Stem Cell Therapy for Spinal Cord Injury
Spinal cord injuries constitute some of the most debilitating of conditions. Trauma to the spine often results in permanent paralysis, such as paraplegia or quadriplegia, and may even cause immediate death.
Many people remember Christopher Reeve for his lead role as “Superman” in the 1978 movie by the same title, although he had also enjoyed early success as a young stage actor prior to achieving widespread fame as a result of starring in this movie. Many people also remember the news announcement that was made in May of 1995 when, at the age of 42, Christopher Reeve was severely paralyzed after being thrown from a horse during an equestrian event. Tragically, he lived the remainder of his life as a quadriplegic, during which time he became a symbol of the devastating nature of spinal cord injuries. Despite the extreme paralysis with which he suffered during the final decade of his life, he was a regular spokesman for others afflicted with such injuries, and indeed he quickly became the most famous, most recognizable individual associated with spinal cord injuries in the American public. His harrowing physical deterioration which resulted from his injuries was a painful sight to his fans, contrasting sharply with the image of his former self, in which his muscular physique and rugged good looks had earned him the role of various heroes on stage and on screen. His mental strength after his injuries proved to be just as great as his physical strength had been prior to his injuries, however, and he made a number of public appearances during the final years of his life, despite being severely incapacitated. After his injuries, he directed, produced and starred in a number of films and television programs, he directed an animated movie, and he authored two books, one of which is an autobiographical account entitled “Still Me”, which was published in 1998, and the other of which is entitled “Nothing is Impossible”, which was published in 2004. He lobbied regularly for stem cell research, he appeared at the 1996 Academy Awards, he spoke at the Democratic National Convention that same year, and he was interviewed numerous times on radio and television. Despite his courageous spirit, fierce determination and vigorous intellect, however, he died in 2004, at the age of 52.
When Christopher Reeve was thrown off of his horse, he incurred one of the worst types of spinal cord injuries possible. The reigns of the horse had caught and tied his hands so that he landed headfirst on the ground, and, at a height of 6 feet 4 inches and a weight of 215 pounds, the full impact of his body landing at this angle resulted in the shattering of his 1st and 2nd vertebrae. Although his helmet prevented direct injury to his brain, all motor function below his head, including the ability to breathe, was severed. His brain had in fact been disconnected from his spinal cord as a result of his fall. He was unable to breathe for 3 minutes before paramedics arrived, and he spent the rest of his life on a respirator, with the exception of one brief period during December of 1995 when he was able to breathe for 30 minutes on his own. He was completely paralyzed below the neck, having lost not only the capacity for movement but also the capacity for sensation throughout his body. By the year 2000, however, he had regained some ability to detect hot and cold as well as the ability to move his left index finger upon command. He strongly believed that the nervous system is capable of regenerating itself, especially when engaged in vigorous physical therapy, and his ability to regain motor control even in one finger was startling proof of such a possibility. His personal physicians and the medical community in general were shocked by this improvement in his condition, which ran contrary to previous scientific dogma which had always held that neuronal connections, once damaged, are irreparable. Such long-standing beliefs have since been repeatedly disproven and are now commonly understood to have been erroneous, thanks in part to Christopher Reeve’s refusal to accept his condition.
Prior to his death, Christopher Reeve had co-founded the Reeve-Irvine Research Center, which is part of the School of Medicine at the University of California at Irvine. He also served as Chairman of the American Paralysis Association and as Vice Chairman of the National Organization on Disability for several years prior to his death. After his death, the Christopher and Dana Reeve Foundation was created out of the American Paralysis Association, which had been established in 1982. Following his equestrian accident, Christopher Reeve had sought the help of the American Paralysis Association, eventually lending his name to the organization. Upon his death in 2004, his widow, Dana Reeve, assumed the role of Chair of the Association, although she died in March of 2006. On the first anniversary of her passing, in March of 2007, the American Paralysis Association formally converted its name to the Christopher and Dana Reeve Foundation. According to its website, the Foundation “is dedicated to curing spinal cord injury by funding innovative research and improving the quality of life for people living with paralysis, through grants, information and advocacy.” The Foundation actively funds a new field of research known as “locomotor training”, in which patients with paralysis walk on a specialized treadmill which mimics the mechanics of walking, thereby “retraining” the neuromuscular system to reestablish the neural connections that are required for such motion. Additionally, the Foundation funds general research in the treatment of spinal cord injury and paralysis as well as other CNS disorders, while also supporting a number of international grants and a research consortium which coordinates the work of nine laboratories. The Foundation operates in collaboration with the Christopher and Dana Reeve Paralysis Resource Center, the mission of which is to teach people with paralysis how to live more independently. More information is available at www.christopherreeve.org and www.paralysis.org
The following description of spinal cord injury is from the website of the National Institute of Neurological Disorders and Stroke (NINDS), a division of the National Institutes of Health (NIH):
“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 (in the U.S.), 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.” (From www.ninds.nih.gov).
According to the World Health Organization, it has been estimated that more than 2 million people worldwide live with a disability related to spinal cord injury. (From www.who.int).
The severity of any particular spinal cord injury depends on the precise location of the injury along the spine, and the extent of the damage. Some injuries are more treatable than others, whereas some are fatal. As Christopher Reeve demonstrated, however, even in the most severe of injuries, as long as one survives the injury itself, intellectual function and creativity remain unimpaired.
Physiology of the Spine:
The spinal cord allows all movement, sensation and autonomic function below the neck to occur. All neurological information that travels in either direction between the brain and the rest of the body must travel via the spinal cord.
The human nervous system consists of 3 distinct yet interrelated parts:
1/ the central nervous system (CNS),
2/ the peripheral nervous system (PNS), and
3/ the cranial nerves.
The cranial nerves emerge directly from the brain and serve the motor and sensory systems of the head and neck. In humans there are 12 cranial nerves that have been formally recognized, although a 13th was hypothesized in 1878 for sharks and in 1913 for humans, but the precise function of this nerve has yet to be fully elucidated and in fact its very status as a cranial nerve is still debated. Of the 12 approved cranial nerves, all but the 1st and 2nd emerge from the brain stem. The 1st cranial nerve serves olfactory stimulation, the 2nd cranial nerve is the optic nerve, and their projections are topographically distinct from the other ten. Since cranial nerves are not part of the spinal cord, nor the CNS nor the PNS, they are not affected by spinal cord injuries unless such injury also involves the brainstem.
The PNS consists of nerves that branch out from the spinal cord to the rest of the body, extending throughout the trunk and the limbs. A subsystem within the PNS is the autonomic nervous system (ANS), consisting of sympathetic and parasympathetic divisions which serve the viscera and control homeostasis.
The CNS consists of the brain and the spinal cord. From the perspectives of developmental biology, philogeny and ontogeny, the spinal cord itself is an extension of the brain. In humans the spinal cord is covered with the same 3 membranes, or meninges, that cover the brain, namely, the pia mater, which is the innermost layer, the arachnoid, which is a delicate middle layer, and the dura mater, which is a tougher outer layer. The human spinal cord is surrounded by a protective vertebral column, namely, the backbone, which consists of 33 vertebrae corresponding to 31 vertebral nerve segments.
A wide subarachnoid space exists between the spinal cord and the vertebrae which contains the cerebrospinal fluid, which provides some shock absorbance as well as a nutritional “milieu” in which the spinal tissue is continuously bathed.
As the primary cable of the body’s neurological communications network, the spinal cord relays two types of information, in one of two directions, namely, commands from the brain to the body, and sensation from the body back to the brain. Descending pathways of neurons and axons deliver signals from the brain through the spinal cord, while ascending pathways deliver sensory information from the spinal cord back to the brain. Networks of nerves then branch out from the CNS to the rest of the body via the PNS, with efferent nerves transmitting signals away from the CNS and afferent nerves conveying signals back to the CNS.
