/. Embryo!, exp. Morph. Vol. 41, pp. 1-13, 1977
Primed in Great Britain <& Company of Biologists Limited 1977
The role of Schwann cells in paradoxical
regeneration in the axolotl
By M. MADEN 1
From the Department of Genetics, University of Birmingham
SUMMARY
The experiments described here examine further the conditions under which paradoxical
regeneration occurs and provide support for the hypothesis that a proximal migration of
Schwann cells is responsible for the phenomenon. When only the hand is shielded from irradition and the limb is denervated, amputation through the forearm or upper arm sometimes
results in regeneration. The effects of variation in the time interval between denervation and
amputation, the level of amputation and the method and number of denervations on the
incidence of regeneration were investigated. The presence or absence of viable Schwann
cells at the amputation plane was deduced from the remyelination of nerves under conditions
which do or do not permit paradoxical regeneration. The nerves of totally irradiated and
denervated limbs remain unmyelinated following regrowth of axons and such limbs do not
regenerate after amputation. When only the hand was shielded from irradiation before the
limbs were denervated, the new axons became completely remyelinated and some of these
limbs regenerated when amputated. It is suggested that under these conditions Schwann cells
can migrate proximally and can then proliferate further to form a blastema, since they would
be the only unirradiated tissue present at the amputation plane.
INTRODUCTION
When amputated through a region which has been irradiated with 2000R
of X-rays, the limbs of young axolotls do not regenerate (Maden & Wallace,
1976). Limbs often do regenerate, however, if they have been denervated shortly
after irradiation and well before amputation (Wallace, 1972). The subsequent
regrowth of nerves from the shielded brachial plexus after denervation restored
the regenerative ability of a proportion of arms when amputated through the
irradiated forearm several weeks later. Regrowth of axons from an irradiated
brachial plexus through a shielded region above the elbow into the irradiated
forearm also permitted regeneration after amputation at the wrist. An even
more striking demonstration ensued when only the hand and wrist were
shielded before denervation; here, amputation in the mid-forearm four weeks
later resulted in the regeneration of half of the specimens after a considerable
delay. The contralateral arms, identical except that they had not been denervated, did not regenerate, confirming that paradoxical regeneration, as Wallace
(1972) termed it, is dependent on the effects of denervation.
1
Authors address: Developmental Biology Group, School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, Sussex, U.K.
2
M. MADEN
Several explanations of these results are apparent, but most do not stand up to
scrutiny. For instance, cells of the unirradiated area could not have reactivated
the rest of the arm because the local nature of X-ray inhibition is well established
(Scheremetjewa & Brunst, 1938; Butler & O'Brien, 1942) and for the same
reason a nervous 'induction' is not feasible. Furthermore, it has been argued
that irradiated tissue is so severely damaged by an X-ray dose of 2000R that
reactivation by unirradiated cells or some 'factor' is impossible (Maden &
Wallace, 1976). One is therefore led to the conclusion that unirradiated cells
migrated into the X-rayed areas and responded to subsequent amputation by
forming a blastema.
Normally after amputation, cells do not travel more than a very short distance (Wolff & Wey-Schue, 1951; Wey-Schue, 1954; Lazard, 1968), so denervation may have promoted the migration of certain cell types from shielded areas.
In the first two cases of paradoxical regeneration mentioned above (unirradiated
shoulder or upper arm section) the cells postulated to carry regenerative competence travelled in the same direction as the incoming nerves and so could have
been carried passively to the site of amputation. But in the third instance, cells
originated in the shielded hand and must have migrated proximally during the
period prior to amputation. It is this line of reasoning that led Wallace (1972) to
propose that Schwann cells are responsible for paradoxical regeneration, since
only this cell type is known to possess the required migratory behaviour. They
would accompany the regrowing axons to provide a new myelin sheath and in
this experimental situation a source of non-irradiated blastemal cells. Other
components of the nerve sheath such as the connective tissue elements do not
respond to denervation in the same fashion (see Discussion).
