Helping Patients to Walk Again: Nerve Regeneration

Researchers at Mayo have completed animal studies that are the first steps in showing that nerves can regenerate in the spinal cord system. Anthony Windebank, M.D., a neurologist, molecular neuroscientist, and director of Mayo's Molecular Neuroscience Program is co-principal investigator of the research team. Michael Yaszemski, M.D., Ph.D., a spine surgeon and chemical engineer who directs the Tissue Engineering and Polymeric Biomaterials Laboratory, is the other co-principal investigator. In October 2003, the research team received a $1.3 million National Institutes of Health grant that underscores the early success of their animal studies.

"I am optimistic that we will be conducting human clinical trials within ten years," says Dr. Windebank. "This project is so exciting that I can't wait to get into the lab."

Dr. Yaszemski agrees. "It is exciting to imagine that we might make a contribution to helping patients with SCI walk again," he says.

The research team that Drs. Windebank and Yaszemski assembled for this project is remarkable for the breadth of its collaboration. Areas involved include:

• Department of Neurology
• Department of Orthopedic Surgery
• Department of Neurologic Surgery
• Department of Physiology and Biomedical Engineering
• Department of Biochemistry and Molecular Biology
• Molecular Neuroscience Program
• Biomedical Engineering Program
• Tissue Engineering and Polymeric Biomaterials Laboratory
• Nuclear Magnetic Resonance Facility

"We have developed the biotechnology that supports nerve regeneration," says Dr. Windebank. "And we've completed animal studies that are the first steps in showing that nerves can regrow in the spinal cord system."

Applying Expertise Beyond the Scientist's Field

Dr. Windebank has devoted most of his research career to understanding the mechanisms of peripheral nerve disease. He has extensive knowledge in how peripheral system Schwann cells stimulate regeneration after injury and was well aware that spinal cord nerve fibers also have the capacity to regenerate, but that many efforts have been thwarted by a cellular process that inhibits regeneration and promotes scarring.

Dr. Yaszemski is an expert in engineering a variety of polymers that are used as scaffolds to support new bone growth. He is also a firm believer in research that is initiated by patients who have a problem that needs to be solved. As a spine surgeon, he is frustrated that the best he can currently offer a person who comes in with an acute spinal injury, is stabilization of the bony spine that will allow the person to function with paralysis.

In casual discussions the two physicians wondered how they could synthesize their combined clinical skills with their cellular biology and engineering expertise and launch an effort to help patients with SCI. Just two years later, they began seeing promising results in studies that evolved from their collaboration.

The Project

Orthopedic surgeon, Bradford Currier, M.D., and neurosurgeons Richard Marsh, M.D., and Robert Spinner, M.D., help plan experiments and keep the team focused on translating the science to humans.

To simulate spinal cord injury a small section of a spine is surgically excised from an anesthetized rat. It is replaced with a trellis-like, biodegradable, polymer scaffold designed to anchor nerve cells, deliver drugs that promote nerve regeneration, and dissolve after a predetermined time to make room for more nerve growth. The goal is to produce a permissive environment that encourages the nerve cells to grow in a predetermined direction.

"We're using polymer chemistry to find the ideal combination of plastics," says Dr. Yaszemski. "And we have designed and constructed a variety of these mini scaffolds. Now we're ready to test them to find the architecture that produces maximal nerve growth."

Another variable is sorting out which compounds do the best job of promoting nerve growth.

"We know that Schwann cells promote nerve growth so we harvest them from the peripheral nervous system and load them into the polymer scaffold," explains Dr. Windebank. "We also introduce neurotrophins - protein growth factors that promote nerve growth by blocking natural cell death. And we are experimenting with compounds that inhibit scar formation."

Three months after they injected Schwann cells into rat spinal cords, the research team observed as many as 5,000 nerve fibers growing throughout the length of the polymer scaffolds. There are hundreds of thousands of nerves in a normal spinal cord but Dr. Windebank estimates that it will be possible to restore function with ten percent of the normal number.

The Next Steps

While encouraged by their progress, Dr. Windebank cautions that they must find a way to guide nerve terminals to make contact with the correct nerve ending before function can be restored.

"Thus far we've successfully implanted a scaffold in animals, shown that it supports and directs growth, and functions as a delivery system for drugs," says Dr. Yaszemski. "That says nothing about the nerve fibers actually functioning."

Slobodan Macura, Ph.D., a biochemist and an expert in nuclear magnetic resonance microscopy and spectroscopy, helps investigators judge their progress by producing images of the tiny polymer scaffold. Together with spectroscopy studies, he is able to provide information on the composition and concentration of metabolites in body fluids, cells, tissues, and organs.

Other basic scientists at Mayo are conducting research that may help when the team is ready to begin the complex process of restoring function.

Helping Patients to Breathe by Themselves

Severe SCI leads to loss of neurological function below the level of injury. When the injury occurs high in the neck, it involves the upper cervical spinal cord and quadriplegia results. When that happens, the patient stops breathing and life can only be sustained by artificial ventilation. Such patients are far more concerned with being able to breathe by themselves than with being able to walk again.

