Roman Reed Research Grants 2001-2002

Armin Blesh, Ph.D. University of California, San Diego	GDNF and BDNF gene therapy after complete spinal cord transection	$80,000
Michael D. Cahalan, Ph.D. University of California, Irvine	Tracking and targeting lymphocytes in spinal cord injury **	$87,329
Nathalie A. Compagnone, Ph.D. University of California, San Francisco	Can neurosteroids restore bladder function after spinal cord injury?	$79,440
Corinna Darian-Smith, Ph.D. Stanford University	Cervical dorsal root lesions in monkeys; neuronal consequences and impairment of voluntary hand function	$76,123
V. Reggie Edgerton, Ph.D. University of California, Los Angeles	Robotic assisted assessment of locomotor physiology after spinal cord injury in transgenic mice	$120,000
David M. Gardiner, Ph.D. University of California, Irvine	Urodele spinal cord regeneration as a model for axonal survival and regrowth	$63,804
Leif A. Havton, M.D., Ph.D. University of California, Los Angeles	Use-dependent plasticity of spinal motorneuron synaptology	$60,000
James G. Hecker, M.D., Ph.D. University of California, Davis	Prevention of secondary injury via non-viral intrathecal delivery of neuroprotective and apoptosis inhibitory genes **	$52,500
Marc Hedrick, M.D. University of California, Los Angeles	Adipose-derived stem cells **	$80,000
Jack W. Judy, Ph.D. University of California, Los Angeles	Spatial and temporal studies of activation in the lumbosacral spinal cord using implantable multimicroelectrode arrays	$105,011
Hans S. Keirstead, Ph.D. University of California, Irvine	Role of T cells in secondary degeneration following contusion injury to the adult spinal Cord **	$75,000
David J. Reinkensmeyer, Ph.D. University of California, Irvine	Robotic outcome assessment in spinal cord injury and regeneration **	$61,293
Michael Sofroniew, Ph.D. University of California, Los Angeles	Genetically targeted astrocyte scar ablation and biopolymer tissue support after spinal cord injury	$158,226
Marylou Solbrig, M.D. University of California, Irvine	Gene therapy of spinal cord injury - adenoviral vector delivery of macrophage/microglia stimulating factors **	$78,480
Lawrence Steinman, Ph.D. Stanford University	Myelin tolerizing DNA vaccination in the treatment of spinal cord injury **	$125,000
Mark Tuszynski, M.D., Ph.D. Shula Stokols, B.S. University of California, San Diego	Synthetic polymer guidance channels for spinal cord injury	$35,000
Marc Tessier-Lavigne, Ph.D. Stanford University	Identification of regeneration-associated genes and their roles in stimulating and inhibiting axonal regeneration	$168,946
Mark H. Tuszynski, M.D., Ph.D. University of California, San Diego	Rolipram for spinal cord injury	$125,000
Mark H. Tuszynski, M.D., Ph.D. University of California, San Diego	University of California consortium to study axonal plasticity and regeneration in the primate spinal cord	$191,500
Jeffery Twiss, MD, Ph.D. University of California, Los Angeles	Mechanisms of activity-dependent conditioning for rapid axon regeneration	$96,694
Richard Vulliet, Ph.D., DVM University of California, Davis	Treatment of spinal cord injury with mesenchymal stromal cells **	$77,000
James A. Waschek, Ph.D.	The role of PACAP in remyelination after experimental spinal cord injury **	$100,000
** Project will be carried out in the Roman Reed Core Laboratory
TOTAL		$2,096,346

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Research Abstracts

A spinal cord injury is one of the most devastating traumas the body can sustain, and for centuries it was considered utterly untreatable. In the last 20 years, however, researchers have made remarkable progress toward understanding the havoc that a spinal cord injury wrecks and identifying potential ways to halt and reverse the damage. Nevertheless, it is unlikely that any one drug or surgical advance will ever cure a spinal cord injury. Instead, victims probably will need a precisely timed series of medications and procedures, starting in the hours after the injury and continuing through rehabilitation -- or longer. Assembling the parts of this treatment package requires a multi-disciplinary effort. Toward that end, the California Spinal Cord Injury Research Fund has awarded 22 Roman Reed grants to researchers from a wide range of specialties. What follows are brief descriptions of some major areas of spinal cord research and the Roman Reed projects in each one.

