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The relation between nerve fiber number and limb regenerative capacity in the lizard Anolis.

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The Relation Between Nerve Fiber Number and Limb
Regenerative Capacity in the Lizard, Anolis '
JOCELYN ZIKA AND MARCUS SINGER
Department of Anatomy and Developmental Biology Center,
Western Reserve University, Cleveland, Ohio
ABSTRACT
The present work provides some quantitative evidence for the view
that the inability of the lizard's limb to regenerate is due to a n insufficiency of nerve
fibers. The number of fibers per unit area of the amputation surface of the lizard's
forelimb is approximately one-half that reported for the limb of the salamander, capable of regeneration. It is known that when the number of fibers in the salamander's
limb is reduced to about half its normal value, regeneration does not occur i n about
50% of the animals. The possibility is also discussed that the lizard's limb, in addition
to being less innervated than that of the salamander, is also more refractory in its
growth response to the nerve. Finally, the comparative poverty of innervation of the
lizard's limb relative to that of the salamander suggests that the evolutionary trend in
the vertebrate series toward elaboration of central nervous mechanisms was accompanied by a decline in richness of peripheral innervation.
The limb of most adult frogs and of the
lizard does not regrow after amputation.
However, if the amputation stump is provided with more nerve fibers by the deviation of a sizable nerve from another source,
regeneration does occur (Singer, '54, '61 ;
Simpson, '61; Kudokotsev, '62). In the case
of the salamander which does regenerate a
limb, the number of nerve fibers normally
available at the amputation surface is more
than adequate to support regrowth; but, if
the number is reduced below a threshold
value, regeneration does not occur (Singer,
'52 review).
From these results the theory was advanced that the absence of limb regeneration in vertebrates above the urodele amphibian is due to an insufficiency of nerve
fibers to satisfy the threshold need for regrowth (Singer, '54). There are three ways
in which such a quantitative inadequacy
could have arisen in the evolution of the
vertebrates : the number of nerve fibers
per unit of limb tissue could have declined
until it became threshold inadequate; secondly, the tissues which respond to the
nerve to produce a regenerate could have
become increasingly refractory until their
nerve needs for regrowth were no longer
satisfied by the available nerve supply;
and, finally, the individual nerve fibers
may have become decreasingly effective as
agents of growth until the combined defiANAT.REC.,152: 137-140.
cit rendered the available nerves an inadequate source of the nerve growth influence. Attempts have been made to assess
these possibilities (Singer, '63; Singer and
Mutterperl, '63; Van Stone, '64).
The present work continues the evaluation and compares the quantity of limb innervation in the non-regenerating limb of
the lizard, Anolis, with that for the limb
of the newt, Triturus, which can regrow.
The results show that the number of fibers
available at the amputation surface of the
lizard limb is comparatively low; indeed,
it would hardly be enough to satisfy the
regenerative needs of the newt's limb.
MATERIALS AND METHODS
The upper arm of the lizard, Anolis carolinensis, was removed and fixed in Bouin's
solution for a few days, decalcified (Decal)
overnight, embedded in paraffin, and crosssectioned serially at 10 u. Sections were
stained for nerve fibers by the Bodian silver method, and then counterstained with
orange G.
A section approximately in the middle
of the upper arm was selected for counting
nerve fibers. The large nerve trunks were
projected onto paper at a magnification of
900 with a camera lucida and the out1 Aided by grants from the American Cancer Society
and the National Institutes of Health, Public Health
Service.
137
138
JOCELYN ZIKA AND MARCUS SINGER
line of each fiber was drawn. Then the
fibers were counted by circling them in
groups of five. The nerve fibers in the
smaller nerves and those scattered among
skin and muscle were counted at the same
magnification by the method of “stripping”
used by Singer (’46).
A Visopan projection microscope (Reichert) was used to obtain the area of the
section. The outline of each section was
traced at a magnification of 50 times and
the area was determined with a planimeter.
