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Research Abstracts

The past 15 years have seen incredible progress in spinal cord injury (SCI) research. The Roman Reed Spinal Cord Injury Research Fund of California has helped move SCI research forward with small "seed" grants. These are funds to take cutting edge ideas and turn them into pilot experiments, the work that demonstrates the idea is a viable one. Researchers have then been able to take the information from these beginning experiments and turn them into large-scale research projects. Roman Reed funds provide the support to get the ball rolling. What you read about below is new and potential the future.

Secondary Degeneration and Neural Protection

A spinal cord injury unleashes a biological tempest that continues to damage and destroy cells for long after the initial insult. This so-called secondary degeneration is fueled by an invasion of inflammatory cells, rising pressure in the spinal cord, dying cells spewing toxins, and healthy cells that self-destruct when something is awry. Seeking to prevent this secondary damage and protect the nervous system from additional damage immediately after SCI is a key area of research.

Motor neurons ROS production and disruption of astrocytic glutamate transport , possible roles in injury progression in the subacute phase after spinal cord injury.
John H. Weiss, MD, 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 an excitatory molecule, called glutamate, from damaged nerve cells. Indeed, spinal trauma is known to cause massive glutamate release, and drugs that block the effects of glutamate do cause a small 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. A vicious cycle develops where glutamate exposure causes the motor neurons to make a toxic substance, reactive oxygen species, which damages the neural support cells that normally remove glutamate from outside the neuron, which results in more glutamate and more reactive oxygen species and more damage. The ultimate results is extensive tissue damage and motor neuron death. We believe that 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, at decreasing the damage and improving recovery.

Is Post-Injury Administration of Estrogen to Male Rats Protective?
Candace L. Floyd, Ph.D.
University of California, Davis

The goal of this research project is to determine if estrogen, given 30 minutes after spinal cord injury (SCI), improves function in male rats. We have recently found that giving estrogen before a SCI to female rats confers significant recovery of function and decreases cell death in the spinal cord. We also have preliminary data that suggests that giving estrogen after injury is protective in female rats. We are now evaluating whether estrogen treatment is beneficial in males.

We plan to give either a low or high dose of estrogen 30 minutes after SCI, and then test estrogen levels in the blood of male rats following treatment. We will also examine the effect of estrogen on post injury cell death, white matter sparing, hind-limb locomotor function, hind-limb pain sensitivity, and bladder function. Interaction of endogenous testosterone and estrogen will be evaluated. After completion of one year of research, we will know whether giving estrogen to male rats after SCI improves outcome. Importantly, we will relate the estrogen blood level to beneficial effects. These data are an important step in evaluating the therapeutic potential of an already FDA approved compound.

Inhibition and Regeneration

In humans, during development, there is massive neuronal growth. Once functional connections are made, this growth is shut off by a variety of inhibitory mechanisms that prevent the nervous system from growing out of control. The problem is, after a spinal cord injury, these inhibitors stop nerve cells from regrowing. The stop signs that are so important in normal development become a major roadblock to regeneration. Over the past decade or so, several of these stop signs have been identified and research programs have been set up to find ways to remove them, go around them, and otherwise change the cellular environment to allow for regeneration.

Myelin inhibitors in CNS axon regeneration: Functional redundancy and an initial application of a genetic tracer
Binhai Zheng, Ph.D.
University of California, San Diego

The lack of any significant axon regeneration after spinal cord injury has been at least partly attributed to the presence of axon growth inhibitors, molecular stop signs, in the central nervous system (brain and spinal cord). Several of these stop sign molecules are found in myelin, the insulation that wraps around axons and allows for electrical messages to be sent. At least 3 of these inhibitors, Nogo, NgR and MAG, interact with the same receptor. Think of the inhibitors as keys and the receptor as the lock. The question is, if we remove all 3 inhibitors, can we improve regeneration. This information will be very important for future treatments, that is designing therapies that target the keys and/or the lock. To remove the inhibitors, mice will be genetically engineered to not have any of the keys. The lock will be there, but the keys will be missing.

