/. Embryo!, exp. Morph. Vol. 53, pp. 315-325, 1979
Printed in Great Britain © Company of Biologists Limited 1979
3] 5
Effects of X-rays on nerve-dependent (limb)
and nerve-independent (jaw) regeneration in the
adult newt, Notophthalmus viridescens
By RICHARD L. WERTZ 1 AND DONALD J. DONALDSON 2
From the Department of Anatomy, University of Tennessee
Center for the Health Sciences, Memphis
SUMMARY
The newt limb requires nerves for successful regeneration, but the jaw appears to be nerve
independent. Among the current hypotheses for the regeneration-inhibitory action of X-rays
is one proposing inactivation of nerves as the main cause. We decided to test this hypothesis
by comparing the irradiation levels necessary for inhibition of limb and jaw regeneration.
Jaws and left front limbs were exposed locally to doses of ionizing X-irradiation ranging
from 250 to 2000 R at least 6 weeks prior to amputation of the jaw and both front limbs.
After 90 days post-amputation all surviving animals were examined grossly for signs of
regeneration. In addition, some of the controls and most of those receiving 250, 500 and
1000 R were processed for histological examination.
All unirradiated limbs and jaws supported regeneration. Those exposed to 250 R also
regenerated, but a third of the jaws were hypomorphic. At 500 R and above, neither jaws nor
limbs regenerated. Since both systems were affected by similar doses of X-rays, it appears
that nerves are not the primary X-ray target in adult newts.
INTRODUCTION
Limb regeneration in adult Notophthalmus viridescens is inhibited by appropriate doses of X-irradiation (Rose, Quastler & Rose, 1955; Stinson, 1963,
1964; Rose & Rose, 1965, 1967; Carlson, 1970). However, after irradiation, the
morphology and function of unamputated urodele limbs remains essentially
unaltered (Brunst, 1950; Conn, Wessels & Wallace, 1971), although some
secondary necrotic wasting occurs (Brunst, 1950; Lazard, 1968; Rose & Rose,
1974ft).
While the effects of nonlethal levels of radiation are not well understood
(Okada, 1970), Maden & Wallace (1976) believe that in regenerating limbs the
inhibitory effects are due to direct nuclear damage to a variety of cell types.
On the other hand, some experimental evidence suggests that X-rays inhibit
1
Author's address: Division of Neurology, Department of Medicine, P.O. Box 2900,
Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
2
Author's address (for reprints): Department of Anatomy, University of Tennessee Center
for the Health Sciences, Memphis, Tennessee 38163, U.S.A.
316
RICHARD L. WERTZ AND DONALD J. DONALDSON
regeneration by interfering with normal nerve function. For example, in larvae,
both denervation and X-irradiation cause extensive regression of the stump
after amputation of the more distal parts (Butler & Schotte, 1941; Schotte &
Butler, 1941; Schotte, Butler & Hood, 1941). Trampusch and associates
(Trampusch, 1964) have shown that shielding of the nerve or nerve cell bodies
can restore regeneration under appropriate conditions, while Rose & Rose
(1967) demonstrated that nerve penetration into the epidermis is blocked in
irradiated limbs, but recurs when the ability to regenerate is restored by providing the limb with unirradiated epidermis. In further studies, inhibitory levels
of exposure failed to prevent regeneration in larval aneurogenic limbs (Rose &
Rose, 1914a). Yntema (1959) previously demonstrated that aneurogenic limbs,
produced by ablation of the presumptive nervous system at an early embryonic
stage, are fully capable of regeneration without nerves, thus providing one of
the few exceptions to the principle that nerves are required for regeneration.
The Roses' aneurogenic study seemed to indicate that nerves are the X-ray
target in larvae. However, a similar study by Wallace and Maden (1976)
produced different results. Instead of obtaining regeneration after irradiation,
they found complete inhibition in 100 per cent of their cases, concluding that
aneurogenic systems are just as sensitive to X-rays as are normally innervated
ones.
The present investigation compares the radiosensitivity of the adult newt
limb (which is nerve dependent) to the adult jaw, which is apparently not
dependent on nerves for its ability to regenerate (Finch, 1969).
MATERIALS AND METHODS
General animal care
Adult common aquatic newts (Notophthalmus (Triturus) viridescens) from
Connecticut Valley Biological were maintained in 10% operating solution
(OS; Rose & Rose, 1965), usually with Aquatonic (0-1 or 0-3 ml/1; Wardley)
added to inhibit fungal growth. At least one week prior to any experimental
procedure, the animals were placed on a 12 h light-dark cycle. They were fed
small strips of beef or calf liver and had the OS changed two to three times a
week.
Irradiation of the jaw and limb
Anesthetized animals (ethyl-m-aminobenzoate methanesulfonate in OS;
Eastman) were placed ventral side up, 15 or 20 at a time, in a circular plastic
container. The animals were positioned so that the jaw and left front limb,
including a portion of the shoulder girdle, were equidistant from the center.
