ANEUROGENIC LIMB DEVELOPMENT
AND MUSCLE DEGENERATION

A Disswfahon
for His Degree of Db. D.

MICHIGAN STATE UNIVERSITY
Heinz Popiela
E975

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93 10491 7913

This is to certify that the
thesis entitled
ANEUROGENIC LIMB DEVELOPMENT
AND MUSCLE DEGENERATION

presented by

Heinz Popiela

has been accepted towards fulfillment

of the requirements for

Ph.D. Zoology

degree in

 

 

 
  
 
 

  
 
 

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Date November 12, 1975

 

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ABSTRACT

ANEUROGENIC LIMB DEVEIOPMENT
AND MUSCLE DEGENERATION

BY

Heinz Popiela

Out of a need for a quantitative study on
aneurogenic limb development and muscle degeneration
the present study was undertaken. Early tailbud
(Harrison's stage 21$) Amblnton maculatum embryos had
their entire presumptive nervous system excised. For
controls . embryos of the same age were operated in an
identical manner except that about a one . segment of
neural tube dorsal to the presumptive limb buds was left
intact. Operated embryos were reared in Steinberg 's
solution at 18°C until processed for light microscopy
and electron microscopy at selected ages. Plastic
embedded. mid-stylopodium regions of limbs were sampled
and the tissues histometrically measured with a MIC
particle counter. The data were statistically analysed.

The results indicated that the humerus was
entirely stable to the absence of nerves whereas
aneurogenic limb myofibers initially developed almost
normally followed by degeneration and muscle disap-
pearance. In 13 day aneurogenic and control limbs the
muscle area (mostly myoblasts) per limb cross section
was determined to be equal: there was then a two to five
fold increase in muscle area in control limbs whereas
the aneurogenic muscle area gradually decreased as the
animal grew older. Myofiber differentiation was measured
by counting the number of peripherally located nuclei
per total number of muscle fiber nuclei. The number of
peripheral nuclei reached a peak at 31 days after the
operation in aneurogenic limbs. The number of peripheral
nuclei in the controls also peaked at the same time but
remained at that maximum.‘with the exception of day 13.
the number of peripheral nuclei was always greater in
control than in aneurogenic muscle.

Total number of muscle nuclei per limb cross section
gradually decreased in aneurogenic muscle reaching a
number significantly lower than in controls by 31 days
after the operation and thereafter. The cross sectional
size of aneurogenic muscle nuclei seemed to decrease at
a faster rate than control nuclei. Reflecting nuclear
density. the myonuclear area per arbitrary muscle area

in aneurogenic limbs reached its lowest value by 21 days

after the operation. Beyond 21 days aneurogenic nuclear
densities progressively increased whereas control
densities remained at the low. day 21 value. From.day 13
to day 21 the muscle nuclear density in control and
aneurogenic limbs was statistically equal.

Ultrastructurally, control larvae were seen to be
normally innervated by the “isolated spinal cord“ and
walking movements were observed in some animals. In
aneurogenic animals nerves were never found in electron
microscopic thin sections. Aneurogenic myofibers in an
advanced state of degeneration incorporated more
3H-leucine or 3HL-uridine than fibers where cross-
striations were still visible with the light microscope.
With the electron microscope acid phosphatase stained
lysosomes were found adjacent to disintegrating myo-
fibrillar material in degenerating aneurogenic myofibers.
Ruthenium.red staining indicated that myofiber basement
laminae developed normally in aneurogenic muscle and
persisted for some time. Ruthenium.red staining assured
the identification of muscle satellite cells which were
routinely detected in aneurogenic. control and normal
muscle. Muscle tendons persisted in aneurogenic limbs
although atrophic in appearance.

ANEUROGENIC LIMB DEVELOPMENT
AND MUSCLE DEGENERATION

BY

Heinz Pepiela

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1975

DEDICATION

In memory of the late Dr. Charles Stead Thornton

who. unfortunately. did not witness the completion of

this work.

ii

ACKNOWLEDGMENTS

I wish to extend my sincere appreciation to the
late Dr. C.S. Thornton, Michigan State University (MSU).
to Dr. M. Singer. Case Western Reserve University (CWRU)
and to Dr. C. Tweedle, MSU. for their advice. criticism
and support. I would like to thank Dr. K.S. Warren,
Division of Geographic Medicine. CWRU. for the use of
the TTMC particle counter and Dr. P.K. Jones, Dept.
Biometry, CWRU. for his help with statistics. I thank
Dr. M. Balaban, MSU. and Dr. S. Bromley, MSU, for
serving on my guidance committee. I am grateful to my
wife Chu for doing the largest part in typing and
editing this dissertation. This investigation was
supported by a grant. number AM 15732, from the National
Institute of Health awarded to Dr. C.S. Thornton and
Dr. S. Bromley.

iii

TABLE OF CONTENTS

Dedication.........................................

Acknowledgments....................................
Table of Contents..................................
List of Tables.....................................
List of Figures....................................
List of Plates.....................................
Introduction.......................................
Material and Methods...............................
Results............................................
Discussion.........................................

App0ndueeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

List Of ROIOrBDCOBeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

iv

Page
ii
iii
iv

vi
vii

12
21
86
100
107

LIST OF TABLES

Table Page

1 (Appendix) ..................................... 100
............................................... 30
............................................... 31

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5 ............................................... 33
6
7
8 ............................................... 36
9

(Appendix) ..................................... 102
10 .............................................. 37
11 (Appendix) .................................... 106
12 eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 38
13 .............................................. 39

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LIST OF FIGURES

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vi

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LIST OF PLATES

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vii

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59
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75
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81
83
85

INTRODUCTION

The idea of studying body parts separated from the
presence of the nervous system has been pursued for a long
time (Zeleni. 1962. review). Harrison in 190k concluded
that the axial muscle develops "perfectly" normally in
the aneurogenic region of a frog embryo. His experimental
strategy was to out .... “a narrow strip extending from
the region of the pronephros to the tip of the tail" off
the dorsal side of early tailbud embryos just above the
notochord. thus removing the developing spinal cord. Six
to seven days later he examined the larval myotome in
section. Harrison wrote that the operated embryos developed
at a slower rate than normal ones and that the larvae are
often edematous. Operated larvae remained motionless even
if "strongly“ stimulated but the muscle contracted locally
if stimulated electrically. The spinal cord was. of course.
absent but he did observe longitudinal nerve bundles to
pass posteriorly into the chordless tissues. The arrange-
ment of myofibers was normal. however he often saw large
clear spaces separating the fibers. Besides clear spaces.
he also found vacuoles along the axes of myofibers which
he attributed to “unfavorable accidents of the operation“
instead of to the nerveless condition. In a brief account
he mentioned having instructed a Ir. Langnecker to make
longitudinal cuts between presumptive limb buds and the

2
notochord in order to separate the buds from their nerve

source. However. he experienced difficulties in keeping
the embryos alive until metamorphosis. Of all Operated
embryos only one specimen survived. In this specimen the
hindlimb had an atrOphic appearance. The hindlimb carti-
lage. bone and muscle were observed to have differentiated
normally but myofiber diameters were smaller than in
muscle of normal forelimbs.

A detailed study on aneurogenic or sparsely innervated
frog hindlimb development was done by Hamburger (1928). He
removed the right posterior segment of the neural folds in
frog embryos in which the neural folds had just closed. The
purpose of the experiments was the study of nerveless hind-
limb development. In another series. he inserted a piece of
mica longitudinally between the hindlimb bud and the neural
tube in tailbud embryos in order to isolate the bud from its
nerve source. In experiments in which the neural fold was
removed he experienced difficulties in obtaining a nerveless
condition since the neural tube regenerated in 70% of the
cases. and in mica implantations nerves often grew around
the slit created by the mica. In the few successful cases
he noted that the right hindlimbs were paralysed. markedly
straphic but perfectly normal in appearance although
miniatures of normal limbs. Morphological development from
limb bud to digit formation occurred normally. However. at
the time when the knee was visible and the hindlimb in the
early five digit stage. growth was gradually retarded

3
although external morphology (Formbildung) occurred

synchronously with the control side. As the animals grew.
the leg muscles on the operated side became progressively
narrower but showed great variation from.animal to animal.
In some cases only one muscle remained behind in their
growth in thickness and in more severe cases the legs were
nothing but ”skin and bones“ at the start of metamorphosis.
In these severe cases the leg skeletal system. although
smaller. was entirely normal except for a couple of cases
in one of which a few distal tarsals were fused and in the
other case the head of the femur was grown to the hipjoint.
All other limb tissues such as skin. skin glands and blood
vessels develOped normally. In most cases Hamburger found
thin nerves either coming from the other side or from
remaining nerves 11 and 12. Hamburger found the musculature
to be the most sensitive component to the absence of
nerves. He did find normal. cross-striated fibers but the
number of fibers and their diameter were smaller. Fibers
were often separated by clear spaces. had a wavy appearance
and their nuclei were distributed randomly in uneven dis-
tances from one another. Many fibers had degenerated the
signs of which were dissolution of cross-striations.
bloated fibers and thicker nuclei. More advanced signs of
degeneration were the wax-like appearance of fibers in
cross section (hyaline degeneration) and disintegration of
fibers where the '... contents of fibers has fallen apart

in large clumps. the sarcolemmal tubes are torn or

h
missing.'* Sometimes fibers were normal looking but ended

in thick. stained clumps. In one case. he fixed a younger
animal in which the musculature had not completely differ-
entiated. The muscle was largely "im.Stadium.der Myo-
blasten' and only few cross-striations existed. In this
particular case he found no signs of degeneration whatso-
ever. As to the causes of muscle degeneration Hamburger
mentioned that he could not tell whether interference with
the nervous system. nutrition or lack of function led to
the poor condition of muscle.

