SURVEY OF OPHTHALMOLOGY
CURRENT
VOLUME 20
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NUMBER 4
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JANUARY-FEBRUARY
1976
RESEARCH
EDWARD GOTLIER, EDITOR
Central Nervous System Regeneration
and
Ophthalmology STEPHEN
GOLDBERG, Department
M.D. of Ophthalmology,
New York Medical
College, New York
Various adult lower vertebrates are capable of optic nerve, and even retinal, regeneration with functional recovery of vision. Possible factors responsible for regenerative failure in mammals are discussed. It is suggested that potentially neuroregenerative agents be tested in the mammalian retina in an attempt to induce visual pathway regeneration. (Surv Opbthalmol 20:261-272, 1976)
Abstract.
Key Words: regeneration
.
axon . central optic nerve -
nervous
system
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degeneration
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neuronal
retina
T
he mammalian central nervous system posteriorly, in the visual cortex.7@-34*87 Why this striking difference between higher (CNS) regenerates poorly following inBecause the retina and and lower vertebrates? Is a relatively small jury. 18*23,43~108 visual pathways are part of the CNS, patients difficulty involved, such as lack of a simple with neuro-ophthalmological problems fre- replaceable substrate or presence of a glial quently cannot be helped. This paper reviews scar at the lesion site, or is there a more inthe possible reasons for regenerative failure in tractable underlying problem involving comthe CNS and discusses the future prospects plex biochemical interactions or inaccessible for the induction of visual pathway regenera- genetic differences between higher and lower tion. vertebrates? The mammalian visual system Following optic nerve section, certain appears very refractory to nerve regeneration, whereas these lower vertebrates appear so lower vertebrates - amphibians and fishes exhibit good return of function.3~s~8~31~47~~o~*i~@~ plastic that one may perform entire ocular The severed optic fibers grow back to their transplants between two species of salamanappropriate destinations in the brain. But in der and achieve recovery of vision.OQ~elThe the mammal, not only does regeneration salamander eye may be refrigerated for a fail, degeneration occurs. Retrograde atrophy week and still be successfully transplanted, and cell loss sets in among the retinal gang- with regeneration of the retina as well!” anterogra& de_ lion cells, 8,*6.61,66.72,97,~07,1~9 Perhaps a comparison between higher and generation damages the lateral geniculate lower vertebrates on many levels of investigaand even more distant tion will eventually help to explain the failure body, ~,*5,28,61.84.107.~0@ degenerative changes are found anteriorly in of mammalian CNS regeneration. A the inner nuclear layer of the retina and knowledge of mechanisms of optic nerve em261
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bryogenesis may also prove of value since regeneration may in large part be a recapitulation of embryonic events. This paper is not an exhaustive review of all aspects of neural regeneration and it does not suggest any single mechanism responsible for regeneration or degeneration. Rather, in the absence of enough data, it simply arranges some of the many possible mechanisms of regenerative failure into a logical framework, as a basis for future research. By understanding why regeneration fails we may find ways to induce regeneration. Growth, Guidance, and Synaptic Factors in Optic Fiber Regeneration and Embryogenesis
Axon regeneration may be generally subdivided into three processes: axonal growth (axonal elongation); axonal guidance (toward the appropriate destination in the brain); and synapse formation. AXONAL GROWTH
Although axonal growth may be defined in various ways, it is used here only in the simple sense of elongation. The normal, mature, uninjured axon is roughly fixed in length from retina to brain terminus. Once the optic nerve is sectioned new axonal sprouts appear which, in lower vertebrates, continue growing until the visual area of the brain is reached. In mammals, initial sprouting also occurs, but results only in an abortive growth. The new fibers do not bypass the glial-connective tissue scar at the site of section and, in time, they degenerate. GUIDANCE
The growing nerve fibers require some sort of guidance mechanism to insure that they reach the brain instead of growing wildly in all directions. Guidance might crudely be mediated mechanically, e.g., via a tissue plane in the region of Wallerian degeneration, or it might involve highly selective chemical or other mechanisms. It is important to distinguish between growth and guidance. The presence of one does not imply the other. Conceivably, an axon might grow quite well, but in the absence of guidance, it might grow wildly without reaching its proper destination. Likewise, the apparatus for axonal guidance
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might be functioning properly, but if the axon lacks the ability to grow, regeneration will fail again. Axon regeneration is defined here as the process wherein an axon not only regrows, but finds its way back to its original terminus. Conceivably, the same factors rendering guidance possible are also responsible for growth. For instance, a chemical released by the central degenerating axon segment may stimulate growth. Sprouts would appear wherever the chemical contacts the axon. Since the chemical would occupy the entire degenerating axon segment, growth would continue back along the original pathway. The same chemical would thus be responsible for both growth and guidance. The growth and guidance mechanisms might, however, be different, For instance, a chemical released at the site of injury might induce growth, but a mechanical tissue plane could function as the guiding factor. Since it is not known whether growth and guidance are mediated by the same factor, it is best to keep in mind the possibility of different mechanisms for each. SYNAPSE FORMATION
When arriving at the appropriate area in the brain, the axon synapses. It is believed to do so highly selectively, choosing only certain cells in a particular predetermined brain locus.31,” Given that axonal growth, guidance, and synapse formation are important to both nerve regeneration and embryogenesis, theoretically, interference with any of these could prevent effective regeneration. Eiectrophysioiogicai, Mechanical and Chemical Factors in Regeneration and Embryogenesis
Throughout this century opinions have fluctuated among three potential mechanisms for nerve regeneration and embryogenesis: the mechanical, chemical, and electrophysiological theories are briefly described below 31.4T,7&80,84.86,94,99,101,102,104,1~ ELECYROPHYSIOLOGICAL THEORY
Proponents of this theory believe that a growing neuron may be guided in the direction of change of electric potential. Marsh and Beams,BS for instance, showed that axons
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The mature visual pathway maintains an orderly map-like point-to-point connectivity between retina and brain.B~31~‘7@ After sectioning the optic nerve, combined with 180” eye rotation, reversed visuomotor responses appeared in the adult amphibian after regeneration of the optic nerve. These behavioral studies suggested that the growing fibers returned to their original synaptic terminals.3+5*77978 Histological and electrophysiological studies have also shown that regenerating axons appear specified as to destination and return to their original brain loci despite the disorientation inherent in neuroma formation and in 180” rotation of the eye and optic nerve.31s47It is difficult to MECHANICAL THEORY account for such specificity except by some Considerable evidence has been compiled chemospecilic mechanism of synapse formafor mechanical factors operating in axonal tion growth and guidance.44@‘~100~104~106 Axons in One must interpret this data with caution. tissue culture tend to grow along etchings on Whereas abundant evidence has shown a high a piece of mica, along the fibrin matrix of a degree of specification in synapse formation plasma clot, or along other mechanical planes by regenerating optic axons, this does not in vitro or in vivo. The mechanical factor necessarily imply specificity of guidance seems necessary not only for axonal guidance, toward those synaptic loci. There could, for but also for sustaining axonal growth. The instance, be initial random, widespread axaxon tip apparently requires some mechanical onal branching with subsequent retraction of framework to hold onto before it can ad- those branches which failed to make apvance. propriate synaptic connections. Or an initial Difficulties arise in trying to explain how overproduction of ganglion cells and axons crudely orienting mechanical factors alone could be followed by the death of those cells can possibly specify highly complex and axons which failed to connect appathways. One can see how fibers might propriately within the brain. Sparse evidence, generally be guided into a region, but though, does suggest some degree of elaborate, consistently appearing turns, specificity in axonal guidance. Arora2,’ branchings and interlacing patterns are diverted the optic tracts of adult goldfish into another matter. The electrophysiological foreign brain regions and then severed the optheory encounters similar difficulties. Both tic nerve and removed a portion of the retina. the electrical and mechanical theories also The surviving optic fibers selected the have trouble explaining synapse formation. originally appropriate optic tract even though How can such grossly orienting influences ac- it had been diverted to a foreign area of the count for the complex and highly orderly brain. In other experiments, when the dorsal point-to-point anatomical projection between half of an optic vesicle was removed in retina and brain? Xenopus laevis and replaced with a ventral half, a biventral eye was formed. ElectrophyCHEMICAL THEORY siological studies demonstrated that each Proponents of the chemical theory, retinal half had communicated with the particularly Sperry and his co-workers, have brain.32*02Histological studies, however, insuggested that axon guidance and synapse dicated that only the medial optic tract, which formation are mediated by select chemical normally contains the fibers from ventral factors, serving to guide axons to their ap- retina, contained optic fibers; the lateral optic propriate destinations. Evidence has largely tract, which normally contains fibers from come from indirect experiments on the visual superior retina, was devoid of fibers (Horder, system of amphibians and fishes.3-6~3’~4’~77~82~*5 T. J. personal communication). This study The reasoning is as follows. thereby supported the findings of Arora in in tissue culture grow in the direction of the cathode. Their diagrams do not appear to indicate an increase in axon growth, but rather a change in direction. Hence, there is some evidence for an electrophysiologic guidance mechanism. Other observers, however, have failed to achieve similar results.g8 Nor has the natural existence of such electrical gradients in the living organism been established, whether in nerve regeneration or embryogenesis. Various data suggest that artificial electric potentials, applied continuously, will not alter embryonic developsignificantly ment.42~ggAs a result, this theory is the least popular of the three.
