Regeneration in botulinum-poisoned forelimbs of the newt, Triturus

Exl'ERIhIENTAL

Regeneration

SEl'IInLoG~

32. 1-11 ( 1971 1

in Botulinum-Poisoned Jriturus

Forelimbs

of the Newt,

Regeneration of an amputated limb \yill take place in the salamander provided that an adequate nerve supply is present in the stump. It has been suggested that acetylcholine (.;ZCh) released by the nerves may he the neural trophic factor \Yhich enables regeneration to take p!ace. In order to test this hypothesis \vveamand then administered high systemic putated the limbs of ‘l’vitutws ~Gvitl~~srt~~~s doses of botulinum toxin. The purified toxin is the most highly specific and potent cholinergic blocking agent knoxvn. In spite of effective cholinergic blockade, regeneration of the amputated limbs took pla:e, and proceeded at a normal rate. It is concluded that :\Ch is not necessary for limb regeneration to occur, and therefore does not fulfill the role of a neurotrophic agent in this situation. Howexr. this does not rule out the possibility that ACh may he the sole neurotrophic agent, or associated lvith the agent. in other biological systems such as skeletal muscle. introduction

Amputation of a limb or of certain other body parts of the salamander normally gives rise to regeneration of the lost appendage. .:1n adequate nerve supply in the stmmp is essential for this process since denervation prevents or halts the regenerative sequence. How the nerve functions in support of regeneration remains unknown in spite of a great nianv expel-imental studies on the subject (reviews : 16, 18, 19). ,4mong the theor&tally possib!e mechnnisms of action. the one most widely favored is that the nerve releases a chemical substance which is important for growth of the regenerate (1s). Since an effective substance has not yet heen isolated or identified. several investigators have speculated that one of the known 1 Supported by grants from the National Institutes of Health, (HD 04817 and NS 07403-lo), the RIultiple Sclerosis Society. and the American Cancer Society.

2

DRACHMAN

AND

SINGER

neurotransmitters may serve this trophic function (6, 11, 23). A great deal of evidence has been marshalled for and against the possible role of acetylcholine (ACh) in regeneration (19). The most compelling evidence supporting the ACh hypothesis is that various cholinergic blocking agents, such as atropine and hemicholinium-3, delay or prevent regeneration in Ambystoma (6)) Triturus (22)) and Hydra (9). iZlthough other studies have cast doubt on the role of ACh in regeneration, we thought it worthwhile to reexamine the problem by blocking ACh transmission with the most highly specific and potent cholinergic blocking agent known: botulinum toxin. Materials

and

Methods

Adult Triturz4s viridescens collected in western Massachusetts or North Carolina were used throughout these experiments. Because of the susceptibility of paralyzed salamanders to infection, special precautions were taken to eradicate pathogens from all newly received shipments. Aquaria and equipment were scrubbed with KMnO* solution (0.1 g/liter). The salamanders were dipped repeatedly in a more dilute solution of KMnO$ (0.03 g/liter), and were stored in water containing 0.002 g/liter of KMn04 prior to, but not after operation. With this treatment subsequent infections were rare, but salamanders were kept isolated, one or two to a container, so as to limit the spread of infections. Animals were anesthetized with “Tricaine” (Sandoz) and their forelimbs were amputated bilaterally be incision through the distal third of the upper forelimb. The stump of the humerus was then trimmed back. Crystalline Type A botulinum toxin, diluted in amphibian Ringer’s solution, was injected intraperitoneally in a volume of 0.1 ml or less per injection. Controt animals received injections of equal volumes of Ringer’s solution. Various treatment schedules were followed, as indicated in Table 1. In all cases treatment was begun on the seventh or eighth day after amputation and each Triturxr received a total of 4G80 X lo-“g of botulinum toxin in one to three divided doses. This amount is equivalent to lo-20 X 10’ mouse lethal doses (10). All the salamanders developed complete or nearly complete paralysis of the skeletal musculature within 24 hr after treatment. Rarely, a treated salamander showed an infrequent movement of the trunk. After the first injection, treated and control animals were placed on cotton moistened with distilled water. Since the paralyzed salamanders could not obtain food, the control animals were also deprived of food for the remainder of the experiment. To reduce the mortality rate, we gently massaged the lower abdomen of the paralyzed salamanders daily, so as to expel wastes. In spite of these precautions, a proportion of the treated salamanders did not survive. In the most favorable group, 11 of 15 botulinum-

treated animals survived to the end of the experiment. Only salamanders which were alive at the termination of the experiment or which were known to have died within a few hours previously were included in the rea sults. Groups of salamanders were killed on days 15-21 after amputation. At that time the limbs were examined under a dissecting microscope, and the stage of regeneration was graded according to the criteria set forth hy Singer (16). Both forelimbs attached to the thorax were fised in Bouin’s solution, embedded in paraffin, and sectioned at 10,~. The sections were stained with silver proteinate according to the method of Bodian and counterstained with orange G or Masson’s trichome stain. Results