Axons, whether in the brain, the spinal cord, or elsewhere throughout the body, are known as “white matter” because they are coated with an insulating myelin sheath, which is white in appearance. Similarly, the nerve cells themselves, which consist of nonmyelinated neurons and dendrites, which are gray in appearance, are known as “gray matter”. Gray matter processes information while white matter transmits information. Gray matter is the predominant tissue in certain areas of the brain, especially in the cerebral and cerebellar cortices, among other regions, but in the spinal cord gray matter is localized primarily in a “butterfly-shaped” region in the center of the spinal cord, in the two halves known as the dorsal and ventral horns.
The spinal cord is organized into segments along its length, such that nerves from each segment connect to specific regions of the body. The functions of these nerves are identified by the region and the number of their corresponding vertebrae as follows:
5 fused sacral
Vertebrae numbered C1 through C8 are located in the neck, known as the cervical region, and nerves in this section of the spinal cord control signals throughout the back of the head and neck, the shoulders, arms, hands, and the diaphragm.
Vertebrae numbered T1 through T12 are located in the thoracic region, in the upper back, and nerves from this section of the spinal cord control signals throughout the chest and torso, as well as some regions of the abdomen and arms.
Vertebrae numbered L1 through L5 are located in the upper lumbar region, in the mid-back, just below the ribs, and nerves from this section of the spinal cord control signals to the lower abdomen and back, the hips and buttocks, some regions of the legs and some regions of the genitalia.
Vertebrae numbered S1 through S5 are in the sacral segments, still in the mid-back region just below the lumbar segments, and nerves from this section of the spinal cord control signals to the legs and feet, the groin and to most regions of the genitalia.
The coccygeal region consists of 3 to 5 vertebrae which are typically fused into 3 or fewer segments, the exact number varying among individuals. A single coccygeal nerve transmits sensory information from the skin of the lower back, although the main function of the coccyx is mechanical rather than neurological, as it provides a place of attachment for the muscles of the gluteus maximus. The cauda equina, just below the conus medullaris, is recognized as the terminal location of spinal cord tissue and CNS nerves in adult humans.
Bundles of nerve fibers from all regions of the vertebrae are responsible for the coordination of sympathetic, parasympathetic and somatic pathways of the nervous system. This collection of sympathetic and parasympathetic ganglion cells, which include pathways of preganglionic input, serve the innervation of the muscles, organs and cells of the entire body. A predominant neuroendocrine axis exists throughout these pathways, with a vast array of hormonal receptors and neuroreceptors in all organ systems, illustrating the intricate interrelationship between the various physiological networks. All such networks and systems throughout the body depend upon the spinal cord for their neurological communication with one another.
An interruption of spinal cord function at any particular level will interrupt sensorimotor function below that level. Paraplegia (paraparesis) therefore occurs when an injury is sustained at the thoracic or lumbar regions of the spine, resulting in a loss of sensorimotor function below the level of the arms. Quadriplegia (quadriparesis) occurs when an injury is sustained in the cervical area, resulting in a loss of sensorimotor function below the neck.
One of the most important aspects of nerve conduction anywhere in the body is myelination. Myelin is a phospholipid with electrically insulative properties and, in healthy individuals, a myelin sheath forms a protective layer that envelops the axons of nerves. This axonal coating is critical to proper neurological function both in the CNS and the PNS.
The myelin sheath allows for the proper conduction of electrical impulses, which travel along properly myelinated nerves at a speed of approximately 100 meters per second. By contrast, in nerves that have been demyelinated, the speed of electrical conduction is reduced to approximately one meter per second. Without myelin, communication between nerve cells, and therefore between parts of the body, becomes interrupted to such a severe extent that it is virtually nonexistent.
Demyelinating diseases are among the most debilitating and include many, though not all, of the neurodegenerative diseases, such as multiple sclerosis and the various leukodystrophies. In their most severe forms, demyelinating diseases are life-threatening. The 1992 movie, “Lorenzo’s Oil”, depicted the true story of Lorenzo Odone who continues to suffer from a specific type of leukodystrophy which is now known as Lorenzo’s adrenoleukodystrophy, or ALD, and which is a severely demyelinating “neurometabolic” disorder of genetic origin. In an effort to save their son’s life and to relieve his suffering, Lorenzo’s parents, Augusto and Michaela Odone, founded the Myelin Project, which is actively involved in promoting research into remyelination. More information is available at www.myelin.org
While spinal cord injuries do not technically fall under the category of demyelinating diseases, they do, nevertheless, often involve some demyelination, among other complicating factors. Remyelination, which is a field of intense research interest in many other diseases, is therefore also of relevance and importance in the treatment of spinal cord injuries.
Although the myelin sheath throughout the PNS is formed from Schwann cells, in the CNS myelin is formed from oligodendrocytes. This distinction becomes important in the type of stem cells that should be used to regenerate damaged tissue in spinal cord injuries, and in the treatment of any disease that involves degeneration of the CNS.
Injury and Repair:
A major difference between the PNS and the CNS is that the neurons of the PNS will often regenerate themselves after injury. The neurons of the CNS, by contrast, do not normally possess such an ability.
In general, nerve cells of the brain and spinal cord respond very differently to insults than do most other cells of the body, for a variety of reasons. Among other distinguishing characteristics, cells of the CNS have a very high rate of metabolism and rely upon serum glucose for energy to a greater extent than do cells throughout the rest of the body. CNS cells are therefore particularly vulnerable to damage from ischemia (reduced blood flow), which often occurs in spinal cord injuries.
The regenerative capacity of CNS tissue is extremely limited in all adult mammals, not only in human beings, and a number of obstacles to CNS regeneration have existed throughout the past. In particular, the body’s response mechanisms trigger the formation of glial scars after an injury to the spinal cord, in an effort to protect the nerve tissue from further damage, but this dense scar tissue also prevents foreign cells, including stem cells, from regenerating the tissue. Now, however, researchers have discovered various ways of employing stem cells to overcome this physical barrier.
CNS Cell Types:
The tissue of the spinal cord consists of neurons, axons, various types of supporting cells and trophic factors, among other components. There are as many as 10,000 subtypes of neurons in the CNS which are specialized to receive and send specific types of information, in addition to detecting changes in their environment while organizing both immediate and long-term responses to such change. Each neuron consists of a cell body, axons, and dendrites.
Among the supporting cells of the nervous system are glial cells, which provide physical support and chemical balance for the surrounding cells, including the blood-brain and blood-spinal-cord barriers. There are 3 types of glial cells, namely, astrocytes, oligodendrocytes, and microglia.
Astrocytes are the largest and most abundant of the glial cells. As the name implies, their shape resembles a star and they are involved in the regulation of the composition of fluids that surround nerve cells. They also provide extra energy and trophic factors that are necessary for the sustenance of neurons.
As previously mentioned, oligodendrocytes produce the myelin that surrounds the axons of the CNS and which is necessary for the conduction of electrical impulses. Paradoxically, oligodendrocytes also manufacture chemicals that prevent axon regeneration in the adult CNS under certain conditions.
Microglia are immune cells which are only activated by injury. They are responsible for removing dead cells and other cellular waste products that accumulate at an injury site. Microglia also manufacture cytokines, which activate other immune cells in the initial responses that occur following cellular trauma.
Axon bundles travel in both ascending and descending pathways. They are insulated by the protective myelin sheath, without which the propagation of nerve signals is virtually impossible. The large motor neurons extending from the brain consist of long axons that control skeletal muscles in the neck, torso, and limbs. In the opposite direction, information is carried from the peripheral regions of the body to the spinal cord via the axons of a specialized type of sensory neuron known as dorsal root ganglion cells. Spinal interneurons, which are contained within the spinal cord, help integrate sensory information and generate coordinated signals that control muscle movement. All such functions of the neurons and axons are supported by the glial cells.
Pathways and Impulse Transmission:
Descending pathways carry information from the corticospinal tract in the brain to the motor neurons in the spinal cord with commands regarding voluntary movement. Ascending pathways carry sensory information to the brain such as body position, temperature, pain, and touch. Mechanisms of impulse transmission and plasma membrane action that occur along these pathways are central to the regeneration of neurons.
