/ . Embryol. exp. Morph. Vol. 28, 2, pp. 419-435, 1972
Printed in Great Britain
419
The components of
regrowing nerves which support the regeneration
of irradiated salamander limbs
By H. WALLACE 1
From the Department of Genetics, University of Birmingham
SUMMARY
Forelimbs of juvenile axolotls do not regenerate when amputated in a previously irradiated
region. They usually do regenerate, however, if they have also been denervated shortly after
irradiation and well before amputation. Five series of experiments are reported which define
the conditions permitting this paradoxical regeneration.
Crushing the nerves of the brachial plexus proved a satisfactory means of causing temporary
denervation. Shielding any region of the arm or shoulder, during an irradiation that preceded
such denervation, permits regeneration to occur at a region which was initially irradiated.
Lengths of brachial nerve implanted into an irradiated arm also support its regeneration.
It is concluded that temporary denervation (including Wallerian degeneration and the
regrowth of axons) mobilizes cells in a shielded region of the arm. These cells migrate both
proximally and distally, so that some come to occupy the site of amputation. Schwann cells
of the myelin sheath are identified as the cells most likely to behave in this way. It thus seems
probable that those non-irradiated Schwann cells which occupy a generally irradiated limbstump can form the exclusive source of a mesenchymal blastema and the various internal
tissues of the regenerate.
INTRODUCTION
It is well known that the regeneration of urodele limbs can be prevented by
prior irradiation of the site of amputation. It is equally well established that
denervation by severing the nerves of the brachial plexus also prevents regeneration until the nerves have grown back to the apex of the limb-stump. A previous
report revealed the apparent paradox that combining these two inhibitory
treatments resulted in normal regeneration (Conn, Wessels & Wallace, 1971).
The regeneration only began when nerves re-invaded the limb-stump, and
occurred most consistently if amputation was delayed to allow for an adequate
reinnervation of the limb. Furthermore, regeneration was only recorded when
the nerves had regrown from a shielded brachial plexus, and thus could provide
non-irradiated tissue within the otherwise irradiated limb. This situation, termed
paradoxical regeneration for brevity, can be explained in two alternative ways,
1
Author's address: Department of Genetics, University of Birmingham, P.O. Box 363,
Birmingham B15 2TT, U.K.
420
H. WALLACE
depending on which components of the non-irradiated nervous tract are able to
return to the site of amputation and support subsequent regeneration.
Nerve axons certainly do grow back into the irradiated limbs in this experimental situation. Intact axons are clearly required for the initial phases of regeneration, as shown both by the coincidental reappearance of nervous coordination and ability to regenerate (Schotte & Butler, 1941) and by the quantitative effects of nerves on regeneration (Singer, 1952). As the axons are cytoplasmic extensions of non-dividing neurons, they cannot constitute a source of
cells in the regeneration blastema. If no other non-irradiated cells accompany the
regrowing axons in this situation, therefore, the blastema would necessarily be
composed of irradiated cells whose regenerative capacity had been restored by
a nervous 'induction'. Trampusch (1964) has argued in favour of this interpretation, but his argument lacks acceptable evidence (Wallace, Wessels &
Conn, 1971).
Schwann cells are known to migrate and might accompany the regrowing
axons to provide eventually both a new myelin sheath and, in this experimental
situation, a source of non-irradiated blastemal cells. As the connective tissue
layers of the nerve sheaths seem to be more persistent structures, the possibility
that they contribute migratory cells after denervation will be neglected for the
moment. According to the weight of evidence, irradiation irreversibly incapacitates cells from participating in regeneration (see reviews by Rose, 1964; Thornton, 1968). It follows that the blastema in this experimental situation must be
derived entirely from Schwann cells, yet can differentiate into all normal limb
tissues.
The theoretical considerations to be derived from either explanation of this
paradoxical regeneration, therefore, justify an attempt to decide which explanation is correct. The experiments described here confirm the occurrence of paradoxical regeneration and provide two independent lines of evidence implicating
the participation of migratory Schwann cells in such regeneration.
MATERIAL AND METHODS
The experiments were performed on young axolotls, Ambystoma mexicanum,
the progeny of a mating between a white female and a dark heterozygous male.
