/. Embryol. exp. Morph. Vol. 62, pp. 1-11, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Regeneration of subnormally innervated axolotl
arms
By H. WALLACE, 1 A. WATSON 1 AND M. EGAR 2
From the Department of Genetics, University of Birmingham
SUMMARY
Forearms of juvenile axolotls contain about 5000 axons, of which only 25 % are myelinated
and visible by light microscopy. Virtually all the axons degenerate after transection of the
brachial plexus, but repeated operations fail to keep the arm completely denervated.
Regrown nerve fibres were detected by electron microscopy after 6 weeks of attempted
denervation and related to the quantity usually considered necessary for limb regeneration.
Such arms regenerated quite normally, provided their innervation had been depleted for
several weeks before amputation. Among other ways of reconciling these observations to
the neurotrophic theory of limb regeneration, it is suggested that tissues can adapt to
deprivation of their nerve supply.
INTRODUCTION
According to the neurotrophic theory of limb regeneration, the establishment
and early growth of a regeneration blastema are dependent upon an adequate
nerve supply but older regenerates are capable of differentiation, morphogenesis and some further growth in the absence of nerves. Both sensory and
motor nerves contribute to the total nerve requirement in adult urodeles,
giving the theory an explicit quantitative basis which is conducive to the
argument that higher vertebrates may be incapable of limb regeneration
because of an inadequate innervation (Singer, 1952-1974). The requirement
for amphibian limb regeneration has been estimated by partial denervation
at about the time of amputation and expressed either as the fibre density at
the amputation surface, the fraction of the surface area occupied by axoplasm
or, more simply, as a percentage of the normal innervation. The frequency of
regeneration exhibited by such partially innervated arm stumps is strongly
correlated to their residual innervation in larval salamanders (Karczmar, 1946),
or correlated within a ' threshold' of about 30 to 50 % of the normal nerve
supply in newt upper arms (Singer, 19466).
There are two clear exceptions to the quantitative and threshold aspects of
1
Authors' address: Department of Genetics, University of Birmingham, P.O. Box 363,
Birmingham B15 2TT, U.K.
2
Author's address: Department of Anatomy and Developmental Biology Center, Case
Western Reserve University, Cleveland, Ohio 44106, U.S.A.
2
H. WALLACE, A. WATSON AND M. EGAR
the neurotrophic theory. Transplanted arms can regenerate when very poorly
innervated (Singer & Mutterperl, 1963); aneurogenic arms of young larvae
regularly regenerate in the virtual or complete absence of nerves (Yntema,
1959; Egar, Yntema & Singer, 1973; Wallace, 1980). Singer (1965) sought to
incorporate these exceptions into an addictive version of the trophic theory by
postulating either that tissue sensitivity to the trophic factor depended on
its prior experience of nerves, or that other tissues could also produce the
trophic factor when stimulated by excessive operational trauma. These modifications seemed to cover all eventualities and were vindicated by an ingenious
test involving the transplantation, innervation and later denervation of
aneurogenic larval arms (Thornton & Thornton, 1970). The test may be
unnecessarily complicated, however, for a tedious argument presented elsewhere (Wallace, 1981) contends it should be possible to demonstrate the
regeneration of denervated arm stumps without resorting to either transplantation or the embryonic operations needed to produce aneurogenic arms.
A preliminary experiment established that juvenile axolotl arms could regenerate while being denervated at weekly intervals, but only if amputation
was delayed for several weeks after the initial denervation. Such arms with
early regenerates were judged to be sparsely innervated or devoid of nerves
when examined by routine histological staining of paraffin sections (Watson,
unpublished). Small unmyelinated axons would have escaped detection by
light microscopy (cf. Egar & Singer, 1971), so we have repeated and refined
the experiment in an attempt to meet this criticism. We find our results are
in better accord with the addictive version of the neurotrophic theory than
with its quantitative or threshold aspects.
MATERIAL AND METHODS
Ten 55-70 mm long axolotls (Ambystoma mexicanum) were selected as an
experimental series. Their right arms were denervated by severing the brachial
plexus close to the head of the humerus. This operation was repeated 2 weeks
later and after a month had elapsed, cutting a more proximal part of the
plexus on each occasion. Both arms were amputated above the wrist at the
time of the third denervation, when the specimens had reached lengths of
66-88 mm. A final resection of the right brachial plexus was performed 10 days
later and both forearms were preserved 7 days after that, 17 days after
amputation.
Ten 60-80 mm long specimens were used as a control series, being subjected
to bilateral amputation above the wrist at the same time as the experimental
series. Their right arms were denervated 10 days later and both forearms were
preserved 17 days after amputation.
