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.