Nerve impulses are transmitted across neurons by a voltage difference that exists across the plasma membranes, and which results from a cytoplasmic fluid inside the membrane that is negatively charged in comparison to the interstitial fluid outside the membrane. This “resting membrane potential” also drives the cellular sodium-potassium pump, which maintains the resting membrane potential across the neuron plasma membrane. Action potentials that attain a minimum threshold level within a neuron will trigger a brief voltage reversal, which in turn triggers another voltage reversal at an adjacent area of the membrane, thereby self-propagating itself along the neuron, during which time the sodium gates at the nodes open, potassium flows out of the cell and sodium flows in.
These action potentials rely upon the myelin sheaths for their transmission along both sensory and motor neurons. As already mentioned, oligodendrocytes are responsible for forming myelin within the CNS, and this particular type of glial cell is critical to the repair of damaged CNS neurons. The cells of neurons are separated by small gaps known as the “nodes of Ranvier”, across which the action potential jumps, from node to node, until it reaches the output zone, where it triggers the opening of gated calcium ion channels that extend across the membrane. Calcium plays an important and complex role in the series of events that occur following a spinal cord injury, and the control of such events is necessary for the improvement and recovery of the patient.
Although individual neurotransmitters that are involved in these cascading events may have either an excitatory or an inhibitory effect, depending on a number of factors, calcium is always excitatory. Following traumatic spinal cord injury, calcium is often found in the presence of glutamate, which is the most common excitatory neurotransmitter, and which is known to damage nerve cells and glia when present in excessive amounts. In fact it is glutamate which overstimulates a specific type of glutamate receptor known as the NMDA (N-methyl d-aspartate) receptor, which in turn allows an excessively high amount of calcium to flow into the cell, as well as excessive amounts of other ions such as sodium and chloride, which are responsible for the uncontrolled swelling that often results from spinal cord injury.
Following acute trauma to the spinal cord, the body’s natural defensive response includes the triggering of an abnormally high amount of excitatory neurotransmitters such as glutamate, in combination with an excessive flow of calcium across the cellular membrane, often to the point of toxicity. This excitotoxicity of nerve cells results in the death of the cells, thereby causing secondary damage to the patient. This phenomenon continues to be a major focus of research in a number of disorders besides spinal cord injury, including stroke, traumatic brain injury and many neurodegenerative diseases.
In healthy individuals, and in the absence of trauma, immune cells do not enter the spinal cord region. Following an injury to the spinal cord, however, immune cells are mobilized and directed in an overabundance to engulf the injured tissue. Neutrophils are the first type of immune cells to enter the CNS, which appear in the spinal cord within 12 hours of injury and may be present for as long as 24 hours. By the third day after injury, T-cells also enter the region. Outside of the CNS, T-cells are well known for the multiple tasks which they perform elsewhere in the body, but within the CNS their function is almost entirely unknown. Following the T-cells, various scavenger cells enter the spinal cord, namely, the macrophages and monocytes. Exactly what any of these cells do within the CNS is not fully understood, nor are the signals which control their entry into a physiological region which they are normally never instructed to enter. Cell adhesion molecules, which exist on the surface of cells, and changes that these molecules undergo during traumatic injury, are being studied for possible clues.
Paradoxically, it is known that the macrophages, monocytes, and microglial cells release a vast array of powerful regulatory substances that further hinder improvement and recovery from injury, such as the cytokines known as TNF-alpha (tumor necrosis factor alpha) and IL-1-beta (interleukin-1 beta), in addition to superoxides and nitric oxide which are also known to contribute to oxidative damage.
The overactivity of calcium in such processes is both directly and indirectly responsible for exacerbating tissue damage. Calcium regulates a number of cellular events, the proper functioning of which depends upon normal calcium activity. Among other agents, calcium activates those proteases known as calpains, which are enzymes that degrade other proteins and are crucial for the metabolic regulation of cells. An imbalance in calcium levels results in an abnormally high activation of such enzymes, which in turn results in cellular damage. Calcium metabolism has also been implicated in oxidative damage, since mitochondria produce energy by the active uptake of calcium, an excessive amount of which triggers the production of oxidizing free radicals.
Scientists are attempting to understand these processes in greater detail, with the hope of being able to control such cellular events and thereby expedite recovery after spinal cord injury by lessening the amount of primary and secondary tissue damage.
Although spinal cord injury is most often caused by trauma to any of the vertebrae, the spinal cord may also be damaged by disease such as poliomyelitis or spina bifida. In the elderly, it is not uncommon for spinal cord injury to occur as the result of skeletal degeneration, such as extreme osteoporosis. Regardless of the precise causes, however, the treatment and recovery of lost spinal cord or CNS function after neuronal and axonal damage is always based upon the same fundamental principles of physiology.
Prior to World War II, serious spinal cord injuries were usually fatal. Medical progress over the past 60 years, especially in the neurosciences and immunology, combined with engineering advances in imaging technology, now offer improved prognoses and therapies.
Immediately following acute trauma to the spinal cord, emergency treatment is necessary during which time three primary conditions must be assessed, namely:
1/ the full extent of vertebral compression, misalignments and structural problems of the spine, which must be relieved to the fullest extent possible,
2/ cellular damage of related tissue, which must be minimized, and
3/ stabilization of the vertebrae, which must be established to prevent further injury.
Often it is the emergency medical services (EMS) personnel, rather than hospital physicians, who must conduct these evaluations and immobilize the individual. Any movement of the injured person, including resuscitation efforts, could cause further damage. Even with the most modern and sophisticated of emergency medical equipment, however, many people with spinal cord injuries still die before reaching the hospital.
Ultimately, through verification with various imaging technologies such as CAT (computed axial tomography) scans, MRI (magnetic resonance imaging), and X-rays, specialists can determine the precise vertebrae that are involved, and the extent of collateral damage.
Conventional medical treatment of spinal cord injuries has typically consisted of physical therapy in combination with medication.
The skull protects the brain, and the vertebrae of the spine protect the spinal cord, but injury to these sensitive organs still occurs whenever these protective skeletal structures are subject to compression, which is usually the case in spinal cord injuries. In addition to relieving such compression, physicians routinely check for “hidden” damage that may have been indirectly induced. Since both the blood-brain barrier and the blood-spinal-cord barrier may be compromised by such injuries, treatment with medication must be carefully assessed in this light.
The first drug to offer improvement in spinal cord injuries was methylprednisolone, which was approved for marketing and standard use in the U.S. in 1990 as a result of clinical trials sponsored by the NASCIS (National Acute Spinal Cord Injury Study). Further NASCIS clinical trials have compared methylprednisolone to the drug naloxone and to a placebo, showing that methylprednisolone improves recovery in humans if given within 8 hours after injury. Naloxone, however, was not found to offer improvement, nor was methylprednisolone if it was administered more than 8 hours after the injury. Further evidence of this “window of opportunity” in the initial hours following trauma would also become apparent with other studies.
Patients who are completely paralyzed and who are given methylprednisolone were found to recover an average of 20% of their lost motor function, compared to an average of 8% recovery of function in untreated patients. Paretic (partially paralyzed) patients who receive methylprednisolone were found to recover an average of 75% of their function, compared to an average of 59% in people who did not receive the drug.
It is now standard procedure for patients with acute spinal cord injuries to be treated with methylprednisolone within 3 hours of injury, especially in severe cases. Hospital emergency personnel and EMS teams are trained in the rapid administration of this drug.
Methylprednisolone, a synthetic corticosteroid, reduces the damage to cellular membranes that contributes to neuronal death after injury. As with all corticosteroids, the mechanism of action involves the blocking of inflammation, and methylprednisolone is also commonly prescribed for other inflammatory diseases such as rheumatoid arthritis, systemic lupus, and Crohn’s disease. In low doses and for short courses the drug is usually well tolerated, although long-term, high dosing may produce serious side effects, which may include weight gain, hypertension, psychotic behavior, glaucoma and cataracts. Long-term use also depresses the body’s natural ability to produce corticosteroids in the adrenal glands, and abrupt withdrawal may induce severe corticosteroid insufficiency, including systemic shock. As with any drug, withdrawal should be tapered gradually. Since inflammation is a natural part of the body’s immune response following spinal cord injury, and corticosteroids work by suppressing such action, complications from immune suppression are among the common side effects. Patients on methylprednisolone are more susceptible to infections in general, including tuberculosis and malaria, which may remain dormant in an individual for years only to become activated by methylprednisolone. Live-virus vaccines should also be avoided, such as those for chicken pox and measles, as these vaccines may cause the disease in patients who are taking methylprednisolone. Corticosteroids also impair calcium absorption and the formation of new bone, thereby presenting an increased risk of osteoporosis. Some people on methylprednisolone experience the destruction of their large joints, which is accompanied by severe pain, and often requires joint replacement surgery. The precise reason for this particular side effect has not yet been discovered.
