“Paralyzed from the waist down.” “A severed spinal cord.” Just a decade ago, they were pronouncements as solemn as fate, and as without hope, for two million people with spinal cord injury. For scientists, spinal cord repair was a research project for visionaries (to put it kindly). There were a few—very few—such visionaries, however, and when their time came, at last, it came with explosive speed and impact. Award-winning science writer Luba Vikhanski journeyed through eight countries, conducting some 150 interviews, to unearth this story of the courage and conviction of a handful of scientists who challenged the impossible and changed brain science forever. This excerpt from the introduction and chapter 14 of In Search of the Lost Cord looks at how the “unthinkable”— the regeneration of the spinal cord—has become a scientiﬁc race for success.
Excerpted from In Search of The Lost Cord: Solving the Mystery of Spinal Cord Regeneration by Luba Vikhanski. Co-published by The Dana Press and Joseph Henry Press, ©2001, Luba Vikhanski. Reprinted with permission. All rights reserved. The full text of this book can be found at the Joseph Henry Press website, www.jhpress.org
NEW HOPE AFTER 3,500 YEARS
“A disease that cannot be treated” was how an Egyptian papyrus referred to spinal cord injury more than 3,500 years ago.1 When this first medical document to describe spinal cord trauma was translated into English in 1930, the verdict was picked up by medical textbooks around the world as still sadly relevant: Several millennia had passed, but physicians were still unable to treat injuries to the spinal cord. Only in the middle of the twentieth century did medicine learn to keep spinally injured people alive. The resulting paralysis, however, was considered irreversible. Dead nerve cells in the spinal cord, scientists believed, could not be replaced; severed nerve fibers would never regenerate; paralyzed people would never walk again. With hardly any research in this area, there was no progress, which in turn reinforced the impression that the problem was intractable.
But in the past two decades, the attitude toward spinal cord injury changed dramatically. In the 1980s, thanks to new discoveries and tools, research in the field of spinal cord regeneration and repair began to gain credibility and momentum. The enormity of the challenge— repairing a structure once believed to be unfix-able—created an appeal some researchers could not resist. It offered an opportunity to leave a mark, to make headway in uncharted waters. At about the same time, the entire thinking about the central nervous system—consisting of the brain and spinal cord—underwent a revolutionary transformation. Once considered a fixed, rigid network of nerves, the system is now viewed as flexible, malleable—and hopefully, repairable. There is now hope that one day, people with paralysis will be able to regain at least some of their lost abilities: breathing, sexual function, bladder and bowel control, walking. Implications for other diseases of the nerves, particularly such brain disorders as stroke and Alzheimer’s and Parkinson’s diseases, can also be great, as spinal cord and brain research are closely intertwined.
The new hope touches the lives of millions of people. In 2001, individuals paralyzed by trauma to the spinal cord numbered 250,000 in the United States alone and an estimated 2 million worldwide.2 Leading causes vary in different parts of the world: traffic accidents in the United States, gunshot wounds in Brazil, earthquakes and coal mine accidents in China, and falls from coconut trees in the Philippines. Some population groups are more vulnerable than others; in many countries, young men, the group most prone to risk-taking behavior, account for more than half of the cases. But anyone anywhere is a potential victim: Some 85,000 new spinal cord injuries around the world—10,000 of them in the United States—put people in wheelchairs every year. The numbers for all neurological illnesses combined are even more staggering. These illnesses annually affect more than 50 million Americans and cost society more than $400 billion, according to the Society for Neuroscience.3
Today hundreds of scientists in different countries are striving to regenerate and repair damaged nerves in the spinal cord, and the pace of their research continues to accelerate. “Now it’s quite accepted that regeneration is a serious scientific field that is going somewhere, not a bunch of lunatics pursuing some crazy notion,” says Dr. Fredrick Seil, who for 15 years organized annual regeneration conferences on behalf of the Department of Veterans Affairs in the United States. But as recently as the mid-1990s, sanctioned human trials for most therapies were only a distant prospect. Then, toward the end of the decade, reports of experiments in humans began to trickle in. Fetal cell trials were launched in Sweden and the United States. In 1999, an Israeli trial got a green light from the U.S. Food and Drug Administration (FDA). In 2000, two other trials, to be conducted in the United States, were approved by the FDA. By 2001, the regeneration field was abuzz with talk about sanctioned human experiments expected to begin in the first decade of the twenty-first century. An FDA approval of a trial does not mean that a therapy is safe and effective—these are precisely the unknowns the trial is designed to resolve. Nor is a single therapy likely to provide a definitive cure for paralysis; scientists believe that if the task is doable, it will take a combination of different approaches to fully repair a complex organ like the spinal cord. The exciting part, however, is that many such approaches are in the works. Will these approaches work in humans? As several regeneration therapies reach the finish line, the answers are nearer, the suspense mounts.
