Genitourinary infections in the patient at risk: An overview

PHASES I N REGENERATION O F THE URODELE LIMB AND THEIR DEPENDENCE UPON T H E NERVOUS SYSTEM OSCAR E. SCHOTTg AND ELMER G . BUTLER Amherst College, Massachusdts mid Princeton Uniueraity, N e u Jersey SEXBNTEEN FIGURES

INTRODUCTION

The present investigation represents a continuation of the authors' earlier studies (Schottd and Butler, '41; Butler and Schottd, '41) on the effects of denervation on regeneration of limbs of urodele larvae. These correlated experimental and histological studies have revealed some hitherto unrecognized features of regenerative activity in the urodele limb. The primary purpose of the present investigation has been to study the dependence of various phases of regeneration upon the nervous system. This purpose has been accomplished by means of experiments in which denervation of limbs was made a t various times after the onset of regeneration. As a convenient approach to a consideration of the problems dealt with here, we shall regard the regeneration of a urodele limb as consisting essentially of three successive phases. After completion of wound healing, there is first a phase of dedifferentiation, which consists primarily in the preparation of the materials of the regenerate at the expense of the old tissues of the limb at,the level of amputation. This process appears to be essentially local in nature and it culminates in the formation of the regeneration blastema. The early blastema is formed from dedifferentiated tissues which represent the totality of the structural components of the limb. It is, however, no more a limb rudiment than the animal pole of a urodele blastula is a medullary plate+before the morphogenetic movements of gastrulation s\et in. The second phase of regeneration is concerned with the transformation of the blastema into a limb rudiment, in which the morphogenetic patterns of the missing parts of the limb are laid down. Little is known about the mechanism underlying this transformation of the blastema into an embryonic organ, but by analogy with what is known from experimental embryology it seems proper to regard it as a phase of in95 TED J O W N A L OI H P a H I N T A L ZOOLWY, VOL. 97. NO. 2 NOVlXBEE, 1944

96

OSCAR E. S C H O T T ~AND ELMER G. BUTLER

duction. In contrast, however, to what occurs in the embryo, which is a self-differentiating morphogenetic system, the “young regenerate is a dependently differentiating system. . . in which, due to the convergent action of many still unknown factors, the form and eventually the type of organ is determined by the nature of the morphogenetic activities inherent in the organ remnant.” (Schott6, ’40, p. 72). The third phase consists essentially of an integrated succession of developmental stages in which proliferation of cells and their transformation into the definitive structures of the limb takes place. This is the period during which growth and visible differentiation lead to the restitution of the parts lost by amputation. We will regard it as the phase of morphogenesis, with no implication that this term provides any explanation of the underlying processes involved. Obviously this last mentioned phase consists of a multiplicity of phases telescoped together. Its complexity can be appreciated when one considers that this phase leads to the physiological integration (functional and correlative adaptation, Weiss, ’35) through which the regenerated organ is incorporated into the general economy of the organism. The two initial phases, those of dedifferentiation and induction, represent regenerative activity in the precise sense of the term, since they include, first, the loss of structural characteristics of the tissues at the amputation surface and, secondly, a retransformation of cells into a limb rudiment. The third phase, however, during which a regenerate grows and differentiates into the visible structures of a limb, must be considered in a different light. Here we deal with morphogenetic activity not specific to regeneration, but related to the more general problem of morphogenesis, with which we are concerned in all developmental processes. It must not be forgotten that all these activities take place at the amputated end of an organ in which, to all intents and purposes, development has already terminated. Moreover, these activities can occur in proper sequence only with the participation of the organism as a whole and are dependent, therefore, on physiological conditions prevailing within the animal. Among the problems which deal with the physiolo,T of regeneration the question of the influence of the nervous system has been a subject of investigation f o r over a hundred years, indeed, since Todd performed his well-conducted experiments of 1823. In view of what we now know regarding the origin of regenerates, their dependence on the organ remnant for differentiation and especially the developmental capacities of regenerates of later stages, it is clear to us, that to ask simply whether or not the nervous system exerts an “influence on regenera-

REGENERATION PHASES AND NERVOUS

SYSTEM

97

tion” is to miss the point. For every thoughtful student of regeneration, it became clear years ago that the problem of the influence of the nervous system on regeneration contained at least two aspects. There is, first, the problem of determining whether the nervous system is necessary for setting into motion the processes which initiate regeneration ; there is another and separate problem, to determine whether nerves are essential for the continuation of regenerative processes already in progress. So far as the importance of the nervous system for setting into mo.tion regenerative activity is concerned, there is overwhelming evidence from the work of numerous investigators. We shall not cite these investigators here, since they have been referred to in our previous publications. For the second part of the problem, namely, whether the nervous system is necessary for ‘the continuation of regenerative processes, evidence is much less unanimous and the conclusions of authors vary. The lack of unanimity is not so much the result of divergent experimental evidence, but rather is due to the fact that various authors have not worked on identical stages. Schott6 ( ’23 and ’26) conducted a systematic investigation in which the effects of denervations on various stages of regeneration in adult newts were recorded. Results of these experiments can be summarized as follows : ( a ) if a limb possessing a young blastema is denervated, then no further regeneration occurs and, in most cases, the blastema disappears;-(b) if a limb possessing an advanced blastema is denervated, the blastema ceases to grow and no further regeneration occurs; (c) if denervation is performed on stages of fairly advanced regeneration, then only slight effects, such as growth inhibition, can be observed and in general further development and growth of the regenerate proceeds. Schneider (’40)has reopened the question of the dependence of regeneration on the nervous system with the following sentence.’ “The present investigation has f o r its purpose to clarify the role of the nervous system in the process of regeneration since, in spite of a whole series of papers concerned with this problem, which have appeared during the last 40 years, it has not been established so far whether or not the nervous system exerts any influence whatsoever on the process of regeneration.” From an examination of Schneider ’s work it is clear that he has made no new contribution to a solution of the problem. His methods are no improvement over the ones which Todd introduced ‘This quotation has been translated by 0. E. Sehotte from the Russian text (p. 388) of Sehneider ’s paper.