Preventing Secondary Damage
Promoting Axon Regeneration
Remyelinating Axons
Replacing Cells
Implanting Artificial Substrates
Retraining and Rewiring the Spinal Cord

Preventing Secondary Damage

A spinal cord injury unleashes a biological tempest that continues to damage and destroy cells for weeks and perhaps months. This so-called secondary cascade 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 they "sense" that something is awry. The California Spinal Cord Injury Fund has awarded Roman Reed grants to the following scientists who are testing ways to prevent or limit the terrible wake of a spinal cord injury:

Tracking and targeting lymphocytes in spinal cord injury
Michael D. Cahalan, Ph.D., and K. George Chandy, M.D., Ph.D.
University of California, Irvine

The Cahalan and Chandy laboratories have pursued a joint research program that focuses on the role of ion channels in immune system function. Ion channels are the gatekeepers of a cell. They permit molecules that are critical for cellular well being and intracellular communication like sodium, calcium, and potassium to pass in and out of cells. Following a spinal cord injury, ion channels malfunction, so the normal concentration of these molecules is disrupted, leading to a widening circle of cell death. If doctors could regulate ion channels, then they could halt the secondary damage that follows an injury.

Drs. Cahalan and Chandy are particularly interested in a group of important immune cells called T lymphocytes, or T cells. In a remarkably successful recent trial, the researchers used a toxin derived from sea anemones to block potassium channels in T cells, reducing paralysis and death in rat models of multiple sclerosis. They believe that this approach also may work for spinal cord injuries and will test this hypothesis in rat models to see if the animals recover function. The scientists also will track T cells within the spinal cord following an injury using two-photon laser microscopy, a powerful new imaging device that enables scientists to observe living cells deep within tissue. If their hypothesis is correct, then blocking potassium channels holds promise as a way to curb the damaging activity of the immune system following a spinal cord injury.

Non-viral Gene Transfer to the Injured Spinal Cord
Leon Hall, Ph.D.
University of California, Davis

Genes contain the instructions for producing the proteins that comprise the structure of the body's cells and determine their functions. Gene therapy is a new technique that one day may help to treat diseases, inherited disorders, and injuries. The approach involves sending therapeutic genes into a patient's cells where, the theory goes, they will either start the production of beneficial proteins or stop the production of harmful ones. Scientists are still testing ways to deliver the desired genes. Some scientists are packaging them in deactivated viruses, but Dr. Hall prefers a lipid molecule, the same fatty substance that forms the membrane of a cell. This lipid capsule is easily incorporated into the body of a target cell, where it dumps its genetic payload. Dr. Hall believes this approach eliminates the risks of using viruses, including an immune response or the inappropriate incorporation of genes.

In this project, Dr. Hall is using rats to test genes that may prevent cell death following a spinal cord injury. He will inject the lipid packages into the cerebrospinal fluid surrounding the spinal cord and then will evaluate how well the genes functioned. Dr. Hall hopes this approach, which mobilizes some of the body's own protective mechanisms, will lead to therapies to minimize secondary damage following a spinal injury. If successful, this project could also pave the way for short-acting gene medicines that doctors would use for other medical problems, such as protecting the spinal cord before certain types of high-risk surgery.

Role of T cells in secondary degeneration following contusion injury to the adult spinal cord
Hans S. Keirstead, Ph.D.
University of California, Irvine

Following a spinal cord injury, immune cells flock to the site causing inflammation and other problems. The reasons for this influx are still poorly understood. Dr. Keirstead has found that a rising concentration of molecules that attract immune cells precedes their arrival. In animal studies, he has prevented the immune cell invasion by injecting an antibody to one of the attractants, a substance known as IP-10. The treatment greatly reduced the second wave of cell damage that normally follows a spinal cord injury and appeared to limit the loss of function. In this study, Dr. Keirstead is testing the antibody treatment on different types of spinal cord injuries and hopes to elucidate further how the immune system contributes to the destructive aftermath of a spinal cord injury.

Myelin tolerizing DNA vaccination: A Strategy To Reduce Post-Traumatic Demyelination in animal models of spinal cord injury
Paulo Fontoura, M.D. and Lawrence Steinman, M.D.
Stanford University

A spinal cord injury causes axons near the lesion and beyond to lose their myelin sheath, a fatty layer that insulates and protects them. This demyelination robs axons of their ability to transmit nerve impulses. Thought to be an autoimmune response, this process resembles what happens in multiple sclerosis when the immune system attacks myelin in the brain and spinal cord. The Steinman laboratory has had encouraging results using DNA vaccines to halt the destruction of myelin in animal models of multiple sclerosis. A new approach to autoimmune disease prevention and treatment, DNA vaccinations involve inoculating animals or humans with the DNA for one protein of a disease-causing organism. The body then produces this harmless protein, provoking a long-lasting immune response to the disease. In this study Dr. Steinman's team is applying the same DNA technology to animal models of spinal cord injury. If successful, this study could lead to new treatments designed to minimize the loss of myelin and, therefore, preserve function in people who suffer spinal cord injuries.