By reducing inflammation near the site of injury and suppressing the activation of immune cells that appear to contribute to neuronal damage, methylprednisolone spares some nerve fibers that would otherwise be destroyed by spinal cord injury, thereby improving the patient’s recovery.
Preliminary clinical trials of another agent, GM-1 ganglioside, have shown that this drug may also be useful in preventing secondary damage in acute spinal cord injury, and other studies suggest that it may also improve neurological recovery from spinal cord injury during the period of rehabilitation. If found to be successful in further clinical trials, GM-1 ganglioside may also be approved for the U.S. market.
In general, once a patient’s condition has been stabilized, long-term protocols include therapy and rehabilitation not only to promote the patient’s physical recovery but also his or her emotional and psychological recovery. Combined therapies are often employed, which may include any of a number of neural prostheses. Since complications from spinal cord injuries are common, with respiratory diseases ranking among the most frequent, therapy often includes treatment of such secondary conditions.
The advancement over recent years in imaging technology such as CAT (computed axial tomography) and MRI (magnetic resonance imaging) scans has greatly aided in the understanding of spinal cord injuries. Despite impressive medical advances, however, most processes of tissue degeneration and regeneration within the CNS are not yet fully understood. As stated on the website of the National Institute for Neurological Disorders and Stroke (NINDS), a division of the National Institutes of Health (NIH):
“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.” (From www.ninds.nih.gov).
The neuronal pathways of the spinal cord are highly specialized and organized, and such tissue has been the object of fascination and experimentation for more than a century. Researchers from a variety of disciplines are still working to unravel the mysteries of the human nervous system, and recent advances in technology are also contributing to a better understanding of this complex subject. Indeed, the intricately intertwined cellular and molecular events that follow spinal cord injury are providing neurologists, immunologists, geneticists and cell biologists, among other specialists, with an ever-expanding list of research topics.
Immunology and Genetics:
After any bodily injury, immune cells play a major role in the repair and recovery of injured tissue. With most injuries, the immune response is a positive one, which expedites the healing process. In spinal cord injuries, however, the body’s natural immune reaction is part of the problem. The resulting inflammation that occurs, and the intense mobilization of immune cells that flood the damaged tissue, only contribute to further damage. The thick scar tissue that forms in the CNS after the initial stages of the injury offers a protective defense against this increasing assault, but the same scar tissue also prevents regenerative cells from healing the tissue. Much of the research related to spinal cord injury is therefore focused on immunology, in an effort to understand such mechanisms more clearly and, ultimately, to control them.
It is known that damage to the spinal cord does not stop immediately after the injury, but in fact continues for some period of time. As already mentioned, a “window of opportunity” has been identified for effective drug treatment, which exists for approximately 8 hours following the trauma, during which time inflammation and the body’s immune response are at their most severe. As already described, however, the full immune response of the body continues intensely for several days. Since the body’s natural immune reactions have been found to cause secondary damage after spinal cord injury, a better understanding of the mechanisms of interaction between the immune system and the nervous system is essential to the development of better therapies for spinal cord patients, especially in the initial stages after injury. Much research is therefore focused on these specific molecular and cellular events that are triggered by trauma to the spinal cord, and on methods by which the resulting oxidative damage and excitotoxicity may be blocked.
Immune cells “speak” to each other via a very specific chemical “language”, which scientists are beginning to decipher. Using cellular markers, for example, researchers have been able to track cytokines, as well as various messenger molecules and cell adhesion molecules on the surface of cells, as they traverse the nervous system, thereby tracking their interaction with the tissue and with each other. Inevitably, such investigations lead to the underlying genetic mechanisms that are at work.
For example, findings from immunology research have indicated that the death of a cell in the spinal cord may occur in a variety of ways. For many years scientists believed that the most common form of cellular death was ordinary necrosis, in which the cells swell and break open. Now, however, apoptosis, which is a form of programmed cell “suicide”, is recognized as a frequent occurrence within spinal cord tissue. While ordinary necrosis is not easily preventable, apoptosis may be blocked by certain methods that “reprogram” the cell. Often this involves the regulation of gene expression as instructed by various “signaling” molecules that may involve transcriptional or translational mechanisms, among other processes.
The specialized differentiation of cells is another focus of ongoing research, as scientists attempt to understand the genetic signals and “cues” that regulate neuronal development, with the hope of ultimately being able to direct or guide such signals. As in other molecular and cellular signaling phenomena, mechanisms of phenotypic and genotypic expression play a vital role. Recently a gene was identified that prevents nerve cells from growing, for example, and the ability to “switch” off this gene is an important step in expediting neuronal regeneration.
As with any disease, before any therapy is tested on humans in clinical trials, animal models serve as the basic proving ground. In the field of spinal cord injury, therefore, mice have been successfully treated by a variety of therapies which have included, among other combinations, the transplantation of stem cells into damaged spinal cord regions in combination with growth factors and the genetic manipulation of apoptosis. Such progress represents the interconnectedness of neurology, immunology and genetics, discoveries in which are already translating into successful human therapies.
The increase in blood flow during reperfusion (the restoration of blood flow following an injury) is often accompanied by an increase in free radical production. In particular, superoxides (oxygen molecules with an extra electron) combine during this process with hydrogen peroxide to form hydroxyl radicals (oxygen-hydrogen molecules with an extra electron), which have been found to be highly reactive in laboratory experimentation where such radicals quickly attack cellular structures and enzymes. In the CNS, however, there are many antioxidant enzymes, such as copper-zinc superoxide dismutase (SOD), which may inactivate some radicals such as the hydroxyl radical. By contrast, nitric oxide is not deactivated by antioxidant enzymes in the CNS, and has been widely implicated in oxidative damage in mammals. By itself it is not destructive and is used throughout the body as a signaling molecule, but nitric oxide can combine with superoxide ions to produce the very toxic compound peroxynitrite, which occurs via a reaction that is approximately one million times faster than the reaction by which hydroxyl radicals are formed, and it exhibits a diffusion rate that is approximately ten thousand times greater. Peroxynitrite cannot be inactivated, and it in turn inactivates antioxidant defenses, such as SOD. Additionally, peroxynitrite is capable of altering the action of NGF (nerve growth factor) from a protective mechanism against apoptosis (programmed cell death) to an agent that accelerates apoptosis. Fortunately, nitric oxide and peroxynitrite damage leave a distinctive molecular “footprint” on cell proteins, which allows researchers a means by which to track the progression of such damage. Such complex interactions are topics of ongoing research.
Most of the lasting damage that results from spinal cord injuries is the result of axonal damage, which is responsible for the loss of motor control and sensation throughout the body. In animal models it has been found that recovery of motor function following spinal cord trauma is closely correlated to the number of remaining axons that survive the injury. In the past, physicians and researchers alike have assumed that such axonal damage is the result of the physical forces of impact, which cause the axon fibers to stretch and tear. It is now understood, however, that such damage does not result exclusively from mechanical trauma but may also occur hours later from the immune response, as the result of chemical trauma.
Changes in the cell membrane surrounding the axons result in an abnormal influx of ions, particularly calcium ions, into the cell, which causes a “compacting” of the cytoskeleton and an interruption of axonal transport. The cytoskeleton is the internal scaffolding that determines the physical structure and shape of cells, and which is necessary for the transportation of substances along the axons. Numerous chemical and molecular events occur as a result of changes in the cytoskeleton, including further impaired axonal transport which further interrupts the vital cellular processes by which molecules flow back and forth between the cell body and the axon terminal. Calpain, the calcium-activated protein-degrading enzyme, participates in this process, which triggers the formation of “reactive swellings” and “retraction balls” along the injured spinal cord. Even in injuries that are not severe, swelling and disrupted transport may still occur in axons in which there is no change in the ion permeability of membranes. In mild to moderate injuries, cytoskeletal neurofilaments within the axons themselves become misaligned, which further impairs transport and thereby aggravates axonal swelling.
