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.
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.
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.
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.
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.
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.
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.
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.