98

OSCAR E.

SCHOTTB

A N D ELMER G. BUTLER

over a century ago. Schneider’s co~~clusion, that the influence of the nervous system on regeneration can neither be affirmed nor denied has no basis in fact2 It was well known before Schneider began his investigation that the influence of nerves varies in respect to the stage of regeneration. This is a point by which he has apparently been confused. There can be no question that nerves are important in regeneration. and the discovery that denervated amputated larval limbs not only fail to regenerate but also undergo regression puts a new emphasis on their role in this process. Our study of the “regression effect’’ together mith the information now available regarding the various phases of regeneration has brought us one step further and leads to the formulation of several specific questions. Is the presence of nerves necessary for the dedifferentiative phase which inaugurates normal regeneration? Are‘ nerves necessary for the formation of the blastema? Does the induction phase, during which the blastema is transformed into a limb rudiment, .require the presence of nerves? Are nerves necessary for morphogenetic activities, which result in the development of the limb rudiment into a visibly differentiated limb? The answer to the first question regarding the role which nerves play in the dedifferentiative phase of regeneration can be made on the basis of our previous work. I n the absence of nerves dedifferentiation begins soon after amputation and wound healing, and it continues unchecked, as long as a limb remains nerveless. For the other questions, we have at the present time only partial answers, and they raise problems which require careful reinvestigation. This study, therefore, is centered around these problems, particularly an investigation of the influence of nerves on the formation of the blastema and on the morphogenesis in more advanced phases of regeneration. MATERIALS AND METHODS

Our experiments were performed on larvae of both Amblystoma punctatum and A. opacum of from 22 to 42 mm. in length. The Amblystoma punctatum were secured from ponds in the vicinity of Amherst; the Amblystoma opacum were collected from Long Island ‘Schneider quotes two books (Korschelt, ’27 and Abeloos, ’32) and a series of papers including those of Weiss ( ’25) and Locatelli (’24), but fail8 to refer to the work of SchotM (’23 and ’ 2 6 ) , which deals specifically with the problems which he was investigating, even though SehottB’s papers are included in the bibliography of every one of the above authors. We wish to express our indebtedness to Dr. John A. Moore, who has kindly secured for us Amblystome opaeum larvae at a time of the year when no other urodele inaterial was available.

REGENERATION PHASES AND NERVOUS SYSTEM

99

The conditions of the experiments made it necessary to obtain early regeneration stages, and also blastemata and regenerates, the age of which was exactly known. Furthermore, it was essential that limbs be maintained nerveless f o r the duration of the experiment. The first prerequisite was realized by keeping all larvae after amputation and throughout the experiment under constant temperature conditions ; the second condition was fulfilled by means of repeated resections of the three spinal nerves (111, IV, V) which provide the innervation for the fore limb. Constant temperature environment of the operated larvae was secured by keeping the individual dishes containing the larvae in BridgesPlunkett Incubators (described in Drosophila Information Service, April, 1936). These incubators were situated in a general constant temperature room, thus enabling us a control of temperature of 2 0.1"C. The majority of our larvae were kept throughout the experiment a t 20°C. When it was discovered, however, that a critical stage in the development of the blastema occurred at from 7 to 9 days, it became advisable further to examine the temperature relationships. For these critical stages exclusively, in addition to the 20" series, several other series were maintained at 21.5"C. It was found that, for the 7-day blastema, in particular, the use of the two temperatures was essential. Bilateral amputations were performed a t one level only, namely through the distal part of the upper arm, slightly proximal to the condyles of the humerus. In every case the bilateral amputation preceded the resection of the brachial plexus by from 2 to 16 days. The number of days by which amputation preceded denervation has been designated by roman numerals in the serial number of each larva. For example, series IV means that amputation was performed 4 days previous to the operation of denervation, series XVI means that the regenerate was 16 days old at the time of denervation. The resection of the brachial plexus and the permanence of denervation were obtained by the methods which have been described in detail in our first paper (Schott6 and Butler, '41). As in the previous investigation, denervation was performed on one limb only of the larva, the other limb serving as control. Permanence of denervation was particularly important in the present investigation and, for that reason, we were careful not to delay the second operation of denervation for more than 7 or 8 days after the first resection of the plexus. The third resection of the plexus was generally performed on either the sixteenth or seventeenth day after the first nerve operation. In general, it was found unnecessary to perform more than three nerve resections. For

100

OSCAR E. SCHOTTI? A K D ELMER

o.

BUTLER

that matter, a fourth operation is often not thoroughly successful, because of the nmze of scar tissues and the profuse bleeding which then occurs in the sub-scapular region. However, three nerve operations have proved to be sufficient in order to achieve our girposes, since, in a few cases only has reinnervation, at once detectable by the beginning of new regeneration, occurred. At the time of the first nerve resection both limbs, whatever their stage of regeneration, were drawn by means of a camera lucida (magnification X 37.5). These drawings were regularly repeated at intervals of 3 or 4 days, in order to secure coiiiplete records of the changes which occurred in the left denervated limb cornpared to the progress- of regeneration of the right control limb. In addition photographic records were made of over forty cases. EXPERIMENTAL

The 252 larvae used in this investigation can be divided into four groups. I n the first belong those cases which had the left limb denervated from 2 to 5 days after amputation, a time at which no real blastema is noticeable. The effects of denervation 011 limbs allowed to regenerate 6 and 7 days before nerve resection, and which exhibited at the time of denervation an unmistakable blastema, represent the second group of cases. A third group of larvae, denervated from 7 to 9 days after the onset of regeneration, includes cases in which the blasteiiia at the time of clenervation was sizable, but in which no signs of differentiation could be detected. The effects of denervation on blastemata of from 10 to 16 days, possessing visible signs of morphological differentiation, are included in the fourth group.