All 40 forearms were processed identically by a standard technique for
electron microscopy: fixation for 2 h in 2-5 % glutaraldehyde in 0-1 M-cacodylate
Chronic denervation in axolotl
3
buffer pH 7-4; 1-5 h in 1 % osmium tetroxide in the same buffer; dehydration
through a series of ethanol steps (70-100 %) and two changes in propylene
oxide; 1 h in equal parts of propylene oxide and epori, before embedding overnight at 60 °C in epon 812. One micron sections were stained with toluidine
blue and azur II, and examined by light microscopy to obtain counts of
myelinated axons. Thin sections were placed on coated single slot grids, stained
with lead citrate and examined with a Phillips 300 electron microscope. A series
of overlapping photographs from two major nerves were printed (ca. x 7000)
and assembled into montages, from which the density of small unmyelinated
axons was estimated relative to the numbers of Schwann cell nuclei. The
diameters of 300 axons in each montage were measured to obtain estimates
of the mean cross-sectional area and thus of the total axoplasmic area at an
amputation surface.
RESULTS
The brachial plexus is easily exposed in these large specimens, so that the
initial operation can be guaranteed to transect all nerves to the forearm except
for a minor sympathetic supply. Each successive operation becomes more
difficult as the amount of scar tissue increases and the nerve trunks become
more transparent. The first three denervations were judged to be successful by
the reduced growth or atrophy and the permanent immobility of the operated
arms throughout the experiment, in contrast to the recovery of movement
expected about 3 weeks after a single denervation (Thornton, 1960; Maden,
1977). The success of the final denervation could only be assessed retrospectively
from sections.
Ten days after amputation, both arms of all the experimental and control
specimens had formed distinct conical blastemata. The control right arms
were denervated then, for a cone blastema is still dependent upon the integrity
of its nerve supply. All the regenerates were examined 1 week later in order
to determine whether or not they had progressed beyond the cone stage
(Fig. 1). Confirming the preliminary test, most of the experimental right arms
had progressed to palette, notch or early digit regenerates under conditions
designed to eliminate their nerve supply. More surprisingly, almost all of the
experimental specimens now showed a more advanced regenerate on the
denervated right arm than on the innervated left arm (Table 1). We can explain
this difference by invoking the Tweedle effect. Tweedle (1971) demonstrated
that denervating or amputating one arm impedes the regeneration of the
contralateral arm by disturbing transneuronal contacts and causing some
chromatolysis of contralateral neurons. Maden (1977) also reported that
denervation causes the degeneration of some contralateral axons. The left arms
of these experimental specimens would be sensitive to the last two operations
on the right brachial plexus, so that their regeneration should be appreciably
delayed. The experimental right arms need not be affected, however, assuming
H. WALLACE, A. WATSON AND M. EGAR
B
C
J
1 cm
Fig. 1. Arms of an experimental (A, B) and a control (C, D) specimen when
scored 17 days after amputation. A, C, the innervated left arms both have palette
stage regenerates. B, the experimental right arm has regenerated a 3-digit hand.
D, the control right arm has regressed from a cone stage, 1 week earlier when it was
denervated, to a small blastema.
Table 1. Stages of regeneration scored at 17 days after amputation
Arm and days denervated Total
Experimental right 45
Experimental left —
Control right
7
Control left
—
10
10
10
10
Blastema
Cone
Palette
Notch
3 digit
0
0
9
0
1
0
1
0
1
9
0
10
6
1
0
0
2
0
0
0
their innervation could not be reduced any further. The right arms of the
control specimens showed a typical response to denervation 10 days after
amputation. None of their cones grew at all in the following week and most
of them shrivelled to a smaller blastema. That would also be expected of any
experimental right arm which contained nerves when it was amputated but
lost them subsequently, as may have happened in a single case (Table 1). The
left arms of the control specimens which were also subject to the Tweedle
effect consistently reached the palette stage after 17 days, duplicating the rate
of regeneration shown by the innervated left arms of the experimental series.
Transverse sections from all 40 forearms were examined by light microscopy,
although a few were too poorly orientated or too close to the elbow to give
quantitative results. The remaining innervated arms of both series provided
a fairly consistent standard for comparison (Table 2). They contained about
1300 large myelinated axons, mostly packed close together in three major
central trunks and two radial nerves with up to 12 minor nerves in the dermis
and others scattered through the muscle, and about 250 Schwann cell nuclei
Chronic denervation in axolotl
Figs. 2-4. Light microscope sections of major nerves in the forearm. 2, normal
myelinated axons in experimental left arm EL 10; 3, degenerating axons and
myelin debris, one week after denervation in control right arm CR 8; 4, absence
of myelinated axons after chronic denervation in experimental right arm ER 1.
