Spinal Cord Injury: Emerging Concepts
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Table of Contents:
Executive SummaryIntroduction
Current Understanding and Treatment
The Normal Spinal Cord
Anatomical and Functional Changes After Injury
Clinical Management
Conclusion
Secondary Damage
Immune System Reactions
Oxidative Damage
Calcium and Excitotoxicity
Necrosis and Apoptosis
Axon Damage
Changes Below the Injury
Conclusion
Regeneration
Implications of Development
Nerve Cell Differentiation
Axon Pathfinding
Synapse Formation
Basic Regeneration Studies
Trophic Factors
Intrinsic Growth Programs
Barriers to Growth
Applied Regeneration Studies
Transplantation
Trophic Factors
Anti-Inhibitory Factors
Combination Therapies
Conclusion
Current Interventions
Drug Therapy
Neural Prostheses
Rehabilitation
Conclusion
Preclinical and Clinical Testing of New Therapies
Preclinical Testing
Clinical Trials
Conclusion
Conclusion
Glossary of Major Terms
Dedication
Acknowledgements
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Executive Summary
Among the most exciting frontiers in medicine is the repair of traumatic injuries to the spinal cord. Improvements in treatment are helping many more people survive spinal cord injury. Yet most spinal cord injuries still cause lifelong disability, and continued research is critically needed. To explore new directions for research on spinal cord injury, the National Institutes of Health sponsored a scientific workshop on September 30 - October 1, 1996. The workshop, Spinal Cord Injury: Emerging Concepts, brought together experts from the field of spinal cord injury research and leaders from other fields such as development, immunology, and stroke.
Current Understanding and Treatment
The normal spinal cord coordinates movement and sensation in the body. It is a complex organ containing nerve cells, supporting cells, and nerve fibers to and from the brain. The spinal cord is arranged in segments, with higher segments controlling movement and sensation in upper parts of the body and lower segments controlling the lower parts of the body. The consequences of injury reflect this organization.
The types of disability associated with spinal cord injury vary greatly depending on the type and severity of the injury, the level of the cord at which the injury occurs, and the nerve fiber pathways that are damaged. Severe injury to the spinal cord causes paralysis and complete loss of sensation to the parts of the body controlled by the spinal cord segments below the point of injury. Spinal cord injuries also can lead to many complications, including pressure sores and increased susceptibility to respiratory diseases.
Clinical management of spinal cord injury has advanced greatly in the last 50 years. Recent advances include improved imaging of damage to the spinal cord and vertebrae and development of the first effective drug therapy for use in the hours just after injury. Current management of acute spinal cord injury involves diagnosing and relieving gross misalignments and other structural problems of the spine, minimizing cellular-level damage, and stabilizing the vertebrae to prevent further injury. Once a patient is stabilized, supportive care and rehabilitation strategies promote long-term recovery.
Secondary Damage
Damage to the spinal cord does not stop immediately after the initial injury, but continues in the hours following trauma. These delayed injury processes present windows of opportunity for treatments aimed at reducing the extent of disability resulting from spinal cord injury.
Most types of immune cells enter the spinal cord only rarely. However, when the spinal cord is damaged by trauma or disease, immune cells engulf the area, eliminating debris and releasing a host of powerful regulatory chemicals, both beneficial and harmful. Scientists know little about the role of these immune cells after spinal cord injury.
Following spinal cord injury, highly reactive chemicals called oxidants or "free radicals" are released. These chemicals attack the body's natural defenses and critical cell structures. Trauma also causes release of excess neurotransmitters, leading to excitotoxicity, or secondary damage from overexcited nerve cells. Understanding how to block oxidative damage and excitotoxicity may provide avenues for reducing damage following spinal cord injury.
New insights about how cells die are affecting many areas of disease research, including spinal cord injury. Until recently, most cell death in spinal cord injury was attributed to necrosis, the common, uncontrolled form of cell death in which cells swell and break open. Recent experiments have shown that some cells die as a result of apoptosis, a form of "cell suicide" in which damaged cells eliminate themselves with less harm to their neighbors. Blocking apoptosis appears to improve recovery after spinal cord injury in rodents.
Damage to axons - nerve fibers that signal to other cells - causes most of the problems associated with spinal cord injury. Until recently, most researchers assumed that the physical forces of spinal cord trauma immediately tear axons. New evidence suggests that many axons deteriorate more slowly because the vital transport of molecules and cell components to and from the ends of axons is disrupted. This delay in axon loss allows time for intervention.
Following injury, nerve cells in the spinal cord below the lesion may die, disrupting spinal cord circuits that help control movement and interpret sensory information. Understanding these changes will be essential for obtaining useful recovery of function following regeneration.
Regeneration
For successful regeneration to occur following spinal cord injury, damaged nerve cells must survive or be replaced, and axons must regrow and find appropriate targets. Axons and their targets must then interact to construct synapses, the specialized structures that act as the functional connections between nerve cells.
Although conditions in the injured adult spinal cord are significantly different from those occurring during development, the requirements for regeneration are similar to those for development. Scientists are beginning to learn how cells specialize, how axons find their correct targets, and how synapses form in the developing spinal cord. Physicians may ultimately be able to manipulate developmental signals to control regeneration.
Central nervous system neurons require combinations of natural chemicals called trophic factors to survive and grow. Understanding which trophic factors are important and how cells respond to these molecules may enable researchers to use trophic factors to foster regeneration after spinal cord injury. Research on ways to administer these factors and avoid side effects will be necessary before they can be used for human spinal cord injury. Scientists are currently studying how nerve cells' innate ability to grow, and the environment that surrounds them, affect regeneration following injury. For example, investigators recently discovered a gene that prevents nerve cells from growing in adults. Methods that control this innate ability to grow may eventually complement other therapies.
Researchers are beginning to apply new knowledge about regeneration in animal models of spinal cord injury. Strategies include grafting of peripheral nerve pieces and fetal tissue into the damaged spinal cord, administering growth factors, genetically manipulating cell death programs, and neutralizing or bypassing natural growth-inhibiting substances. Combinations of such therapies have produced the first evidence that some functional regeneration of completely severed spinal cords in adult mammals is possible.
Current Interventions
Effective drug therapy for spinal cord injury first became a reality in 1990 with the finding that the steroid drug methylprednisolone can significantly improve recovery. Clinical trials of methylprednisolone demonstrated that there is an 8-hour window of opportunity for treatment after injury. This trial also showed that health care systems can provide the rapid treatment necessary in spinal cord injury, and it serves as a model for efficient clinical trials of other therapies. Methylprednisolone has now moved from clinical trials to standard use.
