Studies of the gastrocnemius muscle were carried out in 4 month old cockerels of the laying hybrid after hypokinesis lasting 15 and 30 days. It was found that restricted movement resulted in dystrophic changes of myotibrils, enlargement of the sarcoplasmic reticulum and oedem of interfibrillar spaces. Histochemical  studies revealed focuses of increased activity of non-specific esterase, decreased activity of dehydrogenase of lactic acid and a positive reaction of acid phosphatase

Belak, M.

Boda, K.

Kocisova, J.

Marcanik, J.

Skarda, R.

English

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M
NASA TECHNICAL MEMORANDUM
	 NASA TM-76717
ELECTRON MICROSCOPICAL AND HISTOCHEMICAL STUDIES ON THE
TRANSVERSE STRIATED MUSCLES OF BIRDS AFTER PROLONGED HYPOKINESIS
M. Belak, J. Kocisova,
J, Marcank, K. Bod ► a and
R. Skarda
(NASA-Til-76717) ELECTRC.N MicROSCOPICAZ AND	 N82-11754
HISTOCHEMICAL STUDIBS CN THE 19ANSVERSE
STRIATED MUSCLES OF 13IAIDS AY' PROLCNGED
HYPOKIN''SIS (Naticual Aeronautics and Space 	 Uncles
Administration) 14 p HC A02/ME A01 CSCL 06C G3/51 27782
Translation of "Elektronenmikroscopische and histochemische
Studien an der quergestreiften Muskulatur tier Voegel nach
la;ngfristiger Hypokinese", Archiv fuer Experimentalle Veterin.
armedizin, Vol. 33, No. 1, 1979, PP 37-46.
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NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
WASHINGTON, D. C. 20546
	 AUGUST 1981
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1STOCHEMICAL STUDIES ON THE'TRANSVERSE
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M. Belak, J.	 Xociaova., J. Mareanik,
K. Bod r a, R. Skarda 1t. WV% unit N..
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NAS%-R. Pnrinrmrn, 0eveni.eflon tleme and Address
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Translation
14. Moss•dM A. Mi caw
12. rin, A .w y N^. .n/ AHo
^t^tiona^ ,sronautics
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 and Spam Adiinistratiom
Washington, D.C.
	