The remainder of the body was covered with a 2 mm thick, serrated lead shield.
Additional protection of the head was provided by slipping a small 2 mm thick
piece of lead into the mouth.
Irradiation was achieved with a Westinghouse Medical X-ray Unit set at
X-rays inhibit jaw and limb regeneration
317
100 KVP, 5 MA, 15 cm STD, no filtration, and a half value layer (HVL) of
1 mm aluminum. (The HVL reference means that 1 mm of aluminum would
reduce the dose rate by 50%. It is a measure of the penetrating power of the
beam.) Two studies were done at different times of the year. The respective dose
rates were 187 and 182 R/min. The first group (Group I) had three subgroups
of 15 animals each receiving either 700, 1200 or 2000 R, with 14 animals serving
as unirradiated controls. In group II there were three subgroups of 20 each
receiving 250, 500 or 1000 R, with 20 controls.
Amputation of the limb and jaw
At doses above 500 R, a latent period was followed by varying degrees of
tissue necrosis and limb regression (Wertz, 1978). In contrast, the jaws showed
no signs of tissue degeneration. Amputations were delayed until 6 weeks
following irradiation so that all damaged limbs would have time to heal. The
animals were then anesthetized and the distal half of the jaw was removed
according to the method of Goss & Stagg, 1958). Simultaneously, the left and
right front limbs were amputated 3 mm distal to the elbow, a level usually
proximal to any visible X-ray damage.
Gross observations were made periodically from both Groups I and II. Most
of the surviving members of the second group had their jaws and limbs prepared
for histological observation. These tissues were fixed in alcoholic Bouin's, decalcified with Decal (Scientific Products), dehydrated and embedded in paraffin.
Sections, 10 /tm thick were cut longitudinally in the widest plane and stained
with Masson's Trichrome (Masson, 1929).
RESULTS
Determination of the threshold dose for inhibition of limb and jaw regeneration
The following data represent observations from both experiments. Only the
animals that survived at least 90 days after amputation were used to compile
the data. Mortality was very high and the extent and possible reasons for this
are described elsewhere (Wertz, 1978).
After 90 days, all of the unirradiated limbs were in the late digit stage of
regeneration (staging of Iten & Bryant, 1973). In contrast, of the eight control
jaws, only five regenerated completely (Fig. 1). The remaining three animals
displayed regenerative activity, but were clearly behind the best regenerates.
All six of the surviving animals exposed to 250 R displayed limb regenerates
indistinguishable from controls. However, only four of the six jaws demonstrated signs of regeneration grossly, with all of the regenerates clearly less
advanced than the most advanced controls, but falling in the normal range. In
contrast to 250 R, 500 R completely inhibited limb regeneration. This dose
also affected jaws severely, as five of six appeared completely inhibited. The
remaining jaw displayed advanced regeneration and resembled the more
21
EMB 35
318
A
RICHARD L. WERTZ AND DONALD J. DONALDSON
'
^
B
Fig. 1. (A) This is an example of the best control jaw regenerate 90 days postamputation. Others in the control and irradiated groups had jaws with varying
amounts of new tissue. (B) An example of an irradiated jaw (500 R) showing no
regeneration.
Table 1. Effects of X-rays on limb and jaw regeneration.
Gross observations
Number regenerating
Group
(R)
Controls
250
500
700
1000
1200
2000
Number of cases
Limbs
Jaws
8
6
6
3
3
4
1
8 (100%)
6(100%)
0
0
0
0
0
8(100%)
4(66%)
1 (16%)
0
0
0
0
advanced controls. After exposures in excess of 500 R, there was no gross
evidence of either limb or jaw regeneration. These results are summarized in
Table 1. It is evident that both jaw and limb regeneration are sensitive to low
doses of X-rays.'From the gross observations the limb appears to be slightly
more resistant than the jaw. A most striking result is the response at and above
500 R. Only one jaw and none of the limbs exposed to these levels of
irradiation showed visible signs of regeneration, while most of the jaws and all
of the limbs that received 250 R regenerated.
X-rays inhibit jaw and limb regeneration
319
Histology of irradiated jaw and limbs ninety days after amputation
The jaw response was divided into three categories based primarily on the
amount of new cartilage that had formed. Since there was a direct relationship
between the amount of new cartilage and size of the entire regenerate this
seemed to be a useful approach. A + + response was scored if there was a
massive amount of new mandibular cartilage with the two sides fusing in^the
midline (Fig. 2 A). A + was given for a smaller amount of cartilage which did
not reach the midline (Fig. 3A). In the poorest response ( ± ) , little or no
cartilage had formed (Fig. 4 A). Table 2 shows that all the sectioned controls were
+ + regenerates while at 250 R there was a tendency for a reduced regenerative
response. At 500 R there was a definite X-ray effect with almost all falling in
the ± category.