The quantitative data were then expressed
in number of fibers per (100 p ) z of crosssectional area according to the method of
Singer (’47).
Recounts of nerve fibers were performed
in three cases to check the reliability of the
data. The second count differed from the
first by a n average of 4 . 3 % ,with a range
of differences of 0.4-10.0%.
(100 v ) ~respectively for the normal amputation surface of the newt (see Singer,
’46, ’47). Consequently, the number of
fibers available at the amputation surface
of the forelimb in Anolis is approximately
one-half that found in Triturus.
It is of particular interest to compare the
threshold number of fibers required for regeneration of the upper arm of Triturus
with the full number found in Anolis because the Anolis limb does not regenerate
nor does the newt limb regrow when the
fiber number is reduced below the threshold value. The average threshold in Triturus is 10.8 fibers per (100 u)’ of surface
area of soft tissue, and 9.6 fibers per
(100 v ) of
~ total surface area (Singer, ’47).
These values are comparable to the number of fibers per unit area ordinarily present in the AnoZis forelimb, namely 12.3
and 11.7 respectively.
RESULTS
DISCUSSION
Our results show that the lizards limb
Results are recorded in table 1. The
average number of nerve fibers found in has much fewer nerve fibers per volume
a cross-section of the forelimb in Anolis of tissue than does that of the salamander.
is slightly higher (2,588) than that (2,357) This fact would support the postulate that
reported for TTiturus (Singer, ’46). The the lack of regenerative powers in the
limb of Anolis is, however, more massive; lizard’s limb and in that of vertebrates
and, when the number was expressed per higher than the urodele amphibian is due
unit cross-sectional area, it was found that to a n insufficiency of the nerve supply
in Anolis there is a n average of 11.7 fibers (Singer, ’54). A s noted previously there are
per (100 u ) ~of total area and 12.3 fibers theoretically at least three ways during
when the area of bone is ex- evolution of the vertebrates in which the
per (100
cluded. This compares with values of 21.7 neuronal supply may have become threshfibers per (100 p)’ and 24.5 fibers per old inadequate to evoke growth : numerical
TABLE I
Innervation o f t h e lizard forelimb
Total
Bone
Soft
tissue
Total
number
of nerve
fibers
1
2
3
4
5
6
7
8
9
10
138
318
191
218
109
188
209
350
401
318
7
11
7
8
7
7
7
9
12
9
131
307
184
210
102
181
202
34 1
389
309
Average
Std. dev.
244
2 96.7
k 1.7
235.6
2 95.0
2 342
Animal
Area of surface of
amputation in (100 p ) 2
8.4
Fiber number per (100 & ) 2
of surface area
Total
Soft
tissue
2748
2863
2158
2593
2033
2328
2486
2561
3313
2497
19.9
9.0
11.2
11.8
18.6
12.3
11.8
7.3
8.2
7.8
20.9
9.3
11.7
12.3
19.9
12.8
12.3
7.5
8.5
8.1
2558
11.7
24.3
2 4.6
12.3
NERVE INFLUENCE IN REGENERATION
insufficiency in relation to other tisuses;
decreased potency of individual fibers; and
relative refractoriness of responding tissues. Using the newt limb as a reference
standard since it does normally regenerate
upon amputation, we find that the forelimb of the lizard has much fewer fibers
per unit volume; and, therefore, that the
postulate of numerical insufficiency is,
indeed, one of the causes for absence of
limb regeneration in the lizard. Moreover,
further support may be drawn from the
results of experiments in which the lizard's
limb was caused to regrow after the amputation surface was increased by deviating
a substantial quantity of fibers into the
limb to supplement the normal supply
(Singer, '61; Simpson, '61; and Kudokotsev, '62).