Neural prostheses present another approach for improving the quality of life after spinal cord injury. These electronic and mechanical devices, such as hand-grasp prostheses, connect with the nervous system to supplement or replace lost motor or sensory function. Devices such as prostheses to control bladder function and to help people stand are now in development or planning stages.
Rehabilitation can greatly improve patients' health and quality of life. New knowledge about the factors underlying spasticity, muscle weakness, and incoordination may lead to innovative ways of reducing these problems. In some cases, drugs available for other purposes may be effective for treating problems associated with spinal cord injury.
Preclinical and Clinical Testing of New Therapies
Animal studies point to several avenues for developing new therapies for spinal cord injury, including drugs that promote regeneration and transplantation strategies. Each of the mechanisms of secondary damage offers targets for intervention.
Efficient preclinical tests can ensure that the most promising potential therapies proceed rapidly to clinical testing. New animal models, innovative approaches to testing, and reliable outcome assessments are essential to this process.
Randomized, controlled, clinical trials are the gold standard for revealing the benefits and drawbacks of a particular therapy, but practical and ethical constraints limit large-scale trials to the most promising therapies. Good preclinical data is essential so that researchers can predict which treatments and doses are most useful and which patients might benefit. Combination therapies present special challenges that must be overcome when designing clinical trials for promising therapies.
Conclusion
Spinal cord injury research has now come of age. Because of general progress in neuroscience, as well as specific advances in spinal cord injury research, researchers can now test new ideas about how changes in molecules, cells, and their complex interactions in the living body determine the outcome of spinal cord injury. One of the most exciting messages from the workshop was the confirmation that findings from other fields, such as development, immunology, and stroke research, can be applied to the study of spinal cord injury.
Researchers are wary of giving people false hope that a "magic bullet" for curing spinal cord injury is just around the corner. However, with accelerating progress in basic and applied research, there is renewed vitality and growing optimism among investigators that, with continued effort, the problems of spinal cord injury will be overcome.
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Introduction
As the 20th century draws to a close, advances in scientific understanding of the human body are leading to tremendous opportunities for treating even the most devastating diseases. Among the most exciting frontiers in medicine is the repair of traumatic injuries to the central nervous system (CNS), including the spinal cord. Improvements in treatment are helping many more people survive spinal cord injury, and the time survivors must spend in the hospital is half what it was 20 years ago. Yet most spinal cord injuries still cause lifelong disability, and further research is critically needed.
The injury of actor Christopher Reeve in 1995 drew the nation's attention to the tragedy of spinal cord injury. Accidents and violence cause an estimated 10,000 spinal cord injuries each year, and more than 200,000 Americans live day-to-day with the disabling effects of such trauma. The incidence of spinal cord injuries peaks among people in their early 20s, with a small increase in the elderly population due to falls and degenerative diseases of the spine. Because spinal cord injuries usually occur in early adulthood, those affected often require costly supportive care for many decades. The individual costs may exceed $250,000 per year, placing an often overwhelming financial burden on these individuals and their families. For the nation, these costs add up to an estimated $10 billion per year for medical and supportive care alone. Of course, no dollar figure can describe the human costs to spinal cord injured people and their families.
To explore new directions for research on spinal cord injury, the National Institutes of Health sponsored a scientific workshop on September 30 - October 1, 1996. The workshop, Spinal Cord Injury: Emerging Concepts, brought together experts from the field of spinal cord injury research and leaders from other fields such as development, immunology, and stroke. The organizers hoped that interactions among these experts might bring new interest and new ideas to spinal cord injury research and foster fruitful collaborations between investigators. Because of the remarkable progress in basic and clinical neuroscience, the time is now ripe to apply knowledge from other fields to treatment of spinal cord injury.
The workshop participants discussed four major topics: the current understanding and treatment of spinal cord injury, mechanisms of secondary damage, possibilities for regeneration, and strategies for intervention. The discussions revealed many areas where continued research could yield benefits. For example, in recent years scientists have gained a better understanding of how trauma injures nerve cells and why cells die. They know that secondary damage continues for hours following an initial trauma, presenting windows of opportunity to limit this damage. Other opportunities for therapeutic intervention, including rehabilitation strategies, extend well beyond this time window. Progress in understanding how the spinal cord changes after injury is pointing to new therapeutic approaches.
The ultimate hope, of course, is not just to minimize damage, but to foster recovery. A century of pessimism about the capacity for regeneration in the brain and spinal cord is now giving way to guarded optimism. Scientists recently demonstrated that nerve cells in the spinal cord can regrow under certain circumstances. Insights from animal models of spinal cord injury and from studies of nervous system development are leading to strategies that may foster regeneration. Researchers also are making outstanding progress in devising neural prostheses that can substitute for some of the functions lost after spinal cord injury. While it is unlikely that the complex problem of spinal cord injury will be solved by a single dramatic discovery, small improvements in therapy can combine to improve the quality of life for those who live with such devastating injuries.
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Current Understanding and Treatment
To understand how treatment for spinal cord injury can be improved, it is important to understand the normal spinal cord and its functions, how these functions change after injury, and the status of current treatment.
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The Normal Spinal Cord
The spinal cord and the brain together make up the CNS. The spinal cord coordinates the body's movement and sensation. Unlike nerve cells, or neurons, of the peripheral nervous system (PNS), which carry signals to the limbs, torso, and other parts of the body, neurons of the CNS do not regenerate after injury.
The spinal cord includes nerve cells, or neurons, and long nerve fibers called axons. Axons in the spinal cord carry signals downward from the brain (along descending pathways) and upward toward the brain (along ascending pathways). Many axons in these pathways are covered by sheaths of an insulating substance called myelin, which gives them a whitish appearance; therefore, the region in which they lie is called "white matter." The nerve cells themselves, with their tree-like branches called dendrites that receive signals from other nerve cells, make up "gray matter." This gray matter lies in a butterfly-shaped region in the center of the spinal cord. Like the brain, the spinal cord is enclosed in three membranes (meninges): the pia mater, the innermost layer; the arachnoid, a delicate middle layer; and the dura mater, which is a tougher outer layer.
