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Download by David Zimmerman The ultimate in nerve regeneration
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by David Zimmerman The ultimate in nerve regeneration—a treatment for paraplegiais still far off. But it is vo longer considered beyond question. Following injury, the living cells of skin, liver and most o t h e r b o d y tissues divide rapidly, repair the damage and restore the organ's normal functions. Neurons—nerve cells—restore themselves far less vigorously, least vigorously of all those of the central nervous system—the brain and spinal cord—of mammals. Restoration of nerve tissue occurs in two ways; in both, regenerative ability diminishes the higher one goes on the phylogenetic tree. O n e way is by mitosis—-cell division— which creates wholly new neurons. These nerve cells then can forge n e w connections to old t e r m i n a l s , t h u s r e s t o r i n g n o r m a l function. Nerve cells that can reproduce by mitosis are found in m a n y invertebrates and in some reptiles and amphibians. But the rule is that neuronal mitosis is not found in higher animals. The second process of nerve repair is axonal reextension, in which a nerve cell's axon—the often lengthy, signal-carrying nerve f i b e r will regrow following injury and ultimately reconnect to its target. Axonal reextension occurs in the central nervous systems of lower animals, and even of some vertebrates. But in higher organisms including h u m a n s , axonal reextension commonly occurs only in the peripheral nervous systems—motor and tactile sensory neurons, for example. A s p i n e - i n j u r e d goldfish will regain s o m e f u n c t i o n s mediated by central a x o n s , for instance, but not others. And a newt—a small s a l a m a n d e r — w i l l r e g r o w an e n t i r e tail, extending central nervous system axons into it. A h u m a n may, over time, find motion and sensation returning to a seriously cut finger as severed peripheral axons reestablish their connections. But despite some claims to the c o n t r a r y , axonal r e g e n e r a t i o n a n d functional recovery from injury to spinal n e u r o n s h a v e y e t to be d e m o n s t r a t e d convincingly in any higher vertebrates, including mammals. MOSAIC September/October 1980 11 It once was considered to be impossible. T o d a y , it no longer is. A n understanding of the factors that inhibit or promote mitosis in some organisms but not in others, or axonal reextension in some central nervous systems b u t not in others, has become an important concern of many neuroscientists. And, no matter h o w fundamental their studies, most scientists investigating nerve regeneration readily acknowledge w h a t one calls their "compassionate c o n c e r n " for the paralyzed h u m a n victims of spinal-cord injury. In the United States, there are an estimated 300,000 paraplegics and quadriplegics. About 15,000 n e w victims of spinal-cord injury are added to the list each year. A decade ago, recalls neuroanatomist Lloyd G u t h of the University of Maryland School of Medicine, the treatment of spinal paralysis was not a concern of basic researchers; the possibility of successful treatment seemed t o o r e m o t e . But a s c i e n t i f i c c o n f e r e n c e c o n v e n e d in 1970 revealed to skeptical neuroscientists that there was indeed cause for t h e m to be concerned: the existence of valid scientific clues to an understanding of barriers to neural regeneration and to methods that might be employed to overcome them. Since then, leaders in the field have come to believe that, as G u t h puts it, "experimental studies on the molecular basis of vertebrate regeneration . . . undoubtedly will lead to m e t h o d s which will enable us to control the (neuronal) regnerative process by minimizing the effects of deleterious extrinsic factors a n d b y e n h a n c i n g the n e u r o n ' s intrinsic regenerative capacity." W h y the confidence? O n e part of the the explanation is new research; another is a reinterpretation of old findings. A crushed or severed axon's first response to the injury is to die back from the wound. This axonal degeneration ceases after a week or so. Then, in those neurons that are capable of it, the neuron appears to stabilize itself metabolically and the axon to regenerate, g r o w i n g past the injury site and restoring function. But not all axons can regenerate. As first described by the