Regeneration in The Vertebrate Central Nervous System1

Regeneration in The Vertebrate Central Nervous System1

REGENERATION IN THE VERTEBRATE CENTRAL NERVOUS SYSTEM' By Carmine D. Clemente Department of Anatomy, School of Medicine and the Brain Research Institu...

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REGENERATION IN THE VERTEBRATE CENTRAL NERVOUS SYSTEM' By Carmine D. Clemente Department of Anatomy, School of Medicine and the Brain Research Institute, University of California, 10s Angeles, California and the Veterans Administration Hospital, Sepulveda, California

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I. Introduction . . . . . . . . . . . . . . 11. Developmental Considerations . . . . . . . . . 111. Theories of Nerve Growth and Orientation . . . . . . IV. Regeneration in the Central Nervous System of Primitive , . . . Vertebrates V. Regeneration in the Central Nervous System of Fishes . . A. Spinal Cord Regeneration in Teleost Fishes . . . . B. Optic Nerve Regeneration in Teleost Fishes . . . . VI. Regeneration in the Amphibian Central Nervous System . . A. Studies on Embryonic Urodeles . . . . . B. Studies on Embryonic Anurans . . . . . C. Central Nervous System Regeneration in Amphibian Larvae D. Regeneration in the Central Nervous System in Adult , . . . . . Amphibians . VII. Regeneration in the Reptilian Central Nervous System . VIII. Regeneration in the Central Nervous System of Birds . . . A. Regeneration in the Embryonic Chick Central Nervous System B. Regeneration in the Central Nervous System of Adult Birds . IX. Regeneration in the Mammalian Central Nervous System . . A. Emergence of the Concept of Abortive Growth . . . . B. Theories Offered to Explain Limited Regeneration in the . . . . . . . . Central Nervous System C . More Recent Studies on Central Nervous System Regeneration . . . . . . . in Mammals . . D. Peripheral Nerve Implantation Studies and Cortical Grafts . E. The Biochemical Search . . . . . . . References . . . . . . . .

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The author's research on this subject has been supported by United States Public Health Service Grant number B987. 257

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I. introduction

That severed neurons in the brain and spinal cord of adult mammals do not possess functionally significant regenerative power, has been firmly established in the literature for decades. During the last 25 years, however, experiments involving different approaches to this same problem have indicated the possibility that a limited growth capacity exists following injury to adult mammalian central neurons. Other studies indicate little or no significant growth of CNS fibers in adult mammals. Certainly it must be admitted that an injury in the human central nervous system which destroys or severs large numbers of nerve fibers is not followed by functional repair to the extent observed following a similar injury in a peripheral nerve. This statement does not hold for all submammalian vertebrates as will be seen further in this review. The interesting phenomena encountered during the restorative events following injury in the central nervous system of lower vertebrates must form a preamble to a consideration of problems in the mammal. Since the proximal end of the severed nerve fiber encounters many of the features in its regrowth which are characteristic of initial fiber outgrowth during embryogenesis, additional factors related to developmental mechanisms should likewise be considered. Thus, a phylogenetic analysis of the problem of regeneration in the vertebrate central nervous system might well be introduced by pointing out the factors considered important in leading or guiding a developing nerve fiber to its destination during ontogenesis. II. Developmental Considerations

Experimentalists in the past have forwarded certain hypotheses in attempts to explain the basic nerve patterns that are established in the developing nervous system. The outgrowth theory of neuron development originally proposed by H. F. Bidder and C. von Kupffer and upheld by W. His, Sr. and S. Ram6n y Cajal was most firmly established by the now classical experiments of R. G. Harrison (1907, 1910) which demonstrated that nerve fibers were formed by neuroplasm anabolized in the cell body and distributed to nerve sprouts. The work which Harrison accomplished, by successfully initiating the method of tissue culture, virtually put to an end the century-old problem of nerve fiber origin and disproved such pro-

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posed ideas as the cell chain theory offered in 1839 by Th. Schwann and the protoplasmic bridge theory of Hensen (18f34, 1868), which, although valuable in their day, were shown to be scientifically inaccurate. That the periphery assumes some role in central nervous morphogenesis has long been recognized. Shorey (1909) showed that following the ablation of peripheral fields there resulted alterations in the corresponding segmental spinal ganglia and that the changes observed were dependent on the amount of skin and musculature destroyed and on the stage of development of the embryos at the time when the larvae were preserved. Such experimental conditions resulted in a reduction in numbers of spinal ganglion cells due to a failure in development, since she was unable to demonstrate degenerating nerve cells. Numerous investigators have agreed that the developing nervous system responds in some manner to alterations in the periphery (Braus, 1906; Hamburger, 1934; Detwiler, 1920, 1924a, 1926, 1927, 1936; Bueker, 1943, 1944, 1945; Hamburger and Keefe, 1944; Piatt, 1946). Detwiler (1919) transplanted limbs of AmbZystomu embryos a short distance either cranial or caudal to their normal positions. When this was done, the respective limbs were supplied by spinal nerves which normally would have innervated them. If, however, he placed the limb several segments away, the innervation was derived from segments of the spinal cord other than those which would normally innervate the limb. Histologically, sections of the spinal cord revealed hyperplasias of sensory areas in those segments from which the transplanted limbs were innervated. He noted by making cell counts that motor areas in the spinal cord were uneffected by limb extirpation and concluded that although the periphery exerted an influence on sensory neuron proliferation, the development of motor neurons remained independent of peripheral factors. Hamburger (1934), Hamburger and Keefe (1944) and Bueker (1943), using the chick embryo did not completely agree with Detwiler concerning the reactions of motor cells after wing bud extirpation or after embryos had been overloaded peripherally by implantations of supernumerary limbs. In either type of experiment these latter investigations revealed either decreased or increased numbers of cells in the lateral motor cell areas, i.e., in those areas which specifically innervated limb musculature. When, however, total cell counts,

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including the nonmotor cells, were made in the ventral horn there was little difference in the numbers of neurons in experimentals and controls. In this point, their results agreed with Detwiler’s, however their reasoning differed. It was felt that although the mitotic activity of embryonic motor neuroblasts was not influenced by changes in the periphery, the processes of motor neuroblastic differentiation were affected, and that the ratio of mature motor neurons and of potential motor neurons was a factor of peripheral regulation similar to the observations made in sensory areas of the cord. Not only has it been postulated that the outlying peripheral areas exert some influence on central neurogenesis, but additional experimental data have been presented by various investigators which either uphold or tend to disprove the thesis that intrinsic factors of an intramedullary nature also influence neurocellular proliferation and differentiation. Thus, Detwiler ( 192313) substituted spinal cord segments 3-4-5 with the normally smaller segments 7-8-9 in Amblystoma embryos. Cell counts in the transplanted segments revealed a hyperplasia comparable to the numbers of cells expected in the normal brachial segments. The same type of experimentation in various cord regions led Detwiler to the conclusion that the more rostra1 spinal-cord levels possess a greater inherent capacity for self development than the more caudal segments. Neural proliferation of the more caudal areas was dependent in large extent, therefore, on the developing longitudinal fiber tracts descending from above. That such a dependence existed in A m b l y s t m was also expressed by Nicholas (1930) and experiments by R. G. Williams (1931) tended to confirm the work in chick embroys. Williams removed segments 19-23 and inserted a mechanical block so that the lumbar cord could not receive any descending tracts. The tail was also severed caudal to the twenty-ninth segment, eliminating the ascending fibers. He found a 40% motor hypoplasia in the chick embryos and concluded that cellular proliferation was considerably regulated by stimuli from other regions of the central nervous system. Other research on this problem, however, revealed that descending tracts exerted little influence on neural proliferation and differentiation. Work by Levi-Montalcini (1945) and Hamburger (1946) which involved either extirpation or isolation of cord segments in the chick embryo (but in any event, removal of incoming fiber tracts) re-

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vealed normal motor cell counts in the spinal areas of the experimental animals. The conclusion that the periphery exerts a much greater influence on differentiation and proliferation than intracentral factors has been found in an extensive series of experiments by Bueker (1943, 1944, 1945). He has shown that after radical lower limb extirpation in the chick embryo, neurocellular hypoplasias resulted of up to 90%.Because of such extensive reductions in cell numbers, he concluded that central conduction pathways played little role in the development of lumbosacral motor elements in the cord. For much more complete reviews of the factors concerned in neurogenesis, the reader is referred to the excellent articles by Piatt (1948) and Straus ( 1946). Ill. Theories of Nerve Growth and Orientation

Through observations on the developing nervous system by the methods of experimental embryology, and through observations on nerve regeneration in the adult nervous system, there have evolved three hypotheses regarding the factors involved in eventual guidance of a growing nerve fiber toward its goal. These have been termed, respectively, the chemical, electrical, and mechanical theories of nerve outgrowth orientation. The chemical theory was advanced by Ram6n y Cajal late in the nineteenth century and states that particular specific chemical substances secreted by localized centers attracts the growing nerve fiber. Other neurologists, Marinesco and Lugaro, at the turn of the twentieth century, shared in this opinion (Ram6n y Cajal, 1928). It was postulated that from degenerating myelin and from Schwann cells in the degenerating nerve stump, there emanated chemical agents capable of attracting nerve sprouts which emerge from the proximal stump. Forssman (1900) referred to this theory as neurotropism and Cajal, who had previously discussed this relationship in the embryonic development of a nerve fiber, interpreted it as being chemotactic in nature. It was even suggested that different types of nerve fibers might be guided selectively by different chemical discharges. The electrical theory proposed that differences in electrical potentials have an orienting effect on nerve fibers. Kappers’ ( 1917,