I . E f e c t s of denervation on pre-blastema stages of regeneration I n forty-four Amblystoma larvae ranging from 22 to 38 mm. in length, the limbs were allowed t o regenerate from 2 to 5 days previous to denervation of the left limb. Dedifferentiation processes can be observed to occur at the healed over amputation surface and only in the 5-day group is a conical cellular hillock generally present. Arbitrary as such an assertion might seeni, this precocious accumulation of materials cannot be considered a true blastema. In all these cases the wound epithelium is drawn tightly over the cut end of the humerus and, due to transparency of tissues, we have been able to observe many signs of dedifferentiation still in progress. For animals kept at 20°C. and

101

REGENERATION PHASES AND NERVOUS SYSTEM

at 21.5”C., one more day is required for the formation of a blastenm, which has the size, color and forin so characteristic for that decisive stage of regeneration. The results of denervation in this group are presented in table 1. It can be seen that in all these cases no regeneration whatsoever occurred for the duration of the experiment. I n addition to the absence of regeneration, all cases, which were kept for more than 4 days after deaervation, showed regression. The “accumulation blastem,” noticeTABLE 1

Statua of larvae kept at 20’C. ond amputated from C to 6 days precious t o denervation. The roman numeral of each series indicates the number of days the l i d was allowed t o regenerate before resection of the brachial plexus. ~~~~

ztz

SLRIES

I1 111

IV

i

~~

~

~

BEUENERATION DAYS A m E R DENERVATfON

Previous to denemstion

At end of experiment

BBO&X8810N

absent absent absent absent

absent absent absent absent

not noticeable noticeable pronounced pronounced

6 11 16

4

abeent

absent

noticeable

5

2

absent absent absent absent absent absent

absent absent absent abeent absent absent

not noticeable noticeable noticeable noticeable noticeable pronounced

3 5 7 8 9 14

beginnings of blastema

absent absent abeent

noticeable pronounced pronounced

6 9

2 1

1; 1

4

12

able in the larvae of Series V on the day of denervation, receded and disappeared, and eventually the amputation surface acquired the typical pigmented yellowish tegumental coverings, so characteristic of the denervated non-regenerating regressing amputated limb. All the forty-four cases of this group, therefore, show a behavior very similar to the one exhibited by denervated and simultaneously amputated larval limbs. I n other words, it makes no difference whether a limb be allowed to regenerate for from 2 to 5 days previous to denervation, or whether amputation is effected simultaneously with denervation. Under both conditions denervation is followed by cessation of regeneration, with subsequent onset of the regression effect.

102

OSCAR E. S C H O T T ~AND ELMER G . BUTLER

I I . Effects of den.ervation on early blastema stages To this group belong sixty-one cases in which denervation of the left limb was performed 6 or 7 days after bilateral amputation. Twenty-one larvae were kept at 20°C. and seventeen larvae at 21.5”C. in the series VI; eighteen larvae of series VII were kept at 20°C. for the duration of the e ~ p e r i m e n t . ~

L.

”,

Fig. 1 Amblystoma opacurn larva, VIlm. Camera lucida drawings of right ( R ) and left (L) fore limbs showing arrest and ultimate disappearance of the denervated left blastema a n b t h e development of the normal right blastema over a period of 33 days.

The effects of denervation on a well-developed, though only 6-day old, blastema can best be described through the use of an example (case VI 183, Amblystoma opacum larva, 35 mm., kept at 20.C.). The series of drawings presented on figure 1, which cover a period of 33 days after denervation (39 days after amputation), illustrate the processes occurring in the denervated limb, while in the right control limb the drawings show the progress of regeneration f o r the same period of time. The first drawing ( l a ) , made at the time of the first resection of the brachial Included in this group are only those cases which a t the time of nerve resection exhibited a well-formed blastema. The infrequent cases i n which, for various reasons (injury, protruding humeri, etc.), limbs did not show a well-defined blastema 6 or 7 days after amputation, were simply allowed to regenerate for several more days, thus becoming material f a r the study of the effect of denervation on older stages.

ItEGENERATION PHASES A N D NERVOUS SYSTEM

103

plexus, s i i o w that the left denervated liiiib exhibits a sizable blastema and, in addition, the stump of the upper ariii is nearly cylindrical. F o u r d a p later ( l c ) it can be observed that tlie denervated limb has thickened slightly and the general form of the stump is no longer cylindrical, but exhibits curved lines. The blastema of the denervated limb has ceased to grow and has clearly contracted. In addition, it caii be seen under the dissecting microscope that the surface of the blastema has acquired considerable pigmentation, probably due to some keratiiiizatioii of its epitheliuni. Seven days after deuervatiou, a t a time when the regenerate of the right limb ( I f ) exhibits s i g m of digit formation, the blastema. 011 the denervated side ( l e ) has become further reduced, while the whole of' the limb stump has regressed considerably. F o u r days later the n e s t drawing sliows, not only rapid contiriuatioii of regression, but also a progressive increase in the width of tlie denervated limb (la), while the blastema itself has beconie eniptied of most of its cellular materials. ('lose examiliation under the microscope indicates that it now consists mostly of keratinized epitheliuiii. Finally, 14 days after denervatioii DO trace of a hlasteina caii he detected 011 the left nerveless side ( l i ) . The obserratioii of the left limb for tlie nest 17 days shows the now familiar picture of the regression effect, which eventually leads to the disappearance of tlie u-hole tipper a m , a s can he seen in the last drawing, (lk),made 33 claps after denervatioii. At that time the right control limb ( l p ) ran be coilsidered as fully regenerated. The effects of denervation 011 a 7-day-old blasteina are shown in case PII,,,,,a n Amblystoma opacum larva, 3 i miii. kept a t 20°C. (figs. 2 4 ) . The well-developed blastema of the left denervated limb, visible in figure 3:C a l i be seen to disappear almost conqdetely within the first 'idays of ncrrelessness (fig. 3 ) . Siiiiultaneously with tlie shrinkage of tlie blastenia, regression of this limb was noticeable a s early a s 3 days a f t e r denervation, at a time when the blastema was still fully developed. After clisappearance of the blastema, regression is very rapid and 19 days after clenervation the whole length of the upper arni, together with i t s 7-clap-old hlasteiina has become subject to regression. During the same period, the riglit control limb has virtually completed its normal regeneration (fig. 4). Among the sixty-one cases composing this group, there were five in wliich, after a n early lapse in the growth of the blasteiiia on the deiierrated limb, regeneration proceeded normally. All these were cases in which the nerves were either incoiripletely removed, or in which early reinnerration had occurred. They will be reconsidered in a later section. The behavior of the remaining fifty-sis cases of this group was so sim-

104

OSCAR E. S C H O T T ~ A N D ELMER G. BUTLEI:

Fi;.. '7 AnilJlystoiiin opaeum, VII,,,, 011 the day of denervation of left fore limb, 7 days after ainlnitatioii of both right and left limbs. Fig. 3 Sanir liirva, 7 days after deiiervation, 14 days after amput:ition. Fig. 4 Same I:irva. 1 9 days a f t e r deiiervation, '7G days after amputation.