This hypothesis is examined here from two aspects. Firstly, variations in the
conditions under which paradoxical regeneration occurs are explored, namely
method of denervation, length and number of denervations, amputation level
and time between denervation and amputation. Secondly, the behaviour of
Schwann cells under conditions which do or do not permit regeneration is
deduced from an histological study.
MATERIALS AND METHODS
These experiments were all performed on young axolotls, Ambystoma
mexicanum, kept at room temperature (16-19 °C).
Varied conditions of denervation and amputation: The irradiation pattern used
throughout this study was the third and most significant one described above,
that is with the hands shielded and arms and shoulders irradiated (Fig. 1). The
head, lower body and hands were covered with 3 mm-thick lead plates while
the exposed areas received 2000R from a Pantak Ltd X-ray machine at 300 kV
12 mA, at a distance of 165 mm from the target, giving a dose rate of 520R min"1.
Immediately after irradiation the left arms of the specimens were denervated
Paradoxical regeneration
Fig. 1. The shielding pattern used in these experiments. The position of the specimens
is shown in outline, omitting the gills. The shaded area represents lead plates.
by crushing or severing spinal nerves, 3, 4 and 5 at the brachial plexus. The
return of motor and sensory capabilities to the upper arm, lower arm and
hand of the left limbs was recorded daily by observation of their use during
swimming (motor) and by their response to pricking with a needle (sensory). At
various times after denervation (1-20 weeks) both arms were amputated either
through the forearm or upper arm, the right arms serving as non-denervated
controls. All specimens were kept for at least 20 weeks after amputation and
the incidence of regeneration recorded according to the criterion of three or
more digits.
Histology. The amputated portions of the limbs in the above experiments
were fixed in neutral formalin, decalcified and examined for the presence, in
transverse paraffin sections, of myelinated nerve fibres and Schwann cells at
the amputation plane. The gallocyanin technique of Augulis & Sepinwall
(1971) was used on 10 jum sections to detect the presence of myelin. The experimental conditions to which these limbs had been subjected (shielded hand and
denervation) would be expected to lead to regeneration in a certain proportion
of cases. As a comparison the myelination of nerves and the presence of Schwann
cells was examined, using the same techniques, in a non-regenerating control
series differing only in that the arms had been totally irradiated.
Control right arms sometimes regenerated, possibly because denervation of
the left arms could cause degenerative changes in the right limb nerves. To investigate this, the left limbs of 13 axolotls were denervated by crushing, and at
4
M. MADEN
various intervals afterwards the occurrence of degenerating myelin was detected
in both left and right limb nerves by the method of Gutman & Sanders (1943).
This technique requires a more complex treatment to ensure good penetration
of the fixative without distortion of the nerves: the upper limb was excised,
stripped of skin and most muscle to expose the major nerve trunks, but their
attachment to the skeleton was maintained. This whole preparation was then
immersed in fresh Flemming's fluid for three days. The ends of these nerves
have inevitably been damagedduring excision and usually show signs of degeneration which are not related to the experimental treatments. To overcome this,
only the mid-sections of these nerves were scored for the presence or absence of
degenerating myelin, after it had been established on several control specimens
that this area was free from mechanical damage.
RESULTS
Method of denervation: The progress of functional reinnervation was followed
after either crushing or severing the nerves at the brachial plexus in batches of
50 or more animals, to establish whether there was any difference between
these two operations which could affect subsequent regeneration. Figure 2
shows a typical example of the return of nerves after crushing, taking four weeks
to return to the hand. A delay in reinnervation of about 1 week occurred when
the plexus had been cut. The rate of regeneration of crushed axons was about
0-75 mm/day. This figure is an underestimate, since new axons would be present
some time before the establishment of functional connexions; it therefore corresponds well with the 1 mm/day quoted by Singer (1964) as an approximate
value for amphibian axons.
