TABLE OF CONTENTS
Page
ACKNOW LEDGEM ENT...........................................................................

1

I N T R O D U C T I O N .................................

2

LITERATURE r e v i e w ...........

3

MATERIALS AND I I E T H O D S ......................

11

RESULTS

:FEXPERIMENT I

....................

16

RESULTS

CFEXPERIMENT II

..................

37

RESULTS

OFEXPERIMENT III

..................

51

D I S C U S S I O N ...................................

60

SUMMARV' AND C O N C L U S I O N S ....................

85

B I B L I O G R A P H Y ..............................

87

PLATES

.......................................

1-14

ACKNOWLEDGEMENT

The author wishes to express his sincere thanks
to Dr, Frank Thorp, Jr., u n d e r whose inspiration, con­
stant supervision, and unfailing interests this inves­
tigation was undertaken and the results are herewith
dedicated.
He is also greatly indebted to Dr. E. T. Hallman
for his kind guidance and valuable help in checking the
pathological tissues.
Grateful acknowledgement is also due to urs. R.
F. Langham a n d E. S. Fee n s t r a for their help in taking
the photomicrographs and pictures.

INTRODUCTION

For many years muscular dystrophy due to d i e t a r y
insufficiency has been postulated*

The investigational

work has been for the most part concentrated on the fac­
tors, producing muscular dystrophy*

Although some w o r k

has been done concerning the his to pathology of nutrition­
al muscular dystrophy,

there has been no satisfactory

work showing the complete picture*

To fill this £h-p,

guinea pigs were used as the experimental animal* a t t e n ­
tion was focused on the pathology of muscular dystrophy,
cell differentiation by vital staining and the repair
of damaged muscle fibers.

The findings recorded s h o u l d

be useful in the explanation of pathology of the w h i t e
muscle disease in lambs*

3

LITERATURE REVIEW

The production of generalized muscular lesions by
dietary means has been achieved by many workers in dif­
ferent species of animals.

Two factors have b e e n discuss­

ed as being important in the production of experimental
nutritional muscular dystrophy.
The first factor in question concerns the direct
and toxic effect of cod liver oil in the production of
muscular dystrophy.

This effect of cod liver oil upon

heart and skeletal muscles was demonstrated by Agduhr
(1926, 1927)

in white mice,

and calves; by Hoejer (1927)

rats, dogs, pig,
and Hendriksen

young rats, by Malmberg (1929)
(1931) in guinea nigs.

cat, rabbits,
(1928)

in

in children and Wahlin

These workers assumed that the

cod liver oil fed those animals in large doses over a
long period of time w a s noxious.
Supporting this view, Madsen, McCay, and Maynard
(1935)

demonstrated that cod liver oil added to a syn­

thetic diet or natural diet resulted in dystrophy of skelet
al and heart muscle in guinea pigs,
sheep.

rabbits, goats and

They held the saponifiable fraction of the cod

liver oil responsible for the development of the lesions.

4

fUy also

thought that some factor in the synthetic diet in­

creased the toxic effect of cod liver oil.

Confirming

these results concerning the harmfulness of the saponifi­
able fraction, McCey, Paul,

and Maynard (1928) demon­

strated that the hydrogfcnation of cod liver oil decreased
the production of muscular dystrophy in guinea pigs,
later, Davis, Maynard,

and McCay (1928) confirmed the

results obtained by Madsen _et al (1925); but denied the
synergetic action of the synthetic diet in the production
of dystrophy of skeletal muscles in guinea pigs.
Burack and Zimmerman (1927) reached the conclusion
that cod liver oil in therapeutic doses could not exert
the injurious effect in view of the small percentage of
rats and mice affected and the large amount of oil used.
A similar suggestion was made by Pavis and Maynard (1928)
who postulated that cod liver oil could be fed to dairy
calves in the amount that might be required to supply
vitamin I- without harm.
The second factor involved in the production of
dystrophy of skeletal muscle concerns the deficiency of
vitamin E in the diet which resulted from the presence
of rancid cod liver oil.

The destructive effect of

rancid cod liver oil on vitamin E also takes place in
the digestive tract.

This effect of cod liver oil may

be considered as indirect in the production of dystrophy

I

5

of skeletal m u s c l e .

The destruction of vitamin E by

oxidative reaction initiated toy autoxidation of cod
liver oil was f’ix*st demonstrated by Cummings and Mattill (1931) and Mattill (1938 b) who postulated that the
oxidative rancidity of animal fat may toe the principal
cause of muscular lesions and that the dystrophy can toe
prevented toy t h e

addition of two percent wheat germ oil

to a synthetic d i e t containing no other source of vitamin
E.

Mattill a g a i n

(1939) (cited by Shimotori et al 1940)

explained the production o f dystrophy in herbivora as
follows;

"Hertoivoruus animals have a large cecum where

the food remains

long e n o u g h for autoxidative changes

to progress furt.ta.er and m o r e rapidly than in omnivorous
animals such as rats.

F r o m this point of view the long

search for a t o x i c factor i n Cod liver oil and for cures
of the disorders

produced thereby (cattle, rabbits, poul­

try) may have b e e n following a wrong trail."
tional evidence,

With addi­

Mattill a n d Golumblc (1942) concluded

that no distinction should be made between cod liver oil
induced muscular dystrophy i n rabbits and nutritional
muscular dystrophy produced, by lack of vitamin E.
workers disproved the t h e o r y
a direct toxic action.
failed to produce

These

• that cod liver oil had

However, Cox and Roos (1934)

lesions i n rats even though they fed

them on a diet containing h i g h levels of cod liver oil.

It seems protable that nutritional muscular dys­
trophy is not due to a single dietary factor.

Muscular

dystrophy was produced in guinea pigs and rabbits w i t h
a diet containing no cod liver oil by Goettsch and Pappenheimer (1931), and Woodward and McCmy (1932).

These re­

sults would seem to exclude the fact that the toxicity
of cod liver oil was the only factor in the production
of nutritional muscular dystrophy.
In regard to vitamin £ deficiency being responsible
for muscular dystrophy,

considerable work has been done.

Muscular dystrophy was produced in rats reared on a vita­
min £ deficient diet by Olcott (1938), Evans, Emerson,
and Telford (1938), Knowlton and Hines (1938), Pappenheimer
(1939-1940) and by Mackenzie et al (1941 a) in the rabbit.
Shimotori, et al (1940) confirmed these findings and re­
ported that wheat germ, wheat germ oil and alpha-tocopherol
protected the guinea pig against nutritional muscular
dystrophy which can be produced in these animals w h e n
reared on the Madsen, McCay, and Maynard (1935) cod
liver oil supplemented diet.

Shimotori et al (1940)

suggested that vitamin E was the specific factor which
prevented nutritional muscular dystrophy when the vitamin
E complex was adequately supplied.

A similar suggestion

was made by Mackenzie and McCollum (1940).

Using the modi­

fied dystrophy producing diet 13 of Goettsch and P a p p e n­
heimer through the addition of ten percent defatted wheat

7

germ as a source of the water soluble factor, muscular
dystrophy was produced in rabbits.

The dystrophy was

accompaniedby an increased creatin output which was con­
sidered an accurate index of the inception of dystrophy.
The condition was cured b y administration of alpha-tocopherol.

Mackenzie and McCollum (1940) believed that

the deficiency of fat soluble factor, vitamin E, was
responsible for the dystrophy, and not water soluble
factors.

Mackenzie, Levine, and McCollum (1940) con­

firmed the previous f i n d i n g that the water soluble fac­
tor in wheat germ was not necessary for the treatment
of dystrophic conditions of skeletal muscles and further­
more that no quantitative or synergetic relationship
existed between alpha-tocopherol and the water soluble
factor*

Recently Pappenheimer (1943) in an excellent

literature review, stated £hat vitamin E was required
in the normal metabolism of skeletal muscle.
Contrary to all these facts, Harris (1941) and
Minot, and Frank (1944) reported no therapeutic effect
following the use of various preparations of vitamin E
in the treatment of progressive muscular dystrophy in
man.

These negative results obtained by Harris (1941),

and Minot,

et al (1944) w e r e explained by Milhoret and

Bartel (1945) who stated that the defect in the utiliza­
tion of tocopherol in progressive muscular dystrophy was

8

due to a deficiency in the reaction of the condensation
of tocopherol and inositol in the gas tro-Intestinal tract
and that the degree of this deficiency appeared to deter­
mine the rapidity with w h i c h muscular dystrophy progress­
ed.

They also pointed o u t that in mild cases patients

can synthesize sufficient amounts of the condensation
product if large amounts of both inositol and tocopherol
were given together, but those in which the disease pro­
gressed more rapidly would probably require the condensa­
tion product itself.
Groettsch a n d pappenheimer

(1931), Rogers et al

(1931), Olcott (1938), and Pappenheimer (1939, 1940)
found that the nutritional muscular dystrophy was confined
to skeletal muscle.

No alteration was found in the cen­

tral nervous system, in the large peripheral nerve trunks,
n or in terminal nerves and end plates.
It seems probable that other dietary constituents
were involved in muscular dystrophy.

Some evidence in­

dicates that a vitamin C deficient diet can produce mus­
cular dystrophy.

Such results were observed by Dalldorf

(1929) in guinea pigs reared on a vitamin C deficient
diet and also by HJarre and lilleenngen (1936) in calves.
There may be other factors which produce muscular
dystrophy in lambs and calves,

so called, "white muscle

disease" o r ("stiff lamb" disease).
muscles w e r e involved.

The heart and skeletal

This condition of young lambs three

to ten weeks old was first observed by Metzer and Hagan
(1927) in this country.

Marsh (1932) confirmed Metzer1s

observation and suggested that an Improper calcium-phos­
phorous ratio in the diet played a role in this condition.
Similar observations were made in lambs by tfillman et;
al (1934), Vawter and Records (1939), Sholl (1939),
Thorp (1942), Salyi (1942), Chdng (1945) and Cameron
(1945).

HJarre and Lilleenngen (1936) and Vawter and

Records (1947) reported a similar condition in calves
of ten days to two months of age.
The white muscle disease of lambs was cured by
Sholl (1939) and Willman e t al (1946) by administration
of wheat germ oil and vitamin E respectively.

Recently,

Vawter and Records (1947) reported that the condition
in calves disappeared within a week to ten days after
the diet of breeding cows was changed to green leafy al­
falfa or green pasture.

Both forms of forage**considered

good sources of vitamin E by Hathaway, et al (1932) and
Hathaway, et al (1934).
Muscular dystrophy can be caused by factors other
than dietary.

Toxic, infectious, and traumatic agents

can produce muscular changes.

Stemmier (1914) listed

the conditions under which muscular degeneration occurred:
Typhus abdominalis, diphtheria, pneumonia,
tetanus, peritonitis, venom intoxication,

scarlet fever,
and anaphy-

10

lactic conditions.

Forbus (1926) observed muscular de­

generation following pneumonia in man.

Similar changes

were observed by Steiner et al (1946) and by Clawson et
al (1947) in rhuematoid and rhuematic arthritis of man.
Muscular degeneration was produced experimentally
in various animals by Forbus (1926) following intra m u s ­
cular injection of powerful irritants or by cutting off
the blood supply; by Fishback and Fishback (1932 a, b)
who used different types of trauma; Clark and Blomfield
(1945), Clark (1946) by ligation of the artery to the
muscle groups.

Harman (1947) produced the condition by

acute ischemia induced in the hind limbs of rabbits.
In this laboratory muscular dystrophy was produced
in growing guinea pigs by feeding a synthetic diet ac­
cording to the formula of Davis et al C 1958)together
with a supplement of 0.5 gram per animal daily of u. S. P.
grade cod liver oil.

The pathological changes might be

attributed to the direct toxic effect of cod liver oil
as many workers have pointed out or to the destructive
effect on vitamin £ in the diet by rancidity as many
people have postulated, or finally to a synergism of
both factors.
Our aim was to study the pathology of nutritional
muscular dystrophy, not the etiology.

11

MATERIALS AHD METHODS

The objectives of the experimentswere to induce
the development of muscular dystrophy,

attempt to differ­

entiate the cells participating in the degenerative and
regenerative processes and to study the process of r e ­
generation of muscle cells at different stages in the
developmental process.
The guinea pigs used in the experiments were divided
into three groups of eight animals each.

They weighed

between 220 and 310 grams w h e n placed on the experiments.
Two guinea pigs from each group were kept as controls.
The controls were kept in a pen and fed rabbit pellets,x
lettuce, and grass.

The other six guinea pigs were con­

fined in individual cages with screen bottoms, no bedding
being used.

They were fed the basal synthetic diet used

by Davis, Maynard and Mccay (1938).

It was made up as

follows:
Casein
15 parts
Sucrose
15
M
Starch
33
"
Yeast
7
**
Lard
3
**
Salt mixture
4
*
^ R e g e n e r a t e d cellulose 20
**
x --- Rockland Rabbit Pellets
xx --- provided through the courtesy of i,r. Ralph T.
Cornwell, Sylvania Division American Viscose
Corp. Fredericksburg, Va.

12

The salt mixture was a modification of Osborne
nnft Hendel salt mixture prepared according to Hawk and

Oscr (1931).
pellets.

A H

guinea pigs were started on rabbit

The diet was gradually replaced by the synthetic

diet over a period of 3 to 4 days.

The basal synthetic

diet was kept in a refrigerator (approximately 40° F. )
throughout the entire experiment in order to avoid the
development of rancidity in the lard.
thetic ration was fed ad libitum.

The basal syn­

The guinea pigs on

the experiment received as a supplement 0.5 gram per
animal daily of cod. liver oil which had a potency of
not'than 1

2,000 U. S. P. units (54,600 units per

fluid ounce) of vitamin A per gram and 250 U. S. P.
units (6,800 units per fluid ounce) of vitamin D per
gram.
In addition, each animal,

including control guinea

pigs of Groups II and III received an ascorbic acid tab­
let, dissolved in one ml. of water, every other day.
Each tablet contained 0.39 grains equivalent to 500 in­
ternational units of vitamin C.

The cod liver oil and

aqueous solution of ascorbic acid were given the guinea
pigs separately by pipette.

The cod liver oil and as­

corbic acid tablets were stored in the refrigerator.
The guinea pigs were weighed every other day.
Group I was used to produce muscular dystrophy

13

with the dietary regimes just described.

The guinea

pigs were killed at different stages of the disease.
The symptoms consisted of decreased activity, difficulty
in walking and paralysis of fore and hind legs.
As an aid in the differentiation of the cells
participating in degeneration and regeneration of muscle
fibers, vital staining with trypan blue was carried out
with guinea pigs of Group II.

Following the technic of

Menkin (1929) and Russell ejt al (1943) the trypan blue
was given subcutaneously in the flank of the animal in
a one percent concentration in physiologic solution of
sodium chloride.

The first injection of the dye was made

on the 20th day of the experiment.

At that time the ani­

mals began to show inactivity which may be considered
as the first symptom of muscular dystrophy.

The amount

of dye given in each instance was determined by the weight
of the animal and was administered on alternate days.
The guinea pigs weighing from 400 to 450 grams were
given 35 mg of the dye each time until a total of 175
og had been administered.

Only 30 mg of the dye was

injected each time in guinea pigs weighing from 310 to
360 grams until a total 150 mg of the dye was introduced.
The plan was to kill the .animals at definite in­
tervals after the injections of trypan blue had b e e n
completed.

Howeger,

one guinea pig died after 3 injec-

14

tions and two others succumbed after four injections.
The remaining guinea pigs received five injections, and
were killed one day and ten days later.
The Group III animals were used to study the re­
generation of damaged muscle fibers at different stages
of the disease.

The guinea pigs were fed the same diet

and supplements as used for the Group I and II until
they began to show some difficulty in locomotion.

The

time of development of muscular dystrophy varied from
one animal to another.
closely observed.

Therefore, each guinea pig was

Whenever one began to show the first

symptoms of decreased activity followed b^ slow locomo­
tion, the diet was changed to that of the controls.

A s­

corbic acid was continued at this time but the cod liver
oil was discontinued.

The animals were killed at 1,

3,6,9, 12, 15 days after having been fed the normal diet.
Ether was used to kill the animals.