The outstanding finding in this study was that hotulinum toxin did not prevent limb regeneration in the newt. Fifty-one of 56 limbs of botulimm-treated salamanders showed evidence of regeneration (Table 1 : Fig. 1). -4 comparison of the mean stage of regeneration of botulinurn-treated and control limbs (Table 2) did not reveal a significant difference between the two groups (p> > .25 ) . The stage of regeneration was quite similar for the two forelimbs of a given salamander. but there was considerable variation from animal to animal. In general, there seemed to be less variability within each gronp of salamanders handled at the same time. ,4ge and size of the salamanders, the season of the year, and other details of the experimental procedure may have contributed to the variation. We found that raising the ambient temperature from 21 to 26 C accelerated regeneration in treated salamanders, as has previouslv heen noted in normals. Neither the total dose of hotulimum toxin given- nor the schedule of administration affected the rate of regeneration (Table 1). Histologically, the earliest stage of regeneration in the normally innervated new limb is characterized hy estensive dedifferentiation of old stump tissues. Cells liberated in the histolytic process accnmulate under the thickened wound epithelium to form a mound, the blastema. These cells rapidly undergo mitotic division, resulting in the growth of the hlastema to form a cone-shaped structure. Cltimately. a more or less perfect limb is re-formed. Denervation before or during the mound phase effectively interrupts growth. The regenerate wastes, and the cells of regeneration are converted into a fibrocellular mass. The stage we selected for initiation of botulinurn treatment was before or at the time of accumulation of the blastema. Blockade of the neurotrophic influence by botulinum toxin would be expected to cause cessation of regeneration and regressive changes. as in the denervated stump. 011 the contrary, histological examination (Figs. 2-6) of the l)otu]i~lut~l-treatec]

4

DRACHMAN

AK-D

TABLE

SINGER

1

RESULTS OF BOTULINUM-TREATED SALAMANDERS, IN TERMSOFGKOSS REGENERATION (SURVIVOKS~NLY)

No.

Treatment Dose, ml (0.4 mg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

0.1 0.1 0.1 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.1 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

after

Time (day! 9 9 9, 9, 9, 9, 9, 9, 9, 9. 9, 9, 9’ 9, 9, 9, 9, 9, 7, 7, 7, 7, 7, 7, 7, 7, 7, 7,

14 11 13, 13, 16 16 16 16 16 13, 13, 13, 13, 13, 13, 13, 11 11 11 11 11 11 11 11 11 11

Time killed amputation (day)

17 17

17 17 17 17 17 17 17

16 16 16 16 18 20 20 21 21 21 21 21 21 21 21 21 21 21 1.5 1.5 15 15 1.5 1.5 1.5 1.5 15 1.5

Regen. ~----~-__~~L. limb lf f f 1+ 1+ 3 f

2+ 3+ 3 0 3+ 3+ 4 2 0 + 1 3 3+ 3+ 2 3

2+ 4 4 3 4

stage R. limb 1 f f 0 1+ 2 lf 3 4 3 0 3+ 3+ 3 1 1 0 1 4 3+ 3 3 3 3 3 4 2 4

newts revealed a typical picture of normal active regeneration. The blastema1 cells were abundant, and were accumulated in subepithelial mounds. Nerve fibers extended singly or in fascicles among the blastemal cells, reaching and penetrating the epithelium. Discussion

It has been thoroughly established that of limb regeneration in urodele Amphibia plied by the nerves (16). If this influence any time up to the early bud stage (at Triturz4s), growth ceases completely, and

both initiation and maintenance require a trophic influence supis eliminated by denervation at approximately 2 weeks in adult the newly formed bud shrivels

LIMP,

‘f.ARLE COSTKOL

No. 1 2 3 4 5 6 7 8 9 10 11

Time killed after amputation (day) 16 16 16 16 16 16 21 21 71 21 21

5

REGENERATION

2

SALAMANDERS

State

of regen.

