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3.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 andR. Skarda

(NASA-Til-76717) ELECTRC.N MicROSCOPICAZ AND N82-11754

HISTOCHEMICAL STUDIBS CN THE 19ANSVERSESTRIATED MUSCLES OF 13IAIDS AY' PROLCNGEDHYPOKIN''SIS (Naticual Aeronautics and Space Uncles

Administration) 14 p HC A02/ME A01 CSCL 06C G3/51 27782

Translation of "Elektronenmikroscopische and histochemischeStudien an der quergestreiften Muskulatur tier Voegel nachla;ngfristiger Hypokinese", Archiv fuer Experimentalle Veterin.armedizin, Vol. 33, No. 1, 1979, PP 37-46.

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NATIONAL AERONAUTICS AND SPACE ADMINISTRATIONWASHINGTON, D. C. 20546 AUGUST 1981

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AUGIJST ^ ^$^1STOCHEMICAL STUDIES ON THE'TRANSVERSE

(4 r«he.iwe00"A sells.CodeSTRIATED MUSCLES OF BIRDS AFTER PROLONGEDHyPnKTNRqT _R_3P. AW*00441 •. r«r.n.i"s O l«weefto R.rwr NaM. Belak, J. Xociaova., J. Mareanik,K. Bod r a, R. Skarda 1t. WV% unit N..

II. Contrast « Great No.NAS%-R. Pnrinrmrn, 0eveni.eflon tleme and Address

SCITRAN

Box A 9.2IL T ip. of Report and Posed CNv.4

Translation

14. Moss•dM A. Mi caw12. rin, A .w y N^. .n/ AHo^t^tiona^ ,sronautics

I^r and Spam Adiinistratiom

Washington, D.C. 40546

1s. sopplementerrN .Ns Trans lati on of "Elektronenmikroseopische andhistochemische Studien an der quergeetreiften Muskulatur derVoegel nach langfristiger Hypokinese",Archiv fuer ExperimentalleVeterinarmedizin, Vol. 33, No. 1, 1979, pp 37-46•

M! Abstract Electronmic roscopical and histochemical studies of thegastrocnemius muscle has been carried out in 4 month old cock-erels of the laying hybrid after hypokinesis lasting 15 and 30days. It was found that restricted movement resulted in dys-trophic changes of myotibrils, enlargement of the sarcoplasmicreticulum and oedem of interfibrillar spaces ' L Histochemicalstudies revealed focuses of increased activity of non-specificesterase, decreased activitry of dehydrogenase of lactic acidand a positive reaction of acid phosphat.ase.

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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 ofbirds after 100 dalls of bypokinesIs and bypergravitatlon was des-cribed with referezice to the effect of these stress states on theehanges in the above mentioned organelles.

Since Increasingly large lesions were always found in hypo-kinetic states, in our further study ., we directed our attention tothe 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-9v,e ►e used. The test animals were kept in cages without possibilityor movement, The rood dishes and water oontalnors were in thefront part of the cage so that the animals had free access to thefood. The cockerels were divided Into three groups. The firstgroup Included 10 animals which were kept for 15 days in the abovedescribed cages while the second group included 11 animals keptunder bypokinesis for 30 days. The third group represented con-trol annuals who were permitted free movement. The excisions weretaken 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 modifiedwater Incubation medium were applied (Lojda and Papousek, 1970).

1.2. Results and discussions

In view of the fact that the mitochondria of the skeletalmuscles 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. Thelatter 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 aresporadically 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 dumagedmuscle. 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, Anoverflow 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 their30 300

Mitochondria (MI) concentrated over a varyThey contain dense internal membranes so

structure is not very striking. Enlargement:

Figure 2. Increasedin dystrophic muscle3.2.

activity of the non-specifi c esterasefibers. Enlargement: Obj: 20, ocul.,

3P4 Cz.

Figure 3.closely30300

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 ISr 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 lacticacid 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 MZ-lines bounding the individual sa ►cQmeres lost their straightcourse (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 similarfocus like dystrophic changes In animals exposed to bypokinesiainduced and other stress states wbiab were not defined as specific,

short term and reversible. The dystrophic foci which we found arereversible, 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 wasas posqible to observe a considerable develop-ment of dystrophic changes, manifested in the disintegration of

the muscle fibers (Figure 7). The myofibrils concerned exceededin 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 oedematousIntermediate 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 morphologicallyand histochemically to their initial state when the animal+

return to normal conditions.

t 2 v`.s .r. ^..

,^Ai

i

1 pure 9. I-bands (I) in the damaged sarcomeres losttheir striking structure, in the oedematous area thereare fragments of the sarcotubular system (SR) withIsolated 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

A

144,

110 ft .~^

Figure 10. Increased activit,; of the acid phosphatasein the dystrophic muscle focucEnlargement: 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 Musclesof 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 Musclesof Birds with Experimentally Induced Hypergravltatlono Arch.exper. Vet. med., 31, 749•

Chev.tlle ,, N. F. (1966^: The Pathology of Vitamin E Deficiency inthe Chick. Path. vet. 3 $ 208.

DeRoberts, E., Nowinski, W. W, and Saez, V. S. (1965); CellBiology, 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): DegenerativeMyopatby in Turkey Breeder Hens. A Comparitive Study of Normaland 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; HereditaryMuscular Dystrophy of Chickens., Amer. J. Path. 70, 273.

Melecbln ,, G. P., Orldin ., N. J. (1977): Physiology of AgriculturalBirds, 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 LimitedMobility-, Arch. anat. - gist. embryol. LXI, 82,

Shafiq ,, 6, A., Askanas ,, V. and Milborat ., A. T, (1974); Fiber Typesand Preclinleal Changes In Cblcker^ Muscular Dystrophy, Arch,Neurol. 25, 560.

W

Addresses 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.

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