In a 4- + regenerate (Fig. 2), the non-cartilage cells consisted mostly of
young fibroblasts in a connective tissue containing fine collagen fibres (Fig. 2B).
There were also areas where there seemed to be fewer collagen fibres and the
cells looked more undifferentiated, similar to those in Fig. 3B. In + regenerates
(Fig. 3), there was less cartilage and therefore more non-cartilage cells. Once
again, there appeared to be two populations of non-cartilage cells, young
fibroblasts like those in Fig. 2B, and less differentiated cells (Fig. 3B). While
it is not possible to predict the fate of a regenerate like the one in Fig. 3, the
heavy connective tissue that is beginning to form (arrow, Fig. 3 A) suggests
that regeneration would not have continued, even though some of the cells
were still dividing (arrow, Fig. 3B).
The poorest response ( ± ) showed less cartilage than the + regenerates but had
the same two populations of non-cartilage cells (Fig. 4). The differentiation of
loose connective tissue around the cartilage (Fig. 4B) suggests that no further
cartilage would have formed. The dense connective tissue in the deeper central
tissues (arrow, Fig. 4 A) also suggests that the undifferentiated cells in the more
superficial central region should not be interpreted as a continuation of regeneration.
While the inhibited limbs formed little or no new cartilage, the other tissues
near the amputation surface had the same general appearance as the inhibited
jaws. Fig. 5 includes a region of dense connective tissue in the distal stump of an
inhibited (500 R) limb. The central region of the stump contained less connective
tissue and a small accumulation of relatively undifferentiated cells (Fig. 5B).
By 45 days all controls had 4-digit regenerates. The inhibited limbs, however,
showed little evidence of regeneration, even after 45 additional days. Despite
an occasional mitotic figure (arrow, Fig. 5B) the limb in Fig. 5 will probably
never regenerate.
Another inhibited limb is shown in Fig. 6. This limb looks even less likely
to regenerate than the one in Fig. 5. The apical epidermis is no longer thickened
and the cells in the distal stump all seem to be embedded in dense connective tissue
320
2A
RICHARD L. WERTZ AND DONALD J. DONALDSON
X-rays inhibit jaw and limb regeneration
321
Table 2. Effects of X-rays on jaw regeneration. Histological observations
Extent of regeneration f
Group
(R)
Control
250
500
1000
Number
of cases*
±
+
+ -f
4
5
6
2
0
1
5
2
0
1
1
0
4
3
0
0
* Some of the animals in Table 1 were not available for histological analysis. Of the-four
controls represented, three were judged grossly as advanced jaw regenerates and one was
considered somewhat delayed. The individual missing from the 250 R group was one grossly
having a normal jaw regenerate.
t + + , Much cartilage fused in midline. + , Significant amount of new cartilage yet
falling short of the midline. ±, Little or no new cartilage.
With one exception, the rest of the inhibited limbs resembled the two examples
just presented. The exception is shown in Fig. 7. This limb appeared to have a
healthy blastema. The apical epithelium is thickened, and there is a sparse
accumulation of apparently undifferentiated cells under it. A blastema like
this ordinarily occurs around 15-20 days after amputation. Since this one was
still present at 90 days, the limb is probably not going to regenerate. It is interesting to note that this best example of an attempt at limb regeneration after
500 R of X-irradiation was from the animal also showing the best attempt at
jaw regeneration.
DISCUSSION
In 1964, Trampusch described an experiment by his student Vergroesen in
which irradiated limbs regenerated if the nerves were dissected out, shielded
from the beam and then replaced. Since unirradiated pieces of nerve severed
from the perikarya did not promote regeneration in irradiated limbs, Trampusch
concluded that regeneration in the first experiment was not due to
F I G U R E S 2-4
Fig. 2. (A) A control jaw showing a + + regenerate. (B) A greater magnification of
the fine connective tissue that has formed distal to the cartilage.
Fig. 3. (A) A + regenerate on a jaw irradiated with 250 R. There is less cartilage here
than in the + + regenerate in Fig. 2. Some dense collagen fibres (arrow) have formed
in the central part of this regenerate. Most of the non-cartilage cells are young
fibroblasts in a fine connective tissue like that shown in Fig. 2B. (B) Distal to the
arrow in Fig. 3 A there were some less differentiated cells, one in mitosis (arrow).
Fig. 4. (A) Shows a ± regenerate on a jaw irradiated with 500 R. Very little cartilage has formed, and while there were a few undifferentiated cells like those in
Fig. 3B, most of the 'regenerate' was composed of the usual fine connective
tissue as well as regions containing coarse collagen fibres (arrow). (B) Shows that a
fine connective tissue has differentiated peripheral to the new cartilage on the right.