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suggesting that optic fibers are capable of or electrophysiologic factors necessary for selecting the appropriate optic tract. regeneration may be functioning in the A major difficulty with the chemical theory neuronal environment, but if the severed of axonal guidance and synapse formation neuron lacks the ability to respond to them, has been the failure to find chemicals that will regeneration will fail. Likewise, the neuron attract axons. Various chemicals, e.g., cyclic may possess all the potential for regeneration, AMP, “*” “NR” factof isolated from cor- but if its environment is not conducive, e.g., tical white matter, and nerve growth factors6 because of a glial scar, failure will again have been shown to enhance growth of axons, prevail. Weiss1o3 has outlined the sites within the but it is unclear whether or not they have an effect on the guidance of axons. For instance, neuron pertinent to regeneration. Fig. 1, nerve growth factor, an isolate from mouse drawn after Weiss, illustrates that regeneraa combination of distal sarcoma and mouse submaxillary gland, has tion requires dramatic growth-promoting effects on sym- sprouting (h), axoplasmic synthesis (a), pathetic and sensory nerve cells.1~24~sB-68J12 proximo-distal conveyance of neuroplasm Axons appear to branch extensively on enter(b,c), retrograde regulative influences (d), ing the mouse sarcoma, at first suggesting a structural resistance of the axon wall (e) and a minimum of ectopic, competitive side possible guidance into the tumor. However, other neurons distant from the tumor branch branchings (f, g) and environmental interference (i). Weiss has derived the formula wildly, e.g., penetrating the skin, suggesting mainly a growth effect?’ (a,b,c,d,e,h) In summary, the main theories of growth, P=f guidance and synapse formation involve elec(f,g,i) trophysiologic, mechanical, or chemical mechanisms. Currently, the chemical theory for probability of successful regeneration. seems the best explanation for selectivity in One may add to this a (j), indicating favorable synapse formation and for guidance into the environmental influences on nerve growth, appropriate optic tract. The evidence for a guidance, and synapse formation, whether of chemical theory is indirect, however, and chemical mechanical or electrocomes chiefly from experiments showing a ;hysiologicai The formula for nature. degree of specificity in regeneration and em- regeneration then becomes bryogenesis that is more commensurate with (a,b,c,d,e,h, j) a chemospecific mechanism than with elecP=f trical or mechanical factors alone. (f,g,i) Conceivably, more than one mechanism is operating. in which (i) and (j) represent environmental Failure of regeneration may result, factors and the other letters represent theoretically, from a defect in either a mechanisms operating within the neuron. mechanical, chemical or electrophysiologic Experiments to date have not provided sufmechanism. ficient information to conclude whether a defect in the neuron or its environment is Retinal Ganglion Cell Versus predominantly responsible for regenerative Environmental Factors in failure. Some evidence, though, implicates Regenerative Failure the environment, at least in part. Ortin and Arcuate” substituted a length of rabbit sciatic Nerve regeneration involves an interaction nerve for a length of rabbit optic nerve. They between the growing neuron and its environment. Neuronal environment means glial demonstrated that the severed optic fibers cells, connective tissue, blood vessels, in- grew from the eye for a short distance into the Their experiment tercellular materials, other neurons, etc., any sciatic nerve graft, of which might influence the course of suggested that adult mammalian optic fibers can regenerate when the environment is regeneration. If regeneration fails, the trouble con- favorable. Other experiments also implicate the enceivably may lie with the retinal ganglion cell, vironment, for mammalian peripheral nerve its environment, or both. fibers, which ordinarily are capable of For instance, all the mechanical, chemical,
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FIG. 1. Neuronal regions pertinent to regeneration (after WeisPS). regeneration, will regenerate poorly when directed into the central nervous system. For example, after severing a dorsal spinal root between the spinal cord and sensory ganglion, the nerve fibers regenerate past the lesion site, but on reaching the spinal cord, growth, while continuing to some extent,68 is apparently significantly retarded.‘8~62~B6+S Whereas these experiments suggest a defect in the neuronal environment, they do not rule out the possibility of an additional intrinsic defect in the CNS neuron itself. Recent experiments suggest that adrenergic neurons are capable of significant regeneration in the mammalian CNS.15*18,48,8s,86,8’ The fact that other kinds of neurons do not appear to regenerate as well in a similar environment suggests that a neuronal defect also may be involved in regenerative failure. “Positive” and “Negative” Influences in Regeneration
Axon growth could be induced by two types of mechanisms: the addition of “positive” growth (regeneration stimulating) factors or the elimination of “negative” (regeneration inhibiting) influences. An example of a positive factor would be the production of a growth-inducing chemical at the site of nerve section or in the denervated lateral geniculate body. Analogous situations include the suggestion of a diffusible, nerve-attracting “neurocletin” substance released from denervated muscle,28 nerve growth factor, 56 “NR” Factor,50 denervation supersensitivity, and many examples of the general phenomenon of positive feedback.” Negative growth influences might normally be acting on the neuron. For instance, the lateral geniculate body (LGB) could normally produce a growth-inhibiting chemical which prevents further growth of the optic nerve once the LGB is reached in embryonic development. The optic nerve would from then on remain fixed in length, With section
of the optic nerve, this negative influence would no longer be available and growth would resume. Hence, the elimination of a “negative” factor. Examples of negative factors include mitosis-inhibiting tissue chalones,” regenerationinhibiting factors in tubularia,75 corticosteroid depression of ACTH release, and many other examples of negative feedback.” There is no necessary rigid distinction between the two types of mechanisms. Both increased positive influences and decreased negative influences could go hand-in-hand as part of the same process. A decrease in an inhibiting factor, for example, could trigger changes in gene activity which would increase the production of growth-favoring factors. Conversely, a growth-inducing chemical might function by repressing a growthinhibiting chemical. The Problem of Degeneration: A Cell Maintenance Factor
Based on the preceding summary, it may be inferred that regenerative failure follows failure to supply regeneration inducers or deficiency in the elimination of regeneration inhibitors. One would, in either case, expect the axon to remain stationary and simply not grow well. However, how can one explain degeneration? Degeneration is a step backward from simply “not growing” and two steps back from regeneration. Degeneration can be explained by assuming either: 1. A negative factor, destructive to the maintenance of normal cell form and function, is introduced in mammalian optic nerve section (e.g. vascular compromise, direct neural injury, autoantibody formation, etc.); or 2. A maintenance factor (chemical or electrical) to preserve the retinal ganglion cell, is
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normally produced centrally, e.g. in the lateral geniculate body, and becomes deficient following axonal sectioning. A chemical factor might spread back to the retina by retrograde axoplamic flo~.~~*~’On sectioning the optic nerve, even very close to the LGB,81 this factor would be lost and degeneration would set in. Van BureP has somewhat differently that suggested retrograde degeneration may result from interruption of centrifugal fibers, but the same principle is involved. Specific Mechanisms in Regenerative Failure
The suggestion frequently has been made that the glial-connective tissue scar which occurs at the site of CNS injury “blocks” the advancement of regenerating fibers. Younger mammals, for instance, produce less glial scarring after injury than adults and appear to regenerate better.2z Certain experiments call into question the idea that the “blocking” effect of the scar is the main cause of regenerative failure. In recent experiments wherein small lesions were produced in the adult mouse retina, the severed optic fibers did not regenerate.37 One would have expected the severed fibers at least to grow around the lesion, for the lesion was so minute, but this did not occur. Following comparable sized lesions in the embryonic
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chick retina, the developing optic fibers showed no difficulty in growing around the block. The embryonic optic fibers in the chick grew in well-organized sheets or bundles of parallel fibers. In situations wherein the embryonic chick fibers did not get around the lesion the fibers still continued to grow, but in reverse direction, toward, and out through, the retinal periphery.s8 In the adult mouse retina, however, severed optic fibers exhibited only abortive growth, remaining at the lesion site as twisted, disorganized, unfasciculated fibers. These experiments suggest that something more than simply a block at the lesion site is responsible for regenerative failure in the adult mammal. Either the adult neuron itself is defective or else some other element of its environment, aside from the “block” at the lesion site, is contributing to regenerative failure. The scar that forms after lesions in the adult retina occurs along with a general radial contraction of the immediate environment toward the lesion site (Fig. 2). Within several days after injury, glial fibers and whole fascicles of optic fibers appear drawn in toward the lesion.s7 This contraction effect was not noted following lesions in the embryonic chick retina. One should consider the possibility that such contraction might significantly impede regeneration for it could set up radially oriented mechanical stress
FIG. 2. Contraction of nerve bundles toward the lesion site in the adult mouse retina. Arrow indicates nerve bundle.
