o n e m u c h l a r g e r c l u s t e r s could b e s e e n ( T a b l e 1 ) .
patients
I n p a t i e n t s V. Ch a n d O. R. s m a l l a n g u l a r f i b r e s , w h i c h s t a i n e d o r reacted s t r o n g l y w i t h non-specific s t e r a s e and N A D H t e c h n i q u e s , w e r e a l s o o b s e r v e d ( F i g u r e 2, T a b l e 1). Neither
degenerative
muscle
changes
nor
architectural
abnormalities
were
seen.
DISCUSSION
The present study has shown the presence of scattered at random denervated atrophic muscle fibres and fibre type grouping, both features were found twice in the same sample, while in the other three patients only grouped fibres of either type I or type II were seen. 2
These observations strongly suggest denervation associated with reservation , nevertheless these findings do not imply an associated clinical symptomatology, for fibre type grouping can be the only finding in clinically uninvolved muscles when there has been an adequate compensation in a chronic denervation. 3
It should be mentioned that in a previous study Laguens et cã. found in the human biceps of EVI-positive chagasic patients non specific muscle histological changes, namely isolated hyalinazed fibres, increased number of central nuclei in some fibres and occasionally sprouting buds containing numerous nuclei. They did not find alterations of succinate dehydrogenase and ATPase activities and no clear distinction between fibre types could be done by employing the phosphorilase technique. The changes observed in our histological study, which resulted from denervation, do not point out the site of lesion within the neurogenic component of the m.u., for either the axon or the motoneurone soma may bring about the same histological picture. However, the electrophysiological evidences presented by Sanz et al. hint that the motoneurone soma may be the site of 5
330
ARQ-
NBURO-PBJQUIATRIA
(BAO PAULO)
VOL.
36, Ν* k, DEZEMBRO,
1978
lesion. Their ideas agree with our findings in two aspects: first the observed muscle changes may be the result of motoneurone soma involvement and second, the described anatomical features can be found in* partially denervated subjects who have recovered from a denervatory disease and, eventually, have not any clinical symptomatology. In conclusion, the present investigation has confirmed electrophysiological studies in which an involvement of the m.u. was reported in patients with chronic Chagas' disease ' . Because the approach applied here has been a morphological rather than a physiological one. it has had the advantage of showing anatomical evidences of muscle denervation which are compatible with an old and well compensated denervatory process which, at the time of the investigation, may be below the clinical level of detection. 4 5
SUMMARY
Gastrocnemius muscle biopsies were performed in 7 subjects with chronic Chagas' disease. On clinical and laboratory grounds the selected patients were judged to be healthy, being the only abnormality found the presence of positive serum tests for Chagas' disease. Fibre type grouping of either type I or type II was observed in 5 of the 7 patients. Furthermore, in 2 of the 5 patients showing muscle fibre groupings, angular fibres reacting with NADH and non-specific sterase were also found. These observations strongly suggest denervation associated with reinervation. This picture often can be observed in the skeletal muscle of patients with well compensated denervatory conditions who do not show clinical evidences of denervation.
RESUMEN
Cambios histológicos e histoquimicos del músculo esquelético en la enfer¬ medad de Chagas crónica. En 7 pacientes afectados por enfermedad de Chagas crónica se procedió al estúdio biópsico del músculo gastrocnemio. Los pacientes seleccionados mostraran como única anormalidad la presencia de tests positivos en suero para la enfermedad de Chagas. Agrupamiento de fibras musculares de tipo I y II fué observado en 5 de aquellos; en 2 de estos últimos 5 se encontro, además, fibras anguladas que se teñian intensamente con NADH y esterase no especifica. Las observaciones hechas sugirieron la presencia de denervaçión seguida por reinervación, cuadro que puede ser observado con asiduidad en el músculo esquelético de pacientes con afecciones denervatorias bien compensadas y en los que no existen evidencias clinicas de ese compromiso.
REFERENCES 1.
C A M P B E L L , M. J . ; McCOMAS, A. J . A P E T I T O , F . —- P h y s i o l o g i c a l c h a n g e s In a g e i n g m u s c l e s . J . N e u r o l . N e u r o s u r g . P s y c h i a t . ( L o n d o n ) 36:174. 1973.
