The relation between nerve fiber number and limb regenerative capacity in the lizard Anolis.код для вставкиСкачать
The Relation Between Nerve Fiber Number and Limb Regenerative Capacity in the Lizard, Anolis ' JOCELYN ZIKA AND MARCUS SINGER Department of Anatomy and Developmental Biology Center, Western Reserve University, Cleveland, Ohio ABSTRACT The present work provides some quantitative evidence for the view that the inability of the lizard's limb to regenerate is due to a n insufficiency of nerve fibers. The number of fibers per unit area of the amputation surface of the lizard's forelimb is approximately one-half that reported for the limb of the salamander, capable of regeneration. It is known that when the number of fibers in the salamander's limb is reduced to about half its normal value, regeneration does not occur i n about 50% of the animals. The possibility is also discussed that the lizard's limb, in addition to being less innervated than that of the salamander, is also more refractory in its growth response to the nerve. Finally, the comparative poverty of innervation of the lizard's limb relative to that of the salamander suggests that the evolutionary trend in the vertebrate series toward elaboration of central nervous mechanisms was accompanied by a decline in richness of peripheral innervation. The limb of most adult frogs and of the lizard does not regrow after amputation. However, if the amputation stump is provided with more nerve fibers by the deviation of a sizable nerve from another source, regeneration does occur (Singer, '54, '61 ; Simpson, '61; Kudokotsev, '62). In the case of the salamander which does regenerate a limb, the number of nerve fibers normally available at the amputation surface is more than adequate to support regrowth; but, if the number is reduced below a threshold value, regeneration does not occur (Singer, '52 review). From these results the theory was advanced that the absence of limb regeneration in vertebrates above the urodele amphibian is due to an insufficiency of nerve fibers to satisfy the threshold need for regrowth (Singer, '54). There are three ways in which such a quantitative inadequacy could have arisen in the evolution of the vertebrates : the number of nerve fibers per unit of limb tissue could have declined until it became threshold inadequate; secondly, the tissues which respond to the nerve to produce a regenerate could have become increasingly refractory until their nerve needs for regrowth were no longer satisfied by the available nerve supply; and, finally, the individual nerve fibers may have become decreasingly effective as agents of growth until the combined defiANAT.REC.,152: 137-140. cit rendered the available nerves an inadequate source of the nerve growth influence. Attempts have been made to assess these possibilities (Singer, '63; Singer and Mutterperl, '63; Van Stone, '64). The present work continues the evaluation and compares the quantity of limb innervation in the non-regenerating limb of the lizard, Anolis, with that for the limb of the newt, Triturus, which can regrow. The results show that the number of fibers available at the amputation surface of the lizard limb is comparatively low; indeed, it would hardly be enough to satisfy the regenerative needs of the newt's limb. MATERIALS AND METHODS The upper arm of the lizard, Anolis carolinensis, was removed and fixed in Bouin's solution for a few days, decalcified (Decal) overnight, embedded in paraffin, and crosssectioned serially at 10 u. Sections were stained for nerve fibers by the Bodian silver method, and then counterstained with orange G. A section approximately in the middle of the upper arm was selected for counting nerve fibers. The large nerve trunks were projected onto paper at a magnification of 900 with a camera lucida and the out1 Aided by grants from the American Cancer Society and the National Institutes of Health, Public Health Service. 137 138 JOCELYN ZIKA AND MARCUS SINGER line of each fiber was drawn. Then the fibers were counted by circling them in groups of five. The nerve fibers in the smaller nerves and those scattered among skin and muscle were counted at the same magnification by the method of “stripping” used by Singer (’46). A Visopan projection microscope (Reichert) was used to obtain the area of the section. The outline of each section was traced at a magnification of 50 times and the area was determined with a planimeter. The quantitative data were then expressed in number of fibers per (100 p ) z of crosssectional area according to the method of Singer (’47). Recounts of nerve fibers were performed in three cases to check the reliability of the data. The second count differed from the first by a n average of 4 . 3 % ,with a range of differences of 0.4-10.0%. (100 v ) ~respectively for the normal amputation surface of the newt (see Singer, ’46, ’47). Consequently, the number of fibers available at the amputation surface of the forelimb in Anolis is approximately one-half that found in Triturus. It is of particular interest to compare the threshold number of fibers required for regeneration of the upper arm of Triturus with the full number found in Anolis because the Anolis limb does not regenerate nor does the newt limb regrow when the fiber number is reduced below the threshold value. The average threshold in Triturus is 10.8 fibers per (100 u)’ of surface area of soft tissue, and 9.6 fibers per (100 v ) of ~ total surface area (Singer, ’47). These values are comparable to the number of fibers per unit area ordinarily present in the AnoZis forelimb, namely 12.3 and 11.7 respectively. RESULTS DISCUSSION Our results show that the lizards limb Results are recorded in table 1. The average number of nerve fibers found in has much fewer nerve fibers per volume a cross-section of the forelimb in Anolis of tissue than does that of the salamander. is slightly higher (2,588) than that (2,357) This fact would support the postulate that reported for TTiturus (Singer, ’46). The the lack of regenerative powers in the limb of Anolis is, however, more massive; lizard’s limb and in that of vertebrates and, when the number was expressed