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1921) elaboration of Strasser's (1892) ideas led to the formulation of the theory of neurobiotaxis as an explanation of how the various brainstem nuclei become arranged ontogenetically, and of their differences in anatomical location phylogenetically. In essence, Kappers states that the growth of the chief dendrite of a neuron proceeds in the direction from which the largest number of stimuli are discharging, and that there is an eventual shifting of the neuron cell bodies toward the direction of stimulus. Ingvar (1920) and more recently Marsh and Beams (1946) observed modifications in the growth of nerve processes in tissue culture by subjecting them to galvanic currents. Much direct and indirect evidence contradicts these results. Weiss (1934) and S. C. Williams (1936) did not find instances in which electrical currents resulted in an orientation of growing nerve fibers in tissue culture. Speidel (1933), working on the tail of living tadpoles, at times found nerve fibers growing in the same pathway but proceeding in opposite directions. The third theory, which to date appears to have very convincing experimental data emphasizes the importance of mechanical factors in the development of nerve patterns. The importance of mechanical influences in directing and determining the pathways of growing nerve processes was advocated by His (1887), Hamson (1910, 1914), and Dustin (1910). This theory stresses the role of solid mechanical structures as being largely responsible for nerve orientation. Unquestionably, the most outstanding work in more recent years in support of this theory has come from the experiments of Paul Weiss and his collaborators. In 1934 Weiss showed that the growth of tissue cultures composed of brain fragments or spinal ganglia of chick embryos remained unoriented when subjected to chemical or electrical stimuli. However, when the medium was stroked gently with a brush in a certain direction, the nerve processes developed in parallel paths in the same direction in which the culture medium had been stroked. According to Weiss, the stroking produced an orientation of the fibrillar or ultrafibrillar particles (micellae), and the growing nerve fibers followed the paths of the oriented medium. This phenomenon has been called contact guidance and supposedly acts indirectly on the nerve fiber through the ground substance. In another experimental procedure in which two spinal ganglia

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cultures grew in the same medium, it was observed that the processes of the explants grew in parallel lines toward each other. In this experiment, Weiss reasoned that the proliferation of the cells caused a dehydration in the surrounding medium, resulting in contraction and the eventual formation of tension forces between the two centers of growth. This tension was reflected in the ultrastructure, and the growing nerve fiber followed the tension lines. In order to disprove the chemical attraction of degenerating nerve material for regenerating in vivo peripheral nerve, Weiss and Taylor (1944) severed the sciatic nerve, extirpated the distal stump, and allowed the proximal stump to regenerate into a forked artery. These experiments were performed in the rat, and a piece of abdominal aorta including its bifurcation into the iliac arteries was used as the regenerating site. The proximal stump of regenerating sciatic nerve was placed in the aorta, and when the regenerating fibers confronted the two iliac channels at the bifurcation, the pathway of choice remained to the individual fibers. Into one iliac vessel was placed a “bait” consisting of degenerating nerve fragments. The other channel was left with no “lure” and only a blood clot filled its path. In all cases the regenerating nerve fibers divided themselves about evenly between the two routes and, the evidence did not indicate that the degenerating tissue inserted into the one iliac vessel attracted the regenerating processes. The assumption that mechanical effects are the only influencing factors cannot be established, for, indeed, even though much evidence has been presented, a good deal is indirect or negative evidence. It is conceivable that if chemical and electrical activity exert an effect on the guidance of growing nerve fibers, such influences may not be acting directly on the nerve process but indirectly through an orientation of the growing medium. Thus, the problem is not closed. Factors influencing regeneration in the central nervous system may in some respects be similar to those in the peripheral nervous system, but there are also many differences. It must first be established that regeneration is possible in the central nervous system. The literature is extremely voluminous and contradictory. An evaluation must also be made as to the type of restoration observed. Certain lower forms have a greater innate plasticity within the cen-

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tral nervous system than is evident in many of the higher animals; thus, what is often called regeneration in lower forms is in reality a differentiation of more embryonic cells into neuron types. IV. Regeneration in the Central Nervous System of Primitive Vertebrates

No information is available to my knowledge on the regenerative capacity of the central nervous system in elasmobranch or ganoid fishes, although two interesting recent studies were reported on cyclostomes. Mar6n (1959), from S. Skowron’s active group in Krakow, found that severance of the spinal cord in larvae of the European river lamprey ( Lumpetra fluoiatilis, 3-18-cm long), resulted in reconnection of the severed stumps of the cord by nerve fibers as early as 10 days after severance. Following removal of 2-3-mm segments of the spinal cord in other specimens, there was re-establishment of the cord stumps by neural tissue after 20 days. Mar6n comments on the abundant mitotic reactivity of ependymal elements characterizing the cord lesion sites and on the fact that Muller’s fibers were also restored. Similar findings were reported by Hibbard (1963) using larvae of Petromyzon marinus. V. Regeneration in the Central Nervous System of Fishes

A. SPISAL CORDREGEKERATION IN TELEOST FISHES Experimentation on the regenerative capacity of the central nervous system in fishes has been limited exclusively to work done on the teleost spinal cord, optic nerve, and retina. Forty years ago Koppanyi and Weiss (1922) transected the vertebrae and spinal cord of an adult fish ( Carussiris vulgaris) and demonstrated a functional return in the paralyzed region 6-8 weeks following section. The coordinated swimming movements were correlated with histological evidence that bundles of nerve fibers could be traced across the original site of transection (see Pearcy and Koppanyi, 1924). These latter authors continued the original work by making behavioral observations in large goldfish ( Carussius auratus, 8-14-inch specimens ) that had sustained spinal lesions indicative that “the spinal cord must have been sectioned.” Functional return was described as swimming movements which were rhythmic and coordinate behind the transection site and which required two-and-a-

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half months for maximum recovery. Although no histological studies were done on these fish, Pearcy and Koppanyi entertained the suggestion by A. J. Carlson that undeveloped nerve cells in the spinal cord above the section had retained their embryonic potentialities and had been stimulated to grow as a result of the transection. Because of the older age of the animals the authors preferred, however, to consider that “morphological regeneration” of the sectioned fibers had actually occurred (see also Koppanyi, 1926). Experiments by Nicholas on the regenerative efforts of transected spinal cord neurons in Fundulus embryos reportedly failed to indicate even a trace of morphological reconstitution ( Nicholas, 1927; Hooker and Nicholas, 1930; Hooker, 1930, 1932). This failure was explained by the high degree of determination in FunduZus tissues at an early period of development, even though Morgan (1900) had shown fin regeneration was able to occur in adult Fundulus specimens. These seemingly contradictory results were the background for the more complete studies of Hooker (1932), Tuge and Hanzawa (1937) and Kirsche ( 1950). Each of these three excellent papers established that anatomical and physiological restitution of the transected teleost spinal cord could occur, and that coordinated function returned after varying periods of time in the different species. Hooker (1932) using the young rainbow fish, Lebistes ~ e ticulatus, found that 4 days after spinal transection there was already an anatomical fusion of the severed stumps by masses of fibers issuing from both cut ends. At the transition site there were few cellular mitoses but a large number of “indifferent neural cells that had wandered out from the cord ends.” Complete physiological and anatomical regeneration of the cord usually occurred in 6 days. These studies were repeated in Hooker’s laboratory on adult rainbow fish by Keil (1940) who described functional return 2 weeks after complete spinal cord transection. Tuge and Hanzawa (1937) reported that a somewhat longer period was necessary for both morphological and physiological regeneration to occur in the transected spinal cord of adult Japanese rice minnows (Oryzias latipes). Anatomically, it was found that after 2-3 weeks a connective tissue scar had formed, filling the gap between the cord stumps. By 4 4 weeks there was a gradual infiltration and bridging of scar tissue by increasing numbers of regenerating fiber bundles. Functional recovery was first noted by the

re-establishment of complicated and coordinated movements in the caudal fin. There was then an abolition of spinal reflexes, and finally a restoration of muscle tone and the return of normal behavior. The attainment of maximum recovery was observed 3 months after the operation. Kirsche (1950) described experiments carried out on 150 adult teleost specimens ( Lebistes reticulatus) , The spinal cord was completely transected and physiological and morphological evidence of regeneration was obtained. The first stage of regeneration (4 days after the operation) was characterized by the growth of the severed spinal cord fibers in an aimless fashion. There were cones of growth at the tips of the growing fibers. These were considered truly regenerating elements and not cells restored by mitosis, which was thought to occur later. The second phase ( 7 days after the operation) was characterized by mitotic reproduction of ependymal cells which developed into neuroblasts and glioblasts. This was observed in both stumps of the cord simultaneously; the newly developed neurons sent parallel bundles of fibers across the transection site by the twelfth day, although at this time functional connections had not yet been established. Kirsche electrically stimulated above the lesion and used caudal fin movements below the lesion induced by the stimulation as evidence of established functional connections. By the fifteenth day such functional connections were observed.

B.