Spanish Nobelist Santiago R a m o n y Cajal at the turn of the century, damaged cells in the central nervous systems of higher animals first die back, as do other n e u r o n s . T h e n new nerve sprouts, which m a y come either from the damaged axons or from undamaged collateral fibers nearby, begin to appear. But the sprouts grow only briefly; they soon stop growing and die. M a n y neuroscientists have interpreted this p h e n o m e n o n as a d e m o n s t r a t i o n of the impossibility of regeneration of neurons in the central nervous system. But according to G u t h , it was Cajal's view—which G u t h 12 MOSAIC September/October 1980 n o w shares—that s p r o u t i n g d e m o n s t r a t e s instead the central n e u r o n s ' " i n t r i n s i c regenerative capacity." In this more sanguine view, the task n o w is to define whatever limitations on regrowth are present in order to overcome them, and to understand h o w lower life forms manage to accomplish this task with the hope of applying these findings to repair of h u m a n injury. Tracing the pathways One way to approach this intrinsic capacity, if it indeed is there, is through study of pathways by which some neurons in central nervous systems of some animals do regenerate, tracing them so that their sources, routes and targets can be k n o w n . Frank Scalia and Dan Matsumoto of the Downstate Medical Center in N e w York, among others, are trying to do this through a study of the o p t i c n e r v e s a n d n e r v e p a t h w a y s in t h e leopard frog which, unlike those of most vertebrates, appear capable of regeneration following surgical interruption. T h e tracer they are using is the enzyme h o r s e r a d i s h peroxidase. It has a useful property: W h e n injected into a n e u r o n or taken up by severed axons, it diffuses, Scalia says, or is transported actively t h r o u g h o u t the cell or affected axons, yet does not leak out through the cell membrane into adjacent a r e a s . T h u s , it m a k e s p o s s i b l e a clear delineation for microscopic study of the nerve cell and its axonal pathways. Using this technique, Scalia has been able to pick out individual optic nerve fibers as fine as 0.2 micrometer (micron) in diameter and to trace their paths from the eye to their t e r m i n a l s in t h e b r a i n . By i n t r o d u c i n g horseradish peroxidase into regenerated optic nerves, he and Matsumoto are trying to trace the pathways of axons to see which—and h o w many—of the nerve cells in the retina are responsible for regeneration. Specifically, they hope to learn w h e t h e r the information that dictates the route of an optic-nerve axon's return to its target is distributed all along the p a t h w a y or is available only at or near the target. T h a t there is such a m a p p i n g system is suggested to them by the fact that frogs regain effective vision following optic nerve regeneration. It is believed that this results from precise, point-for-point connections present between the retina and the visual c e n t e r s in the b r a i n . D u r i n g a f r o g ' s embryonic development, these connections are k n o w n to be mediated by embryonic precursors of the optic nerve. A n earlier finding by Scalia and M a t s u m o t o suggests the presence in the adult of what may be a similar system during regeneration. Their studies have s h o w n that, following injury, optic fibers leading to different targets in the brain degenerate at different rates. M o r e i m p o r t a n t , s o m e of t h e s e a x o n s degenerate very slowly; they may remain present and intact d u r i n g recovery. These residual fibers, Scalia speculates, may serve to g u i d e the o t h e r r e t u r n i n g axons back toward their targets. T h e y thus may be an important factor in the u n u s u a l regnerative ability of the frog's optic nerve. If so, finding out how these fibers resist degeneration, as Scalia and Matsumoto now hope to do, could point toward ways to assist regrowth in other injured central n e r v o u s system p a t h w a y s . Crippled fish Another extraordinary model of central nervous system injury, which a principal investigator calls the "paraplegic goldfish," offers s t r i k i n g l y different insights into regeneration. The researcher is neurobiologist S t e v e n J. Zottoli of W i l l i a m s College in Massachusetts. The model is valuable because the fish's dramatic and highly visible ability to perform a sudden tail