The spinal cord is organized into segments along its length. Nerves from each segment connect to specific regions of the body. The segments in the neck, or cervical region, referred to as C1 through C8, control signals to the neck, arms, and hands. Those in the thoracic or upper back region (T1 through T12) relay signals to the torso and some parts of the arms. Those in the upper lumbar or mid-back region just below the ribs (L1 through L5) control signals to the hips and legs. Finally, the sacral segments (S1 through S5) lie just below the lumbar segments in the mid-back and control signals to the groin, toes, and some parts of the legs. The effects of spinal cord injury at different segments reflect this organization.
Several types of cells carry out spinal cord functions. Large motor neurons have long axons that control skeletal muscles in the neck, torso, and limbs. Sensory neurons called dorsal root ganglion cells, whose axons form the nerves that carry information from the body into the spinal cord, are found immediately outside the spinal cord. Spinal interneurons, which lie completely within the spinal cord, help integrate sensory information and generate coordinated signals that control muscles. Glia, or supporting cells, far outnumber neurons in the brain and spinal cord and perform many essential functions. One type of glial cell, the oligodendrocyte, creates the myelin sheaths that insulate axons and improve the speed and reliability of nerve signal transmission. Other glia enclose the spinal cord like the rim and spokes of a wheel, providing compartments for the ascending and descending nerve fiber tracts. Astrocytes, large star-shaped glial cells, regulate the composition of the fluids that surround nerve cells. Some of these cells also form scar tissue after injury. Smaller cells called microglia also become activated in response to injury and help clean up waste products. All of these glial cells produce substances that support neuron survival and influence axon growth. However, these cells may also impede recovery following injury.
Nerve cells of the brain and spinal cord respond to insults differently from most other cells of the body, including those in the PNS. The brain and spinal cord (i.e., the CNS) are confined within bony cavities that protect them, but also render them vulnerable to compression damage caused by swelling or forceful injury. Cells of the CNS have a very high rate of metabolism and rely upon blood glucose for energy. The "safety factor," that is the extent to which normal blood flow exceeds the minimum required for healthy functioning, is much smaller in the CNS than in other tissues. For these reasons, CNS cells are particularly vulnerable to reductions in blood flow (ischemia). Other unique features of the CNS are the "blood-brain-barrier" and the "blood-spinal-cord barrier." These barriers, formed by cells lining blood vessels in the CNS, protect nerve cells by restricting entry of potentially harmful substances and cells of the immune system. Trauma may compromise these barriers, perhaps contributing to further damage in the brain and spinal cord. The blood-spinal-cord barrier also prevents entry of some potentially therapeutic drugs. Finally, in the brain and spinal cord, the glia and the extracellular matrix (the material that surrounds cells) differ from those in peripheral nerves. Each of these differences between the PNS and CNS contributes to their different responses to injury.
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Anatomical and Functional Changes After Injury
The types of disability associated with spinal cord injury vary greatly depending on the severity of the injury, the segment of the spinal cord at which the injury occurs, and which nerve fibers are damaged. In spinal cord injury, the destruction of nerve fibers that carry motor signals from the brain to the torso and limbs leads to muscle paralysis. Destruction of sensory nerve fibers can lead to loss of sensations such as touch, pressure, and temperature; it sometimes also causes pain. Other serious consequences can include exaggerated reflexes; loss of bladder and bowel control; sexual dysfunction; lost or decreased breathing capacity; impaired cough reflexes; and spasticity (abnormally strong muscle contractions). Most people with spinal cord injury regain some functions between a week and six months after injury, but the likelihood of spontaneous recovery diminishes after six months. Rehabilitation strategies can minimize the long-term disability.
Spinal cord injuries can lead to many secondary complications, including pressure sores, increased susceptibility to respiratory diseases, and autonomic dysreflexia. Autonomic dysreflexia is a potentially life-threatening increase in blood pressure, sweating, and other autonomic reflexes in reaction to bowel impaction or some other stimulus. Careful medical management and skilled supportive care is necessary to prevent these complications.
Researchers studying spinal cords obtained from autopsy have identified several different types of spinal cord injuries. The most common types of spinal cord injuries found in one large study were contusions (bruising of the spinal cord) and compression injuries (caused by pressure on the spinal cord). Other types of injury included lacerations, caused by a bullet or other object, and central cord syndrome.
In contusion injuries, a cavity, or hole, often forms in the center of the spinal cord. Myelinated axons typically survive in a ring along the inside edge of the cord. Some axons may survive in the center cavity, but they usually lose their myelin covering. This demyelination greatly slows the speed of nerve transmission. Slowing of nerve impulses can be measured by a diagnostic technique called transcranial magnetic stimulation (TMS).
Another example of a spinal cord injury is central cord syndrome, which affects the cervical (neck) region of the cord and results from focused damage to a group of nerve fibers called the corticospinal tract. The corticospinal tract controls movement by carrying signals between the brain and the spinal cord. Patients with central cord syndrome typically have relatively mild impairment, and they often spontaneously recover many of their abilities. Patients usually recover substantially by 6 weeks after injury, despite continued loss of axons and myelin. Delays in motor responses persist, but permanent impairment is usually confined to the hands.
Complete severing of the spinal cord is rare in humans, but even axons that survive the initial injury often lose their ability to function. Secondary damage, which continues for hours, can cause loss of myelin, degeneration of axons, and nerve cell death. Patients with their spinal cords completely severed often show abnormal reflexes that emerge more than 8 months after injury. These reflexes, such as twitching of muscles in the arm and hand in response to sensory stimulation of the legs and feet, may result from "sprouting" of new branches from sensory fibers just below the lesion. They may also result from activation of nerve pathways that are normally suppressed. Other abnormal responses, such as sweating in response to movement of a hair, may be due to sprouting of nerves in the autonomic nervous system. The autonomic nervous system is the part of the PNS that controls involuntary body functions such as sweating and heart rate.
Since even a small number of nerve fibers can support significant nervous system function, measures that reduce damage could allow much greater function than would otherwise be expected. Devising interventions that will achieve this goal is one of the major challenges in spinal cord injury research today.
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Clinical Management
Medical care of spinal cord injury has advanced greatly in the last 50 years. During World War II, injury to the spinal cord was usually fatal. While postwar advances in emergency care and rehabilitation allowed many patients to survive, methods for reducing the extent of injury were virtually unknown. Although techniques to reduce secondary damage, such as cord irrigation and cooling, were first tried 20 to 30 years ago, the principles underlying effective use of these strategies were not well understood. Significant advances in recent years, including an effective drug therapy for acute spinal cord injury (methylprednisolone) and better imaging techniques for diagnosing spinal damage, have improved the recovery of patients with spinal cord injuries.