This process by which axonal injury progresses is known as “Wallerian” or “orthograde” degeneration, the ultimate result of which is the disconnection of axons from their nerve cell bodies, which in turn is followed by axonal disintegration.
For any nerve cells that survive an injury, in order to become functional once again, they must first regrow axons, which in turn must navigate throughout the cellular “landscape” in search of viable targets, to which they must then reconstruct synapses for the release of neurotransmitters. Since synapses in the CNS are not easily accessible, scientists have turned to skeletal tissue in order to study the neuromuscular junctions (NMJs), which are the synapses by which motor neurons activate muscle cells. These nerve-muscle synapses are capable of regeneration and offer an instructive model in cellular regenerative processes.
A number of studies have yielded new information about specific cellular conditions that are necessary for the formation of NMJs. In various animal models, attempts have been made to bypass spinal cord lesions by connecting descending motor fibers with skeletal muscle. In one such study, muscular nerve branches were inserted into the severed lateral bundle of the spinal cord in a monkey, which resulted in reinnervation and restored motor function, indicating that the descending axons from the central noncholinergic neurons were responsible for functional restoration of the muscle. Further evidence of such axonal reinnervation was found when the distal stump of a transected rat motor nerve was connected via the implantation of an autologous sural nerve graft into the lateral white matter of the spinal cord that had been previously severed, and which was then connected to the transected nerve of the internal obliquus abdominis muscle. The researchers observed new glutamatergic innervation of skeletal muscle, which had replaced the original cholinergic muscle. Further electrophysiological, molecular, and immunohistochemical analyses also revealed that reinnervated muscles were reprogrammed by supraspinal neurons to organize functional glutamatergic neuromuscular junctions (NMJs).
Geneticists typically study diseases by using “knockout” mice, in which particular genes have been deleted, which results in the inactivation of the corresponding protein encoded by the gene in the mice. Studies with knockout mice in which the gene that codes for the agrin protein is missing have demonstrated that agrin is involved in the development of muscle aggregate molecules that serve as acetylcholine receptors at synapses. Mice with inactivated agrin genes therefore exhibit defective NMJs, as expected. Other knockout mice are also being studied for other proteins and the corresponding genes that control the functional development of the NMJ.
Embryologists are studying the developing embryo in order to identify the precise genetic signals that are involved in the early cellular differentiation of CNS tissue. Researchers have discovered two major signaling systems that control the development of embryonic brain and spinal cord cells, one of which controls the specialization of the nervous system along the long vertical axis that extends from the brain through the spinal cord, and the other of which controls specialization along the horizontal axis, or dorsoventral plane, which is represented by a cross-section of the spinal cord.
Like the spinal cord and the brain, the eyes are a part of the CNS. Like the spinal cord, in fact, the eyes are a literal extension of the brain. The retina is therefore a convenient and popular object of research by scientists who wish to understand the CNS in more detail. In particular, the ganglion cells of retinal neurons, which carry signals from the retina to the brain, together with supporting cells and axons, form the optic nerve. Once the optic nerve has been transected, retinal ganglion cells do not normally regenerate, even if new nerve fibers are grafted onto the tissue. Indeed, such cells seem to be obstinately resistant to any attempts at regeneration. Initial experiments with retinal ganglia therefore raised more questions than they answered, especially with regard to the nature of PNS growth, and why the PNS is inherently so different from the CNS, which seems to be so refractory to regeneration. Such questions in turn have led to additional studies focused on growth factors, or “trophic factors”, and the genes that regulate these trophic factors, which have shed some light on the matter.
Trophic factors are chemical signals that promote the survival and growth of nerve cells. Nerve cells are especially dependent on trophic factors during the period when their differentiation is becoming increasingly specialized as they begin to connect with their targets. For example, in utero, the developing nervous system produces many more nerve cells than the adult nervous system needs, and cells at this stage compete with each other to obtain trophic factors that are supplied by the desired target cells. Those neurons that do not succeed in competing for the appropriate connections die through apoptosis.
The first trophic factor to be successfully isolated was NGF (nerve growth factor), which is essential for the survival of some types of nerve cells in the PNS, and its withdrawal is commonly used in laboratory studies of apoptosis. Only recently have scientists discovered the role of NGF in the CNS.
The mechanisms by which retinal ganglion cells establish pathways to the brain are of particular scientific interest, with direct applications to spinal cord regeneration and to the treatment of various neurodegenerative diseases. Researchers have therefore developed methods of isolating retinal ganglia with a 99% purity rate, in order to culture the cells in combination with trophic factors and with an activated form of the cyclic AMP (adenosine monophosphate) “second messenger” system, which has been found to augment the efficacy of trophic factors. Cyclic AMP is a small molecule that transports electrically encoded messages from cell surface receptors that have been activated by “first messengers”, such as hormones or neurotransmitters, to specific target sites within the cell. Like other second messenger systems, this biochemical pathway allows a single first messenger to control several cellular processes, thereby regulating and integrating the numerous signals that cells receive.
Results of such experimentation have indicated that specific combinations of trophic factors are essential for the survival of CNS neurons, and “second messenger” systems play an important role in the electrical signaling and stimulation of cells. Changes in such cells below the level of a spinal cord injury are often dramatic, indicating extensive disruption of such cellular and molecular processes. The controlled stimulation and modulation of neurotrophic factors therefore offers a means by which damage from such disrupted processes may be minimized.
Even prior to modern research techniques, it has commonly been understood that the intrinsic capacity of nerve cells to grow varies according to age. Within any species, very young mammals will recover more quickly and completely from any injury than adults will, and this is especially true of CNS injuries. By studying the mammalian retina and the tectum (the area of the brain to which retinal ganglion cells connect) in culture, scientists have demonstrated that the ability of ganglion cells to grow as an animal ages is dependent upon two basic factors, namely, changes in the growth capacity of the retinal cells themselves, and changes in the target tissue. The bcl-2 gene was identified in retinal ganglion development, and this gene was discovered to have been turned “off” at the same time that retinal cells lose their ability for growth. This gene was already known to be an important regulator of apoptosis, and it is now being studied for its role in ganglia development. Retinal ganglion cells taken from mice with an inactivated bcl-2 gene (bcl-2 knockout mice) did not exhibit the typical sharp decline in growth ability that is normally expressed in control mice. Cells from the adult retina of these knockout mice were also found to be capable of growing if they were given pluripotent stem cell tissue as a target. The bcl-2 gene is now recognized as a “switch” that controls axon growth in the CNS, depending upon whether this switch has been turned “on” or “off”. The ability to control such genetic switches may expedite more efficacious therapies for a number of conditions that require axonal regeneration.
Differences between the PNS and the CNS continue to fascinate researchers, as nerves in the former naturally regenerate themselves whereas those of the latter do not. However, peripheral nerves from the PNS that are grafted directly into the CNS seem to stimulate the regeneration of CNS axons, via progression through the peripheral endoneural tubes. Logically, therefore, it would seem as though the proper “environmental” conditions are necessary for regeneration, in which such an environment offers not only the proper chemical milieu but also the right physical substrate with the appropriate mechanical properties. In fact, as soon as the axons reestablish contact with the CNS milieu, their regrowth ceases.
Corroborating such evidence, numerous grafting studies have demonstrated that CNS axons are capable of elongating within autologous nerves that have been grafted from the PNS into the spinal cord, in which functional synapses with skeletal muscles have formed, which in turn result in motor and sensory recovery. Certain neurons of the CNS seem to be particularly responsive to axonal regeneration from PNS nerve transplants, which include those neurons that originate in the rubrospinal, vestibulospinal, and reticulospinal tracts, which would include neurons throughout the spinal cord as well as in the midbrain and the brainstem.