flip that displaces it sideways out of d a n g e r ' s way is effected via a single pair of h u g e , identifiable axons, called M - a x o n s . W h e n the M-axons are cut, the tail flip d i s a p p e a r s . T h i s m e a n s that Zottoli, unlike Scalia, has the advantage of working with accessible axons that specify easily detectable behavior. T h e M - a x o n s are named for their 19th century discoverer, Bohemian ophthalmologist Ludwig M a u t h n e r . In the goldfish, they run the length of the spinal cord and are large enough to be identified with the aid of a low-power microscope. Each M - a x o n is 100 micrometers in diameter, at least twice the size of the next largest axon in the goldfish's central nervous system. Large axons transmit impulses more rapidly than small ones. And since the huge M-axon transmits an impulse that can be readily identified at the point where it enters the M-cell body in the brainstem, Zottoli is able to distinguish its input in the brain from that of other axons. W h e n its spinal cord is cut, a goldfish will lie on its side; it cannot swim upright and has lost its tail-flip ability. After several weeks' convalescence, the fish may regain the ability to swim upright, a behavior that is believed to be controlled by nearby spinal a x o n s . But its tail flip, mediated by the M-axons, does not return. Zottoli's next round of experiments will be an effort to find out why. He will attempt to determine whether M-axon regeneration is limited by an extrinsic factor—a mechanical barrier or a chemical i n h i b i t o r s e c r e t e d b y n e a r b y cells, for example—or whether it is inhibited by an intrinsic factor, perhaps a metabolite produced within the M - a x o n or the cell body itself. Several hypotheses have been advanced to explain w h y mammalian central nervous L „ 11 ^^ £ 1 bysiein iieuroiitj usuaiiy are no more su^v_essrui in regenerating than is the goldfish M-cell. Scar tissue, which may include glial cells that normally provide structural and metabolic support for the axons, might block regrowth. In a very recent experiment, G u t h and his University of Maryland co-worker, pharmacologist Edson X. Albuquerque, eliminated scar formation as a variable in their work with another animal, the thirteen-lined ground s q u i r r e l . T h e s e s q u i r r e l s are l o n g a n d profound hibernators. T h e y are so damped d o w n metabolically w h e n hibernating that they do not synthesize scar tissue in response to injury. In the experiment, spinal axons were cut while the rodents were hibernating so that no scar tissue would appear. T h e axons nevertheless regenerated only up to the point of injury. There they " s t o p p e d dead in their tracks," G u t h says, indicating that something other than scar tissue inhibited their regrowth. Another possible hypothesis for the failure of c e n t r a l n e r v o u s s y s t e m n e u r o n s to regenerate is that the embryonic stem cells that originally produce central nervous system neurons may no longer be present or are only selectively present in an adult animal. Another is that regrowth may be frustrated by the a b s e n c e of the s u p p o r t cells t h a t originally laid out the pathway and provided the directional cues during embryonic neural development. A l t e r n a t i v e l y , the m y e l i n s h e a t h t h a t surrounds and encloses the M-axon and other central nervous system axons may become impenetrably fused following injury. Or these nerves may be prevented from regenerating by the very slow rate at which the materials required for elongation can be transported d o w n the axon. These latter possibilities, Zottoli suspects, are the most promising; they are the ones he is n o w pursuing. In his current experiments, Zottoli is trying to cut just the M-axon, leaving other, nearby axons intact. T h e ability to do this, he says, will eliminate m a n y of the extrinsic f a c t o r s such as mechanical barriers and vascular interruptions—that have complicated previous studies of this kind. Biochemical cues Some hard data already are available on one of Zottoli's questions: the rate at which cut axons regrow. Axonal reextension closely reflects the rate at which actin, one of the principal cellular building blocks of nerve fibers, is transported from a nerve's cell body d o w n to its axon's growing tip. These findings come from the Case Western Reserve University School of Medicine, where neurobiologist R a y m