105

REGESEHATION PHASES A X D XECVOUS SYSTEM

ilai, to the two above described ones that the results of these experiments have been summarized in the form of table 2. Our results show that in every case, cveii when the experimental animal was allowed to live as little a s 6 or 7 days after denervatiori before being fixed, the blastemata on denervated linibs have always ceased to gro-w and have become greatly reduced in size. I n the cases in which the duration of the experiment exceeded 10 days, the previously welldeveloped blasteinata shrunk to insignificant epithelial flaps and, in most cases, disappeared altogether. Moreover, the regression effect, as proved TABLE

2

Effects of perttianent tlenervaiion on limbs w i t h blastemata aged 6 ( k e p t at SO" a d S l . 5 " C . ) a i d 7 ( k e p t at 20°C.) (Zap. RF&ENER.ITION

NO. O F

SERIES

CASES

DAYS AFTER DENERVATION

Previous to denervation

At end of experiment

REaRESS1oN

Blastema Blastema Blastema Blastema Blastema

decreased absent absent decreased o r disappeared

noticeable pronounced pronounced pronounced pronounced

12 13

Blastema Rlastema Bla sterna Blastema Blastema

decreaeed or disappeared disappeared disappeared

noticeable pronounced pronounced pronounced pronounced

7 11 14 18 20

Blastema Blastema Blastema Blastema Blastema

decreased considerable derrease or disappearance

noticeable pronounced pronounced pronounced pronounced

7 13 14 19 22

56

13 decrease 43 blastema absent

G 8 9

~~

Total

36

56 regression

by conspicuous shortenings of the denervated limb, was observable in every one of the cases of this series and its extent depended, not on the smaller o r greater size of the blastema, but solely on the duration of the experiment. The results of these experiments show coiiclusively that blastematn aged 6 (kept a t 20" and 21.5"C.) and 7 days (kept at 20°C.) cannot grow and develop in the absence of innervation ; these experiments show, also, that well-developed blastemata of these ages can neither check the oiiset nor the continuation of regression.

106

OSCAR E. S C H O T T ~A E D ELMER G . BUTLER

I I I . Efl(2ct.s of dmeruatiot! o i l blnsteinata at the “critical” stage This series coniprises fifty-nine cases which were allowed to regeiierate, previous to denervation, for 7 days at 21.5”C. a i d for 8 or 9 days at 20°C‘. In all these cases the blastenia was fully developed, but not noticeably larger than the one described in the previous section. Iii several larvae of this group the blasteniata reactecl t o denervatioii in a niaiiiier similar, if not identical, to that illustrated by the above

Fig. 5 Amblystonia opacum, VII,. Camera lucida drawings of right ( R ) and left ( L ) fore limbs. Left limb denervated, right limb possessing normal innervation.

described case VI1210(figs. 2 4 ) . Other larvae behave differently from the above and also differ amoiig themselves. They follow roughly three types, which will be illustrated by the description of representative cases. A first type is represented by case VII& (Amblystoma opacum larva, 34 mm., 7-day-old blastema, kept at 21.5”, fig. 5). A comparison of the drawing of the left blastema of this case at the time of denervation (5a) with the blastema of the previously described case VI,,, ( l a ) shows that there is no essential difference in the general features of the two bla-

REGENERATION PHASES Ah'D

NERVOUS SYSTEM

107

stemata. Yet, a s shown by the drawiiig 5c, there is evident, 3 days after denervation, no appreciable shrinkage of the blastema in this case. On the contrary, 6 days after denervation, the blastema (5e) has clearly increased in size, although reniaining considerably smaller than the regenerate on the right control limb (5f). This undeniable increase in sizc of the denervated blastema does not, however, lead to any further development, since drawing 5g, made 4 days later, shows that it has remained stationary. Twenty-one days after denervation, at a time when the normal right limb has foriiied a iiiorphologically fully developed regenerate ( 5 j ) , the blastema of the denervated limb is smaller than it was at the beginning of the experiment. illoreover, the deiiervated blastema has acquired all the characteristic features of a heavily pigmented keratinized blastenia of the type illustrated 011 figures l c and l e for case VIIB3.Other cases of this type, kept for over a month, show that such a hlastema is incapable of further development and can remain in a state of "inactivation" f o r as long a s the status of nervelessness persists. I n spite, however, of the continuous presence of a blastema for the duration of the experiment, the denervated stump had regressed without interruption to nearly half of its original length. A second type possesses a blasteina which persists over a long period and inay grow appreciably in continuous absence of nerves, as illustrated by case VII1214(Amblystoma opacum larva, 34 mm., kept at 20°C., figs. 6-9). The 8-day-old blastema of this case (fig. 6) shrinks after deiiervation of the left a r m to a considerable extent within 7 days (fig. 7 ) , while during this time the now 15-day-old normal regenerate on the right control side shows clear iiiclicatioiis of digital promorphology. The denervated blastema, reduced in size, persists in the stage represented by figure 8 f o r about 15 days. Although careful and repeated examination of the opened sub-scapular area revealed no trace whatsoever of a regeneration of the plexus, the blastema on the denervated stump resumed about 3 weeks after denervation a certain amount of growth and reached the size shown in figure 9, 25 days after denervation. This regenerate, however, was unable to develop further than into a cylindrical structure without any digital differentiation. In this case, as in the above described one, the continued presence of a blastema did not interfere with the regression effect. However, regression proceeded at a slower pace than usual. Lastly, in a number of cases, it is possible to detect only slight effects of denervation on the growth and developiiieiit of a blastema which, in all essential features, such as size aiid general characteristics, is not markedly different from the previously described aiid illustrated blas-