The two remaining postulates, namely
decline in potency of the individual fibers
and increased refractoriness of response
of tissues to the nerve, may still be contributing causes, and therefore must remain as supplementary possibilities to explain the absence of limb regeneration in
higher forms. Indeed, our results may also
be interpreted to suggest that increased
refractoriness of tissues to the nerve may
also be responsible for the inability of the
amputated lizard's limb to regrow. The
number of fibers in the lizard limb is approximately that of the average threshold
value for the newt. The latter value is the
mean of a range of fiber values above
which limb regeneration always occurs and
below which there never is regrowth.
Therefore, the average represents a quantity of fibers which support limb regeneration in the newt in about one-half of the
instances. However, this same value, which
as we have shown is close to the normal
supply in the limb of Anolis, never evokes
regeneration of the lizard's limb. Consequently, in addition to the numerical factor, the non-nervous tissues of the lizard's
limb may be less facile in their response
to the nervous agent of growth. There is
some published evidence that the capacity
of non-nervous tissues to respond to the
nerve plays an important role in establishing threshold fiber requirements (Singer
and Mutterperl, '63). When the limb of
the newt is transplanted to the back, the
139
threshold fiber requirements are greatly
lowered and now much fewer fibers are
needed to support regrowth.
There is no evidence as yet to support
the third postulate, namely that the individual nerve fiber is a less potent source
of the nervous growth factor in vertebrates
higher than the urodele amphibians. Assuming this postulate obtains in fact, it
may be due to a quantitative biochemical
change within the neuron; or, it may be
that the neuraxons in forms above the
amphibian branch more frequently, and
therefore that the nervous agent of growth
is diluted in the many branches.
Finally, the richer innervation of the
limb of the urodele than that of the reptile
suggests that the vertebrate limb was more
highly innervated early in its evolution.
One might have expected that the higher
terrestrial forms which use their limbs in
much more useful and complex ways
would have a richer peripheral nerve supply. The decline in limb innervation may
mean that the saturation of limb tissues
with nerve fibers was of lesser survival
value than the elaboration of central mechanisms for the use of peripheral information. In this way central elaboration
prevailed over richness of peripheral innervation.
LITERATURE CITED
Kudokotsev, V. P. 1962 Stimulation of the regeneration process i n the extremities of lizards
by the method of supplementary innervation.
Dokl. Akad. Nauk SSSR, 142: 233-236.
Simpson, S. B., Jr. 1961 Induction of limb regeneration i n the lizard, Lygosoma laterale, by
augmentation of the nerve supply. Proc. SOC.
Exp. Biol. Med., 107: 108-111.
Singer, M. 1946 The nervous system and regeneration of the forelimb of adult Triturus.
V. The influence of number of nerve fibers,
including a quantitative study of limb innervation. J. Exp. Zool., 101 : 299-338.
1947 The nervous system and regeneration of the forelimb of adult Triturus. VII. The
relation between number of nerve fibers and
surface area of amputation. J. Exp. Zool.,
104: 251-265.
1952 The influence of the nerve i n regeneration of the amphibian extremity. Quart.
Rev. Biol., 27: 169-200.
1954 Induction of regeneration of the
forelimb of the postmetamorphic frog by augmentation of the nerve supply. J. Exp. Zool.,
126: 419-471.
140
JOCELYN ZIKA AND MARCUS SINGER
1961 Induction of regeneration of body
parts in the lizard, Anolis. Proc. SOC.Exp. Biol.
Med., 107: 106-108.
1963 Nervous control of the regrowth
of body parts in vertebrates. In: The Effect of
Use and Disuse on Neuromuscular Functions.
Ed. by E. Gutmann and P. Hnik, Czech. Acad.
Sci., Prague.
Singer, M., and E. Mutterperl 1963 Nerve fiber
requirements for regeneration in forelimb
transplants of the newt Triturus. Dev. Biol.,
7: 180-191.
Van Stone, J. M. 1964 The relationship of
nerve number to regenerative capacity in the
developing hind limb of Rana syluatica. J. Exp.
Zool., 155: 293-302.
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fiber, limba, relations, capacity, lizard, nerve, anolik, number, regenerative
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