o n d J. Lasek and his colleagues have s h o w n that repair materials, packaged in discrete intracellular structures, move d o w n the axon at varying rates of up to 400 millimeters a day. Actin, however, is carried by one of the slower of these packets, which Lasek calls slow component b. It travels at a b o u t two millimeters per day. T h i s neatly—but not surprisingly—turns out to be almost identical to the rate of axonal r e g r o w t h . A n d Irv M c Q u a r r i e , a n e u r o surgeon working with Lasek, has shown that twice-injured axons appear to regrow faster than axons injured but once, suggesting that whatever the repair mechanism turns out to be, it can be stimulated. According to another of Lasek's associates, neurobiologist Scott Brady, these findings u n d e r s c o r e the p o i n t t h a t nerve g r o w t h depends on biochemical cues as well as on p h y s i c a l stimuli and c o n s t r a i n t s . C o n s e quently, it will require new approaches to a field that heretofore has been dominated by morphological, physiological and behavioral studies. Already, for example, Lasek and his colleagues have found biochemical differences in the structure of uninjured axons and those re-elongated following injury. They are now at w o r k t r y i n g to s p e c i f y w h a t t h e s e differences are. Sleeves and channels If growth—elongation—is one fundamental element in nerve regeneration, a second is direction. A n elongating axonal tip must travel toward its severed peripheral or central connections if it is to contribute to functional r e s t o r a t i o n . Since the axon d e g e n e r a t e s MOSAIC September/October 1980 13 backward from both sides of a nerve wound, regenerative repair from one side only may be a long process, even for a fiber that has been injured close to its target endpoint. A pioneer investigator of this problem has been L a s e k ' s chief at C a s e W e s t e r n Reserve, neuroanatomist Marcus Singer, who says he n o w may have part of the answer to h o w the nerve reaches its destination. He rejects the notion that the target tissue secretes a specific substance that the regrowing axon can sniff and h o m e in on like a beagle dog. " N e r v e fibers are not smart e n o u g h to reach their targets by themselves/ 7 Singer says. R a t h e r , he a n d d e v e l o p m e n t a l n e u r o biologist Ruth H . Nordlander suggest, on the basis of a decade's research, the axons are guided from the injury site toward their original connections by a system of "canals" c o m p o s e d of e p e n d y m a l cells, a type of epithelial support cells. These canals develop ahead of—and so guide—the regenerating axon tip. This guidance may be mediated by tactile cues or by substances secreted by the canal walls. T h e existence of an ependymal guidance system first was inferred over a decade ago from electron m i c r o g r a p h s of n e w t tails regenerating after amputation. N e w t s are a principal model for Singer, who long has been a leader in the experimental study of limb regeneration. Cross-sectional electron micrographs of Mr, Zimmerman, author of "Rh, The Intimate History of a Disease and its Conquest" and "To Save a Bird in Peril," has also written articles for Smithsonian, A u d u b o n , Natural History and T h e N e w York Times Magazine. 14 MOSAIC September/October 1980 n e w t - t a i l s p i n a l c o r d , m a d e s o o n after a m p u t a t i o n , s h o w e d m a n y light areas— a p p a r e n t l y e m p t y spaces—between cells. Initially, these were thought to be artifacts of the fixation techniques. However, slides taken days or weeks later showed axons growing within these spaces. "It made us t h i n k , " N o r l a n d e r recalls, " t h a t the pattern of vertical fiber pathways was being directed by the e p e n d y m a l cells. I think there are channels there . . . toward the target." This i n t e r p r e t a t i o n was considerably strengthened in recent years when Nordlander discovered strikingly similar channelization in the n e u r o n a l development of embryonic newts. In n o r m a l development, as in regeneration after injury, it appears that epithelial channels guide the growing axon tips toward the a p p r o p r i a t e targets. The a p p a r e n t similarity between the two events strongly s u g g e s t s to Singer and N o r d l a n d e r that regeneration is a replay of embryogenesis. Injury, in the newt at least, reactivates the developmental