Other regeneration techniques that do not directly involve stem cells have also proven to be effective in spinal cord regeneration. One such technique was developed in Tel Aviv, Israel, by the Proneuron Biotechnologies company. Known as “Autologous Activated Macrophage Therapy”, the technique involves the use of white blood cells which are extracted from the skin and bone marrow of a patient and then transplanted into the region of damaged spinal cord tissue. Dr. Valentin Fulga, vice president of Proneuron, directed the clinical trials. One such patient who participated was an 18-year-old female from the U.S. whose spinal cord had been severed in a car accident. Her own physicians were unable to offer her hope so she traveled with her family to Israel to participate in Proneuron’s first human trial. Within 5 months after receiving the treatment, she began regaining sensation below the site of injury and has since regained bladder control and some arm and leg movement. Macrophages are adult immune cells that are involved in wound healing and in tissue regeneration throughout the PNS, but macrophages exist only in very small quantities within the CNS, where their activity is barely detectable. The results of this clinical trial indicate that the natural “environmental” conditions of the CNS are distinct from those that exist elsewhere throughout the body, and such conditions may be improved by deliberate alteration and augmentation of various chemical and cellular factors.
The emerging field of bionanotechnology also shows great promise as a treatment for a number of neurodegenerative diseases and disorders, in addition to spinal cord injuries. Researchers have developed methods by which nanomaterials with “self-assembling” properties are injected into damaged tissue, in which these materials then stimulate natural mechanisms to heal the injury, according to the particular type of injury that has been incurred. In the case of spinal cord injuries, such nanomaterials would expedite the regeneration of specific neurons. Ideally, therapies could be individually tailored according to the unique needs of each patient. One of the leading scientists in this new field is Dr. Samuel Stupp, professor of materials science, chemistry and medicine, and director of the Institute for BioNanotechnology in Medicine at Northwestern University in Chicago. As he describes,
“Nanotechnology entails the measurement, prediction and construction of materials on the scale of atoms and molecules. A nanometer is one-billionth of a meter, and nanotechnology typically deals with particles and structures larger than 1 nanometer, but smaller than 100 nanometers. A nanometer-size particle is about twice the diameter of a gold atom and a very small fraction of the size of a living cell. Such a particle can be seen only with the most powerful of microscopes.”
Bionanotechnology represents one of the latest advances in regenerative medicine. More information is available at www.nanotechproject.org
Conventionally speaking, traumatic spinal cord injury is still considered to be an irreversible condition. The entire CNS, in fact, is considered to be highly “nonpermissive” in regard to the improvement of injured axons, meaning that the regeneration of tissue is severely suppressed, primarily because of the cascading series of molecular and cellular events that are automatically triggered by spinal cord trauma and which have been described herein. Further compounding this “nonpermissivity” is the total absence of growth factors at the somata, which is the region of the neuronal growth cone.
Stem cell therapy, however, now offers a new and promising treatment for spinal cord injury. Numerous studies with adult stem cells have in fact already demonstrated very positive results.
Whenever the body is injured, whether externally or internally, scar tissue usually forms. On external tissue such as skin, the formation of scar tissue helps expedite the healing process and may also prevent infection or additional physical injury. The formation of internal scar tissue on the spinal cord, however, inhibits healing and interferes with the regeneration of nerve fibers, as already described. Because of this scarring, the simple transplantation of adult stem cells into a damaged spinal cord is not usually enough to stimulate the regrowth of tissue. Initially, this fact was not readily apparent to researchers, but today this scar tissue is commonly recognized as one of the major impediments to spinal cord therapy. However, a natural potential for regeneration does seem to be built in to spinal tissue, but unless the right combination of symbiotic factors exists, the body’s natural mechanisms for healing are ineffective.
In many spinal cord injuries, the spinal cord itself is not severed, and many of the axons remain intact. Furthermore, the body’s natural stem cells are automatically stimulated after injury, and numerous studies have shown that these stem cells successfully migrate to the areas of tissue damage. As early as 1995, Dr. Frissen and colleagues observed that spinal cord injury increases the production of nestin, which is a protein that is naturally expressed by stem cells. The increased presence of nestin in the CNS therefore indicates that neural stem cells have indeed been activated after an injury. In fact, studies with nestin have demonstrated that the brain and spinal cord are automatically stimulated to increase their production of stem cells as a result of acute trauma, yet despite this increase in stem cell number and activity, however, the CNS does not heal itself. It is now understood that the inability of the CNS to repair its own tissue is not due to the absence of an innate stem cell response, but is instead due to confounding factors, such as the multi-staged inflammatory immune reaction of the body which results in complex secondary damage that includes demyelination and the formation of unusually dense glial scar tissue in the absence of neurotrophic factors. Additionally, of those stem cells that are naturally triggered within the body, few of them are oligodendrocytes, which are required for CNS remyelination.
Scar tissue that forms in the CNS is highly impermeable, and the glial scar tissue that forms along the spinal cord after trauma had presented a serious obstacle to regeneration, in the past. Today, however, the precise nature of this scar tissue is understood more clearly, and it no longer represents the insurmountable hurdle that it once did. Current stem cell techniques are now able to penetrate such scar tissue directly, thereby facilitating regenerative processes.
According to Dr. Philip Horner, assistant professor in the Department of Neurosurgery at the University of Washington School of Medicine,
“We’ve found that the axons, the parts of the nerves that transmit signals, try to regenerate after an injury but get caught in the scar. It’s like they’re stuck in the mud. We’re studying ways that this process is regulated to see if it can be manipulated to promote healing. In other words, we’re looking at ways to get the axons out of the mud. One way is to make the mud less sticky by manipulating stem cells that participate in scar formation. Another is to stimulate the axons to push through the scar by providing the cut nerves with molecules that induce elongation. We’re using molecular signals called growth factors to simulate the growth of cultured nerve cells in the laboratory.”
Dr. Horner was joined by Dr. Thomas Reh, a professor in the Department of Biological Structure at the University of Washington, and Dr. Fred Gage from the Salk Institute in California, for a discussion on “Neural Stem Cells in Health and Disease” at the 2004 annual meeting of the American Academy for the Advancement of Science (AAAS). Their recent findings at that time were among the first to indicate that the same stem cells behave differently under different circumstances, and those that create scar tissue in the spinal cord can also create new cells in the retina of the eye, depending upon the presence of trophic factors and corresponding signaling pathways. The retina is the light-sensitive membrane that transmits light signals from the eye to the brain, and it is easily damaged by a number of diseases, including glaucoma and macular degeneration. Some other species, however, do not develop glaucoma or macular degeneration, such as the salamander, the newt, various types of frogs and some fish, because they naturally regenerate damaged tissue from their own abundant supply of stem cells. As Dr. Reh explains,
“We’re trying to understand the remarkable regenerative powers of these lower vertebrates, and through this understanding develop strategies to stimulate regeneration in the human retina.”
Some species, such as salamanders, maintain this regenerative capacity throughout their lives, while most other species lose the ability as they age. Dr. Reh continues,
“At some point in the life cycle of each species, the stem cells in the retina make a transition from a regenerative cell to a cell that will make a scar in response to injury, like the cells that cause scars in the spinal cord. Chickens make the transition a few weeks after hatching in most of their retina, though they retain some limited capacity to regenerate retinal cells throughout life. In rats, it’s only a matter of a few days after the cells are generated that they lose their ability to regenerate other retinal cells.”
In other species, such as humans, retinas are incapable of repairing themselves, at any age, although human retinal cells have exhibited the ability to regenerate neuronal cells under certain laboratory conditions. Dr. Reh adds,
“The hope is that many of the molecular and cellular mechanisms necessary for regeneration that serve amphibians so well are still in place in humans. Future studies from the nervous system, as well as other organ systems, should enable us to define the roadblocks in the regenerative process, and develop strategies to go around them.”
