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Research Abstracts

The projects supported in 2003-2004 cover a board range of research areas, including understanding and preventing secondary cell death immediately following injury, scar formation around the injury site, regeneration of nerve cells, nerve cell replacement treatments with stem cells, locomotor rehabilitation and locomotor treatments to improve recovery of function, and new animal models of spinal cord injury. Of 29 applications, the 17 projects summarized below were chosen as Roman Reed Research Projects. Please note that several of the studies can fit in several research categories, although below each project is described only under one area.

Secondary Degeneration

Following an injury to the spinal cord, several things happen. First the spinal cord, which contains the neurons and axons that allow the brain and body to communicate, is injured, crushing or damaging the axons at the site of the injury. This is followed by a series of events that occur days to weeks after the initial insult and involve significant tissue loss, or secondary degeneration. These 2 projects seek to better understand what those events are and explore ways to prevent further tissue loss after injury.

Motor neurons ROS production and disruption of astrocytic glutamate transport - possible roles in injury progression in the subacute phase after SCI
John Weiss, M.D., Ph.D.
University of California, Irvine

There is a period of time, lasting from hours to days, after a traumatic spinal cord injury, during which tissue damage actually worsens before recovery or regeneration begins. One mechanism that is thought to contribute to this secondary cell death is "excitotoxicity", or injury caused by release of the excitatory nerve cell signaling compound, called glutamate, from damaged nerve cells. Indeed, spinal trauma is known to cause massive glutamate release, and drugs that block effects of glutamate do cause a mild reduction of tissue loss.

Motor neurons are the type of large nerve cell in the spinal cord that control all muscular activity. In recent studies of mechanisms that may underlie motor neuron loss in another spinal disease, amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), we found evidence for a possibly critical interaction between motor neurons and the support cells that surround them, called astrocytes. Motor neurons are highly sensitive to glutamate injury, and in response to glutamate exposure, they produce exceptionally large quantities of highly reactive compounds called reactive oxygen species. We further find that this reactive oxygen species appears to disrupt the function of the molecules in the surrounding astrocytes that are responsible for taking the glutamate back up inside cells (called "glutamate transporters") where it is no longer toxic. When the transporters are damaged, the glutamate level rises further, causing more reactive oxygen species production in the motor neurons, possibly setting in motion a vicious cycle that culminates in extensive tissue damage from the reactive oxygen species, and motor neuron death. This project examines whether an identical cycle gets set off after spinal trauma, which contributes to the worsening of injury. To that aim, we will first examine changes in the spinal cord over time after trauma, to see if damage to tissue and dysfunction of the glutamate transporters is most evident in the areas of the cord where the motor neurons reside. Additionally, we will test the utility of certain drugs, injected into the spinal canal, for decreasing the damage and improving recovery.

Activated Protein C, coagulation and inflammation after spinal cord injury
William Whetstone, M.D.
University of California, San Francisco

Spinal cord injury produces local bleeding, an acute inflammatory reaction and, ultimately, the formation of a scar around the injury site. Blood clotting is one of the first effects following spinal cord contusion injury, and thrombin is the first and most important component of blood clotting. Unfortunately, thrombin also has many detrimental effects within the central nervous system. It has been recently shown that thrombin causes blood vessels in the brain to leak. In addition, thrombin worsens inflammation, changes the shape of scar forming astrocytes, and is directly toxic to neurons. However, there is a natural occurring protein, Activated protein C, that counteracts many of thrombin's effects, and has recently been approved by the FDA to treat the inflammation and blood leakage that occurs with certain overwhelming infections. Given the way Activated Protein C works, we believe that it will decrease the blood-spinal cord barrier leakage and early infiltration of inflammatory cells following spinal cord injury. These events contribute to irreversible cell injury, and thus influence the extent of functional recovery after spinal cord injury. We plan to treat spinal cord injured mice with Activated Protein C 20 minutes after injury. We will determine the extent to which this treatment limits the infiltration of white blood cells and if such treatment protects nerve fiber tracts from the brain, thus improving walking function. Together, these experiments will determine the benefits and mechanism of this clinically approved drug in limiting the secondary injury effects of spinal cord injury.

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Scar Formation

Only 10% of brain and spinal cord cells are neurons. The remaining 90% are support cells called glia. One particularly interesting type of glia are astrocytes. Astrocytes make chemicals, or neurotrophic factors, that are like vitamins for neurons and may play an important role in nerve cell communication. However after injury, astrocytes also create scar tissue. They wall off the damaged part of the spinal cord with a scar. This scar is a major problem for regenerating nerve cells, acting as a physical barrier. Two Roman Reed projects are examining scar formation and ways of helping nerves get through the scar tissue.