Gary Sieck, Ph.D., Chair of the Department of Physiology and Biomedical Engineering, has earned continual funding from the National Institutes of Health for more than 18 years to conduct research that may contribute to freeing people with quadriplegia from being tethered to ventilators.

The nerve, the muscle, and the junction that connects them, all exhibit degrees of plasticity they adapt to accommodate changing levels of activity. Dr. Sieck's Cell Imaging and Physiology Lab studies the plasticity of neuromotor control of the diaphragm muscle. Greater understanding of this process could contribute to pragmatic therapies, such as more effective phrenic nerve pacing. And Dr. Sieck's expertise will be helpful when Drs. Windebank and Yaszemski are ready to focus on coaxing the new nerve terminals to find and connect with the right targets.

How Do Motoneurons Interact with Muscle Fibers?

A motoneuron is simply a nerve with a motor function. It extends out from the spinal cord to innervate skeletal muscle fibers. The drive to breathe resides in the medulla, which is located in the lower brain, while phrenic motoneurons that control the diaphragm are located in the lower cervical spinal cord. So when a patient sustains a severe injury to the upper cervical spinal cord, the connection between the two vital locations is disrupted, and the patient stops breathing.

Our rhythmic pattern of inspiration means that phrenic motoneurons are active almost half the day, all day long, every day, making them some of the most active neurons in the body.

"When SCI imposes sudden and total inactivity, phrenic motoneurons, diaphragm muscle fibers and the synapses between them display plasticity - each adapts to the change in activity," explains Dr. Sieck. "To devise effective therapies, we must understand the basis for this neural plasticity."

The Role of Neurotrophins in Motoneuron Plasticity

While Drs. Windebank and Yaszemski exploit neurotrophins because they help generate nerve growth, Dr. Sieck is intrigued by their role in enhancing neuroplasticity.

The system involves brain-derived neurotrophic factor (BDNF), and Neurotrophin 4 (NT4) neurotrophins that are mediated through a receptor called tyrosine kinase receptor B (TrkB). The receptor sets up an intracellular signaling cascade that causes changes in protein expression, which results in changes in the synapse.

"We've shown that BDNF and NT4 are expressed in phrenic motoneurons and at the neuromuscular junction," says Dr. Sieck. "We know that the TrkB receptors are present on both the pre- and post-synaptic side of the neuromuscular junction both neurons and muscle fibers. We can enhance synaptic transmission by treating the diaphragm and the nerves with neurotrophins. Or we can hinder synaptic transmission by blocking the TrkB receptors or the intracellular signaling cascade induced by TrkB activation. Clearly neurotrophins play a role in neuroplasticity."

Phrenic Nerve Pacing

In SCI, the phrenic nerve remains intact so diaphragm muscle fibers can be stimulated to cause breathing.

"Surprisingly, the diaphragm muscle doesn't atrophy with inactivity as limb muscles do," explains Dr. Sieck. "Since it's not a weight-bearing muscle, it's assumed that gravity exerts some trophic influence."

That's why patients who have been on long-term mechanical ventilation can still generate enough force to breathe when phrenic nerves are electrically paced. Unfortunately, it does not take long for the diaphragm to become fatigued when the phrenic nerves are stimulated. Dr. Sieck's research has contributed to understanding the physiology of why that happens knowledge that may lead to treatments that can lengthen the time a patient can tolerate pacing.

Dr. Sieck's lab has shown that physiological and metabolic properties vary with different types of diaphragm motor units. Larger motor units are susceptible to fatigue and are recruited when the diaphragm needs greater force such as for sneezing and vomiting.

"We have modeled how the nervous system controls motor unit recruitment during different ventilatory and non-ventilatory behaviors of the diaphragm," says Dr. Sieck. "The problem with electrical pacing is that fatigable types of motor units are recruited before the most fatigue-resistant ones so pacing studies need to focus on finding ways to encourage the normal recruitment order."

In animal models, the Sieck lab transects half of the spinal cord at C2. The rodents can still breathe because the phrenic nerve on the other side remains intact. Interestingly, the animals can still ambulate because locomotor patterns are generated in the spinal cord. So the only result of the SCI is paralysis of the diaphragm on the transected side. Then the lab imposes different patterns of stimulation to try to simulate the normal recruitment order of motor units.

Collaboration with the Spinal Nerve Regeneration Project

Mayo researchers devote much of their time to educating future leaders in science and educational duties frequently lead to further scientific collaboration. For example, Drs. Windebank, Sieck and Yaszemski are all members of the thesis committee of a graduate student who is working on the polymer scaffolds an activity that keeps them abreast of each other's projects.

"Neuron target cell interactions have been best characterized in the motoneuron muscle area," says Dr. Sieck. "Potentially, motoneurons can pick any muscle fiber they want to innervate but they choose very specific types of muscle fibers that express the same contractile and metabolic proteins. The same problem exists for nerve axons in the spinal cord as they regrow following SCI. As we increase our understanding of the mechanisms by which neurotrophins mediate neuron-target cell interaction, we can apply that knowledge to developing therapies that will help Drs. Windebank and Yaszemski to steer a newly regenerated nerve in the right direction."