Another cited investigator. Wintrebert (1903).
repeatedly denervated the hindlimbs of 50 frog larvae in
the early hindlimb digit formation stage and observed the
continued gross development of the limbs. However. he did
not examine the operated animals for the presence of nerves
nor did he check on the histological condition of tissues.
According to Wintrebert. the hindlimbs developed normally
in general farmland proportion and with such meagre data
to go by he wrote: ”Nous pouvons donc conclure que le
systime nerveux n'est pas necessaire dans la géniration du
membre. ni pour sa croissance. ni pour sa morphoginie
generals. ni pour sa differentiation.“ In a later experi-
ment (Vintrebert. 1905) he excised a piece of the neural
tube from.frog embryos at the closed neural fold stage and
noted that larvae developed all right but were paralysed.

* This author's translation.

5
Again the observations made were superficial and contained

no histological data.

Hooker (1911). a student of Harrison. examined the
development of aneurogenic voluntary and cardiac muscle in
frog embryos by removing the presumptive spinal cord.
brain. cranial ganglia. and head skin from closed neural
fold stage embryos. He observed that the myotome developed
normally and contracted upon electrical stimulation or by
pricking with a needle between 1 to 3 days after the
operation; spontaneous movement he never detected. The
irritability in a majority of animals lasted only for 1 or
2 days after the operation and no reaction was elicited
after the third day.

Other techniques of isolating organs or tissues from
the influence of the nervous system included transplan-
tation of somites to the chorio-allantoic membrane of
chicks (Hoadley. 1925). transplantation of limb buds to
the chorio-allantoic membrane of chicks (Hunt. 1932).
transplantation of chick limb buds to the coelom
(Eastlick. 1943). and transplantation of somite to ventro-
lateral positions in.Amblystoma embryos (Muchmore. 1968).
In all of these cases early myofibers develOped normally
at first followed by atrophy and degeneration. In the
chick embryo. degeneration of muscle manifested itself by
fat replacement. More modern techniques employed to study
the influence of nerves on muscle tissue include tissue

culture methods (Shimada et a1.. 1967). explant culture

6
methods (Peterson and Grain. 1970; Lents. 1971. 1972;

Askanas et al.. 1972: Grain and Peterson. 197u) and minced
muscle implants (Hsu. 197a: Carlson. 1973. review). These
studies also indicate that muscle does not need nerve until
the late myotube to early myofiber stage after which times
it will not differentiate (Askanas et al.. 1972). Nerves
are absolutely required for the continued maintenance of
:muscle tissue. Emphasizing the importance of nerve for
muscle development and maintenance it was shown that the
degenerative process of nerveless muscle explants in
culture is reversed when pieces of embryonic spinal cord
are presented and neuromuscular connections are established
(Peterson and Grain. 1970; Grain and Peterson. 1974).

By far the most extensive literature exists on 12:1222
denervated adult muscle (Gutmann and Zelena. 1962; Guth.
1968. review). The term. denervated. denotes a tissue which
has had received innervation during ontogeny but in which
the nerve supply was destroyed. One may add that the
observed processes of muscle degeneration in denervated
muscle are quite comparable to the processes of degener-
ation in aneurogenic muscle. a muscle which had never been
innervated during ontogeny (Tweedle et al.. 1974). If adult
cat forelimb muscle is kept denervated for 2 months one
sees a 90% to 50% loss of muscle weight. By 2 months after
denervation myofibers are reduced in diameter by 1/3 to
1/2. by h months the muscle appears vacuolated and granular

7
and by one year muscle fibers are “reduced to the status
of fibrous tissue" (Tower. 1935). In the denervated rat
leg muscle Pellegrino and Fransini (1963) described two
major. overlapping phases of muscle atrOphy. In the first
phase they recognize a degenerative autolysis which
manifests itself by a loss of striations and an almost
50% weight loss. In the second phase. myofibrils are
reduced in diameter. lose their parallel arrangement and
break down within the interfibrillar space. The Z-lines
become irregular and the number of nuclei seem more
numerous and are centrally located. Possibly. the increased
number of nuclei is an illusion due to the disappearance of
contractile material since Cardasis (1975) found no change
in the number of nuclei per isolated myofiber following
denervation. Surprisingly. the sarcoplasmic reticulum is
still well preserved and relatively overdevelOped. Mito-
chondria disappear in parallel with the contractile
material. The reduction in cross sectional fiber area and
myofibrillar area per fiber was confirmed quantitatively
in the adult rat (Engel and Stonnington. 1979). Further-
more. Engel and Stonnington observed a transient increase
in mitochondrial area and total sarcotubular surface
followed by a decrease. In fact. Muscatello et al. (1965)
believe that an increase in sarc0plasmic reticulum and
number of mitochondria in response to denervation reflects
change to a new metabolic pattern for the survival of

cells and muscle repair. The number of sarcomeres is

8
reduced by 35% in denervated muscle (Goldspink et al..

1979). Schiaffino and Settembrini (1970) in the neonatally
denervated rat leg also noted hypertrophy of the sarco-
tubular system and concluded that the continued differ-
entiation of the sarcotubular system is not impaired by
neonatal denervation. Muscle spindles never differentiate
in rat muscle denervated during the myotube stage (Zelena.
1957) the cause of which is the absence of sensory
innervation (Zelena and Soukup. 1979). In rat leg muscle.
denervation 3 days before birth was found to result in
unaffected growth and development initially. but the
differential Z-band pattern of white and red muscle never
developed in extensor digitorum longus and soleus fibers
(Hanzlikova and Schiaffino. 1973). Furthermore. neonatally
denervated rat muscle developed histochemically
identifiable type I fibers but not type II fibers (Engel
and Karpati. 1968). Apparently. fiber types do not differ-
entiate in the absence of nerves (Shafiq et al.. 1972a)
and mature fibers degenerate upon denervation in a pattern
characteristic of their type (Tomanek and Lund. 1973).
Physiologically. denervated muscle shows extra-
junctional sensitivity to acetylcholine but by 29 hours
miniature endplate potentials are no longer detected
(Albuquerque and Mo Isaac. 1970). As to the mechanism of
contractile protein removal lysosomes have been detected

among myofibrils in dystrophic muscle (Shafiq et al..

9
1972b: Milhorat et al.. 1966). acid hydrolases (Pollack

and Bird. 1968) and lysozyme (Krishnamoorthy. 1972) have
been seen to increase in denervated muscle. However.
Schiaffino and Hanzlikova (1972) conclude that myofibril
disintegration in denervated muscle is not a result of
lysosomal activation but is initiated by an extralysosomal
mechanism. In corroboration. Kohn (196“. 1966) does not
find enzymes capable of degrading myosin in denervated
muscle.

The denervated rat diaphragm hypertrophies before it
shows the usual symtoms of atrophy (Sola and Martin. 1953).
The parameters are an initial increase in wet weight. amino
acid incorporation. protein synthesis and DNA synthesis 3
to 6 days after denervation followed by a decrease below
control levels at 12 days after denervation (Zak et al..
1969: Turner and Manchester. 1973). Ultrastructurally the
number of actin and myosin filaments increase during the
hypertrophic period followed by the usual signs of straphy
(Miledi and Slater. 1969). The insulin accelerated uptake
of 14C-leucine or aminoisobutyric acid (AIB) into control
muscle did not occur in denervated hemidiaphragms (Buse
et al.. 1965: Manchester. 197“). When ribosomal activity
was studied $2,!itgg. it was found that the number of
nascent peptide chains and the specific activity in terms
of amino acid incorporation did not differ in denervated
and control diaphragms; however. the ribosomal yield
increased by 80% in diaphragms 3 days after denervation

10
(lanohester. 197k). The increase in ribosomal number was

ultrastructurally confirmed.by‘Manolov and Ovtsoharoff
(197M .

Insect musculature. in addition to the usual signs of
muscle degeneration upon denervation (Teutsch-Felber. 1970.
Rees and Usherwood. 1972) has the unusual feature of con-
taining normally degenerating muscle during development
(Nuesch. 1968. review). In the bug Rhodnius. the interseg-
mental muscle repeatedly breaks down after each moulting
cycle (Wigglesworth. 1956) although normal neuromuscular
Junctions are maintained during the breakdown process
(Anwyl and Pinlayson. 1973). In the cricket the axon ter-
minals looked normal even after the contractile system had
disappeared (Gutmann et al.. 1974); and the normal degener-
ation of muscle is retarded upon injection of ecdysterone
but no effect is observed in denervated muscle (Srihari et
al.. 1975). In the moth lysosomes appear in normally degen-
erating muscle (Lockshin and Beaulaton. 197ha.b). but myo-
protein is thought not to be digested within lysosomal mem-
branes. However. muscle proteins do appear in the hemolymph
(Lockshin. 1975). The control over insect muscle degener-
ation is hypothesised to occur via combined hormonal and
nervous means (Srihari. 197h). The development of dener-
vated cricket thorax muscle was quantitatively studied by
Thommen (19711): the results indicate that growth is
interfered with but not stopped. Growth.in fiber thick-

ness was found to be the most sensitive aspect to

11
denervation. The number of muscle nuclei were calculated to

be more than 100% over normal. Thommen eXplained. that
because of denervation growth in thickness is interfered
with much more strongly than nuclear proliferation thereby
creating a higher nuclear number per fiber volume.