Spinal cord repair involves many goals, but this book focuses mainly on regeneration, a compelling quest to reverse the irreversible, to undo a fatal mistake. It does, however, refer to other aspects of repair, such as “reeducating” the spinal cord after injury, as well as to certain brain research projects, while the appendix offers a review of spinal cord anatomy and several basic concepts of neuroscience. This book is the tale of the turnaround that has occurred in spinal cord research and of the people who made hope real—their failures and successes and their dedication to a scientific pursuit of a goal once considered an impossible dream.
AXON, WHAT GUIDES YOU?
A hot new field of neuroscience seeks to explain how nerve networks come into being and is opening up the intriguing possibility of healing the injured spinal cord with the same tools that nature used to wire it up in the fetus. The study of axonal guidance, as this area of research is known, took off in a major way only in the mid-1990s, but it may very well hold the final key to the future of spinal cord regeneration. Studying axonal guidance may allow scientists to simultaneously go after two major goals that until recently were usually pursued in separation: to stimulate the growth of axons and to eliminate the mechanisms inhibiting this growth.
The fetal nervous system is a busy place. It swarms with billions of hairy nerve fiber tips that crawl about until they hook up into elaborate nerve circuits. The result of this wiring is a network that, in humans, holds the distinction of being the most complex object in the known universe. The human brain alone has more than 100 billion nerve cells, 4 about 10 times more than the number of stars in our galaxy.5 Some simpler neurons have “only” 100 connections with other neurons, but many have 1,000 to 10,000, or even more. How are circuits of such mind-numbing complexity formed? Apparently, growing axons are guided to their new homes by chemicals that have the effect of far-reaching “odors.” The growing axons, like living beings with a strong preference for particular odors, appear to be attracted by some chemicals and repelled by others.
Once the development of the nervous system is completed, the nerve fibers stop their crawling about. Scientists suspect that the same mechanisms that “freeze” the crawling remain present throughout adulthood and may inhibit effective regeneration after injury. When the Swiss scientist Martin Schwab zeroed in on the first growth-inhibiting mechanism in the adult spinal cord in the mid-1980s, he was hoping to find one regeneration-blocking molecule. He identified the protective myelin sheath as a “fixative” blocking nerve growth in the adult spinal cord and spent 15 years scanning myelin for the inhibiting molecule he called Nogo. Now it appears that the brain and spinal cord are also strewn with numerous molecules that helped wire up the system in the fetus but “froze” it after the development was completed. To produce regeneration, scientists would have to “unfreeze” the spinal cord, unleashing the guiding chemicals and inviting the cord to be “born again.” After all, during regeneration the axons need to repeat what they once did in the developing nervous system: grow and arrange themselves into functioning circuits.
The idea that chemicals control the wiring up of the nervous system goes back to Santiago Ramón y Cajal. In 1892, relying entirely on observation and intuition, Cajal postulated that chemicals of an unknown nature guide growing axons in the embryo—and consequently, during regeneration, one of the founders of modern neuroscience. The tools to identify the guidance molecules were unavailable at the time, and a competing theory took over in the 1920s and 1930s.6 It stated that during development growing nerves form random connections that are later transformed by experience and that in the course of this process, only the appropriate connections survive. Scientists were claiming that growing nerve fibers found their way to appropriate targets by following physical pathways, or grooves, in the developing tissue.
In the early 1940s, a future Nobel laureate, Roger Sperry, then at the University of Chicago, revived the research into chemical guidance of growing and regenerating axons. Sperry would later be called “the rebellious graduate student” because he began these studies while still working on his doctorate and challenged the views of his teachers, among them some of the strongest proponents of the physical guidance idea.7 He devoted more than a decade to nerve regeneration studies in fish, frogs, and salamanders and showed that growing nerve fibers did not follow a fixed mechanical path but appeared to carry chemical labels that enabled them to sort each other out.