Indeed, the retinal cells of the eye remain the model of choice for many researchers who are studying the molecular mechanisms of the CNS. An affiliate of Harvard Medical School, the Schepens Eye Research Institute of Boston is the largest independent eye institute in the United States. By studying physiological processes of the eye, scientists at the Schepens Institute have recently identified a key mechanism that is necessary for the successful transplantation of tissue into the adult CNS. It was already known that stem cells induce the natural production of MMP-2 (matrix metalloproteinase 2) but this molecule has now also been shown to break down physical and chemical barriers on the outer surface of CNS scar tissue. In studies with damaged retinas, the administration of MMP-2 in combination with transplanted tissue was found to expedite integration of the new tissue into the damaged fibers. Given in combination with stem cells, MMP-2 facilitates regeneration of the nerves to an even greater extent. Since the eye, like the brain and the spinal cord, is part of the CNS, results of such studies are applicable not only to patients with retinal diseases, but also to those suffering from spinal cord injuries and neurodegenerative disorders.
Researchers at the Rochester Medical Center of New York, in collaboration with scientists at other institutions, have also discovered a method for overcoming the problem of spinal cord scar tissue. The researchers isolated glial-restricted precursor cells, or GRPs, which they cultured under specific laboratory conditions that stimulated the GRPs to differentiate into a particular type of astrocyte, which was then transplanted into the areas of spinal cord injury. Rats who were given this specialized astrocyte cell were found to develop less scar tissue and nerve damage when compared to rats in a control group, and their locomotion improved to such an extent that they regained the ability to walk after receiving the treatment. The brains of the rats also showed general improvement in other areas, with an 80% decrease in the degeneration of neuronal fibers that extend down the spinal cord. Commenting on these results, Dr. Wise Young, a neuroscientist and director of the W.M. Keck Center for Collaborative Neuroscience at Rutgers University, has said,
“The GRPs are of great interest, and the study reaffirms that they are an attractive method for repairing spinal cord damage. The paper shows very compelling data for moving GRPs to clinical trial as soon as compatible human cells can be obtained. This is going to create a lot of excitement in the field.”
Dr. Young, a specialist in the treatment of spinal cord injury himself, has organized clinical trials in China where he is involved in the use of stem cells derived from human umbilical cord blood in the treatment of CNS disorders. As he explains,
“A stem cell is just a cell expressing certain genes, and there’s nothing more mysterious about it than that. We just have to know what the genes are.”
A number of studies have corroborated such findings. Scientists at the Baylor College of Medicine in Houston have also achieved very positive results by using astrocytes to treat spinal cord injury in rats. In a study led by Dr. Stephen Davies, assistant professor of neurosurgery, rats that were given astrocytes exhibited a 40% regeneration of nerve fiber at the sight of injury within 8 days, and they began walking again within 2 weeks of being treated. According to Dr. Davies,
“Stem cell technology is moving at a tremendous pace at the moment, and this represents advances in how to use that technology.”
Results of the research, which was funded in part by the Christopher and Dana Reeve Foundation, were published in the Journal of Biology.
Researchers at Johns Hopkins University in Baltimore, Maryland successfully restored spinal cord function in rats via the localized transplantation of stem cells. Dr. Douglas Kerr, assistant professor of neurology, led a study in which mice had been paralyzed by a virus that disabled the entire spinal cord. After stem cells were injected into the spinal fluid, the mice were able to stand and move their feet. This study represented the first time that motor function had been restored after complete spinal motor atrophy in an animal model.
Although stem cells are believed to exist in other locations not yet discovered, stem cells have already been confirmed in two specific regions of the adult mammalian brain. Specifically, neural stem cells were isolated from the dentate gyrus of the hippocampus and from the ependymal layer of the ventricular system. The progeny of these stem cells are known to differentiate in the granule cell layer into oligodendrocytes, astrocytes and neurons. It is now therefore a well established fact that neurogenesis continues throughout adult life. These stem cells are also known to migrate along the rostral migratory stream to the olfactory bulb, where they further differentiate into neurons and glial cells.
Dr. Fred H. Gage of the Salk Institute for Biological Studies in La Jolla, California, has conducted numerous studies with multipotent progenitor cells in the adult dentate gyrus. These cells have been shown to differentiate into glial cells, which are the precursor cells for neuronal tissue. Dr. Gage has also directed studies examining the survival and differentiation of adult neuronal progenitor cells within the adult brain. Findings from his research were published in the Journal of Neurobiology and in the Proceedings of the National Academy of Sciences. Similarly, Dr. C.B. Johansson and colleagues in the Laboratory of Genetics at the Salk Institute have identified neural stem cells throughout the adult mammalian CNS. The results of their research were published in the journal Cell.
Scientists at the Reeve-Irvine Research Center at the University of California at Irvine School of Medicine, in collaboration with researchers at the Salk Institute of Biological Studies in La Jolla, have successfully used adult human neural stem cells to regenerate damaged spinal cord tissue in mice. Dr. Brian Cummings and colleagues used adult human neural stem cells which were shown to differentiate into oligodendrocytes after transplantation into the mice. The axons of the mice were then shown to have undergone restored myelination, the neurons formed new synaptic connections, and the mice themselves exhibited improved mobility. As Dr. Cummings describes,
“We set out to discover whether these cells would be able to respond to the injury in an appropriate and beneficial way on their own. We were excited to discover that the cells responded to the damage by making appropriate new cells that could assist in repair. This study supports the possibility that formation of new myelin and new neurons may contribute to recovery.”
The mice were given human adult neural stem cells 9 days after spinal cord injury, and improvement in their walking ability continued for more than 4 months. Unlike previous studies with embryonic stem cells, these human adult neural stem cells were not directed by laboratory methods to differentiate into any specific type of cell prior to implantation. The findings were published in the Proceedings of the National Academy of Sciences.
Another study also conducted at the Reeve-Irvine Research Center at the University of California at Irvine showed positive results with embryonic stem cells in rats, but only if administered during the very early stages after injury. Dr. Hans Keirstead and colleagues demonstrated that embryonic stem cells promoted some remyelination in the neuronal tissue of rats that received the stem cells within 7 days after developing a spinal cord injury. If the rats were given the stem cells 10 months after being injured, however, remyelination did not occur, nor did any type of neuronal improvement in the rats, even though the embryonic stem cells had been coaxed in the laboratory to differentiate in oligodendrocytes, which are the specific type of cell required for the formation of CNS myelin. Even in the rats who received the stem cells within 7 days of their injury, they did not begin to show improvement in their walking ability until more than 2 months later. Although this study was limited to a specific examination of myelin, and of the ability of stem cells to remyelinate neurons that had been demyelinated, such findings indicate the importance of the “window of opportunity” that exists for effective treatment only in the very early stages after spinal cord injury. Logically, demyelination would be less pronounced in the period of time immediately following an injury, and the demyelination would progressively worsen over time. Even though the oligodendrocytes were found to migrate to the sites of injury in all the rats, it was also verified that remyelination was prevented by the scar tissue which had formed 10 months after injury, whereas scar tissue had not yet formed as extensively within the first 7 days following injury. Indeed, the extent of demyelination and of scar tissue formation constitute major differences between acute and chronic damage, and such differences will influence to a great extent the nature of the treatment protocol as well as the ultimate prognoses of the patient. In previous studies, Dr. Keirstead and his colleagues had examined the stages of the body’s immune response after spinal cord injury, and the precise cellular and molecular mechanisms by which myelin is destroyed. Their findings were reported in the Journal of Neuroscience.
Since myelin plays such an integral role in neuronal health, the regeneration of neurons must include the appropriate factors for remyelination. At Yale University, Dr. Jeffrey Kocsis and colleagues have tested a number of ways to remyelinate neurons with stem cells. Stem cells were shown to initiate the remyelination of nerves in a variety of animal models that were not limited only to rodents but which also included small primates and monkeys such as the marmoset. Even autologous Schwann cells were shown to expedite remyelination of CNS nerves when the cells were extracted, grown in the laboratory under specific conditions, and transplanted back into the donor at the site of tissue injury.