process, and the mystery of h o w regenerating axons find their way back thus ties to the question of h o w nerves grow and establish connections in the first place. Blueprint hypothesis Since repair may represent reactivation of a developmental blueprint, Singer, Nordlander, and M a r g a r e t Egar w h o demonstrated the same phenomenon in lizard-tail regeneration, call their theory " t h e blueprint h y p o t h e s i s . " They say the ependymal cells in these animals have "retained the capacity to unroll a blueprint that the nerve fibers have to follow to reach their destination. . . . In addition to providing specific highways for regenerating axons, the blueprint hypothesis implies that the individual axons 'recognize' and follow p a r t i c u l a r itineraries, even w h e n challenged by m u l t i p l e h i g h w a y s . " T h i s guidance system, Singer adds, is probably not target-specific. It brings the axon to the ppropriate area, b u t some other,still-to-beelucidated cue then must tell it to stop elongating and make a connection. T o check both of these components of the blueprint hypothesis-—to induce a severed neuron to extend its axon beyond the wound and seek reconnections—a bridge across the wound has been sought. The possibility that a surgically implanted graft of healthy nerve tissue could serve as such a bridge is one approach being tried. N e u r o l o g i s t A l b e r t A g u a y o , of McGill University, and Carl C. Kao, a Georgetown University neurosurgeon, both have used i m p l a n t s of e m b r y o n i c nerve tissue and reported some success in inducing central n e r v o u s system axons to regrow into such " c a b l e g r a f t s " in d o g s . S i n g e r a n d his Cleveland colleagues have introduced a couple of wrinkles into the cable-graft option. Reasoning that the regenerative spinal cord from lower v e r t e b r a t e s may h a v e useful properties that are lacking in the nonregenerative mammalian cord, anatomist Keith Alley, an associate of Singer's, transplanted a "bologna slice" of lizard tail. The slice, with is e p e n d y m a l s u p p o r t - c e l l c h a n n e l s correctly aligned, was inserted into a notch cut into the spinal cord of a nude mouse, a mutant chosen because it lacks immunologic competence and so cannot reject the graft. He later found axons, which he believes are from the mouse, in the cable graft. But, he says, there were disappointingly few of them. He plans horseradish peroxidase studies to see If these axons will reenter the mouse tissue on the far side of the bridge. Peripheral neurons T h e guidance role that support cells play in axonal r e e x t e n s i o n has been further suggested by experiments that University of Utah neurophysiologist Kenneth W. Horch conducts with cats. He focuses his attention on sensory nerves and their receptors in the skin of the cat's hind leg, a peripheral nervous system model. Horch stimulates raised pressure-receptor domes in the shaved skin of a cat's leg in order to delineate the skin area served by a nerve he has exposed surgically. The borders of the region and the domes themselves are marked to produce a m a p of the receptors activated by the nerve. T h e nerve then is either cut or crushed, and the wound is closed. After the nerve has had several months to regenerate, Horch repeats the procedure and compares before-and-after maps. With crush, the before-and-after maps are essentially the same, leading Horch to conclude that reinnervation proceeds along preexisting channels that survive the injury. With cut, which disrupts the channels, the picture is far different; more than half the receptor domes will have disappeared from the skin by the time of the reexamination; they a p p a r e n t l y a t r o p h y for w a n t of a functional axonal contact. Those that remain appear to have been innervated by other than their original axons. This scrambling of the wires, as it were, may well compromise the organization of sensory receptors and their neuronal connections to the brain. Compensating adaptation But functioning may be restored even if circuits remain hopelessly scrambled. There is a current belief, Horch says, that in mammals everything is "hard-wired," that i n a p p r o p r i a t e r e c o n n e c t i o n s are n o t selfrectified. T h e alternative that some investigators are coming to accept, and that Horch n o w is exploring, suggests that everything MOSAIC September/October 1980 15 is not hard-wired, that