Targeting Antibodies to Repair the Myelin Sheath and to Promote Nerve Regeneration

Moses Rodriguez, M.D., a basic scientist in the Department of Immunology, and a neurologist in the Department of Neurology, has developed antibodies that may play a critical role in repair of the myelin sheath - the fatty insulation that surrounds most nerves in the brain and spinal cord. He is optimistic that a greater understanding of the mechanisms that promote remyelination will one day result in non-invasive treatments that promote nerve repair.

Dr. Rodriguez directs the Demyelinating Laboratory and has dedicated 20 years to researching ways to promote nervous system repair. Though his primary interest is multiple sclerosis, much of what he has learned is applicable to SCI.

"Multiple sclerosis results from injury to the myelin sheath and we have developed a series of assays by which we can examine how various antibodies directly stimulate its repair," explains Dr. Rodriguez. "There's strong pathological evidence that demyelination is the cause of dysfunction in many SCI cases. And I believe that some antibodies that promote remyelination will also promote functional recovery."

In addition to the myelination projects, Dr. Rodriguez's lab is now developing a whole new set of antibodies that are specifically targeted at nerve regeneration.

"We have characterized a series of two monoclonal antibodies that are very effective in promoting neural outgrowth in tissue culture," says Dr. Rodriguez. "We are very excited about testing them in animal models."

Next Steps for the Demyelinating Lab

While the nerve regeneration project is in its early stages, the remyelination studies are likely to lead to clinical trials in the near future.

"We have secured patents and worked with industry to purify the antibodies and produce them in high concentration," says Dr. Rodriguez. "We are currently conducting toxicity studies and could begin clinical trials within a year."

As a neurologist, Dr. Rodriguez is most excited that he is focusing on research that could lead to non-invasive treatments for his patients.

"Seeing patients is a very strong motivator for my research," says Dr. Rodriguez. "It is very exciting to be able to take our laboratory successes and apply them to better care for our patients."

Other SCI-Related Research at Mayo Clinic

Adil Bharucha, M.D., a gastroenterologist, and his colleagues in the Enteric Neuroscience Program are developing new approaches to assessing bowel motility. SCI can cause constipation due to an inability to use abdominal muscles or pelvic floor muscles, or both, or it may result from colonic motor dysfunction. Understanding the severity of bowel dysfunction is useful for guiding effective treatment. Dr. Bharucha is also in the process of developing protocols to test newly available therapies that can increase motor function and facilitate the movement of food, gas and stool through the bowels. His colleague, Michael Camilleri, M.D., recently conducted research that played a significant role in the development of one of these therapies, Tegaserod (Zelnorm®)

Kenton Kaufman, Ph.D., an expert in human locomotion, directs the Motion Analysis Laboratory in the Department of Orthopedics and is past president of the Gait and Clinical Movement Analysis Society. His lab is uniquely equipped with specialized testing equipment that objectively evaluates functional impairments while the patient is moving. His lab also evaluates and develops mobility aids. And they help wheelchair users to optimize their efficiency. Dr. Kaufman's expertise would be invaluable if basic scientists can restore function to the limbs of people who been paralyzed.

Ronald Reeves, M.D., a physiatrist in the Department of Physical Medicine and Rehabilitation (PMR) and a spinal cord medicine specialist, studies the epidemiology of SCI and strategies to improve rehabilitation outcomes for people with spinal cord diseases and injuries. Additionally, he is participating in a Phase III multi-center trial to test how the drug 4-aminopyridine affects potassium channels in the central nervous system and enhances transmission of spinal cord nerve signals. PMR conducted 40 SCI-related clinical trials in 2002.

Douglas Husmann, M.D., a urologist in the Department of Urology, studies bladder dysfunction with a special focus on recurrent urinary tract infection.

Isobel Scarisbrick, Ph.D., a scientist in the Department of Physical Medicine and Rehabilitation studies enzymes that are responsible for promoting axon outgrowth and the development of synaptic connections in the brain and spinal cord. In collaboration with Drs. Rodriguez, Windebank and Sieck, she has discovered that the same enzymes are present in excess following injury and contribute to further tissue destruction. Further investigations aim at modulating enzyme activity to prevent further injury and promote development of new synaptic connections in the injured spinal cord.

David Piepgras, M.D., Chair, Department of Neurologic Surgery has participated in phrenic nerve stimulation pacing of the diaphragm multi-center studies in patients with chronic ventilatory insufficiency.

The Future of Spinal Cord Research at Mayo Clinic

Interest in spinal cord research is growing at Mayo Clinic. Mayo has expertise in all the right areas basic science, clinical studies, Physical Medicine and Rehabilitation, a highly specialized Gait and Motion Analysis Laboratory, and a model system of interdisciplinary collaboration to make a significant contribution to SCI research that can benefit patient care.

Mayo provides important base support for SCI research projects by funding necessities not funded by external grants such as space, infrastructure, microscopes, centrifuges, electrophysiological and other critical equipment.