Current care of acute spinal cord injury involves three primary considerations. First, physicians must diagnose and relieve cord compression, gross misalignments of the spine, and other structural problems. Second, they must minimize cellular-level damage if at all possible. Finally, they must stabilize the vertebrae to prevent further injury.
The care and treatment of persons with a suspected spinal cord injury begins with emergency medical services personnel, who must evaluate and immobilize the patient. Any movement of the person, or even resuscitation efforts, could cause further injury. Even with much-improved emergency medical care, many people with spinal cord injury still die before reaching the hospital.
Methylprednisolone, a steroid, has become standard treatment for acute spinal cord injury since 1990, when a large-scale clinical trial showed significantly better recovery in patients who began treatment with this drug within 8 hours of their injury. Methylprednisolone reduces the damage to cellular membranes that contributes to neuronal death after injury. It also reduces inflammation near the injury and suppresses the activation of immune cells that appear to contribute to neuronal damage. Preventing this damage helps spare some nerve fibers that would otherwise be lost, improving the patient's recovery.
A controversial topic in the acute care of spinal cord injury is whether surgery to reduce pressure on the spinal cord and stabilize it is better than traction alone. A study in the 1970s showed that, in some cases, surgical intervention actually worsened the patient's condition. This finding prompted many physicians to become more conservative about using these techniques, although advances in care since that time have reduced the risk of complications due to surgery. While there is no proof that surgeons must operate to decompress the spinal cord within the 8-hour time window established for methylprednisolone, many believe it may help and try to do it then. Early surgery also allows earlier movement and earlier physical therapy, which are important for preventing complications and regaining as much function as possible. Use of imaging methods such as computed tomography (CT) scans to visualize fractures and magnetic resonance imaging (MRI) to image contusions, disc herniation, and other damage can help define the appropriate treatment for a particular patient. Several types of metal plates, screws, and other devices also are now available for surgically stabilizing the spine.
Once a patient's condition is stabilized, care and treatment focus on supportive care and rehabilitation strategies. Attention to supportive care can prevent many complications. For example, periodically changing the patient's position can prevent pressure sores and respiratory complications. Rehabilitation, which focuses on the patient's physical and emotional recovery, is also very important. Almost all patients with spinal cord injuries can now achieve a partial return of function with proper physical therapy that maintains flexibility and function of the muscles and joints. Physical therapy can also help reduce the risk of blood clots and boost the patient's morale, while counseling can help a person adjust emotionally to the injury and its consequences.
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Conclusion
Recent years have seen many advances in understanding and treating spinal cord injury. These include the development of CT and MRI scans to visualize injuries and the use of methylprednisolone to reduce damage. However, many facets of what happens when the spinal cord is injured are still unknown. An exact description of the structural and tissue changes that occur in spinal cord injury is necessary for planning effective interventions. Studies aimed at better describing what happens following spinal cord injury may lead to improved treatments.
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Secondary Damage
Damage to the spinal cord does not stop with the initial injury, but continues in the hours following trauma. Paradoxically, this delayed, secondary damage is not all bad news because secondary injury processes present windows of time in which treatment may reduce the extent of disability. The effects of methylprednisolone demonstrate that such treatment is possible and present a model for the development of other treatments.
Two major themes about secondary damage recurred throughout the workshop. The first theme reflects increasing recognition that similar cellular processes contribute to damage in many different neurological disorders. The second theme mirrors one of the most active areas in all of biology -- how cells die. Cells, including those in the spinal cord, die in two general ways. Necrosis is a relatively uncontrolled process in which cells swell and break open, leaking substances that can be toxic to their neighbors. However, in apoptosis, or programmed cell death, cells activate a "cell suicide" program, an ordered sequence of events that leads to cell death with relatively little damage to surrounding cells. The relationship between apoptosis and necrosis, the role that each plays in spinal cord injury, the signals that regulate cell death, and the potential to halt death programs are now being explored to find ways of minimizing secondary damage following spinal cord injury.
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Immune System Reactions
There is no single point at which to begin describing the intricately intertwined cellular and molecular events that follow spinal cord injury. However, the immune reaction is a good place to start because of its importance. Most types of immune cells enter the CNS only rarely unless it has been damaged by trauma or disease. It is not always clear to what extent immune reactions help or harm prospects for recovery, although immune reactions do appear to cause some secondary damage.
The last decade has brought extraordinary advances in understanding the immune system and its interactions with the nervous system. Using newly developed markers, scientists can identify subsets of immune cells with different functions and can monitor these cells in the nervous system. They are also beginning to understand the chemical language immune cells use to communicate. Cytokines, for example, are a diverse group of diffusible messenger molecules that control many aspects of immune cell function and also enable immune cells to influence other cells such as neurons. Cell adhesion molecules on the surfaces of cells control the traffic of immune cells into the brain and spinal cord and have other wide-ranging influences. Epithelial cells of blood vessels and various types of immune cells normally display certain cell adhesion molecules on their surface. These adhesion molecules change when blood vessel and immune cells encounter foreign molecules, sense damaged tissue in the vicinity, or detect cytokines. Advances in understanding the immune system are now being applied to learn how immune cells influence recovery from spinal cord injury.
Microglial cells, which are normally found in the CNS, have some immune functions and become activated in response to damage. Following trauma, other types immune cells react to signals from damaged tissue and changes in endothelial cells by entering the CNS. Neutrophils are the first type of immune cells to enter the CNS from the rest of the body. These cells enter the spinal cord within about 12 hours of injury and are present for about a day. About 3 days after the injury, T-cells enter the CNS. T cells have many functions in the body, including killing infected cells and regulating many aspects of the immune response; however, their function in spinal cord injury is totally unknown. The key types of immune cells in spinal cord injury appear to be macrophages and monocytes, which enter the CNS after the T-cells. These cells scavenge cellular debris. One type of macrophage, the perivascular cell, may also mediate damage to the endothelial cells that line blood vessels. It is not clear which signals control the entry of immune cells into the CNS, but changes in cell adhesion molecules most likely play an important role.