H. WALLACE, A. WATSON AND M. EGAR
Table 2. Light microscope counts of nerve constituents
{mean ± standard deviation) from forearm sections
Schwann cell nuclei
Arm and days denervated Specimens
Experimental right 45
Experimental left —
Control right
7
Control left
—
10
8
10
9
Myelinated
fibres
Counts
Mitotic index
8±10
1396 ±228
18±7
1283 ±163
191+40
282 ±37
303 ±75
265 ±48
6-3%
1-3%
0-3%
1-3%
(Fig. 2). The control right arms contained less than 30 apparently undamaged
myelinated fibres, interspersed among empty myelin sheaths and debris (Fig. 3).
Many of the Schwann cell nuclei had enlarged at 7 days after denervation, but
very few were dividing and the number in each section had not increased
appreciably. The experimental right arms contain 0-24 relatively small and
lightly myelinated axons, scattered among enlarged intercellular spaces but
without much debris, suggesting that material had been lost earlier during
the prolonged denervation (Fig. 4). These sections contained significantly
fewer than the normal number of Schwann cell nuclei but nuclear divisions
were relatively common (Table 2). The counts of myelinated fibres shown
in Table 2 reveal that at least 97-5% of the major axons are degenerating
7 days after denervation, while the apparently intact residue may only be
resistant internodes of severed axons. The presence of a few lightly myelinated
axons in about half of the experimental right arms indicates that repeated
denervations were not uniformly successful in excluding regrown fibres or those
invading the limb from adjacent spinal nerves. These fibres never amounted
to more than 2 % of the counts in normal arms, so the experimental right
arms must be considered as sparsely innervated unless they contain an excessive
number of unmyelinated axons.
Electron microscopy of innervated and recently denervated control arms
confirmed the preceding observations and extended them to unmyelinated
fibres. All the major nerves normally contained bundles of small axons
embedded in the folds of scattered Schwann cells. The vast majority of all
axons showed signs of degeneration 7 days after denervation, and this applied
to all the nerves examined of control right arms. The six experimental right
arms examined by electron microscopy presented a more confusing picture,
for their major nerve trunks contained large spaces with fine debris between
the Schwann cells whose processes enveloped typical axonal profiles (Fig. 5).
Although the Schwann cell processes in severed nerves can fold round each
other to give a very similar appearance (Payer, 1979), the elongated profiles
seen in oblique and longitudinal sections convince us that most of them are
genuine axons. A montage of prints from a major nerve of the smallest experi-
Chronic denervation in axolotl
•>*>*„
Me
Fig. 5. Detailed structure from a major nerve in experimental right arm ER1,
showing unmyelinated axons (A) enveloped in a process (P) of Schwann cell
cytoplasm; Schwann cell nucleus (N).
mental right arm yielded an average ratio of 16 axons to each Schwann cell
nucleus in the area. Averaging two perpendicular diameters of 300 axons,
their mean diameter was 0-96 ju,m (range 0-3-3 /im) and the mean crosssectional area was calculated to be 0-885 /*m2. A similar montage from a major
nerve of the smallest innervated arm yielded a lower density of unmyelinated
axons, 13 per Schwann cell nucleus, of much the same size range with mean
values of 1 /im diameter and 0-995 /*m2 area. The myelinated fibres had a mean
diameter of 3-7 /«n (range 2-9 /*m) and area of 11-8 /im2. These average values
misrepresent the irregular distribution of unmyelinated axons, which occurred
in bundles in both montages, but they can be combined with light microscope
data to calculate the relative innervation of the experimental right arm. As
shown in Table 3, this arm contained a substantial number of axons, amounting
to almost half the normal supply and nearly 90 % of the normal density of
axons at an amputation surface. That degree of innervation might be exceeded
slightly in other experimental right arms which contained a few remyelinated
axons. These arms can still be considered as sparsely innervated in the restricted
sense that the total mass of axoplasm (or axoplasmic area in a section) is
much less than that present in a normally innervated arm.
H. WALLACE, A. WATSON AND M. EGAR
Table 3. Characteristics of the two smallest arms from the experimental
series, combining data from thick sections and EM montages
Denervated arm (ER 1)
Normal arm (EL 10)
No. of Schwann nuclei and
section area
Unmyelinated axons
Myelinated axons
Total axons
- as % of normal arm
Density per mm2 of section
- as % of normal arm
2
dumber
Area (/*m )
276
3-0 xlO 6
139
3588
1186
4774
—
1591
—
3570
13995
17565
—
5855
—
2224
0
2224
47%
1390
87%
Number
Area (/*m2)
l-6xlO 6
1968
0
1968
11%
1230
21%
DISCUSSION
Prolonged denervation of an axolotl arm prior to amputation clearly allows
it to regenerate through all the stages which are usually dependent upon a nerve
supply. If we had been content to assess the residual innervation of these
arms by light microscopy, we should have been satisfied they were virtually
devoid of nerves and thus concluded nerves were dispensible agents in regeneration. The substantial numbers of fine axons in these arms 17 days after
amputation compel us to moderate that conclusion, but not to abandon it
altogether.