In Amblystoma larvae the development and subsequent
degeneration of limb musculature in aneurogenic or sparsely
innervated larvae were studied with the electron microscope
by Tweedle et al. (197a). The results indicate that early
development and differentiation of this muscle occurs in
a comparable manner to normal larvae. Muscle differentiated
to an early myofiber stage but soon atrophied in a
characteristic pattern similar to the one described above.

The present study was undertaken out of a need for a
quantitative description of aneurogenic limb development
and muscle degeneration. The results showed. in addition.
that the aneurogenic larvae in this study indeed had no
nerves eliminating doubts about ”sparsely innervated“
conditions (Egar et al.. 1973). More over. further details
of ultrastructural and cytochemical characteristics of

deterioration in aneurogenic muscle are presented.

MATERIAL.AND METHODS

1. Surgical procedures.

Amblygtoma maculatum embryos. obtained from Tennessee.
were allowed to develop at 18°C. At Harrison's stage 2“ the
embryos had their entire presumptive nervous system removed.
down to the notochord. by excision with modified watch-
maker's forceps. Any remaining presumptive nerve cells could
be easily detected due to their different pigmentation and
were therefore removed with a Spemann hairloop. The
operative procedure is schematically shown in Figure 1. The
stippled region indicates the level of excision of
presumptive neural tissue. Figure 2 schematically shows the
Operation performed to produce control animals. fibre. the
excision of presumptive neural tissue paralleled that of
the aneurogenic embryos except that a segment of neural
tissue about 1 mm long dorsal to the limb bud anlagen was
left intact. Control larvae had to be produced
experimentally since normal. unfed and freely walking and
swimming larvae deplete their yolk supply at a faster rate
[than.aneurogenics. develOp only three digit forelimbs.
and died at an earlier time than aneurogenics. Stage 24
embryos were chosen because they permitted easy removal of
the neural tube and assured removal of the neural crest

cells before their beginning migration (Detwiler. 1937).

12

13

Figure 1

Surgical procedure to produce aneurogenic larvae.
Harrison's stage 2# embryos had their presumptive neural
tissue. including the neural crest. excised (stippled
region). Operated embryos were allowed to develop
immersed in Steinberg's saline. Photograph (a) shows a
38 days (Stage 2“ + 38 days) old aneurogenic larva.
Arrow in (a) indicates mid-stylopodium region of fore-
limb where cross section samples were taken for
measurement.

Photograph (a) 15X

11+

Fig. I

Discarded

3‘0 g e 24 '-
embi‘yo s§§§§2

 

 

Aneurogenic Animal (38 days)
Four digit forelimbs

15

Figure 2

Surgical procedure to produce control larvae.

Harrison's stage 2“ embryos had their presumptive neural
tissue excised (stippled region) except for an about

1 mm segment dorsal to the limb bud anlagen. Operated
embryos were allowed to develop immersed in Steinberg's
saline. Photograph (b) shows a 38 days old (Stage 2“ + 38
days) control larva. Arrow in (b) indicates mid-
stylopodium region of forelimb where cross section
samples were taken for measurement.

Photograph (b) 15X

16

Fig.2

Discarded ,.. Discarded

.
. .‘r
94:34.}
'-'-:;:-. ._

 

Canlrol Animal (38 days)
Four digil forelimbs

17
After the operation. embryos were placed in previ-

ously aerated sterile Steinberg's solution containing 0.25
g of Na-sulfadiasene. 0.2 mg of penicillin and 0.5 mg of
streptomycin per liter. Operated embryos were allowed to
develop at 18°C in an incubator. Clearly. aneurogenic and
control animals had to rely on their stored yolk for food;
they were unable to feed even when mature. Normal animals
were fed brine shrimp. Daphnia. and immture snails.

2. Tissue processing.

At appropriate times whole animals were killed in ice
cold. 2% purified glutaraldehyde solution in a 0.1 I caco-
dylate buffer. pH 7.4. Limbs were dissected with the ani-
mals immersed in the fixative and then placed into fresh.
ice cold fixative solution for 1 to 2 hours. Entire limbs
were postfixed in ice cold 2% osmium tetroxide in a 0.1 I
cacodylate buffer. pH 7.“. containing 0.15 M sucrose for
3 hours. After fixation. tissues were washed three times
in cacodylate buffer containing 0.2 I sucrose. Next. the
tissues were dehydrated in ethanol. treated with 1%
p-phenylenediamine in 70% ethanol (Ledingham and Simpson.
1972) for 10 minutes and stained 2g blgg with 2% uranyl
acetate in 100$ ethanol. Following dehydration and pre-
staining. the tissues were infiltrated with Spurr's
plastic (Spurr. 1969). For light microscopy. micrometer
thick sections were cut on a LKB or Porter Blum MT2
ultramicrotome. mounted on microscope slides. and stained

with 1% azure B containing 1% sodium borate. For electron

18
microscopy. grey sections were cut with the same

instruments. placed on copper grids and stained with lead
citrate for about 20 minutes. Grids were viewed with a
Hitachi 11E or a Zeiss 9A electron microscope.

3. Ruthenium red staining (myofiber basement laminae).
For ruthenium red staining. the method of Luft
(1971a. b) was employed except that ruthenium red was not
added to the glutaraldehyde fixative solution. Prior to
ruthenium red - osmium tetroxide fixation. the limb skin

was peeled from fixed limbs since the skin blocks
penetration of ruthenium red. The dehydration and
infiltration procedure was followed as described above.
a. Acid phosphatase staining (lysosomes).

Glutaraldehyde fixed limbs denuded of their skin
were incubated for 15 minutes according to the method of
Novikoff et a1. (1971). For control incubations. 10 mmol
sodium fluoride was added to the incubation mixture or
the substrate was omitted. Sodium fluoride was shown
quantitative-histochemically (Pfeifer and Witschel. 1972)
and biochemically (Boyer et al.. 1961. 1971) to be an
extremely potent (98%) inhibitor of acid phosphatase.

5. Autoradiography.

Aneurogenic animals. whose forelimbs were in the
early four digit stage of development. were treated
with 0.02 mCi of tritiated uridine or 0.001 mCi of
tritiated leucine in a volume of 0.02 ml. The tritiated

precursors were injected intraperitoneally with a

H' _*. I

‘4" ‘ “Vt ‘-

‘4'

19
Hamilton syringe to which a glass micrOpipette was

mounted with paraffin to the syringe needle. After 3
hours of incorporation the animals were killed while
immersed in the fixative and processed for light
microscopy and electron microscopy as described above.
Stained sections were dipped in Ilford K-S emulsion
diluted 3:1 with distilled water. After a 3 week exposure
time the stained slides were photographically processed.
6. Quantitative procedure.

Plastic embedded limbs were marked at the proximal-
distal center of the upper forelimb and several 1 um
cross sections per slide were mounted and stained as
described above. Before the measurement. all slides with
13 to 65 day aneurogenic and control as well as #3 day
and 65 day normal section samples were shuffled for
randomization. Cross sectional limb tissue areas were
measured with a TTMC particle measurement computer
system (Millipore Corp.) which consisted of a light
microscope with an attached television scanner. and a
small electronic computer. Whole limb areas and solid
tissue areas (whole limb minus clear spaces) were measured
at 10X: muscle and humerus areas were measured at uox. For
myonuclear area measurements a region of a muscle bundle
for each limb was selected. Measurements were performed
under oil (100x) and the electronically selected muscle
frame was placed at random.near the periphery of a muscle

bundle (C. Table 13). The same muscle bundle was chosen

20
for each limb. The total number of myonuclei per limb

and the peripheral or central location of myonuclei
within a myofiber were determined with a tally. Lengths
of myonuclei were measured with an ocular micrometer.
7. Statistics.

For statistical calculations program BMD07M. stepwise
discriminant analysis. and program BMDOiD. single data
description. from the Health Sciences Computing Facility.
University of California (Dixon. 1973) were used. In
addition. t-tests were done on a programmable Olivetti

calculator.

RESULTS

In contrast to previous studies (Thornton and Steen.
1962; Egar et al.. 1973: Tweedle et al.. 1974). the
methodology employed in this investigation produced true
aneurogenic limbs; i.e. nerves were never (even with the
electron microscope) seen in these aneurogenic creatures.
The control animals. on the other hand. developed nerve
strands; and typical neuromuscular junctions were
detected (Plate 1. Arrows). Furthermore. several mature
control animals were seen to use their forelimbs in
walking movements. The development of aneurogenic and
control forelimbs occurred at exactly the same rate
(Tweedle. personal communication).

Table 1 (Appendix) shows the cross sectional areas.
in squared micrometers. of the various aneurogenic.
control and normal limb tissues and Table 2. which was
derived from Table 1. shows the average limb tissue
areas including their standard errors. A statistical
time series analysis (Table 3. b. 5) by computer revealed
that during the 13 to 65 day time span (Table 3) the
whole aneurogenic limb underwent the greatest area change;
less but still statistically significant change is seen
in muscle and in the number of muscle nuclei during this
time. The least change in time is seen in the solid tissue

area (all stained tissues exclusive unstained clear

21

22
spaces). In fact. the change is statistically

insignificant even at P<0.05. That the solid tissue
area does indeed show an insignificant change during the
13 to 65 day period is graphically obvious in Figure 4.
In comparing the Y-axis positions of the 13 day to the
65 day plot. one appreciates the negligible distance
between the two points on the Y-axis. The control whole
limb tissue area also shows the greatest change during
the 13 to 65 day time period immediately followed by
muscle. However. the change in total number of muscle
nuclei per limb cross section is barely significant at
P<0.01. and the change in time of solid tissue and
humerus area is not significant at all at the 5% and 1%
level of significance.