108

OSCAR E. S C H O T T ~A N D ELMER G . BUTLER

temata. Case VII,,, (Amblystoma opacum larva, 32 mni., kept a t 21.5"C., figs. 10-12) illustrates the general behavior of larvae of this type. At the time of denerration, the left blastema, although slighjly larger than the one 011 the control limb, was otherwise typical of a 7- to %day development (fig. lo]. Our records show that 3 days after a complete removal of all t h e e components of the nerve plexus, up t o their spinal

Fig. 6 Amblystonia opaeum, VII121,,on the day of denervation of the left fore limb, 8 days after amputation of both right. and left limbs. F i g . 7 Samc larva, 7 days after denervation, 15 days after amputation. Fig. 8 Same larva, 18 days after denervation, 26 days after amputation. Fig. 9 Same larva, 35 days after denerration, 33 days after amputation.

REGENERATIOX PHASES AND NERVOUS SYSTEM

109

Fig. 10 Amblystoma opncum, VIILc7,on day of denervation of left fore limb, 7 days after amputation of both right and left limbs. Fig. 11 Same larva, 6 days a f t e r dcuerration, 13 days after amputation. Fig. 12 Same larva, 23 days after denervntioii, 30 days after amputation.

110

OSCAR E. S C I - I O T T ~A N D ELMER G . BVTLER

origins, the denervated blastema preseilts a definite increase in size. Six days after deiiervation of the left limb, as the photograph in figure 11 shows, altliougli the left regenerate in size and in development is appreciably less advanced than tlie one of the control limb, it has unmistakably grown and differentiated. The further development of the nerveless regenerate during the 23 days of the experiment (in spite of two careful reopenings of the sub-clavian area aiid painstaking removal of every suspicious fiber in that region) is characterized by an uninterrupted advance, in so far a s both growth and differentiation of the regenerate a r e coiiceriied (fig. 12). Moreover, in contrast to all previous cases, there a r e 110 indicatioiis in this case of any appreciable regrcssion. Several examinations during the 33 days of the experiment verified tlie absence of innervation. TABLE 3

Effects of permanent nrrvelessness on limbs possessing b k s t e n w t a agrd 7 , 8 a n d :I days. The roman ,?iztnierfllof eoclk series indicates ill days the age of t h e blostenm brforc defiervation. Szinrbew in. vertiral colicmiis iiidimie ?iit~inbersof individ,tcnls of the series ]raving s1iou.n ir7enticcrl resztlts. (Redirction i n nirntber of cases u7ilhin a srrirn iitdicatrs that SOnie harvne were fi.xed f O T histological st.itilirs.) ~ _ _ ~ _ _ _ ~ S E RI ES T I 1 21.50

WERIBS V I I I 2 0 0

(12 individuals)

( 2 5 individuala)

22 individuels)

Increased Stationary Decreased

4 5

20 3

3

>

16 3 3

Sizo of blastem? 10 days after denervatinn

Increased Stationary Decreased

4 1

19

19

1

..

3

1

3

Final state of denervated blastema

Increased Morphopresent genesis absent Stationary Disappeared

4

18 9 3

18 18

Present Ahsent

6 4

Sizo of blastenis 3 or 4 days after denerva tinn

Regression

3 1 1 4

SERIES IX 2 0 0

..

..

..

4

3

4

3

18

18

The results from all the fifty-nine individuals with blasteniata aged froin 7 to 9 days have been suniinarized in table 3, in which are recorded the initial changes in the size of the deiiervated blastemata and the later morphological consequences of denervation. It can be seen from this table that, the older tlie blastema at the time of denervatioii the less a r e the effecis of this operation on its survival and further developiiient. Another important feature is the remarkable fact that, although in the 7-day-old hlastema ( a t 21.5"C.) the regression effect still occurs in

REGENERATION PHASES A N D NER,VOUS SYSTEM

11s

60% of the cases, we observe that in both the 8- and 9-day-old blastemata (out of 43 larvae which have been kept long enough) only seven individuals, or 16.3%, show any regression at all. In conclusion, it can be said that for blastemata of 7 days, if kept at 21.5”C., and of 8 or 9 days, if kept at 20°C., the consequences of denervation manifest themselves in one of four different ways: (1)The blastema on the denervated side decreases and eventually disappears altogether. Under such circumstances, regression sets in as a matter of c o u r ~ e(13 cases among the 59 individuals of this group, or 2270 ) ; (2) The blastema ceases to grow and remains stationary; a comparatively rare occurrence, since only two among fifty-nine, or 3.4%, have thus behaved. In this event, in spite of the continuous presence of a blastema, regression sets in and exerts its effects as long as the state of nervelessness persists : (3) After an initial arrest in development, the blastema, even in absence of nerves, recuperates its capacity for growth. I n such a case, however, the regenerate, having been inactivated for a considerable lapse of time, is incapable of further normal regeneration, and abortive regeneration, typified by case VII1214,ensues ( 4 cases out of 59, or 6.8%, behaved in this manner). I n a last group of blastemata, particularly among those aged 8 and 9 days, deneryation fails to exert a decisive action. While in every case differences in the size of the denervated blastema compared to the normal one can be detected, it has been observed that for forty cases out of fifty-nine, or for 67.8%, the supprespion of all innervation neither prevents growth of the blastema nor its differentiation.

IV. Eflects of deraervation on older, but aot yet visibly diferentiated blmtemata, am?om regenerates showing beginning morphogenesis This group includes ninety-three larvae, kept at 20”C., which were allowed to regenerate for 10 to 16 days previous to denervation. At 10 days a blastema ordinarily shows no visible signs of differentiation, but its size consistency and the frequent occurrence of some strands of precartilage clearly characterizes it as an “old” blastema. We have also included in this series regenerates which, before denervation, were as old as 16 days and in which, in every case, precartilage and even digital differentiations were clearly discernible under the microscope. On the basis of individual differences within each age group, and particularly in accordance with the characteristics of the age group themselves, we can distinguish essentially three types.