OPTIC

NERVEmGENER4TION

ZnT TELEOST FISHES

From the fascinating studies of Sperry (1948), functional optic nerve regeneration following its severance was shown to result in 5 different species from 3 families of marine teleost fish and in 2 fresh-water species ( Sperry, 1955)- Microscopic examination revealed copious regeneration through the site of severance in the optic nerve, which led to the re-establishment of anatomical connections between the retina and the brain. Further, he described other experiments in which optic nerve severance was combined with 180" rotation of the eye. Functional return in these fish indicated reversed vision, and 18 days after the operation, the fish exhibited optokinetic responses in the direction opposite from normal. Thus, not only was restoration of vision accomplished in these specimens, but the re-establishment of functional connections of the regenerated optic nerve fibers was orderly and systematic and seemed

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destined along some predetermined scheme. Good visual recovery in still another series of marine teleosts (Bathygobius soporator) was also reported by Sperry (1949, 1955). Furthermore, Sperry (1955) reported success in tests of color perception following regeneration of visual neurons in fishes that had been trained in a color discrimination task prior to optic nerve section. Other evidence that optic nerve fibers bypass many neurons in order to make functionally appropriate connections has come from this same laboratory ( Attardi and Sperry, 1963; Sperry, 1963). Some of the earliest experiments on the regenerative capacity of teleost optic nerves were carried out by Przibram ( 1923), Koppanyi (1923a, b, c ) , and Kolmer (1923), and these were discussed more recently by Koppanyi ( 1955). Although these early experiments leave something to be desired from the standpoint of control observations and detailed histological analysis, nevertheless, they indicated long ago the possibility that regeneration of teleost optic nerve could occur. Blatt ( 1924), impressed with Koppanyi’s “sensational communications,” reported on 340 eye transplantation experiments and 60 eye re-implantation experiments done on 3 species of fish, the carp (Cyprinus carpio), Barbus fluviatilk, and Scardinius eythrophthulmus. From the 400 fish, there were 19 cases of anatomical healing of transplanted eyes and 7 cases of healing of reimplanted eyes. In no case did Blatt feel that he had observed functional restitution and he stated that all of the transplanted eyes in his fish were blind, On the other hand, F. Ask (1926), F. Ask and Anderson (1927) and Anderson and 0. Ask (1933) described unquestionable evidence of a copious and forceful regenerative capacity of optic nerve fibers following reimplantation experiments in goldfish and in a European fresh-water cyprinoid tench (Tinca

uulgaris).

Meanwhile Matthews (1933) in Philadelphia offered a very plausible answer to the contradictory results of the various authors cited above. Using Fundulus heteroclitus (4-6 cm in length) he showed that when the optic nerve was cut in such a way that the blood supply to the eyeball was left intact, no degeneration of the retina was observed. This was followed by an extensive “neuromalike” growth of optic nerve fibers that had grown from the stumps of the severed nerve. On the other hand, when the optic nerve and the blood vessels to the eyeball were both cut, the pars optica

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retinae degenerated completely, obviating the possibility of regrowth of the original ganglion cell processes. The pars caeca of the retina in the ischemic eyes, however, did not degenerate (results similar to those of Fugita, 1913, in Triton) and within this rudiment, mitotic figures appeared and proliferated into variable amounts of new retinal tissue. Rasquin (1949) also stressed that vascularization must take place prior to degeneration of the retina and lens. She also considered it necessary for the severed stumps to be very close €or regeneration of severed optic nerves to occur in the characin, Astyanux mexicanus. Thus, she reported good functional recovery of visual feeding reactions in 5 of 12 fish with severed optic nerves. Less successful, however, were those with transplanted eyes in which the vascularization was maximally interrupted. VI. Regeneration in the Amphibian Central Nervous System

Taxonomically the amphibia are subdivided into three orders: ( a ) the Apoda ( Gymnophiona) comprised of small headed, virtually tailless members which are also limbless and almost eyeless and which are found in tropical parts of the Old and New World; ( b )the Urodela (Caudata), whose species have long tails which are retained throughout life and long bodies with short weak limbs (represented by the salamanders and newts); and ( c ) the Anura (Salientia), which are distinguished by the complete absence of a tail in the adult stage and which possess long strong hind limbs (the frogs, toads, and tree toads). Since no information could be found on the regenerative capacity of central neurons in any specie of Apoda, this discussion will be limited to findings in urodeles and anurans during their embryonic, larval, and sexually mature adult periods. A. Smm ox EMBRYONIC URODELE~ Information is plentiful on the central nervous system’s restoration potential following lesions in the brain and spinal cord of embryonic salamanders and newts. During the early phases of development there is quick and orderly return of function following spinal transecting lesions. Resumption of normal swimming movements occurred as early as the eighth postoperative day when the spinal cords of developmental stage-40 A m b l y s t m punctatum were com-

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pletely cut. Severance at the earlier stage 32 (the time of first neuromuscular reflex response to light touch) did not result in an alteration of the normal developing behavioral pattern, and the spinal-sectioned embryos began swimming at the same time as unoperated specimens (Piatt, 1955). For information on the restorative character of the embryonic urodele central nervous system at even earlier stages, one can refer to the studies of Lewis (1910), Hooker (1922, 1930), Wieman (1922, 1925a), and Detwiler (1923a, b, 1924a, 1925, 1929). Although the primary objective of these experiments for the most part dealt with the further understanding of the underlying mechanisms involved in amphibian neurogenesis, the techniques utilized and the results obtained pointed clearly to the conclusion that complete restitution can be expected following CNS lesions in embryonic Amblystoma from stage 21 (completely closed neural folds) to the stage 32 animal used by Piatt (1955). Not only could certain segments of developing spinal cord from a donor embryo be successfully transplanted into different regions of the host neuraxis, but developing donor brainstem could also be successfully transplanted to host spinal levels. Additionally, even heteroplastic transplantation of spinal segments from Amblystm punctatum to corresponding regions in the cord of Amblystoma tigrinum and vice versa were shown to be successful at these early stages ( Wieman, 1925b, 1926; Detwiler, 1931). A more recent group of papers by Detwiler (1944a,b, 1945, 1946a, b, 1947), Harrison ( 1947), Piatt (1949, 1951), Holzer (1951, 1952), Hollinshead (1952), Sibbing (1953), and others have indicated to some extent the limits and the qualitative features of this restorative process in embryonic urodeles. Thus, maximal recovery could be expected when unilateral portions of the medulla, mesencephalon, or spinal cord had been removed, whereas less complete restitution occurred following bilateral removal of the same regions. With respect to higher centers, Detwiler (1945) stated that “when the right half of the forebrain (including the optic and olfactory rudiments) is removed from embryos in stage 21, there is no regeneration.” These findings essentially confirm those of Burr (1916) that the cerebral hemispheres of Amblystoma will not regenerate in the absence of the developing nasal placode. Thus, factors other than those inherent within the developing urodele brain are also of

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significance in the restoration process observed following CNS lesions. The exact nature and the extent of these influences are still to be determined. B. STUDIESox EMBRYONIC ANURANS Similar to the process described in embryonic urodeles, a very favorable degree of restoration is observed in the central nervous system of anurans following the production of lesions during the embryonic period. From experiments as early as those of Harrison (1898) on three species of American frogs, it can be said that successful restoration of the spinal cord occurs following complete amputation of the tail bud in anuran embryos of 4 mm. Harrison, utilizing the methods of G. Born (1896, 1897), essentially confirmed and extended earlier studies of Vulpian (1859) and showed that growth of the spinal cord from one species of frog would take place into a grafted tail bud of another species. During the early part of this century, Bell (1906, 1907), utilizing 2.5-4-mm embryos of RUM fusca showed that an entire half of the brain of young frog embryos regenerates readily if the remaining half is intact. Removing the brain entirely, however, in 33-mm specimens did not result in restitution (Schaper, 1898; Rubin, 1903a, b ) . Bell ( 1907) argued that these somewhat varying findings might be explained on the basis that the central nervous system in these embryos is better capable of regenerating laterally from intact cerebral masses but its regrowth craniocaudally is more limited. Lewis (1910) removed the anterior end of the neural plate in frog embryos and noted that even after “large pieces” were removed, regeneration was practically complete and that all of the cranial nerves were present. Spirit0 (1929, 1930) called attention to the fact that increased mitotic activity over relatively long periods was an important feature of the restoration process. More recently Terry (1956) has studied the regenerative capacity of the midbrain in R a m pipiens embryos. He found that reorganization and restitution occurred in all cases of partial optic lobe excision. Animals of a slightly older developmental stage regenerated incompletely. Ferguson ( 1957) unilaterally excised the medulla in frog embryos ( R u m pipiens and R a m cutesbianu) at the neural-fold stage and earlier, and obtained complete morphological restitution of the missing half. Migration from the intact side