Table 4 shows a time series statistical analysis
of the various aneurogenic limb tissues added
sequentially. For example. aneurogenic muscle (M) alone
shows no statistically significant change (at P(0.01)
during 13 to 43 days except for the day 21 to day 31
time period. The least change occurs during 54 to 65
days followed by 31 to 43 days. The greatest change
occurs in aneurogenic muscle during the day 21 to day
54 time period. If two aneurogenic variables. muscle
area and limb area. are considered together (Table 4.
M+L) no statistically significant change is apparent
during the day 13 to day 43 time period. However. from

23
day 21 to day 31 the change in muscle plus limb area

is statistically significant. The greatest change in
the muscle plus limb area is seen during the day 54 to
day 65 time period. The bottom horizontal segment of
Table 4 considers all five variables together (M+L+S+H
+N). i.e. whole limb (L). solid tissue (S). humerus

(H). muscle area (M) and number of muscle nuclei (N).
This entire aneurogenic limb system. defined by the five
variables. shows the greatest single time period (54 to
65 days) change during 54 to 65 days. The greatest over-
all change occurs from 21 to 54 days and statistically
insignificant change at the 1% level of significance is
seen during the 13 to 31 and 31 to 43 day time periods.

Table 5 shows the same statistical time analysis
for control limb tissues. Here. the muscle alone
changes the most during 13 to 31 days and least during
43 to 54 days. Considering the entire control limb
system. one finds the greatest area change to occur
during 13 to 21 days and statistically insignificant
changes at P<0.01 are seen during the day 21 to day 43
time span.

The gross. cross sectional limb areas for
aneurogenic. control and normal animals presented in
Table 2 are graphically shown in Figure 3. At 13 days
there is no statistical difference between aneurogenic

and control means (Table 6. Figure 3). In fact. the two

 

24
means are statistically equal even at the 5% level of

significance. Beginning with 21 days after the Operation
aneurogenic and control limb areas are different: the
aneurogenic limbs remain smaller in cross sectional area
beyond 13 days in comparison to control limbs. Both kinds
of limbs. however. seem to increase in area from day 13 to
day 21 and then gradually decrease in size except for
aneurogenic limbs which drastically increase in cross
sectional area from day 54 to day 65. The control solid
limb tissue. measured as the cross sectional limb area
minus the clear spaces. shows a gradual increase in area
from 13 days to 31 days (Figure 4. Table 1. 2) followed
by a decline. Aneurogenic solid areas seem to diminish
in time from day 13 except for the period of days 54 to
65 where the solid areas seem to return to day 13 levels.
The control and aneurogenic humeri (Table 1. 2. Figure 5)
gradually increase in cross sectional area in time
reaching a maximum at 43 days. Statistical comparison of
aneurogenic with control humeri (Table 6) indicates no
difference between means at the 5% level of significance
throughout the entire time period. Another structure
exhibiting a remarkable stability inspite of the absence
of nerves is the muscle tendon. With each limb cross
section a single tendon is seen (Plate 3). Even 65 day
old aneurogenic limbs contain one clearly visible tendon

which becomes more obvious since all muscle had virtually

25
disappeared. Plate 4 shows an electronmicrograph of a
tendon from a 65 day aneurogenic forelimb. For
comparison. a tendon from a normal forelimb is shown in
Plate 5 and further magnified in Plate 5a. However. one
does notice that the aneurogenic tendon is somewhat
reduced in diameter and that the amount of collagen.
which fills the intercellular spaces. is reduced.

The greatest change within the limb area occurs in
the muscle (Plate 2. 3. Figure 6. Table 3. 4. 5). In
fact. two aneurogenic animals have no light micro-
SCOpically detectable muscle whatsoever at 54 and 65
days respectively (Table 1). However. small remnants of
myofibers could always be found with the electron
microscope (Plate 6). The aneurogenic limb musculature
at day 13 is not significantly different from controls
(Table 6). At this stage of development the upper arm
limb muscle is in the early myotube stage. But beginning
with day 21. Figure 6 and Table 6 show a statistically
significant separation between control and aneurogenic
muscle. The control muscle reaches a plateau at the four
digit stage (31 days) of development and decreases in
area from 54 to 65 days (Figure 6). Aneurogenic muscle
seems to increase from 13 to 21 days. then gradually
diminishes with time. The great difference between
aneurogenics and controls in cross sectional limb muscle

area is readily apparent in Plates 2 and 3. Furthermore.

‘

26
Plates 2b. 3b and 3c show large nerve bundles. whereas

the aneurogenic limbs do not have any nerves whatsoever
(Plates 2a. 3a). Far from being metabolically inactive.
degenerating aneurogenic limb muscle readily incorporates
tritiated leucine (Plate 7) or tritiated uridine. In
fact. by visual inspection one notices in autoradio-
graphic preparations the presence of more disintegration
silver grains over partially degenerated muscle than over
muscle where cross striations are still visible (Plate 7.
arrows). The number of aneurogenic peripheral myonuclei.
as a measure of myofiber differentiation. reaches its
peak at the four digit stage in the upper arm (Tables 7.
8. Figure 7). In the control larvae. peripheral nuclear
numbers plateau at the four digit stage. The plateau is
statistically sound taking into consideration that at
the 5% level of significance the control 31 and 43 day
means are equal whereas for aneurogenics they are not
(Figure 7. Table 8). At 13 days after the operation
there is no statistical difference between aneurogenic
and control groups at 1% or 5% level of significance
(Table 8). However. at 21. 31 and 43 days nuclear
numbers are statistically quite different between
aneurogenic and control larvae.

In addition to the disappearance of muscle. the
total number of muscle nuclei (myonuclei and satellite

cell nuclei) declines also with time in control and

2?
aneurogenic limb muscle (Figure 8. Tables 1. 2). Inhially.
at days 13 and 21 one does not detect a statistical
difference between aneurogenic and control mean number of
muscle nuclei. but from day 31 on a separation between
controls and aneurogenics at the 0.1% level of signifi-
cance becomes established. Also. beginning with day 31
the total number of muscle nuclei per limb section in
aneurogenic limbs diminishes from less than two thirds to
as much as one fourth as compared to control limbs. The
cross sectional area of muscle nuclei also diminishes with
time (Table 9 - Appendix. Table 10. Figure 9). However.
only on days 43 and 54 does one see any statistical
difference between aneurogenic and control groups. From
day 13 to day 31 and on day 65 one finds no statistical
difference between the two groups. The length of
aneurogenic and control myonuclei at 43 days of
deve10pment is presented in Table 11 (Appendix) and
Table 12. Analysis of variance (Table 12) indicates that
all myonuclei are statistically equal at the 1% level of
significance. The control sum nuclear area per selected
muscle area (Table 13) reaches a level minimum by 21
days (Figure 10). whereas the aneurogenic muscle attains
a peak at 54 days. This peak sum nuclear area is
approximately at the same level as in day 13 muscle. At
day 13 and 21 no statistical difference between

aneurogenic and control sum nuclear area is found (Table

28
14). However. from.day 31 on the difference between

aneurogenics and controls is significant at the 1% and
5% level of significance.

In a previous study (Tweedle et al.. 1974) it was
suggested that macrophages may engulf degenerating
aneurogenic muscle fragments thereby removing waste
sarcOplasmic material. In corroboration Plate 8 is
presented showing a macrophage in the process of
engulfing a muscle fragment (Arrows). Plate 9 shows an
acid phosphatase (Acpase) preparation depicting a macro-
phage with several digestion vacuoles and lysosomes.
Employing the Acpase technique several lysosomes were
found next to myofibrillar material within degenerating
aneurogenic muscle. Plate 10 shows a sample electron-
micrograph of such an Acpase preparation. Here. a
lysosome is situated adjacent to some myofibrillar
material. Phagosomes. similar to the ones seen in Plate
9 were never found in aneurogenic myofibers or remnants
of myofibers. Also. developing aneurogenic muscle or
control muscle never exhibited Acpase stained lysosomes.
In ruthenium red stained muscle it was found that
aneurogenic myofibers develOped basement laminae similar
in appearance as in normal animals. Plate 11 shows
basement laminae of normal muscle and Plate 12 depicts
a portion of a 21 day aneurogenic myofiber with its

ruthenium red stained basement lamina. Ruthenium red

29
stain was also effective in the identification of
muscle satellite cells. Plate 13 is an example of the
numerous satellite cells seen in normal and aneurogenic
Amblystoma muscle. In addition to limb musculature. the
axial musculature also disappears in time although the
degeneration of axial muscle was not specifically

studied here.

30

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analysis-~Hsa1th Sciences Computing
Facility. UCLA. 1970.

Statistics: BMDO7M-Stepwise discriminant
P-valucs : Freund . Livermore .

1960.

Manusclc: sthole limb: Sssolid tissues

Miller--Mannsl of
N-nnnber of muscle nuclei: Hflhnncrus.

Experimental Statistics.