per higher than the urodele amphibian is due unit cross-sectional area, it was found that to a n insufficiency of the nerve supply in Anolis there is a n average of 11.7 fibers (Singer, ’54). A s noted previously there are per (100 u ) ~of total area and 12.3 fibers theoretically at least three ways during when the area of bone is ex- evolution of the vertebrates in which the per (100 cluded. This compares with values of 21.7 neuronal supply may have become threshfibers per (100 p)’ and 24.5 fibers per old inadequate to evoke growth : numerical TABLE I Innervation o f t h e lizard forelimb Total Bone Soft tissue Total number of nerve fibers 1 2 3 4 5 6 7 8 9 10 138 318 191 218 109 188 209 350 401 318 7 11 7 8 7 7 7 9 12 9 131 307 184 210 102 181 202 34 1 389 309 Average Std. dev. 244 2 96.7 k 1.7 235.6 2 95.0 2 342 Animal Area of surface of amputation in (100 p ) 2 8.4 Fiber number per (100 & ) 2 of surface area Total Soft tissue 2748 2863 2158 2593 2033 2328 2486 2561 3313 2497 19.9 9.0 11.2 11.8 18.6 12.3 11.8 7.3 8.2 7.8 20.9 9.3 11.7 12.3 19.9 12.8 12.3 7.5 8.5 8.1 2558 11.7 24.3 2 4.6 12.3 NERVE INFLUENCE IN REGENERATION insufficiency in relation to other tisuses; decreased potency of individual fibers; and relative refractoriness of responding tissues. Using the newt limb as a reference standard since it does normally regenerate upon amputation, we find that the forelimb of the lizard has much fewer fibers per unit volume; and, therefore, that the postulate of numerical insufficiency is, indeed, one of the causes for absence of limb regeneration in the lizard. Moreover, further support may be drawn from the results of experiments in which the lizard's limb was caused to regrow after the amputation surface was increased by deviating a substantial quantity of fibers into the limb to supplement the normal supply (Singer, '61; Simpson, '61; and Kudokotsev, '62). The two remaining postulates, namely decline in potency of the individual fibers and increased refractoriness of response of tissues to the nerve, may still be contributing causes, and therefore must remain as supplementary possibilities to explain the absence of limb regeneration in higher forms. Indeed, our results may also be interpreted to suggest that increased refractoriness of tissues to the nerve may also be responsible for the inability of the amputated lizard's limb to regrow. The number of fibers in the lizard limb is approximately that of the average threshold value for the newt. The latter value is the mean of a range of fiber values above which limb regeneration always occurs and below which there never is regrowth. Therefore, the average represents a quantity of fibers which support limb regeneration in the newt in about one-half of the instances. However, this same value, which as we have shown is close to the normal supply in the limb of Anolis, never evokes regeneration of the lizard's limb. Consequently, in addition to the numerical factor, the non-nervous tissues of the lizard's limb may be less facile in their response to the nervous agent of growth. There is some published evidence that the capacity of non-nervous tissues to respond to the nerve plays an important role in establishing threshold fiber requirements (Singer and Mutterperl, '63). When the limb of the newt is transplanted to the back, the 139 threshold fiber requirements are greatly lowered and now much fewer fibers are needed to support regrowth. There is no evidence as yet to support the third postulate, namely that the individual nerve fiber is a less potent source of the nervous growth factor in vertebrates higher than the urodele amphibians. Assuming this postulate obtains in fact, it may be due to a quantitative biochemical change within the neuron; or, it may be that the neuraxons in forms above the amphibian branch more frequently, and therefore that the nervous agent of growth is diluted in the many branches. Finally, the richer innervation of the limb of the urodele than that of the reptile suggests that the vertebrate limb was more highly innervated early in its evolution. One might have expected that the higher terrestrial forms which use their limbs in much more useful and complex ways would have a richer peripheral nerve supply. The decline in limb innervation may mean that the saturation of limb tissues with nerve fibers was of lesser survival value than the elaboration of central mechanisms for the use of peripheral information. In this way central elaboration prevailed over richness of peripheral innervation. LITERATURE CITED Kudokotsev, V. P. 1962 Stimulation of the regeneration process i n the extremities of lizards by the method of supplementary innervation. Dokl. Akad. Nauk SSSR, 142: 233-236. Simpson, S. B., Jr. 1961 Induction of limb regeneration i n the lizard, Lygosoma laterale, by augmentation of the nerve supply. Proc. SOC. Exp. Biol. Med., 107: 108-111. Singer, M. 1946 The nervous system and regeneration of the forelimb of adult Triturus. V. The influence of number of nerve fibers, including a quantitative study of limb innervation. J. Exp. Zool., 101 : 299-338. 1947 The nervous system and regeneration of the forelimb of adult Triturus. VII. The relation between number of nerve fibers and surface area of amputation. J. Exp. Zool., 104: 251-265. 1952 The influence of the nerve i n regeneration of the amphibian extremity. Quart. Rev. Biol., 27: 169-200. 1954 Induction of regeneration of the forelimb of the postmetamorphic frog by augmentation of the nerve supply. J. Exp. Zool., 126: 419-471. 140 JOCELYN ZIKA AND MARCUS SINGER 1961 Induction of regeneration of body parts in the lizard, Anolis. Proc. SOC.Exp. Biol. Med., 107: 106-108. 1963 Nervous control of the regrowth of body parts in vertebrates. In: The Effect of Use and Disuse on Neuromuscular Functions. Ed. by E. Gutmann and P. Hnik, Czech. Acad. Sci., Prague. Singer, M., and E. Mutterperl 1963 Nerve fiber requirements for regeneration in forelimb transplants of the newt Triturus. Dev. Biol., 7: 180-191. Van Stone, J. M. 1964 The relationship of nerve number to regenerative capacity in the developing hind limb of Rana syluatica. J. Exp. Zool., 155: 293-302.