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Promoting Axon Regeneration

Unlike nerve cells in the body's extremities, neurons in the brain and spinal cord cannot regrow their axons, the snaky extensions of the cell body that transmit electrical impulses from one neuron to another. Scientists have recently identified naturally occurring substances that become active following an injury and turn the area around it into hostile territory for axon regeneration. The body also manufactures growth boosters known as neurotrophins, or growth factors, that spur neurons to replace their lost axons and help those new axons to flourish. Treating spinal cord injuries may require a two-step approach to reduce or neutralize growth inhibitors and amplify the action of neurotrophins. The following scientists have received Roman Reed grants to focus on how axons can be helped to regenerate:

Gene therapy of spinal cord injury- adenoviral vector delivery of macrophage/microglia stimulating factors
Marylou Solbrig M.D.
University of California, Irvine

Dr. Solbrig is interested in how two immune system scavenger cells, macrophages and microglia, set the stage for repairing a spinal cord injury. As the first responders of the immune system, macrophages quickly surround and engulf invading microorganisms or release toxins to poison them. Following a spinal cord injury, macrophages improve the conditions for spinal cord regeneration by mopping up cellular debris and secreting helpful substances, including neurotrophins and interleukins, which are signaling molecules that suppress inflammation. Microglia are tiny versions of macrophages in the brain and spinal cord. After a spinal cord injury, both macrophages and microglia protect surviving cells and promote regeneration, but precisely how they accomplish these important tasks is unclear.

In this study, Drs. Solbring first wants to learn more about how these scavenger cells behave following a spinal cord injury and then hopes to maximize their beneficial effects by trying to increase their number and activity. Using rat models, she will transplant into a spinal cord lesion viruses that have been genetically engineered to deliver cytokines, a family of growth factors that stimulate the immune response. The goal is to activate existing microglia and to attract new macrophages and activate them. At two and four weeks after injury, the researchers will evaluate changes in the animals' spinal cords and assess their function. These experiments may show that using gene therapy to manipulate the immune system can protect surviving neurons and create a fertile environment for spinal cord repair.

GDNF and BDNF Gene Therapy after Complete Spinal Cord Transection
Armin Blesch, Ph.D.
University of California, San Diego

Genetic engineering turns cells into microscopic pharmacies that deliver therapeutic substances directly to the site of a spinal cord injury. In this study, Dr. Blesch is modifying skin cells or fibroblasts so they produce several types of growth factors that have promoted axon regeneration in earlier experiments on rats with incomplete spinal cord injuries. Transplanted cells also provide a scaffolding, or substrate, to support new axons as they grow.

Dr. Blesch will test whether using this form of gene therapy to deliver two neurotrophins, GDNF and BDNF, will promote axon regeneration and restore function in rats with complete spinal cord injuries. Control animals will receive cells that produce only an inert green fluorescent marker protein. At one and three months after the cells are transplanted into the rats, Dr. Blesch will examine the animals to see if neurons have grown new axons that extend into or through the transplanted cells. He will look for changes especially in the reticulospinal and rubrospinal pathways, which are critical to the recovery of walking and limb use. He will also see if the experimental animals regain more limb function than do the controls. If successful, this approach could be part of a series of treatments to rebuild the damaged spinal cord and reverse paralysis.

Urodele spinal cord regeneration as a model for axonal survival and regrowth
David Gardiner, Ph.D.
University of California, Irvine

Adult salamanders, or urodele amphibians, are unique among vertebrates because they can regenerate body parts, including their spinal cord. Although little is known about the molecular mechanisms that control spinal cord regeneration in these remarkable animals, Dr. Gardiner and his colleagues are applying to the spinal cord the techniques they developed to study the salamander's ability to replace lost limbs. Dr. Gardiner's research tools include microarray analysis, which can determine the activity of hundreds of important genes simultaneously, and genetic engineering, which enables him to test the function of any genes that appear, from micro array analysis, to be important in spinal cord regeneration.

In the second phase of this project, Dr. Gardiner will compare the genetic findings on salamanders to the newly available genetic data on the mouse, which cannot regrow its spinal cord. Spinal cord researchers who work with other mammals may then compare the genetic profiles of those animals with the results from this project. Such comparisons could produce a blueprint for new forms of gene therapy to enhance spinal cord regeneration in humans.

Genetically targeted astrocyte scar ablation and biopolymer tissue support after spinal cord injury
Michael V. Sofroniew, M.D.,Ph.D., V. Reggie Edgerton, PhD., and Ben Wu, Ph.D.
University of California, Los Angeles

Using a mouse model, these researchers are testing a two-step approach to repairing the injured spinal cord. They plan to prevent the formation of scar tissue, which blocks new axons from rebuilding damaged nerve circuits, and then to implant biodegradable "scaffolding" that will both promote and support the growth of those replacement axons.