Matrix metalloproteinases and spinal cord injury
Linda Noble, Ph.D.
University of California, San Francisco

The degree of functional recovery after spinal cord injury is related to 3 major factors: the magnitude of the initial injury, secondary events that occur early after injury and contribute to additional tissue damage, and complex wound healing events that involve remodeling of the tissue including the formation of a scar. The latter is the focus of this project and is based on the fundamental belief that certain wound healing events are beneficial and if amplified could improve functional recovery. In fact, a particular protein, matrix metalloproteinase-2 or MMP-2, seems critical to those wound healing processes that foster motor recovery. Evidence for this comes from mice that lack MMP-2, who showed impaired motor recovery after injury, suggesting that MMP-2 is indeed important to wound healing. This project will explore the biologic basis MMP-2's role in recovery. We will focus on scar formation, which is thought to impede recovery processes, and sparing and/or regrowth of those nerves in the cord that are essential for voluntary movement. We plan to evaluate how MMP-2 influences the formation of a scar and if it protects nerve fibers and/or promotes their regrowth in the injured spinal cord.

Developing therapies to optimize recovery after spinal cord injury is dependent on a clear awareness of those biologic events that cause tissue damage as well as those that may protect against injury and promote recovery. The long-term objective of these studies is to develop a therapeutic regimen that is specifically designed to not only minimize early tissue damage, but also maximize those wound healing events that foster regeneration.

Genetic manipulation of scar forming astrocytes, and biopolymer tissue support after SCI
Michael V. Sofroniew, M.D., Ph.D.
University of California, Los Angeles

Regenerating axons face many challenges, but two major ones are the scar formed by astrocytes and the lack of something to grow along. Axons need support, like a bridge or tunnel, to grow along and in the injury site where tissues has been lost, that support has also been lost. This project will be looking at both of these problems independently and then in combination. First, we will use mice that do not have reactive astrocytes or mice that are have astrocytes that are not able to make specific scar forming responses. We want to see if after spinal cord injury these animals show significant levels of axon regeneration. Second, a support system will be provided to help regenerating axons. Microspheres coated with a molecule, laminin, that supports and stimulates axons to regenerate will be injected into the injury site and regeneration through the injury will be examined. Finally, we plan to combine the two treatments. If successful, this information could lead directly to potential treatment strategies for humans.

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Regeneration Studies

There are many road blocks to regeneration after spinal cord injury, including formation of a scar around the injury site, chemical stop signals for axon growth, and a reduction in molecules that encourage axon growth to name a few. Five Roman Reed research projects are exploring ways to overcome these barriers and improve axon regeneration.

Regeneration of injured dorsal column fibers: the contribution of re-priming
Allan Basbaum, Ph.D. and Simona Neumann, Ph.D.
University of California San Francisco

There is an interesting regeneration phenomenon in the central nervous system, which normal does not show good axon growth following injury. If an injury is made to a nearby peripheral nerve a week before the spinal injury in rats, axons in the spine grow through the injury site. This effect is called priming, the peripheral injury seems to get the central nervous system ready to repair itself.

In recent studies, our laboratory has identified a naturally occurring molecule that can significantly promote regeneration of injured spinal cord nerve fibers. Our studies suggest that this molecule, cycle AMP or cAMP, which is critical for daily cellular function, increases the inherent growth capacity of injured nerve fibers when administered prior to the spinal cord injury, and appears to promote growth in the presence of molecules that normally inhibit regeneration. It appears that cAMP plays a role in the priming effect. In the present study, we propose to extend our analysis to investigate whether priming has an effect only on the cell bodies of the neurons that receive the priming or whether there are changes in the environment that can affect other fibers in the spinal cord. Taken together, our studies will provide important new information on the ability of priming to maintain a growth state that could lead to anatomical and functional recovery after spinal cord lesion.

Combining Ex Vivo and In Vivo gene delivery to promote axonal regeneration
Laura Taylor, B.S. and Armin Blesch, Ph.D.
University of California, San Diego

Gene therapy is a potentially useful means of providing growth factor support to injured axons after spinal cord injury. Cells are genetically engineered to make proteins that, in this case, are thought to be helpful to regenerating axons. Numerous studies have shown that the delivery of nervous system growth factors, such as neurotrophin-3 (NT-3), by cells transplanted into the lesion site promotes regeneration in rats with spinal cord injury. The transplanted cells provide a substance to fill the lesion cavity at the site of injury and growth factors support axonal regeneration into the cell graft. The problem so far is that axons like the transplant environment so much that they do not continue on through the injury site and make connections with intact neurons on the other side of the lesion.