This study will also evaluate a new tool to allow us to actually see neurons that regenerate. A yellow fluorescent protein will be put into the genes of mice and will make parts of their spinal cord glow yellow. In this way we can follow neurons that are injured and regrow. Current methods for looking at neurons have flaws, and this new technique has the potential to serve as a powerful tool to help expedite target validation and drug discovery in spinal cord regeneration and repair.

Structure of Proteins that Inhibit CNS Repair: Nogo and It's Receptor
Melanie Cocco, Ph.D.
University of California at Irvine

Although nerves in the peripheral nervous system will regrow after damage, nerves in the central nervous system (CNS) do not. Understanding why the CNS does not repair itself as the peripheral nervous system does may lead to a treatment for spinal cord injury. Recently proteins that block CNS nerve repair have been discovered. One of these, called Nogo, is a small protein found in myelin (the insulating sheath surrounding the axon). There is some evidence that inhibiting Nogo results 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, NMR can participate in the development of therapeutic agents designed specifically to bind Nogo as part of a treatment to enable cellular repair.

Neutralization and conversion of netrin-1 signaling from repulsion to attraction to promote axon regeneration after spinal cord injury
Mark Tuszynski, M.D., Ph.D.
University of California, San Diego

Oligodendrocytes, the cell responsible for making the insulation, or myelin, around axons, give off various molecular stop signs, called myelin inhibitory proteins such as Nogo and MAG, in the adult spinal cord. These proteins have been held responsible for the lack of regeneration after spinal cord injury. We have identified netrin-1 as another oligodendrocyte expressed inhibitor.

The role of netrin changes during development. Netrin is an inhibitor in the adult central nervous system (CNS), but promotes axon outgrowth during development of the spinal cord. This changes has to do with the receptors, or locks, in which the netrin key fits. In adults, the netrin key fits with the repulsion mediating receptor Unc5. During development, netrin fits with a different receptor subtype, DCC.

We propose 2 strategies to over-come netrin-1 mediated inhibition in the adult spinal cord: 1) Neutralization of netrin-1 by quenching with a soluble extracellular Unc5 receptor, so that netrin-1 can no longer bind to endogenous Unc5 receptors expressed on the axon of CNS neurons and does therefore no longer induce repulsive signaling. 2) Recapitulation of the developmental growth state in adult CNS neurons by over-expression of a modified version of DCC in adult neurons. This latter strategy not only neutralizes inhibition but also makes netrin-1 an attractant that should promote axon outgrowth on formerly inhibitory myelin.

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.

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

Astrocyte scar tissue after spinal cord injury is thought to be a major impediment to nerve fiber regrowth, and thus be a major contributor to poor functional outcome after spinal cord injury (SCI). The long-term goal of this project is to improve axon regeneration and recovery of function after SCI by safely decreasing astrocyte scar formation and by providing a synthetic biomaterial tissue matrix that supports and stimulates axon regeneration. The aims of this ongoing project are to (i) use transgenic (genetically modified) mice to identify and characterize both positive and negative roles of scar forming astrocytes after SCI; (ii) test the effects on axon regeneration of either removing or reducing reactive astrocytes after SCI in transgenic mice; (iii) test the ability of degradable synthetic biomaterials such as amphiphile hydrogels and biopolymer microspheres and microfibers loaded with growth-promoting molecules to support and stimulate axon regeneration; (iv) examine the effects on axon regeneration of combining biomaterial implants and genetic decrease of reactive astrocytes after acute and chronic SCI.

Our findings thus far show modest, but significant increases in axon regrowth into areas where scar-forming astrocytes have been killed by transgenic manipulation. However, we also found that this astrocyte removal leads to pronounced local tissue disruption, death of local nerve cells and loss of motor function, indicating that astrocytes perform beneficial as well as negative roles after SCI. One of our current goals is to use transgenic mice to identify different molecular signaling pathways involved in triggering different aspects of astrocyte reactivity. We have already developed and are studying new transgenic models to do so.