Astrocytes are cells that nourish and support neurons, or nerve cells. In response to a spinal cord injury, however, astrocytes become a liability as they proliferate near the lesion and wall it off with scar tissue. The Sofroniew laboratory has developed a novel way to eliminate scar-forming astrocytes after a brain or spinal cord injury in genetically modified mice. Preliminary findings show this technique increases the sprouting of new axons and yields modest regrowth of nerve fibers from the brain across the injury site in the spinal cord.

To maximize the amount of regrowth, Dr. Sofroniew and his colleagues also will implant tiny, porous, biodegradable beads that offer regenerating axons a foothold as they travel across the gap in the injured spinal cord. The beads also are impregnated with growth factors that will gradually be released into the injury site and nurture specific types of axons. Because the beads are suspended in a liquid, Dr. Sofroniew predicts that they will not cause additional tissue damage, which can occur with rigid implants. If successful, this project will improve the understanding of the cell biology of spinal cord injury and could provide new strategies for treatments.

Identification of Regeneration-Associated genes and their roles in stimulating and inhibiting axonal regeneration
Mark Tessier-Lavigne, Ph.D.
Stanford University

A new tool known as microarray analysis enables scientists to analyze thousands of genes from a tissue sample at once to see which genes are active or expressed and which are silent. This technology helps spinal cord researchers to learn, for example, how gene expression changes after a spinal cord injury in tissue near a spinal cord lesion and beyond. In this project, Dr. Tessier-Lavigne is using microarray analysis to study a fascinating mechanism in nerve cells called primary spinal sensory neurons, which have a peripheral branch that extends into, say, a muscle and a central branch that leads to the spinal cord. Like other axons in the body's extremities, the peripheral branch can regenerate after an injury; the central branch, like axons in the brain and spinal cord, cannot. However, scientists have found that, following an injury in the peripheral branch of a sensory neuron, the central branch will regrow across a spinal cord injury. Recently researchers have reproduced this "priming" effect by injecting a signaling molecule called cAMP into the body of a sensory neuron.

Dr. Tessier-Lavigne wants to identify the genes that are expressed and those that go silent in the spinal sensory neurons of rats in two scenarios: following an injury to the peripheral branch and the injection of cAMP in others. Once he pinpoints the genes that seem to encourage axon regeneration and those that appear to block it, he will run additional laboratory tests to characterize the functions of these genes. The next step will be to test the most promising genes in animal models to identify those that might one day be used to treat human spinal cord injuries.

Rolipram for spinal cord injury
Mark H. Tuszynski, M.D., Ph.D., Karin Lowe, Ph.D., and Marie Filbin, Ph.D.
University of California, San Diego

Signaling molecules influence the behavior of a cell by carrying information from its surface to the proteins inside, where enzymes quickly break down the signaling molecules to end their transmission. One important signaling molecule, Cyclic AMP or cAMP controls a host of metabolic processes in cells and has been shown to enhance nerve cell growth in tissue culture. Preliminary studies in animals indicate that drugs that increase the activity of cAMP may promote axon regeneration following a spinal cord injury. Dr. Tuszynski and his colleagues are trying to achieve similar results with an oral drug called rolipram, which blocks the action of the enzymes that destroy cAMP.

In this three-part project using rat models, Dr. Tuszynski will try different treatment protocols and then will examine both the treated animals and the untreated controls after fours weeks to assess whether axons have regrown. He first will give rolipram one week before he creates the spinal cord lesions and then administer the drug for three more weeks. In the second phase, he will start the four-week rolipram therapy eight hours after cervical spinal cord injury. This schedule mirrors what might happen after a human spinal cord injury. Finally, he will test whether he can improve upon the results from phase two by adding a complementary treatment. He will follow the same rolipram schedule as he did in phase two, but he also will transplant cells into the injury site that have been genetically engineered to produce several growth factors. The transplanted cells also will provide a framework to support regenerating axons. Successful results in this study could pave the way for a new first-line treatment for spinal cord injuries.