We propose to examine whether we can get the growing axons through the graft by injecting nerve growth expressing cells below the lesion. A source of NT-3 beyond the graft may attract axons across the graft to intact tissue on the other side of the lesion. In addition to providing a potentially clinically applicable method for inducing axons to bridge a lesion site, these experiments provide a means to examine whether axonal regeneration into intact tissue will lead to formation of contacts with intact neurons.

Structure of proteins that inhibit CNS repair: Nogo and it's receptor
Melanie Cocco, Ph.D.
University of California, Irvine

Although nerves in the peripheral nervous system will regrow after damage, nerves in the central nervous system, brain and spinal cord, do not. Understanding why the central nervous system does not repair itself as the peripheral nervous system does may lead to a treatment for spinal cord injury. Recently proteins that block spinal cord nerve repair have been discovered. One of these, called Nogo, is a small protein found in the myelin (the insulating sheath surrounding the axon). It appears that inhibiting Nogo will result in regrowth of damage axons. The structure of Nogo has eluded detection by the most common structural technique, crystallography, because it resists forming crystals. Nuclear magnetic resonance (NMR) spectroscopy offers a unique means to determine the structure of Nogo because it looks at proteins in solution and does not require solid crystals to form. Knowledge of the protein structure should enable us to design a molecule that targets Nogo's surface and blocks its action. Most importantly, Nuclear magnetic resonance imaging can be used in the development of therapeutic agents designed specifically to stop Nogo as part of a treatment to enable cellular repair.

Nerve Guidance Scaffolds for Spinal Cord Injury
Shula Stokols, B.S. and Mark Tuszynski, M.D., Ph.D.
University of California, San Diego

In order for nerves to grow through a spinal injury site, they need several things. First of all, after injury a big fluid-filled hole or cyst usually forms at the injury site. This creates a major problem for nerves trying to grow -- they can't grow through fluid alone. Regenerating nerves need some sort of bridge or scaffold to grow along. Nerves also need help to regenerate. They need molecules like nerve growth factors that help them move them grow. In this project, we are helping nerves get through a spinal cord injury by providing supporting molecules and a bridge. We do this by implanting "nerve guidance channels" in to the injury site. These channels have several properties that should support regeneration and so treatment of SCI: they can 1) provide a physical bridge in a lesion site to provide guidance to the regrowing axons, 2) contain permissive axon growth factors to support nerve growth, and 3) release factors, such as neurotrophins, to stimulate growth. This study will test whether a novel material, made from a polymer, can be formed into nerve guidance channels that will support and guide axonal regeneration across sites of SCI.

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Cell Replacement Therapies

After a spinal cord injury, spinal cord tissue is lost. It is destroyed by the trauma or it is destroyed by the body's own response to the injury. One potential way to restore function after injury is to replace the missing tissue, in the hopes that it will be able to restore functional connections with uninjured tissue above and below the injury. Three of the 2004 Roman Reed Research Awards were given to projects exploring the use of stem cells to replace lost tissue.

Implantation of adult human neural stem cells in a chronic cauda equina/conus medullaris injury model
Leif Havton, M.D., Ph.D. and Harley I Kornblum, Ph.D.
University of California, Los Angeles

Injuries to the lower part of the spinal cord may lead to significant impairments and disabilities. Many individuals with these forms of injuries develop leg weakness and numbness, gait impairment, and bladder and bowel dysfunction. Recent animal studies have suggested that one significant contributor to these dysfunctions is the prominent death of injured spinal cord neurons. In the proposed studies, we will attempt to replace the lost nerve cells by implanting adult human stem cells into the spinal cord in a rat spinal cord injury model. We will also investigate whether the implanted stem cells may be able to extend their processes to outside the spinal cord as well, hopefully making functional connections with the peripheral nervous system, which will be an important part of recovery. The proposed studies may contribute to the development of a new strategy for patients with chronic conus medullaris and cauda equina injuries.

The functional consequences of remyelination following transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into the injured spinal cord
Hans S. Keirstead, Ph.D.
University of California, Irvine

Spinal cord injury does not end immediately after the initial injury. An injury to the spinal cord is followed by a secondary phase of injury, in which the initial injury site expands, and regions of injured tissue are removed and may evolve into a cavity. One of the components of this secondary phase of injury is the loss of the insulation around some of the nerves that transmit electrical signals within the spinal cord. When the insulation is lost, the nerves are impaired in their ability to conduct electrical signals and send messages to other neurons. Our laboratory has developed a way to repair the lost insulation, by implanting human cells that produce the insulation in the injured spinal cord. We have evidence that animals in which the insulation has been repaired regain some walking ability. The transplanted cells are derived from federally approved human embryonic stem cells, which are coaxed in our laboratory to become insulation-producing cells called oligodendrocytes.