What immune cells do once they enter the damaged spinal cord is poorly understood. Some cells engulf and eliminate debris as they do during inflammation in other parts of the body. Macrophages, monocytes, and microglial cells release a host of powerful regulatory substances that may help or hinder recovery from injury. Potentially beneficial substances released by these cells include the cytokines TGF-beta and GM-CSF (transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor) and several other growth factors. Apparently detrimental products include cytokines such as TNF-alpha and IL-1-beta (tumor necrosis factor-alpha and interleukin-1-beta) and chemicals such as superoxides and nitric oxide that may contribute to oxidative damage. Again, it is unclear what is helpful and harmful about many of these powerful substances in the context of the injured spinal cord.
Several workshop participants emphasized how important it is to learn about the role of the immune response in spinal cord injury. Understanding the possible links between the immune system and oxidative damage, apoptosis of nerve cells, and demyelination is an important area for research. Other critical areas for study include the signals controlling the traffic of immune cells into the spinal cord following injury and the time course and subsets of immune cells involved. Progress in understanding the immune system now makes answering these questions technically possible.
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Oxidative Damage
After a spinal cord injury, the body's inflammatory cells, among others, produce highly reactive oxidizing agents including "free radicals." Oxidizing agents attack molecules that are crucial for cell function by modifying their chemical structures. This process is called oxidative damage. Oxidative damage occurs in disorders ranging from slow neurodegenerative diseases like amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and Parkinson's disease to acute events like stroke and trauma. Thus, it has been the focus of intensive research. Scientists are learning which chemicals are responsible for oxidative damage in the nervous system, how they are generated, and what role the natural antioxidant defense systems play.
Free radicals are produced as a byproduct of normal metabolism. The brain and spinal cord normally have a high rate of oxidative (energy-producing) metabolism. The increases in blood flow during "reperfusion," when blood flow is restored following injury, may raise free radical production even more. Inflammation can also accelerate the production of free radicals. Many scientists believe that superoxides (oxygen molecules with an extra electron) can escape from the normal antioxidant defenses of the CNS and combine with hydrogen peroxide, also normally present, to form hydroxyl radicals (oxygen-hydrogen with an extra electron). In the test tube, hydroxyl radicals are extremely reactive and quickly attack crucial cellular structures and enzymes. However, evidence suggests that this scenario may be different in the living CNS. For one thing, the CNS has concentrations of enzymes that can safely inactivate free radicals. The antioxidant enzyme called copper-zinc superoxide dismutase (SOD), for example, is abundant in the CNS.
Although hydroxyl radicals are the most reactive molecules in the test tube, nitric oxide may be a more important cause of oxidative damage in living animals. Nitric oxide itself is not very destructive -- in fact the body uses it as a signaling molecule -- but it can combine with superoxide ions to produce a very toxic compound called peroxynitrite. Nitric oxide forms peroxynitrite by a reaction that is a million times faster than the one that forms hydroxyl radicals, and it diffuses ten thousand times farther. Peroxynitrite increases its range of damage even more by inactivating some antioxidant defenses, such as SOD. This free radical also can change how cells respond to natural growth and survival factors; for example, it can change the effect of NGF (nerve growth factor) from protecting against apoptosis to accelerating this type of cell death.
The complex actions of nitric oxide illustrate how the interactions between oxidants and biological systems influence how toxic the oxidants' effects can be. These results focus attention on harmful chemical agents that elude antioxidant defenses and attack critical cell molecules. One useful finding is that nitric oxide damage leaves a characteristic molecular "footprint" on cell proteins. This footprint may allow researchers to identify targets of oxidative damage following spinal cord trauma and help in developing therapeutic and protective measures.
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Calcium and Excitotoxicity
Following trauma, an excessive release of neurotransmitters - chemical messengers that travel between neurons -- can cause secondary damage by overexciting nerve cells. This phenomenon, called excitotoxicity, has been a major focus of research on stroke and traumatic brain injury, and it may also contribute to neurodegenerative diseases and spinal cord injury. Researchers know about excitotoxicity (and calcium-mediated damage, which often follows) from both cell culture experiments, in which relevant variables are simplified and controlled, and from experiments in the much more complex living animals. Insights about excitotoxicity are now being applied to understanding secondary damage following spinal cord trauma.
Glutamate is the neurotransmitter most often used by nerve cells to activate, or excite, one another. Excitotoxicity caused by excessive release of glutamate contributes to damage following traumatic CNS injury and stroke. Excessive glutamate can damage nerve cells and glia in several ways. One harmful sequence begins when glutamate overactivates a type of glutamate receptors called NMDA receptors, allowing high levels of calcium to enter the cell. Calcium regulates many cellular processes. For example, calcium activates certain proteases called calpains. Proteases are enzymes that degrade other proteins and have important regulatory roles in cells. Inappropriate activation of these enzymes can damage important parts of the cell. Calcium metabolism is intimately related to oxidative damage as well. Mitochondria--structures within cells that are responsible for producing energy by oxidation -- actively take up calcium. Mitochondria damaged by excessive calcium may produce even more oxidizing free radicals. Excitotoxicity can also damage cells through processes that do not involve calcium. For example, glutamate allows entry into cells of ions such as sodium and chloride that can cause water to enter, leading to uncontrolled swelling.
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Necrosis and Apoptosis
New insights about how cells die are dramatically affecting many areas of disease research, and spinal cord injury is no exception. Until recently, scientists believed that necrosis, or uncontrolled cell death, was the only way cells die after CNS trauma. Findings presented at the workshop now suggest that apoptosis (programmed cell death) occurs in parallel with necrosis, and that delayed apoptosis contributes to secondary damage following spinal cord trauma. Cell death programs and experimental interventions to halt them were major themes of the workshop.
Apoptosis occurs in many contexts other than disease. For example, it plays a key role in the developing nervous system. The embryonic spinal cord and brain generate many more neurons than are found in the adult organism. Neurons compete for natural chemicals called trophic factors that are supplied by target cells, and nerve cells that do not make proper connections die by apoptosis.
Many forms of damage can trigger cell death. Cells undergoing apoptosis exhibit changes very different from those of cells dying from necrosis, reflecting the more controlled nature of programmed cell death. Necrotic cells swell and break open, leaking their contents into the surrounding area and provoking an inflammatory response. In apoptosis, cells go through a series of characteristic structural changes. During apoptosis, bubbles or "blebs" form in the outer cell membrane, and membrane-enclosed fragments of the cell may break away. The cell nucleus also condenses and fragments, and polyribosomes (the cellular machinery for synthesizing proteins) break up. In most cells, enzymes cut DNA into unequal pieces. This DNA degradation may have evolved as a defense against viruses that attempt to establish residence within cells. Chemicals released from dying cells then induce surrounding cells to scavenge the debris. Apoptosis eliminates damaged cells without releasing dangerous molecules like proteases and glutamate that might harm neighboring cells.
