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 limb-stump can form the exclusive source of a mesenchymal blastema and the various internal tissues of the regenerate.

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, 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 co-ordination and ability to regenerate (Schotté & 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 inter-pretation, 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 para-doxical regeneration and provide two independent lines of evidence implicating the participation of migratory Schwann cells in such regeneration.

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 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 al. 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.

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.

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.

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.

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.

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. 1A). 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 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.

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.

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.

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.

Table 1

Numerical summary of results, scored 24 weeks after amputation

Numerical summary of results, scored 24 weeks after amputation
Numerical summary of results, scored 24 weeks after amputation

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 re-innervated within 3 weeks, but amputation at that time did not provoke regeneration. 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.

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.

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.

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 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 inter-pretation. 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 the 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. 1D). 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.

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.

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.

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 & Harrebomée, 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.

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 et al. 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 & Inoué (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 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 (Schotté & 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, 1946 b). That would explain why paradoxical regeneration did not occur as consistently in these experiments as it did with younger A. opacum (Conn et al. 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 re-growing 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 motor innervation. (Singer, 1946 a); 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 ‘neuro-trophic theory (Singer, 1965) has recently found some experimental support (Thornton & Thornton, 1970).

The experiments described here confirm a previous report (Conn et al. 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, demon-strating 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 Schotté & 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 (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 al. 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,cannot restore regenerative ability to irradiated cells. It follows that other non-irradiated cells probably cannot do so either, and hence must proliferate to provide all the internal tissues of the regenerate.

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.

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.

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 the melanophores 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 fibro-blasts, 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 that specific cell-types 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 tissue-specificity (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 Guyénot & Schotté (1926, p. 35) observed: ‘Il parait même y avoir un rapport génétique entre les cellules de la gaine des nerfs et les éléments cellulaires du blastème. En effet, les noyaux de la gaine présentent, vers l’extrémité des nerfs, des mitoses anormalement nombreuses et l’on observe tous les passages entre les noyaux allongés des gaines nerveuses et les noyaux de plus en plus gros des cellules conjonctives. II ne serait donc pas impossible que les gaines nerveuses participassent, pour une part importante, à la formation du blastème et que ce mécanisme jouât un röle important dans la genèse des pattes supplémentaires. ‘

Allen
,
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