Crushing the nerves after irradiation with a shielded hand and amputating in
the forearm four weeks later, Wallace (1972) obtained five left arm regenerates
out of ten. Cutting instead of crushing the nerves reduced the frequency of
regeneration of left arms (Table 1). A range of time intervals between denervation and amputation was explored in case the delay in reinnervation after
severing (Fig. 2) caused a delay in cellular migration. Increasing the time interval did not make up the deficiency; thus it seems that rapid reinnervation may
promote the survival or migration of shielded cells. No control right arms regrew, confirming that denervation is necessary for paradoxical regeneration.
The development of paradoxical regenerates was both delayed and slower
than normal: blastemas appeared in 5-14 weeks compared to 1-2 weeks normally, three digits in 11-20 weeks compared to 3-4 weeks normally.
Effect of amputation level: The effect of amputating at a different level was
investigated on a total of 50 animals, to determine whether increasing the distance from the shielded region altered the frequency of paradoxical regenerates.
To do so the standard shielding pattern was employed, the left arms denervated
by crushing, and amputated through the distal humerus. The time interval
Paradoxical regeneration
Table 1. Number of left arm regenerates after two different methods of denervation
and amputating through the forearm at various times. For each time interval a
group of 10 specimens was used, the actual number of survivors varying from 8-10.
Here and in the succeeding tables regeneration is defined according to the criterion
of three or more digits, and — means no experiment performed
Interval between denervatiorl
and amputation in w<;eks
Series
Wallace
(1972)
1
Method of
denervation
Amputation
site
crush
forearm
sever
forearm
100
2
3
4
5
6
8 10
50%
5
0
1 0
Overall
frequency
1 2 0
0
6%
n
50-
1
2
3
4
5
6
Weeks after denervation
Fig. 2. The return of functional innervation to the left arms after denervation by
crushing (A, B, C) or severing (D) the nerves at the brachial plexus. % reinnervation
refers to the percentage of animals showing both a sensory and a motor response.
This experiment was performed on 70-90 specimens.
A - return to shoulder, indicated by movement of the entire arm as a unit. B - return
to elbow, indicated by movement of this joint. C - return to hand, indicated by
movement of wrist and digits. D - return to hand after severing the nerves. Note the
delay of about I week compared to C.
between denervation and amputation was varied from 1-5 weeks. Upper arm
amputation reduces the number of regenerates from 50 % to an overall frequency of 8 % (Table 2), a value close to that obtained by altering the method of
denervation. This would be consistent with the notion that the unirradiated
cells present at the amputation plane had migrated proximally: the further the
distance to be travelled the less chance there is of successful regeneration. No
control, non-denervated right arms regrew, confirming that denervation is
necessary for paradoxical regeneration.
6
M. MADEN
Table 2. Number of left arm regenerates after amputation at two different levels.
For each time interval a group of 10 specimens was used, the actual number of
survivors varying from 6-10
Series
Method of
denervation
Amputation
site
Wallace
(1972)
crush
forearm
2
crush
upper arm
Interval between
denervation and
amputation in weeks
A
,
^
12 3 4 5
_
0
1 0
Overall
frequency
5—
50%
1 1
8%
Table 3. Number of left arm regenerates after amputating at various times after
denervation. For each time interval a group of 10 specimens was used, the actual
number of survivors varying from 5-10
Interval between denervation
and amputation in weeks
Series
Method of
denervation
Amputation
site
2 4 5 6 7 8 10 15 20
Overall
frequency
3
crush
upper arm
0 2 0 0 0 1 1 0 2
9%
A.
Effect of delayed amputation: Increasing the time interval between denervation and amputation might raise the incidence of regeneration when amputation
is performed in the upper arm, since more migrating cells might have time to
arrive. Ninety axolotls were irradiated except for the shielded hand, the left arm
denervated and both limbs amputated after 2-20 weeks. This procedure did
not significantly increase the frequency of paradoxical regenerates (Table 3). The
low overall frequency confirms that amputation further from the shielded hand
decreases the incidence of regeneration, which is consistent with the view that
proximal migration takes place rapidly to repopulate the entire arm with unirradiated Schwann cells. Once completed no additional movement seems to
occur, and those cells that are present at proximal limb levels remain and can
be induced to divide when amputation is performed much later.