1.. limb

R. limb

2 2+ 1+ 3 1+ 2 3 1 f 4 3

2+ 3 1+ 3 ‘4 2 3 2 zk 1+ .3

(18). In the present study, botulinum treatment was begun on the seventh before the development of a regenerative or ninth day after amputation, blastema. If the treatment had interrupted the nerve’s trophic influence, regenerative changes would not have occurred. On the contrary, our results clearly demonstrate that l~otulinuni toxin does not prevent early limb regeneration in Tvit~r~s. Furthermore. the rate of regeneration was not significantly slowed throughout the time period studied, in spite of large and effective systemic doses of the toxin. A11 understanding of the implications of these experimental findings requires further knowledge of the pharmacological properties of botulinurn tosin. The Type 11 toxin used in these esperiments is a purified crystallizable protein derived from the anaerobic bacterium Closfr-itliunL botuli~~z~~r (10). It is an extremely potent neuromuscular blocking agent, and the respiratory paralysis which results from the ingestion or injection of a fraction of a microgram is lethal to most animals (7). Certain animals, including the newt (23 ) , can effect respiration by passive diffusion of gases through a moist skin without muscular contractions, and therefore are able to survive complete muscular paralysis. Pharmacologically, botulinum toxin prevents the release of ACh from peripheral nerve endings, but the detailed mechanism of its action is not presently known ( 13). Most of the pharmacological studies of botulinurn have utilized mammalian preparations, but neuromuscular blockade has been described in fowl and frogs as well (1, 2). In the present study we found that intraperitoneally injected toxin was effective in blocking neuromuscular transmission in Trit~us. h~uscular contractions were absent or minimal in all the treated animals. Further, electrical stimulation (2 msec, up to 40~) of the tlerves of the

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DRACHMAN

AND

SINGER

FIG. 1. Limb regeneration in botulinum tl Injections of botulinum were given on days tion in both limbs.

FIG.

2. Section

through

actively

growing

blastema

of newt.

16 davs

a4t-v

n-n*.+,-+;-,

I~azhial plexus fai!ed to elicit a muscle response, while direct stimulation of the limb musculature resulted in brisk contractions. Since effective hloc!iade of neural &ZCh transmission did not prevent limb regeneration, we are led to the conclusion that ACh does not play a significant neurotrophic role in this process. Our findings and conclusions are con&tent with most of the available inforn-ation concerning amphibian limb regeneration and cholinergic mechanisms. Previous inquiries into this subject have taken three general lines of approach : measurement of .Ch : replacement of ACh : and elimination of cholinergic transmission. Afrasurcwr~t of A3Clz. Singer (17) measured the ACh content of the amputated Trjfl/rl/.r limb throughout the sequence of regenerative stages. He found that the .‘iCh levels were high prior to the “digital regenerate” stage (about 21 days) and dropped thereafter to adult levels. The high .\Ch levels appeared to coincide with the time when the nerve was eserting its greatest trophic influence on regeneration. The subsequent fall in ACh could he accounted for hy an increase in acetylcholinesterase (ChE) activity (21) with resulting hydrolysis of ACh. It seemed that the L9Ch and ChE levels correlated nicely with the events of regeneration. However, by changing the experimental conditions, this apparent correlation could be broken. For example, complete denervation of a regenerating limb caused immediate cessation of regeneration, with a much more gradual decline in ACh levels (19‘). Further, selective destruction of the motor nerves but preservation of the sensory innervation in the limbs did not suppress regeneration (11) in spite of the loss of ACh. Conversely, destruction of the sensory nerves alone did not lower the ACh levels significantly, hut invnriably halted regeneration ( 15 >. Evidently, the motor nerve supply, which is quantitatively smaller than the sensory supply, is incapable of sustaining regeneration alone, in spite of its high .4Ch content. It is now clear that the quantity of innervation is the critical factor in determining whether regeneration will occur (20)) rather than the ACh content of the nerves. Rrplacrwent of AClt. In this type of experiment, ,2Ch was applied to the denervated stump externally l)y immersion (23) or internally by microinfusion ( 19) in an effort to substitute for the trophic influetlce of the nerve. This approach has never met with success. While the te&liques of continuous application of ACh may be criticized as being “u~lpl~ysiological,” Botulinum toxin Masson trichrome

was injected on day 9. Note abundance of stain. Reduced 167% from X 300. approximately.