It is not obvious that preventing apoptosis would be beneficial in spinal cord injury. Cells rescued from apoptosis might go on to die by necrosis and damage their neighbors. Nerve cells that survive a "suicide attempt" might have impaired function and be more disruptive than beneficial. In many cases, necrosis and apoptosis probably occur in parallel. In experiments reported at the workshop, necrosis from excitotoxicity killed most cultured cells from the mouse cerebral cortex. Blocking this excitotoxic necrosis with glutamate antagonist drugs and extending oxygen-glucose deprivation to overcome the protective effect led to apoptosis. Some drugs had opposite effects on necrosis and apoptosis. For example, certain chemical signals promoted necrosis but reduced apoptosis.
Recently, scientists have found that apoptosis contributes to spinal cord cell death and dysfunction after trauma. Necrosis was prominent in rats subjected to severe spinal cord trauma. However, following milder trauma, cells died by apoptosis. Mapping the positions of apoptotic cells within these spinal cords revealed interesting patterns. Apoptosis of nerve cells was largely restricted to sites near the impact zone itself and generally occurred within about 8 hours of the trauma. Apoptosis in glial cells was much more prolonged, and a second wave of apoptosis occurred in the white matter -- probably among oligodendrocytes -- at about 7 days after injury. This wave of secondary death rippled out much further than the original site of injury. In one experiment, moderate-impact contusions in the rat spinal cord caused little apparent structural damage to myelinated axons in the first few hours, but led to extensive demyelination, probably because of delayed apoptosis of oligodendrocytes, by 3 weeks after injury. These results are important in defining the time windows during which therapeutic intervention might be beneficial. Optimal strategies for saving nerve cells may be different from optimal strategies for saving oligodendrocytes.
Much of what we know about the cellular mechanisms that underlie apoptosis comes from studies of the nematode worm C. elegans. This tiny worm has only about 300 nerve cells, each of which is individually recognizable, unlike the uncountable billions of neurons in a mammalian nervous system. These worms also allow genetic manipulations that are much more difficult to perform in mammals. Scientists studying C. elegans have begun to understand the basic elements of the cell death program by observing worms with mutations in genes that control apoptosis. These include death-suppressor genes, killer genes, genes that control engulfment of cell debris, and genes for degrading DNA. Crucial cellular processes are highly conserved in evolution, that is, they don't change much between lower and higher animals. The detection of cell death genes in higher organisms, based on their resemblance to genes in worms, has been key to understanding cell death in mammals.
The best-studied models of mammalian nerve cell apoptosis are cultures of sympathetic nerve cells (a type of PNS cell) from which the critical trophic factor NGF, or nerve growth factor, has been removed. The cell death program initiated by removing NGF includes five stages: activation, propagation, commitment, execution, and death. Scientists have now defined each stage by cellular events such as the activation of specific genes and enzyme systems. Up until the commitment stage, interrupting the synthesis of new proteins needed for the program to proceed can halt apoptosis. Even after that stage, blocking the actions of certain enzymes, especially a group of protein-degrading enzymes called the ICE (interleukin converting enzyme) family of proteases, can interrupt the death program. Cell death programs may differ among cells; for example, some cells apparently do not require new protein synthesis for apoptosis. Different cell death programs may occur even in the same type of nerve cell in response to different types of injury. In all cases, however, the cells actively participate in the process that leads to their demise.
Using cultured PNS neurons, scientists have tested two strategies for interrupting programmed cell death. One method used drugs that inhibit the ICE family of proteases, proteins that are crucial for the cell death program. The other method used genetically engineered cells lacking bcl-2, a regulator gene needed for the apoptosis program to go forward. In other words, scientists bred mice in which the cell death program was genetically suppressed. Scientists found that regardless of the strategy tested, nerve cells deprived of NGF were arrested in a metabolically quiescent "undead state" for long periods. When subsequently given NGF, these cells were "resurrected" -- they appeared normal and grew.
Similar strategies have been used to block apoptosis in animal models of cerebral ischemia (stroke) and spinal cord injury. In rodent models of stroke, blocking apoptosis, either with drugs or by genetic manipulations, reduced brain damage after blood flow was interrupted. Improved movement in these animals showed that surviving brain cells could still function. Rats with spinal cord injuries that were given an inhibitor of protein synthesis for 1 month were able to retain some use of their hindlimbs. This radical treatment blocked apoptosis by preventing the synthesis of new proteins necessary for the cell death program to go forward.
These studies collectively suggest that blocking cell death programs might buy time that will allow some cells to survive the initial trauma of spinal cord injury. However, the methods used to block cell death in these experiments are not practical for human application: The drugs can be toxic, and genetic manipulation to create humans resistant to injury is obviously not a viable solution. In addition to developing better drugs to block apoptosis, scientists need to answer several key questions about the nature of cell death. These questions include what triggers apoptosis, how developmental apoptosis resembles (or differs from) injury-related apoptosis, how cell death programs and timing vary in different cell types, and to what extent this form of cell death contributes to the functional losses seen in patients with spinal cord injury.
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Axon Damage
With the current scientific excitement about cell death, it is important to emphasize that damage to axons causes most of the problems associated with spinal cord injury, including loss of motor control and sensation. In rat spinal cord contusion injuries, for example, recovery of function correlates closely to the number of remaining axons. Until recently, most researchers assumed that the physical forces of spinal cord trauma immediately tear axons. Recent studies of axon damage following traumatic brain injury are changing this view.
Within several days of traumatic brain or spinal cord injury, grossly swollen axons, termed "reactive swellings" or "retraction balls," appear. Many scientists believe that physical forces of trauma stretch axon fibers, causing them to tear and swell. Studies using multiple animal models and various anatomical tracers now have shown that much of the axon damage following CNS trauma is not immediate. Instead, it occurs hours later from swelling caused by impaired axonal transport. Axonal transport is a vital cellular process that moves molecules and cell components from the cell body toward the axon terminal and from the terminal back to the cell body.