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OSCAR E. SCHOTTI? A N D ELMER G. BUTLER

To the first type belong most of the thirty-three larvae of the 10-dayold blastema group, among which case XI,, can be considered to be representative (Amblystoma punctatum larva, 42 mm., blastenia a t denervation 10 days old, animal kept a t 20°C., fig. 13). From the drawing of the blastema, made at the time of denervation (13a), it can be seen that the blastema was still small, without the slightest indication of any differentiation. Yet, the next drawing, made 3 days later, shows considerable increase in the size of the denervated blastema (134. In contrast

Fig. 13 Amblystoma punctatum, X,,,. Camera lucida drawings of right ( R ) and left (L) fore limbs. Left linib denerrated, 'right limb possessing normal innervation.

to previously described cases, there are no indicatioiis of difference in size between the denervated and control blastemata (13c and 13d). Only 7 days after denervation, a certain lag in growth, but not in development, becomes noticeable on the denervated side (13e and 13f), and this lag persists until the end of the experiment (13g and 13h). Besides the :light difference in size between the denervated and norinal regenerates,

REGENERATION PHASES A N D NERVOUS SYSTEM

113

tlie progress of regeneration on both limbs of this larva, and of most other larvae of the group, is strikingly idcntical, botli from the point of view of tlie time of appearance of the first perceptible streaks of precartilage and from the point of view of the external morphogenesis of tlie regenerates. A iiiore aclvanced type of regenerate is represcnted by case XIIlo9 (Aiiiblystoiiia opacuiii larva, 33 mm., 13-day blastema, kept a t 20°C., fig. 14). I n this case the blastenm lias the appearance of the flattened shovel, so cliaractcristic for the early morpliogeiicsis of the regenerate (14a, 14b). I n addition, there can he obscrved within tlie regenerate

1 DAYS

17 DAYS

distinct streaks of precartilage, representing. tlie foundatioiis of the future skeleton of the fore arm. Curiously enough, in this case, aiid in all the other cases of denervated regenerates in which precartilagenous formations a r e distinguishable, the difference in size between the denervated a n d tlie control blastemata appears as early as 4 or 5 days (14e aiid l 4 d ) . The further history of such cases is uneventful. Both growth and differentiation of the dcnervatcd blastema proceed regularly (14e, 14f, 14g and 1 4 1 ) . The study of this case and of similar cases of this group, denioiistrates that, although the niorphology of the dciiervatecl limb develops in a n essentially normal manner, a noticcable rcduction in the size of thc deiiervated rcgencratcs always is evident.

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OSCAR E. S C H O T T ~AND ELMER G. BUTLER

Finally, in case XIV,,,, (Amblystoina punctatum larva, 33 miii., figs. 15 and 16) the 14-day-old regenerate shows (fig. l 5 ) , at the time of denervation, distinct indications of digital differentiation. I n comparative clevelopriient of both the denervated and cont rol regenerates the history of this rase is similar to the previous one (fig. 14.) The photograph in figure lG, Iiiade 19 days after denervation, shows clearly the perfect form of the regenerate on the deiiervated left limb. There is, however, considerable clifference in size between the normal and the denervated regenerates.

Fig. 15 Ainblgstoma punetaturn, XIV,,, on day of denervation of left fore limb, 14 days after aniputation of both right and left limbs. Fig. 16 Same larva, 19 days after denervation, 33 days after amputation.

In cases of still older regenerates with three or four digits clearly indicated (16-day-old, Series XVI), denervation has little effect on subsequent growth and none at all on differentiation. Moreover, in the case of regenel-ates as old as this, the lag in growth on the denervated side, as compared to the control side, is much less conspicuous-in some cases there is scarcely any difference at all. Regression, so conspicuous i n the previous series, is totally absent among sixty individuals possessing 12- to 16-day-old blastemata and has been observed in only three individuals among the thirty-three cases of the 10-day-old group.

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7. Reinnervation of previously denervated limbs I n spite of overwhelming evidence demonstrating the necessity of the nervous system for the initiation of regenerative processes, it still seems legitimate to be concerned with the problem of whether, after disappearance of a blastema with concomitant regression, a limb is capable of starting new regeneration as a consequence of reinnervation. Case VIIIla4(Amblystoma opacum larva, 31 mm., fig. 17), provides such conditions.

0-DAY

4 DAYS

A 3 15 DAYS

10 DAYS

Fig. 17 Arnblystorna opaeum, VIII,.

7 DAYS

DAYS

Camera lueida drawings of left fore limb denervated

8 days (a) after amputation. Drawings g, h and i show progress of regeneration after reinner-

vation.

The series of six drawings of the left denervated limb of case VIIIla4 shows that, within 17 days after denervation, the 8-day blastema entirely disappears (17a t o 17f). Simultaneously, the left limb loses about three-fifths of its initial stump to the regression effect. The last resection of the nerves of the plexus, effected in this case with doubtful results, because of profuse bleeding, was obviously not entirely successful. Eighteen days after the initial denervation visible signs of regeneration appeared on the left stump. The establishment of a large blastema within an interval of 5 days (17f and 17g) is so unusual that it seems highly probable that the dedifferentiated materials of the previously denervated limb have been used in the building up of this

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OSCAR E. S C H O T T ~AND ELMER Q. BUTLER

blastema. Within the next 14 days (17h and 17i), the left limb has reached a stage of regeneration in which it differs from a normal limb only in size. An examination of the left brachial plexus at the end of the experiment showed that prominent first and third branches of the plexus (spinal nerves I11 and V) had regenerated, the second branch (sp. n. IV) was still missing. Early reinnervation of previously denervated blasternata accounts.for the above mentioned five cases in section 11, among a total of fifty-nine, in which, after a short initial lag in growth of the “denervated” blastema, sudden growth and differentiation set in. In every one of these cases dissection of the sub-scapular area revealed the presence of regenerated or incompletely severed portions of the p l e ~ u s . ~ Concerning the whole problem of reinnervation, it can be said that neither the cessation of growth of a blastema nor its ultimate resorbtion, nor even the extent of regression, have anything to do with the ultimate fate of a once denervated stump. Because we regularly keep our larvae, with their individual numbers, under observation long after the main-purpose of the experiment has been accomplished, we are able to report that, in all cases, regeneration eventually sets in, unless the larva has in the meantime metamorphosed. The situation is slightly different with adult newts, where the thickness of the tegumental coverings of the amputation surface can be such that regeneration, due to reinnervation, cannot take place without reamputation (Schotti5, ’26).