there is some innate plasticity in the mammalian nervous system. Such plasticity would enable a cat, or a human, to use higher centers in the brain to learn other methods for processing information delivered through an altered p a t h w a y . (See "The Neural Net: Change in a Fixed System/ 7 Mosaic, Volume 9, N u m b e r 2.) Vanderbilt University neurobiologist Jon H. Kaas is pursuing similar possibilities in experiments with a primate on which the mapping is being performed higher in the central nervous system—in the brain. The mapped target is an area of the cerebral cortex that is activated by sensory impulses carried from the palm of the h a n d via the median nerve. T h e owl m o n k e y was the p r i m a t e c h o s e n for t h e s e e x p e r i m e n t s , because, in this South American species, the cortical area activated b y the palm is a large area on the surface of the brain; it can be easily mapped before and after the nerve is damaged at the level of the wrist. W h e n the median nerve is crushed, says Kaas, it regenerates correctly; points on the palm reactivate their former target points in the cortex. With cut injuries, however, he finds, as did Horch, " m a n y mislocations" on the map; sensory points on the palm now target different points in the cortex. Kaas now is looking for changes in cortical organization in a follow-up experiment in which the nerve serving the palm is cut and then prevented from regenerating. He has found that in such a case the cortical area previously served by the palm via the median nerve becomes reactivated by another nerve—either the ulnar or the radial—which carries sensory impulses from the back of the hand to its own parts of the brain. To learn the effects of such anomalous connections, Kaas has conditioned his owl monkeys to reach u p w a r d in response to stimulation on the back of the h a n d and downward when the palm is stimulated. The q u e s t i o n is w h e t h e r a m o n k e y , after the severing of the median nerve serving the palm and after takeover of the palmar part of the cortex by either the radial or ulnar nerve, will continue to move the h a n d up, as it has been taught, w h e n the back of its hand is stimulated. If it doesn't, if it reaches d o w n in the palmar direction in response to back-ofthe-hand stimulation, the implication is that the reactivated palmar or median area of the cortex—which, because it is as m u c h as 20 times the size of the area served by the radial or u l n a r nerves—overrides a n d p r o d u c e s sensations on the palm no matter h o w it got its message. T h e inference is not conclusive, because the radial or ulnar nerves would 16 MOSAIC September/October 1980 still be performing their normal function as well and might not be overridden. But the implication w o u l d be t h a t the b r a i n is hard-wired rather than plastic. T h e palmar region remains the palmar region however it is stimulated. But if such a monkey later correctly reaches u p w a r d in r e s p o n s e to b a c k - o f - t h e - h a n d stimulation, despite the fact that the brain's dominant palmar area is being stimulated, Kaas adds, then the absence of hard-wiring is suggested; there is plasticity in the cortex that permits its palmar area—or presumably any newly i n n e r v a t e d region—to serve whatever neuron brings messages to it. T h e brain would be adapting itself to respond a p p r o p r i a t e l y e v e n to s c r a m b l e d i n p u t pathways. "This would be very exciting from the point of view of recovery from nerve d a m a g e , " Kaas says. His experiment has not yet been completed; his hypotheses are still hypotheses. Though training has begun and the necessary surgery has been scheduled, several months m u s t elapse after surgery for the animals to heal before testing. An exciting exception In these experiments, Kaas, like many of his fellow investigators of n e u r o n a l regeneration, is s t u d y i n g a x o n a l r e g r o w t h of peripheral n e u r o n s . But from the medical viewpoint, and perhaps scientifically too, the u l t i m a t e c h a l l e n g e is t h e m a m m a l i a n central nervous system, where injured axons sprout and die back without ever reconnecting and where the power of mitotic division appears to have been lost; mitotic division and recovery of function for the most part simply do not appear to occur in the mammalian central n e r v o u s system. Recently, h o w e v e r , a s u r p r i s i n