University of California consortium to study axonal plasticity and regeneration in the primate spinal cord
Mark Tuszynski, M.D., Ph.D.*, Reggie Edgerton, Ph.D.†, Jeff Roberts, D.V.M.††, Leif Havton, M.D., Ph.D.†, Bruce Dobkin, M.D.†, Os Steward, Ph.D.** Corinna Darian-Smith, Ph. D ¥
*UCSD, †UCLA, †† UC-Davis, ** UC-Irvine, ¥ Stanford University
(See Creating Models and Research Tools)

Mechanisms of activity-dependent conditioning for rapid axon regeneration
Jeffery Twiss, M.D., Ph.D. and Fernando Gómez-Pinilla, Ph.D.
University of California, Los Angeles

When disease or injury damages nerve cells in the brain or spinal cord, the cells do not regrow. However, injured nerves in the legs, arms, or other parts of the peripheral nervous system do regenerate, and scientists have been studying the process, hoping to find a mechanism that might be reproduced to repair spinal cord injuries. In fact, scientists have learned that the injured peripheral nervous system can regenerate better than can the uninjured nervous system, and regeneration of a nerve can even be accelerated by a so-called conditioning injury. An initial injury seems to set the stage for repair, and it appears this is true even in the central nervous system. In theory, creating a new injury would spur regeneration in an old injury.

Because it is neither ethical nor practical to create a second injury to heal a first, Dr. Gómez-Pinilla looked for other ways to prime the nervous system for self-repair. To their surprise, they found that exercise also creates favorable conditions for regeneration. The researchers focused on dorsal root ganglia neurons, or DRGs, nerve cells that lie just outside the spinal cord and receive information from both the sensory nerves and the spinal cord. They took DRGs from animals and kept the cells alive in cultures. They discovered that the cells from animals that had exercised were more likely to regenerate than the cells from rats that had not exercised.

This project will determine whether exercise-conditioned neurons also show increased nerve regeneration in living animals, and if exercise-conditioned neurons can defy the growth inhibiting substances that occur naturally in the brain and spinal cord. Finally, the researchers will determine whether exercise after injury can also increase regeneration in the nervous system. If successful, these experiments could point to new ways to promote regeneration after a spinal cord injury.

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Remyelinating Axons

Some axons that survive a spinal cord injury still lose their protective sheath of myelin, the fatty substance that insulates and protects them. Without myelin, the axons stop functioning. How to induce remyelination is one of the challenges in developing treatments. The following researcher has a Roman Reed grant to test an approach to remyelination:

The role of PACAP in remyelination after experimental spinal cord injury
James A. Waschek, Ph.D.
University of California at Los Angeles

Dr. Waschek's project is based on the recent discovery of a neuropeptide called PACAP. Neuropeptides are chemical messengers that enable cells in the nervous system to communicate with one another. PACAP, which also promotes the growth and survival of nerve cells, is active during the development of the brain and spinal cord and becomes active again after a spinal cord injury. The Waschek laboratory recently found that PACAP regulates the production of oligodendrocytes, the cells that produce the fatty protective layer called myelin that enwraps axons in the brain and spinal cord and is critical for the transmission of nerve impulses. A spinal cord injury causes axons near the lesion and beyond to lose myelin, so remyelination is an important step in restoring both nerve circuitry and lost function. If scientists can learn precisely how PACAP works, they might be able both to summon more PACAP to an injury site and to enhance its beneficial effects.

In this two-part project, Dr. Waschek first will use animal models to study the role of PACAP in remyelination following a spinal cord injury. Then he will add PACAP to laboratory dishes containing neural progenitor cells, primitive cells that spin off all the cells in the nervous system, to see if the PACAP will program the cells to become oligodendrocytes. In a future study, Dr. Waschek plans to use a form of gene therapy to increase the amount of PACAP in mice to see if it promotes remyelination after a spinal cord injury. If Dr. Waschek's hypotheses are correct, PACAP could be used to treat injuries as well as diseases, like multiple sclerosis, that attack myelin.

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Replacing Cells

One strategy for repairing the damaged spinal cord and restoring function is to replace the lost neurons and the glial cells that support, protect, and nourish them. Some scientists are working on transplanting primitive stem cells that will give rise to the cells needed to repair the spinal cord. Other researchers are concentrating on how best to pretreat stem cells so that they become nerve cells or glia. Still others believe the body has the potential to repair itself and are focusing on restarting the mechanisms that first created the brain and the spinal cord. The following researchers who are working on cellular replacement have received Roman Reed grants:

Application of adipose-derived stem cells to the repair of spinal cord injury
Marc Hedrick, M.D.*, Hans Keirstead, Ph.D. †, Gregory R. Evans, M.D. †
* University of California, Los Angeles, † University of California, Irvine

Primitive master cells can evolve into neurons and have enormous potential for treating spinal cord injuries and degenerative nervous system disorders, including amyotrophic lateral sclerosis (Lou Gehrig disease) and Parkinson's disease. Both embryonic stem cells and neural progenitor cells extracted from the adult central nervous system have therapeutic potential. However, logistical, ethical, and immunilogic factors limit their practicality. Earlier studies by these researchers suggest that human fat, or adipose tissue, also contains stem cells that spawn neural progenitors. These progenitor cells have several advantages over cells from other sources, including that they can be easily extracted in large numbers from each patient and, when transplanted back, will not provoke an immune response. In this project, the collaborators are investigating how adipose-derived stem cells can be coaxed to differentiate into neurons or glia the cells that nourish and support neurons both in tissue cultures and in animals with spinal cord injuries. A detailed understanding of how these cells differentiate and survive might enable scientists one day to control this process well enough to use these cells to rebuild a damaged spinal cord and restore function.