40546
1s. sopplementerrN .Ns 	 Trans lati on of "Elektronenmikroseopische and
histochemische Studien an der quergeetreiften Muskulatur der
Voegel nach langfristiger Hypokinese",Archiv fuer Experimentalle
Veterinarmedizin, Vol.	 33, No.	 1,	 1979, pp 37-46•
M! Abstract
	 Electronmic roscopical and histochemical studies of the
gastrocnemius muscle has been carried out in 4 month old cock-
erels of the laying hybrid after hypokinesis lasting 15 and 30
days.	 It was found that restricted movement resulted in dys-
trophic changes of myotibrils, enlargement of the sarcoplasmic
reticulum and oedem of interfibrillar spaces ' L	 Histochemical
studies revealed focuses of increased activity of non-specific
esterase, decreased activitry of dehydrogenase of lactic acid
and a positive reaction of acid phosphat.ase.
ORIGINAL PAGE IS
OF POOR QUALITY
17. Row Words (MiN1N O/ A Molo$ stotonew
Os►cLssitisfd - Onli^it^
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Unclassified Unclassified 14
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LZBOTMN MICROSOOPIOAL AND UISTOCIMMIOAL STUDIES ON THE TARNS-
"VERSE STRIATED MUSOLES OV WRDS AFTER PROLONGED •YPOKINESTS
M, Belak ., a. Kocisovao a. Maroaniks K. Bod l ao R. Skarda
* LU
In the previous studies (Sulk et al o 1977) 0 the ultrastruo-
ture or the mitochondria of the transverse striated muscles of
birds after 100 dalls of bypokinesIs and bypergravitatlon was des-
cribed with referezice to the effect of these stress states on the
ehanges in the above mentioned organelles.
Since Increasingly large lesions were always found in hypo-
kinetic states, in our further study ., we directed our attention to
the efreet of the limited movement on the overloaded tibia muscles.
1. Our studies
I.I. Material and methods
In the experiment, tour-montb old cockerels of layins hybrid-9
v,e ►e used. The test animals were kept in cages without possibility
or movement, The rood dishes and water oontalnors were in the
front part of the cage so that the animals had free access to the
food. The cockerels were divided Into three groups. The first
group Included 10 animals which were kept for 15 days in the above
described cages while the second group included 11 animals kept
under bypokinesis for 30 days. The third group represented con-
trol annuals who were permitted free movement. The excisions were
taken immediately after killing from the M. gastronemius and pro-
cessed according 'to the metbode indicated in earlier publications
(Belak et al,. 1977).
Numbers in margin indicate p4gination of foreign text,
In order•
 to establish the activity of 'che acid pbospbatase
and non-speoific esterase, in the bistochemical study, asnocopu-
lation methods, aebydrogenasis of lactic aeld and a modified
water Incubation medium were applied (Lojda and Papousek, 1970).
1.2. Results and discussions
In view of the fact that the mitochondria of the skeletal
muscles form an accumulation center through their membrane arrange-
ment, and their main function consists in cell respiration, it was
possible in a disturbance of the normal processes in the first
phase of bypokinesis to find quickly the unfavorable effect. The
latter was manifested in the first group by the multiplication
and concentration over very large areas and in a thickening of the
internal membranes (Figure 1). Themitochondrial crests are very
numerous and show a continuous variation. We find only in the	 J'
later stages a mild transparency of the matrix with small vacuQles.,
Part of the mitochondria assume larger dimensions, while they are
sporadically bloated. Often mitochondria are affected whose length /40
exceeded the saroomere. The occurrence of increased ., concentrated
and gigantic mitochondria, supports the hypothesis of increased
energy needs and that of an increased metabolism in the dumaged
muscle. To this corresponded also the histological and chemical
finding of an increased activity of the non-specific esterase Mg-
ure 2). At the same time in this group we alsQ established grey and
as a rule round fat particles (Figure 3)., regarding whose occur-
rence we know, that they represent a source of energy for muscular
activity. The non-specific esterase hydrolyzes the esters of the
lower fatty acids which are then burnt up in the Krebs cycle of the
trioarbqnic acids, taking place in the undamaged mitoebondria, An
overflow product of the fat disintegration is water and it is there-
fore possible that under certain conditions or increase deQomposi-
t1on of esters of the fatty acids and with insufficient,fluid elim-
InatIon from the tissues, there should be accumulation in the
tissues and oedema should arise,
2
Figure 1 .
large area.
that their
30 300
Mitochondria (MI) concentrated over a vary
They contain dense internal membranes so
structure is not very striking. Enlargement:
Figure 2. Increased
in dystrophic muscle
3.2.
activity of the non-specifi c esterase
fibers. Enlargement: Obj: 20, ocul.,
3
P4 Cz.
Figure 3.
closely
30300
Squeezed fat particles (F) are found between
The dystrophic process developing in the 15 days od hypokin-
etic animals involved almost all the components of the muscle
tissue. Extended cisterns of the sarcoplasmic reticulum (Figure
4) Nere observed. also changes in the myofibrils.
^r
9	 4
Figure 4. Extended cisterns of the ^;arcoplasmic reticulum
(SR) are visible near the Z-line. Fat particles (P)
Enlargement: 30300
CRI:'^ r.:. FACE IS
r C'. QUALITY
Figure 5. Disturbed Z lines (arrow) designated as zig-
zag line and vague boundary between I and A lines.
Enlargement 30300 x
.
F!gure 6. Reduced activity of dehydrogetiase of lactic
acid in the dystrophic muscle focus (arrow).
Enlargement.: Obj . 16, ocul . 's
5
n
The loss or transverse striation with no established bgund-
ary of actin and myocin fibers was particularly striking, The 	 M
Z-lines bounding the individual sa ►cQmeres lost their straight
course (Figure 5) which was described by many authors as an accom-
panying phenomenon of the dystrophic processes and is designated
as the so-called zig-zag (Z-) line. The dIfferenoes described by
us in the myofibrils, sarcomeres, Z-1ines and rtiitochondria ,  which
differed from the normal undamaged muscular tissue are still rever
sible.	 This is also confirmed by the reduced activity observed
by as in the dobydrogenase of lactic acid (Figure 6). Portugalov
(1971, 1976) and Ilyina Kakeuva et al,.(1976) describe similar
focus like dystrophic changes In animals exposed to bypokinesia
induced and other stress states wbiab were not defined as specific,
short term and reversible. The dystrophic foci which we found are
reversible, since they occurred as a rule only in isolated muscle
fibers with preserved saroolemma so that a relative extensive regen-
eration of the transverse striated muscles is possible (Novotny,
1966). This hypothesis is also favored by the studies of JQzsa
(1974) who after traumatization of the skeletal muscles in rats
found, similarly to our results, a reduced dehydrogenase aotivJty
of the lactic acid.
In the group of animals whQ were kept for 30 days in conditions
of hypQkinesis I t was posqible to observe a considerable develop-
ment of dystrophic changes, manifested in the disintegration of
the muscle fibers (Figure 7). The myofibrils concerned exceeded
in number the undamaged fibers while they were mostly concentrated
in the process over large areas. Frequent interspaoes were also
noted between the myofibrils, while the latter were oedematic (Yig-
ure 8). The sarcomeres remained preserved but the Z-1ines showed,
less visible outlines ,  while the 1-band., lost their characteristic
structure. This ended in a destruction of the muscular tissue,
In the oedematous area there were fragments of the sarcQtubular
system with isolated myosin fibers (Figure 9)• The described changes
also included modifications in the tubular system.
Figure 7. Focus-like decay of the muscle fibers, mito-
chondria (MI) vacuolized (arrow). Enlargement: 30300 x
The positive reaction of the acid phosphatase was observed
In the h,ypokinetic animals in regularly defin ed areas in the muscle /42
tissue and was manifested in a darker color of the cell elements
which were generally arranged radially. Some of them formed foci
penetrating irregularly into the surrounding medium (Figure 10).
It seems that there is decomposition of the basic muscular sub-
stance. In muscular dystrophies, several authors (deRobertis, 1965;
David, 1967; Pitt, 1975) describe, beaide increased number of mito-
chondria, a multiplication of the lysosomes, whose missions appears
more clearly in the tissues with damaged function. Acid phosphates
cr,I ;'; Al FACE IS
C' r F'%OVR QUALITY
	