Each animal

was necropsied immediately after death or if living was
killed.

Materials were taken for histological study

from the heart, abdominal muscles,

tongue, masseter, and

the various muscles of the front and rear legs.

The

specimens were fixed both in Zenker*s fluid and formalin
(10 percent).

Formalin fixation was used only for vitally

stained material and those to be frozen.

Paraffin sec­

tions were cut 4 to 5 miera in thickness and stained

15

with Hematoxyline-eosin and Mallory*s aniline blue stain.
Eosin was used as a contrast in vitally stained sections.
The frozen sections were cut 15 mi era in thickness and
stained with Sudan IV.

RESULTS OF EXPERIMENT I

The body weights of the guinea pigs are shown in
Table I and Graph I.

A study of the table and graph

disclosed that there was a gradual increase in body weight*
However, a drop in the body weight occurred during the
first four days of the experiment.

This resulted from

the replacement of the rabbit pellet ration by the basal
synthetic diet.

The guinea pigs consumed very little of

the ration during the first four days of the trial.
ing with individuals,

Vary­

the body weights declined again

during the 2 2 n d to 20th day of the experiment.

The d e ­

crease in body weight occurred synchronously with the
appearance of clinical symptoms, which were decreased
activity,

slow locomotion,

difficulty in rising when

placed on the back, lack of body tone, and finally paralysis
of the fore and hind legs followed by death.

However,

some of the guinea pigs showed these symptoms and died
without drop in body weight.
Unfor t u n a t e l y , a comparison could not be made
between the body weights of the guinea pigs on the ex­
periment and the controls.

The controls did not show

regular and gradual increases in weight because supple­
ments of lettuce and grass were neglected for a few days.
The body weights declined during this time.

This was

remedied by supplementing the diet with an ascorbic acid
tablet, dissolved in one ml* of water, on alternate days.

17

Case reports of individual guinea figs used in
the experiment follow.

18
TABLE Is

Days

The Body Weight of the Guinea Pigs.
(grams)

Guinea nigs on the exne rlment
No. 1 No. 2 No. 3 No. 4 No. B

No. 6

Controls
No. 7 No. 8

Initial

293

289.6

294

301

291

310.6

288. 4

295.5

2

297

263.4

264

301

274

318.4

301

306

4

273.8

258.6

242.6

290

253

319

296

271

6

276

275

261

311

274.5

322.4

295

270

_ 8

291

290

274

319

288

329

271

253

10

303

308

283

329

290

348

268

237

12

314

312

302

327

302

359

262

223

14

317.6

321

208

342

306

368

247

211

16

329

338

324

360

323

382

268

221

18

342

349

328

372

322

391

288

244

20

361

351

335

381

329

289

308

260

22

361

352

336

391

338

406

323

283

24

369

364

342

397

340

398

348

313

26

384

378

352

387

344

384

353

341

26

384

360

360

400

died

400

376

359

30

370

killed 371

409

384

401

371

32

319

353

393

349

410

380

33

killed

344

390

killed 429

380

killed killed

killed kille<

■$r

Group
No.

Animal
No.

Survi val
period
(d a y s )

I

5

26

B o d y
W e i g h t
Initial" Maximal’ Pinal
(g r a m s )
(g r a m s )
(g r a m s )
291

344

344

Clinical observation;
Guinea pig 5 reared on the basal synthetic diet
with supplements of 0.5 gram cod liver oil and Vitamin
C gained weight until found dead in the cage.

Toward

the end of the experiment the guinea pig became inactive
which was accompanied by general flabbiness,

decreased

body tone and difficulty in rising when placed on the back.
Autopsy findings:
The carcass was in good physical condition.

Prac­

tically all muscles of the fore and hind legs, especially
the gracilis, adductors, biceps femoris,

triceps brachii,

suprascapularis as well as the tack, abdominal and inter­
costal muscles were involved.

However,

and masseter seemed to be normal.

the diaphragmatic

The pathological change

appeared to be degeneration of the skeletal muscles.

The

degeneration in some muscles was localized and involved
only a part of the muscle, in others, the degeneration

20

was generalized and affected the entire muscle*

The

degenerated muscles were pale, yellowish gray streaked,
patchy and had a cooked and moist appearance (Figs. 1, 2).
They were easily torn and friable.

The abdominal, muscle

were so thin and atrophied that the viscera could be
observed through the abdominal wall*

In the longitudinal

view, the degeneration appeared as grayish streaks which
were mottled or continuous throughout the length of the
muscle*

The grayish streaks and patches were distributed

symetrically and bilaterally.
The right ventricle of the heart was dilated and
the muscle was pale.
The liver appeared fatty and mottled,

pneumonia

and congestion of the small intestine were present.

The

other viscera did not reveal any pathological changes.
Microscopic f indings;
Primary changes consisted of coagulation necrosis
in which the contractile substance acquired a hyaline,
waxy appearance.

(Fig. 3)

The muscle fibers involved

showed partial or total waxy degeneration.
of muscle fibers was taking place.

Regeneration

The histological pic­

ture was not uniform throughout a single fiber.
The affected fibers became swollen,

stained weak­

21

ly with •osin and were separated by edematous fluid.
The nuclear activity of affected fibers was increased.
The number of nuclei increased and were hyperchromatic.
Muscle fibers, undergoing degenerative changes, lost
first the cross and later the longitudinal striations,
became homogeifbus, translucent, and stained deeply with
cosin.

Some affected fibers at this stage broke into

small pieces, giving a granular appearance to the fiber.
The granular mass filled the sarcolemma of the fiber when
the sarcolemma was intact (Fig. 7, 8).

Some other af­

fected fibers did not break into granules but formed
clumps which were homogerfous and sometimes contained
vacuoles (Fig. 4, 7, 10).
Nuclei of affectedfibers were pyknotic,

staining

dark blue with hematoxyline-eosin and many of them had
undergone karyo6*i&s and karyolysis.
colemma was involved.

Usually,

the sar­

In some instances, however, the

destructive process was not severe -—

the contractile

substance was only hyalinated and the sarcolemma appeared
not to be affected.
The changes stimulated infiltration of histiocytes,
polymorphonuclear leucocytes, lymphocytes and plasma
cells; proliferation of fibroblasts and giant cell form­
ation, and finally multinucleated syncytial cell masses
(Figs. 5, 9, 10, 12, 13).

22

The Histiocytes were numerous; playing an active
role in removal of necrotic and hyalinated material*

The

necrotic part of the fiber became enveloped in giant cells
which engulfed the tissue debris which was observed in
these cells as blue-purple granules when sections were
stained with l£allory*s aniline blue stain.

The multinu-

cleated syncytial cell masses which were abundant and
formed islands in the fibroblastic tissue appeared to
be similar to the giant cells,

as

far as could be seen,

the multinucleated syncytial cell masses did not engulf
debris.

They appeared to be nonphagocytic and possibly

of muscular origin,

Sarcolemma which escaped destruction

were completely filled with muscle spindle cells, multinucleated syncytial masses, giant cells and histiodytes,
giving rise to a "muskelzellenschlauche” of Waldyer
(Fig, 10),

The number of infiltrating polymorphonuclear

leucocytes, lymphocytes and plasma cells was small*
some instance,

In

the older degenerated tissues were partially

calcified (Figs,

6, 11),

The regeneration of muscle fibers occurred syn­
chronously with degenerative alteration of the contrac­
tile substances

(Fig* 9).

The plasmodial outgrowths

were formed^ from the old damaged fibers.
their attachment to the old fiber.

They retained

Where the sarcolem-

mal sheaths were preserved these outgrowths extedded

23

through it in an even course, forming a cone-shaped
mass with a pointed tip.

The nuclei within the plasmodial

outgrowths were arranged in rows parallel to the long
axis of the old fiber and were centrally located.

The

nuclei were bladder shaped and contained one or two nucleo­
li.

A granular cytoplasm surrounded the nuclei.

These

outgrowths were stained purplish with hematoxyline-eosin.
Such outgrowths gave origin to new muscle fibers.
generation was a progressive process; therefore,

Re­
the

new fibers exhibited different stages of development.
Some of them had only longitudinal striation and the
nuclei were in rows centrally located.

Others showed

cross striation with peripherally located nuclei,
cross section,

in

the new fibers were of different diameters.

When the pointed tip of the sarcoplasmic strands
met an obstruction (necrotic tissue) in the growth pro­
cess they became expanded and formed large multinucleated
syncytial masses of sarcoplamm.
The complete regeneration of the muscle fibers
necessitates the presence of at least a single muscle
cell which developed new fibers by continuous growth, of
cytoplasm accompanied by division of nuclei.

The nuclear

division in regenerating muscle fibers took place by amitosis.
Although the diaphragmatic,

the masseter, and the

£4
tongue muscles were macroscopioally normal* microscopi­
cally degenerative changes w e r e revealed.

However* these

charges were not extensive* being confined to a small
number of muscle fibers.
The heart muscle showed hydropic degeneration.
Careful examination of the heart did not reveal hyaline
degeneration*
Case £

Group
No.

Animal
No.

Survival
period
(days)

1

2

28

B o d y
W e i g h t
Initial
Maximal
Final
(grams)
(grams)
(grams)
289

378

360

Clinical observ a t i o n :
Guinea pig 2 gained weight until the 26th day of
the experiment.

The body w e i g h t then declined f r o m 578

grams to 360 grams in two days.

A t the same time there

was a loss of activity* accompanied by weakness of the
fore and rear leg muscles*

slow locomotion,

lack of body

tone and finally difficulty in righting itself w h e n placed
on the back.

The toes of the r ear legs curled backward*

Indicating severe involvement of the extensor muscles.
Respiration w a s abdominal and labored.

The guinea pig

vomited on £7th and 28th days of the experiment.
animal was killed on the 28th day.

The

25

Autopsy findings:
The carcass was in fair physical condition*
skeletal muscles on both sides of the body,

The

especially

the tibialis anterior, biceps brachii and pectoral muscles
were light in color and appeared parboiled*
some whitish patches were observed.
cross cut muscles was moist*

Here and there

The surface of the

The diaphragmatic, masseter

and tongue muscles were apparently normal.
The right ventricle of the heart was dilated and
flaccid.
The liver showed some fatty degeneration.

The

stomach and small intestine were highly congested, being
involved with catarrhal inflammation.

The peyer*s patches

were swollen and projected into the lumen.

The lung

showed anthracosis.
Microscopic findings;
The skeletal muscles were primarily involved.

The

pathological changes were more or less similar to the
changes described for Case 1.

The dystrophy was well

pronounced especially in the abdominal,
oris, gracilis,

and triceps brachii.

quadriceps fem-

The diaphragmatic,

masseter and tongue muscles showed the least degenerative
changes.

The muscle fibers w ere involved partially or

totally.

Some fibers were swollen,

stained weakly with

26

eosin, while others were shrunken.
tudinal striations were lost.

The cross and longi­

However, in some fibers,

the cross striations were accentuated and appeared close
to each other, persisting even in the completely degener­
ated part of the muscle fiber, which had a homogenous
appearance.
The nuclei of necrotic fibers were pyJcnotic or
had disappeared.
not constant.

The presence of the sarcolemma was

In some instances, the sarcolemma retain­

ed its integrity.
The necrotic fibers were invadedby histiocytes.
A small number of polymormonuclear leucocytes, and lympho­
cytes were observed among the histiocytes.

The process

of removal of the necrotic areas was not as active as
in Case 1.

The giant cells, enveloping the necrotic

tissues, and the fibroblastic activity of the interstitial
connective tissue were also less.

Multinucleated syncy­

tial masses were rarely observed.
Myoregeneration was not pronounced.

Here and there

the formation of some new muscle fibers from the old fibers
was observed.
Some of the cardiac muscle fibers were swollen
and showed granular changes.

27

Case 3

Group
No.
I

Animal
No.
1

Survival
period
(d a y s )
32

B o d y
W e i g h t
Initial
Maximal
Final
(g r a m s )
(g r a m s )
(g r a m s )
293

384

319

Clinical observation:
Guinea pig 1 was doing well and grained weight
until the 27th day of the experiment.
activity of the animal decreased.
aggravated day by day.

At this time the

This condition became

Three days later the guinea pig

was scarcely able to w alk and the toes of the rear legs
curie a backward and the animal could not reach the food
and water containers.

The body v/eight dropped sharply

during this period of time (from 384 to 319 grams).

The

animal was killed on the 32nd day of the experiment.
Autopsy fin d i n g s ;
The carcass was in fair physical condition.

The

skeletal muscles on both sides of the body and the diaphragm
were involved and lesions appeared as whitish patches
or streaks scattered throughout the muscles.

However,

the masseter was apparently normal.
Both ventricles of the heart were dilated.

The

walls of the ventricles appeared thinner than normal,

88

and flaccid.
The liver showed fatty degeneration and was fragile.
The small intestine was highly congested and showed
catarrhal inflammation.

The lung, spleen, kidney and

other organs were apparently normal.
Microscopic findings:
Myodegeneration and well pronounced regeneration
of muscle fibers were observed.
swollen.

The muscle fibers were

The nuclei of the fibers were increased in

number, were hyperchromatic, bladaer-shaped, and located
centrally and peripherally.
fibers was pronounced.

The cross striation of some

There were other fibers which

had lost the cross and longitudinal striations and were
homogenous.

Some degeneratedfibers were fragmented and

contained deposits of calcium salts as evidenced by
staining dark blue with hematoxyline.
either pyknotlc or karyolytic.
ma was not constant.

The nuclei were

The presence of sarcolem­

Where the sarcolemma existed it

was filled with new muscle cells, histiocytes, giant cells
and multinucleated syncytial masses.
The necrotic part of the muscle fiber was invaded
by histiocytes and surrounded by large numbers of giant
cells.

Polymorphonuclear leucocytes, and lymphocytes

were observed to a lesser extent.

The interstitial con­

nective tissue was stimulated,
ic cells.

giving rise to fibroblast-

Multinucleated syncytial masses were scattered

throughout the fibroblastic tissue.
Myoregeneration was well pronounced.

The plasmodial

outgrowths from the remains of the fibers were at differ­
ent stages of development.

Some of these outgrowths were

very young and did not show striation, stained basophilic
and contained bladder-shaped nuclei which were centrally
located and surrounded by a granular sarcoplasm.

Others

were more mature and showed longitudinal striations.
The heart showed degenerative changes with cellu­
lar infiltration (Fig. 14).

Case 4

Group
No.

Animal
No.

I

6

Survival
period
(d a p s )
32

B o d y
We
Initial
maximal
(g r a m s )
(g r a m s )
310

406

i g Ji t
Final
(grams)
349

Clinical observation;
Guinea pig 6 gained weight gradually until the 21st
day of the experiment at which time the body weight started
to decline.

At the same time, the activity and locomotion

of the animal decreased.

This condition was more aggravated

so

day toy day.

Finally the animal developed paralysis of

the hind legs.

The fore legs were still functioning.

The guinea pig could not raise itself when placed on the
tack.

On the 32nd day, paralysis was generalized and the

animal refused to move.
dominal.

Respiration was labored and ab­

The diarrhea that developed on the 29th day

continued for 3 days.

The animal was killed on the

32nd day and autopsied.
Autopsy findings;
The carcass was in fair physical condition.

The

whitish patches and streaks were scattered throughout
the skeletal muscles with exception of the masseter.
The changes were well pronounced in the intercostal,

ab­

dominal, pectoral, gracilis, adductors and the triceps
brachii muscles.

The diaphragmatic muscle showed a few

whitish patches.

The degenerative changes were bilateral

and symmetrical.
Theright ventricle of the heart was dilated and
flaccid.

The wall of the right Ventricle was thinner

than normal.
The lung showed pneumonia.
fatty and was friable.

The liver appeared

The stomach and small intes­

tine were highly congested and showed catarrhal inflamma­
tion.

The urinary bladder was distended with urine.

31

The other organs appeared normal.
Microscopic f iridings ;
Practically all skeletal muscles were involved.
The type of cellular changes were more or less similar
to the cases described above.

The extent of degenerative

and regenerative processes varied from one muscle to an­
other, even between fibers.