Rolipram for spinal cord injury
Mark H. Tuszynski, M.D., Ph.D.
University of California, San Diego
(See description under Promoting Axon Regeneration)

Treatment of Spinal Cord injury with mesenchymal STROMAL CELLS
Richard Vulliet, Ph.D., DVM
University of California, Davis

Doctors routinely harvest bone marrow from patients in a relatively simple and painless procedure, and the marrow is a rich source of primitive stem cells. Dr. Vulliet is studying whether these bone marrow stem cells - mesenchymal stromal cells or MSCs - can be transplanted and replace the cells destroyed in spinal cord injury. Other studies have shown that pretreating the bone marrow cells programs them to become either neurons or supporting glial cells. In this project, Dr. Vulliet will investigate whether adult bone marrow stem cells can differentiate into neurons or glial cells in the rat spinal cord, and then evaluate whether these cells promote recovery of function after a spinal cord injury. He predicts that the cells will become glial cells and prevent further damage to neurons following traumatic injury. To test this theory, he plans to inject the stem cells directly into the area of injury and then determine if this approach improves both the survival of neurons and functional recovery. If bone marrow stem cells are beneficial in rat models, Dr. Vulliet's treatment protocol might move to human trials, since bone marrow is readily available from all patients.

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Implanting Artificial Substrates

Some scientists believe that therapies to repair nerve circuits will work better if they are combined with a device that actually spans the gap in the injured spinal cord. Researchers are testing tiny bridges, tunnels, and scaffolding or substrates that would be placed between the two stumps of the injured spinal cord to support and guide regenerating axons as they travel toward their target connections. The following researchers received a Roman Reed grant to explore this approach:

Synthetic polymer guidance channels for spinal cord injury
Mark H. Tuszynski, M.D., Ph.D. and Shula Stokols
University of California, San Diego

A spinal cord injury leaves a breach in the nerve circuitry that connects the brain with the rest of the body. Coaxing new axons to bridge that gap is a major step in rebuilding nerve pathways and restoring function. In experiments with animals, scientists have had success when they implant some tiny structure for axons to grow through or over. In this project, these researchers are using rat models of spinal cord injury to test nerve guidance conduits fabricated from natural biomaterials. The cellular response to the conduit, the extent to which axons growth into the conduit and the linearity of the regenerated axons is evaluated.

This research is encouraging on three levels. First, the conduit provides scaffold-like support and guides the regenerating axons as they travel across the injury toward their target connections. Second, the implant provides a barrier between the spinal cord and the environment around it, minimizing the infiltration of fibrous tissue and thwarting the inflammatory response. Finally, the conduits can be used to provide a source of helpful substances, such as anti-inflammatory agents or growth factors. These researchers hope to develop a practical, convenient, and inexpensive nerve guidance conduit that could be used to restore nerve pathways and lost function in people with spinal cord injuries.

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Retraining and Rewiring the Spinal Cord

Certain forms of rehabilitation appear to do more than maintain bone mass, muscle strength, and cardio-vascular fitness in someone with a spinal cord injury. In fact, recent research has shown that some training protocols including progressive weight bearing and repetitive stepping routines may restore function by promoting axon regeneration and the creation of new neuron-to-neuron connections, or synapses. These routines may also condition the lower spinal cord to take over some brain function and activate the muscles in the legs and feet that are used in walking. With Roman Reed grants, the following researchers will continue to explore this promising approach:

USE-DEPENDENT PLASTICITY OF SPINAL MOTONEURON SYNAPTOLOGY
Leif A. Havton, M.D., Ph.D., and V. Reggie Edgerton, Ph.D.
University of California, Los Angeles

A complete severing of the spinal cord at the mid-chest level leads to paralysis of the legs. This laboratory has shown that animals with these injuries can regain their ability to stand and step through a repetitive training regimen. These results suggest that specific activities and training actually change the connections, or synapses, between nerve cells in the injured spinal cord. How this process works, however, is unknown.