Observations on axolotl arms subjected to a single denervation (Egar, unpublished) indicate that axons regrow into the upper arm within a week and
increasing numbers of them gain a thin myelin sheath by 11-14 days. The
scarcity of myelinated fibres in the repeatedly denervated arms considered here
thus implies that most of the detected axons only entered the arm after
amputation, perhaps during the final week. Others might have been present
earlier and degenerated after a subsequent denervation, of course, but our
experience of the increasing difficulty in finding nerves to resection convinces
us the arms were more sparsely innervated during the early stages of regeneration
than at the end of the experiment. In that case, Table 3 probably over-estimates
the residual nerve supply at the actual amputation surface 17 days previously.
Assuming axons of all sizes dispense equal amounts of trophic factor, then
its concentration should be proportional to the density of axons at an amputation surface. Partly due to the cessation of growth and muscular atrophy
during prolonged denervation, regrown axons amounting to only half the
normal innervation achieved about 90 % of the normal density (Table 3). We
cannot pretend that estimate is accurate to within 10% or that it is valid
for all the experimental right arms, but the kind of effect predicted by the
neurotrophic theory here is a greater delay of regeneration than that ascribed
Chronic denervation in axolotl
9
to the Tweedle effect on the contralateral innervated arms. Karczmar (1946)
noted a reduced rate and frequency of regeneration by arms of A. maculatum
larvae when he reduced their innervation to 90 % at the time of amputation,
although axolotls do not respond so clearly to partial denervation (Egar,
unpublished). We conclude there is a discrepancy between the present results
and previous ones which is probably related to the relative timing of denervation
and amputation.
The main alternative version of the neurotrophic theory assumes large
axons secrete more trophic factor than small ones and consequently relates
the frequency of regeneration to the fraction of the amputation surface occupied
by axoplasm (Singer, Rzehak & Maier, 1967; Singer, 1974). Table 3 reveals
a serious discrepancy from this assumption, for arms which had regenerated
rapidly and perfectly only contained about 11 % of the normal axoplasmic
area or 21 % when normalized to equal sized arm cross sections. If the calibre
of nerve fibres is an important parameter of the trophic factor then prolonged
denervation before amputation certainly disturbs its expected relationship to
regeneration.
Curiously enough, some of the most persuasive evidence adduced for the
quantitative neurotrophic theory also comes from experiments involving delayed
amputation. A regenerated motor supply provides sufficient innervation for
about 50% of the tested newt arms to regenerate (Singer, 1946 a; Sidman &
Singer, 1960), or for regeneration in all the larval salamander arms tested by
Thornton (1960). It is usually argued that a hyperplastic reinnervation by
motor nerves must have boosted the supply of trophic factor above a threshold
value in order for regeneration to occur several weeks or months later. No
fibre counts were reported for these experiments, however, and no attempt was
made to determine if the timing of amputation influenced the results. The
present results supply this missing control, suggesting the known delay of
amputation provides a more valid explanation of regeneration than the presumed
degree of innervation.
The data considered here and reviewed in more detail by Wallace (1981)
oblige us to reformulate the neutrotrophic theory in a more flexible way.
Nerves undoubtedly have a trophic influence on amputated limbs and young
regenerates, for an initial denervation then reduces a variety of synthetic
activities, cell division and growth (e.g. Singer & Caston, 1972). The regeneration
of aneurogenic arms and the present results, however, suggest there can be
no fixed demand for a particular limiting threshold quantity or density of
nerve fibres. Apparently, an arm can become accustomed in a matter of weeks
to a depleted residual innervation, or to none at all, and then regenerates
perfectly normally as long as that degree of innervation is maintained. The
simplest interpretation of the nervous control over regeneration, therefore, is
that tissues are sensitive (or addicted) to their current supply of neutrotrophic
factor. A marked reduction of innervation, causing an abrupt step-down of
10
H. WALLACE, A. WATSON AND M. EGAR
the trophic supply, results in temporary withdrawal symptoms which either
prevent regeneration or delay it until the tissues have adapted to the new
level. Adaptation could be achieved by a local production of trophic factor
to compensate for that normally supplied by the nerves. It is not related to
operational trauma, however, for Karczmar (1946) often prevented or delayed
regeneration by a sequence of repeated denervations like those employed here.
There is no evidence for any alternative source of the trophic factor at present,
but compensatory production may offer one way of reconciling the present
results to the quantitative aspects of the neurctrophic theory.
We are conscious of how much this study depends upon investigations conducted by
Marcus Singer over the past 40 years, even if our conclusions make this seem a rather
back-handed compliment. M. Egar was supported by N1H Grant NS-07403-19 to Dr Singer,
and thus feels doubly grateful. A. Watson acknowledges receipt of an SRC Studentship.
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11
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{Received 8 September 1980; revised 7 October 1980)