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lines which guide the fibers toward the lesion, preventing them from either extending around the lesion or traveling in reverse direction. Weiss”‘*104 and others have provided strong evidence for the influence of stress lines in the guidance of nerve fibers. The relative paucity of extracellular space in the adult mammalian CNSz7 could also be implicated in regenerative failure. With no space for axonal growth, regeneration would fail. When a CNS axon is severed, the distal end of the axon undergoes Wallerian degeneration, thereby opening up a potential space for new axonal growth. In fact, axons from nearby, uninterrupted fiber tracts can develop new sprouts which grow into these vacant areas.22~38~40~61~8Z These findings would suggest that there is extracellular space available for axonal growth even in the adult. However, it should be noted that these studies have not demonstrated growth of axonal sprouts for any significant distances. Peripheral nervous system axons regenerate well in adult mammals. It has been suggested that Schwann cells, which are present in the peripheral, but not the central, nervous system, may be a necessary prequisite for regeneration for, following axon sectioning, they form tube-like structures which are believed by some to form guides for regenerating axons. This argument, however, is apparently deficient because the CNS in many lower vertebrates regenerates well without the presence of Schwann cells. Some evidence suggests that myelin debris is cleared more slowly in the CNS than in the peripheral nervous system,4B thereby suggesting the possibility that myelin debris may significantly impede regeneration. However, it should be stated that the goldfish optic nerve is virtually 100% myelinated17 and yet it regenerates well; optic fibers in the mammalian retina are unmyelinated but regenerate poorly.37~87~6a~110~111 An autoimmune hypothesis for regenerative failure has recently been advanced.” Injury to the CNS would trigger an autoimmune reaction to antigens specific for the lesion site. Embryos and lower vertebrates, which have poorly-developed immune systems, would therefore regenerate better than adult mammals. Moreover, successes reported in using steroids to induce regeneration would result not from a reduction in glial-connective scarring but from a suppression of the immune system.21*30
Although the hypothesis is ingenious, it remains to be determined whether or not powerful immunosuppressive agents will allow significant functional regeneration. Preliminary experiments in our laboratory have failed to show any significant effect of antilymphocyte serum, given locally and systemically, in inducing regeneration of optic fibers in the adult mouse retina. Recent work involving spinal cord lesions in goldfish has pointed to another potential mechanism of regenerative failure. Severed fibers in the goldfish spinal cord normally regenerate well past the lesion site. Such regeneration was prevented by introducing tantalum blocks at the lesion site. The severed fibers then synapsed on nearby inappropriate neurons. On subsequent removal of the block, the inappropriate synapses remained and no regeneration occurred. In order to induce regeneration, it was necessary to perform an additional procedure in which the inappropriately synapsing axons were once again severed. The axons then regrew past the original lesion site.10m’3Conceivably, such inappropriate synapses are made in the adult mammalian CNS following injury because the axon or its environment lacks the necessary information to direct the axons to their appropriate synaptic endpoints. In applying this hypothesis to the visual system one should recall that the optic nerve contains virtually no neuronal cell bodies. Hence, on severing the optic nerve, inappropriate synapses would have to be either axon-axonally or between axons and cells which are nonneuronal. Summary: Reasons for Failure of Mammalian Optic Nerve Regeneration
In review of the preceding sections, one can derive a number of generalizations to account for failure of optic nerve regeneration. 1. The fault may lie in the retinal ganglion cell or in the extraganglionic environment. 2. The deficit may be in cell maintenance, or in axonal growth, guidance or synapse formation. 3. Axonal growth, guidance or synapse formation may involve chemotactic, mechanical, or electrophysiological mechanisms or some unknown mechanism or combination thereof. 4. In general, failure of nerve regeneration in mammals results from an increase in
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FAILURE OF REGENERATION
RETINAL GANGLION CELL DEFICIENCY
MAINTENANCE; GROWTH
CHEMICAL; MECHANICAL tNEGATIVE FACTOR;
ENVIRONMENTAL DEFICIENCY
GUIDANCE; SYNAPSE FORMATION
ELECTROPHYSIOLOGIC;OTHER?
iPOSITIVE FACTOR
FIG. 3. Summary diagram of the potential mechanisms for failure of mammalian optic nerve regeneration. negative influence on regeneration or from a decrease in positive regenerative influence, relative to amphibians and fishes. These thoughts are summarized in Fig. 3. One may readily discover many potential mechanisms of regenerative failure on piecing together varying combinations of factors from each row in the diagram. For example, regarding the glial scar hypothesis, the glial scar represents a difficulty at the level of the neuronal environment. It involves growth and guidance interference of a mechanical nature. One may look upon this failure as either an increase in negative factor (glial scar) or a decrease in (disruption of underlying positive factor guiding mechanical framework). The autoimmune hypothesis represents difficulty at the ganglion cell level (antigenic stimulus) and in the environment (antibody response). Interference occurs with axonal growth and involves an increase in a negative factor of a chemical nature (antibody response). And so on. Cell Body Regeneration in the Retina
Most of the preceding discussion has been concerned with the problem of axonal regeneration. Far more difficult is the problem of how to induce the proliferation of new cell bodies to replace those that have been lost through injury. This problem will not be resolved by decreasing glial scars, decreasing the immune response, or any other measures aimed simply at improving axonal growth and guidance. In broadly considering regeneration, one is struck by the observation that regeneration of other organ systems generally is poorer in
mammals than in lower vertebrates. Certain adult lower vertebrates, for instance, can regenerate not only optic nerves, but whole retinas, lenses, limbs and tails. Rather than thinking narrowly in terms of secondary mechanisms such as glial scars and decreased extracellular space, perhaps there is a broad primary mechanism of regenerative failure in mammals which encompasses axonal and cell body regeneration and regeneration in general. Various data suggest that gene derepression occurs less and less the farther the advance in phylogeny.‘8 According to one view of development, genes normally are unrepressed in the zygote but gradually become more and more repressed as development proceeds and cells become more specialized. Regeneration would largely consist of a derepression of genes, in a sense a recapitulation of embryonic development. If so, then perhaps axons and neuronal cell bodies fail to regenerate in mammals because the genes do not become derepressed following injury; there is no return to the embryonic state?O This hypothesis, while speculative, is important because it raises the possibility of using gene derepressors as a means of fostering regeneration in mammals. In fact, much of present regeneration research in the Soviet Union is based on the presumption that regeneration may be induced by the application of specific tissue extracts which act as gene derepressors. Recent work along this line has been encouraging.” Prospects for Regeneration in the Human Visual Pathways
Will we someday be able to induce regeneration in the mammalian visual system? When we know the mechanisms of regenerative failure we will be much closer to
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knowing whether regeneration can be induced. Presently we do not even have sufficient evidence to decide whether to attribute the failure predominantly to the neuron or its environment. Any therapeutic approach will depend on the mechanism of regenerative failure. Various agents have been tested for either reducing the glial-connective tissue scar or for fostering axoplasmic synthesis to provide a more forceful “push.” These include ACTH, cortisone, desoxycortisone acetate, bacterial piromen, malononitrile, succinonitrile, hexen01actone,28~33~60~03thyroxine,46 RNA,’ “NR” factor,” cyclic AMP and its dibutyrl derivative,73v74 nerve growth factor,7B xirradiation,0s and more careful surgical union. These have met with varying and largely controversial degrees of success, particularly regarding the issue of functional regeneration. Apparently there are no reports testing the potential value of these or other agents in promoting visual pathway regeneration. This lack of research is surprising, for the retina may well be the best model system in this area of research for the following reason. Testing large numbers of potential agents in different dosages, combinations, routes of administration, vehicles and frequencies of administration requires thousands of animals. The spinal cord, on which most of the work has been done, is an inadequate model system for dealing with the large numbers of animals required for this work. It may take months for functional spinal recovery to occur, and histology employing silver stains and serial sectioning is cumbersome, time-consuming and often inconclusive. The retina, on the other hand, is virtually the only area of the CNS that can be prepared as a whole mount. We recently described a simplified, consistent whole mount staining technique for the adult mammalian retina.s6-s7 Several days after producing a lesion in the adult mouse retina, the eye is fixed and the retina is prepared as a whole mount that is quickly stained. Evidence can then be sought for induced optic axon elongation around or through the lesion site following application of potential neuroregenerative agents. Many eyes can be examined in a short time at comparatively little expense, and, since the technique employs whole mounts, it avoids the delay, inconvenience and error inherent in the reconstruction of serial sections. This in vivo approach provides a more physiologic