The larvae were first reared in mass cultures on a constant supply of chopped
tubificid worms, isolated when about 50 mm, and used when 60-80 mm long.
Narcotized larvae were partially covered with 3 mm thick lead plates to
provide the different shielding patterns shown in Fig. 1. The exposed parts
received 2 krad (i.e. 20 J/kg) X-rays at 300 rad/min, from a 250 kVp General
Electric Maximar machine with a 1 mm aluminium filter. The lead plates transmitted less than 5 % of the incident dose, so that shielded regions received about
50-100 rad. Temporary denervations were performed within 3 h of irradiation,
by exposing the brachial plexus on one side only and either severing or crushing
Paradoxical regeneration
All
its three major nerve tracts. These nerves were severed with irridectomy scissors,
or crushed by repeatedly pinching a short segment with fine forceps. Re-innervation of the arm was monitored by observing its position, movement and
sensitivity. Both arms were amputated above the wrist, after a delay of 3 weeks
which permits re-innervation of the forearm and thus limits the extent of
regression. This procedure is described and justified in more detail elsewhere
(Conn et ah 1971). Brachial nerves were transplanted between sibling donors and
hosts of the different colour genotypes - dark Dd and white dd. The major nerve
tract of the upper arm of each donor was excised in Barth's solution (Barth &
Barth, 1959) and picked free of adventitious tissue. A length of nerve was then
inserted through a hole in the skin of each host's left forearm, which had been
irradiated less than 3 h previously, and pushed distally to prevent its expulsion
from the contracting wound opening. The implants could still be detected as
swellings with some attendant haemorrhage under the host skin 3 days later,
when the arms were amputated just distal to the implant. Other brachial nerves
were excised and cleaned in the same manner and then fixed. These nerves were
examined in sections to assess the purity of the implants.
Regeneration or regression of the amputated arms was followed by weekly
camera lucida drawings. Regeneration was assessed according to a preselected
criterion of obvious growth with the formation of at least three of the normal
four digits. The speed of regeneration was not strictly reproducible between all
series of experiments, but each series was compared to the regeneration of
normal limbs amputated at the same time. A variable amount of regression
usually occurred before regeneration began. In some series, where the upper arm
was shielded, any failure of paradoxical regeneration resulted in a continuation
of regression into the upper arm. Later regeneration from the shielded tissues
there is of no interest in this study. The two types of regeneration could be
distinguished, as the elbow marked the proximal boundary of definitely irradiated tissue. Only regeneration from the irradiated forearm was classed as paradoxical in these series. The delayed amputation and the age of the specimens
reduced the extent of the initial regression, but introduced a third type of result:
a stabilized stump which neither regressed perceptibly nor regenerated.
RESULTS
The shielding patterns shown in Fig. 1 and subsequent operations were designed to produce novel experimental conditions with appropriate controls,
where the regenerative response could be predicted on the basis of previous
results from larval marbled salamanders, Ambystoma opacum (Conn et al. 1971).
Several of the controls, of course, merely demonstrate that the general conditions
of regeneration or of its inhibition by irradiation have been satisfactorily duplicated in this study. The numbers of specimens involved in each series of operations are shown in the Table and ignored in the following description.
422
H. WALLACE
A
B
I cm
Graft
D
Fig. 1. Shielding patterns for the six series of operations. The positions of specimens
during irradiation are shown in outline, omitting the gills. The shaded area represents
lead shielding.
(A) Followed by severing the left brachial plexus for series 1; or by crushing the left
brachial plexus, with the right arm entirely shielded (broken outline) for series 2.
(B) Followed by crushing the left brachial plexus for series 3, or the right brachial
plexus for series 4.
(C) Followed by crushing the left brachial plexus for series 5.
(D) Followed by implanting a non-irradiated nerve trunk (arrow) for series 6.
Note the frequent use of an elbow to mark the most proximal irradiated region of the
arm.