r
F, .
s
Figure. 8. Separated myofibrils (My) with oedematous
Intermediate spaces (arrow). Erilargement: 30300
Is located in these organelles and its in,^reared activity sup-
ports the hypothesis of its effect in the dystrophic muscle.
Morphometric calculations gave data concerning the number
and size of the mitochondria surfaces in the ind i vidually followed
time intervals. Thus, the average number of mitochondria was
lower for 15 days old hypokinetic birds, while the area was larger,
whereas the contrary happened in the group of 30 days old animals.
This finding is explained by the fact that in animals exposed to
hypokinesis for 30 days, which was manifested in increased require-
ments on the organism, a multiplication of young mitochondria) 	 X43
forms '.s obtained (Portugalov, 1971; Frol.ov, 1972) which can test-
ify to an adaptation in the e..fort at compensation of this stress
situation.
All the changes described here reached their maximum on the
30th day of hypokinesis which proves that the organism has dealt
with the new effects and compensa-ed for them, adjusted to them and
gradually reached the same level. Portugalov (1976) and Melechin
8
and origin (1977) estab,ished that thes e, • hanges observed have
disappeared and the muscles have been re ster •ed morphologically
and histochemically to their initial state when the animal+
return to normal conditions.
t	 2 v`.s	 .r. ^..
,^A
i
i
1 pure 9. I-bands (I) in the damaged sarcomeres lost
their striking structure, in the oedematous area there
are fragments of the sarcotubular system (SR) with
Isolated myosin fibers (arrows)
:nlargement: 30300
In conclusion, it may be stated that our observations cor-
respond to the typical picture of a dystrophic process, as was
described by many authors for different nutritional deficiencies
(Cheville, 1966; Shafiq, 1971), hereditary dystrophies ;Julian,
1973), myopathies (Jones, 1974) and in the administration of toxic
substances. The lack of activity of an organ or its weakened
function caused by limitation of movement or total hypokinesis,
Cl- ,,,,R QUALITY
	 9
A144,
110
	