Although we have demonstrated that the transplanted human cells promote the repair of insulation within and around the site of injury, we do not know whether the transplanted cells actually produce the insulation, or whether they promote the injured spinal cord to produce insulation. Further, we do not know if the repair of insulation is responsible for the return of some walking ability. These studies will address these two questions, and in so doing, further the pre-clinical development of this repair strategy.

Novel microfluidic technologies for sorting and differentiating neural stem cells
Edwin S. Monuki, M.D., Ph.D.
University of California, Irvine

Neural stem cells (NSCs) hold tremendous promise for cell-based therapies in spinal cord injury and other neurologic disorders. However, this promise is currently tempered by our inability to purify NSCs and to control their differentiation into desired cell types. In addition to traditional cell biology approaches to these problems, novel technologies that allow individual cells to be manipulated precisely, rapidly and efficiently in minimal volumes of media will be needed. Microfluidic systems represent just such a technology.

Together with the laboratories of Abraham Lee and Noo Li Jeon in the UCI Department of Biomedical Engineering, our goal is to develop new and existing microfluidic devices to sort human and mouse NSCs and to control their differentiation. We are currently developing an integrated cell sensor and sorter, which will be used to isolate cell subpopulations based on distinctive electrical signatures. In addition, we are culturing NSCs in an existing device that allows combinations of growth factor gradients to be applied with spatial and temporal precision, which should greatly improve our control over cell differentiation. If successful, this level of control over NSC sorting and differentiation using microfluidic technologies should be a significant advance towards the ultimate goal of cell-based therapy for human neurologic disorders.

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Retraining and Rewiring the Spinal Cord

One obvious result of spinal cord injury is disruption of locomotion, the ability to walk. There is now evidence that the mechanics of walking, lift your knee -- extend your leg -- plant your foot, are actually carried out by the spinal cord, not the brain. Indeed, several of the Roman Reed awards are exploring ways to retrain the spinal cord control walking in a step training paradigm, where the body is supported and various means are used to move the legs on a treadmill. Other projects are trying to better understand the basics of walking, an incredibly complex movement to say the least, or how the locomotor centers in the brain reorganize after injury.

Combining bicuculline treatment and robotic locomotor training
Ray de Leon, Ph.D.
California State University, Los Angeles

Body weight supported treadmill (BWST) training is a successful therapy for improving walking function following a spinal cord injury. However, providing effective BWST training can be a challenge for therapists because of the difficulties involved in assisting leg movements particularly for an individual with full paralysis in the legs.

We propose to study the combined effects of robotic training and a drug treatment that enhances neuronal activity in a rodent model of spinal cord injury. In the first year of the project, we successfully developed and implemented a robotic training algorithm that trains the hindlimbs of complete spinally transected rats to step on a treadmill. We also determined that a drug (quipazine) can be used to boost neuronal activity during training. We now propose to expand our studies and use the robotic training and pharmacological interventions to enhance locomotor recovery in a model of incomplete spinal cord injury. We will administer quipazine to rats that have undergone moderate spinal cord contusions. We will then use the robotic device to guide the hindlimbs in a walking pattern. We hypothesize that through the combination of training and quipazine treatment, the effectiveness of repetitively practicing walking movements will be increased. The results of this project will have important clinical implications as it could indicate that combining these two approaches would enhance and/or accelerate gains in walking function that occur with BWST training.

Combining pharmacology and epidural electrical stimulation to induce locomotion in adult spinal rats
V. Reggie Edgerton, Ph.D. and Ronaldo Ichiyama, Ph.D.
University of California, Los Angeles

Neuroprostheses are electronic devices used to help improve functionality of damaged structures in the body. Some of these devices have been successful in stimulating muscles and help prevent loss of bulk and spasticity after a spinal cord injury. Using electrical stimulation of the spinal cord, we have successfully induced stepping in severely injured adult rats without the necessity of activating individual muscles. Our implants are placed on the surface of the spinal cord and do not cause any further damage to the cord. In this project, we propose to study the interaction between pharmacological agents and electrical stimulation in the recovery of locomotor function after spinal cord injury. We proposed to study the doses of the drug necessary to induce stepping, and the effects of long-term step training using the combination of the pharmacological agent and electrical stimulation on the recovery of locomotor function. The implications of this study are substantial for the future development of novel therapies for the treatment of spinal cord injuries.