In one of his most famous regeneration studies, Sperry showed that even when regenerating fibers are artificially deflected from their course, they adopt tortuous routes and find ways to grow back to their normal targets. In one experiment, he cut the optic nerve of a frog, which, unlike that of mammals, can regenerate and restore normal vision. He then rotated the eye 180 degrees, waited for the nerve to regenerate, and submitted the frog’s vision to a simple test. When presented with a bug, the frog flicks its long tongue to snap its favorite delicacy; if it can see well, it won’t miss. Sperry found that the regenerated optic fibers from the frog’s rotated eye followed a roundabout route to their original targets in the brain. For example, the axons from what had originally been the bottom side of the retina restored connections with the part of the brain normally responsible for receiving signals from the bottom of the retina. As a result, the frog now saw the world upside down and backwards; the floor appeared to be above its head and the ceiling below, and on the vision test the frog acted accordingly: It erroneously flicked its tongue downward to catch the bug above its head and upward to catch the bug below. The preprogrammed wiring had been restored with ruinous consequences for the frog’s ability to feed itself. These results suggested that the axons did not follow a predetermined mechanical path but searched out the appropriate target, apparently guided by chemical cues.
Such biochemical recognition is now known to lie at the basis of connections formed by neurons in development and in regeneration. In fact, some of Sperry’s devotees believe his contributions to this field were worthy of a Nobel Prize, 8 but he won the award in 1981 for a different achievement. (That research, conducted by Sperry later in his career at the California Institute of Technology, focused on the so-called split brain and showed that although the brain’s two hemispheres work closely together, consciousness and awareness seem to exist separately and independently in each hemisphere.) Sperry’s research did not immediately help revive Cajal’s ideas, and chemical attraction remained extremely unpopular for several decades despite accumulating evidence in its support. It was only in the early 1990s that the search for guidance chemicals began in earnest.
IF A FETUS CAN DO IT...
One of the studies was initiated in 1990 by Marc Tessier-Lavigne, a neurobiologist in his 30s who had just set up his own lab at the University of California in San Francisco. The Canadian-born Tessier-Lavigne had started out studying mathematics, then become captivated by the circuitry of the brain, and done postdoctoral studies in neuroscience at Columbia University. When he announced his intention to look for the guidance molecule, some seasoned researchers were skeptical.9 The chemical attraction idea was still controversial, and finding one of the guiding molecules, if they existed, seemed like a long shot.
Tessier-Lavigne decided to focus on the favorite spot for axonal guidance studies, the midline of the embryo. This is an area where growing axons behave in a dramatic and stereotypical manner. On a particular day of development, some of the axons on each side of the embryo turn their growth cones toward the midline, as if responding to an irresistible appeal, and head in its direction until they cross over to the other side. The result is a crisscrossing pattern of axons in the middle of the brain and spinal cord. In the brain, some axons from the left hemisphere cross over to the right, while some of those from the right cross over to the left. This is why in stroke, for example, people with damage to one side of the brain have impairment on the opposite side of the body. No one knows why the nervous system is organized in this manner, but hypotheses abound. One plausible explanation is that the crisscrossing of axons enables the right and left halves of the nervous system to communicate. A simpler, somewhat unromantic, theory states that the crisscrossing keeps the two halves from falling apart.
Tessier-Lavigne wanted to identify the guidance signal that attracts axons to the midline in vertebrates and decided to conduct his research on chick embryos. In case the guiding molecule was scarce, he reasoned, it would be easier to obtain large numbers of embryonic brains from chicks than from rats or mice. This turned out to be a wise decision because eventually it would take 25,000 brains to identify the guidance signal. Once a week for several months, a truck carrying cartons with 1,000 fertilized eggs would climb up the scenic streets of San Francisco and pull up behind the gray, 14-floor university building housing Tessier-Lavigne’s lab. All the eggs contained embryos that were 10 days old, an age at which embryonic brain tissue was found to attract axons in a laboratory dish. The research team included two postdoctoral fellows and three graduate students, but when an egg shipment arrived, everybody—other students, friends of students, family members—was recruited to help. On what was called “the Bastille day” of the week, some 10 people would line up to crack the shells of 1,000 eggs, extract the embryos, and dissect out their brains. The decapitation created a formidable mess after which the lab required a thorough cleaning, but it produced half a beaker containing 1,000 embryonic brains for the study.
Within two years—an extraordinarily short period considering the novelty of the field and the enormity of the challenge—the team purified one protein, out of some 50,000, that was making the attraction happen in the chick embryonic nervous systems. “They say luck favors a prepared mind, and that may have been true in our case, but it would be silly to deny there is some luck involved in research—and we were obviously incredibly lucky,” says Tito Serafini, former postdoctoral fellow in Tessier-Lavigne’s team. The scientists then cloned the gene that carries the code for the protein, and called it netrin, after the Sanskrit root netr, meaning “one who guides.” The netrin report appeared in the journal Cell in August of 1994,10 roughly 100 years after Cajal had formulated the chemical guidance idea.