Series 1 was intended merely to confirm the routine occurrence of paradoxical
regeneration. The left forearms were irradiated and their nerves were then
severed in the shielded brachial plexus (Fig. 1 A). After 3 weeks, when nerves
had regrown into the forearm, the arms were amputated above the wrist. Only
one of the surviving specimens regenerated a hand from the irradiated forearm,
after a surprisingly long delay (Fig. 2). Regression continued to above the elbow
Paradoxical regeneration
423
Table 1. Numerical summary of results, scored 24 weeks after amputation
Specimens
Series Operated Scored
Arm
Irradiated region
(A, B, C, or D of Fig. 1)
Number of arms
Operation
Regenerated* Inhibited!
3(0)
1
A. Forearm
Nerves cut
Left
—
4(2)
0
A. Forearm
Right
A. Forearm
Left
5
10
2
Nerves crushed
3
2(1)
5
0
A. —
Right
—
12
Left
0
B. Arm, shoulder
Nerves crushed
9
9
3
—
9(3)
0
Right
B. Forearm, shoulder
—
Left
12
12
12
0
B. Arm, shoulder
4
8
Right
B. Forearm, shoulder
Nerves crushed
4(1)
Left
C. Wrist to shoulder
Nerves crushed
5
5
10
10
5
—
0
Right
C. Wrist to shoulder
10
Left
D. Arm, shoulder
Nerve implant
1
10
8
9
6
9
—
Right
D. Shoulder only
0
* Regeneration occurred from the forearm in all cases.
f No regeneration occurred from the irradiated region; regression into the upper arm sometimes led
to regeneration from shielded tissue (the number of such cases is given in parentheses).
1
6
4
Fig. 2. Best examples of regeneration in series 1 and 2. Each column shows the same
limb in ventral view, reading downward at 5, 10, and 20 weeks after amputation.
Vertical lines mark the position of the elbows. Regenerated tissue is stippled.
(A) Right arm from series 1 which regressed to above the elbow and then regenerated
from the shielded upper arm.
(B) Left arm from the same specimen beginning to regenerate from the irradiated
forearm. This reached the criterion of 3 digits at 24 weeks after amputation.
(C) Right arm from series 2 which was shielded during irradiation and regenerated
completely in 5 weeks after amputation.
(D) Left arm of the same specimen which regressed considerably but regenerated
from the irradiated forearm.
424
H. WALLACE
in the other specimens. The right forearms were also irradiated and amputated
3 weeks later. No regeneration occurred on these forearms, which gradually
regressed until only the shielded upper arm remained. This establishes that
2 krad of X-rays was sufficient to inhibit regeneration.
Series 2 was performed simultaneously with the previous series to test another
means of causing denervation. Studies on mammalian nerves (Young, 1942;
Weiss & Hiscoe, 1948) show that crushing damages axons sufficiently for the
distal part to disintegrate - the process known as Wallerian degeneration; after
some delay, the proximal part regrows down the intact nerve sheath. The
persistence of the intact nerve sheath after crushing is expected to confine the
regrowing axons, which had an opportunity to fray out from the severed brachial
plexus in series 1 (cf. Weiss, 1937). Consequently, re-innervation of the forearm
might be achieved more rapidly, completely and consistently after crushing than
after severing the nerves. A comparison of the rates of recovery of arm movement in these two series supports this interpretation.
The left forearms of this series were irradiated exactly as in the previous one,
but temporary denervation was achieved by crushing each nerve of the shielded
brachial plexus several times with fine forceps. Both motility and sensitivity of
the arm were lost for at least a week. After 3 weeks, when nerves had usually
reached the hand, the arms were amputated above the wrist. More than half of
these specimens regenerated a hand from the irradiated forearm after a protracted period of slow regression (Fig. 2). Although the number of specimens
was so greatly reduced by escapes during this period that the conclusion must
remain tentative, crushing seemed to be a superior method of causing temporary
denervation and was therefore employed in subsequent series. The right arms of
the same specimens were shielded during irradiation and also amputated 3 weeks
later. The perfect regeneration of these arms (Table 1) confirms the normal
regenerative ability of axolotls, demonstrates that the shielding was an effective
protection (in comparison to the right arms of series 1), and provides a control
of the onset and rate of regeneration in the left arms of these two series. On this
basis, it is clear that paradoxical regeneration only occurs after a noticeable
delay, at least 4 weeks in these series, and proceeds more slowly than normal.