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Table 5
STATISTICS FOR TIME SERIES. CONTROLS

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34

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Table 7 LOCATION OF HONUCLEI
A B
Number of Number of
Age of centrally peripherally
animals Animal located located B
(days) I.D. nuclei nuclei I33
“5‘158 8 3 0e27
“5-178 17 3 0e15
13 b5-16s 11 1 0.08
#5-158 3 2 0.#0
45-188 h 2 0.33
#6-3 2“ 5 0.17 0
#6-6 21 3 0.12 i
21 h6-10 16 8 0.33 .d
46-1} 20 9 0.21 5
h6-5 12 10 O. 5 o
h7-2 6 8 0.57 '3
07-3 1 5 0.83 e
31 1.7-5 6 6 0.50 3’
#7-8 10 10 0.20 g
b7-12 10 7 0. 1 o
b8-1 3 2 0.ho ‘5
#8-16 18 5 0.21
#3 h8-6 15 7 0.32
#8-15 13 7 0.35
h8-9 11 6 0.35
05-1 10 6 0.3?
45-3 12 9 0. 2
13 45-5 1 5 0.25
06-93 11 26 0.70
h6-88 h 29 0.88 ,3
21 h6-6s 8 39 0.83 a
h6- s 10 30 0.75 .a
#6 s 12 2“ 0.66 5
h7-7s 5 30 0.87 .4
07-1 8 8 30 0.79 8
31 h7-1 s k 35 0.90 'g
#7-“8 h 27 0°37 °
07-13: 2 36 0.95 0
08-38 h 32 0.88
h8-10s h 30 0.88
43 08-53 h 34 0.89
08-153 h 29 0.88
“8‘93 9 26 0e?“

 

 

 

 

 

 

 

36

Table 8

LOCATION OF MYONUCLEI

 

Age of

Experimental

 

 

 

 

 

 

 

3:38 am: °‘ 12:: °‘ — 3:35. .
13 zggfiigéenic g 3:232 338;: 1'“29°
21 :ggzigéenic g 8:332 3:323 6.902“
31 :zgtggéenic g giggg 328;? “.1978
“3 :zgfiigéenic 2 3:32: 3.3%: 13-200

3 333353 0.5882
3% 333.533: 3.0490

 

 

 

 

 

 

XB - Average fraction of number of peripheral

A+B

P‘eOS 3 2.306
P<-01 ' 3-355

Statistics: t-test for equality of two means.

myonuclei per total

 

37

Table 10

MYONUCLEAR AREA

 

 

 

 

 

 

Age of Number Number Nuclear area D/C

animals of of lzgg Std. Std.

(days) limbs nuclei ( ) error lean error
13 0 26 53.050 10.133 0.502 0.028 g
21 5 03 06.66 0.591 0.276 0.051 O
31 5 09 30.25 0.128 0. 76 0.035 E
z i 1 28.201 1.187 0. 22 0.033 g
5 2 18.382 1.257 0.097 0.062 6
65 5 27 27.363 1.350 0.023 0.000 P
13 32 03.833 0.301 0.39 0.033
21 15. 36 00.326 2.9 2 0.15 0.020 9
31 6 53 37.2 0 2.5 0 0.156 0.018 =3
a 5 0 32.518 2.513 0.153 0.025 if
5 5 3 33.826 1.860 0.177 0.007 3
65 5 05 28.113 1.826 0.196 0.018
03 5 1 01.257 2.172 0.217 0.019 2
65 5 1 36.001 0.970 0.110 0.018 Be,

1

 

 

 

 

 

 

 

 

D/C 1 See Table 13

 

Table 12

MYONUCLEAR LENGTH \

 

 

 

 

 

 

 

 

Mean

length of
Animal myonuclei Standard Standard
I.D. (um) error deviation
08-10 06.408 2.000 6.301
08-2 37.030 1.905 6.160 Aneurogenic
08—13 26.690 2.328 7.027 #3 day old
#8— 0.820 1.238 3.909 animals
08- 39.601 2.181 6.897
#B-Bs 36.690 1.591 5.570
08-6s 39.090 1.739 .511 Control
48-148 38.757 1.415 .539 03 day old
08-123 8.020 1.356 4.362 animals
08-28 0.378 1.238 3.909

 

F=2.66 for equality of all means
P<.01=2.80
P< e 0532 e 00

 

39

Table 13
MYONUCLEAR AREA

 

 

 

 

 

 

 

 

 

 

 

c D
Selected Sum
Age of muscle Number of nuclear
animals Animal areg nuclei area
(days) I.D. (um ) per C (umz) D/C
“5’15d “Dec 5 1 4. 0.402 I
13 05-186 080.0 6 226.8 0.556
45-168 6 5.0 8 376.6 0. 02
05-170 12 8.3 7 583.9 0. 68
06-3 2810.1 10 003.5 0.158
06-6 1585.7 6 370.5 0.230
21 02' 2 23.; 13 271.0 0.057
- . 559-5 0.227
06-10 983.8 6 301.7 0.307
07-2 1830.2 11 068.2 0.2
07-12 070.6 6 193.0 0.038 #3
31 07-5 853.5 12 308.8 0.362 a
07-3 765.0 10 357.8 0.068 a
07-8 700.0 6 271.1 0.387 0
08-16 726.8 11 270.6 0.378'2
“8-1 579.0 10 313.1 0. 01 $0
48-6 867.0 10 301.5 0. 08 §
08-15 025.2 7 183.1 0. 31 2
03-9 560.9 17 361.0 0-639 ‘0'
03-10 09.6 15 229.7 0.561
50 03-3 259.8 5 96.6 0. 72
03-6 211.0 5 88.3 0. 18
03-13 0.0 0 0.0 0.0
50-2 005.7 5 126.2 0.311
50-5 97.8 6 169.1 0.025
65 50-9 38.5 5 161.3 0.368
08-12r 1620.0 10 016.3 0.257
”8.101. 1905.5 12 ”2801 0e225 O
03 08-7r 2006.5 7 300.2 0.170 3
08- r 2282.1 10 588.6 0.258 g
08- r 1706.? 8 306.7 0.176 s
50-8r 2085.6 13 390.0 0.157 H
50-10: 32 3.5 9 337.1 0.100 g
65 50-15r 28 2.8 6 187.7 0.066 0
50-91. 2552-0 7 196.0 0.07? z

 

 

 

 

 

 

 

 

#0

Table 13(cont.)
MYONUCLEAR AREA

 

 

 

 

 

 

 

 

c D
Selected Sum
Age of muscle Number of nuclear
animals Animal ares nuclei area
(days) I.D. (um ) per C (um )
75-1 1533.9 10 583.8
05-9 090.0 7 232.9 0. 53%
13 05-5 067.0 6 221. 0. 073
75-13 632.3 2 225. 0 0. 356
05-3 593-? 182. 0 0. 307
06-06 3811. 0 10 391.7 0 103
2106-53 3330. 0 11 532. 0.160
06-93 1776. 8 8 272.6 0.153
06-66 1387 2 7 277.9 0.200
“7'78 5590e0 15 558e2 0.100
07-10s 2390.2 10 2 7.8 0.120 .
07-as 12 5. 5 7 200.5 0.193 8
07-153 991. 2 6 210.2 0.216 g
07.0: 1660. 3 6 291.0 0-175.5
08-33 2385. 7 16 028.0 0.180 3
08-153 2600. 7 8 228.2 0.086-9
03 08-53 2316. 2 13 078. 0.207 8
08-93 2366.0 6 239.7 0.1010
08-10: 1120.0 7 213.7 0.191
09-10 3318.0 10 533.3 0.161
49-1“ 535“. 0 9 307.5 0.057
50 49-9 3528.1 9 336.8 0.09
09-2 509. 0 5 156.0 0.28
09-13 587. 7 6 169.5 0.288
50-7: 1960. 9 10 330.3 0.168
50-168 17023 .3 10 22.3 0.201
65 50-“8 7 15909 0.19“
50-103 763.1 7 175.9 0.221
50-13 1022.0 7 205.8 0.1 5

 

 

 

 

 

 

 

 

41

Table 14

STATISTICS FOR MIONUCLEAR AREA

 

 

 

 

 

t for
Age of mean
animals nuclear t for
(days) area mean D/C P<.05 P<.01
13 0.95 2.30 2.36 3.50
21 1008 2002 V V
ii -0.22 5.8 2.26 3.25 Aneurogenic
z -20 605 2031 3036 V80 contrOI
5 -6.09 0.18 2.36 3.50
65 -O.33 0.75 2.31 3.36
03 -5.28 5.39 Aneurogenic
65 -1.75 6.51 vs. normal
03 2.63 -2.05 L Control vs.
65 -1.57 3.29 i 0 normal

 

 

 

 

 

 

 

 

Statistics: Two tailed t-test for equality of two means

P-valuess Freund. Livermore. Miller- Manual of
Experimental Statistics. 1960.

 

#2

Figure 3

Gross cross sectional limb area (see Table 2, 6).

n.d. a No statistical difference between aneurogenic and
control means at P<0.05

Numbers between graphs = Difference between aneurogenic
and control means at the indicated level of significance.

24.

0—0

Limb area (umzx IO4)

3

'5

O)

 

“3

Figure 3

WHOLE LI MB anormal

 

   

 

 

T
‘
1
Eoneurogenic
.00:
.Ol\ ___._o
n.d. 3...... ———§ control
W W W 4
I8 2] 3| 48 5A4 65

Days after 3999-24

Figure 0

Gross cross sectional limb area minus clear spaces (see
Tables 2, 6).

.3 Aneurogenic

OI Control

[La Normal

n.d. a No statistical difference between aneurogenic and
control means at P<0.05.

Numbers between graphs = Statistical difference between
aneurogenic and control means at indicated level of
significance.

'fissue area (umleOA)

l8*

K)

(D

A

 

“5

Figure 4

SOLID TISSUE

 

 

 

I
l
o—-—-’2\I\
n..d$\ ‘0' 1M\l\l};n.d
§\T/§ \Ol f
W X"? S”? 4
I3 2| 31 48 5‘4 65

Days after stage 24

06

Figure 5

Cross sectional area of the humerus (see Tables 2, 6).