In this experiment some control arms regenerated at 4, 5, 7, 8, 10 and 20
weeks, giving an overall frequency of 17 %, almost double the experimental
frequency. This was the first time that controls had regenerated, which cast
doubt on the validity of this particular experiment. It could have been due to
an ineffective irradiation dose resulting from instrument error, movement of
the animals or inaccurately placed shields. To check that these control regenerates were not due to epidermal or any other cellular migration independent of
denervation, 10 larvae were irradiated identically with shielded hands and
2000R, but not denervated. Half were amputated through the upper arm four
Paradoxical regeneration
7
Table 4. Frequency of regeneration of left arms after
different types of denervation
Series
Method of denervation
Amputation site
Control
4
5
single crush
2-3 crushes
3 week
prolonged
upper arm
upper arm
upper arm
Incidence of
regeneration
10 %
13 %
5%
weeks later and the other half at six weeks. None of these 20 limbs regenerated; all had rounded, smooth ends characteristic of inadiated stumps. The
possibility of contralateral nervous effects due to left arm denervation will be
examined in greater detail later.
Repeated or prolonged denervation: Two further aspects of temporary denervation were explored by experiments in which the previous techniques of
denervation and amputation in the upper arm remained constant. Firstly, the
effect of repeated denervation was tested by repeated crushes at four week
intervals, allowing reinnervation to occur in the intervening period. Secondly,
the effect of prolonged denervation was examined by reoperating at weekly
intervals for three weeks, then permitting reinnervation and amputating. A
group of specimens subjected to the standard single denervation served as a
common control and duplicated previous results by producing a regeneration
frequency of 10 % (Table 4). The proportion of regenerates shown in Table 4
fall within the extremes found after a single denervation. Thus it seems that
neither repeated nor prolonged denervation has any enhancing effect on paradoxical regeneration. One control right arm regenerated in series 4.
Behaviour of Schwann cells: The amputated arms obtained from the experiments described above were examined for the presence of myelinated axons at
the level of amputation. They had been irradiated except for the shielded hands
and the left arms denervated and were compared with a control series differing
only in that the arms had been entirely irradiated including the hands, a situation in which no regeneration occurs after amputation. The observations of the
four sets of limbs are summarized in Table 5 and typical nerve trunks are
illustrated in Figs. 3, 4 and 5.
The intact nerves in the right arms of all specimens retained prominent myelin sheaths without any obvious change for at least 12 weeks after irradiation
(Fig. 3). The denervated left arms showed a progressive loss of myelin associated
with Wallerian degeneration of the axons which was complete by two weeks
after denervation. Axons grew into these upper arms during the second and
third weeks according to the recovery of sensitivity and movement (Fig. 2).
These axons remained naked for at least 12 weeks and perhaps permanently
when the arm had been totally irradiated (Fig. 4), but became remyelinated in
M. MADEN
A
A '
n
Paradoxical
regeneration
Table 5. The percentage of upper arms with major nerves
myelinated after various treatments
Both hands shielded
Entire arm irradiated
A
Weeks after
treatment
Left (denerv.)
Right
Left (denerv.)
Right
(%)
(°/o)
(%)
(%)
0-1
2
3
4
100
0
—
0
70
100
100
100
100
—
100
100
100
100
100
0
0
0
0
0
0
100
100
100
100
100
100
100
' 5
6
7-12
5-6 weeks when the hand had been shielded from X-rays (Fig. 5 and Table 5).