Activity-dependent plasticity after human spinal cord injury
Susan Harkema, Ph.D.
University of California, Los Angeles
Recovery of standing and walking is generally considered unattainable following a clinically complete spinal cord injury. A previous study, in people with a complete spinal cord injury, showed that by practicing a specific standing task (on one leg or on two legs) we could improve the muscle output for standing but did not improve stepping. Weight bearing on both legs provides signals that activate the muscles and entrain standing and improve bone. In these studies, we examine whether the same people who learned to stand, if trained to step can improve both. This study will address whether the combination of stand and step training can also increase bone mineral density of the legs more than either alone. Understanding how best to train people to stand or step can help design better rehabilitative approaches and also may be important when nerve repair strategies reach clinical applications. Further, maintaining higher bone mineral density after human SCI would reduce the incidence of fracture.

Intrinsic dynamics of the vertebrate locomotor pattern generator: A computational study
Allen Garfinkel, Ph.D.
University of California, Los Angeles

Based on our preliminary results, the network responsible for vertebrate locomotion requires only three components: a simple excitatory network where activity of one neuron leads to activity of its neighbors, a simple inhibitory network where activity of neurons on the left side of the spinal cord decreases activity on the right side of the spinal cord and vice versa, and a mechanism to initiate each step at the region of the network closest to the head. The first two components have been experimentally verified. In this project, we will develop a computational model that accurately depicts the anatomy and physiology of the spinal cord network responsible for locomotion. We will scrutinize this model by comparing its behavior to the behavior of a variety of experimental models of locomotion.

Does mental practice of foot movement improve corticospinal conduction and motor status after spinal cord injury?
Steven Cramer, M.D. and Michael Lacourse, M.D.
University of California, Irvine

Spinal cord injury remains a major source of disability. In most patients, there are some residual connections between brain and spinal cord, even if no voluntary movement can be performed. Several researchers have suggested that patients might be able to improve function by strengthening surviving connections that run from brain motor areas through the region of spinal cord injury. This suggestion is also likely to be useful for future therapies that aim to reconnect the two ends of the spinal cord injury, as the brain normally drives--and listens closely to--all movements of the legs. However, there has been limited study in human patients of interventions that might strengthen surviving connections.

In previous studies, methods to activate the motor cortex with imagined movements have been refined. Though studies to date have been in healthy subjects, a brief session focused solely on imagining hand movement activates motor cortex, and improves hand motor function. Here we will adapt this approach to imagining foot movements. We expect that this will activate the brain area related to foot movement in patients with a weak or paralyzed foot after spinal cord injury, as all patients who will be in the proposed study will have a normal brain (as is usually the case after spinal cord injury). The brain remains a great resource in this population, and the spinal cord may often have useful, albeit extremely thinned, connections to drive leg muscles. We will administer the imaginary leg movement therapy to patients with total, and to patients with moderate-severe, leg weakness after spinal cord injury. The purpose of the proposed study is to see whether such an intervention improves the flow of electrical signals, and improves foot movements, in such patients.

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Spinal Cord Injury Models

Before taking some treatments into humans, it will be necessary to test them in other animal models besides rats or mice. This may not be required in all cases, but we need to have a good way of testing future treatments in a primate model.

UC Consortium to study axonal plasticity and regeneration in the primate spinal cord
Mark Tuszynski, M.D., Ph.D.
University of California, San Diego

This is a collaborative project among three UC campuses (UC San Diego, UCLA, and UC Davis) that aims to enhance our understanding of mechanisms of axonal plasticity and regeneration in the primate spinal cord, and to test potential therapies that may be candidates for clinical translation.

Progress in the field of SCI research has been substantial in the last decade, and several experimental manipulations have been reported to enhance functional recovery after SCI in animal models. Among recently reported advances are growth factor gene delivery to stimulate axonal growth, use-dependent plasticity to activate pattern generators in the spinal cord, stem cells, and neutralization of inhibitors to regeneration. The relevance of these findings from rodent models to the larger human spinal cord remains unclear. This research program examines injury, plasticity and regeneration in the adult primate spinal cord so that we can initiate a rational movement of potential therapies, and synergistic therapies, to humans. Thus, work of this consortium will both focus on enhancing our mechanistic understanding of injury to the spinal cord, and aim to develop practical and validated therapies that could undergo human testing in the future.

In the last year of this project we have developed a C5-6 hemisection model of injury that results in quantifiable and consistent anatomical disturbance, and functional deficits in hand use and, to a lesser extent, locomotion. We have now begun studies of autologous primary cell grafting to the lesion site combined with growth factor gene delivery into and beyond the lesion site, in an attempt to determine whether outcomes can be improved on anatomical and functional levels. In the coming year we also expect to begin studies of use-dependent plasticity in combination with the above.