In this animal the diaphrag­

matic and tongue muscles were more necrotic and showed
more deposits of calcium salts than the other guinea
pigs.
The pathological changes consisted of a coagulation
necrosis characterized by a hyaline waxy appearance fol­
lowed by myoregeneration.

Muscle fibers underwent coagu­

lation necrosis, breaking up into granular masses; in
some instances the necrotic fiber became homogei&us,
translucent, and stained evenly with eosin.

These necro­

tic fibers showed loss of nuclei and sarcolemma.
ally intact sarcolemma was observed.

Occasion­

The necrotic tissues

were invaded by histiocytes that developed into giant
cells which engulfed the necrotic debris.

When the sec­

tions were stained w i t h M a l l o r y ’s aniline blue, the en­
gulfed debris took the dark blue stain.

Such debris

was easily demonstrable in the giant cells.

The degenera­

tive process stimulated fibroblast proliferation which
was infiltrated by a small number of polymorphonuclear

32

leucocytes#

Tills fibroblastic tissue contained a large

number of multinucleated syncytial masses.

As far as

could be seen these cells had nothing to do with removal
of the necrotic debris#

They stained evenly with the

Mallory's aniline blue stain.
a dark red color.

The cytoplasm was stained

The nuclei were red in color.

When

the sarcolemma of the fiber was intact, it was complete­
ly packed with histiocytes,

spindle shaped muscle cells,

giant cells and multinucleated syncytial masses, giving
rise to the "muskelzellenschlauche" of Waldyer.
In some instances, for example in the quadriceps
femoris, the necrosis dominated the process of myoregeneration, while in other instances,

in the biceps femoris

and the biceps brachii, the myoregeneration predominated
ovtr the dystrophic changes.

The myoregeneration took

place synchronously with the degenerative alteration of
the contractile substance.

Here and there new plasmodial

outgrowths sprouted from the old fibers.

Some of them

had longitudinal striations with bladder-shaped nuclei,
centrally located.
The heart showed hyaline degeneration to some
extent in the right ventricle.

33

Case 5

Group
Animal
Burvlval
B o d y
h e i g h t
No.
No.
period
Initial
Maximal
Final
____ _______________ (days_)_____(g r a m s )
(g r a m s )
(grams )
I

3

33

294

371

344

Clinical observation:
Guinea pig 3 gained weight until the 29th day of
the experiments

Then,

during three days.

the body weight declined gradually

The activity and locomotion of the

animal decreased at the same time.

At the 30th day the

guinea pig was confined to the corner of the cage and
did not walk.

When the animal was placed on its back,

it did not attempt to rise and kept this position for
some minutes.

Finally,

the body lost tone.
labored.

general paralysis developed and

The respiration was abdominal and

The guinea pig was killed on the 33rd day of

the experiment.
Autopsy findings:
Practically all skeletal muscles were involved
and showed whitish streaks and patches throughout.

How­

ever, the tongue muscles and the masseter appeared normal.
Both ventricles of the heart were dilated.

The

34

wall8 of the ventricles were thin and flaccid.
The liver was friable and showed fatty degeneration.
The lungs and small intestine were highly congested.
The other viscera were apparently normal.
Microscopic fi nd i ng s:
The skeletal and diaphragmatic muscles were de­
generated and showed coagulation necrosis.

The masseter

and the tongue muscles were also involved to a minor ex­
tent.

Seme necrotic fibers were broken into small pieces;

others were homogenous; translucent and stained dark
red with eosin.

Calcium salts were deposited in some

necrotic tissues as evidenced by the dark staining.
the other hand,

On

some muscle fibers were swoxlen, the

diameter being increased two or three times.

The nuclei

were active, hyperchromatic and increased in size.
removal of the necrotic part was in progress.

The

The giant

cells and histiocytes engulfed the necrotic debris which
were noticeable in these cells as blue granules when
stained with the Mallory's aniline blue.

The formation

of fibroblasts and multinucleated syncytial masses was
very active where the removal of the necrotic tissues
was almost complete.
The regeneration of new fibers occurred simultan­
eously.

The other changes were similar to the cases de-

scribed above.
The heart showed fatty degeneration.

Case 6

Group
No.
I

Animal
No.

Survival
period
(d a y s )

4

B o d y
w e i g h t
Initial
Maximal
Final
(grams)
(grams)
(grams)

33

301

409

390

Clinical observation:
Guinea pig 4 increased in weight gradually until
the 29th day.

The animal showed decreased activity which

became aggravated day by day.
ly declined*
wal£.

The body weight concurrent­

O n the 31st day the animal could hardly

Paralysis of the fore and hind legs developed.

The guinea pig could not rise when placed on its back.
Respirations were abdominal and labored.

The animal was

killed on the 33rd day*
Autopsy f i n d i n g s ;
The animal was in good physical condition.

All

skeletal rauScles and the diaphragmatic showed whitish
patches and streaks.

The masseter was apparently normal.

The liver was fatty and friable.

The small intes-

36

tine was highly congested,
to the right apical lofce.

Pneumonia was confined
The other organs were normal.

Microscopic f i n d i n g s :
The pathological changes consisted of hyaline de­
generation and myoregeneration.

The type of reaction

was similar to that described for the previous cases.
The only difference was the extensive necrotic changes
in the skeletal muscles.

The masseter was also involved.

The regeneration was active,

resulting in some new fibers

at different stages of development.
The heart muscle showed hyaline degeneration.

37

RESULTS OF EXPERIMENT II

The body weights of all guinea pigs on this ex­
periment are shown in Table II, and Graph II.

It can

readily be seen that the body weights increased gradu­
ally until the 20th day of the experiment.

Increase in

body weights of controls during the same period of time
was more than that of the animals reared o n the basal
synthetic diet supplemented with cod liver oil and
ascorbic acid.
Body weights declined sharply after the guinea
pigs had received injections of a one percent solution
of trypan blue.
the dye.

Each animal responded differently to

Guinea pig 10 died after three injections,

while guinea pigs 9 and 11 succumbed after four doses.
Others remained alive after five injections had been
given.

The controls also responded to injections of

the dye by lo sing weight.

Due to the weight loss of

the controls it might be concluded that trypan blue in­
terfered with the gain in body weight, and was possibly
toxic for the guinea pigs.
of the animals.

Trypan blue hastened the death

38

TABLE IIs

4

H
e

Guinea nigs on the experiment
No. 9 No. 10 No. 11 No. 12 No. 13

0

Days

®ie Body Weights Of The Guinea Figs.
(grams)

Contro:Ls__
No. 15

Initial 282

281

272

280

316

272

279

292

£

£76

£98

£77

270

318

262

281

293

4

£79

£99

277

279

327

274

301

307

6

£86

304

£79

£94

332

292

314

317

8

£89

294

£69

299

339

299

329

324

10

£91

306

278

314

341

309

339

341

1£

304

321

£94

324

341

324

261

352

14

307

£97

304

327

339

312

370

363

16

316

324

299

344

356

332

387

374

18

32£

331

311

359

357

341

397

4

4

£0

337

££

340

346
317

24

337

301

321

£6

306

died

£8
30

311
321

4

372
384

4

4

391
4

367*
357

354
351

372

359

349

421

444

287

367

339

332

416

416

£74

264

349

342

302

399

409

died

died

killed

324

killed

389

409

killed

419

414
437

411S<
426

3£

289

34

266

416

killed

kill

*The guinea pigs received the first injection of trypan
blue*

...

* ft

+CT

i/

ir-.L'

// /

39

Case 7

Group
No.

Animal
No.

Survival
period
(d a y s )

2

10

26

B o d y
Initial
(grams)
281

W~ e 1 g h t
Maximal
Final
(grams)
(grams)
346

301

Clinioal observation;
Guinea pig 10 gained weight gradually until the
20th day at w h ich time the first Injection of the dye
was made.

The body weight declined after w h i c h the a c ­

tivity and appetite of the animal decreased.
Injections,

After two

the guinea pig stayed in a corner of the cage,

developed diarrhea which continued until the guinea pig
succumbed.

The skin, conjunctiva and feces were stained

intensely blue.

This was accompanied by flabbiness of

the muscles decreased a c tivity and difficulty on rising
when placed on its back.

The animal received three in­

jections of trypan-blue and died on the 26th day of the
experiment .
Autopsy fl ndi n g s :
The carcass w a s in f a i r physical condition.
skin, conjunctiva,

The

all skeletal muscles and viscera were

stained blue in color.

The lungs w ere purplish.

blue

coloration in the heart was confined to the sulci coronaria.
The right ventricle of the heart appeared dilated.

40

The liver was friable and appeared fatty.

The

stomach and small intestine were inflammed.
Microscopic f i n d i n g s :
A H

skeletal muscles were more or less involved.

The changes consisted of coagulation necrosis in which
the contractile substance acquired a hyaline waxy appear­
ance.

The degree of degeneration varied from one muscle

to another.

The pathological picture was similar to find­

ings recorded for experiment I.
len and weakly stained.
had disappeared.

Muscle fibers were swol­

cross and longitudinal striations

In some of the fibers,

cross striations

were accentuated, becoming closer to each other.

The

cross striations persisted in some of the necrotic fibers.
The contractile substance became homogenous,
breaking up into small pieces.

Nuclei of the affected

fibers were either pyknotic or karyolysed.
ma of the fibers was involved, however,
persisted.

The sarcolem­

it sometimes

These changes stimulated the infiltration

of histiocytes,
cytes.

translucent,

polymorphonuclear leucocytes and lympho­

The two last mentioned cells were few in number.

Hyaline clumps were invaded by histiocytes and often
enveloped in giant cells.
increased.

The fibroblastic activity

Spindle shaped muscle cells and multi-nucleated

syncytial masses were formed.

W hen the sarcolemma of

an affected fiber persisted it was completely filledby

41

histiocytes, giant cells,

spindle shaped muscle cells,

and multinucleated syncytial masses.

These cells phago-

cytosed the injected dye (Figs. 15, 16, 17).

However,

not all spindle shaped muscle cells and multinucleated
syncytial masses engulfed the trypan blue (Fig. 18).
The dye was present in these cells in the form of fine
granules.

In addition to these cells endothelial cells

of the capillary vessels p.nd fibroblasts also engulfed
the trypan blue (Fig. 19).
Myoregeneration was taking place at the same time,
but it had not progressed very far.
outgrowths were observed,

Some plasmodial

showing nuclei arranged in

rows and granular sarcoplasm.
The heart muscle had undergone hydropic degeneration.

Case 8

Group
No.
2

Animal
No.
9

Survival
period
(d a y s )
28

B o d y
W e i g h t
Ini ti al
Maximal
f i n a l '"
(grams)
(grams)
(grams)
28 2

340

274

Clinical o b s e r v a t i o n ;
Guinea pig 9 lost activity on the 22nd day and deve­
loped general flabbiness,

slow locomotion and difficulty

42

in righting itself when placed on the back.
time, the body weight declined.

At the same

This condition became

progressively worse and the animal died on the 28th day
of the experiment.

The guinea pig received four injections

of the dye, which was followed by intense coloration of
skin and conjunctiva*

The feces voided were also blue

in color.
Autopsy find i n g s :
The carcass was in poor physical condition.

prac­

tically all skeletal muscles and viscera were stained
blue.

The masseter and the latissimus dorsi were dark

purple in color.
to be swollen.

The right axillar lymph node was found
A small abscess was present in the right

masseter region.
Both ventricles of the heart appeared dilated.
The viscera were apparently normal.
Microscopic f i n d i n g s ;
All skeletal muscles showed more or less dystrophic
changes.

In comparison w ith case 7, it was found that

necrosis of the muscle fibers was more advanced.
wise the changes were similar to case 7.
giant cells,

Other­

Histiocytes,

some spindle shaped muscle cells and a few

of multinucleated syncytial masses phagocytosed the trypan

43

blue (Figs. 15, 16, 17, 18, 19).

T h e coarse granules

of the dye were observed in these cells.

The sections

of the popliteal lymph node showed that the reticulocytes,
small lymphocytes and macrophages h a d also absorbed the
dye.

(Fig*

22).

The heart showed no pathological changes.

Case 9

Group
No.

Animal
No.

Survival
period
(d a y s )

2

11

28

B o d y
W e i g h t
Initial
Maximal
Final
(g r a m s )
(g r ams)
(g r a m s )
27 2

321

269

Clinical observation:
Guinea pig 11 received four injections of the dye.
After two injections the increase in body weight ceased
and declined on the 24th day.

The decline in weight was

accompanied by loss of activity,

paresis of the hind legs

and bacJcward curling of the toes of r ear extremities.
The animal stopped eating aftt.r the 3 r d injeotion of the
dye, developed diarrhea which was greenish colored and
of an offensive odor.
day.

The guinea p i g died on the 28th

44

Autopsy fin di n gs ;
All skeletal muscles and viscera were stained
blue.

The gracilis and abdominal muscles appeared atrophied

and friable.
The heart was flaccid and the walls were thinner
than normal.
ariae.

It was stained blue along the sulci

coron-

The small intestine showed catarrhal inflammation.

Microscopic findings:
Prr.ctically all muscles,

especially the quadriceps

femoris, gracilis, gastrocnemius,

triceps brachii, ab­

dominal muscles and the tongue muscles were involved.
The pathological changes consisted of extensive coagula­
tion necrosis of the muscle fibers.

Cellular reaction

and myoregeneration were minor in extent.

Histiocytes

anu giant cells w iich phagocytosed the dye were rarely
observed.

The dye granules which appeared in these cells

were fine.

The spindle shaped muscle cells and multinu-

cleated syncytial masses were also scarce.

No trypan

blue could be seen in the cytoplasm of these cells.
Myoregeneration was almost nil.
The heart showed hyaline degeneration to a limited
extent.

None of the cells engulfed the dye.

45

Case 10

Group
No.

Animal
No.

Survi val
period
(d a y s )

12

28

2

B o d y
W e i g h t
Initial
Maximal
final
(g r a m s }
(g r a m s )
(g r a m s )
280

384

349

Clinical observation:
The body weight of guinea pig 12 increased gradu­
ally until the 22nd day of the experiment.

The animal

lost weight after two injections were applied.

At this

time the activity of the guinea pig decreased,

and loco­

motion became slow.

The animal received 5 injections of

the dye and was killed one day after the last injection
was made.
Autopsy f indings:
The carcass was in good physical condition.

The

skin, conjunctiva, and skeletal muscles were tinged blue.
The muscles especially the abdominal, gracilis, and ad­
ductors muscles appeared atrophied and were easily torn.
The heart was dilated and stained blue along the
sulci coronariae.
The viscera were blue in color.
stained blue.

The feces were

46

The liver was enlarged and Triable*
Microscopic f i n d i n g s ;
The skeletal muscles showed hyaline degeneration*
The degenerative processes were not extensive, being con­
fined

to a small number of fibers which were surx'ounded

by fairly normal muscle fibers.

The character of the de­

generative change was similar to the cases heretofore
mentioned.
Histiocytes and giant cells were filled with the
dye.

(Fig* 15, 16).

granules.

The engulfed dye appeared in coarse

Some spindle shaped muscle cells and a few

multinucleated syncytial masses phagoeytosed trypan blue
(Fig8 * 16, 18).

When the sarcolemma of fibers persisted

it was packed by these cells giving rise to the "muskelzellenschlauche w of Waldyer (Fig. 16).
In addition to these cells, histiocytes,

some fibro­

blastic cells of interstitial tissue and endothelial
cells o f

capillaries engulfed the dye.

A large number

of his-tiocytes were observed around blood vessels.

(Fig.

19, 2 0 ).
liJyoregeneration was in progress.
The heart showed hyaline degeneration and was in­
filtrated by histiocytes w h i c h had engulfed the trypan

47

blue (Fig. 21).

Case 11

Group
No.

Animal
No.

Survival
period
(d a y s )

2

14

28

£ o d v
W e i g h t
Initial
Maximal
Final
(g r a m s )
(g r a m s )
(g r a m s )
27 2

354

302

Clinical o b s e r v a t i o n ;
The increase in body weight of guifaea pig 14 con­
tinued gradually until the 20th day of the experiment.
The animal started to lose weight after the first injec­
tion of trypan blue.

This condition continued and became

aggravated when the animal had received four injections
of the dye.
blue.
parent.