These two series of operations confirm that paradoxical regeneration occurs
in axolotls, but at a lower frequency and after a longer delay than was previously
found for larval A. opacum. These differences may merely reflect the fact that
older and larger specimens were used here in order to increase the precision of
the shielding pattern.
Series 3-was designed as a control to demonstrate that paradoxical regeneration
only occurred if some part of the arm had been shielded from irradiation. The
entire left arms and shoulders were irradiated and then denervated by crushing.
This operation produces the condition where all tissues, including the origin of
the regrowing axons, have been irradiated (Fig. 1B). The forearms became reinnervated within 3 weeks, but amputation at that time did not provoke re-
425
Paradoxical regeneration
A
B
CD
Fig. 3. Typical examples of regeneration from series 3, 4 and 5. Each column shows
the same limb in ventral view, reading downward at 6, 10 and 14 weeks after
amputation. Vertical lines mark the position of the elbows. Regenerated tissue is
stippled.
(A) Right arm from series 3 which rapidly regressed to the elbow, then formed
a twisted regenerate originating in the anterior of the shielded upper arm. The left
arm did not regenerate.
(B) Right arm from series 4 which regressed slowly before regenerating from the
irradiated forearm. The left arm did not regenerate.
(C) Right arm from series 5 showing continued regression.
(D) Left arm from the same specimen showing regeneration from the irradiated
forearm.
generation. In comparison to this failure of regeneration, it is concluded that
cases of paradoxical regeneration in the two earlier series must be attributed to
the localized shielding employed there. Exactly as found previously with
A. opacum, paradoxical regeneration has only been obtained when the axons
have regrown from a shielded brachial plexus.
The unoperated right arms of the same specimens were also amputated through
irradiated tissue of the forearm after a 3-week delay. All these arms regressed
slowly, either to the elbow or well into the upper arm. Several of these arms
regenerated eventually, but always from above the elbow and so presumably
from shielded tissue (Fig. 3). This result approximates to the demonstrations on
adult Triturus (Scheremetjewa & Brunst, 1938) and on larval Eurycea (Butler &
O'Brien, 1942) that neighbouring shielded tissue does not affect the local inhibition of regeneration at the irradiated amputation site.
Series 4 was performed simultaneously with the previous series, to determine
if paradoxical regeneration can occur if some other region of the arm than the
shoulder has been shielded from irradiation. The shielding pattern was identical
426
H. WALLACE
to that of series 3, but here the right arms were denervated. The entire left arms
and shoulders were irradiated and then amputated 3 weeks later, with no intervening operation. These arms regressed at about the same rate as both the left
and right arms of series 3; none of them regenerated. The right forearms and
shoulders were irradiated and then denervated by crushing to create a novel
situation. The axons regrew from an irradiated brachial plexus through a shielded
region above the elbow, before penetrating the irradiated forearm (Fig. 1B).
Amputation above the wrist 3 weeks later frequently provoked regeneration
from the irradiated forearm, after a considerable delay (Fig. 3).
The significance of this result emerges by comparison with the consistent
failure of regeneration in either forearm of the specimens in series 3. That series
demonstrates that neither regrowth of axons from an irradiated source, nor
a shielded region proximal to the amputation site, are independently sufficient
to overcome the local X-ray inhibition of regeneration. The combination of these
two conditions, achieved in the right arms of this series, is expected to permit
regeneration only if the regrowing axons mobilise cells in the shielded region
and if these cells migrate into the forearm. The nerve sheath cells, particularly
the Schwann cells, are the most obvious candidates to respond in this way to
regrowing axons. The regeneration observed in this series, therefore, supports
the proposed interpretation implicating nerve sheath cells as the migratory agents
of paradoxical regeneration.