.8 Aneurogenic
()8 Control

AMI Normal
n.d. I No statistical difference between aneurogenic
and control humeri at P<0.05.

07

Figure 5

HUMERUS IN CROSS SECTION

 

l87
W\T I

AlG‘ l
'9 T °‘\E\\a
QM ° E/fl 1 it]
x l\
“’92 T/E/L‘
3 1/

IO- 2
o
0)
L8.
0 nd nd nd n.d nd nd
86-
35:34, I7 W fi"? 4
E
02*

0 I3 2| 3| 43 as

 

 

Days after stage 24

08

Figure 6

Cross sectional total upper forelimb muscle area (see
Table 2, 6).

n.d. = No statistical difference between aneurogenic
and control means at P<0.05

Other numbers within graphs 2 Difference between
aneurogenic and control means at indicated level of
significance.

“9

Figure 6

TOTAL MUSCLE PER LIMB
60| a

 

 

 

 

 

52 »
J.
38F
'9" ’
Q ,
Nx | |
30 . . norma
s I 1
V F? V“? X“? 2
C
(D .
5 20 T
. O
.02 ’ I/i\§—"'“’
8 I6’ 1
3 ’ .L.
2 . .0| .00| 00|
'0 ’ T 0' Econtrol
’ nd ' -
6 1/‘\ -.
A n 9 aneurogenic

 

0 I3 2| 3| 43 5‘4 65
Days after stage 24

 

50

Figure 7

Number of peripheral myonuclei per total number of
myonuclei (see Table 8).

vertical arrows 2 statistics aneurogenic vs. control.
horizontal arrows = statistics 31 days vs. 43 days.
n.d. = No statistical difference between means at
the indicated level of significance.

Other numbers within graph 8 Statistical difference
between means at the indicated level of significance.

51

Figure 7

MYOFIBER DIFFERENTIATION

LO

3'

Number . peripheral nuclei/Total

00

'0)

 

 

T
Hnd.
; .05 T
/l\Icontrol
I. 1
.0l
I z.
. l
| I |
| .(lDI
O T .
05+
n..d :OIT * Ianeurogemc
I/
17 3’7 1“?-—*
2| 3|

Days after stage 24

52

Figure 8

Total number of muscle nuclei per limb cross section
(see Table 2, 6).

n.d. = No statistical difference between aneurogenic and
control number of muscle nuclei at P<0.05

Other numbers between graphs 2 Statistical difference
between aneurogenics and controls at indicated level of
significance.

 

Number of muscle nuclei xIO

53

Figure 8

MUSCLE NUCLEl/LIMB

N
O

5

I?)

(I)

O)

A

N

O

 

A normal

 

”\

 

 

i
Id\o/.°L/§\
. §"'d\ 00| .00| §
"‘— \§ \§ control

' .OOI

\ .00|

I\§ aneurogenic

Y} {‘7 5‘9
I3 2| 3| 43 54 65

Days afler slage 24

54

Figure 9

Czoss sectional area of muscle nuclei (see Tables 10,

1 .

.8 Aneurogenic

08 Control

[‘9 Normal

n.d. a No statistical difference between control and
aneurogenic means at P<0.05.

Numbers between graphs 3 Statistical difference between

aneurogenic and control means at indicated level of
significance.

55

Figure 9

SIZE OF MUSCLE NUCLEI

 

 

 

 

.0 .0
\To.. nlu. .e. . Mu
s / m.\ 3
TalOToi; . 4
\\ E
JxOhMIJ w a
\.

.\lo..WWoll W ad
T e d rlpll. W B
0 w O mu 0 O O

4 3 2 I

E36 was: 90sz V5 85

Days ofler stage 24

56

Figure 1 0

Sum mzonuclear area per selected muscle area (see Table
10. 1 .

Myonuclear area/Muscle area = D/C in Table 10

n.d. a No statistical difference between control and
aneurogenic means at P(0.05

Numbers between graphs 3 Difference between aneurogenic
and control means at indicated level of significance.

0r 0re0/ lVluscle 0re0

Myonucle

.0
on

.0
4s

.0
o:

.0
N

.0

 

57

Figure 10

NUCLEAR DENSITY

‘l

n..d T/ \"
" . aneurogenic

 

 

 

v normal
ll ll 9? a
:3 2] 3] 43 5‘4 6‘5

 

Days 0“” SW39 2‘4 _ - -

58

Plate 1

Control three digit (21 days) forelimb muscle.
Two axons are seen to contact a myofiber (Arrows).
Electronmicrograph 13680X

 

 

 

60

Plate 2

Cross section at mid-stylopodium region at 54 days of
develOpment.

a a Aneurogenic forelimb

b = Control forelimb

Note absence of nerves in aneurogenic limb and virtual
absence of muscle.

a Blood vessel

Epidermis

Humerus

Muscle

Nerve bundle

Tendon

hotomicrograph 80X

tn

E
H
M

N
T
P

61

 

 

62

Plate 3

Mid-stlepodium cross section at 65 days of deve10pment.
a = Aneurogenic forelimb

b = Control forelimb

c = Normal forelimb

Note virtual absence of muscle in aneurogenic limb.
a Blood vessel

Epidermis

Humerus

Muscle

Nerve bundle

= Tendon

hotomicrograph 80X

B
E
H
M
N
T
P

 

64

Plate 4

Tendon from 65 day aneurogenic forelimb muscle in cross
section.
Electronmicrograph 12300x

 

66

Plate 5

Tendon from normal forelimb muscle in cross section.
Electronmicrograph 4980K

67

Plate 5

 

 

 

68

Plate 5a

Higher magnification of Plate 5.

C = Collagen
Note collagen fibrils within intercellular spaces.

Electronmicrograph 77200X

 

70

Plate 6

Remnants of forelimb muscle after 65 days in aneurogenic
condition. This thin section was taken from a specimen
in which muscle could no longer be detected with the
light microscope.

Electronmicrograph 12890X

 

72

Plate 7

Longitudinal forelimb section from 26 days old
aneurogenic larva. Grains are sites of disintegrations
from incorporated tritiated leucine. Tritiated uridine
incorporations show a similar grain pattern.

E a Epidermis

H = Humerus

M = Muscle

Note that more grains are visible over degenerated
myofibers (arrows) than over fibers where cross
striations are still visible.

Photomicrograph 2250K

ell
0‘
l
l
.
U
,
1.4..

w.

1v.
5...):

 

74

Plate 8

Macrophage from aneurogenic four digit forelimb with
muscle fragment in the process of being engulfed
(arrows).

MaN = macrophage nucleus

Electronmicrograph 25800x

 

76

Plate 9

Acid phosphatase preparation of a portion of a macro-

phage found among degenerating four digit aneurogenic
muscle.

D = Digestion vacuole
L = Lysosome
Electronmicrograph usooox

 

at; .

2'

 

 

 

 

78

Plate 10

Acid phosphatase preparation of degenerating aneurogenic
muscle.

L = Lysosome

MN = Myofiber nucleus

My 2 Myofibrillar material

Note the position of the lysosome adjacent to some
disintegrating myofibrillar material.

Electronmicrograph “SOOOX

r.” .r

 

80

Plate 11

Portion of normal myofibers.

BL a Basement lamina

F1. F2 2 Myofiber

Ruthenium red stain

Compare basement lamina with aneurogenic in Plate 12.
Electronmicrograph 23760X

 

82

Plate 12

Portion of a three digit (21 days) aneurogenic myofiber.
BL = Basement lamina

F = Myofiber

Ruthenium red stain

Compare basement lamina with that in Plate 11.
Electronmicrograph hOOOOX

. 5.4,,

vi.
7 ms

 

 

 

84

Plate 13

Satellite cell in normal Amblystoma forelimb muscle.

BL - Basement lamina

F1. F2 8 Myofiber

N a Satellite cell nucleus

Ruthenium red stain

Note that the basement lamina envelOps the myofiber
and its satellite cell. The satellite cell cytoplasm is
separated from the sarcoplasm by a double plasma
membrane (arrow) (Mauro. 1961).

Electronmicrograph 25080X

,J‘ .
Yné‘l'fifi

“a“.

mag ’-
"' “fl 3415‘; ‘ ._

 

DISCUSSION

The most striking visual difference between
aneurogenic and control larvae is the complete absence
of pigmentation in the aneurogenic ones (Figure 1a)
which is not surprising since melanocyte precursors'
were removed with the neural crest (Detwiler. 19373
Balinsky. 1970). Control larvae are pigmented (Figure
2b). As in previous studies (Wintrebert. 1905' Hooker.
1911), aneurogenic larvae were never seen to move
spontaneously and nerves were never detected in the
aneurogenics even with the electron microscope.
Aneurogenic limbs extended from the body and the only
movement visible was in the heart and circulating
blood. However. control larvae were seen to use their
forelimbs spontaneously in walking movements attesting
to the functional innervation of these limbs by the
”isolated spinal cord”. Furthermore. control limbs and
control mid-trunk regions showed reflex movements upon
stimulation by touch. The control limb musculature was
seen in the electron microscOpe to be normally inner-
vated although a myofiber has been seen with two
neuromuscular junctions (Plate 1). It is not known
whether or not multiple innervation in Amblystoma is
the rule or appeared only under these experimental

conditions. With the exception of one case (Plate 1),

86

87

control sections showed one neuromuscular junction per
fiber which, of course, does not rule out the possibility
of further innervation elsewhere along the myofiber.