The skin,

conjunctiva ana feces were stained

Decreased activity and slow locomotion became a p ­
The respirations were abdominal and labored.

The animal was killed one day after the fifth injection
had been administered.
Autopsy f i n d l n g s ;
The carcass was in good physical condition.
skeletal muscles,

The

skin and conjunctiva were stained blue.

The abdominal and pectoral muscles took on a deep blue
color.

They were thin and atrophied.

The masseter was

48

dark purple in color.
Both ventricles of the heart were dilated and blue
stain was present along the sulci caronariae.
The viscera were tinged blue.
larged and friable.

The liver was en­

The other organs appeared normal.

Microscopic find i ng s:
The skeletal muscles, especially the abdominal,
quadriceps, femoris, gastrocnemius, intercostal and tri­
ceps brachii were primarily involved.

The changes con­

sisted of coagulation necrosis of the muscle fibers and
was similar to the cases described heretofore.

The necro­

tic tissues were infiltrated by histiocytes and surround­
ed b_ giant cells.

These cells had phagocytosed the try­

pan blue which was coarsely granular.

Some spindle shaped

muscle cells and a few multinucleated syncytial masses
appearedto engulf the trypan blue.

The dye granules

were also observed in the cytoplasm of fibroblasts, histio­
cytes of connective tissue and endothelial cells of cap­
illaries.
The heart showed hyaline degeneration and was in­
filtrated with histiocytes, and showed absorbed fcye (Fig.

21).

49

12

C^ise

•roup
Ho.

Animal
SurvivKl
B o d y
/ / e i g h t
Ho.
period.
Ini ti al
Kaximal
Final
___________ (ddys )_____(grama)
(grams)
(grams)
17;

-4

£16

767

702

Clinical observation!
Guinea jIq 1^ g a i n e d weight gradually
20th day of the experiment;.

until the

The body weight declined

and the activity of the a n i m a l decreased on the c.ays fol­
lowing the first inje::t i o n s
shin,

of the try pan blue.

conjunctiva, -n' f e c e s were

animal had difficulty i n

6

completed.

a^s

after

The

rising w h e n placed on the bach

and locomotion was slow.
continued

stained blue.

The

"Decrease of the tody weight

t B e five injections had bee n

The aninm 1 v/c s hilled on the 74th dcy of the

experiment.
Autopsy f i n d i n g s :
The carcass wrs i n
shin, skeletal muscles

poor physical condition.

anc

The

conjunc iva were staine^ blue.

The abdominal and rear -Leg muscles showed atrophy.

The

madibula was easily fractured.
The right ventricle of tne heart was dilated.
heart was stained deeply

The

blue a l o n g the sulci coronariae.

4

50

The viscera, w e r e blue.
The l i v e r w as

enlarged

and

The l o n g s

shov/ed p n e u m o n i a .

friable.

M i c r o s copic f i n d i n g s ;
The

skeletal muscles,

especially

the

femoris , s e m i t e n d i n o s u s , g a e t r o c n e m i u s ,
a b d o m i n a l , and d i a p h r a g m a t i c
necrosis w h i c h
These

acquired

changes were

detail f o r the
ference

being

muscles

a hyaline

similar

to

showed

ca ses h e r e t o f o r e r e p o r t e d .
the

extensive

necrosis

was not

and

here

engulfed

observed.

A

syncytial

fi b r oblasti c tiss ue.
The

dy e

of

a lso

Some
had

01

T ne

in

only d i f ­

J/y©regeneration

s ome h i r t i o c y t e s

fine granules

sma ll n u m b e r

end m u l t i n u e l e a t e d

the dye.

tnere

appearance.

of m u s c l e f i b e r s

of c a l c i u m salts.

active.

coagulation

cnanges described

and large d e p o s i t i o n s

cells w i t h

triceps b r a e h i i ,

a nd w a x y

the

quadriceps

and

giant

of t r y p a n b l u e w e r e

spindle

shapec: m u s c l e

masses were
t h e se

scattered

c e l l s hso

ac u m u l a t e d

cells
in

the

phago c y t o s e d

i n the n e c r o t i c

tissues.
The
cium salts.
of the

aer.rt s h o w e d n e c r o s i s
Histiocytes

c: r d iac

muscle.

and

infiltrated

r e p o s i t i o n of
the

cal­

degenerated

a r e as

51

RESULTS

The bod^' w e i g h t s

CF o-Xrli.INEKT I I I

of all guinea p i g s

iment rie shown i n Table III ana Graph

XIX*

readily be s e e n that the tody weight o f
gradually.
of the

The

di f f erence between the

guinea p i g s

of the

final

ing w h e n

placed

slov; locomotion and

the

ox*

this e x p e r i m e n t

night be attributed tc

difference

ind i v i d u a l s

the sam e banal

s y n t h e t i c diet, cod liver*

acid w e r e

t h r o u g h o u t the three e x p e r i m e n t s .

ana outside

Experiment III w a s

set up during N o v e m b e r

and

in

s u p p l e m e n t e u cod liver oiJL

resulted in a u t o x i d o t i v e destruction of

i n y sus­
because

For
The

ex ­

October.

continued

s e ason

peristalsis of tne i n t e s t i n e

in

ascorbic

and

and

The h o t

stayed longer i n gastro-intestinal t r a c t
the s e p a r r tely

oil

conducted during A u g u s t

until the first w e e k of February.
decreased

ris­

Is *7 "th

tempercturc

dietary factors were c o n s t a n t .

periments I, II w e r e

caused

in

-tire d i s e a s e

ceptibility of

the

of

of the experiment.
o u r a t i c n of the course

this reason,

course

"t :a m

difficulty

The l o n g

used

con­

consisted

on tne La. el, appeared b e t w e e n

and the 44th d ay

-tlie

longer

The early symptoms v;hi e h

decreased act ivity,

of

weights

that t h e

c_iser.se i n this experiment was u.u.ch

in previous ones.

increased

body

that

It was noti c e d

exper­

It c a n

animals

on the experiment a n d

trols w : s not significant.

on t h i s

and

possibly
food

contact

which

with

probably

-vitamin E

i n ' the

58

aiet.

This v i t a m i n E deficient diet and cod liver oil

together s h o r t en ed the course of the disease and re sulted
in early ap pe ar an ce of symptoms in th$ experiments I and II.
C a s e reports of uguinea pigs u s e d in this experiment
follow.

53

TABLE III.

Days
Initial
2
4
6
8
10
18
14
16
10
80
82
84
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
62
64
66

The Body Weights of the Guinea Pigs.
(grams)

_ Gui n e a pigs on the experiment
ifo. ifr No. 18 No. 19 No. 4 6
no. 81 ^No.
859
272
284
282
296
312
324
337
324
336
356
374
376
401
410
422
439
452
469
494
507
501
504
514
509
514*
542
569
566
581
killed

22 5
246
256
262
272
255
266
269
279
287
296
311
324
331
329
344
339
349
371
390
394
369
402
417
414
426*
434
446
463
484
502
516
killed

274
279
272
284
293
306
312
326
340
332
351
353
351
351
372
398
394
403
421
431
439
449
447
died

222
226
242
251
257
875
285
295
317
331
324
346
359
375
387
409
416
424
418
404
426
432
438*
449
446
469
killed

284
288
289
300
312
327
329
336
339
349
354
369
377
377
369
378
387
394
403
403
422
434
444
454
449
459*
466
492
506
521
527
537
557
566
killed

22

251
255
266
270
279
288
301
311
322
314
322
321
334
352
369
372
374
380
392
416
424
426*
399
372
killed

Conirols
No.ftS N o TST
294
262
306
284
315
291
319
297
329
315
327
343
351
344
356
356
369
367
377
384
386
393
404
402
427
404
401
432
407
442
424
462
477
426
434
489
497
446
434
510
527
454
537
471
481
561
486
559
566
496
579
509
506
584
509
595
600
524
587
610
615
532
539
killed
542
547
killed

’♦'The g u i n e a pigs w e r e plac e d on rabhit pellets, supple­
mented b y ascorbic acid, the cod liver oil was discontinued.

»!'*

*/

ry

*p

**

72-

Group
No.

Animal
No.

Sur vi val
period
(d a y s )

is o
Initial
(grams )

S

19

44

274

d y

W e i g h t
""" Pinal
(g r a m s )
447

Clinical o b s e r v a t i o n :
Guinea pig 19 gained weight gradually until the
20th u ay of the experiment*

The increase of body wei gh t

was almost insignifleant during the 20th to the 26th days.
Then,

the animal started to gain weight a g a in until it

was found dead o n the 4 6 t h day.

However,

the animal w a l k ­

ed slowly and its activity decreased.
A utopsy f i n d i n g s ;
The skeletal muscles,

especially the pectoralis,

i n t e r c o s t a l i s , tibialis anterior andthe triceps brachii
muscles showed whitish sti-eaks.

Some degenerative whitish

patches were also observed in the abdominal,

gracilis

and the adductors muscles.
The right ventricle of the heart appeared dilated
and flaccid.
The l i v er was fatty.
ently normal.

The o ther organs were a ppar­

55

Microscopic f i n d i n g s :
The skeletal muscle fibers had undergone coagula­
tion necrosis and showed a hyaline, waxy appearance*

These

changes were more or less similar to previous cases here­
tofore described*
extensive*
colemma*

The necrosis of muscle fibers was

The fibers lost striations, nuclei and sar—
However,

in some instances,

the cross striations

were accentuated and the sarcolemma persisted*
crotic fibers appeared as small granules,
a homogenous appearance.

Some n e ­

others showed

Cellular reaction was not active.

Here end there some histiocytes,

polymorphonuclear leuco­

cytes and lymphocytes were observed in necrotic tissues.
K y or eg en e ra ti on was i n s i g n i f l e a n t .
The cardiac muscle showed hyaline degeneration
and was infiltrated by histiocytes,

polymorphonuclear

leucocytes and lymphocytes.

Case 14

Group
Animal
Survival
B o d y
W e i g h t
No.
No.
period
Initial
J^inal
_________________________ (days )_______ (grams )________ (grams )
3

22

46

251

372

56

Clinical o t a e r v & t i o n :
Guinea pig 22 increased in body weight gradually
until the 42nd day of the experiment.

The body weight

declined fr o m 426 grams to 372 grams during the last four
days.

The animal showed slow locomotion and difficulty

in rising w he n placed on the b ac k the 37th day ani-i developed
diarrhea on the 38th day.

This condition disappeared

on the 42nd day of the experiment.
pig was placed on rabbit pellets,
acid,

Then,

the guinea

supplemented by ascorbic

end the cod liver oil was discontinued.

The animal

was killed on the 46th day of the experiment.
A utopsy bindings:
The skeletal muscles,
triceps brachii,

especially the adductors,

quadriceps femoris,

s u b s c a p u l a r i s , biceps brachii,

tibialis anterior,

pectoralis muscles,

abdominal,

and intercostal muscles showed extensive w h i t i s h streaks.
The gluteus,

s e m i m e m b r a n o s u s , semitendinosus and biceps

femoris muscles w e r e light in color.

The masseter and

diaphragmatic muscles were apparently normal.

The d egenera­

tive changes were symmetrical and bilateral.
The right ventricle of the heart appeared dilated.
The liver was fatty.
gested.

The small intestine was highly con­

The lungs showed pneumonia.

57

Microscopic f i n d i n g s ;
The pathological changes of the muscle fibers
consisted of coagulation necrosis and myoregeneration
w hich were more or less similar to the findings her et o­
fore described.

Necrosis of muscles fibers was extensive.

Some necrotic fibers were b r o k e n into small pieces, giving
rise to a gra nu la r appearance in the regenerated areas.
Others were homogenous and translucent,
w ith eosin.

staining evenly

The nuclei of necrotic fibers were either

pyknotic or karyolytic.

Persistence of sarcolemma of

affected fibers was not constant.
was very active.

The cellular reaction

Fibroblast proliferation,

infiltration

of histiocytes and giant cell formation were active.
The necrotic fibers w e re invaded by histiocytes and en­
veloped in giant cells.

The removal of tissue debris

was taking place by these last two types of cells.
process was followed by active myoregeneration.

This

However,

degenerative and regenerative processes went hand in hand.
R eg en er at io n was a progressive process;

therefore,

the

new fibers exhibited different stages of development.
The most successful regeneration took place in those i n ­
stances where only the contractile substance was destroyed
while sarcolemma and the peripheral layer of sarcoplasm
with nuclei w e r e intact.
The first sign of formative activity of muscle

58

fibers a fter injury appeared in the muscle nuclei of the
old damaged fiber.

This process was followed by formation

of plasmodial outgrowths from the stumps of old fibers*
These plasmodial outgrowths containing nuclei arranged
in rows retained their attachment to the old fiber and
usually showed pointed tips which penetrated into the f i b ro ­
blastic tissue

(Fig.

23).

The pointed tips became pear-

shaped in those instances where the tips met obstructions
(possibly a necrotic tissue).

The thickened tips gave

rise to multin uc le at ed syncytial masses which lost
their attachment to the old fibers and were observed in
fibroblastic tissue as islands morphologically resembling
giant cells.
The nuclei withi n plasmatic strands became h y p e r ­
trophied,
division.

bladder shaped and multiplied by continuous
The divisional process was limited to nuclei

not sarcoplasm.
by amitosis.
observed.

The nuc le ar division took place mostly

Here and there some mitotic figures were

Nuclei were centrally located in the plasmodial

outgrowths and were formed in rows parallel to the axis
of the old fiber.

In some instance*, the nuclei were

separated f r o m each other by vacuoles.
Cytoplasm of the plasmodial outgrowth appeared
granular in the early stages and was basophilic.

As

the n e w muscle fiber approached maturity the basophilic

59

s t a i n i n g c h& ra cteristic of cytoplasm decreased and they
stained more i n t e n s el y with, eosln.

Hyofibrils appeared

in the c y t o pl as m at e arly stages of growth,
the l o n g striated appearance to the

and gave

cytoplasm.

The m y o ­

fibrils o c c u p i e d the periphery of plasmatic strands in
the beginning.

D u r i n g lat er stages the nuclei moved

toward the periphery,

so that the central parts became

o ccupied by, myofibrils.

The appearance of myofibrils

in the c y t o p l a s m did not talte long after the plasmatic
strands had b e e n pushed out from the old fibers.
l on gi tu di na l

The

striations were followed by, the appearance

of c r os s -s tr ia ti on s in the fibers.
The n e w l y for me d muscle fibers w e r e parallel to
each other and they f o l l o w e d the usual muscle pattern.
The r e g e n e r at iv e processes were pronounced in
the abdominal,
ever,
ment.

pectoral and gastrocnemius muscles.

How­

the fibers formed w er e at different stages of deve lo p­
The diameter of the new fibers was still small.
The c.'rdiac m u s cl e showed hydropic degener at io n

Case 15

Group
No.

Animal
No.

Survival
period
(d a y s )

3

20

50

B o d y
Initial
(g r a m s )
222

y / e i g hHE
T i na l’ ■
(grams)
469

60

Clinical o b s e r v a t i o n ;
Guinea pig SO gained weight gradually until the
50th day of* the experiment and developed diarrhea on the
S5th day w h i c h continued for three days.
in rising w h e n placed on the back,
appeared on the 38th day.

The difficulty

and slow locomotion

This condition became aggravated

day by day until the 44th day at w h i c h time the animal
was placed on rabbit pellets,

supplemented by ascorbic

acid and the cod l iver oil was discontinued.

The guinea

pig was fed this diet for si* days and was killed on
the 51st day of the experiment.
Autopsy findings;
The carcass was in gq.od physical condition.
skeletal muscles appeared light in color.

The

Some whitish

patches were observed in the pectoral and rhomboideus
mascles•
The right ventricle of the heart was dilated.
The other organs w er e apparently normal..
Microscopic f i n d i n g s ;
The skeletal muscles were involved.

Coagulation

necrosis of the muscle fibers and myoregeneration w e re
taking place.

Necrotic fibers showed similar changes as

61

heretofore described, but to a minor extent.

The r e­

moval of n ecrotic debris was apparently taking place
by histiocytes and giant
cells was very small in

cells.