Series 5 provides a more striking demonstration in support of this interpretation. Both arms and shoulders were irradiated, while both hands and wrists
were shielded (Fig. 1C). The left arm was denervated by crushing and gradually
became re-innervated during the following 3 weeks. In an attempt to ensure that
regrowing axons penetrated the shielded hands, these specimens were left 4 weeks
before amputating both arms in the irradiated mid-forearm. Several of the left
arms regenerated after a considerable delay. None of the right arms regenerated,
demonstrating that all shielded tissue had been removed by the amputation.
A typical case is illustrated in Fig. 3. The regeneration of the left arm can be
attributed to the same factors established for the previous series, where regrowing
nerves either originated in or traversed shielded tissue. The cells postulated to
carry regenerative competence travelled in the same direction as the nerves in
those series, and so could have been carried passively to the site of amputation.
The left arms of this series, however, were only shielded distal to the site of
amputation. Competent cells could only originate in this shielded region (cf.
series 3 left arms), and were mobilized as a result of temporary denervation
(cf. right arms of the same specimens). These cells, therefore, must have migrated
proximally from their original shielded location during the 4-week period prior
to amputation. It is likely that regeneration would have occurred more frequently
if the amputations had been delayed even more.
The general conclusion emerges from these last three series of experiments
that some cells in any shielded region of the arm are mobilized, either by th(
Paradoxical regeneration
427
local degeneration of severed axons or by the regrowth of new ones, and these
cells migrate actively up and down the arm. Since nerves are apparently indispensable for regeneration in these conditions, the experiments provide the
clearest available demonstration that temporary denervation and the regrowth
of axons are indirect agents of paradoxical regeneration, in that they permit or
elicit a movement of non-irradiated cells which does not occur in normal limbs.
Only Schwann cells are known to possess the required migratory character
which appears in response to axonal changes (cf. Weiss & Wang, 1945). These
experiments thus implicate Schwann cells as the direct agents of paradoxical
regeneration.
Series 6 was performed to provide direct evidence that nerve sheath cells can
support the regeneration of a limb whose other tissues have been irradiated.
Lengths of brachial nerves from non-irradiated donors were implanted into the
irradiated left forearms of differently coloured hosts 70-80 mm long (Fig. ID).
After a delay of 3 days, to allow the host skin to heal, the arms were amputated
just distal to the still visible implants. Most of these arms regenerated perfectly.
The right arms of the same specimens which had been shielded from irradiation
and carried no implants were amputated at the same time as the left arms. They
all regenerated to reach the criterion of three digits within 5 weeks; the left arms
took 10 weeks on average to form similar regenerates, and remained smaller than
the controls for several weeks after that (Fig. 4). This series repeats a preliminary
trial (Conn et al. 1971), where three out of five axolotls regenerated irradiated
arms bearing non-irradiated nerve implants. That result required confirmation,
especially as Vergroesen (1958) had reported the failure of regeneration to occur
in axolotl hind-limbs in essentially the same condition. In comparison to the
uniform local inhibition of regeneration caused by this dose of irradiation, this
series demonstrates that non-irradiated nerve implants do support the regeneration of irradiated arms. As argued at length by Conn et al. (1971), that ability
cannot be attributed either to operational trauma or the axon fragments in the
grafts and must therefore be a property of the nerve sheath cells.
The fixed nerves corresponding to those implanted in this series were found
not to be contaminated by blood vessels or pigment cells, but perhaps retained
some strands of connective tissue outside the perineurial sheath. Ignoring the
anucleate axons, the implants consisted exclusively of Schwann cells and fibroblasts. Both of these cell types have been identified as components of the early
blastema during normal limb regeneration (Chalkley, 1954; Trampusch &
Harrebomee, 1965). In this experimental situation, where the implant is the only
local source of non-irradiated cells and is demonstrated to be required for
regeneration, these Schwann cells and fibroblasts must certainly contribute to
the blastema and probably generate the entire blastema. This conclusion is
supported by the observation that the general coloration of the experimental
regenerates was consistently that of the donor specimens.