These researchers hypothesize that the training alters the composition of synaptic contacts on motoneurons, the nerve cells that control muscles in the rats' hind limbs. To test this theory, these researchers use a robotic system to train rats after a spinal cord injury. They also use an electron microscope to examine the synapses of the motoneurons that control standing in uninjured animals, in injured but untrained animals, and in injured animals that undergo training to stand. Understanding how the training actually modifies important synapses will help scientists design treatments to enhance or duplicate the benefits of training, first in animals and eventually in humans.

Spatial and Temporal Studies of Activation in the Lumbar Spinal Cord Using Implantable Multimicroelectrode Arrays
Jack W. Judy, Ph.D.
University of California, Los Angeles

A specific group of neurons in the lower spinal cord, called spinal pattern generators, coordinates the movements required in locomotion. The brain sends signals to initiate walking, but it is the spinal pattern generators that actually coordinate lifting the knee, extending the foot, planting the heel, and the other movements involved in taking one step after another. Even after an injury disconnects the brain from the spinal cord below the lesion, spinal pattern generators retain some control over locomotion. This laboratory has shown in animals with training routines that involve repetitive stepping on a treadmill improve pattern generating following a spinal cord injury. Now Dr. Judy and his colleagues seek to learn more about pattern generating in rats with and without spinal cord injuries. These researchers also want to determine whether electrical stimulation might enhance stepping and standing following a spinal cord injury. By implanting an array of tiny electrical probes into the pattern generator circuitry, researchers will be able to stimulate the pattern generators and to record electrical activity in the lower spinal cord while the animals are stepping.

With sophisticated new tools for analyzing the electrical activity of neurons over time and through space, researchers hope to learn precisely how pattern generators activate muscles. They also will look at other factors that may contribute to the control of locomotion, such as signals from the brain and sensory cues from the hind limbs. From all this data, Dr. Judy will determine just how much current to deliver to each electrode. He then will stimulate the injured spinal cord and look for improvement in locomotion from both the current and the treadmill step training. This work might provide the foundation for the development of simple electrical devices to restore locomotion in people with a spinal cord injury.

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Restoring Concomitant Function and Eliminating Complications

The complications and loss of function that accompany spinal cord injuries not only impair quality of life but also can be life threatening. In addition to paralysis, people living with spinal cord injuries can, among other problems, suffer infections, spasticity, irregularities in temperature and blood pressure, and intractable pain. Moreover, spinal cord injuries almost always interfere with bowel, bladder, and sexual function. A Roman-Reed award will support the following researcher who is trying to restore bladder function:

Can neurosteroids restore bladder function after spinal cord injury?
Nathalie A. Compagnone, Ph.D.
University of California, San Francisco

Most spinal cord injuries cause a loss of bladder control. This is one of the most resented problems that plague people living with these injuries, and restoring bladder control would greatly improve their quality of life. When normal voiding cannot occur, the walls of the bladder become distended, which correlates with a change in the composition of the bladder wall. Using a model of moderately bruised spinal cord in the mouse Dr. Compagnone has show that the use of neurosteroids as therapeutic adjuncts shortens the time it takes mice to regain the ability to control their bladders after injury. In this project, she is looking at the effect of this neurosteroid treatment on the composition of the bladder wall. She is comparing bladder walls in animals with spinal cord injuries that received treatment, in untreated animals with spinal cord injuries, and in control animals that under go sham operations on their spinal cords and have normal functioning bladders. In addition, Dr. Compagnone proposes to correlate these measurements to functional assessment of the bladder control in order to devise a quantitative measurement of bladder function, which will help her and other scientists to measure the efficacy of future treatments to restore bladder control.

Using Robotics for Assessment and Training

Neuroscientists and engineers are collaborating to create clever robotic devices that help researchers to make objective, quantitative assessments of the loss of function and its recovery following a spinal cord injury. Robotics also can assist animals and humans during training routines by initiating and controlling limb movements. The following researchers who focus on robotic devices received Roman-Reed awards:

Robot Assisted Assessment of Locomotor Physiology after Spinal Cord Injury in Transgenic Mice
V. Reggie Edgerton, Ph.D.
University of California, Los Angeles

Since the publication of the mouse genome, some spinal cord researchers have begun switching from rat to mouse models for their studies. Although objective and quantifiable tools are available for evaluating walking ability in rats, no comparable devices exist for mice. Instead, researchers have relied on subjective and qualitative behavioral tests to measure the effects of both spinal cord injuries and experimental treatments. To remedy this situation, Dr. Edgerton and his colleagues are adapting for mice the robotic technology they developed to assess locomotion in rats and will test a prototype of the hardware and software in this project. The prototype is a two-armed device that attaches to the hind limbs of a mouse. The arms can function passively, merely recording limb movements; or motors attached to the arms can be set to define the movements the mouse can generate. The device also has a weight-supporting element to record and control the load the limbs bear.