g and provocative exception has come to light: neurons of the olfactory nerve. These are part of the central nervous system and are often called an extension of the brain outside the cranial cavity. U n d e r w h o l l y n o r m a l conditions, these n e u r o n s c o n t i n u o u s l y die and are replaced t h r o u g h mitotic division of neuroepithelial stem cells. W h e n olfactory n e u r o n s are d e s t r o y e d , t h e y n o t o n l y regenerate, b u t they avidly search out and forge n e w connections. T h e y will seek out the olfactory bulb in the brain, their normal t e r m i n a l , or they will seek deeper b r a i n structures if the bulb has been surgically ablated. These remarkable regenerative powers are present in mature as well as immature animals, in the "morphologically identical" olfactory systems of mice, rats, cats and m o n k e y s , and very likely also in h u m a n s . T h e y are present as well in the sensory apparatus of lower animals, including the octopus in which they were first elucidated. T h e researchers who n o w have confirmed the admittedly wild hypothesis that the prim a r y olfactory neurons continuously turn over, doing something no other mammalian n e u r o n appears able to do, are neuroanatomists Pasquale P. C. Graziadei and Giuseppina A. M o n t i Graziadei—husband and wife—of Florida State University. A colleague, biochemist Frank L. Margolis of the Roche Institute of Molecular Biology, has isolated and characterized a low-molecular-weight protein that occurs in the olfactory neurons of all vertebrate species studied, but apparently not in other central nervous system neurons. This olfactory marker protein, Pasquale Graziadei says, is unique in that "it is the only protein that has been found that appears to be specific to a class of n e u r o n s . " This suggests, he adds, that " m a y b e we have a golden egg inside." T h e possibility, which he and other researchers now are avidly p u r s u i n g , is t h a t t h i s s i n g u l a r p r o t e i n stimulates or de-represses mitotic regeneration in olfactory neurons. So far, efforts to crack the "golden e g g " have been hindered by the scant supply of the marker protein. The marker, says Pasquale Graziadei, has thus far been " v e r y difficult" to isolate and purify. O n e of the Graziadeis' first experimental efforts, using an antibody to the olfactory marker protein supplied by Margolis, is to try to block the protein in living olfactory neurons. They hope thereby to see if such blocking compromises the ability of the cells to r e g e n e r a t e . " W e ' r e working, and we do have leads," is all that Pasquale Graziadei will say at this time of this provocative work in progress. The olfactory neuron's unique regenerative properties reflect its unique—and Pasquale G r a z i a d e i believes quite primitive—traits. He first explored these more than two decades ago in the analogous neuronal elements of the octopus, an animal with olfactory-like sentivity a thousand times that of h u m a n s . He located sensory receptors on the octopus's lips and on the rims of the suckers on each of its arms. He found that in octopuses, as in the higher animals that he has studied since, c h e m o s e n s o r y n e r v e cell b o d i e s — s o m e animals may have as many as 50 million of them—are spread t h r o u g h o u t the sensory tissue, or n e u r o e p i t h e l i u m , a n d are not collected in groups or ganglia as is the case with other types of sensory n e u r o n s . A n d unlike other central nervous system sensory neurons, the dendrites and ciliary appendages of these cells penetrate the epithelial surface and are in direct contact with the environment. T h e regenerative capacity of vertebrate olfactory neurons had been suspected by some 1 9 t h c e n t u r y a n a t o m i s t s , b u t this possibility was not fully appreciated by their 20th century successors, Pasquale Graziadei says, until he and his wife demonstrated its existence. They showed that the nucleoside thymidine, which is incorporated by cells only during mitotic division, is present in the cytoplasm and nucleus of olfactory stem cells. It was then seen in the olfactory neurons' d a u g h t e r cells, after the s t e m cells were incubated with thymidine. "For the first time," Pasquale Graziadei exclaims, " w e have a neurogenic matrix that replaces the neurons that die out—and the new n e u r o n s regrow their axons. W e have a neural system that