Packard Animal Studies Show Motor Nerve Circuits Can Be Repaired

June 20, 2006

In a dramatic display of stem cells' potential for healing, a team of Packard Center scientists reports that they've engineered new, completed, fully-working motor neuron circuits — neurons stretching from spinal cord to target muscles — in paralyzed adult animals.

The research, in which mouse embryonic stem (ES) cells were injected into rats whose virus-damaged spinal cords model nerve disease, shows that such cells can be made to re-trace complex pathways of nerve development long shut off in adult mammals, the researchers say.

"This is proof of the principle that we can recapture what happens in early stages of motor neuron development and use that to repair damaged nervous systems," says Douglas Kerr, M.D., Ph.D., who led the Packard team based at Johns Hopkins in Baltimore.

"What we've created amounts to a potential 'cookbook' recipe to restore lost nerve function," Kerr adds. Principles uncovered in the study could one day lead to repair of the injury from such diseases as ALS or multiple sclerosis, once the basic source of damage is stopped, the researchers say. "With small adjustments keyed to differences in nervous system targets," Kerr adds, "the approach may also apply to patients with Parkinson's or Huntington's disease."

In a report on the study that will appear in this month's online Annals of Neurology, the Packard team says 11 of 15 treated rats gained significant, though partial, recovery from paralysis after losing motor neurons to an aggressive infection with Sindbis virus — one that, in rodents, specifically targets motor neurons and kills them. The animals recovered enough muscle strength to bear weight and step with the previously paralyzed hind leg.

Kerr likens the approach to electrical repair. "Paralysis is like turning on a light switch and the light doesn't go on. The connectivity is messed up but you don't know where. We've asked stem cells to go where needed to fix the circuit.”

For a brief period after a nerve dies, it leaves behind what's essentially an empty shell, with some scaffolding and non-nerve substances remaining. But with ES injections at the right time and place, and by adding the right cues, we've learned to restore the biological 'memory' for growing neurons, which is clearly still in place," he added.

The motor circuit engineering combines recent discoveries on stem cell differentiation, a growing understanding of early development of the nervous system, and insights into behavior of the nervous system in traumatic injury.

"As adults, our cells no longer respond to early developmental cues because those cues are usually gone," says Kerr. "That's why we don't recover well from severe injuries. But that's what we believe we have changed. We asked what was there when motor neurons were born, and specifically what let motor neurons extend outward. Then we tried to bring that environment back, in the presence of adaptable, receptive stem cells."

In the study, the team first pre-treated cultures of mouse embryonic stem cells with growth factors that both increase survival and prompt specialization into motor neurons. Adding retinoic acid and sonic hedgehog protein — agents that direct cells in the first weeks of life to assume the proper places in the spinal cord — readied the conditioned ES cells for the motor neuron circuit that starts in the spinal cord. Then, stem cells were fed into the paralyzed rats' spinal cords.

Extending new motor neurons in an adult nervous system, however, meant overcoming hurdles. One involved myelin, the fatty material that insulates mature motor neurons. Like the coating on electrical wire, myelin prevents weakening of the traveling electrical impulse and lets it continue long distances. In humans, the myelinated sciatic nerve, for example, exits the spinal cord and extends to the leg muscles it activates, carrying impulses several feet.

Once laid down, however, myelin inhibits further nerve growth — nature's way to discourage excessive wiring in the nervous system. "We had to overcome inhibition from myelin lingering in the dead nerve pathways," Kerr explains. Earlier studies by Packard scientist Marie Filbin suggested drugs that could do that work and the team used them.

The assorted treatments let the new motor neurons survive, grow through the spinal cord and extend slightly into the outlying nervous system. A second hurdle remained in getting the neurons to skeletal muscle targets.

As suggested by recent work by Packard researcher Ahmet Hoke on repair in the outlying, peripheral nervous system, the researchers applied GDNF, a powerful stimulator of neuron growth, to the remains of the newly-dead sciatic nerve at a point near its former leg muscle contacts.

GDNF attracted the extending motor neurons, "luring" them to the muscles. To ensure a continuous supply of GDNF, the researchers relied on injected mouse fetal neural stem cells, a known source of the molecule. (Earlier studies by Packard scientist and collaborator Jeffrey Rothstein first showed the usefulness of GDNF- secreting stem cells in encouraging motor neuron growth.)

Of some 4,100 new motor neurons created in the spinal cord, roughly 200 exited the cord and 120 reached skeletal muscle, forming typical nerve-muscle junctions, with appropriate, typical chemical markers. Microscopically, the neurons and their muscle associations appear identical to natural ones in healthy animals. Fifty of the new neurons were found to carry electrical impulses. (Such testing is time and labor intensive, so only a small area of leg muscle was assayed. Because of the improved ability of treated rats, however, more functional neurons are likely.) The rats gained weight, were more mobile in their cages and measures of muscle strength increased .

Animals treated without even one component of the "cocktail" experienced no such recovery. Novel ways of tracing the neurons back to their source assured the scientists that they indeed had come from the injected stem cells, not from lingering host neurons.

Research begins this summer to see how well the technique applies to human nerve recovery by using federally-approved human ES cells in larger mammals like pigs. Each of six academic institutions in a new collaboration will tackle a different major question of safety and effectiveness. Fundamental, important questions of tumor-formation, a concern with ES cells, of the safety of surgery needed to inject the cells and of the cells' ability to form healthy motor circuits in people are critical, time-consuming questions to answer before the FDA would consider approval of human trials.

“Overall, this research sets the stage for ways we might fashion an approach for ALS patients,” says Packard Director, Jeffrey Rothstein, who was also on the research team. “It reflects the truly collaborative nature of stem cell research at Johns Hopkins and the Packard Center; it supports our central mission of collaborative, translatable science.”

The study was supported by The Robert Packard Center for ALS Research at Johns Hopkins, the ALS Association, the Muscular Dystrophy Association, Wings Over Wall Street® and a grant from the NIH, among others.

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