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medium than can be obtained in tissue culture, although the latter approach is also to be greatly encouraged. The problem of regenerative failure in the visual pathways is far from hopeless, but little effort has been made to try to reverse the situation. The future course of research will involve a further narrowing down of the list of possible mechanisms of regenerative failure as well as emperical attempts to test potential neuroregenerative agents. References 1. Angeletti PU, Levi-Montalcini R, Calissano P: The Nerve Growth Factor: Chemical properties and metabolic effects. Adv Enzymol 31:51-75, 1968 2. Arora HL: Effect of forcing a regenerative optic nerve bundle toward a foreign region of the optic tectum. Anat Ret 145:202, 1963 3. Arora HL, Sperry RW: Studies on color discrimination following optic nerve regeneration in the cichlid fish Astronotus ocellatus. Anat Ret 131:529, 1958 4. Arora HL, Sperry RW: Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Am Zoo1 2:389, 1962 5. Arora HL, Sperry RW: Color discrimination after optic nerve regeneration in the fish Astronotus ocellatus. Dev Biol 7~234-243, 1963 6. Attardi DG, Sperry RW: Preferential selection of central pathways by regenerating optic fibers. Exp Neural 7:46-64, 1963 I. Barron KD, Doolin PF: Ultrastructural observations on retrograde atrophy of lateral geniculate body. I. Neuronal alterations. J Neuropathol Exp Neurol 26:300-326, 1967 8. Barron DK, Oldershaw BJ: Cytochemistry of lateral geniculate body: Transneuronal and retrograde degeneration. Neurology 15:289, 1965 9. Batkin S: The effect of RNA on the recovery of spinal sectioned carp. Proc Nat Acad Sci 56:1689-1691, 1966 10. Bernstein JJ, Bernstein ME: Effect of glialependymal scar and teflon arrest on the regenerative capacity of goldfish spinal cord. Exp Neurol 19:25-32, 1967 11. Bernstein JJ, Bernstein ME: Ultrastructure of normal regeneration and loss of regenerative capacity following teflon blockage in goldfish spinal cord. Exp Neurol 24:538-557, 1969 12. Bernstein JJ, Bernstein ME: Axonal regeneration and formation of synapses proximal to the site of lesion following hemisection of the rat spinal cord. Exp Neurol 30:336-351, 1971 13. Bernstein ME, Bernstein JJ: Regeneration of
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axons and synaptic complex formation rostra1 to the site of hemisection in the spinal cord of the monkey. Int J Neurosci 5:15-26, 1973 14. Berry M, Riches AC: An immunological approach to regeneration in the central nervous system. Br Med Bull 30:135-140, 1974 15. Bjijrklund A, Katzman R, Stenevi U, West K: Development and growth of axonal sprouts from noradrenaline and 5-hydroxytryptamine neurons in the rat spinal cord. Brain Res 31:21-33, 1971 16. Bjijrklund A, Nobin A, Stenevi U: Regeneration of central serotonin neurons after axonal degeneration induced by 5,6-dihydroxytryptamine. Brain Res 50:214-220, 1973 17. Bruesch SR, Arey LB: The number of myelinated and unmyelinated fibres in the optic nerve of vertebrates. J Comp Neurol 77:631-665, 1942 18. Bullough WS: The Evolution of Differentiation. London, New York, Academic Press, 1967 19. Cajal S Ramon y: Degeneration and Regeneration of the Nervous System. (May RM, translator and editor) London, University Press, 1928 20. Cankovic JG: Contribution to the study of regenerative-degenerative qualities of the fasciculi optici in mammals under experimental conditions. Acta Anat (Basel) 70:117-123, 1968 21. Cavanagh JB, Joseph J: The effects of cortisone, . ACTH, nitrogen mustard and colchicme on the cell population of degenerating spinal tracts in the rabbit. Guys Hosp Rep 107:144-150, 1958 22. Chambers WW: Structural regeneration in the mammalian central nervous system in relation to age, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 23. Clemente CD: Regeneration in the vertebrate central nervous system. Int Rev Neurobiol 6:257-301, 1964 24. Cohen S: Purification of a nerve growthpromoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum. Proc Nat Acad Sci 46:302-311, 1960 25. Cook WH, Walker JH, Barr ML: Cytologic study of transneuronal atrophy in cat and rabbit. J Comp Neurol 94:267-292, 1951 26. Cragg BG: The fate of axon terminals in the visual cortex during tram-synaptic atrophy of the lateral geniculate nucleus. Brain Res 34:53-60, 197 1 27. Del Cerro MP, Snider RS, Oster ML: Evolution of the extracellular space in immature neurons. Experientia (Basel) 24:929-930, 1968 28. Edds MV: Collateral nerve regeneration. Q Rev Biol 28:260-276, 1953
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JA, Seyan SSAS: 29. Fertig A, Kiernan Enhancement of axonal regeneration in the brain of the rat by corticotrophin and triiodothyronine. Exp Neurol33:372-385, 1971 30. Flint G, Berry M: A quantitative investigation of the response to injury of the central nervous system of rats treated with ACTH and triiodothyronine. Experientia 29:566-568, 1973 31. Gaze RM: The Formation of Nerve Connections. London, New York, Academic Press, 1970 32. Gaze RM, Keating MJ, Straznicky K: The retinotectal projection from a double-ventral compound eye in Xenopus. J Physiol (Lond) 214:37P-38P, 1971 33. Gerard RW: A perspective evaluation of central nervous system regeneration in mammals, in Windle WF (ed.): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 34. Gills JP, Wadsworth JAC: Retrograde transsynaptic degeneration of the inner nuclear layer of the retina. Invest Ophthalmol 6~437-448, 1967 35. Goldberg S: Silver staining, featuring rapid reduction, for whole mounts of retina and optic pathways in chick embryos. Stain Technol 47:65-69, 1972 36. Goldberg S, Coulombre AJ: Topographical development of the nerve fiber layer in the chick retina. A whole mount study. J Comp Neurol 146:507-518, 1972 37. Goldberg S, Galin MA: ‘Response of retinal ganglion cell axons to lesions in the adult mouse retina. Invest Ophthalmol 12:382-385, 1973 38. Goldberg S: Studies on the mechanics of development of the visual pathways in the chick embryo. Dev Biol 36:24-43, 1974 39. Goodman DC, Bogdasarian RS, Horel JA: Axonal sprouting of ipsilateral optic tract following opposite eye removal. Brain Behav Evol 8:27-50, 1973 40. Goodman DC, Horel JA: Sprouting of optic tract projections in the brain stem of the rat. J Comp Neurol 127:71-88, 1966 41. Goss RJ: Adaptive Growth. New York, Academic Press, 1964 42. Gray P: Experiments with direct currents on chick embryos. Arch Entw Mechan 139:732-779, 1939 43. Guth L. Windle W: The eniema of central nervous. regeneration. Exp -Neural suppl 5:1-43, 1970 44. Harrison RG: The reaction of embryonic cells to solid structures. J Exp Zool 17:521-544, 1914 45. Harvey JE, Srebnik HH: Locomotor activity and axon regeneration following spinal cord compression in rats with L-thyroxine. J
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Neuropathol Exp Neurol 26:661-668, 1967 46. Hirose G, Bass NH: A quantitative histochemical study of early cellular events associated with destruction of myelinated axons in rat optic nerve. Exp Neurol 44:82-95, 1974 47. Jacobson M: Developmental New York, Holt, 1970
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48. Katzman R, BjGrklund A, Owman Ch, et al: Evidence for regenerative axon sprouting of central catecholamine neurons in the mesencephalon following electrolytic lesions. Brain Res 25:579-596, 1971 49. Kimmel DL, Moyer EK:
Dorsal roots following anastomosis of the central stumps. J Comp Neurol 87:289-321, 1947 50. Koechlin BA: The neuroregenerative factor “NR”, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 51. Kupfer C, Palmer P: Lateral geniculate nucleus: Histological and cytochemical changes following afferent denervation and visual deprivation. Exp Neural 9:400-409, 1964 52. Lampert P, Carpenter
S, Cressman M: Axonal regeneration in the dorsal columns of the spinal cord of adult rats - an EM study.
Lab Invest 13:825-839, 1964 53. LaVail JH, LaVail MM: Retrograde axonal
transport
in the central nervous system. 1972 54. LaVail JH, Winston KR, Tish A: A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS. Brain Res 58~470-477, 1973 55. Leinfelder PJ: Retrograde degeneration in the optic nerve and retinal ganglion cells. Tr Science 176:1416-1417,
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ing of dorsal root axons. Arch Neurol (Chicago) 79:46-6 1, 1958 62. Lynch G, Deadwyler S, Cotman C: Postlesion axonal growth produces permanent Science 180: connections. functional 1346-1366, 1973 63. Marsh G, Beams HW: in vitro control of growing chick nerve fibers by applied electric current. J Cell Comp Physiol 27: 139-157, 1946 64. Mathews MR, Cowan WM, Powell TPS: Transneuronal cell degeneration in the lateral geniculate nucleus of the macaque monkey. J Anat (Lond) 94:145-169, 1960 65. Moore RY, Bjiirklund A, Stenevi U: Plastic
changes in the adrenergic innervation of the rat septal area in response to denervation. Brain Res 33: 13-35, 197 1 66. Moyer EK, Kimmel DC, Winborne LW: Regeneration of sensory spinal nerve roots in young and in senile rats. J Comp Neurol 98:283-308, 1953 67. Mukai N: Axonal reaction of the optic nerve following heat coagulation. Histochemical evidence for antidromic convection. Can J Ophthalmol 5:78-90, 1970 68. Nathaniel EJH, Nathaniel
DR: Regeneration of dorsal root fibers into the adult rat spinal cord. Exp Neurol 40:333-350, 1973 69. Okun E, Collins EM: Histopathology of experimental photocoagulation in the dog eye. II. Production of cytoid body lesions. Am J Ophthalmol 54:786-793, 1962 70. Ortin L, Arcuate LR: Procesos regenerativos
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R: The Nerve Growth Factor: Its mode of action on sensory and sympathetic nerve cells, in The Harvey Lectures, Series 60:217-259, 1964 Levi-Montalcini R, Angeletti PU: Nerve Growth Factor. Physiol Rev 48:534-569, 1968 Levi-Montalcini R, Booker B: Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein. Proc Nat Acad Sci 46:384-391, 1960 Levi-Montalcini R, Caramia F, Luse S, Angeletti P: In vitro effect of the Nerve Growth Factor on the line structure of sensory nerve cells. Brain Res 8:347-362, 1968 Littrell JL: Apparent functional restitution in piromen treated spinal rats, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 Liu CN, Chambers WW: Intraspinal sprout-