recovers from injury." T h e Graziadeis have c o n f i r m e d these f i n d i n g s m o r p h o l o g i c a l l y , by d i s c o v e r i n g regenerated cells that reestablished synaptic c o n n e c t i o n s in m o u s e b r a i n s . T h e y h a v e confirmed morphological recovery following surgical injury to the olfactory nerve even in monkeys. Functional and behavioral studies are continuing in the Graziadeis' Tallahassee laboratory and in several others. " W e have found a neuron that is exceptional," Pasquale Graziadei says. " W e must n o w try to find the parameters of its exceptionality." Postulates for nerve research M u c h current research is directed to the elaboration of models that may point to factors t h a t r e p r e s s or p r o m o t e n e r v e g r o w t h . Experimental rigor is s o u g h t because m a n y of these experiments are difficult to conduct and interpret, given the n u m b e r of neurons a n d t h e w e l t e r of t h e i r a x o n s in m o s t physiologically significant nerve tracts. T h e frog optic nerve, to cite one such model, carries almost half a million axons emanating from a like number of nerve cell bodies. Experimental rigor is sought also because claims have been made that several experimental surgical and pharmacologic therapies h a v e successfully p r o m o t e d s p i n a l cord regrowth in higher animals, even h u m a n s . " N o n e of these therapies has produced regeneration," says neuroanatomist Lloyd G u t h . But "these misleading claims have had the unfortunate effect of confounding the scientific literature, promoting wasteful duplication of scientific effort and d i s a p pointing the paraplegic c o m m u n i t y . " T o limit m i s l e a d i n g claims, G u t h , a n d associates in the Stroke and Trauma Program of the National Institute of Neurological and Communicative Disorders and S t r o k e , recently published a set of "Koch's postulates" or rules for proving regeneration, w h i c h , G u t h says, have thus far not been w h o l l y satisfied in any single experiment. (Robert Koch, the t u r n - o f - t h e - c e n t u r y G e r m a n bacteriologist and Nobelist, established a set of rules—Koch's postulates—for confirming the connection between a particular m i c r o organism and a particular disease. T h e t e r m has been generalized to signify rules of proof in any scientific inquiry.) To conclude that functional regeneration of spinal neurons has occurred, Guth's postulates stipulate, a researcher must demonstrate: • T h a t the e x p e r i m e n t a l lesion c a u s e d disconnection of the nerve processes. • That the new central nervous s y s t e m fibers bridge the level of the injury and make j u n c t i o n a l contacts as d e m o n s t r a t e d b y anatomical studies. • T h a t the regenerated fibers also can be shown to generate nerve responses across the injury. • That some functional change—preferably for the better—can be s h o w n to result from new connections. These postulates thus become, in a sense, an agenda as well as a challenge a n d a c o n c e p t u a l f r a m e w o r k for r e g e n e r a t i o n research in the 1980s. T h e propriety of s u c h a f r a m e w o r k is u n d e r l i n e d by d e c i s i o n s reached, in the spring of 1980, at a conference in B e r m u d a u n d e r t h e aegis of D u k e University and the Paralysis Cure Research Foundation. Just a decade after the earlier conference, which Guth recalls as the turning point of the field, participants in the Bermuda meeting identified central nervous s y s t e m r e g e n e r a t i o n r a t h e r t h a n , for i n s t a n c e , improved acute care of spinal injury, as a reasonable research target for the future. Such a target is b o u n d to breed u n d e r s t a n d a b l e b u t unrealistic e x p e c t a t i o n s in persons afflicted by nerve injuries a n d in those who care about them. T h e " c o m p a s sionate concern" that investigators of neural regeneration express for the victims of spinal cord injury requires that the scientists avoid to the extent possible the e n c o u r a g e m e n t of such premature or u n w a r r a n t e d o p t i m i s m . Such strictures as Koch's postulates for nerve research may well be an appropriate screen. • The National Science Foundation contributes to the support of the research discussed in this article through its Neurobiology and Sensory Physiology and Perception Programs, MOSAIC September/October 1980 17