Aneurogenic and control limbs developed at exactly
the same rate. Notch forelimbs developed at 13 days after
the operation with the musculature largely in the early
myotube stage of development. three digits developed at
21 days and four digits at 31 days (note developmental
stages schematically indicated on all figures). On the
other hand, normal, unfed larvae never develOped beyond
the three digit stage and were observed to have depleted
their yolk supply at that time. Presumably lack of food
arrested the deve10pment of normal, unfed larvae at the
three digit stage. The total aneurogenic and control
larvae in this study had an adequate yolk supply until
the four digit stage and beyond and presumably quiescence
in aneurogenics and reduced movement in controls
conserved food for complete develOpment. It is for these
reasons that normal, unfed larvae were not used as
controls and experimental controls were produced. Also,
control larvae were more equal to aneurogenics since
their nervous system was removed in a similar fashion
(brain, pituitary etc.) except for the segment of spinal
cord dorsal to the limbs.

The entire limbs (Table 2, Figure 3) in control and

aneurogenic larvae were identical in thickness (Table 6)

88
at 13 days after the operation. Starting with day 21.

aneurogenic limbs were thinner except for day 65 when
aneurogenic limbs were actually thicker than control
limbs. Possibly the sharp increase in limb thickness from
day 54 to 65 was due to a general edema as seen previously
for whole animals (Hooker. 1911). Surprisingly, even if
the fluid (unstained clear spaces) was electronically
subtracted from limb cross sections one sees an increase
in aneurogenic limb tissues from day 54 to 65 (Figure 4).
Muscle nuclei increased in cross sectional size also
during the same time (Figure 9). Possibly, in addition to
the influx of more intercellular fluid during the last
nine days of the experimental series, intracellular fluid
increased also.

The aneurogenic humeri grew in size from day 13 to
43 but remained constant in size from day 43 on (Figure 5.
Table 2). It is important to note that the humeri were
completely insensitive to the absence of nerves either
during the humerus growth period (day 13 to 43) or during
the maintenance period (day 43 to 65, Table 6). Even limbs
from normally feeding animals, where limb tissues were
twice as abundant as in aneurogenics. contained humeri of
aneurogenic or control size. Apparently. even normalcy and
feeding had no effect on the cross sectional size of
humeri.

The tendons also showed a remarkable insensitivity to

89

the absence of nerves (Plate 4). This insensitivity was
dramatized when tendons became extremely conspicuous in
limbs where the muscle had almost completely disappeared
(Plates 2. 3). Normalcy. on the other hand, did have an
effect on tendons as for example in 65 day normal
tendons (compare Plate 4 with Plates 5, 5a). The normal
tendons are much thicker at day 65, contain more
collagen within intercellular spaces and have more
nuclei than aneurogenic tendons.

A single limb tissue most susceptible to the
aneurogenic condition was musculature. In two cases all
traces of muscle, as seen with the light microsc0pe. had
disappeared by 54 and 65 days (Table 1. Appendix).
Although Table 3 indicates that whole aneurogenic limbs
underwent a slightly greater change during the 13 to 65
day period than muscle, it probably is a reflection of
the additive effects of all limb tissues. Figure 6
indicates an insignificant (Table 4) increase in
aneurogenic muscle mass from 13 to 21 days whereas
ultrastructural studies have shown deposition of
contractile material at that time (Tweedle et al.. 1974).
In the neonatally denervated rat limb muscle Zelena
(1962) observed an initial three to five fold increase in
weight. Cricket thorax muscle denervated at juvenile
stages also grew slightly in cross sectional area and

fiber number (Thommen. 1974). The fact that the nuclear

90
density (Figure 10) decreased by about 50% in aneurogenic

muscle from day 13 to 21 (D/C ratio, Table 10) combined
with the statistical probability that the muscle area per
limb remained constant during this time period leads one
to conclude that some growth of aneurogenic muscle must
have occurred from day 13 to 21. As mentioned above.
aneurogenic muscle growth was detected with the electron
microscOpe; aneurogenic early myotubes possessed much
less contractile protein than fully differentiated, four
digit. aneurogenic myofibers (Tweedle et al.. 1974).
Control muscle grew most rapidly during the 13 to 31 day
period (Table 5. Figure 6) but also declined later in
time. Aneurogenic and control larvae had depleted their
yolk supply by approximately 54 days. Therefore, one
could assume that the decline of control muscle from 54
to 65 days was due to starvation. Aneurogenic muscle
disappeared in time beginning with day 21 and finally
reached amounts one tenth of controls at 54 days. A
couple of reference plots for normal muscle at 54 and 65
days indicate the enormous effect of normalcy and
feeding on muscle growth. By 65 days normal muscle was
six times the amount of control muscle. Statistically.
the initial amounts of control and aneurogenic muscle
per limb were equal at 13 days but soon thereafter the
amounts of aneurogenic and control muscle diverged and

remained statistically separated throughout the

91
remaining time period. Except on day 13. aneurogenic

muscle was always much less than control muscle. Control
muscle grew rapidly whereas aneurogenic muscle grew only
feebly and then degenerated soon. Clearly. the only
difference between the two groups of animals were the
pieces of spinal cord left in control embryos in time
innervating control limbs.

The total number of muscle nuclei per limb cross
section gradually declined in aneurogenic limbs (Figure
8) shown statistically in Table 3. However. such a
decline during the 13 to 65 days period was not evident
at the 1% level of significance in control muscle.
Statistical equality between aneurogenic and control
number of muscle nuclei from 13 to 21 days suggest that
total number of muscle nuclei in aneurogenics and
controls are the same during that time. Beginning with
day 31. however. total number of muscle nuclei are
different in control versus aneurogenic limbs (Table 6)
suggesting that survival and possibly proliferation of
nuclei was interfered with in aneurogenics but less in
controls. By counting the numberof muscle nuclei in the
'denervated thorax musculature of the cricket. Thommen
(1974) also saw a reduction in the number of nuclei per
individual muscle. For example. two of the thorax
muscles contained 6.4% and 15% of normal numbers of
nuclei after denervation at stage 3 or 5 and measurement

at adult stages. However. a shorter denervation period

92
led to a survival of a higher number of nuclei. Gutmann

and Zelena (1962) wrote that nuclear changes are still
controversial but claim a temporary 40% increase in DNA
during the first few months of denervation in adult rat
limb muscle followed by a return to normal levels
thereafter. In the present study. an increase in total
number of nuclei per aneurogenic limb even from notch
to the three digit stage. a time when myotubes
differentiate. is doubtful.

The amount of nucle0plasm in cross section per
arbitrary aneurogenic muscle area shows an increase to
notch stage levels by day 54 (Figure 10. Tables 10. 13.
14). This increase in nuclear density does not seem to
have occurred by swelling of nuclei (Gutmann and Zelena.
1962) since the size of muscle nuclei actually decreased
with developmental age (Figure 9. Table 10). Apparently.
the increase in nuclear density of aneurogenic muscle is
a reflection of greater rate of disappearance of
myofibrillar material relative to nuclear numbers
combined with a greater survival rate for myonuclei. A
similar survival of muscle nuclei relative to denervated
muscle was hinted at by Gutmann and Zeleni (1962). If
one rearranges the data obtained by Thommen (1974) into
a form similar to the one employed in this study one
sees the same increased number of muscle nuclei per

muscle area in all of the denervated cricket thorax

 

93

muscles. Figure 10 furthermore shows a parallel decrease
in nuclear density in control and aneurogenic limb
muscle from 13 to 21 days of development. During this
period. the difference between aneurogenics and controls
is not statistically significant (Table 14) allowing one
to conclude that nuclear densities are the same in
aneurogenic and control muscle at 13 and 21 days. Such a
conclusion implies some growth of aneurogenic muscle
during the early part of limb development. Starting with
day 21. control nuclear density reaches a plateau of
about 15% nucle0plasmic area per muscle area whereas
aneurogenic muscle shows a sharp increase in nuclear
density as mentioned previously. From the three digit
stage of development until the termination of the
experimental series aneurogenic nuclear densities were
distinctly and statistically significantly (Table 14)
separated from control densities.

It was previously mentioned that individual nuclear
areas decrease with progressing developmental age (Figure
9. Table 10). Since no statistical difference between
aneurogenic and control muscle is apparent until the four
digit stage (Table 14) it seems that nuclei decrease in
cross sectional size regardless of whether the muscle is
innervated or not during this time. However. at 43 and 54
days aneurogenic nuclei are statistically smaller in

cross section than control nuclei. A decrease in cross

94
sectional area. of course. does not necessarily mean a
decrease in volume since it is well known that myonuclei
elongate as muscle differentiates. However. Table 12
shows the length of muscle nuclei for aneurogenics and
controls at 43 days of development and analysis of
variance indicates at the 1% level of significance that
all nuclei (aneurogenic and control) are of the same
length. So. one can conclude that at least 43 day
aneurogenic nuclei are smaller in volume than control
nuclei making it improbable that a differential elonga-
tion of aneurogenic vs. control muscle nuclei occurs.
Armed with this piece of information. one could
interpret the steeper slope of aneurogenic nuclear size
as compared to the control slape (Figure 9) to mean a
greater rate of nuclear shrinkage for aneurogenic
muscle nuclei. At 65 days aneurogenic. control. and
normal muscle nuclei are statistically the same; the
apparent size increase in aneurogenic nuclei from day
54 to 65 is probably due to a general limb edema as
discussed previously. When the number of peripherally
located nuclei were counted as a measure of myofiber
differentiation (Figure 7. Table 8). it was found that
aneurogenic myofibers from the upper forelimb reached
their peak differentiative state at the four digit
stage. Control myofibers differentiated at the same

time and remained differentiated whereas aneurogenic

95

fibers appeared to return to an undifferentiated state.
Possibly the apparent return of aneurogenic myofibers
to an undifferentiated state is an illusion since. as
already discussed. contractile material disappears at a
greater rate than nuclei leaving nuclei with a thin rim
of cytoplasm in the latter part of the experimental
series (Plate 6). That control fibers reached a plateau
in their differentiation and aneurogenic fibers did not
is indicated by the statistical comparison of
aneurogenic and control fibers (Table 8) during the 31
to 43 day time span. Control fibers do not differ
statistically from 31 to 43 days at the 5% level of
significance whereas aneurogenic fibers do during the
same time period and at the same level of significance.
At notch stages of development control and aneurogenic
fibers were equally undifferentiated; however.
subsequent to day 13 control fibers were always more
differentiated than aneurogenic fibers. Both conclusions
are confirmed by statistical inference (Table 8). Even
at their peak differentiative state. the aneurogenic
myofibers were less differentiated than control fibers.