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and increased mitotic activity on the defective side were characteristics of the restitution. Somewhat similar results were obtained by Stevens (1959) in the brachial spinal cord. From the foregoing studies and those of Hooker (1915, 1916, 1917) and Spemann (1912), it can be concluded that the central nervous system of the embryonic anuran is at least equal to that of the embryonic urodele in its restorative capacity. Following simple severance, healing occurs per primam. If the stumps of the severed cord are intentionally not placed in direct apposition, fibers grow from each end of the cord and from the epithelioid cells of the central canal in order to establish anatomical continuity of the severed stumps. Furthermore, the elements which enter into this restorative process are derived from the cord itself and not from surrounding connective tissue. The development of behavior in such operated embryonic anurans appears generally to keep pace with that of normal animals. C. CENTRAL NERVOUS SYSTEMREGENERATION IN AMPHIBIAN LARVAE Successful regeneration following lesions in the central nervous system of urodele larvae has been described by many authors. Piatt (1955) observed both morphological and functional recovery following spinal cord transection in Amblystoma punctatum larvae of 35 and 4550 millimeters. In the younger larvae, nerve fibers had already bridged the transection site on the fifth postoperative day, and normal swimming movements occurred by the twentieth day. The older larvae (45-50mm) required a somewhat longer interval for the return of coordinated hind-limb function and of normal swimming movements but, “structurally,” Piatt states that “no essential difference in the regenerative capacity of the spinal cord was observed between the two larval series.” The source of the regenerating fibers at these stages appeared to be from already differentiated neurons, since mitotic activity was not observed at the transection sites. Interestingly, the Mauthner fiber did not regenerate in these larvae, whereas Baffoni (1952) and Stefanelli (1952) did describe Mauthner fiber regeneration in larval newts following tail amputation. On the other hand, Stefanelli (1951, 1952) did not find this fiber capable of regeneration in the caudal spinal cord of adult newts. Regeneration in the larval urodele central nervous system was

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also studied by Koppanyi and Weiss (1922) and by Weissfeiler (1924, 1925). The former reported functional recovery in larval newts 5-7 weeks following spinal cord transection while the latter quite thoroughly described the regeneration of olfactory lobes and portions of the cerebral hemispheres in the Axolot2. Actually, as early as 1864 research studies have described the regenerative capacity of urodele larval spinal cords. Muller (1864-1865) noted regeneration of the spinal column and spinal cord in triton larvae (Triton taeniatzcs and T . cristatus) and in 1885 Fraisse described regeneration in the lower spinal cord of various larval urodeles (both salamanders and newts) following tail amputation. Even more reports are available on the regenerative capacity of larval anurans. Lorente de N6 (1921) described an extensive regeneration capacity of spinal cord fibers in frog larvae (Rana, 2035 mm) and, furthermore, claimed that regenerated dorsal root fibers were capable of penetrating the spinal cord and of growing in both directions within its substance. A few years later Hooker (1925) utilized tadpoles of R a m syluatica and Rana catesbianu ranging in length from 6.75 mm (tail-bud stage) to 25 mm (hindlimb stage; in process of metamorphosis ) and carried out a complete transection of the spinal cord in the cervical region. Animals in the hind-limb series demonstrated the return of completely normal behavior 20 days to 3 months after the operation and neural transmission through the severed segment of the cord was “always accompanied by at least a fairly complete restitution of the form of the cord.” Hooker describes the outgrowth of neuraxes from both ends of the cord, the establishment of a central canal, and the proliferation and migration of indifferent cells in the original cord stumps. Recent studies have been reported from a group of Polish investigators utilizing tadpoles of the African tongueless frog, X m q u s laeois. Jordan (195!5, 1958) and Srebro (1957) reported a remarkable regeneration of the extirpated telencephalon of tadpoles and metamorphosed forms. Srebro (1957) states that “after four days the cut end of the brain is covered with a layer of ependymal cells.” Large numbers of mitoses can be observed as long as 20 days after the operation, and by this time the telencephalon is almost completely redeveloped. Jordan (1958) felt that 8 weeks was required to accomplish the same process. If, however, the olfactory organs are also removed bilaterally, regeneration of the telencephalon is

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abortive (Kosciuszko, 1958). The studies from this group have reported a lack of regeneration in diencephalic areas ( Srebro, 1959). “After total removal of the between brain regeneration of this organ is insignificant,” Srebro states, “and the regenerating optic nerves unite with the midbrain.” Kwaitkowski (1959) studied the regenerative capacity following transections in various areas of the neuraxis in Xenopzcs tadpoles and concluded that distinct connections could be found as early as the fourteenth day after section in the more rostra1 parts of the brain, whereas it required a longer interval (up to 6 weeks) for this to occur in the lower brainstem. Regeneration of the caudal spinal cord and other tail tissues following operations on and amputations of the tail in larvae of the true toad (Hyla urbmea) has been described ( Stefanelli, 1950b; Stefanelli et al, 1950a; Stefanelli et al., 1950b; Themes, 1950a, b; Santa, 1951). In the studies of Stefanelli, incipient hind-limb budstage specimens were subjected to removal of 24-mm segments of the caudal spinal cord. As early as 8 hours after the operation, undifferentiated ependymal elements created ampullae at both stumps and by the fourth day, these outgrowths came into anatomical continuity. D. REGENERATIONIN AMPHIBIANS

THE

CENTRAL NERVOUS SYSTEMOF ADULT

1. Urodeles Although many experiments have been reported on the regeneration capacity of the adult urodele tail and caudal spinal cord, only a few studies have dealt with other regions in the adult urodele neuraxis. Piatt (1955) using the Japanese water newt (Triturus Pywhogastm) carefully and completely severed the spinal cord in the middle trunk region. These animals lost their ability to swim and there was no functional use of the hind limbs 24 hours after the operation. In 8-10 days, first placement movements on land were noted in the hind limbs and, thereafter, walking improved rapidly. Coordinated fore-limb and hind-limb motion was noted 70 days postoperatively. Between 90 and 120 days, rhythmic swimming coordination was achieved and “the behavior of the oldest animals (175 days) was normal in all respects.” Piatt retransected above the level of original severance in one regenerated cord and

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noted an immediate resulting paralysis, stressing that true functional recovery had, indeed, occurred. There was no obvious evidence of cellular proliferation or increased mitotic activity. Successful regeneration had, therefore, occurred from the severed fibers and not by neocellular replacement. Another very complete study on 6-14-cm specimens of a different species of urodele ( Arnblystma m a a n u r n ) was published by Kirsche (1956). This investigator severed the spinal cord in the lower trunk region and, in addition to studying his animals histologically, he used electrical stimulation techniques. Kirsche reported that 14 days postoperatively he obtained the first unequivocal evidence of restored function. Out of 112 animals, 85 gave a clear picture of restored motor function below the site of severance, between 12 and 95 days after the operation. Anatomically, nerve fibers were observed to have regenerated across the transection site 2 weeks after the operation. The findings of Piatt and Kirsche, although thorough and important, were not surprising in the light of many previous studies on the tail and caudal spinal cord of adult urodeles. Stefanelli and Cervi (1946) extirpated 5-6-mm segments of the spinal cord near the base of the tail in adult tritons and noted that after 2 months the newly formed spinal cord segment appeared normal. Earlier Stefanelli (1944) and Stefanelli and Capriata ( 1944) had amputated the tails in the adult newts, Triton cristatus and Triton taeniatus. Three processes were thought successful in accounting for the attainment of a regenerated caudal cord: first a process of degeneration, then a process of cellular migration of ependymal elements from the remaining cord and finally a process of proliferation and multiplication of the cellular elements. Many investigators, before the turn of the twentieth century, were interested in the process of tissue regeneration. The urodele tail served well as an experimental model and, although many observations were made on the consequent regenerative capacity of the spinal cord in these studies, the primary objectives were often broader and extended to tissues other than the nervous system. Amongst the earlier workers that described regenerated urodele tail spinal cord were Fraisse (1885), Colucci (1884), Barfurth (1888, 1891), Caporaso ( 1889), Sgobbo ( 1890), Goldfarb ( 1909), S’imoes-

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Raposo (1922, 1923, 1925), Duesberg (1922, 1924a, 1924b) and McCreight ( 1924, 1931). Others ( SchottB, 1926; Locatelli, 1924, 1925, 1929) were interested in the influential role of the nervous system within the general question of tail regeneration. 2. Anwuns Studies on regeneration in the postmetamorphic anuran central nervous system have been limited in number and those that have been published deal primarily with spinal cord or optic nerve. Masius and Vanlair (1869, 1870a, b) reported partial regeneration of the severed spinal cord in adult frogs following the excision of 2-mm segments. Physiological regeneration resulting in a return to “normal function as well as anatomical restitution of neurons which successfully developed neural processes” were reported by these authors. Certain subsequent studies were unable to confirm these results (Sgobbo, 18%; Marinesco, 1894) setting the background for Hooker’s (1925) finding that there was a gradual reduction in the regenerative capacity of the frog spinal cord as the animal approached metamorphosis. More recent studies (Piatt and Piatt, 1958; Jordan, 1958; Roguski, 1959) have shown clearly that although significant intraspinal regeneration can occur in the early postmetamorphic and adult frog (see Figs. 8 and 13in Piatt and Piatt, 1958),it is more the exception than the rule. In this respect perhaps adult X m w p u s Zumi.~ responded somewhat better than Runa pi@em. Why does significant regeneration occur only in a few adult frogs? What factors endow the CNS fibers of some animals with a higher capacity for growth while in others of the same species and age there results a virtually complete failure? Are these factors genetic, biochemically metabolic, or environmental? What combination of factors leads to success? No one really knows the answers to these questions although extensive regeneration of the optic nerve usually does occur in adult urodeles (Matthey, 1927; Stone and Chance, 1941) and anurans (Sperry, 1944), and functional return to a remarkable degree of performance following optic nerve severance has been observed. Since an excellent review stressing the ontogenetic implications of the visual system problem has been published in this journal recently (Gaze, 1960), the author will not comment further on this subject.