The number of these

those cases where the re­

moval of debris was accomplished*
aue filled the gap left behind.

The fibroblastic tisHere and there* some poly­

morpho nu cl e ar leucoc,ytic and lymphocytic infiltration
was observed.
R eg enerative processes dominated the necrotic
changes.

The regenerating fibers w er e in different stages

of development.

The old damaged fibers sprouted,

giving

rise to plasmodial outgrowths which penetrated the f i b ro ­
blastic tissue.

The nuclei of the plasmodial outgrowths

were arranged in rows parallel to the long axis of the
original muscle and w ere located in the central part.
The sarcoplasm was granular (Fig#

24).

The central part

of the s a rc oplasm showed some vacuoles wh ich extended
between adjacent nuclei#

Some fibers showed longitudinal

striation at this stage.

Others disclosed a faint dross

striation.

Nuclei had migrated toward the periphery (Fig.

myofibrils occupied the central portion of the fibers.
The numb er of nuclei in these fibers was still abundant.
The end part of the regenerating fiber was usually pointed.
Nuclear div is io n was active in this part.
a while,

Every once in

a pointed tip became tuickened and pear-shaped,

24);

62

and contained many nuclei which w ere arranged in the form
of clusters

(Fig.

25).

The raultinucleated syncytial m a s s ­

es were disconnected fr o m the old fibers.

It was not u n ­

common to f ind that the end part of the regenerating fiber
showed bifurcation.

In such fibers two rows of nuclei

were present and located at the periphery as though they
were goi ng to give rise to two separate fibers.
The re ge ne ra t in g fibers were parallel to the old
ones and r etained their attachment to the stump of pre­
existing fibers.

In cross sections the newly formed

fibers were of different diameter.
No noticeable

changes were found in the myocardium.

Case 16

Group
No.

Animal
No.

Survival
period
(d a y s )

b o d y
Initial
(g r a m s )

3

17

58

259

W e i g h t
Final
(g r a m s )
581

Clinical o b s e r v a t i o n :
The b o d y weig ht of guinea pig 17 gradually increased.
The animal showed diffic ul ty in righting itself w he n placed
on the b acb and w a l k e d slowly on the 44th day of the
experiment.

This condition became aggravated during the

63

following days.

On the 50th day of the experiment the

guinea pig was placed on rabbit pellets,
by ascorbic acid.

supplemented

The cod liver oil was discontinued.

The disorders mentioned above disappeared in three
days.

The animal was killed 9 days after the diet had

b een changed.
Autopsy f i n d i n g s :
The skeletal muscles, especially the pectoralis,
triceps brachii,

quadriceps femoris and biceps femoris

appeared u n i f o r m l y light in color.

The abdominal muscle

showed some w h i t is h patches.
The heart and viscera were apparently normal.
llieroscdpic f indings ;
The skeletal, muscles,

especially the abdominal

and quadriceps femoris showed degenerative and regenera­
tive changes.

De generative change was minor in extent.

H ere and there some limi te d necrotic areas were observed.
The histiocytes and giant cells were very scant.
generative processes were very active.

Uyore-

The n e w muscle

fibers extended into the fibroblastic tissue and were
parallel to each other.
nounced.

The cross-striation was pro­

The nuclei in some fibers were still numerous

and formed rows and they migrated more or less to the

64

peripheral part of the fibers*
looked more mature*

However,

some others

The nuclei had decreased in size

and number, b e i n g found u n d e r the sarcolemma (Fig.

26).

The heart muscle did not .show any change*

Case 17

Group
No.

Animal
No.

Survival
period
(days)

5

18

62

B o d y "r e i"«TTT
Initial
..
Final
(g r a m s )
(g r a m s )
225

516

Clinical o b s e r v a t i o n ;
The b ody weight of guinea p i g 18 increased g r a du ­
ally u n t i l the end of the experiment.

However,

the ani­

mal showed d i f f ic ul ty in r i si ng w h en placed on the back
and w a l k e d slowly on the 4 6t h day.

These disorders were

aggravated until the 50th day and disappeared in three
days a f te r the animal was placed on rabbit p e l l e t s , sup­
plemented by ascorbic acid and the cod liver oil d i s ­
continued.

The guinea pig was fed this diet for twelve

days and it was killed on the 62nd day of the experiment.
Autopsy f i n d i n g s ;
The skeletal muscles appeared light in color.

65

No w h i t i s h patches or streaks were found in the muscles.
The h e a r t showed some dilatation.

The other

organs w e r e apparently normal.
Microscopic findings:
Some necrotic changes of the muscle fibers were
observed after a long search of the sections.
tive processes were active.

Regenera­

The muscle fibers were m a ­

ture and extended into the fibroblastic tissue.

The

cross striations of n e w fibers we r e pronounced.

The

nuclei in these fibers w e r e large and abundant.

They

were located peripherally.

Some of them were in the

central part of the fiber.

{Fig.

27).

The diameter

of n e w fibers w e r e different.
The he art muscle showed no changes.

Case 18

Group
No.
3

£ o’ d y ' W e i g n t
'
Final
(g r a m s )
(grams)

Ani ma l
No.

Survi val
period
(d a y s )

InTlTal

21

66

284

566

Clinical o b s e r v a t i o n :
Guinea pig 21 gained weight gradually and showed

66

difficulty in r i g h t i n g itiolf wh e n placed on the hack
and wa lk e d slowly on the 47th day of the experiment*
This di sorder almost disappeared in three days after

•

the animal had b ee n placed on rabbit pellets supplement—
ed by ascorbic acid and the cod liver oil discontinued
on the 50th day.

The guinea pig was killed 15 days after

substituting the new ration.
Aut op s y findings:
The skeletal muscles were apparently normal..

The

pectoral and biceps brachii muscles were lighter in color
than the others.
The heart and viscera appeared to be normal.
Microscopic findings:
The muscle fibers exhibited necrotic and regenera­
tive changes.
observed.

Here and there small necrotic areas were

The regenerative processes were active.

The

n ewly formed muscle fibers were of different diameter
(Fig.

28).

The fibers were more or less mature, but

some fibers contained a large number of nuclei.

The

muscle fib er s w e r e extending into fibroblastic tissue
and were parallel to each other.

There were certain

areas in w h i c h fibroblastic activity dominated over the
for mation of n e w fibers.

The gap left after the necro­

tic fibers had b e e n removed was filled by fibroblastic

67

c on nective t i ss ue in w h i c h fat lymphocytes,

and some

p o l y m o r p h o n u c l e a r leucocytes had infiltrated*

These

cells w e r e o f t e n o b s er ve d around small vessels.
The h e a r t did not reveal any microscopic changes*

68

DISCUSSION

Skel et al m u s c u l a r d ys trophy was produced in grow­
ing g u i n e a pigs by f e e d i n g a synthetic d iet according to
the f o r m u l a of Davis et al (1938) together with a supple­
m ent of 0 . 5 g ra m per animal da ily of U. S. p. grade cod
liver oil.
T h e e xperimental design was not concerned part ic u­
larly w i t h the e t i o l o g y of m u s c u l a r dystrophy.

However,

the p a th ol og ic al changes produced might be attributed to
the direct toxic effect of cod liver oil as pointed out
by A g d u h r (1926,

1927),

Hoejer (1927), Ilendriksen (1928),

M a l m b e r g (1929), W a h l i n (1931),

Madse n et al (1935),

McCay

et al (1938), and Davis et al (1938), or to the d e s tr uc ­
tive effect of cod l i v e r oil on vitamin E in the diet b y
r a n c i d i t y as p o i n te d out by Cummings et al
(1938 b, 1939),

and Mattill et al (1942),

a s y n e rg is m of b o t h factors.

(1931),

Mattill

or f i n a l l y to

A n attempt wa s m ade to m i n i ­

mize the develo pm en t of r an cidity in the fat by k e e p in g
the synthetic d i e t and cod liver oil in the refrigerator.
A l t h o u g h the cod liver oil was given separately,

no steps

were t a k e n to prevent m i y i n g of the cod l i v e r oil and diet
in the g a s t r o - i n t e s t i n a l tract and subsequently to m ini­
mize the probable d e s t r u c t i o n of vitamin E.
circumstances,

U n d e r these

w e were not able to h ol d any particular

substance r esponsible f o r the condition produced.

Irre—

69

spective of w h e t h e r cod liver oil acted directly or
indirectly there was evidence that the addition of cod
liver oil to the synthetic diet resulted in muscular
d ystrophy in gui ne a pigs which confirmed the results
o btained by M a d se n et al (1935) and Davis et al (1938).
These changes in skeletal muscles were not specific for
a cod liver oil containing synthetic diet.

The muscular

changes were also produced in guinea pigs and rabbits
w ith a diet containing no cod liver oil by Goettseh and
P a pp e nh ei me r

(1931), Woodward and McCay (1932), in rats

reared on a vitamin E deficient diet by Olcott (1938),
Evans et al (1938),
heim er (1939,

Knowlton and Hines (1938), Pappen-

1940) and in the rabbit by Mackenzie at

al (1941 a).
Some evidence indicates that a vitamin C deficient
diet can also produce muscular dystrophy.

Such results

were observed by D a l ld or f (1929) in guinea pigs and by
Hjarre and Hi ll ee n ng en (1936) in calves.
There may be other factors which cause muscular
dystrophy in lambs and calves,
disease".

the so called "white muscle

This condition was observed by Metzer and Hagan

(1927),

Marsh (1932),

tfillman et al (1934),

Records

(1939),

(1939),

Sholl

Vawter and

Thorp (1942), Chen g (1945)

and Cam er on (1945) in lambs; by HJarre and Lilleenngen
(1936) and Vawter and Records

(194?) in calves.

70

In addition mus c u l a r dystrophy can h e caused by
factors other than dietary, that is, b y toxic,
and traumatic agents.
Stemmier

(1914)

Such Instances w ere reported by

in different --- diseases, Forbus

in pneumonia, S t e i n e r et al (1946)
(1947)

infectious

(1926)

and Clawson et al

in rhu e matold and rhuematic arthritis of man.

lhe c o n dition was also produced in various animals by
Forbus

(1926) f o l l o w i n g intramuscular injection of powerful

irritants or b y cutting off the b l o o d supply, by Fishback and F l s h b a c k (1932 a, b) w i t h different types of
trauma, b y Clark and Blomfield

(1945), Clark (1946) and

H a r m a n (1947) f o l l o w i n g ligation of the ertery to the muscle
groups.
U n d e r the

conditions described above, the guine%

pigs fed the synthetic diet,

supplemented b y 0.5 g r a m cod

liver oil d a i l y per animal and ascorbic acid on the alter­
nate days gained w e i g h t gradually.

After a period of growth

w hich r a nged f rom 22 to 36 days there was usually a decline
in w e i g h t w h i c h occurred synchronously with the appearance
of clinical symptoms.
decreased activity,

The clinical response consisted of

slow locomotion, difficulty in rising

when plac e d on the back, w h i c h sometimes preceded the de­
cline in b ody weight, l ack of body tone, and finally paraly­
sis of the fore and h ind l egs followed b y death.

As long

as the a d m i n i s t r a t i o n of cod liver oil w a s continued no
spontaneous r e c o v e r y took place.

However,

if dystrophic

71

changes h a d not p r o g r e s s e d too Tar, and the a n i m a l s w e r e
t

! placed o n rabbit pellets,

supplemented by ascorbic a d d

%

, a l t e r n a t e days,

and the

o o d l i v e r oil d i s c o n t i n u e d ,

on

a remark­

ably- q u i c k a l l e v i a t i o n or c l i n i c a l s y m p t o m s a n d r e c o v e r y ,
f o l l o w e d b y i n c r e a s e in b o d y w e i g h t ,

o c c u r r e d i n a short

p e r i o d of time w i t h o u t u s i n g w h e a t germ, w h e a t g e r m oil a nd
a l p h a t o c o p h e r o l as s u g g e s t e d b y S h l m o t o r i et a l

(1940)

M a c k e n z i e and M c C o l l u m

of the c o n ­

dition.

(1940) f o r the t r e a t m e n t

an d

The course, d u r a t i o n , a n d i n t e n s i t y of the c o n d i ­

t ion v a r i e d c o n s i d e r a b l y i n e a c h animal.

The d u r a t i o n of

the c o u r s e of t h e d i s e a s e m i g h t be i n f l u e n c e d b y o u t side
t e m p e r a t u r e a s o b s e r v e d in e x p e r i m e n t s X end II,
dietary f a c t o r s w e r e kept constant.

If o t h e r

The h o t s e a s o n s e e m e d

to s h o r t e n the c o u r s e p o s s i b l y b y oau s i n g d e c r e a s e d p e r ­
i s t a l s i s of the i n t e s t i n e w h i c h m a k e s f o o d s t a y lon g e r i n
the g a s t r o - i n t e s t i n a l t r a c t i n c o n t a c t w i t h s e p a r a t e l y s u p ­
p l e m e n t e d cod l i v e r oil w h i c h p r o b a b l y r e s u l t s in autoo x i d a t i v e d e s t r u c t i o n of v i t a m i n E in the d iet if
.

Mattill*s hypothesis

(1939) w a s correct.

E deficient d i e t a n d cod l i v e r oil

The vitamin

t o g e t h e r r e s u l t e d in the

e a r l y a p p e a r a n c e of clinical symptoms.
Pappenheimer

(1939)

a t t r i b u t e s the d y s t r o p h i c c h a n g e s

p r o duced in the y o u n g f e m a l e r a t b y r e s t r i c t e d v i t a m i n E
d iet to e x c e s s i v e c o n t r a c t i o n of
tal r u p t u r e

the f i b e r w i t h a s e g m e n ­

subsequent necrosis,

a direct and

s e l ec­

tive t o x i c a c t i o n a n d f i n a l l y to a n g i o s p a s t i c o c c l u s i o n
w h i c h r e s u l t e d in a n o x e m i a a n d infarction.

D u r i n g the

78

d e g e n e r a t i v e p r o c e s s of ske l e t a l m u s c l e s the m y o f i b r i l l s
u n d e r g o d y s t r p p h i e c h a n g e s fir s t .

T h e y lose their i n t e g r i t y

end b e c o m e h o m o g e n o u s .

these changes i n te r ­

Of course,

f ere w i t h the n o r m a l f u n c t i o n of the m u s c l e f i b e r ,
other words,

in

i n a b i l i t y in c on tract ion o f c o n t r a c t i l e sub­

stance de v e l o p e d .

F u r t h e r m o r e I t is h a r d to b e l i e v e that

f i b e r s c o n t r a c t e x c e s s i v e l y w h e n they l o s e t h e i r a b i l i t y
far contract i o n .
t i v e changes,

N o t all m u s c l e f i b e r s u n d e r g o d e g e n e r a ­

some of

them are p a r t i a l l y involved.

It

s eems im p r o b a b l e t h a t a n g i o s p a s t i c o c c l u s i o n r e s u l t s in
a n e c r o s i s o f one p a r t of a f i b e r and
of t h e s a m e f i b e r sound.
clear.

leaves another part

Pathogenesis

so d e s c r i b e d is not

It s e e m s r e a s o n a b l e to ass u m e t h a t t h e r e w a s a

d i s t u r b a n c e i n m e t a b o l i s m of m u s c l e c e l l s d u e to t h e toxie
e f f e c t of c o d liver o i l a n d l a c k o f vlfcamln E i n diet a s
a r e s u l t of r a n c i d i t y .

H a w k et al

(1947)

s t a t e d the

import­

a nce of v i t a m i n E i n t h e o x i d a t i v e p r o c e s s e s i n m u s c l e cells.
The p a t h o l o g i c a l

changes a p p e a r e d to b e c o n f i n e d p r i ­

m a r i l y to skeletal mu s c l e s .

In a d v a n c e eases, p r a c t l o a l l y

all skele t a l m u s c l e s w e r e m o r e o r less involved.