Recapitulation

A digest of the effects of denervation on the ultimate fate of limbs amputated from 2 to 16 days previous to denervation is presented on table 4. The data in this table based on observations made on 252 individuals are self-explanatory. They clearly demonstrate that the action of nerves on regeneration depends on the age and, therefore, the degree of development of regenerates. These experiments also show a clear-cut correlation between the regression effect and the stages of * I t may appear strange that after so much experience the operator still finds himself compelled to admit several unsuccessful denervations. The reasons for such failures lie, in the opinion of the operator, in the unpredictable distribution of the nerves of the plexus. Generally, the two main stems (sp. n. 111 and IV) are clearly distinct and do not form a plexus until they reach the area situated approximately halfway between their emergence from the spinal column and the shoulder region. Sometimes, however, there are several fine branches of both of these nerves which detach themselves early from the main stems and lie close to the subclavian artery. J t has seemed preferable, in this investigation, to take the risk of a few incomplete denervations, rather than to sever the subclavian artery, since changes i n the size of a blastema might easily result from alterations in its blood supply.

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REGENERATION PHASES AND NERVOUS SYSTEM

blastemata, in so f a r as their dependence upon the nervous system is concerned: when limbs with young blastemata are denervated and when, a s a consequence of this denervation, blastemata either disappear or are simply arrested in their development, then, always, regression sets in and its extent is only a matter of the duration of the state of nervelessness; when on the contrary, older blastemata remain unaffected by the severance of their nerve supply then, also, regression fails to appear. TABLE 4 AOO~or BLALSTEYATA

No of days

2-7

(20") 7 (21.5") 8 and 9 (20")

10 (20") 12-16 (20")

SIATUB

NO. OF CASES

Increased

OF BUWFEYATA AT DND or PXPEBIYENT

Stationary Decreased

100 59 33

..

..

42

6

30

..

60

60

..

13

.. 3

..

REOR18810N

Absent

Absent

Present

87

4

96

11

43

..

30 60

11 3

..

DISCUSSION

I n the introductory part of this paper we raised several specific questions regarding the action of nerves on the various phases of regeneration. The data presented in the foregoing pages enable us to answer these questions with reasonable completeness and provide us, also, with a more comprehensive knowledge than we heretofore possessed of the manner in which nerves influence the whole regenerative process. Concerning the question of the necessity of nerves for the dedifferentiative phase of activity, which inaugurates normal regeneration, a great deal of information is available from previous research. Denervation, when made simultaneously with amputation, always results in inhibition of regeneration. I n larval limbs, not only is regeneration prevented, but also extreme dedifferentiation and regression ensues. If denervation is delayed till the first phase of regeneration is well started, our present experiments show that nervelessness results in a great disorganization of this dedifferentiative phase. Conditions are such as we have described in our previous papers. I n other words, it appears to make no difference whether amputation and denervation are made simultaneously, or whether denervation is performed during the short period of dedifferentiative activity which normally takes place after amputation. Tf a limb is denervated after a young visible blastema has been established, at a time which marks the beginning of the second great phase of regeneration, the conditions are considerably'different, but the results

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OSCAR E. SCHOTTE AND ELMER G . BUTLER

are surprisingly similar to those of simultaneous denervation and amputation. Our experiments show that, in every case, a blastema less than 8 days old (if larvae are kept at 20°C.) begins to decrease in size as soon as denervation is effected, loses the characteristics of a normal active blastema, and eventually becomes subject to the regression which overtakes the entire limb. Thus, it is evident that tbe consecutive processes of dedifferentiation and blastema formation are dependent upon the nervous system, both for setting these processes into motion and also for their normal continuation. We regard, therefore, this portion of our research as completed, and feel that further efforts on this special subject should be concerned, not with an endeavor to show that nerves are necessary for the initiation of regeneration, but rather with an elucidation of the mechanism of the nervous action involved. The relation of nerves to later phases of regeneration, the period of morphogenesis, is in great contrast to the clear effects which nerves exert on early regenerative processes. Our results fully corsfirm previous observations made by Schott6 ( '23, '26), Weiss ('25), and more recently by Schneider ( '40),that the suppression of innervation in later stages of regeneration exerts no fundamental effects on growth and further development of a regenerate. It cannot be denied, however, that nerves may exert some type of trophic, or possibly tonic, action. Yntema ('43) has clearly shown that muscular atrophy occurs in anefferented limbs. We have also demonstrated (Schott6 and Butler, '41) that absence of nerves in the limbs of Amblystoma larvae results in general physiological atrophy. Still, the fact remains that completely denervated'regenerates grow and differentiate into morphogenetically well-defined limbs.6 There is a period of regeneration, an intermediate stage, during which, in some cases, denervation results in changes similar to those described for young blastemata; in other cases, the same operation of denervation causes little or no disturbance. In these latter cases, the result is similar t o that obtained when old blastemata or regenerates are deprived of their innervation. I n still other cases, blastemata denervated during this intermediate stage neither regress nor develop morphologically ; occasionally they grow slightly, but in general they remain inactive over long periods. Such conditions prevail in blastemata aged 7 days, for larvae of 2 5 4 0 mm. kept a t 21.5"C., and in blastemata aged 8 to 9 days, f o r larvae kept at 20°C. During this intermediate period of * Although the limbs obtained froni denrrvnted blastemata in our experiments possess an apparently normal morphology, it ,is possible that detailed histological examination will reveal morphological defects similar to those previously reported by Brunst ( '27).