28
EMB 28
428
H. WALLACE
Paradoxical regeneration
429
DISCUSSION
Paradoxical regeneration occurred in more than half the appropriate cases
summarized in Table 1. This means that only small numbers of specimens were
required in each series to distinguish the occurrence of paradoxical regeneration
from the uniform failure of regeneration in irradiated control arms. The frequency of paradoxical regeneration found here and previously (Conn etal. 1971)
is sufficient to establish it as a genuine phenomenon. It cannot be dismissed as
an artifact of occasional shielding errors, any conjectural threshold effect of the
radiation dose, or a result of operational trauma. The radiation dose used was
more than twice that which routinely inhibits regeneration in young axolotls.
The operational trauma was reduced by delaying amputation for several weeks,
while identical operational damage has never provoked regeneration in irradiated
control arms (see Series 3, cf. Conn et al. 1971). Ignoring the effects of hormones
and morphogenetic fields which were not deliberately altered in these experiments, the major requirements for limb regeneration are held to be a wound
epithelium covering the amputated surface, an adequate supply of intact axons,
and some non-irradiated limb tissue close to the site of amputation. The present
results can be interpreted in terms of these three requirements and help to clarify
their interaction.
The wound epithelium
The essential characteristic of the wound epithelium, shared by the apical
epithelium of regressing limbs (Thornton & Kraemer, 1951), is apparently that
it lacks the normal dermal layer of the skin and is consequently vulnerable to
infiltration by regrowing axons. Perhaps as a result of hyperinnervation, the
epithelium thickens as an apical cap resembling that of an embryonic limb and
generally held to have some morphogenetic influence on the blastema cells
which collect underneath it (Thornton, 1965). The relationship between the
apical cap, nerves and blastema outlined here has been questioned by Singer &
Inoue (1964), who cite examples of apical cap formation without epithelial
innervation and without any subsequent accumulation of blastemal cells. Perhaps
apical caps are not so easily recognized, just as the apex of a regressing limb can
FIGURE 4
Typical examples of regeneration from series 6.
(A) White dd specimen whose left arm was irradiated and then amputated just
distal to an implanted Dd branchial nerve.
(B) Dark Dd specimen whose left arm was irradiated and amputated just distal to an
implanted dd brachial nerve.
These two photographs were taken against the same background when the pigmentation of the regenerates had stabilized 24 weeks after amputation. The donor
coloration is shown by the entire left regenerate and extends some way into the irradiated host arm.
28-2
430
H. WALLACE
resemble an early blastema. The wound epithelium is initially formed by a mass
spreading of the epidermis adjacent to the site of amputation, but the later
thickening of the apical cap also involves cell division. Both these processes
apparently occur equally well in irradiated and normal limbs (Rose & Rose,
1965). The simple conclusion that epidermis is relatively insensitive to radiation
may be justified in the following terms. Cells of the proliferative basal layer are
capable of lateral displacement, so that lethally irradiated cells may be displaced
by survivors; the principal differentiation of the outer layers, a suicidal conification, is perhaps achieved as easily by lethally irradiated cells as by any others.
The present results include examples where regression continued into a shielded
and normally innervated upper-arm. Previous experience would predict the
spontaneous regeneration of larval limbs in this situation (Schotte & Butler,
1941), but often the epithelium healed and regression ceased - leaving a stable
stump which did not regenerate. It must be remembered that the experimental
axolotls were more than 100 mm long by this time, and both regression and
regeneration occurred more slowly than in younger specimens. Apparently
adolescent axolotls become more comparable to postmetamorphic stages of
other urodeles; simultaneously denervated and amputated arms of adult Triturus
viridescens show very little regression and rarely regenerate (Singer, 19466). That
would explain why paradoxical regeneration did not occur as consistently in
these experiments as it did with younger A. opacum (Conn et ah 1971).