However,

the abdominal,

adductors

intercostal, peotoralls,

gracilis,

end t r i c e p s bra o h l l m u s c l e s w e r e a f f e c t e d b e f o r e the others.
The lesions i n the m u s c l e s w e r e
by Forbus
(1931)

(1986)

i n pneumo n i a ,

similar to t h o s e d e s c r i b e d
G o e t t s c h and P a p p e n h e l m e r

f o l l o w i n g the f e e d i n g of diet 13, M a d s e n et j^L (1935),

D a v i s et a l

(1938)

b y f e d d l n g a s y n t h e t i c diet,

supplemented

toy c o d l i v e r oil, E v a n s a t al
1940)

toy a v i t a m i n 2

(1938), P a p p e n h e i m e r

de f i c i e n t diet*

(1939,

There w a s some s i m i ­

l a r i t y b e t w e e n the l e s i o n s p r o d u c e d I n t his l a b o r a t o r y
and t h o s e d e s c r i b e d toy M e t z e r and H a g a n
and C o h r s

(1931), Will m a n

(1936), S a l y l
(1947)

et al

(1942), C h e n g

(1987), N l e b e r l e

(1934) H J a r r e , L i l l e e n n g e n

(1945), V a w t e r and R e c o r d s

I n s o c a l l e d w h i t e m u s c l e d i s e a s e of l a m b s and

calves*

The p a t h o l o g i c a l

m u s c l e dege n e r a t i o n .

The

c h a n g e s c o n s i s t e d of

S k eletal

d e g e n e r a t i o n In some m u s c l e s w a s

l o c a l i z e d a n d I n v o l v e d o n l y a p art of the mus c l e ;

In others,

t he d e g e n m r a t l o n w a s g e n e r a l i z e d and a f f e c t e d t h e e n t i r e
m u s cle*

The m u s c l e s w e r e pale, y e l l o w i s h g r a y st r e a ked,

p a t c h y and
They were

s h o w e d a cooked, m o i s t a p p e a r a n c e
easily t o m ,

i tudinal view,

friable

(Figs* 1, 2)•

and atrophied*

In the l o n g ­

the degeneration appeared a s grayish streaks

w h i c h w e r e m o t t l e d or c o n t i n u o u s t h r o u g h o u t the l e n g t h of
the m u s c l e .

The g r a y i s h str e a k s

and p a t c h e s w e r e d i s t r i ­

b u t e d s y m m e t r i c a l l y a n d b i l a t e r a l l y a s s t a t e d b y M a d s e n et
al

(1935).

H o w e v e r , P a p p e n h e i m e r (1939)

m u s c l e s w e r e i n v o l v e d in the

s t a t e d t hat all

d e g e n e r a t i o n p r o d u c e d in y o u n g

r a t s on a r e s t r i c t e d v i t a m i n E diet, b u t w e r e not n e c e s s a r i ­
l y aymiLetrlcal*
T he h i s t o l o g i c a l p i c t u r e f o u n d on e x a m i n a t i o n of the
S keletal m u s c l e s d e p e n d e d o n the i n t e n s i t y and p r o g r e s s of
t he d e g e n e r a t i v e p r o c e s s .

P r i m a r y changes c o n s i s t e d of c o ­

a g u l a t i o n n e c r o s i s i n w h i c h the

contractile

s u b s t a n c e ao-

74

q u l r e d a h y a l i n e , w a x y a p p e a r a n c e f o l l o w e d toy m y o r e g e n e r a tlon.

Muscle fibers

degeneration

Inv o l v e d s h o w e d p a r t i a l or total w a x y

(Fig. 3).

The h l s t l o l o g l o a l p i c t u r e w a s not

u n i f o r m t h r o u g h o u t a single fltoer.
Hie a f f e c t e d fltoers b e c a m e swollen,

stained w e e k l y

w i t h e o s i n and w e r e s e p a r a t e d toy e d e m a t o u s fluid, w h i l e
o t h e r s w e r e shrunken.

The n u c l e a r a c t i v i t y o f a f fected

fifcers w a s I n c r e a s e d *

The n u m b e r of n u c l e i w e r e i n c r e a s e d

and a p p e a r e d h y p e r o h r o m a t l c *

M u s c l e f i b e r s f i r s t lose the

c r o s s and l a t e r t h e l o n g i t u d i n a l
lar

(1933)

striationa.

A l t h o u g h M ll-

s t a t e d t h a t d u r i n g d e g e n e r a t i v e p r o c e s s e s tooth

the ^ and the

J b a n d s s e e m e d s i m i l a r l y a f f e c t e d and d i s a p ­

p e a r e d a t an e a r l y stage in t h e dystrophic

c h a n g e w h i l e the

Z l i n e o r K r a u s e ' s m e m b r a n e r e m a i n e d I n t a c t e v e n in a d v a n c e
stages of h y a l l n l z a t l o n *
i nto g r a n u l e s *

A t t h i s time the m u s c l e f i b e r s b r o k e

The author

c o n c l u d e d t h a t the Z line w a s the

m o s t r e s i s t a n t c o m p o n e n t o f t h e cell a s f a r a s s t a r v a t i o n
w a s concerne d *

A s f a r as c o u l d b e s e e n i n o u r s e c t i o n s the

Z line s e e m e d to d i s a p p e a r w i t h the Q and the
However,

in same fibers,

J bands*

the c r o s s s t r i a t i o n s w e r e a c c e n t u ­

a ted and a p p e a r e d c l o s e to e a c h other, p e r s i s t i n g e v e n in
the c o m p l e t e l y d e g e n e r a t e d p a r t of the m u s o l e fiber*
w a s o b s e r v e d b y C l a r k (1946)

and Harman

(1947)

This

in m u s o l e

n e c r o s i s ind u c e d b y l i g a t i o n of the a r t e r y to t h e m u s o l e
groups*

Hie m u s c l e f i b e r s b e c a m e h o m o g e n o u s , t r a n s l u c e n t

a nd s t a i n e d d e e p l y w i t h e o s i n *

S o m e a f f e o t e d f i b e r s at

75

"this stage b r o k e

Into s m a l l p i e c e s . g i v i n g a g r a n u l a r a p ­

p e a r a n c e t o the fiber.
oolemma of
8 )•

The g r a n u l a r m a s s f i l l e d t h e sar-

the f i b e r w h e n this s h e a t h w a s int a o t (Figs. 7,

O t h e r e f f e c t e d f i b e r s did not b r e a k into g r a n u l e s

b u t f o r m e d c l u m p s w h i c h w e r e h o m o g e n e s a n d sometimes c o n ­
tained vacuoles

(Fig. 4, 7, 10).

The n u c l e i

of a f f e c t e d f i b e r s w e r e p | k n o t i o f s t a i n ­

ing d a r k b l u e w i t h h e m a t o x y l i n - e o n l n a n d m a n y of t h e m h a d
u n d e r g o n e k a r y o r r k e x l s and ksryol y s l s .
c o l e n m a w a s involved.

I n some

st r u c t i v e p r o c e s s w a s not

instances, h o w e v e r ,

severe,

w a s onl y h y a l l n a t e d and the

U s u a l l y t h e sarthe d e ­

the c o n t r a c t i l e substano e

s a r o o l e m m a a p p e a r e d n o t to be

affected.
The c h a n g e s s t i m u l a t e d the i n f i l t r a t i o n of p o l y m o r p h o ­
nuclear leucocytes,

lympho c y t e s ,

p l a s m a cells and h i s t i ocytes,

also the p r o l i f e r a t i o n of f i b r o b l a s t s ,

g i a n t cell f o r m a t i o n

and d e v e l o p m e n t of m u l t i n u c l e a t e d syncytial cell m a s s e s
5. 9, 10.

18. 15).

(Figs.

The n u m b e r of the f i r s t three cells w a s

f e w and t h e y w e r e o b s e r v e d in the interstitial

tissue.

The

h i s t i o c y t e s w e r e n u m e r o u s a n d I n v a d e d t h e n e c r o t i c fibers,
playing

f>n a c t i v e role i n the r e m o v a l of n e c r o t i c ma terial.

The n e c r o t i c p a r t
cells w h i c h
cytial

of the f i b e r became e n v e l o p e d I n g i a n t

e n g u l f e d t i s s u e debris.

The multlnuoleated syn­

cell m a s s e s w h i c h a p p e a r e d t o b e s i m i l a r t o giant cells

m o r p h o l o g i c a l l y w e r e of m u s c u l a r origin.

W h ere t h e p o i n t e d

tip of a r e g e n e r a t i n g f i b e r m e t a n obstruction, t h e f i b e r

76

b e came p e a r shaped,
in c l u s t e r form*

'thickened and c o n t a i n e d m a n y nuclei

T h e multi n u c l e a t e d s y n c y t i a l m a s s e s w e r e

d i s c o n n e c t e d f r o m the o l d fibers.

A s f a r as c o u l d b e seen

the m ulti n u c l e a t e d syncytial m a s s e s d i d not e n g u l f debris.
Xn some Instances,
ly calcified
The

the o l d e r d e g e n e r a t e d a r e a s w e r e p a r t i a l ,

(Figs. 6, 11).

saroolemma which escaped destruction w e r e com­

pletely filled with muscle

spindle

cells, m u l t i n u o l e a t e d

s yncytial m a s s e s , g i a n t c e l l s and h i s t i o c y t e s , g i v i n g rise
to " M u s k e l z e l l e n s c h l a u c h e ” of W a l d y e r (Fig. 10)•
TOiere w a s n o u n i f o r m i t y of o p i n i o n c o n c e r n i n g the o r i ­
g i n of the p h a g o c y t i c cells

in the

"Uuskelzellenschlauohe"

of W a l d y e r w h i c h i n v a d e d t h e n e c r o t i c m a t e r i a l .
Forbus

(1986)

c o n d u c t e d an e x p e r i m e n t a l study o n m y o ­

d e g e n e r a t i o n a n d m y o r e g e n e r a t i o n using vital

staining.

As

a r e s u l t , h e c o n f i r m e d his p r e v i o u s w o r k and s t a t e d "the
p h a g o c y t i c cells f o u n d w i t h i n the p e r s i s t e n t sarcoleama,
w h i c h t o g e t h e r w i t h the m u s o l e cells d e r i v e d f r o m the m u s c l e
nuclei f o r m the " M u s k e l z e l l e n s c h l a u c h e ” of Waldyer, a r e w a n ­
dering c e l l s of e x t r a m u s o u l a r orig i n a n d h a v e n o c o n n e c t i o n
w ith the m u s c l e cells,

a l t h o u g h he m e n t i o n e d t h a t " m u s c l e

cells a s wel l as p h a g o c y t i c cells of the
l auche" are

capable

of vital d yes
ment,

"Muskelzellensch-

of b e i n g s t a i n e d b y i n t r a v e n o u s I n j e c t i o n

(Carmln o r t r y p a n blue)

and h e n c e s u c h v i t a l

during t h e i r d e v e l o p ­

s t a i n i n g alone w a s i n s u f f i c i e n t

i

77

f o r d i f f e r e n t i a t i n g b e t w e e n c e l l s of m u s c l e o r i g i n and
c e l l s of e x t r a m u s c u l a r origin".

W e f o u n d that the "Husk-

elzellensohlauohe** of W a l d y e r c o n t a i n e d histio c y t e s , g i a n t
cells w h i c h p h a g o e y t o s e d the
spindle-shaped musole
masses were not

dye in l a r g e amounts.

cells and multinucleated

The

syncytial

a c t i v e l y p h a g o c y t i c ; h o w e v e r some of t h e m

e n g u l f e d th e dye to a l i m i t e d e x t e n t

(Figs. 15,

16, 17, 18).

F i b r o b l a s t s a n d e n d o t h e l i a l c e l l s of c a p i l l a r i e s took u p
t he t r y p a n blue

(Fig. 19).

On t h i s b a s i s it w a s difficult

to d r a w an a c c u r a t e c o n c l u s i o n c o n c erning the o r i g i n of
t h e s e cells.
G o e t t s c h and P a p p e n h e i m e r

(1931)

reported their

r e s u l t s on the s t u d y of n u t r i t i o n a l m u s c u l a r d y s t r o p h y in
g u i n e a n i g s and r abbits.
the

o r i g i n of the

T hey agreed w i t h For b u s as to

so c a l l e d i n v a d i n g " histiocytes".

also found large multlnuoleated plasmatic masses,

They

lying

a g a inst the n e c r o t i c r e m a i n s of the m u s c l e substance.

Xn

fact, a c c o r d i n g to o u r i n t e r p r e t a t i o n the large m u l t i n u c l e ­
a te d p l a s m a t i c m a s s e s a r e n o t h i n g m o r e
In the

than giant

s ame year, N i e b e r l e a n d C o hrs

s t u d y of h y a l i n e d e g e n e r a t i o n of muscle,

cells.

(1931),

in the

o b s e r v e d the p r o ­

l i f e r a t i o n of h i s t i o c y t e s in the " m u s k e l z e l l e n s c h l e u c h e ,"
b u t the y did n o t s t e t e a n y t h i n g a b o u t the o r i g i n of the
c e lls a n d a l s o p o i n t e d o u t that the g r a n u l a t i o n tissue s u r ­
r o u n d i n g the n e c r o t i c t i s s u e s w a s

infiltrated by polymorpho—

78

n u c l e a r l e u c o c y t e s and f o r e i g n b o d y giant; cells*
cells w e r e also o b s e r v e d at th<

These

end or the m u s c l e fiber,

f o r m i n g h o o d - s h a p e d m a s s e s w h i c h a c c o r d i n g to t h e m lost
their a t t a c h m e n t to

the fibers*

It s e e m e d to u s that the

m a s s e s f o r m e d a t the end of the

f i ber w e r e s i m i l a r to our

multinucleated

s y n c y t i a l m a s s e s d e s c r i b e d a b o v e w h i c h were

form e d w h e n the g r o w i n g e n d o f a r e g e n e r a t i n g f i b e r m e t a n
o b s t r u c t i o n ( p o s s i b l y n e c r o t i c material)
ly concerned w i t h
ever some

and

are n o t active­

the r e m o v a l of n e c r o t i c m a t e r i a l *

of these cells p h a g o c y t i z e

the

How­

dye to a m i n o r

extent.
M a d s e n et a l
vario us

(1935)

p r o d u c e d m u s c u l a r d y s t r o p h y in

a n i m a l s w i t h a c o d liver oil c o n t a i n i n g diet and

found m y o d e g e n e r a t i o n f o l l o w e d b y an a c t i v e m y o p h a g o c y t o s i s .
They d i d not m e n t i o n ,

however,

the type

of c e l l s r e s p o n s i b l e

for m y o p h a g o c y t o s i s .
In the

s t u d y of the p a t h o l o g i c a l

changes of skeletal

m u s c l e s i n du c e d in r a t s b y v i t a m i n E r e s t r i c t e d diet, P a p penheimer

(1939) f o u n d t hat the s e g m e n t s of n e c r o t i c m u s c l e

f i b e r s b e c a m e e n v e l o p e d in p l a s m a t i c m u l t i n u c l e a t e d m a s s e s
and p o i n t e d o u t t h a t these p l a s m a t i c m u l t i n u o l e a t e d m a s s e s
p o s s i b l y w e r e d e r i v e d f r o m the p e r s i s t i n g m y o c y t e s .
y ea r l a t e r the s a m e a u t h o r
iment

(1940)

One

conducted another e xper­

and p r o d u c e d m u s c u l a r d y s t r o p h y in g u i n e a p i g s and

r a b b i t s b y a v i t a m i n E d e f i c i e n t d i e t a n d s t a t e d t hat the
necrotic fibers become

i n v a d e d b y p o l y m o r p h o n u c l e a r leuco-

79

cytes an d Illstiocytes w h i c h o f t e n fused to f o r m p l a s m a t i c
m u l t i n u c l e a t e d m a s s e s a b o u t the d e g e n e r a t e d remains*

It

Is p r o b a b l e that b o t h of these cells a r e giant c e l l s w h i c h
o r i g i n a t e d f r o m h i s t i o c y t e s a n d w e r e a c t i v e i n the removal
of n e c r o t i c m a t e r i a l .
syncy tial m a s s e s ,

I n our se d t i o n s the m u l t i n u c l e a t e d

originating from muscle

actively phagoeytio,

cells w e r e n o t

e v e n t h o u g h some of these cells e n ­

gulfed the t r y p a n b l u e to a slight degree.
Cheng
of lambs,
stain,

(1945),

In t h e study of w h i t e m u s c l e d i s e a s e

s t a t e d that **by m e a n s of a n i l i n e blue and e o s i n

the m o r p h o l o g y of nuc l e i and the s t aining a f f inity

of b o t h g i a n t cells and r e g e n e r a t i n g m u s c l e f i b e r s w e r e
e x a c t l y the same,
r i v e d f r o m the

i n d i c a t i n g c l e a r l y that they w e r e ell de ­

same parent tissue,

not of e x t r a - m u s c u l a r origin", and
f u n c t i o n of the m u s c l e
m u s c l e tissue

i. e., m u s o l e a n d w e r e
also a d d e d t h a t "the

giant cells w a s to r e m o v e the d e a d

t h a t o f the r e g e n e r a t i n g m u s c l e f i b e r s

to fill the g a p s l e f t b y the d e a d tissue**.