In addition, we are developing degradable biomaterials in the form of viscous gels, microspheres and microfibers to support and stimulate axon growth. The rational behind this approach is that many studies have shown that when nerve fibers are presented with two environments in tissue culture they chose to grow into an environment with attractive molecular cues. Biomaterial implants represent a means of presenting axons with an attractive growth matrix that may enable axon regeneration without the need to inhibit endogenous processes that may be providing essential tissue protection after SCI. Our work thus far indicates that microspheres loaded with laminin, a protein that supports axon growth in tissue culture, can induce nerve fibers to grow into the inhibitory environment of CNS white matter. In addition, we have begun to develop biopolymer microfibers to support directed axon growth. These approaches may be useful after both acute and chronic spinal cord injury. Over the past year we have been working on implanting these microspheres and filaments in vivo in mice with SCI and have learned that delivery of sufficient amounts of spheres requires an appropriate suspension medium. We found such a medium in the form of an amphiphile hydrogel generated in the UCLA department of biomedical engineering that works well in vitro (in a dish) and are currently conducting in vivo (in the animal) tests.

There are few approaches that currently investigate the potential of achieving axon regeneration at long times after chronic spinal cord injury. Implantable biomaterials have strong theoretical potential for providing an extracellular growth matrix and delivering growth factors after chronic SCI. Our current goal is to test the ability of biomaterial implants to deliver different growth promoting molecules that may support axon growth after both acute and chronic SCI. Biopolymers of the type we are testing are already in use for drug delivery outside of the CNS. If these materials can be adapted to use in the CNS, they have the potential to lead to treatment strategies for humans.

Improving Motor Recovery

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.

Possible Role for BDNF in the Locomotor Recovery of Spinal Rats Following Robotic-Assisted Treadmill Training
Ray de Leon, PhD
California State University, Los Angeles

Brain derived neurotrophic factor (BDNF) has been shown to be effective in enhancing connections between neurons in the central nervous system. There has been much interest in developing treatments that deliver BDNF in the hopes of improving connections in the injured spinal cord. Recent evidence suggests that treadmill exercise stimulates specific cells in the spinal cord, i.e. motor neurons, to produce BDNF. It is possible, therefore, that motor neurons could serve as an important source of endogenous BDNF to help restore local connections after a spinal cord injury.

We propose to study the benefits of BDNF that is naturally produced by motor neurons. We will use a robotic device to train spinal cord injured rats to step on a treadmill. As a first step toward understanding the role of BDNF, we will examine the extent that motor neuronal synthesis of BDNF is dependent on hindlimb activity levels. If our studies are successful, the findings could lead to new activity-based strategies that unlike transplantation or exogenous delivery, enhance BDNF levels from within the spinal circuitry. These studies could also aid in the development of robotic devices for gait rehabilitation.

Mapping the spatial-temporal pattern of neuronal activity in an injured spinal cord
Jack Judy, Ph.D. and V. Reggie Edgerton, Ph.D.
University of California, Los Angeles

The spinal cord contains groups of motor neuron, called motor neuron pools, that are connected by cells called interneurons, which are intrinsically capable of coordinating muscle group activation for locomotion. Microelectrodes are implanted into locomotor regions of the spinal cord to record simultaneous electrical activity produced by the interneurons involved in rhythm generation for locomotion. Electrodes are placed in the muscles that move the ankle to collect electromyographic recordings (EMG). The correlated neuronal electrical activity in the spinal cord and the EMG activity of the ankle muscles will show which neurons at a particular site in the spinal cord are active during each phase of a step. In our previous work, we have created these spatiotemporal maps of intact spinal cord neural activity during stepping towards the identification of target for intraspinal stimulation therapies. However, previous studies have demonstrated considerable reorganization of the neurons that generate locomotion following an injury. For that reason we would like to look at how activity in L1 and L2 change immediately following a spinal cord injury and after a partial recovery. Spinal cord maps will be produced one hour post spinal cord lesion and six to eight weeks post lesion and compared to those maps that were acquired from the intact spinal cord preparations. Identification of these changes will provide important targets for rehabilitative efforts that utilize regeneration and pharmacological approaches to enhance posture and locomotor recovery.