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VII. Regeneration in the Reptilian Central Nervous System

The works published by Stefanelli and his collaborators have stressed the events coincident with regeneration of the tail in lizards following the periodic tail amputation that occurs naturally in these animals. These studies followed their observations on amphibia and have been limited to animals from two suborders of the order Sauria, the Geckones, and the Lacertae. Marrotta (1946) and Stefanelli (1951) in Lacerta mudis and Zannone ( 1953) in the gecko, Tarantola muuritunica, concluded that regeneration of the caudal spinal cord following amputation commences by an invasion of the blood clot with connective tissue elements, and an ameboid migration of ependymal elements which effectively obstructs the lumen of the central canal. This is followed by a proliferation of the ependymal cells and a regrowth of nerve fibers from above to form a regenerated caudal spinal cord. This grows into the center of the cartilaginous spinal column, which forms the skeleton of the lizard's new tail. Since both the motor and sensory innervation of the regenerated tail is derived from ganglia rostra1 to the site of amputation ( Temi, 1920; Stefanelli, 1950, 1951; Zannone, 1953), the spinal regenerate begins to involute, probably because of a lack of peripheral connections. The once regenerated caudal spinal cord regresses to a thin-walled filamentous ependymal tube by the third month after tail amputation. Marrotta (1946) concluded that regeneration of the caudal spinal cord in Lacertae occurs and that the mechanism of ependymal proliferation, differentiation and regrowth are similar to that seen in the amphibian urodele. To this author's knowledge a systematic study of the regenerative capacity of the adult reptilian spinal cord following transection at various levels and utilizing acceptable physiological and neurohistological techniques does not exist. A short description of spinal transection experiments again in the tail of Lacertn niuralis by Themes ( 1950), however, provides reason to believe that care must be taken in such experiments to insure an adequate blood supply in both spinal stumps. If this is done, there is reason to predict that successful regeneration might occur in the adult reptilian spinal cord (Rossi, 1910a, b). Effective experiments in these species, however, await investigation. It has been recognized since the studies of Gegenbaiir ( 1862), MiiIIer (1863, 1864-

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1865) and Fraisse (1885) that the spinal cord in the lizard tail is in continuity with the more rostra1 spinal cord, but it is composed simply of ependymal epithelium with some nerve fibers and neuroglia (Hughes and New, 1959). It seems that higher spinal transections or cerebral lesioning experiments would reveal more conclusive information on the capacity of adult reptilian central neural elements to regenerate. VIII. Regeneration in the Central Nervous System of Birds

A. REGENERATIONIN

THE

EMBRYONIC CHICKCENTRAL NERVOUS

SYSTEM

Although a number of studies have been carried out on the restorative capacity of the central nervous system during the early developmental stages in the chick, very little work has been done in older embryos ( Hamburger, 1955). During the early developmental stages, experimental evidence warrants the statement that some degree of regeneration occurs. Waddington and Cohen (1936) removed one lateral half of the forebrain in embryos of 5-25 somites and showed that the organ could remodel itself into a complete forebrain. The repaired forebrain even induced the formation of a nasal placode from the overlying nonpresumptive nasal epidermis, but the formation of the optic evagination did not occur if the optic vesicle had been entirely removed. These findings were confhned and extended by Spratt (1940) who also pointed out that the restorative powers diminished in older specimens. Less success was obtained in operations involving complete removal of the forebrain, following which there was no replacement from the midbrain, but simply a nonneural healing anteriorly. Regeneration within the chick brain during early embryonic development, thus, mimics the situation observed in other areas of the body. If a complete presumptive region is removed, there is no regenerative replacement, but if a portion is left, it may be capable of replacing the entire region (Weiss, 1939; Waddington, 1952). Bearing this premise in mind, it is not surprising that when extensive bilateral ablations in developing chick spinal cord or brainstem have been performed, regeneration has been rarely described. On the other hand, when Kallen (1955) extirpated one or two hindbrain neuromeres on one side in embryos and allowed them to survive to

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an age of 4.5-7.5 days, almost half of the cases showed a regenerating cell mass developing in a normal way. The regeneration was correlated with an increase in mitotic activity on the intact side, but bilateral extirpation was wt followed by regeneration. The positive results obtained following hemiablation in the forebrain (Waddington and Cohen, 1936) and in the spinal cord (Watterson and Fowler, 1953) were confirmed by Kallen (1955), and were contrary to the contention of Wenger (1950) who claimed that lateral halves of the chick neural tube were incapable of regulation or regeneration. Other pertinent studies on the regenerative capacity of the developing chick central nervous system include the earlier findings of Hoadley (1925) who transplanted the midbrain of 48-hour chick embryos to the choriallantoic membrane. Growing central tract fibers emerged from the transplant and large fascicles innervated the host tissue in the vicinity of the implantation. R. G. Williams (1931) found that the 56-hour chick embryo spinal cord “showed remarkable powers of reconstitution.” In attempts to produce isolated areas in the cord, he resorted to the use of mechanical blockades such as packed egg shelling instead of merely transecting the spinal cord. Rhines (1943) and Rhines and Windle (1944), producing lesions in the midbrain and hindbrain of chick embryos of 30-40 hours incubation, also reported a case in which regeneration had occurred in a descending bundle of the medial longitudinal fasciculus and the individual fibers mingled freely with the elements in the posterior stump. In over half of their experimental specimens central nervous system regeneration was evidenced by emergence of nerve fibers from the brain into the surrounding mesenchyme. In an extensive series of experiments, Clearwaters (1946, 1954) produced transections in spinal cord areas of embryonic and newborn chicks. Her operations were performed behind the wing-bud region at the levels of the twenty-first and twenty-third somite and in some cases one or two segments of the cord were removed. Embryos operated on after 48 hours incubation showed essentially a complete repair through the lesion site 6 days after transection. When operations were performed on incubated embryos of 72 hours, longitudinal spinal cord sections taken on the fifth day revealed bundles of nerve fibers crossing the gap to join the two stumps of the cord. An animal operated on after 96 hours of incubation and

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sacrificed after hatching showed “no impairment of voluntary function of its legs and appeared to be perfectly normal in its reactions.” Histological examination of the cord revealed a scar in the dorsal cord region and many regenerating fibers in the ventral areas. Interestingly enough, the dorsal scar area was composed of neuroglial tissue, and very few fibers succeeded in penetrating this portion of the scar, Other animals operated on after hatching and sacrificed up to 5 weeks later revealed that the ends of the cord were completely rounded off by neuroglial tissue. Regenerating nerve fibers turned laterally or medially when this barrier was reached and the physical resistance offered to regeneration by the neuroglial proliferation was evident, When certain fibers did succeed in breaking through this barrier, they could be traced into the inter-stump region which consisted of a wedge-shaped connective tissue scar continuous with the dura. Thus, it was shown in these prenatal birds that a forceful regeneration process commenced, and that the majority of the regenerative vigor was spent through the blocking property of the neuroglial tissue. Glial proliferation to injury appeared only in animals studied after the sixth day of incubation and embryos studied in later stages showed a marked decrease in success of spinal cord fibers bridging the site of transection even though the forceful innate regenerative or reparative properties of the neurons were still present.

B. REGENERATION IN THE CENTRAL NERVOUS SYSTEMOF ADULTBIRDS Among his many studies dealing with the central nervous system, Brown-Sequard (1848, 1850, 1851) also studied the regenerative capabilities of the adult pigeon spinal cord. He reported that if the cord was severed “immediately behind the wings” the animals appeared to recover and would show voluntary movements between the third and sixth postoperative month. He claimed that by the fifteenth month the gradual restoration of function resulted in a motor and sensory return that was almost normal. Voit (1868) ablated a portion of the cerebral hemispheres in a pigeon and after 5 months, he found within the area of ablation a cystic mass whose walls consisted entirely of nerve cells and fibers. His observation might be questioned since the cystic mass described may have become larger than the original lesion site and may have invaded areas of undamaged tissue. Grunert (l899), after hearing of Voit’s often

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quoted findings, made lesions in the cerebral hemispheres of 14 pigeons and was unable to detect any regeneration of central elements in his animals. He described neuroglial scars which did not contain nerve fibers or neuron cell bodies. A few years earlier Sgobbo (1890) was unable to confirm the older findings of BrownSequard and found neither anatomical nor physiological evidence of regeneration in the adult pigeon spinal cord. A description of the results of Sgobbo also appeared two years later in Archives de Physiologic (Gley, 1892). To this Brown-Sequard ( 1892) responded that the reasons for the success of his experiments were the meticulous postoperative care that he rendered his animals and the fact that he was patient enough to await the long period of time that functional return required. Furthermore, Brown-Sequard claimed that 4 anatomists had assisted him and each had agreed that regeneration must have occurred in the adult pigeons. It should be remembered, however, that very limited histological procedures were in vogue in the midnineteenth century, when Brown-Sequard carried out his experiments, methods not precise enough by today's standards to be considered critical. During the twentieth century, very few significant studies have been carried out on the regenerative capacity in the central nervous system of adult birds, although Foster (1911) in Ram6n y Cajal's laboratory described degenerative and regenerative events following spinal cord lesions in newly hatched chicks and pigeons. Since her animals were allowed only to survive for about a week following the production of lesions, her conclusions were more relevant to degenerative events than regenerative phenomena. A more thorough study was reported by Cattaneo (19.23) on optic nerve regeneration in birds and rabbits. He reported experiments on 18 chickens, 4 pigeons, and 1 falcon, and in some animals he not only severed the optic nerve but also inserted between the cut stumps excised pieces of the trigeminal nerve or the peripheral end of a severed branch of the trigeminal to act as a guiding path for the newly regenerated fibers. It is interesting that the greatest success was achieved in animals in which a peripheral nerve graft had been employed. Regenerated optic nerve fibers were described emerging from both the retinal and central stumps of the severed optic nerve in chickens, 23,35, and 40 days after operation. Instances of complete traversing of the lesion site by regenerating optic fibers were observed with