322
5A
RICHARD L. WERTZ AND DONALD J. DONALDSON
X-rays inhibit jaw and limb regeneration
323
unirradiated cells adherent to the nerves but occurred because X-rays inhibit
regeneration by damaging a radiosensitive neural agent. More recent experiments have challenged this analysis (Wallace, 1972). For example, nerves can
regenerate even when irradiated. They therefore appear to be radioresistant.
Furthermore, Wallace found that unirradiated pieces of nerve, will in fact,
promote regeneration in irradiated limbs (contrary to the results of Trampusch).
In Wallace's experiments, nerve implants were made from black donors to
white hosts and vice versa. The regenerates were consistently the colour of the
donor, suggesting strongly that they came primarily from unirradiated Schwann
cells or fibroblasts adhering to the implant. There is therefore no compelling
reason to accept Trampusch's evidence as proof that X-rays inactivate nerves.
The demonstration by Rose & Rose (1974a) that aneurogenic limbs are
relatively radioresistant at first appeared to be a strong indication that X-rays
interfere with nerve function. However, based on aneurogenic experiments with
a slightly different protocol, Wallace & Maden (1976) have taken issue with
the Rose's conclusion. The investigators in this later study used a different
species to produce aneurogenic limbs, waited until the limbs had reached the
3- or 4-digit stage, and then irradiated with a smaller dose of X-rays (2000 R),
exposing the entire limb and shoulder. Under these conditions there were no
signs of regeneration. Wallace & Maden have suggested that the aneurogenic
limbs in the Rose's study may not have been irradiated far enough proximally
to prevent unirradiated cells from producing a regenerate. In any case, radioresistance does not appear to be a general feature of nerve-independent systems.
We chose to investigate this question by comparing the effects of X-rays on
limb and jaw regeneration. Since Notophthalmus']?ws will regenerate even though
they normally have less than half the threshold number of nerve fibres needed for
limb regeneration, and reduction of the number normally present by 85-99 %
during the time of blastema formation has no effect on regeneration, it appears
that this process is nerve independent (Finch, 1969). The selective X-ray
inactivation of the nervous system proposed by Trampusch (1964) and Rose &
Rose (1967, 1974a) predicts that the jaw would be more radioresistant than the
limb. On the contrary, we found that jaws and limbs are inhibited by similar
FIGURES
5-7
Fig. 5. An inhibited limb given 500 R of X-irradiation. (A) While the controls and
250 R limbs all produced 4-digit regenerates, this limb has not regenerated. (B)
Shows the coarse collagen (c) that has formed in this limb as well as a small pocket
of cells that appear less differentiated than the cells in Fig. 6B. Note the mitotic
figure (arrow).
Fig. 6. (A) An inhibited limb given 500 R of X-irradiation. (B) The distal part of the
stump has formed coarse and fine connective tissue.
Fig. 7. (A) The best attempt at regeneration on a limb given 500 R. There appears
to be a relatively normal blastema with perhaps fewer cells than normal. (B) Shows
a higher magnification of the 'blastema' cells. Note the mitotic figure (arrow).
324
RICHARD L. WERTZ AND DONALD J. DONALDSON
doses of X-rays. In fact, the data suggest that jaws may actually be slightly more
sensitive than limbs. These results and the aneurogenic experiments of Wallace
& Maden show that regeneration-inhibiting doses of X-rays probably affect
non-nerve tissues even in nerve-dependent systems. In fact, morphological
evidence of radiation damage in larval urodeles has been observed in nuclei of
cartilage, perichondrium, epidermal and other cells (Maden & Wallace, 1976).
In addition, mitotic and DNA synthetic studies in irradiated (Wertz, Donaldson & Mason, 1976) and denervated (Mescher & Tassava, 1975) adult limbs
indicate that separate mechanisms of action are responsible for these two types
of inhibition. When the adult limb is denervated at the time of amputation the
potential blastema cells synthesize normal levels of DNA but do not divide,
indicating a specific G2 block. In contrast, in the irradiated limb both DNA
synthesis and mitosis are affected. These results in conjunction with the observations previously discussed demonstrate that regeneration failure following
X-irradiation is not due primarily to an effect on neural structures.
The technical assistance provided by Dr Hassain Omar in all aspects of radiation treatment and theory is appreciated. The preparations of histological specimens by Ms Mary Jo
Winbigler and the manuscript by Ms Bequi Bradford are also acknowledged. Portions of
this paper appeared in a dissertation by Richard L. Wertz presented to the Faculty Council
for partial fulfillment of the requirements for the degree of Doctor of Philosophy, and a
preliminary report was presented to the American Association of Anatomists (Anat. Rec.
187, 746-747, 1977). Funds and support to R.L.W. were provided by U.S.P.H.S. Grant
GM 00202.
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(Received 20 March 1979, revised 15 May 1979)