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de1 neuro optic y retina con ocasion de ingertos nerviosos. Trabaj Lab Investig Biolog Univ Madrid XI(62 W XVI) s 239, Madrid, 1913 in Polezhaev LV: Organ Regeneration Animals. Springfield, Ill, Charles C Thomas, 1972 Polyak SL: The Retina. Chicago, Univ. of Chicago Press, 1941 Roisen FJ, Murphy RA: Neurite development in vitro. II. The role of microfilament and microtubules in dibutyryl adenosine 3’, 5’-cyclic monophosphate and nerve growth factor stimulated maturation. J Neurobiol 4:397-412, 1973 Roisen FJ, Murphy RA, Braden WG: Dibutyrl cyclic adenosine monophosphate stimulation of colcemid-inhibited axonal elongation. Science 177:809-811, 1972 Rose SM: Regeneration. New York, Appleton-Century-Crofts, 1970 Scott D Jr: Influence of NGF on spinal regeneration in kittens. Anat Ret 145:283,
1963 77. Sperry RW: Patterning
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78. Sperry RW: Regulative factors in the orderly growth of neural circuits. Growth Symp 10:63-87, 195 1 79. Sperry RW: Developmental patterning of neural circuits. Chicago Med School Q 12:66-73, 195 1 80. Sperry RW: Mechanisms of neural maturation, in Stevens SS (ed): Handbook of Experimental Psychology, New York, John Wiley, 195 1 81. Sperry RW: Functional regeneration in the optic system, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 82. Sperry RW: Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc Nat Acad Sci 50:703-710, 1963 83. Sperry RW: Evidence behind chemoaffinity theory of synaptic patterning. Anat Ret 145:288, 1963 84. Sperry RW: Embryogenesis of behavioral nerve nets, in DeHann RL, Ursprung H (eds): Organogenesis, New York, Holt, 1965 85. Sperry RW: A selective communication in nerve nets: Impulse specificity versus connection specificity. Neurosci Res Prog Bull 3:37-43, 1965 86. Stenevi U, Bj6rklund A, Moire RY: Growth of intact central adrenergic axons in the denervated lateral geniculate body. Exp Neurol 35:290-299, 1972 87. Stenevi U, Bjijrklund A, Moire RY: Morphological plasticity of central adrenergic neurons. Brain Behav Evol 8:110-134, 1973 of 88. Stone LS: Heteroplastic transplantation eyes between two species of Amblystoma. J Exp Zoo1 55:193-261, 1930 89. Stone LS: Normal and reversed vision in transplanted eyes. Arch Ophthal (Chicago) 49:28-35, 1953 90. Stone LS: Polarization of the retina and development of vision. J Exp Zool 145:89-93, 1960 91. Stone LS, Ussher NT, Beers DN: Reimplantation of larval eyes in the salamander (Amblystoma punctatum). J Exp Zool 77:13-48, 1937 92. Straznicky K, Gaze RM, Keating MJ: The retinotectal projection from a double-ventral compound eye in Xenopus laevis. J Embryo1 Exp Morphol 31:123-137, 1974 93. Stuart EG: Tissue reactions and possible mechanisms of piromen and desoxycortisone acetate in central nervous system regeneration, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 94. Szdkely G: Embryonic determination of neural connections. Adv Morphog 5: 18 l-2 19, 1966 95. Tower SS: A search for trophic influences of posterior spinal roots on skeletal muscle,
GOLDBERG with a note on the nerve fibers found in the proximal stumps of the roots after excision of the root ganglia. Brain 54:99-110, 1931 96. Turbes CC, Freeman LW, Gastineau DC: Xray radiation of experimental lesions of the spinal cord. Neurology 10:84-89, 1960 97. Van Buren JM: Transsynaptic retrograde degeneration in the visual system in primates. J Neurol Neurosurg Psychiatry 26:402-409, 1963 98. Weiler IJ: Restoration of visual acuity after optic nerve section and regeneration in Astronotus. Exp Neurol 15:377-386, 1966 99. Weiss P: Principles of Development. New York, Holt, 1939 100. Weiss P: Nerve patterns: The mechanics of nerve growth. Growth suppl 5:163-203, 1941 101. Weiss P: Genetic Neurology: Problems of the Development, Growth and Regeneration of the Nervous System and its Function. Chicago, Univ Chicago Press, 1950 102. Weiss P: Central versus peripheral factors in the development of coordination. Proc Assoc Res Nerv Mental Dis 30:3-23, 1952 103. Weiss P: Parameters of nerve regeneration, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 104. Weiss P: Nervous system (neurogenesis), in Willier BH, Weiss PA, Hamburger V (eds): Analysis of Development, Philadelphia, Saunders, 1955 Int Rev Cytol 105. Weiss P: Cell contact. 7:391-423, 1958 106. Weiss P: Chairman’s summary. Neurosci Res Prog Bull 3:5-36, 1965 107. Wiesel TN, Hubel DH: Effects of visual deprivation on morphology and physiology of cells in cat’s lateral geniculate body. J Neurophysiol 26:978-993, 1967 108. Windle W: Regeneration in the Central Nervous System. Springfield, Ill, Charles C Thomas, 1955 109. Wisniewski HM, Ghetti B, Horoupian DS: The fate of synaptic membranes of degenerating optic nerve terminals, and their role in the mechanism of trans-synaptic changes. J Neurocytol 1:297-310 10. Wolter JR: Axonal enlargements in the nerve-fiber layer of the human retina. Am J Ophthalmol 65:1-12, 1968 11. Wolter JR, Lund OE: Reaction of centrifugal nerves in the human retina. Am J Ophthalmol 66:221-232, 1968 12. Zanini A, Angeletti P, Levi-Montalcini R: Immunochemical properties of the Nerve Growth Factor. Proc Nat Acad Sci 61:835-842, 1968 Reprint requests should be addressed to Stephen Goldberg, M.D., Dept. Biological Structure, University of Miami School of Medicine, Miami, Fla. 33152.
CURRENT
VOLUME 20
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NUMBER 4
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JANUARY-FEBRUARY
1976
RESEARCH
EDWARD GOTLIER, EDITOR
Central Nervous System Regeneration
and
Ophthalmology STEPHEN
GOLDBERG, Department
M.D. of Ophthalmology,
New York Medical
College, New York
Various adult lower vertebrates are capable of optic nerve, and even retinal, regeneration with functional recovery of vision. Possible factors responsible for regenerative failure in mammals are discussed. It is suggested that potentially neuroregenerative agents be tested in the mammalian retina in an attempt to induce visual pathway regeneration. (Surv Opbthalmol 20:261-272, 1976)
Abstract.
Key Words: regeneration
.
axon . central optic nerve -
nervous
system
??
degeneration
??
neuronal
retina
T
he mammalian central nervous system posteriorly, in the visual cortex.7@-34*87 Why this striking difference between higher (CNS) regenerates poorly following inBecause the retina and and lower vertebrates? Is a relatively small jury. 18*23,43~108 visual pathways are part of the CNS, patients difficulty involved, such as lack of a simple with neuro-ophthalmological problems fre- replaceable substrate or presence of a glial quently cannot be helped. This paper reviews scar at the lesion site, or is there a more inthe possible reasons for regenerative failure in tractable underlying problem involving comthe CNS and discusses the future prospects plex biochemical interactions or inaccessible for the induction of visual pathway regenera- genetic differences between higher and lower tion. vertebrates? The mammalian visual system Following optic nerve section, certain appears very refractory to nerve regeneration, whereas these lower vertebrates appear so lower vertebrates - amphibians and fishes exhibit good return of function.3~s~8~31~47~~o~*i~@~ plastic that one may perform entire ocular The severed optic fibers grow back to their transplants between two species of salamanappropriate destinations in the brain. But in der and achieve recovery of vision.OQ~elThe the mammal, not only does regeneration salamander eye may be refrigerated for a fail, degeneration occurs. Retrograde atrophy week and still be successfully transplanted, and cell loss sets in among the retinal gang- with regeneration of the retina as well!” anterogra& de_ lion cells, 8,*6.61,66.72,97,~07,1~9 Perhaps a comparison between higher and generation damages the lateral geniculate lower vertebrates on many levels of investigaand even more distant tion will eventually help to explain the failure body, ~,*5,28,61.84.107.~0@ degenerative changes are found anteriorly in of mammalian CNS regeneration. A the inner nuclear layer of the retina and knowledge of mechanisms of optic nerve em261
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bryogenesis may also prove of value since regeneration may in large part be a recapitulation of embryonic events. This paper is not an exhaustive review of all aspects of neural regeneration and it does not suggest any single mechanism responsible for regeneration or degeneration. Rather, in the absence of enough data, it simply arranges some of the many possible mechanisms of regenerative failure into a logical framework, as a basis for future research. By understanding why regeneration fails we may find ways to induce regeneration. Growth, Guidance, and Synaptic Factors in Optic Fiber Regeneration and Embryogenesis
Axon regeneration may be generally subdivided into three processes: axonal growth (axonal elongation); axonal guidance (toward the appropriate destination in the brain); and synapse formation. AXONAL GROWTH
Although axonal growth may be defined in various ways, it is used here only in the simple sense of elongation. The normal, mature, uninjured axon is roughly fixed in length from retina to brain terminus. Once the optic nerve is sectioned new axonal sprouts appear which, in lower vertebrates, continue growing until the visual area of the brain is reached. In mammals, initial sprouting also occurs, but results only in an abortive growth. The new fibers do not bypass the glial-connective tissue scar at the site of section and, in time, they degenerate. GUIDANCE
The growing nerve fibers require some sort of guidance mechanism to insure that they reach the brain instead of growing wildly in all directions. Guidance might crudely be mediated mechanically, e.g., via a tissue plane in the region of Wallerian degeneration, or it might involve highly selective chemical or other mechanisms. It is important to distinguish between growth and guidance. The presence of one does not imply the other. Conceivably, an axon might grow quite well, but in the absence of guidance, it might grow wildly without reaching its proper destination. Likewise, the apparatus for axonal guidance
GOLDBERG
might be functioning properly, but if the axon lacks the ability to grow, regeneration will fail again. Axon regeneration is defined here as the process wherein an axon not only regrows, but finds its way back to its original terminus. Conceivably, the same factors rendering guidance possible are also responsible for growth. For instance, a chemical released by the central degenerating axon segment may stimulate growth. Sprouts would appear wherever the chemical contacts the axon. Since the chemical would occupy the entire degenerating axon segment, growth would continue back along the original pathway. The same chemical would thus be responsible for both growth and guidance. The growth and guidance mechanisms might, however, be different, For instance, a chemical released at the site of injury might induce growth, but a mechanical tissue plane could function as the guiding factor. Since it is not known whether growth and guidance are mediated by the same factor, it is best to keep in mind the possibility of different mechanisms for each. SYNAPSE FORMATION