A second aim of this project is to explore the role of astrocytes, cells that support and nourish nerve cells, in repairing spinal cord injuries. With one group of normal mice and a second group bred without astrocytes, the researchers will use the same training techniques with the mice that they have been using with rats to improve the recovery of walking following various spinal cord injuries. The training involves suspending the animals over a treadmill so that their feet move in stepping patterns. Dr. Edgerton will use the robotic prototype to assess the walking capability of the mice after a mild spinal injury. He then will apply electrophysiology techniques to record the muscle activation patterns generated by spinal circuitry, both during treadmill walking and in response to stimulation from the brain. The results of this study could lead to combination therapies that would be more effective than just training for restoring the ability to walk after some types of spinal cord injuries.

ROBOTIC OUTCOME ASSESSMENT IN SPINAL CORD INJURY AND REGENERATION
David J. Reinkensmeyer, Ph.D.
University of California, Irvine

In evaluating their efforts to restore function, researchers now rely on visual observations of rat behavior. Although this approach provides a good indication of gross recovery, it is imprecise, revealing little about which movements in the series of motions that add up to walking actually improves and by how much. As a result, scientists often cannot tell how their interventions affect important aspects of locomotion, such as the strength for stepping, postural stability, and step variability.

In this project, Dr. Reinkensmeyer is perfecting a small robotic device for rats that accurately measures and controls thousands of hind limb movements. In addition, the device can determine how strong a rat's hind limbs are and can vary the amount of weight the animal bears during stepping. To see how different challenges affect both stepping and the nerve circuitry in the lower spinal cord that control leg and foot motions, researchers also can program the device to challenge the animal's legs as if it were walking on, say, steep or rocky terrain. This robotic tool will enable scientists to identify which of their therapies restores limb movements when the spinal cord injured animal has help bearing its weight. The new device will be available for shared use at the Roman Reed Core Laboratory at the University of California at Irvine.

Creating New Models For Spinal Cord Research

Before promising therapies can be tested on humans, scientists need to learn as much as possible about precisely what happens following a spinal cord injury and how well potential treatments work in animals These researchers have won Roman Reed Grants to develop new animals models of spinal cord injuries:

Cervical dorsal root lesions in monkeys: neuronal consequences and impairment of voluntary hand function
Corinna Darian-Smith, Ph.D.
Stanford University

Dr. Darian-Smith and her team are studying how the nervous system responds to injuries of the cervical dorsal roots, the part of the sensory nerves from the arm and hand that enters the spinal cord. Any damage to these key junctures greatly impairs manual dexterity and may cause permanent loss of arm and hand function. Dr. Darian -Smith has developed a monkey model of a dorsal-root injury that disrupts sensory information from only the index finger and thumb of one hand. The model enables Dr. Darian-Smith and her colleagues to study how the peripheral nervous system adapts structurally and functionally following this type of disabling injury. This work is important because cervical dorsal root injuries, in which the brachial plexus - the major nerve in the upper arm - is torn away, often occur in motorcycle accidents and traumatic births.

Dr. Darian-Smith has already found that, several months after the dorsal root is cut, the animals recover significant function in the hand and the underlying nerve pathways reorganize. Although the severed dorsal roots do not regrow, a number of dramatic changes do occur following the injury, and in this project Dr. Darian-Smith is trying to pinpoint why. She plans to correlate anatomical and physiological changes within the nervous system with the recovery of function she observed during the three to four months following a spinal cord injury. A better understanding of the post-injury reorganization of the underlying neural pathways in monkeys would pave the way for more effective treatments for people with these injuries.

University of California consortium to study axonal plasticity and regeneration in the primate spinal cord
Mark Tuszynski, M.D., Ph.D.*, Reggie Edgerton, Ph.D.†, Jeff Roberts, D.V.M.††, Leif Havton, M.D., Ph.D.†, Bruce Dobkin, M.D.†, Os Steward, Ph.D. ** Corinna Darian-Smith, Ph. D ¥ *UCSD, †UCLA, †† UC-Davis, ** UC-Irvine, ¥ Stanford University

In the last decade, spinal cord researchers have made substantial progress working with rodent models, and several experimental therapies appear to enhance the recovery of function after an injury. However, it remains unclear whether these findings from rodent models will apply to the larger spinal cord in humans, so research on primates is a logical next step. This project is a collaboration among noted scientists from five universities, who are developing a practical but humane model of a partial primate spinal cord injury. Next they plan to test potential therapies in the new model. This project should enhance the understanding of how nerve cells regrow their axons after an injury and how spinal circuitry changes in response to activity. This group of scientists hopes to validate in adult primates therapies that have been successful in smaller animals, moving research closer to treating humans. .