U s i n g the same

s t a i n i n g m e t h o d w e f o u n d that the g i ant c e l l s c o n c e r n e d w ith
r e m oval of f o r e i g n m a t e r i a l s h e w e d e n g u l f e d debris.
as far as could b e

s e e n the s p i n d l e shape m u s c l e

However,

cells and

m u l t i n u c l e a t e d s y n c y t i a l m a s s e s (oalled b y C h e n g m u s c l e
giant cells)
us t h a t

the

d i d n o t r e v e a l e n g u l f e d debris.

I t s e e m e d to

s t a i n i n g m e t h o d s u g g e s t e d b y C h e n g w a s not c on­

c lusive r e g a r d i n g the o r i g i n of the cells.

80

C l a r k (1946), w o r k i n g on r e g e n e r a t i o n of m a m m a l i a n
striped m u s o l e

in r abbits,

did not a c c e p t tbe v i e w concern*

ing the p h a g o c y t i c f u n c t i o n of r e g e n e r a t i n g m u s c l e c ells
and t h e i r a c t i v e role

in the r e m o v a l of n e c r o t i c material*

H o w e v e r h e m e n t i o n e d t h a t in c e r t a i n c i r c u m s t a n c e s t h e re*
m a ins

of a d a m a g e d m u s c l e f i b e r m i g h t give r i s e by b u d ding

to I n d e p e n d e n t c e l l u l a r e l e m e n t s and h e added that there
w a s n o e v i d e n c e that t h e s e i n d e p e n d e n t cells f o r m e d m y o *
blastic

t i s s u e or t hat

t h e y p l a y e d a n y part i n the r e m o v a l

of n e c r o t i c m u s c l e fibers.
M a x i m o w and B l o o m (1947)

a s s u m e d that the

s a r o o blasts

p o s s e s s e d p h a g o c y t i c f u n c t i o n s and d i g e s t e d the d e g e n e r a t e d
fibers.
C o n f i r m i n g the f i n d i n g of fforbus (1926) A l t e c h u l ,
and F r i e s e n

(1947)

r e p o r t e d that d i s s o c i a t i n g m u s o l e

cells

of d a m a g e d ,

b u t n o t c o m p l e t e l y n e c rotic, m u s c l e f i b e r s phag o *

cytosed the dye f o l l o w i n g the i n s e r t i o n of catgut i m p r e g n a t e d
w i t h e i t h e r t r y p a n blue, carmine o r hemato x y l i n .
found tha t in the r a b b i t w h i c h
peri t o n eally,

He also

r e c e i v e d t r y p a n b l u e intra-

the p r o l i f e r a t e d m u s c l e nuclei

did not s h o w

g r a n u l e s of t r y p a n blue i n the j u x t a n u o l e a r p l a s m a and he
a s s u m e d tha t e i t h e r the s a r c o l e m m a p r e v e n t e d the p e n e t r a t i o n
of the d y e or t h a t the m y o c y t e s w e r e n o t s u f f i c i e n t l y i n d i v i d u ­
alized.

It s e e m e d to us that t h e spi n d l e s h a p e d m u s c l e

cells and m u l t i n u c l e a t e d syncytial m a s s e s h a v e the same
origin, a n d s ome of t h e m p h a g o c y t o s e the v i t a l l y in j e cted

81

dye to a m i n o r ext e n t ,
h ist i o c y t e s ,

h i s t i o c y t e s a n d f u s e d f o r m s of

i. e., g i a n t cells,

ingu l f the d y e i n large

a m o u n t s and are e x t r a m u s c u l a r i n origin.
vremont

(1940)

However,

Che*

r e p o r t e d the d l r e d t t r a n s f o r m a t i o n i n v i t r o

of m y o b l a s t s i n t o typical histio c y t e s .
R e g e n e r a t i o n of m u s c l e f i b e r s o c c u r r e d s y n c h r o n o u s l y
w i t h d e g e n e r a t i v e a l t e r a t i o n of the c o n t r a c t i l e substance
as o b s e r v e d b y F o r b u s (1926), G o e t t s c h and P a p p e n h e i m e r
(1931), M a d s e n et al (1935), D a v i s et a l
haimer

(1939)

a n d Cheng

(1945).

(1938), P a p p e n *

The m o s t successful r e*

g e n e r a t i o n t o o k p l a c e in those I n s t a n c e s w h e r e o n l y the eon*
tractile s u b s t a n c e w a s destroyed,

the s a r c o l e m m a a n d the

p e r i p h e r a l 3-ayer of sarco p l a s m w i t h n u o l e l r e m a i n e d intaot.
This o b s e r v a t i o n w a s a l s o

stated b y N l e b e r l e and G o h r s (1931).

The f i r s t s i g n of f o r m a t i v e a c t i v i t y of m u s c l e f i b e r s a f t e r
I n j u r y a p p e a r e d i n the m u s c l e n u c l e i of the o l d d a m a g e d fibers.
This p r o c e s s w a s f o l l o w e d b y f o r m a t i o n of p l a s m o d l a l out*
g r o w t h s f r o m the s t u m p s of old fibers.
o u t g r o w t h s r e t a i n e d t h e i r attachment

These p l a s m o d l a l

to the o l d f i b e r and

u s u a l l y s h o w e d p o i n t e d t i p s w h i c h p e n e t r a t e d the f l b r o b l a s t l o
tissue

(Fig.

However,

23).

B i f u r c a t i o n o f the tip w a s n o t uncommon.

t he p o i n t e d tip b e c a m e t h i c k e n e d and p e a r * s h a p e d

w h e n it m e t a n o b s t r u c t i o n ,
syncytial m a s s

(Fig.

25) •

g i v i n g rise to a m u l t i n u c l e a t e d
Similar observations were m a d e by

Lewis

(1943)

in t r a n s p l a n t s of r h a b d o m y o a a r o o m m a and b y

Clark

(1946)

in the s t u d y of the r e g e n e r a t i o n of m u s c l e

in

82

rabbits.

Nuc l e i w i t h i n p l a smatic

by amitosis.

H o w e v e r here and there some m i t o t i c figures

were also observed.
and C l a r k * s

(1946)

This f i n d i n g confirmed K i t t

(1929)

observations.

U a x i m o w and B l o o m (1947)
nuclei,

strands m u l t i p l i e d m o s t l y

stated also t hat "The

duri n g the g r a d u a l g r o w t h of the muscle fibers,

increased in n u m b e r b y m i t o s i s and in l a t e r stages, p e r ­
haps by anltosls*.

C o n t r a r y to our finding. .Forbus (1926).

G o e t t s c h and F a p p e n h e l m e r
heimer

(1939).

(1931). S p e l d e l

Che n g (1945)

(1938), P a p p e n -

claimed that the n u c l e a r division

in r e g e n e r a t i n g muscle fibers w a s mitotic.

Nuclei w ere ce n ­

t rally l o c ate d in y o u n g p l a s m o d l a l o u t g rowths and surrounded
by a g r a n u l a r c y t o p l a s m w h i c h stained ba s o p h i l i c (Fig. 24).
A s the n e w f i b e r s a p p r o a c h e d m a t u r i t y the basophilic stain­
ing c h a r a c ter i s t i c of the c y t o p l a s m d e c r e a s e d and t h e y
stained m a r e intensely w i t h eosin.
the p e r i p h e r y of plasmatic

M y o f l b r i l l s occupied

strands in the beginning.

D ur­

ing l a t e r sta g e s the nuclei moved toward the periphery,

so

that the c e n t r a l p a r t s bec a m e occ u p i e d b y m y o f l b r i l l s
(Figs.

26. 27).

The ap p e a r a n c e of m y o f l b r i l l s

in the c yto­

p l a s m did not take l o n g after the pb a a m a t i c strands h a d
p u shed out f r o m the o l d fibers.

The l o n g i t u d i n a l s t r l a t i o n s

ap peared f i r s t and w e r e f o l l o w e d d b y c r o s s - s t r i a t i o n of the
fibers.

R e g e n e r a t i n g f i b e r s a r o s e f r o m the stumps of p r e ­

existing f i be r s b y co n t i n u o u s outgrowth a s o b served b y Clark
(1946).

There w a s no e v i d e n c e to indicate that m u s c l e cells

o r iginated f r o m fibroblastic tissue.

Levande*

(1945)

olaimed

83

that m y o b l a s t ic element a a r o s e b y d i f f e r e n t i a t i o n s of mesenchymatous

tissue by a p r o c e s s of Induction.

C l a r k and B l o m f l e l d r e p o r t e d

(1945)

Also,

tfa.at f i b r o b l a s t i c elements

m i g h t c o n t r i b u t e to m y o g e n e s l s , b u t t h e y did n o t m e n t i o n w h e t h ­
er f i b r o b l a s t s o r i g i n a t e d f r o m n e w m u s c l e fibers o r not.
It s e e m s t o us that the f i b r o b l a s t i c t i s s u e f o r m e d s t r e n gthens
w e a k e n e d m u s c l e d u e to n e c r o s i s a n d it h e l p s in the f o r m a ­
t ive p r o c e s s .
The o r i g i n of s a r c o l e o m a h a s been di s c u s s e d by
m a n y w o r kers .

Forbus

(1926)

of a n e w sarcol e m m a , w e are
Kitt

(1929)

s t a t e d t h a t "of the o r i g i n
able to say nothing", w h e r e a s

b e l i e v e d that the

ssroolemmal sheath probably

w a s f o r m e d b y c o n n e c t i v e t i s s u e w h i c h e n v e l o p e d the n e w
fiber.

In s t u d y o f l i v i n g m u s c l e of the f r o g

Speidel

(1938) f o u n d t h a t the s a r c o l e m m a w a s d e r i v e d f r o m

myoblasts.

(tadpoles),

It s e e m e d t h a t s a r c o l e m m a w a s f o r m e d b y m u s c l e

cells.
T h e new f i b e r s f o r m e d w e r e p a r a l l e l t o e a c h other.
The f i b e r s f a r m e d w e r e a t d i f f e r e n t

s t a g e s o f growth.

f o r e we w e r e not a b l e to m e a s u r e the r a t e of gro w t h .
study of r e g e n e r a t i o n of m a m m a l i a n s t r i p e d m u s c l e ,
(1946)

There­
In a

C l a rk

f o u n d t h a t the r e g e n e r a t i n g m u s c l e f i b e r s g r e w a t the

rate of at l e a s t 1 - 1 * 5
findings,

mm.

p e r day.

C l a r k and Yfejda (1947)

rate of g r o w t h w a s

Confirming previous

r e p o r t e d t hat the a v e r a g e

cst the o r d e r of 1 . 2 mm.

a d a y f r o m the

84

4th to 1 4 t h day and i n s o m e o a ses t h i s r a t e r e a c h e d as
m u c h a s 1.7 mm. tier day,
of the p a r t

end a l s o a d ded that i m m o b i l i z a t i o n

s e v e r e l y i m p a i r e d the p r o c e s s of r e p a i r and

regeneration .

M y o r e g e n e r a t i o n ap p e a r e d complete.

85

S U M M A R Y A N D CONCLUSIONS

1 - D y s t r o p h y of s k e l e t a l m u s c l e s w a s p r o d u c e d

in growing

g u i n e a p i g s b y f e e d i n g a synthetic d i e t a c c o r d i n g to
the f o r m u l a of D a v i s e t al (1938)

together with a dally

s u p p l e m e n t of 0 . 5 g r a m p e r animal of U. S. P. g r ade
cod l i v e r oil.
2 - The s e t u p of the e x p e r i m e n t s d i d not p e r m i t a c o n c l u s i o n
c o n c e r n i n g etiology.

H o w e v e r , the p a t h o l o g i c a l c h a n g e s

m i g h t be a t t r i b u t e d to a d i r e c t toxic e f f e c t of cod liver
o i l or to the d e s t r u c t i v e e f f e c t of cod l i v e r oil o n v i ­
t a m i n E in the diet b y r a n c i d i t y ,

or to a s y n g d g l s t l c

a c t i o n of b o t h f a c t o r s .
3 - Pathological

c h a n g e s c o n s i s t e d of c o a g u l a t i o n n e c r o s i s

of s k e le t a l m u s c l e s w h i c h
pearance,

acq u i r e d a waxy, h y a l i n e a p ­

f o l l o w e d b y m y o r e g e n e r a t i on.

4 - N o t all spi n d l e

shape m u s c l e

syncytial m a s s e s ,

cells and m u l t i n u c l e a t e d

of m u s c u l a r o r i g i n p h a g o c y t o s e d v i ­

t a l l y I n j e c t e d t r y p a n b l u e d y e d u r i n g t h e i r development.
5 - R e g e n e r a t i o n of m u s c l e w a s complete.
a r o s e f r o m the

R e g e n e r a t i n g f i bers

stu m p s of p r e - e x i s t i n g m u s o l e f i b e r s b y

c o n t i n u o u s outgrowth.

In some

instances,

the pointed

tip of r e g e n e r a t i n g f i b e r s b e c a m e t h i c k e n e d and p e ars h a p e d g i v i n g r i s e to m u l t i n u c l e a t e d syncytial masses.
6 - N u c l e a r d i v i s i o n in r e g e n e r a t i n g m u s c l e f i b e r s took place

86

m o s t l y b y a m l tosia.
7 - T he s a r c o l e m m a a p p e a r e d to be a p r o d u c t o f r e g e n e r a t i n g
muscle cells•
8 - Hyaline

d e g e n e r a t i o n of c a r d i a c m u s c l e d i d not o ccur

I n all c a s e s studied.

87

BIBLIOGRAPHY

Agduhr, E r i k .
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of n u t r i t i o n and c i r c u m s t a n c e s cf f u n c t i o n i n g .
I.
The c h a n g e s in the h e a r t t h r o u g h the p r e s e n c e of
c o d - l i v e r oil (oleum jecoris aselll) in the food.
A c t a P a e d i a t r i c s 5:319-410, 1926.
C h a n g e s i n the o r g a n i s m c a u s e d b y c o d - l i v e r oil a d d ­
e d to the food.
A c t a P a e d i a t r i c s 6:165-179, 1927.
A l t s c h u l , R . , a n d ifriesen, A. M.
Studies on phagocytosis.
Am.
17:4 4 4 - 4 4 6 , 1947.

Jour.

Cline. Path.

Burack, Eth e l , and Zimmerman, H. M.
S t u d i e s o n the a l l e g e d toxic a c t i o n of cod l i v e r
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in a n i m a l s fed l a r g e m o u n t s of cod l i v e r oil.
Jour. N u t r i t i o n 1 4 : 5 3 5-551, 1937.
Cameron, H. S.
M u s c u l a r d y s t r o p h y (white m u s c l e disease).
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Calif. Ag. Ext. Serv. ulr. 130,
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Cheng,

S h eep
22-23,

Ching- t u a n .
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C h e v r e m o n t , M.
Le m u s c l e s a u e l e t t i q u e c ultive i n vitro.
Transforma­
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51:313-333, 1940.
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Clark,

W. T.
V o l k m a n n * s ischemic contracture.
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54:339-341, 1946.
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s t u d y of the r e g e n e r a t i o n of m a m m a l i a n
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Clark, W. E. L., a n d Wajda, M . S.
The g r o w t h a n d m a t u r a t i o n of r e g e n e r a t i n g striated
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Jour. Anat. 81:56-63, 1947.