Models and Techniques

In order to find treatments for spinal cord injury, researchers need good tools. These tools allow us to ask questions like did the treatment result in better functional recovery, is a specific molecule responsible for blocking regeneration, and which cells survived after transplantation. Tools include the correct animal models. Rats and mice are excellent animal models for spinal cord injury research. Rats have much the same physical response to injury that humans do, and we know a great deal about mouse genetics and so are able to manipulate their genes to ask all manor of questions. Of course there are major differences between rodents and humans, not the least of which is size, so models using animals closer in size and structure to humans will be needed before going to clinical trial with some therapies. In addition to models, research tools also include techniques that allow us to ask new questions, examine problems in a new way, or get information that was previously inaccessible. Development of such tools is an essential part of spinal cord injury research and will play a critical roll in all treatments.

Novel Microfluidic Technologies for Sorting and Differentiating Neural Stem Cells
Edwin Monuki, MD, PhD
University of California, Irvine

Stem cells hold great promise for treating spinal cord injury (SCI) and other disorders. To help keep this promise, however, the methods used to culture stem cells must be improved. Current methods do not control how stem cells change in culture with enough precision, and once they change, the sorting tools needed to isolate the right cells are limited. Microfluidics is an emerging technology with clear-cut advantages over traditional culture and sorting tools, which should help us overcome these issues surrounding stem cells. To culture more precisely, we designed a microfluidic device that bathes stem cells in a culture environment that is tightly and continuously controlled. In this device, we find that the behaviors of neural stem cells (NSCs) from mice and humans are quite predictable. To sort different cell types, we are developing a new microfluidic method that detects differences in the electrical properties that cells have. Recent tests of this method have been highly encouraging.

In this proposal, our collaborative team of bioengineers and biologists will use microfluidics to produce a cell type damaged by SCI, the cortical neurons that project into the spinal cord and control movement. In microfluidic cultures, we will test a molecule known as BMP4, which regulates cortical neuron production in the developing brain. We will also test cells that should glow when they become cortical neurons, which will expedite and empower the microfluidic work even further. These studies should also reveal how to produce NSCs that are predisposed to generate cortical neurons, which would be useful to test in transplants along with the neurons themselves. These projects should bring us closer to producing the cell types we want with precision and reproducibility, which should ultimately lead to better and more reliable therapies using stem cells.

Microfluidic Platform for High Throughput Screening of Agents for Spinal Cord Axonal Regeneration
Noo Li Jeon, Ph.D.
University of California, Irvine

This research further develops and tests a micro-scale neuronal culture device that can be used to perform reproducible axotomy, cutting of axons, such that the effects of various growth factors and drugs can be quantitatively characterized and compared with each other. We use state-of-the-art engineering approaches to direct the attachment of neurons on defined positions in the culture device and isolate axons from the cell body to perform reproducible axotomy. The device is compatible with optical microscopy methods such that the rate of axon elongation, degree of branching, and growth cone morphology can be followed by time-lapse microscopy and immunocytochemistry. If successful, this project should not only advance our understanding of different growth factors' influence on axonal regeneration- and of growth factor gradients on regeneration- but should also provide a versatile culture platform with a number of potential basic and clinical applications.

University of California Consortium to Study Axonal Plasticity and Regeneration in the Primate Spinal Cord
Mark H. Tuszynski, M.D., Ph.D., Reggie Edgerton, Ph.D., Leif Havton, M.D., Ph.D., Os Steward, Ph.D., Nick Lerche, D.V.M. UC San Diego, UC Los Angeles, UC Irvine, UC Davis

This is a collaborative project among four UC campuses (UC San Diego, UCLA, UC Irvine 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. However, the relevance of findings from rodent models to the larger human spinal cord remains unclear. There are distinct differences between rodent and primate spinal cord systems, including: 1) Size. Regeneration of an injured axon in the rat over several millimeters might be sufficient to generate functional recovery, whereas distances of many centimeters might be required in the primate spinal cord. 2) Anatomy. There are differences in the number, location and termination patterns of important axonal systems such as the corticospinal tract in primates compared to rodents. 3) Function. Some important axon systems have different functional roles in primates and rodents. 4) Inflammatory responses and secondary injury. Although not well characterized, the intensity and nature of inflammation and secondary damage may differ in rodent and primates species due to differences in immune complexity and molecular (e.g., cytokine) recruitment to injury sites.