ft .~^
Figure 10. Increased activit,; of the acid phosphatase
in the dystrophic muscle focuc
Enlargement: Obi. 20; ocul. 3.2
cause a larKe numbt• r of cytochemical indices, which Is mani-
fested In the last phase in morphological changes. The primary
cause resides probably in the inadequate o: interrupted perfu-
sion of the muscle which when c3ntinued very long leads to the
development of pathological changes. It is necessary to find
the limits, for which the changes are still reversible since we
were not able to rind any similar stage in the degree of the
pathological changes. In some cases, these alterations encom-
passed larger areas of muscle tissues, or again only individual
groups of fibers.
13,
LITERATURE	 C.5
Belak $ M., Kocisova, ►1, and Bod'a. X, (1977): Study of the Ultra-
structure of Mltoebondria in the Transverse Striated Muscles
of Birds In Case of Experimentally Induced Hypokinesis, Arab,
exRer. Vet. med. 31,. 537.
Belak, M., Kocisova, J. and Bod'a ., K. (1977): Electron Micro-
scopical Study of Mitoebondria in the Transverse Striated Muscles
of Birds with Experimentally Induced Hypergravltatlono Arch.
exper. Vet. med., 31, 749•
Chev.tlle ,  N. F. (1966^: The Pathology of Vitamin E Deficiency in
the Chick. Path. vet. 3 $ 208.
DeRoberts, E., Nowinski, W. W, and Saez, V. S. (1965); Cell
Biology, p. 146. W. B, Saunders Company, Philadelphia, London..
Frolov, V. A. (1973): Possible Role of Lysosomes in the Reproduc,
tIon Processes of Hyocardl al. Mitochondria ., Arab. Rat. 32 ,  22.
Ilyina-Kakueva ., E. I., Portugalov, V. V. and Krivenkova, N. P.(1976): Space Environ, Med. 47, 700.
Jones, J. M., King, N. R. and Mulliner, M. M. (1974): Degenerative
Myopatby in Turkey Breeder Hens. A Comparitive Study of Normal
and Affected Musule, Br. Poult.,Sci., 15, 194.
Jozsa, L. (197 4 ): Histoobemical Study of Traumatized Muscle,
Folia Histoobem. Cytocbem• 12, 157,
Julian, 1. M. (.1973): Animal Model of Human Disease; Hereditary
Muscular Dystrophy of Chickens., Amer. J. Path. 70, 273.
Melecbln ,  G. P., Orldin ., N. J. (1977): Physiology of Agricultural
Birds, Kolos Publ. House, Moscow.
Novotny, E. Boebm, R., Geissel, V. and Holman ., J. (1966); Veter-
inary Histology Stat. zemed. nakl., Prague4
Pitt, D. (1975): Lysosomes and Cell Function. page 165, Longman,
Lor.don, New York.
Portugalov., V. V., Ilyina-Kakueva lE,1., Starostin, V. 1,, RQcb-
lenko, K. D. and Savik, Z. F.	 97
1
 ): Structural andCytQ-
chemi,., al Changes In the Skeletal Muscles of Rats with Limited
Mobility-, Arch. anat. - gist. embryol. LXI, 82,
Shafiq ,  6, A., Askanas ,  V. and Milborat ., A. T, (1974); Fiber Types
and Preclinleal Changes In Cblcker^ Muscular Dystrophy, Arch,
Neurol. 25, 560.
WAddresses of the authors:
M`; Dr Milan Belak, DrSc. Central Laboratory of Electron Micro-
scopy of the University of Veterinary Medicine, 04181, Kosice,
Komenskeho 73, CSSR; MIIDr Jolana Kocisova, GSc and Prof. MVDr
Koloman Bod'a,, DrSc, Physiology Institute for Domestic Animals of
the Slovak Academy of Sciences, 04000 Kosice, Palackebo, 36, CSSR;
MVDr Jozef Marcanik, CSc, and Doc. MVDr Rudolf Skarda, CSc,
Instlt! to of Pathological Anatomy and Histology of the University
of Veterinary Medicine, 04181 Kosice Komenskeho 73 CSSR.
/46


Electron microscopical and histochemical studies on the transverse striated muscles of birds after prolonged hypokinesis

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