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the Cajal silver-nitrate impregnation methods. Cattaneo appeared to be influenced toward a neurotropism interpretation of his successful experiments. A detailed analysis of spinal cord regeneration in adult birds still remains to be done. IX. Regeneration in the Mammalian Central Nervous System

A. EMERGENCE OF THE CONCEPT OF ABORTIVE GROWTH

Regeneration in the central nervous system of mammals has interested some of the greatest experimental neurologists that have lived. Brown-Sequard, Bielschowsky, R a m h y Cajal, Tello, Marinesco, and Ranson, to name only a few, each contributed to some extent to an understanding of the basic problems involved. An extensive literature in the latter half of the nineteenth century developed around this fashionable research problem. Papers written during this period usually contained some of the most dramatic claims of success or the most dogmatic denials and negations. Many of these works, however, must be viewed critically because of the inadequacy of the histological procedures used. One of the earliest and more frequently quoted articles is the one of Dentan (1873). He performed lumbar cord severances in young dogs, but of four operated animals, two died after 2 days, one died after 3 days, and still another died after 7 days. Despite such poor postoperative success, Dentan reported complete active motility in the seven-day animal. Histologically he observed “normal” nerve fibers between the two stumps in the anterior cord, while in the region of the posterior cord, he described a connective tissue and glial scar containing no nerve fibers. His histological description coupled with his reported functional restoration by the seventh day after transection casts doubt on the operative procedure and the completeness of bansection. During the last quarter of the nineteenth century many German, Italian, and French investigators studied the results of spinal cord lesions in mammals. The works of only a few will be reviewed. Eichhorst and Naunyn (1874) crushed the spinal cord of rabbits and dogs. The gap was ‘infiltrated with neuroglial tissue after three to five weeks.” They found nerve fibers in the scar tissue but denied spinal cord regeneration believing that the fibers originated from the

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spinal ganglia. It has since been shown that the method of intradural spinal crushing is not satisfactory to insure a complete cord transection; additionally, it results in excessive traumatic degeneration. Schiefferdecker (1876) reported no signs of regeneration in the cord of spinal dogs up to 300 days following transection. He explained early functional restoration such as that described by Brown-Secjuard in the pigeon on the basis of spinal reflexes below the transection level. While Schiefferdecker examined traumatic and secondary degeneration most profoundly, the regenerative events were treated quite superficially. Kahler (1884) also refuted regeneration in the spinal cord of the dog and reasoned that the central nervous system contained no cells of Schwann. Taking a different view a decade later, Stroebe (1894) described fine nerve fibers within the scar tissue between the stumps of the transected spinal cord in rabbits. A few fibers appeared to bifurcate and cross the scar tissue to gain access to the opposite dorsal columns. Using the anilin blue staining method, Stroebe thought that transected spinal cord fibers commenced to regenerate. However, a true restitution of spinal cord tissue does not occur (“es aber zu einer eigentlichen Regeneration von Riickenmarksgewebe nicht kommt.”). Stroebe’s studies received important confirmation 12 years later by one of the most outstanding neurological scientists of the day, Max Bielschowsky. Working in Berlin under the direction of Oskar I’ogt, Bielschowsky (190s) described newly grown fine nerve fibers in the peripheral areas of brain tumors. He noted that these fibers had terminal boutons and other forms of terminal arborizations and, therefore, concluded that true regeneration of central fibers had occurred. In 1909 Bielschowsky extended his brain tumor studies to include similar observations on tumors of the spinal cord which apparently also attracted regenerated intraspinal fibers. At about this same time, Fickler (1901, 1905), Nageotte (1906), Marinesco and Minea (1906a,b) and the Spanish school headed by Cajal established once and for all the concept that regeneration to some degree was capable of occurring following lesions in the mammalian central nervous system. Rambn y Cajal (1906a, b ) transected the spinal cord of young cats and dogs and using his finer histological methods reported that large numbers of the severed intraspinal fibers sprouted new proc-

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esses with cones of growth similar to those observed in his studies with peripheral nerves. After 4 or 5 weeks, however, the numbers of regenerated fibers diminished, and he concluded that the processes of regeneration were followed by atrophy and absorption. In other experiments, he also described regeneration of fibers in the white matter of the cerebral cortex in newborn dogs. The work on degeneration and regeneration by Cajal and his associates in Madrid culminated in the publication of a most extensive and scholarly experimental treatise, which in many respects still stands as Cajal’s greatest work ( R a m h y Cajal, 1928). The experiments of Stroebe, Fickler and Cajal and the astute observations of Bielschowsky, thus, were especially responsible for the thesis that the regenerative efforts in the central nervous system of adult mammals resulted in abortive growth. In their opinions central nerve fibers commenced to regenerate, but for some reason, the newly formed sprouts would not continue across the transection site and make functional connections in the opposite stump.

B. THEORESOFFEREDTO EXPLAIN LIMITED REGENERATION IN THE CENTRAL NERVOUS SYSTEM One can ask: If regeneration of central nerve fibers commences,

what factors are responsible for aborting the growth? In the first place, it must be established that replacement of lost neurons in the adult mammal does not occur to any significance as a result of a differentiation of new cells from existing, less differentiated elements. Nor does any neuronal division occur to any significant degree. This latter phenomenon is so rare that if it is ever considered to be encountered, investigators feel obliged immediately to report the findings (Altman, 1962). In mammals, it is generally stated that mitotic division of neurons ceases either during prenatal development or shortly after birth. Addison (1911) reported that mitosis did not occur in the rat cerebellum after the twenty-first postnatal day, and using tritiated thymidine, Sidman and Miale (1959) and Miale and Sidman (1961) found thymidine incorporation only through the tenth postnatal day in mouse cerebellum. Essentially the same facts have been reported for the spinal cord and for other areas in the central nervous system of mammals (Buchholtz, 1890; Sclavunos, 1899; Allen, 1912). It becomes evident that without the availability of new neurons, provided by differentiation and mitosis

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in lower forms, the central nervous system of adult mammals must depend on the ability of the severed fibers to sprout and to develop new processes, In other words, regeneration in the mammalian central nervous system means the regrowth of severed neuron processes rather than a restoration or restitution of new neuron cell bodies. This lack of cells, potentially capable of differentiation into new neurons, certainly places the mammalian brain and spinal cord to a distinct disadvantage with respect to functional repair in comparison to lower forms. Other investigators have compared central nervous system regeneration events with phenomena that occur in the peripheral nervous system, and have reasoned that in the brain and the spinal cord there are no Schwann cells and, hence, little or no regeneration. Yet the exact metabolic role of the Schwann cell in the peripheral nerve function is poorly understood and little evidence exists to support the idea that peripheral nerve fibers fail to regenerate in the absence of the Schwann cells. It must be pointed out that the neurilemma tubes and Schwann elements in the peripheral nervous system do afford the regrowing fibers with a means of parallel alignment and physical guidance according to the theories of Weiss ( 1934, 1936). In addition, regenerating peripheral nerve fibers guided through neurilemma tubes regenerate about 3 times faster than those fibers oriented away from these distal elements (S. C. Williams, 1930). Nevertheless, peripheral nerve fibers oriented away from neurilemma tubes still regenerate. The outgrowth of embryonic, maturing, and adult central neurons in tissue culture and the reports of so many investigators on limited regeneration of central fibers in vivo strongly indicate that the Schwann cell is not indispensable for some fiber growth to occur. Others have hypothesized that functional regeneration within the central nervous system does not occur because of damage inflicted on the neural tissue through disturbances in the vascular pattern following injury. Adequate vascularity is unquestionably an important feature in regeneration of tissue anywhere in the body. Hunter and Royle (1924) discussed this question following the production of lesions in the central nervous system of adult animals, pointing out that vascular disturbances following spinal cord transection may bring about an “isolation dystrophy” leading to chromatolysis in the ischemic zone. Hooker and Nicholas (1930) also