When arriving at the appropriate area in the brain, the axon synapses. It is believed to do so highly selectively, choosing only certain cells in a particular predetermined brain locus.31,” Given that axonal growth, guidance, and synapse formation are important to both nerve regeneration and embryogenesis, theoretically, interference with any of these could prevent effective regeneration. Eiectrophysioiogicai, Mechanical and Chemical Factors in Regeneration and Embryogenesis
Throughout this century opinions have fluctuated among three potential mechanisms for nerve regeneration and embryogenesis: the mechanical, chemical, and electrophysiological theories are briefly described below 31.4T,7&80,84.86,94,99,101,102,104,1~ ELECYROPHYSIOLOGICAL THEORY
Proponents of this theory believe that a growing neuron may be guided in the direction of change of electric potential. Marsh and Beams,BS for instance, showed that axons
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The mature visual pathway maintains an orderly map-like point-to-point connectivity between retina and brain.B~31~‘7@ After sectioning the optic nerve, combined with 180” eye rotation, reversed visuomotor responses appeared in the adult amphibian after regeneration of the optic nerve. These behavioral studies suggested that the growing fibers returned to their original synaptic terminals.3+5*77978 Histological and electrophysiological studies have also shown that regenerating axons appear specified as to destination and return to their original brain loci despite the disorientation inherent in neuroma formation and in 180” rotation of the eye and optic nerve.31s47It is difficult to MECHANICAL THEORY account for such specificity except by some Considerable evidence has been compiled chemospecilic mechanism of synapse formafor mechanical factors operating in axonal tion growth and guidance.44@‘~100~104~106 Axons in One must interpret this data with caution. tissue culture tend to grow along etchings on Whereas abundant evidence has shown a high a piece of mica, along the fibrin matrix of a degree of specification in synapse formation plasma clot, or along other mechanical planes by regenerating optic axons, this does not in vitro or in vivo. The mechanical factor necessarily imply specificity of guidance seems necessary not only for axonal guidance, toward those synaptic loci. There could, for but also for sustaining axonal growth. The instance, be initial random, widespread axaxon tip apparently requires some mechanical onal branching with subsequent retraction of framework to hold onto before it can ad- those branches which failed to make apvance. propriate synaptic connections. Or an initial Difficulties arise in trying to explain how overproduction of ganglion cells and axons crudely orienting mechanical factors alone could be followed by the death of those cells can possibly specify highly complex and axons which failed to connect appathways. One can see how fibers might propriately within the brain. Sparse evidence, generally be guided into a region, but though, does suggest some degree of elaborate, consistently appearing turns, specificity in axonal guidance. Arora2,’ branchings and interlacing patterns are diverted the optic tracts of adult goldfish into another matter. The electrophysiological foreign brain regions and then severed the optheory encounters similar difficulties. Both tic nerve and removed a portion of the retina. the electrical and mechanical theories also The surviving optic fibers selected the have trouble explaining synapse formation. originally appropriate optic tract even though How can such grossly orienting influences ac- it had been diverted to a foreign area of the count for the complex and highly orderly brain. In other experiments, when the dorsal point-to-point anatomical projection between half of an optic vesicle was removed in retina and brain? Xenopus laevis and replaced with a ventral half, a biventral eye was formed. ElectrophyCHEMICAL THEORY siological studies demonstrated that each Proponents of the chemical theory, retinal half had communicated with the particularly Sperry and his co-workers, have brain.32*02Histological studies, however, insuggested that axon guidance and synapse dicated that only the medial optic tract, which formation are mediated by select chemical normally contains the fibers from ventral factors, serving to guide axons to their ap- retina, contained optic fibers; the lateral optic propriate destinations. Evidence has largely tract, which normally contains fibers from come from indirect experiments on the visual superior retina, was devoid of fibers (Horder, system of amphibians and fishes.3-6~3’~4’~77~82~*5 T. J. personal communication). This study The reasoning is as follows. thereby supported the findings of Arora in in tissue culture grow in the direction of the cathode. Their diagrams do not appear to indicate an increase in axon growth, but rather a change in direction. Hence, there is some evidence for an electrophysiologic guidance mechanism. Other observers, however, have failed to achieve similar results.g8 Nor has the natural existence of such electrical gradients in the living organism been established, whether in nerve regeneration or embryogenesis. Various data suggest that artificial electric potentials, applied continuously, will not alter embryonic developsignificantly ment.42~ggAs a result, this theory is the least popular of the three.
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suggesting that optic fibers are capable of or electrophysiologic factors necessary for selecting the appropriate optic tract. regeneration may be functioning in the A major difficulty with the chemical theory neuronal environment, but if the severed of axonal guidance and synapse formation neuron lacks the ability to respond to them, has been the failure to find chemicals that will regeneration will fail. Likewise, the neuron attract axons. Various chemicals, e.g., cyclic may possess all the potential for regeneration, AMP, “*” “NR” factof isolated from cor- but if its environment is not conducive, e.g., tical white matter, and nerve growth factors6 because of a glial scar, failure will again have been shown to enhance growth of axons, prevail. Weiss1o3 has outlined the sites within the but it is unclear whether or not they have an effect on the guidance of axons. For instance, neuron pertinent to regeneration. Fig. 1, nerve growth factor, an isolate from mouse drawn after Weiss, illustrates that regeneraa combination of distal sarcoma and mouse submaxillary gland, has tion requires dramatic growth-promoting effects on sym- sprouting (h), axoplasmic synthesis (a), pathetic and sensory nerve cells.1~24~sB-68J12 proximo-distal conveyance of neuroplasm Axons appear to branch extensively on enter(b,c), retrograde regulative influences (d), ing the mouse sarcoma, at first suggesting a structural resistance of the axon wall (e) and a minimum of ectopic, competitive side possible guidance into the tumor. However, other neurons distant from the tumor branch branchings (f, g) and environmental interference (i). Weiss has derived the formula wildly, e.g., penetrating the skin, suggesting mainly a growth effect?’ (a,b,c,d,e,h) In summary, the main theories of growth, P=f guidance and synapse formation involve elec(f,g,i) trophysiologic, mechanical, or chemical mechanisms. Currently, the chemical theory for probability of successful regeneration. seems the best explanation for selectivity in One may add to this a (j), indicating favorable synapse formation and for guidance into the environmental influences on nerve growth, appropriate optic tract. The evidence for a guidance, and synapse formation, whether of chemical theory is indirect, however, and chemical mechanical or electrocomes chiefly from experiments showing a ;hysiologicai The formula for nature. degree of specificity in regeneration and em- regeneration then becomes bryogenesis that is more commensurate with (a,b,c,d,e,h, j) a chemospecific mechanism than with elecP=f trical or mechanical factors alone. (f,g,i) Conceivably, more than one mechanism is operating. in which (i) and (j) represent environmental Failure of regeneration may result, factors and the other letters represent theoretically, from a defect in either a mechanisms operating within the neuron. mechanical, chemical or electrophysiologic Experiments to date have not provided sufmechanism. ficient information to conclude whether a defect in the neuron or its environment is Retinal Ganglion Cell Versus predominantly responsible for regenerative Environmental Factors in failure. Some evidence, though, implicates Regenerative Failure the environment, at least in part. Ortin and Arcuate” substituted a length of rabbit sciatic Nerve regeneration involves an interaction nerve for a length of rabbit optic nerve. They between the growing neuron and its environment. Neuronal environment means glial demonstrated that the severed optic fibers cells, connective tissue, blood vessels, in- grew from the eye for a short distance into the Their experiment tercellular materials, other neurons, etc., any sciatic nerve graft, of which might influence the course of suggested that adult mammalian optic fibers can regenerate when the environment is regeneration. If regeneration fails, the trouble con- favorable. Other experiments also implicate the enceivably may lie with the retinal ganglion cell, vironment, for mammalian peripheral nerve its environment, or both. fibers, which ordinarily are capable of For instance, all the mechanical, chemical,
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FIG. 1. Neuronal regions pertinent to regeneration (after WeisPS). regeneration, will regenerate poorly when directed into the central nervous system. For example, after severing a dorsal spinal root between the spinal cord and sensory ganglion, the nerve fibers regenerate past the lesion site, but on reaching the spinal cord, growth, while continuing to some extent,68 is apparently significantly retarded.‘8~62~B6+S Whereas these experiments suggest a defect in the neuronal environment, they do not rule out the possibility of an additional intrinsic defect in the CNS neuron itself. Recent experiments suggest that adrenergic neurons are capable of significant regeneration in the mammalian CNS.15*18,48,8s,86,8’ The fact that other kinds of neurons do not appear to regenerate as well in a similar environment suggests that a neuronal defect also may be involved in regenerative failure. “Positive” and “Negative” Influences in Regeneration