What disrupts axonal transport and causes delayed axon damage? There appear to be multiple causes, but changes to the cytoskeleton play a critical role. The cytoskeleton is the internal scaffolding that determines the shapes of cells. It is necessary for transport of substances along the axons. In severe injuries, changes in the cell membrane that surrounds axons can allow an abnormal influx of ions, particularly calcium. This leads to compacting of the cytoskeleton and interruption of axonal transport. Calpain, a calcium-activated protein-degrading enzyme, probably participates in this process. Swelling and disrupted transport also occur in axons whose membranes show no change in ion permeability. In these axons, which predominate in mild to moderate injuries, neurofilaments (one component of the cytoskeleton) become misaligned. This, again, impairs transport and leads to swelling of axons.
Damage to axons has several consequences within the spinal cord. Following axon injury, axons disconnected from their nerve cell bodies disintegrate by a process called "Wallerian" or "orthograde" degeneration. Nerve cell bodies with damaged axons, and the axon segment that remains attached, may die by retrograde degeneration, that is, degeneration that begins at the site of injury and progresses back toward the cell body. From a functional point of view, the delayed death of oligodendrocytes and the resulting demyelination of axons are also critical events, because unmyelinated axons do not conduct electrical impulses normally. The death of these glial cells may result partly from the degeneration of damaged axons because oligodendrocytes apparently require contact with axons to remain healthy. The removal of normal nerve connections also has important consequences. The diverse effects of axon injury suggest that more than one therapeutic approach may be needed to overcome this damage.
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Changes Below the Injury
While the most dramatic changes in the spinal cord occur at or near the site of injury, the spinal cord also changes below that point. Understanding these changes is becoming more important as researchers learn how to foster axon regeneration. A key question is what regenerating axons will find when they reach the spinal cord below the injury. Changes below the injury site also influence clinical symptoms, such as reflex changes and spasticity, and they may be a factor in the success of future neural prostheses that might rely upon spinal reflexes or motor control circuits.
The spinal cord is not just a passive conduit carrying signals to and from the brain. It helps to control movement and to interpret sensory information flowing in from the body. Walking, for example, includes three neural processes. First, networks of nerve cells within the spinal cord (central pattern generators) generate the basic motor patterns that activate muscles in the sequence appropriate for walking. Second, sensory feedback from the limbs into the spinal cord modifies this basic motor pattern. Third, control signals from higher centers in the brain modulate the spinal circuits. These higher centers turn the spinal pattern generators on and off, shift between different types of locomotion, control sensory influences according to the type of movements, and govern posture and balance. Scientists are beginning to learn how these systems work and how they come together during development.
How the spinal cord circuitry below the trauma site changes following injury is poorly understood, but scientists are beginning to recognize that these changes are important. In one series of experiments, scientists transected the spinal cords of chick embryos and removed a segment. Spinal cords in very young chick embryos regenerated remarkably well. In older embryos, however, axons did not regenerate and many motor neurons, interneurons, and sensory neurons died below the injury. This cell death resulted from the injury rather than from the programmed cell death that normally occurs during development. These experiments suggest that death of cells below the site of injury may be a factor in human spinal cord injury as well.
Spinal cord injury also may alter connections among nerve cells that survive below the injury. The adult CNS is much more plastic, or changeable, than scientists believed just a few years ago. One interesting discovery is that some of the cellular mechanisms that allow the nervous system to adapt with experience, such as glutamate signaling and calcium-mediated events, are the same as those that go awry after injury and cause secondary damage. This discovery may explain why some of these apparently harmful mechanisms have persisted in evolution.
Immediately after spinal cord trauma, nerve cells below the site of injury are excited by trauma-induced release of neurotransmitters. A loss of normal excitatory and inhibitory signals follows when the severed axons die. In many parts of the CNS, including the spinal cord, strong excitation of neurons modifies the strength of synapses. This form of plasticity might alter the remaining circuits of the spinal cord in unpredictable ways. The removal of normal signals also provokes sprouting of nearby axons into the territory vacated by degenerating axons. The consequences of this rewiring are hard to predict. They may include the changes in reflexes often seen in people with spinal cord injury. These complex and diverse consequences suggest that attention to the changes below the site of spinal cord injury may be essential for successful regeneration and rehabilitation.
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Conclusion
Scientists who have been studying spinal cord injury for many years say that spinal cord injury research has now come of age. Because of progress in neuroscience, as well as in spinal cord injury research, researchers can test specific ideas about how changes in cells and molecules affect spinal cord injury. Not long ago, only descriptive studies were possible. Oxidative free radicals, calcium-mediated damage, proteases, cytoskeletal dysfunction, excitotoxicity, immune reactions, apoptosis, and necrosis all come into play following spinal cord injury. These sources of secondary damage interact in complex ways that scientists are just beginning to understand. What is encouraging is that each of these harmful processes offers targets for developing therapies.
Much of the workshop discussion about secondary injury processes relied upon experiments in fields other than spinal cord injury, especially stroke and traumatic brain injury. The potential for application of such findings to spinal cord injury was one of the most exciting aspects of the workshop. While scientists do not agree about how directly this information will apply to the specifics of spinal cord trauma, most believe that studying other disorders can provide insights that will improve understanding of spinal cord injury. Most importantly, studies in other experimental systems can provide hypotheses to test in models of spinal cord injury itself.
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Regeneration
For successful regeneration to occur following spinal cord injury, several things must happen. First, damaged nerve cells and supporting cells must survive or be replaced, despite the acute effects of trauma and the conspiracy of processes that cause secondary damage. Replacement of lost cells in the CNS is unlikely without intervention because adult nerve cells in the brain and spinal cord cannot divide. Nerve cells that survive the injury often must regrow axons, despite tissue changes such as cavity formation that obstruct growth. Axons also must navigate among the myriad possibilities to find appropriate targets. Once the axons locate their targets, they must construct specialized structures to release neurotransmitters at synapses, while target cells must assemble and precisely locate the structures needed to respond to neurotransmitters. Finally, the neural circuitry may have to compensate for changes that have occurred in the spinal cord circuitry following injury.
Until recently, most scientists believed that nerve cells in the CNS of adult mammals could not regenerate. Dramatic findings, some presented at this workshop, are now changing that pessimistic outlook. For example, some studies have demonstrated that nerve cells in the brain and spinal cord make unsuccessful attempts to regenerate and can regrow under some conditions. New findings also demonstrate that the spinal cord has more active repair mechanisms than previously suspected. Although researchers recognize the many obstacles to obtaining regeneration in the human spinal cord, they believe successful regeneration of even a small percentage of nerve fibers will produce significant recovery of function.