In a previous study (Tweedle et al.. 1974) ultra-
structural changes during aneurogenic muscle
degeneration were described. In the present examination
of aneurogenic muscle degeneration it was found that

the ultrastructural characteristics of degeneration as

96
described by Tweedle et al. applied here also. although

the methodology employed differed and larvae were
totally aneurogenic. The most striking characteristics
of aneurogenic muscle at its peak differentiative
period is the abnormal dilation of the sarcoplasmic
reticulum. Otherwise. aneurogenic myofibers looked
quite normal at that time.

Basement laminae develOped in aneurogenic myo-
fibers in a similar fashion as in normal limbs (compare
Plate 11 with 12) indicating that the absence of nerves
did not affect the development of fiber sheaths. Typical
muscle satellite cells (Mauro. 1961) were detected in
aneurogenic. control. and normal Amblystoma muscle
although satellite cells are characteristically found in
mammalian muscle only and have not been seen heretofore
in amphibian muscle except in.Amblystoma mexicanum
(Flood. 1964; Flood. 1971).

The mechanism by which muscle atrophies and
contractile protein disappears has remained a mystery
to this day. Thus. Kohn (1964. 1966) did not find
enzymes capable of digesting myosin in denervated muscle
nor has contractile protein been seen within lysosomal
membranes. In the aneurogenic limb muscle stained for
acid phosphatase. lysosomes were seen between disinte-
grating myofibrils (Plate 10) but as others have noted
(Lockshin and Beaulaton. 1974a.b3 Kohn. 1966) besides

97
the presence of lysosomes no evidence of lysosomal

digestion of contractile material has been observed.
Some macrOphages were seen to ingest muscle fragments
(Plate 8) but did not seem numerous enough to account
for the massive disappearance of aneurogenic muscle.
Interestingly. Lockshin (1975) observed the appearance
of muscle antigens in the hemolymph of silkmoths during
normal degeneration of intersegmental muscle. Concomi-
tantly. enzymes. which have been shown to appear in
dystrOphic muscle. also appeared in hemolymph. In
addition. Goldberg et al. (1974) claimlan increased rate
of protein degradation and decreased rate of synthesis
in denervated or disused muscle. The reverse is claimed
to take place during work induced muscle hypertrophy
i.e. synthesis increases and degradation decreases;
however. no values are given. Possibly. as in other
muscle systems (Goldberg et al.. 1974). so in aneurogenic
.Amblystoma larvae the homeostatic turnover balance
between catabolism and metabolism of myoprotein is
shifted towards the catabolic mechanism: protein and
amino acids liberated from aneurogenic muscle end up in
the bloodstream to be reutilized or metabolized elsewhere.
At this point it is interesting to note that aneurogenic
muscle was far from.being metabolically inactive but
incorporated tritiated leucine (Plate 7) and tritiated
uridine vigorously. In fact. by visual inspection more

98
disintegration grains were found over myofibers where

cross-striations had disappeared than over fibers where
cross-striations were still clearly visible. Possibly
myofibers well underway in their atrophy synthesize more
protein and RNA than relatively unatrophied fibers.

That aneurogenic muscle degeneration is readily
reversible was shown in experiments in which 70 to 90
day old aneurogenic limbs were orthotopically
transplanted. Most animals regenerated a new limb next
to the transplanted one but in one case the animal
failed to regenerate its own limb and the transplanted
limb became entirely functional. Piatt (1942) determined
that aneurogenic limbs became approximately normally
innervated in a three dimensional pattern after ortho-
tOpic transplantation to normal larvae and in the one
successful case in this study normal use of the
transplanted forelimb spoke for normal innervation.
Fifty days after transplantation the larva was fixed and
the limb examined in cross section. The muscle tissue
was almost as extensive as in the contralateral normal
limb indicating that aneurogenic. greatly atrophied
muscle had grown back to almost normal cross sectional
dimensions after innervation and use. Another indication
of easy reversibility of aneurogenic muscle degeneration
has come from regeneration studies. Aneurogenic limbs

were amputated and regenerated normally (Yntema. 1959a.

‘ 99
b; Thornton. 1969). When the limb muscle was studied in

longitudinal section it appeared as if proximal to the
amputation plane muscle continued to atrophy but that
distal to the amputation plane muscle had regenerated
from degenerating muscle remains. However. the results
were not clear cut and therefore are not discussed

‘further in this report.

APPENDIX

100

Table 1
LINB TISSUE AREAS

 

 

 

 

 

 

 

 

 

 

Whole Solid Total Total
Age of limb tissue Humerus muscle number
animals Animal are: are! areg are? of myo4
(days) I.D. (um ) (um ) (um ) (um ) nuclei
45-153 82100 66372 9443 8767 57
45-15d 87172 69685 10201 6779 60
13 45-179 78275 53816 9685 8239 41
45-183 75260 59900 11306 4253 40
45-168 82772 65696 10087 5539 38
46-5 123840 63510 11454 6081 49
46-6 115 40 42940 13385 5089 41
21 46-4 87 60 65510 10787 10910 21
46-10 90300 57130 11301 10746 9
46-3 82610 56270 14009 7944 49 ,9,
47-8 87020 3800 12765 5172 51 8
47.5 86870 0500 14671 039 32 *1
31 47-3 70510 36970 11110 082 '1 5
47-12 65680 33840 12323 4271 36 :1
47-2 87020 51230 151 5080 1 '3'
48-15 57686 46952 15584 5642 40 3o
48-1 102300 53860 19843 211 29 §
43 48-6 92370 58710 14953 7318 44 ,
48-9 108330 68180 15 21 6221 33 5
48-16 632 0 42390 17 30 4603 7
43-13 44270 27040 13910 0 0
43-3 61770 29960 15324 664 12
54 43-6 62460 41770 17041 1050 23
43-9 87510 47980 19111 782 28
43-14 55740 41560 13280 610 28
50-5 158866 69560 10291 655 11
50-8 150180 11200 16278 0 0
65 50-7 163000 80660 19332 1170 21
50-9 145580 81480 16537 675 7
50-2 136900 68060 14557 1085 18
48-4r 173350 140367 14998 25 72 126
48-7r 181020 9 870 1148 28 92 125 '3
43 48-3r 197640 13 960 1887 39898 145 l
48-12r 151 20 108890 11431 26571 135 ..
48-10r 17 30 135430 20499 32355 133 5
50-11r 188260 143700 12419 38915 130 .4
50-9r 267120 206940 11194 82417 200 g
65 50-8r 236430 146640 20252 54882 187 a
50-101- 318790 257980 18115 83395 266 g
50-15r 154180 119210 12538 37335 93

 

 

 

 

 

 

 

 

 

 

101

Table 1
LIMB TISSUE AREAS (Cont.)

 

 

 

 

 

 

 

 

Whole Solid Total Total
Age of limb tissue Humerus muscle number
animals Animal are; are: area area of myo-
(days) I.D. (um ) (um ) (umz) (um ) nuclei
45-5 71900 56260 12639 6965 106
45-13 88946 82312 122 3 6375 56
13 45-1 80520 66900 9 6 9917 67
45-3 96350 68770 13304 10092 51
45-9 80635 54972 11248 8432 77
40-6s 125310 84860 14800 18405 81
46- s 120900 83520 12701 16452 49
21 46- s 128420 95690 17473 22 76
46-88 140140 61230 11443 13100 50
46-9s 144660 72600 14366 13754 57
47-4s 103140 7 922 11490 16518 68
47-153 111580 9 500 14100 25381 51
31 47-13s 116350 75180 15467 32259 61 :
47-Bs 126530 78420 8768 14310 66 a
47-7s 121870 78720 14599 26138 78 .4
47.14: 118600 84510 12958 22691 73 5
48-103 110200 81765 17873 19602 65 2
48-58 88250 41300 12056 14897 70 a
43 48-153 104930 64490 18674 19611 79 g
48-9s 141310 72910 1 413 17588 60 8
48-3s 146700 99840 1 026 29119 74
49-13 104370 75050 10570 9096 41
49-2 95280 73220 13850 9792 48
54 49-14 89710 32310 17900 2271 62
49-9 112050 930 1 267 3635 63
49-10 99700 52050 1 890 15356 52
50-1s 94180 38014 15969 7299 36
50-16s 112020 71130 13181 11908 57
65 50-14s 116540 74120 12816 8909 48
50-4s 1012 0 66390 13496 9096 5
50-78 895 0 55910 16987 11375 8

 

 

 

 

 

 

 

 

 

 

Table 9

MYONUCLEAR AREA (umz)

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