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PHASES AND NERVOUS SYSTEM

119

from 7 to 9 days, therefore, critical changes take place, within the blastema, in so f a r as its dependence on the nervous system is concerned. Our experiments clearly show that there are many cases in which blastemata, although prevented from growing and differentiating, continue to exist for a long time. Presented in a simplified manner, we regard this critical intermediate period a s consisting essentially of at least three phases of activity. During the early part of the period the blastema can be regarded a s an “accumulation blastema,” in other words, an aggregation of cells lately dediff erentiated. Such a young blastema, when denervated, regresses. In the other extreme, during the latter part of the critical period, when a blastema has become, in our opinion, a well-determined regenerate, denervation has little o r no effect. Between these two extremes a phase of imbalance prevails, during which the essential processes of induction undoubtedly occur, resulting in the transformation of the “accumulation blastema” into a morphologically determined regenerate. The reality of this latter phase is clearly indicated by those numerous cases which have shown persistent blastemata, regardless of how long the state of nervelessness prevailed. Our experiments show, therefore, that the presence of nerves is essential for the phase of regeneration which we regard as the “induction phase.” It would be arbitrary to declare that the nervous system serves as an active agent during the induction phase of the hlastema, simply because the presence of nerves is necessary for the survival bf a young blastema, and nerves are unnecessary when a blastema becomes capable of selfdifferentiation. We are fully aware that it remains to be demonstrated that the stage of development of a blastema at which it is still dependent on the nervous system is, in actuality, the stage at which it is morphologically indifferent. Furthermore, evidence must still be presented that it is precisely during the 8- to 9-day period (at 20°C.) that a blastema becomes morphologically determined. It can only be concluded, that the preseiice of nerves is indispensable for setting into motion the normal process of dedifferentiation, for its regulation and for the establishment of an “accumulation blastema.” Likewise, nerves are essential for the maintenance of an accumulation blastema and for its transformation into a true regenerate, possessing morphological determination. However, once a morphologically determined regenerate has been established, nerves are not prerequisite f o r growth and differentiation. It is of particular significance, that nerves are essential f o r those activities which are peculiar to regeneration. They are not essential

120

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E. S C H O T T ~A N D ELMER

o.

BUTLER

for these other activities, ordinarily regarded as a part of regeneration, but which are essentially morphogenetic in nature and, in commoii with other morphogenetic activities, are independent of nervous inffuence. SUMMARY

1. The influence of nerves on regeneration of the limb of the larval urodele has been investigated from the standpoint of the effects which they exert on particular phases of the regeneration process. 2. To obtain comparable stages of regenerating limbs, temperature conditions and age of larvae have been carefully controlled. Two hundred and fifty-two cases of Amblystoma punctatum and A. opacum have been studied. 3. Permanent nervelessness of limbs has been effected by repeated resection of the brachial plexus. 4. Denervation followed amputation and was delayed by from 2 to 16 days. This procedure provided a means of determining the importance of nerves with relation to the various phases of regeneration. 5. The presence of nerves has been found to be necessary: (a) for thc whole of the dedif'ferentiative phase which inaugurates regeneration and leads to the establishment of the blast,ema; (b) f o r the transformation of the young blastenla into a regenerate possessing morphogenetic determination. 6. The presence of nerves has been found to be unnecessary for growth and differentiation of the regenerate (phase of morphogenesis). 7. The nervous system is an important agent only for those activities specifically regenerative in character : it is of no more importance for morphogenesis during regeneration than for morphogenesis in embryonic limb development. LITERATURE CITED ABELOOS, M. 1932 La rbgbn6ration et les problbmes de la morphogbn6se. Gauthier-Villars. Paris. BRUNST,V. 1927 Zur Frage nach dem Einflum des Nervensystems auf die Regeneration. ROUX'Arch., Bd. 109, 8. 41-53. BUTLEE, E. G., AND 0. E. S C H 1941 ~ Histological alterations in denervated non-regenerating limbs of urodele larvae. J. Exp. Zool., vol. 88, pp. 307-341. E. 1927 Regeneration und Transplantation. Borntraeger. Berlin. RORSCE~ELT, LOCATELLI,P. 1924 L'influenza del sistema nemoso sui processi di Rigenerazione. Archivio di Scienze Biol. 5, (362-376). SCHNEIDER, G . 1940 Der Einfluss des Nervensystems auf die Regeneration der Gliedmassen der Axolotl. Bull. de 1'Acad. d. 8c.i. d. U.R.S.S., SBr. Biol. 1940, S.385-404. (Russian, with German abstract).

REGENERATION PHASES AND NERVOUS SYSTEM S C H d ,

12 1

0.E. 1923 Influence de la section tardive des nerfs sur 11% pattea d e Tritons en rhgh5ration. C. R. SOC.Phys. e t Hist. nat. Genhe., vol. 40, pp. 86-88.

1926 Systhme nerveux e t r6gCnbration chee le Triton. Rev. Suisse de Zool., vol. 33, pp. 1-211. 1940 The origin and morphogenetic potencies of regenerates. Growth, Suppl., pp. 59-76. ScHo~Tlt,0.E.,AND E. 0.BUTLER 1941 Morphological effects of denervation and amputation of limbs in urodele larvae. J. Exp. Zool., vol. 87, pp. 279-322. TODD,J. T. 1823 On the process of reproduction of the members of the aquatic Salamander. Quart. J. Bci., vol. 16, pp. 84-96. WEISS, PAUL 1925 Abhangigkeit der Regeneration entwickelter Amphibienextremitaten vom Nervensystem. Arch. f. Mikr. Anat. und Entw.-Mech., Bd. 104, S. 317-358. 1935 The so-called organizer and the problem of organization in amphibiaIl development. Phys. Reviews, vol. 15, pp. 639-674. YNTEYA,C. L. 1943 Deficient efferent innervation of the extremities following removal of neural crest in Amblystoma. J. Exp. Zool., vol. 94, pp. 319-349.