The axons
The mechanism by which axons promote the formation of a blastema remains
largely unexplained, despite continued efforts to identify a neurotrophic substance (Lebowitz & Singer, 1970; Burnett, Kary & Lagorio, 1971). Regrowing
axons are involved in virtually all known cases of limb regeneration, either following temporary denervation or merely because they are also severed by the
amputation. Severing sensory axons in the brachial plexus prevents the formation
of a blastema in adult newts, but severing the central connexions of the same
neurons does not (Sidman & Singer, 1951). This convincing demonstration that
regeneration is dependent upon intact axons incidentally shows that cells of the
nerve sheath are not concerned in the neurotrophic control of regeneration, and
perhaps again implies that the neurotrophic factor is a property of local regrowing axons. Singer's (1952) quantitative neurotrophic theory incorporates his
earlier demonstrations that the normal sensory innervation alone, or a hyperinnervation by regrown motor fibres, is sufficient to support regeneration. Since
motor nerves normally innervate only internal tissues, their neurotrophic action
detracts from the credibility of either a nervous control over the formation of the
apical cap or any epithelial-nervous control over regeneration, as postulated by
Rose (1962, 1964) and Trampusch (1964). It is worth noting, however, that the
crucial test concerns regeneration supported by an abnormally dense regrown
Paradoxical regeneration
431
motor innervation (Singer, 1946a); Weiss (1937) records that such regrowing
axons penetrate tissues at random - and thus might reach the wound epithelium.
The discovery that aneurogenic limbs regenerate perfectly well with only
a minute fraction of the normal nerve supply (Yntema, 1959) has been exploited
by Steen & Thornton (1963) to show that later innervation of these limbs slowly
makes their regenerative ability dependent on the nerves - and that dependency
is apparently a characteristic of the skin. A suitably amended ' addictive' neurotrophic theory (Singer, 1965) has recently found some experimental support
(Thornton & Thornton, 1970).
The experiments described here confirm a previous report (Conn et ah 1971)
that a conventional dose of X-rays, which inhibits regeneration, does not
detectably impede the regrowth of severed axons. Similarly irradiated axons and
even those that have regrown from irradiated neurons retain their physiological
functions, including the neurotrophic property of promoting regeneration. Yet
regeneration only occurs when some non-irradiated tissue is present, demonstrating that the neurotrophic factor cannot elicit the recovery of irradiated cells.
There is no interaction, therefore, between the neurotrophic factor and the effect
of irradiation on regeneration, despite the similarity of response to X-rays and
denervation emphasized by Schotte & Butler (1941, p. 281).
Localized irradiation
It is generally agreed that irradiated urodele limbs do not recover their former
ability to regenerate over a period of years, and no certain evidence has been
obtained that irradiated cells of internal tissues can participate in regeneration
(see reviews of Brunst, 1950; Thornton, 1968). The several observed effects of
irradiation on growing or regenerating limbs are consistent with the general
explanation that X-rays cause genetic lesions and chromosome aberrations
which are usually lethal to dividing cells (see Davies & Evans, 1966). Differentiated cells of the limb survive and function normally after doses of X-rays
considerably higher than used in this study, but 2 krad may kill the vast majority
of dedifferentiated cells after a few divisions at most. The most pertinent observations on amphibian limbs are that irradiated and amputated larval limbs
regress, suggesting that dedifferentiation occurs normally but the potential
blastemal cells vanish (Puckett, 1936); dividing cells are destroyed but growth
and cellular differentiation continue for a short while after irradiation of larval
limbs (Allen & Ewell, 1959). The 'genetic lesion' explanation is consistent with
the apparent insensitivity to X-rays of all axons, including regrowing ones; but
fails to explain the continued division of irradiated epidermal cells, except as
suggested earlier in this discussion.
The genetic lesion explanation outlined in the previous paragraph presupposes
a direct effect of irradiation on each exposed cell of the limb. The same assumption was made and justified in the classical demonstrations that blastemal cells
originate in a strictly localized region adjacent to the site of amputation
432
H. WALLACE
100 i -
5
50
12
Weeks after amputation
24
Fig. 5. Cumulative incidence of regeneration to the criterion of three digits in 17
shielded control arms from series 2 and 6 (A), 9 irradiated arms bearing nerve grafts
from series 6 (B), and 22 locally irradiated arms after temporary denervation from
series 4 and 5 (C). The shading before each curve stretches to the first certain record
of blastemata and thus indicates the estimated duration of regeneration. Both the
delay and duration of regeneration can be related to the scarcity of non-irradiated
cells at the site of amputation: cells of all tissues, Schwann cells and fibroblasts of
the graft, and probably Schwann cells only.