2.
K A R P A T I , G. & E N G E L , W . K . — T y p e g r o u p i n g i n s k e l e t a l e x p e r i m e n t a l reinervation. N e u r o l o g y ( M i n n e a p o l i s ) 18:447, 1968.
3.
L A G U E N S , R. P . ; COSSIO, P . M.; D I E Z , C ; S E G A L . Α . ; VAZQUEZ, C ; K R E U T ¬ ZER, E . ; K H O U R Y , E . & A R A N A. R. Μ — I n m u n o p a t h o l o g i c a n d m o r p h o l o g i c s t u d i e s of s k e l e t a l m u s c l e i n C h a g a s ' d i s e a s e . A m J . P a t h o l o g y (Chicago) 80:163. 1975.
4.
P A G A N O , Μ. Α . ; A R I S T I M U Ñ O . G. G.; B A S S O . S U S A N A ; COLOMBI, A. & SICA. R. E . P . — E l e c t r o m y o g r a p h i c a l f i n d i n g s i n chronic C h a g a s ' d i s e a s e . A r a . N e u r o ¬ P s i q u i a t . ( S ã o P a u l o ) 36:316, 1978.
5.
SANZ, OLGA, P . ; R A T U S N U . A. F . : A R I S T l M U Ñ O . G. G.; O ' N E I L L , Ε . M. & SICA, R. E . P . — A n e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n of s k e l e t a l m u s c l e in h u m a n chronic Chagas' d i s e a s e . Arq. N e u r o - P s i q u i a t . ( S ã o P a u l o ) 36:319, 1978.
6.
SICA, R. E . P . ; SANZ, OLGA P . & COLOMBI, A. — T h e effect of a g e i n g the human soleus muscle. Medicina ( B u e n o s A i r e s ) 36:443, 1976.
Reprint request:
Dr.. R. Hospital
muscle
after
upon
E. P. SICA — Sección de Electroneurofisiologia Clinica ·— Ramos Mejia — Urquiza 609 -— Buenos Aires — A r g e n t i n a .
TARATUTO FUMO
Μ. A. PAGNO OLG P. SANZ R.
E. P.
SICA
In a recent electrophysiological investigation Pagano et al. demonstrated the presence of mild denervation in the skeletal muscle of patients with chronic Chagas' disease. Later on, the Ramos Mejia Hospital's group confirmed the findings of Pagano et al. and pointed out the spinal alpha motoneurone soma as the site of the lesion within the motor unit (m.u.) . 4
e
The aim of the present study has been to obtain information concerning the eventual histological changes of the skeletal muscle of patients with chronic Chagas' disease. MATERIAL AND
METHODS
Open b i o p s i e s w e r e carried o u t u n d e r local a n a e s t h e s i a on t h e g a s t r o c n e m i u s m e d i a l h e a d m u s c l e i n 7 s u b j e c t s , 5 m a l e s a n d 2 f e m a l e s , a g e d b e t w e e n 25 and 44 y e a r s . A t h o r o u g h h i s t o r y w a s o b t a i n e d f r o m e a c h p a t i e n t and a c o m p l e t e clinical a n d laboratory examination w a s also performed; on these b a s e s the selected subjects w e r e j u d g e d to b e h e a l t h y , b e i n g t h e o n l y a b n o r m a l i t y d e t e c t e d t h e p r e s e n c e of 2 o u t of 3 p o s i t i v e s e r u m t e s t s for Chagas* d i s e a s e . N o n e of t h e m h a d b e e n p r e v i o u s l y t r e a t e d w i t h specific d r u g s . C o i n c i d e n t a l c a u s e s of m u s c l e d e n e r v a t i o n w e r e a v o i d e d b y r e j e c t i n g p a t i e n t s over t h e a g e of 60 y e a r s ι,β a n d t h o s e w i t h a h i s t o r y of t o x i c o r m e t a b o l i c d i s o r d e r s k n o w n to b e a s s o c i a t e d w i t h m u s c l e d e n e r v a t i o n . Also were eliminated those patients w i t h p e r s o n a l o r f a m i l y h i s t o r y of m u s c l e d