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felt that vascular disturbances potentially determined the postoperative results in their animals. They ascribed to this factor the primary reason for their failure to observe forceful regeneration of spinal cord fibers in rat fetuses. There is no question but that neural tissue is delicately sensitive to interference with its nutrition and homeostatic conditions, and it is common knowledge that neurons cease to function normally within a few seconds following alterations in circulation. An interesting thought in the light of present day electron microscopy was the hypothesis commented on by Clark (1943) that the density and texture of the tissues of the central nervous system are unsuitable for the growth of nerve fibers. It has been shown that brain tissue is much more compact than was once considered. Membrane physiologists postulated the existence of extracellular tissue spaces comparable to that seen in most other organs. Such spaces, however, do not exist (Schultz et al., 1957; Maynard et al., 1957) and the neuroglia with their cytoplasmic processes fill all of the interstices of the neuropil. The intracellular aqueous cytoplasm of certain neuroglia may act as the extraneuronal fluid of the brain. Perhaps the delicate tip of a regenerating fiber cannot travel far before it meets a glial membrane that offers resistance to further advance. This resistance may even become exaggerated if reactive neuroglia begin to divide to form glial scars following the production of lesions in the CNS. Might not abortive growth be primarily the result of the physical resistance of membranes rather than an incapability of the CNS fiber to grow? C. MORERECENTSTUDIESON CENTRALNERVOUS SYSTEM REGENERATION IN MAMMALS During the last three or four decades, a number of investigators, employing methods different from those of Cajal and his contemporaries, have broadened our views of the regenerative capacities in the CNS. Most of these experiments have been carried out in rodents and carnivores. The studies of Gerard and Koppanyi (1926), in which the spinal cord of rat fetuses in utero, neonatal rats, and young adult rats was transected, indicated the possibility of functional return, although histological proof of spinal cord regeneration was lacking. Hooker and Nicholas (1927, 1930) and NichoIas and Hooker (1928) using an electrocautery as well as a scapel to tran-

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sect the spinal cords of rat fetuses concluded that there was no evidence of functional regeneration even though “some fibers may begin to regenerate only to atrophy before becoming functional.” Shortly thereafter, Gerard and Grinker (1931) conceded that there was little crucial evidence of spinal cord regeneration in young rats and attributed the return of function in the hind limbs to the initiation of spinal reflexes below the level of transection. A further contribution by Gerard, nine years later (Sugar and Gerard, 1940), stimulated interest in the subject once again. These investigators demonstrated successful regeneration of intrinsic nerve fibers in transected spinal cords of rats, most of which had been operated on at an early age. They observed the return of spontaneous hind limb movements and then succeeded in obtaining contractions of the leg muscles upon electrical stimulation of the cerebral peduncles, Correlated with the physiological studies were histological preparations which demonstrated regenerated nerve fibers through the area of transection. Much of the functional return was noted during the second postoperative month. Bundles of regrown fibers bridged the lesion between spinal cord tracts of both cut stumps. In some animals, these authors placed muscle and nerve implants in the gaps at the site of transection, and they reported the greatest success when the implants were oriented in a longitudinal direction. Although some have questioned Sugar and Gerard’s positive observations of regeneration in the rat spinal cord (Bernard and Carpenter, 1950; Feigen et al., 1951) much confirmation for this earlier work exists in the thousands of animals studied by L. W. Freeman and his group over the past fifteen years (Freeman et al., 1949; Freeman, 1949, 1952a, b, 1954, 1955). In young rats, kittens and puppies it was concluded that functional regeneration could occur and that some of these processes could be influenced by meticulous postoperative care, drug administration ( Gokay and Freeman, 1952; Stokes and Freeman, 1951) or ionizing radiations (Turbes ef al., 1960). At about the same time that Freeman and his collaborators were studying regenerative features in the rat spinal cord, Windle and Chambers ( 1950a, b, 1951 ) described regeneration of nerve fibers in the transected spinal cord of adult cats and dogs which had been used in experiments designed to determine the central site of action of bacterial pyrogenic induced fever. The spinal cords of 4 animals

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living 20 to 59 days after the operation revealed new growth of intraspinal neurons into and across the cut. Confirmation and neurophysiological extension of these earlier studies by Windle and his collaborators soon followed (Clemente et al., 1951a; Windle et al., 1952b, 1953; Clemente and Windle, 1954; Scott and Clemente, 1951, 1952,1955). These were summarized in a monograph edited by Dr. Windle in 1955. It was shown that regenerated intraspinal fibers in adult cats were capable of anatomical regeneration, of traversing the site of transection, and of conducting impulses into the opposite stump for distances of up to 30 mm. Ascending tracts have also been shown to react similarly (Liu and Scott, 1958). In these studies significance was attached to the fact that pyrogen-treated animals revealed scars at the transection site which were more vascular and of a looser connective tissue matrix than in untreated animals. It was reasoned that these conditions presented to the regenerating fibers an environment more favorable for growth. In no animal, however, could it be established that transynaptic regeneration had resulted in useful functional regeneration. An interesting observation in these studies was the fact that pyrogen-treated animals showed a variable but consistent diminution in glial scarring at the lesion site which appeared to be beneficial to the growth potential of intraspinal nerve fibers. Recent advances in neurosurgical procedures have also been reported to be of benefit to nerve regeneration, both peripherally and in the spinal cord. Campbell and Bassett with their associates have described the use of a porous membrane-like filter sheet called Millipore, already well known to tissue culturists, to assist in the alignment of severed stumps of peripheral nerves (Campbell et al., 1956; Campbell and Bassett, 1957; Noback et al., 1958; Campbell et al., 1961). The linear arrangement of regenerated peripheral fibers and their success in the bridging of interstump gaps led to the use of these techniques in transected spinal cord studies in adult cats (Campbell et al., 1957a, b; Bassett et d.,1959). Encasing the spinal stumps with Millipore tended to orient regenerating spinal fibers longitudinally, and pial and glial cells migrated cephalad and caudad along the inner surface of the Millipore, thus, eliminating a dense scar between the cord stumps. Electrical stimulation of spinal cord fibers in these animals was capable of evoking conducted potentials through and beyond the site of transection (Thulin,

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1960a, b). In many respects the results reported by Thulin were similar to those of Scott and Clemente (1955), but once again the return of function below the lesion was never established. Experiments by Lance (1954) did not reveal even an attempt at regeneration of pyramidal fibers severed at the medullary level, despite the almost universal finding of at least abortive regeneration of central neurons by others. Lance noted no differences in scars of pyrogen-treated animals and controls with the exception of the 32day animals of his series. He suggested either a variance in regenerative capacity of different central neurons of a deterioriation of the pyrogenic substances which had been used in his experiments. Arteta (1956), using Pyrogens 5 and 3895 (Merck) demonstrated modifications in central scar formation with these substances. The cicatrix in the spinal cord lesions of his pyrogen-treated cats was made up of a matrix rich in reticular tissue and macrophages and “better vascularized than the controls.” The scars in the spinal cords of his treated animals resembled those of pyrogen-treated animals in the series of Windle and Chambers (195Oa) and Clemente and Windle ( 1954). Arteta { 1956) felt that regeneration of central nerve fibers was impossible because of the lack of an adequately arranged guiding system and not because central nerves were incapable of growth. The lack of neurilemma and of an “adequately arranged guiding system” in the spinal cord and brain certainly does not help a central regeneration process, but it cannot explain why neuroma formation is not observed in a severed spinal cord. It has been the author’s experience in the past that intrinsic spinal fibers do regenerate (Clemente and Windle, 1954), but even in the most successful animals the rate of regeneration was much slower than in peripheral nerve. Scott and Clemente (1955) were able to record impulses along regenerated spinal fibers only as far as 3 cm below a site of transection, even though animals were treated and allowed to live for as long as 17 months following complete spinal transection. Perhaps no sites existed on the postsynaptic neuron membrane at the time of need by the presynaptic regenerating fibers. A new means of producing controlled lesions in the central nervous system was recently described by Malis et al. (1957). By the use of monoenergetic, heavy, ionizing particles, such as those

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emitted from the 60-inch cyclotrons at Brookhaven and Berkeley, laminar lesions of the cerebral cortex were produced. Sharply defined narrow cortical zones could be completely destroyed with a minimum of injury above and below the lesions (Malis et al., 1960). Several weeks following radiation injury, abundant sprouting from adjacent cortical fibers was observed (Rose et al., 1960). Prolific regrowth of severed apical dendrites and other cortical fibers often created an “artificial zonal lamina” when higher radiation doses caused necrosis to occur in the more superficial cortical layers. These authors present the alternative hypothesis that their results may represent an expression of a normal continuous growth of all neurofibrils that may be characteristic of these cells rather than a mere regenerative response to injury. D. PERIPHERAL NERVEIMPLANTATION STUDIESAND CORTICAL GRAFTS

Cajal interpreted that success in peripheral nerve regeneration resulted from the existence of some neurohumor, probably emanating from the cells of Schwann, which positively influenced the outgrowing fibers in a neurotropic sense. Although he did not express that abortive regeneration in the central nervous system was due to a lack of Schwann cells, he suggested to Tello that grafts of peripheral nerves placed in the brain may attract regenerating central fibers. Tello (1911a, b) predegenerated pieces of sciatic nerve, implanted them into the cerebrum, and noted extensive growth of “central” fibers into the graft after 2 weeks. These signs of regeneration later vanished, however, as the implant was resorbed. Ortin and Arcuate (1913) and Cattaneo ( 1923) investigated the regenerative possibilities of optic nerve fibers under conditions somewhat similar to Tello. Clark ( 1942) used homographs and inserted predegenerated peripheral nerve stumps into the brains of adult rabbits, but concluded that the fine fibers which were observed reinnervating the grafts came from meningeal nerve branches or perivascular nerves. He was unable to convince himself that intrinsic central nervous system fibers had grown into the graft. In other animals (1943) he severed peripheral nerves and placed the central end (regenerating stump) into the brain. Although in many animals the regenerating peripheral nerve fibers remained within the confines of the implant,