Axon growth could be induced by two types of mechanisms: the addition of “positive” growth (regeneration stimulating) factors or the elimination of “negative” (regeneration inhibiting) influences. An example of a positive factor would be the production of a growth-inducing chemical at the site of nerve section or in the denervated lateral geniculate body. Analogous situations include the suggestion of a diffusible, nerve-attracting “neurocletin” substance released from denervated muscle,28 nerve growth factor, 56 “NR” Factor,50 denervation supersensitivity, and many examples of the general phenomenon of positive feedback.” Negative growth influences might normally be acting on the neuron. For instance, the lateral geniculate body (LGB) could normally produce a growth-inhibiting chemical which prevents further growth of the optic nerve once the LGB is reached in embryonic development. The optic nerve would from then on remain fixed in length, With section
of the optic nerve, this negative influence would no longer be available and growth would resume. Hence, the elimination of a “negative” factor. Examples of negative factors include mitosis-inhibiting tissue chalones,” regenerationinhibiting factors in tubularia,75 corticosteroid depression of ACTH release, and many other examples of negative feedback.” There is no necessary rigid distinction between the two types of mechanisms. Both increased positive influences and decreased negative influences could go hand-in-hand as part of the same process. A decrease in an inhibiting factor, for example, could trigger changes in gene activity which would increase the production of growth-favoring factors. Conversely, a growth-inducing chemical might function by repressing a growthinhibiting chemical. The Problem of Degeneration: A Cell Maintenance Factor
Based on the preceding summary, it may be inferred that regenerative failure follows failure to supply regeneration inducers or deficiency in the elimination of regeneration inhibitors. One would, in either case, expect the axon to remain stationary and simply not grow well. However, how can one explain degeneration? Degeneration is a step backward from simply “not growing” and two steps back from regeneration. Degeneration can be explained by assuming either: 1. A negative factor, destructive to the maintenance of normal cell form and function, is introduced in mammalian optic nerve section (e.g. vascular compromise, direct neural injury, autoantibody formation, etc.); or 2. A maintenance factor (chemical or electrical) to preserve the retinal ganglion cell, is
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normally produced centrally, e.g. in the lateral geniculate body, and becomes deficient following axonal sectioning. A chemical factor might spread back to the retina by retrograde axoplamic flo~.~~*~’On sectioning the optic nerve, even very close to the LGB,81 this factor would be lost and degeneration would set in. Van BureP has somewhat differently that suggested retrograde degeneration may result from interruption of centrifugal fibers, but the same principle is involved. Specific Mechanisms in Regenerative Failure
The suggestion frequently has been made that the glial-connective tissue scar which occurs at the site of CNS injury “blocks” the advancement of regenerating fibers. Younger mammals, for instance, produce less glial scarring after injury than adults and appear to regenerate better.2z Certain experiments call into question the idea that the “blocking” effect of the scar is the main cause of regenerative failure. In recent experiments wherein small lesions were produced in the adult mouse retina, the severed optic fibers did not regenerate.37 One would have expected the severed fibers at least to grow around the lesion, for the lesion was so minute, but this did not occur. Following comparable sized lesions in the embryonic
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chick retina, the developing optic fibers showed no difficulty in growing around the block. The embryonic optic fibers in the chick grew in well-organized sheets or bundles of parallel fibers. In situations wherein the embryonic chick fibers did not get around the lesion the fibers still continued to grow, but in reverse direction, toward, and out through, the retinal periphery.s8 In the adult mouse retina, however, severed optic fibers exhibited only abortive growth, remaining at the lesion site as twisted, disorganized, unfasciculated fibers. These experiments suggest that something more than simply a block at the lesion site is responsible for regenerative failure in the adult mammal. Either the adult neuron itself is defective or else some other element of its environment, aside from the “block” at the lesion site, is contributing to regenerative failure. The scar that forms after lesions in the adult retina occurs along with a general radial contraction of the immediate environment toward the lesion site (Fig. 2). Within several days after injury, glial fibers and whole fascicles of optic fibers appear drawn in toward the lesion.s7 This contraction effect was not noted following lesions in the embryonic chick retina. One should consider the possibility that such contraction might significantly impede regeneration for it could set up radially oriented mechanical stress
FIG. 2. Contraction of nerve bundles toward the lesion site in the adult mouse retina. Arrow indicates nerve bundle.
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lines which guide the fibers toward the lesion, preventing them from either extending around the lesion or traveling in reverse direction. Weiss”‘*104 and others have provided strong evidence for the influence of stress lines in the guidance of nerve fibers. The relative paucity of extracellular space in the adult mammalian CNSz7 could also be implicated in regenerative failure. With no space for axonal growth, regeneration would fail. When a CNS axon is severed, the distal end of the axon undergoes Wallerian degeneration, thereby opening up a potential space for new axonal growth. In fact, axons from nearby, uninterrupted fiber tracts can develop new sprouts which grow into these vacant areas.22~38~40~61~8Z These findings would suggest that there is extracellular space available for axonal growth even in the adult. However, it should be noted that these studies have not demonstrated growth of axonal sprouts for any significant distances. Peripheral nervous system axons regenerate well in adult mammals. It has been suggested that Schwann cells, which are present in the peripheral, but not the central, nervous system, may be a necessary prequisite for regeneration for, following axon sectioning, they form tube-like structures which are believed by some to form guides for regenerating axons. This argument, however, is apparently deficient because the CNS in many lower vertebrates regenerates well without the presence of Schwann cells. Some evidence suggests that myelin debris is cleared more slowly in the CNS than in the peripheral nervous system,4B thereby suggesting the possibility that myelin debris may significantly impede regeneration. However, it should be stated that the goldfish optic nerve is virtually 100% myelinated17 and yet it regenerates well; optic fibers in the mammalian retina are unmyelinated but regenerate poorly.37~87~6a~110~111 An autoimmune hypothesis for regenerative failure has recently been advanced.” Injury to the CNS would trigger an autoimmune reaction to antigens specific for the lesion site. Embryos and lower vertebrates, which have poorly-developed immune systems, would therefore regenerate better than adult mammals. Moreover, successes reported in using steroids to induce regeneration would result not from a reduction in glial-connective scarring but from a suppression of the immune system.21*30
Although the hypothesis is ingenious, it remains to be determined whether or not powerful immunosuppressive agents will allow significant functional regeneration. Preliminary experiments in our laboratory have failed to show any significant effect of antilymphocyte serum, given locally and systemically, in inducing regeneration of optic fibers in the adult mouse retina. Recent work involving spinal cord lesions in goldfish has pointed to another potential mechanism of regenerative failure. Severed fibers in the goldfish spinal cord normally regenerate well past the lesion site. Such regeneration was prevented by introducing tantalum blocks at the lesion site. The severed fibers then synapsed on nearby inappropriate neurons. On subsequent removal of the block, the inappropriate synapses remained and no regeneration occurred. In order to induce regeneration, it was necessary to perform an additional procedure in which the inappropriately synapsing axons were once again severed. The axons then regrew past the original lesion site.10m’3Conceivably, such inappropriate synapses are made in the adult mammalian CNS following injury because the axon or its environment lacks the necessary information to direct the axons to their appropriate synaptic endpoints. In applying this hypothesis to the visual system one should recall that the optic nerve contains virtually no neuronal cell bodies. Hence, on severing the optic nerve, inappropriate synapses would have to be either axon-axonally or between axons and cells which are nonneuronal. Summary: Reasons for Failure of Mammalian Optic Nerve Regeneration
In review of the preceding sections, one can derive a number of generalizations to account for failure of optic nerve regeneration. 1. The fault may lie in the retinal ganglion cell or in the extraganglionic environment. 2. The deficit may be in cell maintenance, or in axonal growth, guidance or synapse formation. 3. Axonal growth, guidance or synapse formation may involve chemotactic, mechanical, or electrophysiological mechanisms or some unknown mechanism or combination thereof. 4. In general, failure of nerve regeneration in mammals results from an increase in
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FAILURE OF REGENERATION
RETINAL GANGLION CELL DEFICIENCY
MAINTENANCE; GROWTH
CHEMICAL; MECHANICAL tNEGATIVE FACTOR;
ENVIRONMENTAL DEFICIENCY
GUIDANCE; SYNAPSE FORMATION
ELECTROPHYSIOLOGIC;OTHER?