i s e a s e . F r e s h m u s c l e s p e c i m e n s w e r e ffrozen r o z e n i n i s o p e n t a n e c o o l e d t o -150C i n liquid n i t r o g e n . C r y o s t a t s e c t i o n s w e r e c u t a t t h i c k n e s s o f 2 to 10 urn and w e r e s t a i n e d or reacted w i t h t h e f o l l o w i n g t e c h n i q u e s : h e m a e t o x i l i n - e o s i n , periodic acid Schiff, m o d i f i e d Gomori trichrome, rreduced educed nicotinamide adenine dinucleotide dehidrogenase ( N A D H ) , miofibrillar a d e n o s i n e t r i p h o s p h a t a s e ( A T P a s e ) a t p H 9.4 a n d 4.6. a l k a l i n e phosphatase and non-specific esterase. RESULTS T h e m o s t c o m m o n f i n d i n g w a s t h e p r e s e n c e of c l u s t e r s of u n i f o r m t y p e of m u s c l e fibres, e i t h e r t y p e I or t y p e I I , i n 56 of t h e 7 s t u d i e d p a t i e n t s . B o t h t y p e s of c l u s t e r s , p l a c e d s i d e b y s i d e , c o u l d o f t e n b e f o u n d i n t h e s a m e s p e c i m e n ( F i g u r e 1. Table 1).
Section Argentina.
of
Clinical
Electroneurophysiology,
Hospital
Ramos
Mejia,
Buenos
Aires.
328
ARQ-
NEURO-PSIQUIATRIA
(8Ã0
PAULO)
VOL.
36 No k, DEZEMBRO, Λ
1978
T h e c l u s t e r s w e r e c o m p o s e d of a b o u t 16 t o 30 fibres, b e i n g s m a l l i n 4 w h i l e i n t h e r e m a i n i n g o n e m u c h l a r g e r c l u s t e r s could b e s e e n ( T a b l e 1 ) .
patients
I n p a t i e n t s V. Ch a n d O. R. s m a l l a n g u l a r f i b r e s , w h i c h s t a i n e d o r reacted s t r o n g l y w i t h non-specific s t e r a s e and N A D H t e c h n i q u e s , w e r e a l s o o b s e r v e d ( F i g u r e 2, T a b l e 1). Neither
degenerative
muscle
changes
nor
architectural
abnormalities
were
seen.
DISCUSSION
The present study has shown the presence of scattered at random denervated atrophic muscle fibres and fibre type grouping, both features were found twice in the same sample, while in the other three patients only grouped fibres of either type I or type II were seen. 2
These observations strongly suggest denervation associated with reservation , nevertheless these findings do not imply an associated clinical symptomatology, for fibre type grouping can be the only finding in clinically uninvolved muscles when there has been an adequate compensation in a chronic denervation. 3
It should be mentioned that in a previous study Laguens et cã. found in the human biceps of EVI-positive chagasic patients non specific muscle histological changes, namely isolated hyalinazed fibres, increased number of central nuclei in some fibres and occasionally sprouting buds containing numerous nuclei. They did not find alterations of succinate dehydrogenase and ATPase activities and no clear distinction between fibre types could be done by employing the phosphorilase technique. The changes observed in our histological study, which resulted from denervation, do not point out the site of lesion within the neurogenic component of the m.u., for either the axon or the motoneurone soma may bring about the same histological picture. However, the electrophysiological evidences presented by Sanz et al. hint that the motoneurone soma may be the site of 5
330
ARQ-
NBURO-PBJQUIATRIA
(BAO PAULO)
VOL.