Since the presence of a shielded hand constituted the only difference between
these denervated arms, shielded Schwann cells must migrate proximally from the
hand to ensheath axons in the irradiated upper arm. This migration could not
be observed directly as irradiated Schwann cells persist, albeit in reduced numbers, even though they are clearly incapable of remyelination and there was no
means of distinguishing different populations of Schwann cells. Detection by
remyelination implies that the migration must have occurred considerably earlier;
the results in Table 2 suggest that two weeks is long enough. If so, then the reason for the 3 to 4-week delay between arrival and remyelination is not clear.
Contralateral effects: Finer details of the changes which follow unilateral
denervation were obtained by scoring nerve trunks for the presence of degenerating granules of myelin. Table 6 shows that the denervated limbs contained
progressively fewer myelin sheaths from the first appearance of degeneration
granules at five days until the complete disappearance of stainable myelin after
about three weeks. Although the contralateral, non-denervated limbs retained
the bulk of their myelin, some degenerating granules were detected sporadically
in samples at 17-35 days (Table 6). These granules occurred considerably later
than in the denervated limbs and only in a few nerve fibres. They must have been
FIGURES
3-5
Fig. 3. Nerve bundles of the upper arm of normal axolotls showing myelin as purple
rings (r) after staining with gallocyanin. Schwann cell nuclei (sc) are present within
the nerves and often appear crescent-shaped as they envelop the axons. Bar = 10 /*m
Fig. 4. A typical nerve bundle of the upper arm 10 weeks after total irradiation and
denervation. Note that the new axons which return to the limb remain unmyelinated.
Irradiated Schwann cells are still present, although in reduced numbers, and are
thus non-functional. Bar = 10 fim
Fig. 5. A typical nerve bundle of the upper arm eight weeks after irradiation and
denervation, but in this case the hand had been shielded. A large proportion of these
axons have been remyelinated, as evidenced by the reappearance of rings. Bar = 10/tm.
10
M. MADEN
Table 6. Presence ( + ) or absence (0) of myelin breakdown granules which could
not be attributed to mechanical damage in the sciatic nerves of denervated and
contralateral limbs. Cases where all the myelin had disappeared are shown
by x
Sample time in
days since
denervation
Limbs
A
\
Denervated
1
3
5
8
11
14
17
20
23
26
29
32
35
0
0
+
+
+
+
+
+
+
X
X
X
X
Contralateral
0
0
0
0
0
0
+
0
0
0
+
+
+
due to the operation, however, as control specimens which had not been
denervated did not reveal any myelin granules in corresponding areas. It seems
that denervation of one limb has a minor, delayed effect on the contralateral
limb nerves as reported by Tweedle (1971) on the basis of different parameters.
This phenomenon could provide an explanation of the regeneration seen in the
controls.
DISCUSSION
In the first part of the work reported here, variations in the conditions under
which paradoxical regeneration occurs were explored. Crushing the nerves was
followed by more rapid reinnervation than severing, presumably because crushing leaves intact the connective tissue sheaths which then guide regrowing
axons relatively quickly through the brachial plexus. After severing the nerves
the number of regenerates was reduced upon subsequent forearm amputation
when compared to previously published results (Wallace, 1972): rapid reinnervation seems to be beneficial for paradoxical regeneration. Neither lengthening
the period of total denervation nor increasing the number of denervations
increased the incidence of regeneration. Amputating further away from the
shielded hand decreased the regeneration frequency; increasing the time interval
between denervation and amputation had no significant effect. The conditions
which promote a high frequency of paradoxical regenerates thus seems to
involve crushing the nerves and amputating in the forearm, as Wallace (1972)
originally described.