(Scheremetjewa & Brunst, 1938; Butler & O'Brien, 1942). Two exceptions to this
strict localization have now been examined - the regeneration following skin
incisions on locally irradiated limbs (Conn et ah 1971) and the experiments
reported here on paradoxical regeneration. Defining the conditions under which
regeneration can occur demonstrates that each of these exceptions can be
explained by an abnormal migration of shielded cells to the previously irradiated
site of amputation. The experiments described here show that regrowing axons
are only able to support regeneration at an irradiated region of the limb if they
have regrown from or through a shielded region. Some cells of the shielded
region are apparently mobilized and migrate distally with the axons, or independently in the opposite direction, to reach the site of amputation. Paradoxical
regeneration is thus interpreted in conformity to the direct local effect of X-rays,
as the equivalent of small graft of non-irradiated tissue into a generally irradiated
region of the limb. The observed delay in establishing a blastema suggests that
the original population of competent cells may be exceptionally small (Fig. 5).
Larger grafts of several tissues, among which brachial nerves can now be included, are well known to support regeneration in these circumstances. Whether
such grafts provide all the cells of the blastema or somehow revive the surrounding irradiated cells remains the vexing question. These experiments involving
local irradiation show that physiologically normal and neurotrophically active
axons, which permeate the limb-stump and whose axonal flow should ensure
a continuous supply of material from the non-irradiated nucleus and cell-body,
Paradoxical regeneration
433
cannot restore regenerative ability to irradiated cells. It follows that other nonirradiated cells probably cannot do so either, and hence must proliferate to
provide all the internal tissues of the regenerate.
The genetic difference between hosts and nerve implants, described in the
results, was intended to produce direct evidence concerning the origin of the
regenerated tissue. Although the pigmentation is not determined by the genotype
of themelanophores themselves, but rather by the genotype of their environment,
the results obtained fit the usual expectation for reciprocal transplantations. The
epidermis of each regenerate was pigmented like the host, from which it was
presumably derived. The internal tissues of each regenerate carried a pigment
pattern like the graft-donor. This result holds out some hope of finally proving
that the mesenchymal blastema originates exclusively from non-irradiated graft
tissue. The implications of such a proof demand a detailed analysis which will be
described separately.
The experiments described here provide unusually clear examples of grafts
restricted to few cell-types and thus, following the preceding argument, of their
probable transformation into different cell types during regeneration. The
grafted segments of non-irradiated nerves only provide Schwann cells and fibroblasts, yet the regenerate which is apparently derived from them includes cartilage
and muscle. It appears quite probable that these tissues can be obtained during
paradoxical regeneration by the transformation of Schwann cells alone. A careful examination of which cells are mobilized during the regrowth of severed axons
is still required to establish this point. The general conclusion trfat specific celltypes genuinely dedifferentiate and can transform to other tissues is already
a strong probability, reinforcing the conclusions of Steen (1968) that some
marked cells in muscle grafts are converted into cartilage during regeneration
(cf. Thornton, 1942), even though implanted cartilage tends to retain its tissuespecificity (cf. Eggert, 1966).
The regenerative capacity of nerve sheath cells advocated here could even
reflect, with some exaggeration, their activity in normal limb regeneration.
Chalkley (1954) estimates their contribution to blastemal mitoses as 4 % in adult
newts, but this might be greater in larval limbs. While clearly dispensable in the
regeneration of aneurogenic limbs, the abundance of nerve sheath cells in early
blastemata has been noticed on several occasions since Guyenot & Schotte (1926,
p. 35) observed: 'II parait meme y avoir un rapport genetique entre les cellules
de la gaine des nerfs et les elements cellulaires du blasteme. En effet, les noyaux
de la gaine presentent, vers l'extremite des nerfs, des mitoses anormalement
nombreuses et Ton observe tous les passages entre les noyaux allonges des gaines
nerveuses et les noyaux de plus en plus gros des cellules conjonctives. II ne serait
done pas impossible que les gaines nerveuses participassent, pour une part
importante, a la formation du blasteme et que ce mecanisme jouat un role
important dans la genese des pattes supplementaires.'
434
H. WALLACE
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{Manuscript received 1 March 1972, revised 27 April 1972)