FIG. 3. Silvered section of blastema of newt, injected Fig. 2. N o t e abundance of intact axons. Bodian stain. approximately.

mesenchymatous

with botulinum Reduced 16%

toxin from

cells. as in X 500,

8

DRACHMAN

ASD

SINGER

LIMB

the negative conclusions drawn supported by other approaches.

9

REGEiXERXTION

f ram

these

experiments

are abundantly

Elituination of Cholinevgic Transu~issio~z.Singer,

Davis, and Scheuing infused various neuroactive drugs into the regenerating limbs of Trifzcms in an effort to block the neurotrophic influence and interrupt regeneration. The list of agents used included atropine sulphate, procaine, hydrochloride, tetraethylammonium hydroxide, physostigmine. pilocarpine nitrate, and salicylate. In unpublished experiments, curare in paralyzing doses was also used. Of the lot, only atropine interfered with regeneration. The simultaneous administration of ACh or its more stable analog, mecholyl, appeared to protect against the action of atropine. The interpretation of these experiments is open to question since the concentration of atropine required to block regeneration was unduly high (19). While the action of atropine as a postsynaptic cholinergic blocking agent is quite specific at ordinary concentrations, it is known to act less selectively at higher levels (4). For example, it inhibits a variety of tissue responses to histamine, serotonin, and norepinephrine. The possibility that its effect on regeneration may have been a nonspecific one was further suggested by the finding of tissue damage in the treated limbs (19). Recently, Hui and Smith (6) have studied the effect of hemicholininum-3) on amphibian regeneration ; HC-3 is a synthetic agent which blocks neuronal uptake of choline, and therefore interferes with synthesis of ,:\Ch( 12). At higher concentrations it exerts postsynaptic blocking actions as well. The growth of the regenerating limb of the larval salamander, Antbysto~a, was retarded during a lo-week exposure to HC-3, but regeneration was not prevented. In order to resolve the questions raised by these pharmacological experiments, the present study utilizing botulinum tosin was undertaken. Recause of the extraordinary potency and specificity of botulinum toxin as a cholinergic blocking agent, it is the most appropriate agent available for (22)

FIG. 4. Silvered section of sarcolysing muscle near amputation wound, 16 days after amputation. Botulinurn toxin was injected on day 9. Note the nerve fiber endings on muscle. Bodian stain. Reduced 16% from X 500, approximately. FIG. 5. Blastema of newt treated as above. Silvered preparation to show numerous intact growing axons scattered among blastema cells. Bodian stain. Reduced 16% from X 500, approximately. FIG. 6. A region of the blastema adjacent to that in Fig. 5, showing intact nerve fibers in thickened epithelium (arrows) and in underlying blastema. This is the characteristic distribution of nerves in the normal regenerate. Rodian stain. Reduced 16% from X 500, approximately.

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DRACHMAN

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SINGER

this purpose (3). As indicated above, our findings showed that complete blockade of ACh transmission did not prevent limb regeneration. From the foregoing, it is concluded that ACh is neither necessary nor sufficient for limb regeneration to occur. Nevertheless, some influence derived from nerves is essential for regeneration to take place. The nature of this trophic factor remains a subject for speculation at present. The view most widely favored is that the nerves release a chemical substance which is capable of promoting regeneration. Recently, Lebowitz and Singer (8) found that infusions of homogenates of nerves from regenerating limbs, or of liver homogenates, were capable of enhancing protein synthesis in denervated newt limbs. It remains to be seen whether these tissue extracts are capable of substituting for the neurotrophic influence on regeneration or whether they merely accelerate biosynthetic processes in a nonspecific fashion. Although the regenerating limb of the newt is a particularly favorable preparation for experimental work, it is only one of many in which the nerve exerts a trophic influence on a target organ (5). Other examples include some taste buds, the barbel of certain fishes, and of special interest, skeletal muscle. It must be emphasized that the trophic influence of a nerve in one such system may differ from that of a different nerve in an entirely different system. Indeed, while ACh may be ruled out as the trophic factor in amphibian limb regeneration, it is possible that it may be the sole agent or actively associated with the agent in other systems (5, 19). References 1.