In addition, the suggestion that Schwann cells are responsible for paradoxical
regeneration was examined and it was found that they experience a proximal
Paradoxical regeneration
11
migration under precisely the conditions which permit regeneration. When the
hand had been shielded during irradiation, denervated arms became reinnervated and the new nerves were myelinated: a low proportion of such limbs
could regenerate when amputated through the upper arm. In contrast, totally
irradiated and denervated arms only became reinnervated by naked nerve fibres:
such arm do not regenerate. Similarly, in the absence of temporary denervation
irradiated Schwann cells and myelin sheaths persist: these arms do not regenerate
either. It can therefore be concluded that under the conditions which permit
paradoxical regeneration, unirradiated Schwann cells are present at the amputation plane. The following sequence of events might be envisaged: denervation
isolates all the axons distal to the brachial plexus from their cell bodies. The
consequent Wallerian degeneration causes the breakdown of the axonal
neurilemma into individual Schwann cells which are then stimulated to divide.
Irradiated Schwann cells - those in the forearm and upper arm - will die as a
result of damage revealed during division (Maden & Wallace, 1976), leaving a
void which can only be filled by migration of the nearest shielded Schwann cells
- in the hand. As all other cells have been heavily irradiated and reactivation by
unirradiated cells is unfeasible, these Schwann cells are likely to be responsible
for the regrowth of the limb. Other factors must intervene, however, as remyelination of irradiated upper arms occurs consistently, yet amputation there rarely
causes more than 10 % of the limbs to regenerate.
The mechanics of Schwann cell migration is well documented and involves
what Ramon y Cajal (1959) called a 'process of rejuvenescence'. The nucleus
enlarges and becomes rich in chromatin after only two days and nucleoli
appear. Mitosis soon begins and increases in frequency over the first week after
sectioning mammalian nerves. Migration results in the peripheral glioma, a
chain of Schwann cells down which new nerve fibres enter. More recent work on
peripheral (Singer & Steinberg, 1972) and optic (Turner & Singer, 1975a, b)
nerves of Triturus viridescens confirms that these events are not restricted to
mammals. This latter work added new information on the rejuvenescence of
Schwann cells, in that their cytoplasm becomes rich in ribosomes and the Golgi
apparatus is extensive, events very reminiscent of normal blastemal cell dedifferentiation. The importance of cell division in thisprocess has been demonstrated
since inhibition of Schwann cell proliferation prevents remyelination (Hall &
Gregson, 1974). In this state of rejuvenescence, Schwann cells are remarkably
similar to blastemal cells and upon amputation might proliferate further to
form a blastema. The other cellular elements of the nerve bundle, the connective
tissue sheath cells, do not seem to show any response to lesion (Nathaniel &
Pease, 1963«, b). Unirradiated fibroblasts would not therefore be present at the
amputation plane.
In series 3 a proportion of control limbs regenerated. Investigations into
contralateral nervous degeneration after unilateral denervation showed that
these anomalies could be explained ia exactly the same terms as the paradoxical
12
M. MADEN
regeneration of deliberately denervated limbs. Contralateral effects have previously been observed in axolotls (Tweedle, 1971), and changes in nerve number
(Dunn, 1909), diameter (Greenman, 1913), cell death (Nittono, 1923) or the
progress of myelination (Tamaki, 1933) have been reported in contralateral
limbs. It seems therefore that contralateral limbs may be inadequate controls
when the experimental procedure involves nerve damage. An alternative explanation of the control regenerates is to postulate that an ineffective irradiation dose
was delivered, owing to some experimental error.
The above argument implies that Schwann cells, of neural crest and hence
ectpdermal origin, can undergo metaplasia and form all the mesodermal tissues
of the limb, supplementing the recent demonstration of chondrocyte metaplasia
(Maden & Wallace, 1975). However, this suggested germ-layer transformation
should be viewed against the background of the remarkable range of other
differentiated cell types which neural crest cells give rise to during the normal
course of development: cartilage, melanophores, muscle, feather parts and
secretory cells (Le Lievre & Le Douarin, 1975).
I am deeply indebted to Dr H. Wallace for the intellectual guidance and encouragement
he gave me during the course of three years and for introducing me to the axolotl. My thanks
also to the S.R.C. for financial support.
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{Received 27 September 1976, revised 6 April 1977)
EMB 41