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Implications of Development
Scientists favor the spinal cord for studying the CNS because it is simpler than the brain. The long tradition of anatomical and physiological research on the cord provides a solid framework for studying development. Developing nerve cells perform the same steps needed for regeneration -- they grow, navigate, and make appropriate connections. Regenerating nerve fibers face problems that are quite different from those faced by developing nerve cells, however. For example, the tissue through which axons move is more loosely connected during development, and an injured spinal cord may become quite disorganized near the injury site. Also, distances in the adult CNS are much greater than in the embryo, and chemical signposts for navigating axons may have changed in the adult. While the extent to which regeneration resembles development is uncertain, research about nervous system development is a source of crucial insights about how to promote regeneration following spinal cord injury.
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The adult spinal cord is an intricate assembly of cells and nerve fibers arrayed in specific locations with very precise interconnections. Nerve cells in the spinal cord include several types of motor neurons, sensory neurons, and interneurons, each of which varies in shape, electrical activity, neurotransmitter release, and many aspects. Glial cells also include several specialized types of cells in the mature CNS, and the major nerve pathways of the spinal cord white matter are highly organized anatomically. How all of this comes about has been a subject of speculation and experiments for more than a century. The mystery is finally giving way to traditional neuroscience research methods, augmented by new technologies such as molecular genetics.
The factors causing cell types in the spinal cord to become distinct from one another are cell lineage (which cells arise from which by cell division) and cues from within the developing embryo. Research is now identifying these chemical cues and discovering how cells respond to them. Two major signaling systems control the fate of embryonic brain and spinal cord cells. One system controls the specialization of the nervous system along the long axis from the brain down through the spinal cord. The other system controls specialization along the dorsoventral plane, that is, in a cross-section of the spinal cord ("dorsal" refers to the back portion and "ventral" denotes the abdominal direction). So far, the general operating principles of the two systems appear to be the same.
The control of cell identity along the dorsoventral axis of the spinal cord illustrates how these developmental systems operate. Among the essential tools scientists developed to study this process are chemical markers that stain specific cell types before they fully specialize in the embryonic spinal cord. Three cell types form in the ventral part of the early embryonic spinal cord. Glial cells form in the most ventral part, called the floor plate; motor neurons and interneurons form more dorsally. Experiments have shown that the key signal that determines the fate of all three cell types is a protein called sonic hedgehog. (The name arises because this mammalian molecule was identified by its resemblance to the "hedgehog" protein of the fruitfly. Flies with a mutation in the hedgehog gene have a peculiar prickly appearance.) To simulate the situation in the developing embryo, scientists placed pieces of ventral spinal cord in cell culture and exposed them to different concentrations of sonic hedgehog protein. These pieces produced motor neurons, glia, or interneurons depending on the concentration of protein to which they were exposed. In the embryo, a structure called the notochord releases the sonic hedgehog protein signal. Spinal cord cells that lie closest to the notochord are exposed to the highest concentration of the signal and become glial cells. Those in more dorsal positions are exposed to lesser concentrations and become, respectively, motor neurons and interneurons.
Although scientists are rapidly identifying the signals that drive the generation of cell types in the developing spinal cord, many basic questions remain. Many signals have yet to be discovered, and it is not yet clear how cells sense small differences in concentrations and respond to become specialized cell types. Interactions among the various signaling systems are likewise obscure.
Answers to these questions may have implications for spinal cord regeneration. In the last few years, scientists have discovered that even the mature CNS may harbor latent progenitor cells that can divide and specialize to form new nerve and glial cells. In a rat model of spinal cord trauma, the single layer of cells lining the central canal of the spinal cord expands to multiple layers of cells about 48 hours after a contusion lesion. The central canal is continuous with the brain ventricles, large fluid-filled spaces inside the brain. During development, new nerve cells arise from cells lining these structures. Cells from the expanded central canal of injured animals appear to stream out into the spinal cord; these may be neural progenitor cells attempting to repair damaged tissue. It is important to know whether developmental signals that might guide neuron growth persist in the adult. Another reason studies of cell specialization may be relevant to spinal cord injury is that the molecules involved in this developmental process may have other important functions in the adult. Understanding the signals that control cell specialization in development may be critical for learning how to help them repair damaged spinal cords.
Many new findings presented at the workshop reflected the ways researchers now study the molecular machinery by which cells operate. Knowing the genetic code for proteins allows scientists to detect similarities among proteins. By comparing genes among different species, researchers can rapidly apply insights from lower organisms to mammals. Comparing newly identified genes and proteins to known ones within the same animal can also help scientists understand what a newly discovered protein does. Identifying one protein often helps reveal other members of the same protein family that have related functions, as in families of growth factors, cell adhesion molecules, and neurotransmitter receptors. Scientists are also learning to recognize functional regions that many proteins share in different combinations. Gene sequences predict many aspects of a protein's function, such as whether it will respond to certain regulator molecules. Thus, the chemical language that orchestrates development provides crucial clues about regeneration, even if the processes differ.
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Developing nerve cells of the brain and spinal cord grow axons over long distances, along specific routes, and to precise targets. The tip of a growing axon forms a specialized structure called a growth cone. These growth cones sense cues, integrate that information, and make choices that steer the axon in one direction or another. Scientists have identified attractants and repellents that diffuse over long distances, as well as chemical attractants and repellents with fixed locations. Together, these cues allow axons to navigate through the developing brain and spinal cord. The identification of one family of long-distance attractants, the netrins, illustrates this area of research and its potential relevance to spinal cord regeneration.
A century ago, the Spanish neuroanatomist Ramón y Cajal speculated that diffusible chemical signals might guide growing axons. The first such signals, called netrins, were discovered just a few years ago in the chick spinal cord. "Commissural" neurons in the dorsal part of the spinal cord send axons from the front of the cord around toward the back. When the growth cones of these axons approach the midline of the developing spinal cord, they make a beeline for the floor plate, a specialized region of the embryonic spinal cord at the ventral edge of the midline. When scientists placed pieces of developing spinal cords in various arrangements in culture, they found that something in the ventral floorplate attracted growing commissural axons from a distance. They isolated the attractants and named the identified proteins netrins. When scientists further examined the effects of netrins, they found that these molecules also repelled growing axons from other types of developing nerve cells. This finding was predicted by studies in worms of molecules that closely resemble netrins. Experiments in normal and mutant mice confirmed that these molecules guide developing axons in living mammals.