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he did observe growth of peripheral fibers into brain tissue when an inflammatory process had occurred around the implant. Also. peripheral fibers grew well when the stumps were inserted into the ventricular system. Success in this experiment has been achieved, however, when the temporal or mandibular branch of the facial nerve was inserted into the brain (Clemente et al., 1951b; Windle ct nl., 1952a; Clemente, 1952, 1955, 1958). By using drugs which decreased the amount of scarring around the implanted stumps, regenerated peripheral nerve fibers were observed to blend with central fibers and cells. It was felt that these experiments confirmed the hypothesis that the central nervous system is capable of maintaining regenerating fibers within its substance. There were indications that primitive mesodermal elements and reticular cells were somewhat beneficial to regenerating nerve fibers, whereas, neuroglial proliferation presented impenetrable scars to regrowing fibers. This view is also shared by Noback et al. (1958, 1959, 1962). Turbes and Freeman (1958) reported on peripheral nerve/spinal cord anastomoses made 1-2 months following complete spinal cord transection in adult dogs. Intercostal nerve trunks were dissected rostra1 to the lesion site and then severed. The proximal nerve stump was then implanted into the spinal cord caudal to the transection site. They claimed that 2 weeks after insertion of the nerve trunk. most of the animals attempted to stand and walk. In 5 dogs this progress was reversed by subsequent surgical sectioning of the inserted nerve. These same authors claim to have evidence of the reestablishment of functional transynaptic connections with motor neurons in the ventral cord (Turbes and Freeman, 1961; Jacoby et al., 1960). The fate of implanted nonneural grafts and of the implantation of spinal ganglia into the central nervous system has recently been reviewed by Glees (1955). It had been noted by Erikson and Glees ( 1953) that grafts of skin implanted into the cerebral cortex of rabbits at times contained regenerated cortical nerve fibers that had grown into the graft from the surrounding brain tissue. On the other hand, muscle grafts appeared to degenerate after a while and become replaced by connective tissue. If muscle grafts survive, however, large numbers of intracortical nerve fibers are observed to penetrate the scar surrounding the graft, perhaps in an effort to reinnervate it (Nathaniel and Clemente, 1959). These findings should

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be viewed in the light of the recent experiments of Rose et al. (1960) in which a prolific growth of cortical fibers was described. E. THE BIOCHEMICAL SEARCH

That biochemical substances exist which are capable of inducing or attracting the growth of nerve fibers has been a question that has interested experimental neurologists since the days of Cajal and Forssman. It was felt by these investigators that degenerating nerve tissues possessed these qualities, although the experiments of Weiss and his collaborators (especially those of Weiss and Taylor, 1944) cast serious doubt on the neurotropic nature of degenerating nerve. Weiss’ evidence, however, should not be interpreted as meaning that other biological systems may not possess nerve growth promoting humors. As a matter of fact, recent evidence points to just the reverse. It was found that when certain mouse sarcomas were transplanted to the body wall (Bueker, 1948) or into the allantoic membrane of the 3-4-day chick embryo (Levi-Montalcini, 1952; LeviMontalcini and Hamburger, 1953) the sensory and sympathetic systems of the host were radically affected. These appeared to be stimulated into excessive growth both with respect to increases in cell numbers and increases in neuronal size. Large numbers of nerve fibers were induced to grow, far in excess of those seen in control animals. These authors then reported that the sarcoma growth-substance was diffusable and effective in vitro. Cultures of chick spinal and sympathetic ganglia, when placed close to sarcoma explants, showed excessive numbers of nerve fibers radiating in all directions from the nerve cell culture, whereas in controls only normal growth was observed (Levi-Montalcini et al., 1954). It has since been found that even more potent nerve growth promoting properties exist in the poisonous venom of the moccasin snake, Agkistrodun piscivms ( Cohen and Levi-Montalcini, 1956; Levi-Montalcini and Cohen, 1956) and in the salivary glands of the mouse and rat (LeviMontalcini, 1958; Cohen, 1958). The growth substance is a protein and has been purified, and it appears to act directly on the nerve cell. These authors have determined that the presence of glucose or mannose is required for continued nerve growth and of the necessity of at least one amino acid, phenylalanine. Scott (1963) has indicated

some success in the use of this substance in the transected spinal cord of kittens, Exactly how the growth factor stimulates the nerve fiber, however, is not known as yet. The fascinating observations of Levi-Montalcini and her c01laborators on tumor and salivary gland extracts is a progressive step which might be considered to have started from the observation of Bielschowsky (1906) over 50 years ago. He observed from neuropathological material that nerve fibers grew into the edges of brain and spinal cord tumors. Duncan and Bellegie (1948) made similar observations after they had transplanted pieces of rat sarcoma into the pia-glial membrane and at the severed end of the spinal cord in rats. Inferred from studies on collateral peripheral nerve regeneration, nerve fibers appear to have a dynamic association with their peripheral end organs such that the fibers may compensatorily respond into new growth, following destruction of neighboring axons (Edds, 1953). The evidence indicates that the sprouting is due to the action of a humoral agent released by adjacent degenerating nerve fibers or from the cells of Schwann. Thus, reinnervation of partially denervated muscle and restoration of function by collateral regeneration in autonomic ganglia have been described (Murray and Thompson, 1957a, b ) . Anatomical and physiological evidence has also been put forward that collateral sprouting occurs in the spinal cord below the level of a partial section, presumably as a response to the injury (Liu and Chambers, 1958; McCouch et d., 1958; Teasdale et d.,1958). Attempts to modify this experiment by severing the dorsal roots in kittens and studying changes in the monosynaptic reflex in the operated segments after the animals had become adult, however, did not reveal evidence of functional colIateraI sprouts (Eccles d d.,1962). This latter evidence does not mean, however, that collateral sprouting had not occurred earlier. Yet another agent has been considered effective in stimulating nerve regeneration. A factor isolated from the white matter of the brain by von Muralt and his associates (Koechlin and von Muralt, 1945, 1947; Jent, 1945; Jent d al., 1945; Koechlin, 1955) was shown capable of increasing the rate of regeneration of corneal nerve fibers. Konig (1953) and Martini and Pattay ( 1954) felt that malononitrile and succinonitrile were also able to increase the speed of peripheral nerve regrowth and they explained it on the basis that malononitrile

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tends to increase the rate of neuronal nucleic acid production (Hydhn and Hartelius, 1948). Thus, the search continues for substances which are capable of stimulating nerve growth or of inhibiting scar formation. An understanding, however, of neuronal metabolism at the cellular level is basic to the problem. Windle (1962) points out that the answer may lie in the comparative anatomy and neurochemistry of lower forms where central regeneration comes about as an innate matter of fact with none of us realizing the biochemical stimulants involved in neural differentiation. REFERENCES Addison, W. (1911). 1. Comp. Neurol. 21, 459. Allen, E. (1912). 3. Comp. Neurol. 22, 547. Altman, J. (1962). Science 135,1127. Andersson, E., and Ask, 0. (1933). Acta Ophthalmol. 11,411. Arteta, J. L. (1956). 3. Comp. Neurol. llos, 171. Ask, F. ( 1926). Acta Ophthulmol.3, 12. Ask, F., and Andersson, E. (1927). Acta Ophthulmol.4, 97. Attardi, D. G., and Speny, R. W. (1963). Exptl. Neurol. 7, 46. Baffoni, G. M. (1952). Rend. accad. nuzl. Lincei sci. j%. e mat. e nut. 8, 189. Barfurth, D. ( 1888). Anat. Anz. 3,403. Barfurth, D. (1891). Arch. mikroskop. Anat. u. Entwicklungmech. 37, 406. Barnard, J. W., and Carpenter, W. (1950). 3. Neurophysiol. 13,223. Bassett, C. A. L., Campbell, J. B., and Husby, J. (1959). Exptl. Neurol. 4, 386. Bell, E. T. (1906). Arch. mikroskop. Anut. u. Entwicklungsmech.68, 279. Bell, E. T. (1907). Arch. Entwicklungsmech.Organ. 23,457. Bielschowsky, M. (1906). J. Psychol. u. Neurol. 7, 101. Bielschowsky, M. (1909). 1. Psychol. u. Neurol. 14, 131. Blatt, N. (1924). Arch. Ophthulmol.Graefe’s 114, 47. Born, G. (1896). Arch. Entwicklungsmech.Organ. 4,349. Born, G. (1897). Arch. Entwicklungsmech.Organ. 4 517. Braus, H. (1906). Morphol. Jahrb.35, 509. Brown-Sequard, C. E. (1849). Compt. rend. SOC. biol. 1, 17. Brown-Sequard, C. E. (1850). Compt. rend. SOC. biol. 2,3. Brown-Sequard, C. E. ( 1851 ). Compt. rend. SOC. biol. $77. Brown-Sequard, C. E. (1892). Arch. physiol. norm. pathol. Ser. V , 4, 410. Buchholz, Dr. (1890). Neurol. Zentr. 9, 140. Bueker, E. D. (1943). 3. Erptl. Zool.93,99. Bueker, E. D. (1944). Science 100, 169. Bueker, E. D. (1945). 1. Comp. Neurol. 82,335. Bueker, E. D. (1948). Anat. Record 102,369. Burr, H. S. (1916). 3. Comp. Neurol. 26,203. Campbell, J. B., and Bassett, C. A. L. (1957). Surg. Forum 7,570.

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