iPOSITIVE FACTOR
FIG. 3. Summary diagram of the potential mechanisms for failure of mammalian optic nerve regeneration. negative influence on regeneration or from a decrease in positive regenerative influence, relative to amphibians and fishes. These thoughts are summarized in Fig. 3. One may readily discover many potential mechanisms of regenerative failure on piecing together varying combinations of factors from each row in the diagram. For example, regarding the glial scar hypothesis, the glial scar represents a difficulty at the level of the neuronal environment. It involves growth and guidance interference of a mechanical nature. One may look upon this failure as either an increase in negative factor (glial scar) or a decrease in (disruption of underlying positive factor guiding mechanical framework). The autoimmune hypothesis represents difficulty at the ganglion cell level (antigenic stimulus) and in the environment (antibody response). Interference occurs with axonal growth and involves an increase in a negative factor of a chemical nature (antibody response). And so on. Cell Body Regeneration in the Retina
Most of the preceding discussion has been concerned with the problem of axonal regeneration. Far more difficult is the problem of how to induce the proliferation of new cell bodies to replace those that have been lost through injury. This problem will not be resolved by decreasing glial scars, decreasing the immune response, or any other measures aimed simply at improving axonal growth and guidance. In broadly considering regeneration, one is struck by the observation that regeneration of other organ systems generally is poorer in
mammals than in lower vertebrates. Certain adult lower vertebrates, for instance, can regenerate not only optic nerves, but whole retinas, lenses, limbs and tails. Rather than thinking narrowly in terms of secondary mechanisms such as glial scars and decreased extracellular space, perhaps there is a broad primary mechanism of regenerative failure in mammals which encompasses axonal and cell body regeneration and regeneration in general. Various data suggest that gene derepression occurs less and less the farther the advance in phylogeny.‘8 According to one view of development, genes normally are unrepressed in the zygote but gradually become more and more repressed as development proceeds and cells become more specialized. Regeneration would largely consist of a derepression of genes, in a sense a recapitulation of embryonic development. If so, then perhaps axons and neuronal cell bodies fail to regenerate in mammals because the genes do not become derepressed following injury; there is no return to the embryonic state?O This hypothesis, while speculative, is important because it raises the possibility of using gene derepressors as a means of fostering regeneration in mammals. In fact, much of present regeneration research in the Soviet Union is based on the presumption that regeneration may be induced by the application of specific tissue extracts which act as gene derepressors. Recent work along this line has been encouraging.” Prospects for Regeneration in the Human Visual Pathways
Will we someday be able to induce regeneration in the mammalian visual system? When we know the mechanisms of regenerative failure we will be much closer to
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knowing whether regeneration can be induced. Presently we do not even have sufficient evidence to decide whether to attribute the failure predominantly to the neuron or its environment. Any therapeutic approach will depend on the mechanism of regenerative failure. Various agents have been tested for either reducing the glial-connective tissue scar or for fostering axoplasmic synthesis to provide a more forceful “push.” These include ACTH, cortisone, desoxycortisone acetate, bacterial piromen, malononitrile, succinonitrile, hexen01actone,28~33~60~03thyroxine,46 RNA,’ “NR” factor,” cyclic AMP and its dibutyrl derivative,73v74 nerve growth factor,7B xirradiation,0s and more careful surgical union. These have met with varying and largely controversial degrees of success, particularly regarding the issue of functional regeneration. Apparently there are no reports testing the potential value of these or other agents in promoting visual pathway regeneration. This lack of research is surprising, for the retina may well be the best model system in this area of research for the following reason. Testing large numbers of potential agents in different dosages, combinations, routes of administration, vehicles and frequencies of administration requires thousands of animals. The spinal cord, on which most of the work has been done, is an inadequate model system for dealing with the large numbers of animals required for this work. It may take months for functional spinal recovery to occur, and histology employing silver stains and serial sectioning is cumbersome, time-consuming and often inconclusive. The retina, on the other hand, is virtually the only area of the CNS that can be prepared as a whole mount. We recently described a simplified, consistent whole mount staining technique for the adult mammalian retina.s6-s7 Several days after producing a lesion in the adult mouse retina, the eye is fixed and the retina is prepared as a whole mount that is quickly stained. Evidence can then be sought for induced optic axon elongation around or through the lesion site following application of potential neuroregenerative agents. Many eyes can be examined in a short time at comparatively little expense, and, since the technique employs whole mounts, it avoids the delay, inconvenience and error inherent in the reconstruction of serial sections. This in vivo approach provides a more physiologic
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medium than can be obtained in tissue culture, although the latter approach is also to be greatly encouraged. The problem of regenerative failure in the visual pathways is far from hopeless, but little effort has been made to try to reverse the situation. The future course of research will involve a further narrowing down of the list of possible mechanisms of regenerative failure as well as emperical attempts to test potential neuroregenerative agents. References 1. Angeletti PU, Levi-Montalcini R, Calissano P: The Nerve Growth Factor: Chemical properties and metabolic effects. Adv Enzymol 31:51-75, 1968 2. Arora HL: Effect of forcing a regenerative optic nerve bundle toward a foreign region of the optic tectum. Anat Ret 145:202, 1963 3. Arora HL, Sperry RW: Studies on color discrimination following optic nerve regeneration in the cichlid fish Astronotus ocellatus. Anat Ret 131:529, 1958 4. Arora HL, Sperry RW: Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Am Zoo1 2:389, 1962 5. Arora HL, Sperry RW: Color discrimination after optic nerve regeneration in the fish Astronotus ocellatus. Dev Biol 7~234-243, 1963 6. Attardi DG, Sperry RW: Preferential selection of central pathways by regenerating optic fibers. Exp Neural 7:46-64, 1963 I. Barron KD, Doolin PF: Ultrastructural observations on retrograde atrophy of lateral geniculate body. I. Neuronal alterations. J Neuropathol Exp Neurol 26:300-326, 1967 8. Barron DK, Oldershaw BJ: Cytochemistry of lateral geniculate body: Transneuronal and retrograde degeneration. Neurology 15:289, 1965 9. Batkin S: The effect of RNA on the recovery of spinal sectioned carp. Proc Nat Acad Sci 56:1689-1691, 1966 10. Bernstein JJ, Bernstein ME: Effect of glialependymal scar and teflon arrest on the regenerative capacity of goldfish spinal cord. Exp Neurol 19:25-32, 1967 11. Bernstein JJ, Bernstein ME: Ultrastructure of normal regeneration and loss of regenerative capacity following teflon blockage in goldfish spinal cord. Exp Neurol 24:538-557, 1969 12. Bernstein JJ, Bernstein ME: Axonal regeneration and formation of synapses proximal to the site of lesion following hemisection of the rat spinal cord. Exp Neurol 30:336-351, 1971 13. Bernstein ME, Bernstein JJ: Regeneration of
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axons and synaptic complex formation rostra1 to the site of hemisection in the spinal cord of the monkey. Int J Neurosci 5:15-26, 1973 14. Berry M, Riches AC: An immunological approach to regeneration in the central nervous system. Br Med Bull 30:135-140, 1974 15. Bjijrklund A, Katzman R, Stenevi U, West K: Development and growth of axonal sprouts from noradrenaline and 5-hydroxytryptamine neurons in the rat spinal cord. Brain Res 31:21-33, 1971 16. Bjijrklund A, Nobin A, Stenevi U: Regeneration of central serotonin neurons after axonal degeneration induced by 5,6-dihydroxytryptamine. Brain Res 50:214-220, 1973 17. Bruesch SR, Arey LB: The number of myelinated and unmyelinated fibres in the optic nerve of vertebrates. J Comp Neurol 77:631-665, 1942 18. Bullough WS: The Evolution of Differentiation. London, New York, Academic Press, 1967 19. Cajal S Ramon y: Degeneration and Regeneration of the Nervous System. (May RM, translator and editor) London, University Press, 1928 20. Cankovic JG: Contribution to the study of regenerative-degenerative qualities of the fasciculi optici in mammals under experimental conditions. Acta Anat (Basel) 70:117-123, 1968 21. Cavanagh JB, Joseph J: The effects of cortisone, . ACTH, nitrogen mustard and colchicme on the cell population of degenerating spinal tracts in the rabbit. Guys Hosp Rep 107:144-150, 1958 22. Chambers WW: Structural regeneration in the mammalian central nervous system in relation to age, in Windle WF (ed): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 23. Clemente CD: Regeneration in the vertebrate central nervous system. Int Rev Neurobiol 6:257-301, 1964 24. Cohen S: Purification of a nerve growthpromoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum. Proc Nat Acad Sci 46:302-311, 1960 25. Cook WH, Walker JH, Barr ML: Cytologic study of transneuronal atrophy in cat and rabbit. J Comp Neurol 94:267-292, 1951 26. Cragg BG: The fate of axon terminals in the visual cortex during tram-synaptic atrophy of the lateral geniculate nucleus. Brain Res 34:53-60, 197 1 27. Del Cerro MP, Snider RS, Oster ML: Evolution of the extracellular space in immature neurons. Experientia (Basel) 24:929-930, 1968 28. Edds MV: Collateral nerve regeneration. Q Rev Biol 28:260-276, 1953
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JA, Seyan SSAS: 29. Fertig A, Kiernan Enhancement of axonal regeneration in the brain of the rat by corticotrophin and triiodothyronine. Exp Neurol33:372-385, 1971 30. Flint G, Berry M: A quantitative investigation of the response to injury of the central nervous system of rats treated with ACTH and triiodothyronine. Experientia 29:566-568, 1973 31. Gaze RM: The Formation of Nerve Connections. London, New York, Academic Press, 1970 32. Gaze RM, Keating MJ, Straznicky K: The retinotectal projection from a double-ventral compound eye in Xenopus. J Physiol (Lond) 214:37P-38P, 1971 33. Gerard RW: A perspective evaluation of central nervous system regeneration in mammals, in Windle WF (ed.): Regeneration in the Central Nervous System, Springfield, Ill, Charles C Thomas, 1955 34. Gills JP, Wadsworth JAC: Retrograde transsynaptic degeneration of the inner nuclear layer of the retina. Invest Ophthalmol 6~437-448, 1967 35. Goldberg S: Silver staining, featuring rapid reduction, for whole mounts of retina and optic pathways in chick embryos. Stain Technol 47:65-69, 1972 36. Goldberg S, Coulombre AJ: Topographical development of the nerve fiber layer in the chick retina. A whole mount study. J Comp Neurol 146:507-518, 1972 37. Goldberg S, Galin MA: ‘Response of retinal ganglion cell axons to lesions in the adult mouse retina. Invest Ophthalmol 12:382-385, 1973 38. Goldberg S: Studies on the mechanics of development of the visual pathways in the chick embryo. Dev Biol 36:24-43, 1974 39. Goodman DC, Bogdasarian RS, Horel JA: Axonal sprouting of ipsilateral optic tract following opposite eye removal. Brain Behav Evol 8:27-50, 1973 40. Goodman DC, Horel JA: Sprouting of optic tract projections in the brain stem of the rat. J Comp Neurol 127:71-88, 1966 41. Goss RJ: Adaptive Growth. New York, Academic Press, 1964 42. Gray P: Experiments with direct currents on chick embryos. Arch Entw Mechan 139:732-779, 1939 43. Guth L. Windle W: The eniema of central nervous. regeneration. Exp -Neural suppl 5:1-43, 1970 44. Harrison RG: The reaction of embryonic cells to solid structures. J Exp Zool 17:521-544, 1914 45. Harvey JE, Srebnik HH: Locomotor activity and axon regeneration following spinal cord compression in rats with L-thyroxine. J
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DR: Regeneration of dorsal root fibers into the adult rat spinal cord. Exp Neurol 40:333-350, 1973 69. Okun E, Collins EM: Histopathology of experimental photocoagulation in the dog eye. II. Production of cytoid body lesions. Am J Ophthalmol 54:786-793, 1962 70. Ortin L, Arcuate LR: Procesos regenerativos
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