Scientists now know that stem cells alone are not usually sufficient for healing damaged CNS tissue, but must be combined with the proper type and amount of neurotrophic factors, which are of major importance in promoting the efficacy of stem cells. The stem-cell-stimulating molecules known as brain derived neurotrophic factors (BDNF) and neurotrophin 3 and 4 (NT-3 and NT-4) have shown particular promise in regenerating injured spinal cord tissue. The natural production of NT-3 especially increases in response to spinal cord injury, and experiments with animal models have demonstrated positive improvement both in the regeneration of partially severed spinal cords after treatment with NT-3, and in the recovery of motor function. Such regeneration is most noticeable in the corticospinal tract, which is the pathway responsible for voluntary movements, in which NT-3 has been found to be highly effective in promoting regrowth of corticospinal axons. Professor Mark Tuszynski and colleagues at the University of California at San Diego have demonstrated that the cellular delivery of NT-3 specifically facilitates corticospinal axonal growth and at least partial functional recovery after a spinal cord injury. His findings were reported in the Journal of Neuroscience. In related studies, Dr. Masaya Nakamura and colleagues have described differences in neurotrophic factor gene expression profiles between the neonatal and adult rat spinal cords after injury. Gene expression itself is a particularly fertile field of research, not only for spinal cord injury but for numerous other disorders, and various neurotrophic factors have been found to influence gene expression in a variety of ways. Dr. Nakamura’s findings were reported in the Journal of Experimental Neurology.
Researchers at the Karolinska Institutet in Stockholm, Sweden, have demonstrated that the transcription factor known as neurogenin-2 facilitates the action of stem cells in the treatment of spinal cord injury. Neurogenin-2 regulates the activity of various genes during the stem cell maturation process, and its presence is believed to inhibit the development of astrocytes while simultaneously stimulating the formation of oligodendrocytes, which is the type of glial cell responsible for the formation of myelin within the CNS. The researchers added neurogenin-2 to the stem cells while the cells were developing in culture, after which the stem cells were then transplanted into damaged spinal cord tissue in rats which resulted in the significant improvement of motor and sensory function below the injury. Some cases of pain superior to the injury were also observed, however, which are believed to result from the low number of astrocytes which had formed and which constitute a type of glial cell that may promote nociception and the formation of nociceptors (pain receptors) in the spinal cord by the secretion of stimulating chemicals that trigger this type of neuronal development. The total number of astrocytes was relatively low, however, while the number of oligodendrocytes was relatively high, which correspond respectively to the inhibited growth of nociceptor axons and to a greater volume of myelinated nerve fibers within the regions of damaged tissue. Restored sensory function following each spinal cord injury was confirmed via functional magnetic resonance imaging (fMRI).
The mere discovery of stem cells in the adult human brain revolutionized neuroscience and paved the way for many advancements in regenerative medicine, as this discovery disproved earlier erroneous beliefs that regrowth of adult neurons is impossible. As previously mentioned, the progeny of neural stem cells originating in the dentate gyrus of the hippocampus and in the ependymal layer of the ventricular system are now know to migrate along the rostral migratory stream into the olfactory bulb, where they further differentiate into neurons and glial cells. The discovery of these stem cells in the olfactory bulb is now revolutionizing neuroscience even further.
Tissue within the human olfactory bulb is continuously regenerating itself. The olfactory bulb transmits scent signals to the hippocampal dentate gyrus, which is an area of the brain that processes olfactory information while organizing short-term memory. Neurons within the olfactory bulb are automatically being renewed and replaced on a continuous basis by an abundant supply of olfactory stem cells. The olfactory bulb is, in fact, one of the richest sources of pluripotent stem cells, in one of the most easily and safely accessible locations of the body.
Olfactory mucosa transplantation is a procedure by which the adult stem cells are removed from the nasal mucosa and transplanted into the specific areas of damaged tissue. Such a procedure has already shown very positive results in the treatment of spinal cord injury. Since this technique represents an autologous transplantation (in which the donor and recipient are the same person), the risk of immune rejection does not exist. Dr. Carlos Lima, a neuropathologist at Egaz-Moniz Hospital in Lisbon, Portugal, developed the procedure and has successfully treated approximately 30 patients who were suffering with spinal cord injury.
In one case, a female individual was paralyzed from the neck down with a C6 vertebral burst fracture after surviving a car accident at the age of 16. Although doctors at several hospitals in the U.S. emphatically assured her that she would never walk again, one specialist informed her of a new procedure that had been developed in Portugal. Within 6 months of receiving a stem cell transplantation from Dr. Lima that involved the use of her own stem cells derived from her own olfactory bulb, she was told by her physical therapists that her spinal cord had begun healing, and MRI scans confirmed that 70% of the lesions that developed after her injury had resolved into normal spinal tissue. She continued to acquire sensation in her extremities and eventually was able to stand and walk with the aid of a walker.
Another patient who was left paraplegic after a car accident with vertebral injuries to T7 and T8 was able to walk again, with braces, within one year after receiving Dr. Lima’s stem cell transplantation. Yet another patient, who was left quadriplegic from a spinal cord injury, regained bladder control and arm and leg movement after being injected with stem cells from her own nasal mucosa by Dr. Lima.
Dr. Jean D. Peduzzi-Nelson, a professor of psychological optics at the University of Alabama in Birmingham, has collaborated with Dr. Lima on this new technique and has testified before the U.S. Senate Committee on Commerce, Science and Transportation regarding the scientific efficacy and safety of this procedure. Dr. Peduzzi-Nelson as well as other scientists, physicians and neurologists in the U.S. are now seeking Congressional and FDA approval for this treatment to be legalized and made available in the United States.
Other therapies have shown positive results when patients with spinal cord injuries were treated with mesenchymal stem cells in combination with CD34. One patient in particular who was paralyzed below the mid-chest level with a T5 injury regained feeling and muscle control in the pelvis, along with restored sensation in his feet, after receiving 3 such treatments. The patient continues to improve. Mesenchymal stem cells are adult pluripotent progenitor cells with the ability to self-renew indefinitely and with differentiation capacities that yield a variety of tissue types including cartilage, bone, muscle, tendon, ligament, fat and nerve. CD34 refers to a group of clustered differentiation molecules, also known as transmembrane glycoproteins, that function as cell-to-cell adhesion factors and which also mediate the attachment of stem cells to the extracellular matrix. They are found in abundance in umbilical cord blood, endothelial cells and in bone marrow hematopoeitic cells, from which they may be isolated via immunomagnetic methods. Undifferentiated CD34+ mononuclear cells are pluripotent hematopoietic stem cells and as such exhibit a vast range of differentiation capabilities.
Adult stem cells offer the same pluripotency as embryonic stem cells, but without the danger of forming teratomas (tumors), which remains a serious risk from embryonic stem cells. It is neither necessary nor desirable to use embryonic stem cells in the treatment of spinal cord injuries or other disorders, since a growing number of studies are showing increasing success with adult stem cells. In fact, the only stem cell studies that have ever shown success in the treatment of any human disease have involved adult stem cells, since no study has ever been conducted in which a disease was successfully treated with human embryonic stem cells, although this fact is not generally reported by the media. Ever since researchers first isolated human embryonic stem cells in 1998, there has never been a successful treatment for any human disease in a human being by embryonic stem cells. Embryonic stem cells have in fact proven to be very problematic, whereas bone marrow and cord blood stem cells, by contrast, have been safely used by doctors for over 40 years. Human umbilical cord blood in particular is now known to be a rich source of growth factors and cytokines, both of which are necessary for the regeneration of tissue, and stem cells that are derived from human umbilical cord blood have been shown to be more effective at tissue regeneration than are other types of stem cells that lack such additional factors. Ethics and politics aside, adult stem cells are highly preferable to embryonic stem cells purely for scientific reasons. (Please see the section entitled “Stem Cell Primer” for an explanation of the different properties of the different types of stem cells).
Spinal cord research has progressed more in the past five years than in the previous 50, thanks primarily to advances in neurology and immunology which have evolved in parallel with a more detailed understanding of the various types and stages of stem cell differentiation. While the entire field of regenerative medicine is still in its infancy, and the greatest discoveries are undoubtedly yet to be made, people who suffer with spinal cord injuries now have a choice of several therapeutic modalities which did not exist at all until recently, and which represent vast improvements over the severely limited therapeutic choices that were available in the past.
Adult stem cell therapy offers a safe and potentially effective treatment of a very severe type of injury which previously has been considered irreversible.