-4. S. V., F. DICKENS, and L. J. ZATMAN. 1949. The action of botutoxin on the neuromuscular junction. J. Physiol. Londm 109 : 10-24. DRACHMAN, D. B. 1964. Atrophy of skeletal muscles in chick embryos treated with botulinum toxin. Science 145 : 719-721. DKACHMAN, D. B. 1971. Botulinum toxin as a tool for research in the nervous system, pp. 325347. Ilt “Neuropoisons.” L. Simpson [ed.]. Plenum Press, New York. Goodman, L. S., and A. GILMAN. 1965. “The Pharmacological Basis of Therapeutics.” Macmillan, New York. GUTH, L. 1969. Trophic effects of vertebrate neurons. Neurosci. Res. Progrmrr Bull. 7 : 1-731. HUI, F., and A. SMITH. 1970. Regeneration of the amputated amphibian limb: Retardation by hemicholinium-3. Sciercce 170 : 131&1314. LAMANNA, C., and C. J. CARR. 1966. The botulinal, tetanal and enterostaphylococcal toxins : A review. Clb. Pharmacol. Ther. 9 : 286-332. LEBOWITZ, P. and M. SINGER. 1970. Neurotrophic control of protein synthesis in the regenerating limb of the newt, Triturus. Nature London 225 : 824G327. LENTZ, T., and R. BARRNETT. 1963. The role of the nervous system in regenerating hydra: The effect of neuropharmacological agents. J. E.@. 2001. 154 : 305-328.

BURGEN,

linum

2.

3.

4. -.i fj. 7.

8. 9.

LlhIR 10.

11.

12.

11

E. J. 1964. Purification and characterization of C. hotulinum toxins, pp. 91-103. Irr “Botulism.” U.S. Dept. of Health, Education, and Welfare, Public Health Service Publication No. 999 FPI. SCHOTTE, 0. I?. 1926. Nouvelles preuves physiologiques de l’action du syst&ne C. R. SC~IIIL~CSSm. l’hys. Hist. nerveux sympathique dans la r&en&ration. .\-otrw. GCIIEZ’C 43 : 140. SCHI.ELEK, I;. \$T. 1960. The mechanism of action of the hemicholit~iutns. Irft.

SCHASTZ.

Rcz~. Ncurobiol. 13. 14.

REGESERATIOS

Srnlrsox,

L.

2 : 77-96.

(Ed.). 1971. “Neuropoisons.” Plenum Press, New York. SIKGER. hf. 1943. The net-x-ous system and regeneration of the forelimb of adult triturus. II. The role of the sensory supply. J. Esp. ZooI. 92: 297-315. 1 .i. SIXGER. hf. 1965. The nervous system and regeneration of the forelimb of adult including a note on the anatomy of triturus. III. The role of the motor supply, the brachial spinal nerve roots. J. Exp. Zoo/. 98 : l-21. 16. SISGER, 1I. 1952. The influence of the nerve in regeneration of the amphibian estremity. Qw~t Rrzv. niol. 27 : 169-200. 17. SISGER, M. 1959a. The acetylcholine content of the normal forelimb regenerate of the adult newt, Triturus. Dczvlop. Biol. 1 : 603-620. 18. SISGER, M. 1959b. The influence of nerves on regeneration, pp. 59-80. III “Regeneration in \-ertebrates.” C. Thornton Led.]. Univ. of Chicago Press, Chicago. 19. SISGER, M. 1960. Nervous mechanisms in the regeneration of bxly parts in vertebrates, pp. 115-132. 11% “Developing Cell Systems and their Control.” Ronald Press, New Yorli. 20. SISGEH, hf. 1968. Some quantitative aspects concerning the trophic role of the nerve cell, pp. 233-245. 111 “Systems Theory and Biology.” M. D. hlesarovic [WI.]. Springer-Verlag. New York. 21. SISGEK, M.. M. H. DAVIS, and E. S. ARKOWITZ. 1960. Acetylcholinesterase activity in the regenerating forelitnb of the adult newt, Triturus. .I. Ewbr-~~~l. Esp. :~forjdd. 8 : 9X-110. 22. SIKGER, II., M. H. DAVIS. and M. R. Scrr~urn-c. 1960. The influence of atropine and other neuropharmacological substances on regeneration of the forelimb in the adult urodele, Triturus. J. Esp. Zovl. 143 : 33-46. 23. TABAX, C. 1955. Quelques problemes de ri.genCration chez les urodbles. I