36, Ν* k, DEZEMBRO,
1978
lesion. Their ideas agree with our findings in two aspects: first the observed muscle changes may be the result of motoneurone soma involvement and second, the described anatomical features can be found in* partially denervated subjects who have recovered from a denervatory disease and, eventually, have not any clinical symptomatology. In conclusion, the present investigation has confirmed electrophysiological studies in which an involvement of the m.u. was reported in patients with chronic Chagas' disease ' . Because the approach applied here has been a morphological rather than a physiological one. it has had the advantage of showing anatomical evidences of muscle denervation which are compatible with an old and well compensated denervatory process which, at the time of the investigation, may be below the clinical level of detection. 4 5
SUMMARY
Gastrocnemius muscle biopsies were performed in 7 subjects with chronic Chagas' disease. On clinical and laboratory grounds the selected patients were judged to be healthy, being the only abnormality found the presence of positive serum tests for Chagas' disease. Fibre type grouping of either type I or type II was observed in 5 of the 7 patients. Furthermore, in 2 of the 5 patients showing muscle fibre groupings, angular fibres reacting with NADH and non-specific sterase were also found. These observations strongly suggest denervation associated with reinervation. This picture often can be observed in the skeletal muscle of patients with well compensated denervatory conditions who do not show clinical evidences of denervation.
RESUMEN
Cambios histológicos e histoquimicos del músculo esquelético en la enfer¬ medad de Chagas crónica. En 7 pacientes afectados por enfermedad de Chagas crónica se procedió al estúdio biópsico del músculo gastrocnemio. Los pacientes seleccionados mostraran como única anormalidad la presencia de tests positivos en suero para la enfermedad de Chagas. Agrupamiento de fibras musculares de tipo I y II fué observado en 5 de aquellos; en 2 de estos últimos 5 se encontro, además, fibras anguladas que se teñian intensamente con NADH y esterase no especifica. Las observaciones hechas sugirieron la presencia de denervaçión seguida por reinervación, cuadro que puede ser observado con asiduidad en el músculo esquelético de pacientes con afecciones denervatorias bien compensadas y en los que no existen evidencias clinicas de ese compromiso.
REFERENCES 1.
C A M P B E L L , M. J . ; McCOMAS, A. J . A P E T I T O , F . —- P h y s i o l o g i c a l c h a n g e s In a g e i n g m u s c l e s . J . N e u r o l . N e u r o s u r g . P s y c h i a t . ( L o n d o n ) 36:174. 1973.
2.
K A R P A T I , G. & E N G E L , W . K . — T y p e g r o u p i n g i n s k e l e t a l e x p e r i m e n t a l reinervation. N e u r o l o g y ( M i n n e a p o l i s ) 18:447, 1968.
3.
L A G U E N S , R. P . ; COSSIO, P . M.; D I E Z , C ; S E G A L . Α . ; VAZQUEZ, C ; K R E U T ¬ ZER, E . ; K H O U R Y , E . & A R A N A. R. Μ — I n m u n o p a t h o l o g i c a n d m o r p h o l o g i c s t u d i e s of s k e l e t a l m u s c l e i n C h a g a s ' d i s e a s e . A m J . P a t h o l o g y (Chicago) 80:163. 1975.
4.
P A G A N O , Μ. Α . ; A R I S T I M U Ñ O . G. G.; B A S S O . S U S A N A ; COLOMBI, A. & SICA. R. E . P . — E l e c t r o m y o g r a p h i c a l f i n d i n g s i n chronic C h a g a s ' d i s e a s e . A r a . N e u r o ¬ P s i q u i a t . ( S ã o P a u l o ) 36:316, 1978.
5.
SANZ, OLGA, P . ; R A T U S N U . A. F . : A R I S T l M U Ñ O . G. G.; O ' N E I L L , Ε . M. & SICA, R. E . P . — A n e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n of s k e l e t a l m u s c l e in h u m a n chronic Chagas' d i s e a s e . Arq. N e u r o - P s i q u i a t . ( S ã o P a u l o ) 36:319, 1978.
6.
SICA, R. E . P . ; SANZ, OLGA P . & COLOMBI, A. — T h e effect of a g e i n g the human soleus muscle. Medicina ( B u e n o s A i r e s ) 36:443, 1976.
Reprint request:
Dr.. R. Hospital
muscle
after
upon
E. P. SICA — Sección de Electroneurofisiologia Clinica ·— Ramos Mejia — Urquiza 609 -— Buenos Aires — A r g e n t i n a .