“THE LIKENESS OF BEING”
The scientific community was spellbound by the discovery of a molecule that attracted growing axons in the embryo. But the true bombshell was dropped when the scientists realized that netrin was strikingly similar to an axonal guidance gene identified two years earlier in a microscopic worm. The similarity suggested that some of the same mechanisms are used to put together the nervous system in worms and in vertebrates!
Many early studies of axonal guidance, from the 1960s through the 1980s, had been conducted on invertebrate creatures, such as insects and worms, but that research seemed hardly applicable to vertebrates, including humans. One object of study had been the microscopic worm Caenorhabditis elegans. Its nervous system, which consists of only some 300 neurons, provided a convenient research model, yet it seemed hardly a match for the awesome neural arrangements of more complex organisms. Although some of the scientists conducting this research believed their work would eventually be relevant for all organisms, few colleagues took them seriously. When Edward Hedgecock, of Johns Hopkins University, who had performed some of the first axonal guidance studies in worms, once expressed the idea in writing, a colleague who reviewed his paper suggested removing the passage because, he said, the parallel between worms and vertebrates was too far-out. Hedgecock’s collaborator, Joe Culotti, of the University of Toronto, whose lab welcomes visitors with the sign “We have worms,” recalls: “When we talked about it people would sort of chuckle, ‘Oh, right, sure,’ but we were quite serious. We always thought there would be similar genes that would play similar roles in worms and in the vertebrate spinal cord. What we didn’t imagine was that they’d be discovered so soon in vertebrates. We thought we had more years to play around with the genetics of the worm and just live in our own little corner of the universe, building the story up.”
The worm gene found to be similar to netrin belonged to a group of genes named uncoordinated—or unc for short—because when these genes were missing or did not function properly, the worm’s axons became messed up and the animal did not wiggle properly. Teams of scientists headed by Hedgecock and Culotti had established that several of the unc genes could account for axonal guidance problems in the C. elegans worm. When the netrin report came out, findings from the netrin and unc studies were found to complement each other in many ways, and together they created a fuller picture of axonal guidance. The report prompted a spate of other comparisons among guidance molecules that were beginning to be discovered in different organisms, and numerous similarities emerged. The same genes, with minor variations, were found to play similar roles in the nervous system’s wiring in worms, insects, birds, and mammals.
It was a humbling but also a fantastic discovery. Corey Goodman, of the University of California at Berkeley, a veteran investigator of axonal guidance, whose own work had dealt with fruit flies, summed up the discovery’s implications in the title of an editorial as the “likeness of being.”11 Worms seem light-years away from vertebrates—particularly the species engaged in neuroscience experiments—yet some of the tools used to guide developing axons have been preserved in the course of evolution and are strikingly similar in these different creatures. The finding was all the more remarkable considering that worms and vertebrates diverged on the evolutionary tree some 600 million years ago. Scientists studying different organisms could now trade insights and make rapid progress in deciphering how the nervous system is formed.
As befits a mechanism perfected over 600 million years, axonal guidance is a smoothly scripted affair. Axons travel a long distance, sometimes more than a thousand times greater than the diameter of their cell bodies, before settling into their assigned spots. They negotiate this challenging journey by breaking it up into short segments, each perhaps a fraction of a millimeter long. At the end of each segment, the growth cone appears to pause, like a traveler at a crossroads, making navigating decisions. What helps it choose its course is the presence of guidance chemicals. Each growth cone is equipped with numerous receptors that allow it to sense attractive and repulsive guidance molecules released by distant tissues. Scientists have found support for Cajal’s idea that the chemical guidance is “graded”: If an appealing “odor” is diffused throughout the tissue, axons are attracted toward its greatest concentration. If, for example, only an attractive chemical is present in the vicinity, the axon will migrate all the way to the highest concentration of this chemical. If it senses both attractive and repellent chemicals, it will migrate to the point where attraction precisely balances out the repulsion.
In addition, the axon is sensitive to the underlying surface, which is studded with molecules that also attract or repel the axon. This surface needs to be sticky, or adhesive, to the proper degree. The fibers get bogged down on a material that is too sticky, but neither can they grow over a smooth surface like glass, which provides them with nothing to grab onto. Because the molecules in the surface material are fixed in place, they do not operate over a long distance, only upon contact or when the axon is within a short range. “Thus, an individual axon might be ‘pushed’ from behind by a chemorepellent, ‘pulled’ from afar by a chemoattractant, and ‘hemmed in’ by attractive and repulsive local cues. Push, pull and hem: these forces appear to act together to ensure accurate guidance,” Tessier-Lavigne and Goodman wrote in a 1996 review.12
Where is the blueprint for this orchestrated deployment? Its main lines are encoded in the genes, but one will never find an entire map of the nervous system in the genetic code. This is because the system’s blueprint is not a static map with all details spelled out in advance. Rather, it is more like an interactive program that unfolds gradually, constantly responding to cues from the environment and making sure different genes are switched on and off at the right times.