88

Clark,

W. E. L. and Blomfield, L. B.
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U n c o m p l i c a t e d v i t a m i n E d e f i c i e n c y in the r a b b i t
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S y n t h e t i c d i e t s for H e r b i v o r e , w i t h s p e c i a l r e f e r e n c e
to the t o x i c i t y o f c o d - l i v e r oil.
C o r n e l l Univ. Agr.
Exp. Sta. M e m o i r 178, 1-53, 1935.
M a l m b e r g , Nils.
S o m e h i s t o l o g i c a l o r g a n i c c h a n g e s a f t e r c o d - l i v e r oil
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Hadleigh.
D i s e a s e s of You n g L a m b s .
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Jour. Am. Vet. Med. A s soc.

M a t t ill , H. A.
V i t a m i n E, The Vi t a m i n s :
A summ a r y of the present
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b y S h i m o t a r l et a l . Jour. N u t r i t i o n , 1 9 : 5 4 7 — 553, 1940.
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M a t t ill, H. V., and Golumbio, C.
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Jour. N u t r i t i o n , S3:685-631, 1942.
M axi mow, A l e x a n d e r A., and Bloom, W i lliam.
A T e x t b o o k of H i s t o l o g y , 4 t h ed. W. B. S a u n d e r s
Comp a n y , P h i l a d e l p h i a , p. 171, 1947.
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C. M . , P a u l , Henry, and M a y n a r d , L. A.
The i n f l u e n c e o f h y d r o g e n a t i o n and o f y e a s t in
c o u n t e r a c t i n g cod l i v e r o i l i n j u r y in H e r b i v ore,
and the i n f l u e n c e of salmon oil on m i l k f a t s e c r e ­
tion.
Jour. N u t r i t i o n , 1 5 : 3 6 7 - 3 7 5 , 1938.

Menkin, Valy.
S t u d i e s on inflam m a t i o n .
I.
F i x a t i o n of v i t a l dyes
in i n f l a m m e d areas.
Jour. Exp. Med. 5 0:171-180, 1929*
Me t zer, H.
The

J . , and ^agan, W. A.
s o - c a l l e d stiff Lambs.

Cor n e l l Vet. 1 7 : 35-44, 1 9 2 7

Mi l horat, A. T. , a n d Bartels, W. E.
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m u s c u l a r d y s t rophy.
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Observation on striated muscle.
37: 1 2 7 - 1 3 5 , 1933.

Jour. Path, and Bact.

Mi not, A. S., and E r a n k , H e l e n E.
S e r u m tocoph e r o l :
its r e l a t i o n to failure o f v i t a m i n
S t h e r a p y f o r p s e u d o h y p e r t o p t i e m u s c u l a r dystrophy.
Am. Jour. Dis. Child, 67:371-375, 1944.
Nleberle, Karl, and Cohrs, Paul.
Lehrbuch der Spezlellen Pathologeachen Anatomic
der Baustftere.
V e r l a g V o n u u s t o v -t'ischer, Jena,
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Olcott, H. S.
The p a r a l y s i s in the Y o u n g of *itamin E d e f i c i e n t
f e m a l e rats.
Jour. N u t r i t i o n , 1 5 : 2 2 1 - 2 2 7 , 1938.
P a p p e n h e i m e r , <<*>• M.
'fhe p a t h o l o g y of n u t r i t i o n a l m u s c u l a r d y s t r o p h y in
y o u n g rats.
Am. Jour. Path. 1 5 : 1 7 9 - 1 8 4 , 1939.
C e r t a i n n u t r i t i o n a l c& sorders o f L a b o r a t o r y a n i m a l s
due to v i t a m i n * d eficiency.
Jour. Mt. S i nai H o s p i ­
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M u s c u l a r d i s o r d e r s a s s o c i a t e d w i t h d e f i c i e n c y of
v i t a m i n E.
P h y s i o l . Kev. 23:37-50, 1943.

92
Rogers, W. M . , P a p p e n h e i m e r , A. M . , a n d ^oettsoh, M a r i anne.
N e r v e e n d i n g a In nutriti onal m u s c u l a r d y s t r o p h y in
g u i n e a pigs.
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P a t h o l o g i c c h a n g e s In the l i v e r a n d k i d n e y s of g u i n e a
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J•
C a r d i a c and s k e l e t a l m u s c l e d y s t r o p h y in y o ung Lambs.
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Abst. in Vet. Bui. 13, 136; 1942.

S h i m o t o r 1, N. B u k . , E m e r s o n , ^la d y s A.,* and jsvans, H e r b e r t M.
The p r e v e n t i o n of n u t r i t i o n a l m u s c u l a r d y s t r o p h y
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Jour. N u t r i t i o n .
1 9 : 5 4 7 - 5 5 3 , 1940.
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Jour. Am. Vet. Med.

Speldel, C. C.
S t u d i e s of l i v i n g m u s c l e .
I.
Growth, i n j u r y and
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o b s e r v a t i o n of i n d i v i d u a l f i b e r s In l i v i n g frogtadpoles.
Am. Jour. A n a t . 62:179-235, 1938.
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L e s i o n s of skeletal m u s c l e s in r h e u m a t o i d arthritis
n o d u l a r nolymyosi tes.
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1942.
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Preventing Lamb Losses.
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Mich. Agr. Ex.

Sta.

Quart.

Vawter, L. R., and Rec o r d s , E dward.
O b s e r v a t i o n s o n the sti f f l a m b p r o b l e m w i t h special
r e f e r e n c e to w h i t e m u s c l e disease.
Jour. Am. Vet.
Med. A s s o c . 489 - 4 9 1 , 1939.
iMuscular d y s t r o p h y (White M u s c l e Disease) In Y o u n g
Calves.
J'our. Am. Vet. Med. A s s o c .
1 1 0 : 1 5 2 - 157,
1947.

93

W a h l I n , Bernard.
C o n c e r n i n g the toxic e f f e c t of cod l i v e r o i l in
the o r g a n i s m .
Acta. Med. Scand. 7 4 : 4 3 0-45©, 1931.
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A n i n v e s t i g a t i o n of the c a u s e of the s t i f f - l a m b
disease.
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and 0 1 ofson, P e t e r .
V i t a m i n E p r e v e n t s and cures the "stiff-lamb** disease*
C o r n e l l Vet. 34:200 - 2 0 4 , 1946.
W o o d w a r d , J. W . , e n d McCay, C. M.
S y n t h e t i c d i e t s f o r H e r b i v o r a . i'roc. Soc. Exp. Siol.
a nd M e d . 3 0:241-242, 1932.

Fig-

1.

Cr.se 4.

(Autopsy

K o . 9461)

Whitish patches and s t r e a k s
are s c a t t e r e d t h r o u g h o u t the
muscle of the hind legs and
the abdomen*

Fig.

£.

Case

4.

(Autopsy

Whitish patches
a.re s e e n i n t h e
and intercostal

U o • 9461)

and strerle
aldoraina.l
muscles.

P L A T E

1

Fig.

S.

Case 6.

( A u t o p s y No.

9455)

C o a g u l a t i o n n e c r o s i s of the
h i c e p s f e m o r i s muscle.
In*
filtration with phagocytic
cells is s e e n (H&E), xl30.

Fig. 4.

C a s e 6.

( A u t o p s y No.

9455)

Extensive coagulation necrosis
of the quadriceps f e m o r i s
muscle.
The h y a l i n e clumps
and g r a n u l a r b r e a k d o w n of
n e c r o t i c f i b e r s are s e e n in
the c e n t e r of p h o t o m i c r o g r a p h .
( H & E ) , xl03.

P

I* A

T E

2

Fig*

5.

C a s e 4.

( A u t o p s y No.

9461)

T h e m u s c l e fibers of the q uad­
riceps femoris were replaced
b y f i b r o b l a s t i c t i s s u e into
which had infiltrated histio­
cytes a n d giant cells.
Ne­
crotic islands are also observed.
(Mallory's aniline blue), xlOS.

Fig.

6.

C a s e 1.

( A u t o p s y No.

94 54)

D e p o s i t s of calcium i n n e ­
crotic tissue of the p e c ­
toral muscle. (H*
% to3 .

P L A T E

S

m m

Fig*

7.

C a s e 1*

( A u t o p s y No.

9454)

Vacuolization of degenerated
f i b e r s of the abdominal muscle,
and h y a l i n e clumps s u r r o u n d e d
b y p h a g o c y t i c c e l l s (H&E), x l O S .

Fig#

8.

C a s e 6.

( A u t o p s y No.

9455)

The f i b e r s of the q ua dri cep s f e m o r i s
show coagul at io n necrosis w h i c h as­
sumed a g r a n u l a r appearance.
Histio­
c y te s w i t h e n g u l f e d d e b r i s a re seen.
( H & E ) , x625.

P L A T E

4

Fig*

9.

Case

6.

( A u t o p s y No.

9455)

C o a g u l a t i o n n e c r o s i s of the
f i b e r s o f th e b i c e p s f e m o r l a
muscle together with histio­
cytes, e n g u l f e d debris, a n d
f o r m a t i o n of some mu s c l e cells
(H&E), x 6 2 5 .

Fig*

10*

C a s e 1*

( A u t o p s y No.

9454)

Vacuolization of degenerated
fibers of the a b d o m i n a l m u s c l e
and cells w i t h s t ain in g c h a r ­
a c t e r i s t i c s of t h e " M u s k e l z e l l e nschlauche" of Waldyer are
seen.
M u s c l e cells and m u l t i ­
nucleated syncytial masses pos­
sess pale nuclei w i t h i n d e f i n ­
itely outlined cytoplasm.
Histiocytes and giant cells
have deeply stained nuclei
with definite outline.
(H&E), x 6 2 5 .

P

X A T £

5

Fig.

XI.

C a s e 1.

( A u t o p s y No.

9454)

Deposits of calcium in the
n e c r o t i c t i s s u e o f t he p e c ­
toral mu s c l e and p e r s i s ­
tence of c r o s s - s t r i a t i o n
i n the d e g e n e r a t e d f i b e r
a r e seen.
( H &E ) , x 6 2 5 .

Fig.

12.

C a s e 1.

( A u t o p s y No.

9454)

D e p o s i t s of calcium, giant
cells, a n d h i s t i o c y t e s are
p r e s e n t i n the center of
photomicrograph.
(E&E), x625.

t

Fig*

13-

C a s e 4.

( A u t o p s y No.

9461)

Giant cells w i t h engulfed
debris are present.
(Mal­
lory's a n i l i n e blue), x625.

Fig.

14.

C a s e 3.

( A u t o p s y No.

9462)

The heart showed degenerative
changes and cellular i n f i l t r a ­
tion.
(H&E), x6 25.

Fig*

25*

Case

10.

( A u t o p s y No*

9559)

Histiocytes and giant cells
show trypan blue granules
which are scarce in spindle
shaped m uscle cells and multinucleated syncytial masses*
The l a t e r two cell types are
seen in the center of p h o t o ­
micrograph (vital staining
with trypan blue & eosin
contrast), x800*

Fig*

16*

Case

10*

( A u t o p s y No*

9550)

Histiocytes and giant cells
e n g u l f e d the t r y p a n blue.
(Vital s t a i n i n g w i t h t r y p a n
b l u e & e o s i n c o n t r a s t ) , x l O 20*

P L A T E

8

Fig.

17.

C a s e 7.

( A u t o p s y No.

9 5 56)

G i a n t cells and h i s t i o c y t e s
e n g u l f e d t r y p a n blue.
They
a r e se e n i n the c e nt er o f
p h o t o m i c r o g r a p h w h e r e t he
muscle fibers had undergone
n e c r o s i s ( v i ta l s t a i n i n g
w i t h t r y p a n b lu e & e o s i n
c on trast), x625.

Fig.

18.

Ca s e 10.

( A u t o p s y No.

95 59)

The d e g e n e r a t e d part of t he
muscle fibers were infiltrat­
ed b y n i s t i o c y t e s and g i a n t
cells.
The muscle nuclei
h a v e i n c r e a s e d in numbers,
f o r m i n g sin gl e a nd m u l t i n u c l e
a t e d s y n c y t i a l masses.
T he
former phagocytosed trypan
b lu e in l a r g e amounts.
Some
of the m u s c l e cells and m u l ­
tinucleated syncytial masses
also e n g u l f e d the dye to a
l e s s e r extent.
(Vital s t a i n ­
i n g w i t h t r y p a n bl u e & e o s i n
contrast), x!020.

P L A T E

9

Fig.

19.

C a s e 10.

( A u t o p s y Ho.

9559)

H i s t i o c y t e s , f i b r o b l a s t s , and
endothelial cells show engulf
ed t r y p a n blue.
(Vi t al s t a i n
ing w i t h trypan blue & eosin
contrast), xl050.

Fig.

20.

C a s e 10.

( A u t o p s y No.

9559)

Histiocytes (which engulfed
t h e dye) a r e o b s e r v e d a r o u n d
the b l o o d v e s s el .
(Vit al s t a i n ­
ing with trypan blue & eosin
c on trast), x565.

P L A T E

10

Fig.

21.

C a s e 10.

( A u t o p s y No.

9559)

The h e a r t shows h y a l i n e d e g e n ­
e r a t i o n a n d is i n f i l t r a t e d w i t h
histiocytes which had engulfed
t r y p a n b l ue .
(Vital s t a i n i n g
with trypan blue & eosin con­
trast), x640.

Fig.

22.

C a s e 8.

( A u t o p s y No.

9556)

T h e p o p l i t e a l l y m p h n o d e.
The reticulocytes, small
lymphocytes and macrophages
t o o k u p t h e dye.
(Vital
staining w i t h trypan blue
& eosin contrast), xl0S5.

p l a t e

11

Fig*

£3.

Case

14*

( A u t o p s y No.

9719)

Plasmodial outgrowths originate
f r o m the stum ps of old p r e e x i s t ­
ing muscle fibers.
The picture
w a s taJcen t h r e e d a y s a f t e r cod
liver oil had b e e n discontinued.
(H&E), x 6 25.

Fig*

£4.

C a s e 15.

( A u t o p s y No.

9 7 £4 )

Young muscle fibers are show­
ing longitudinal striations.
Nuclei are centrally l o c a t e d
a n d s u r r o u n d e d by g r a n u l a r
cytoplasm.
The picture shows
r e g e n e r a t i o n of m u s c l e f i b e r B
6 d a y s a f t e r co d l i v e r o i l
had been discontinued.
(H&E),
x 6£5.

PLATE

12

Fig-

25

C a s e 15.

( A u t o p s y No-' 9 7 2 4 )

Y o u n g m u s c l e f i b t r s at d i f f e r ­
ent stages of d e v e l o pmen t.
Multinucleated syncytial mass­
es at t h e end o f the f i b e r a r e
seenThe pic t u r e was taken
6 days after cod liver oil
had been discontinued.
(H&E),
x625.

Fig.

26.

Case

16.

( A u t o p s y Ho.

9752)

Y o u n g m u s c l e f i b e r s at d i f ­
ferent stages of growth.
They
are still m u l t i n u e l e a t e d , l o ­
c a t e d c e n t r a l l y and p e r i p h e r ­
a l ly .
The c r o s s - s t r i a t i o n s
are pronounced.
The g u i n e a
p i g w a s K i l l e d 9 d a ys a f t e r
t h e c od l i v e r o i l h a d b e e n
discontinued.
(H&E), x 6 2 5.

P L A T E

13

&
*

%

*

/

h

§
I

>

Fig-

27.

Case

17-

( A u t o p s y No.

9759)

The m u s c l e f i b e r s are mature.
T h e c r o s s - s t r i a t i o n s of* n e w
f i b e r s are pronounced.
The
n u m b e r of nu cl ei are decreased.
The guinea p i g was killed
tw e l v e days a f t e r the cod
l i v e r oil h a d b e e n d i s c o n ­
tinued.
(H&E), x625.

Fig.

28.

Case

18.

( A u t o p s y No.

9786)

The cross s e c t i o n shows d i f ­
ferent diameters of ne w mu s c l e
fibers.
The guinea pig was
killed f i f t e e n days after the
cod-liver oil had been dis­
continued.
(H&E), x 6 2 5 .

P

Z* JL T E

14