The goal of this research program is to examine 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. The work of this consortium focuses on both enhancing our mechanistic understanding of injury to the primate spinal cord, and on developing practical and validated therapies that could undergo human testing in the future.

Myelin inhibitors in CNS axon regeneration: functional redundancy and an initial application of a genetic tracer
Binhai Zheng, Ph.D.
University of California, San Diego

See Inhibition and Regeneration.

Autonomic Function

Paralysis is the most obvious result of SCI, however according to a recent survey, people with SCI reported that return of bladder / bowel and sexual function would most improve quality of life. These are autonomic functions, functions over which we have little conscious control. A major autonomic dysfunction after spinal cord injury involves the development of pain. For largely unknown reasons, some people develop debilitating pain below the level of their injury even though they may have no ability to feel touch, hot or cold, or a pin prick. Clearly, there is an urgent need for better understanding and management of autonomic functions.

Effect of Spinal Cord Transection Injury on the Synaptology of Autonomic and Motor Neurons Innervating the Lower Urinary Tract in Rats
Leif A. Havton, M.D., Ph.D.
University of California, Los Angeles

Spinal cord injuries cause significant neurological disability with paralysis, sensory loss, pain as well as loss of bladder, bowel, and sexual functions. The bladder symptoms include incomplete emptying of the bladder with need for bladder catheterizations to avoid bladder infections and other complications.

In this study, we will use anatomical labeling techniques to mark spinal cord nerve cells, which control bladder and urethral sphincter contractions. For this purpose, spinal cord tissues will be studied in the electron microscope to determine what type of nerve connections are made with the bladder and sphincter nerve cells after a spinal cord injury. We will also use an antibody technique to determine which transmitter chemicals are present in the contacts made with the labeled nerve cells. We believe that there will be detectable signs of significant re-organization taking place in the spinal cord, both in the acute and chronic state after a spinal cord injury.

Findings from the proposed studies may assist in identifying mechanisms underlying the abnormal bladder reflexes, which emerge after spinal cord injuries. A better understanding of these mechanisms may, in turn, assist in the development of novel treatment strategies for bladder control after a spinal cord injury.

The Contribution of Ca_vα₂δ₁ Protein to Spinal Cord Injury Pain
Zhigang David Luo, Ph.D., M.D.
University of California, Irvine

Chronic pain in SCI patients is difficult to manage, and decreases the quality of the daily-lives of SCI patients dramatically. Current medications can only provide partial pain relief and are often associated with intolerable side effects. The development of target specific, and safer pharmacological agents depends on identification of new targets that mediate the development of chronic SCI pain. This proposal is to define the contributory role of a novel protein, the calcium channel alpha-2-delta-1 subunit, in the development of chronic SCI pain.

This protein seems to be involved in pain development following injury outside of the central nervous system. There is a tight correlation between increases in this protein and development of neuropathic pain after peripheral nerve injury. We suggest that SCI may alter the expression of this protein in the cells of the spinal cord that mediate the development of chronic SCI pain. To test this hypothesis, we proposed to examine whether changes in the spinal cord levels of this protein correlate temporally with the development of chronic SCI pain, and if so, in what cell types these changes occur. Finally, we plan to examine whether reversing the elevated alpha-2-delta-1 protein after SCI with a drug can provide analgesic effects in SCI rats with chronic pain. Findings from these studies will provide us with critical information for the development of the next generation of analgesic agents for better chronic pain management in SCI patients.