Each axon does not have a specific gene-encoded “address” telling it in advance where to go. Instead, at different times, the “program” prompts the axon to display different receptors on its surface, altering its response to the surroundings. The changeover in receptors may not be necessarily preprogrammed in advance; it can occur in response to signals arriving from the environment. This means that in identical twins, who have the same genes, the final arrangement of the nerve circuits will be at least somewhat different. The midline and other tissues releasing guidance signals also appear to change their personality at different stages of development by displaying different cues and affecting axonal navigating decisions. Thus, as the axons approach the midline, they find it irresistibly alluring, apparently because they are drawn by netrins. But once they have crossed the midline, the axons turn sharply and run along the length of the body on the other side or towards a different destination, and they never cross back. Not only do they no longer find the netrins attractive, they now find them repulsive.
COLLAPSIN AND CONNECTIN
Scientists are only beginning to uncover the details of this intricate topography. No one knows how many guidance molecules exist, but several dozen have already been found and others keep being discovered. Their names often reflect their function. One family is called semaphorins, after the word “semaphore,” meaning a signaling system. A guidance gene robo, for “roundabout,” the British term for a traffic circle, is so called because when mutated, it causes fruit fly axons to cross the midline back and forth. Mutation in another gene— commissureless, or comm for short—leads to the opposite problem, a lack of axonal crossovers, or commissures. Examples of other evocative gene names include beaten path, sidestep, razzled, collapsin, and connectin.
Axons have no problem following the attractive and repulsive cues in the embryo, but by adulthood something happens to the environment in the central nervous system, making it hostile to growth. Scientists hypothesize that the “go” signs that guided axons to their places in the fetus turn into “stop” signs in the adult organism. To produce regeneration, the guiding signs would have to be switched from “stop” back to “go.” This idea is based on a fascinating property of all guidance molecules: They can be attractive or repellent at different times, or attractive for some growth cones but repulsive for others, which suggests that their guiding properties can be manipulated.
Scientists have proposed that the molecular “stop-go” switch may be the same for all guidance molecules. No one has yet proved this hypothesis, but in several studies a manipulation of guiding properties has already been accomplished. In one series of experiments, a team led by Dr. Mu-ming Poo, from the University of California at San Diego, in collaboration with Tessier-Lavigne’s lab, achieved a feat that sounds like an episode from a romantic novel. The scientists managed to transform repulsion into attraction. Using a molecular switch they identified, they altered the effect of two guidance molecules on the tips of growing nerve fibers: Instead of repelling the growing fibers, the molecules started to attract the fibers in a laboratory dish.
Scientists are trying to identify the key targets for such manipulation. Netrins, first identified in worms and chick embryos but later found to be present in fruit flies and in humans, are among the potential candidates. Timothy Kennedy, former postdoctoral fellow from Tessier-Lavigne’s San Francisco netrin team, who now runs his own lab at McGill University’s Montreal Neurological Institute, has shown with his colleagues that netrins are present in large amounts in the adult spinal cord of rats, and that their amounts change after injury. During development, netrins have been shown to attract the growth of some axons and repel the growth of others, but Kennedy believes that their role in the adult nervous system may be to block nerve fiber growth. By cranking up the attractive of properties of netrins, he suggests, it may be possible to encourage spinal cord axons to regenerate in adults.
Producing massive growth in the spinal cord, however, will only be useful if the growing fibers are properly rewired. The chances for such rewiring would increase if the fetal road map for growing axons were preserved throughout an organism’s life. Several studies have suggested that when fetal neurons or stem cells are transplanted into the injured adult brains or spinal cords of rats, they deploy themselves just as they would have in an embryonic nervous system. It is possible that the transplanted cells and their fibers rely on navigating instructions that are still present in the adult system and can follow these instructions in order to stay on the right track, at least when in close proximity to their targets. This navigating savvy, in turn, increases the chances that the new nerve fibers may be capable of making the right connections for restoring function after injury. “I think we will be able to help rewire parts of the brain or spinal cord following injury,” says Tessier-Lavigne. “Our best hope is that enough guidance information remains in the adult nervous system to guide regenerating fibers back without us having to assist in this process. Our major task will be to get lots of regrowth, and the nervous system can then make sure it’s the right kind of regrowth, so it can be translated into an effective therapy.”