BALTIMORE—Axonal sprouting and the formation of new patterns of connections in the cortex near the stroke site causally mediates recovery, reported S. Thomas Carmichael, MD, PhD, at the 134th Annual Meeting of the American Neurological Association. Dr. Carmichael and colleagues identified a novel molecular signaling system in the brain that controls axonal sprouting and recovery, EphrinA5, and developed a drug delivery system to target the stroke cavity and produce more substantial patterns of reconnection and recovery after stroke.

“The adult brain is capable of a high degree of rewiring after stroke, and this rewiring mediates aspects of recovery,” Dr. Carmichael told Neurology Reviews. “The brain normally locks down, or inhibits, the potential to form these new connections in an age-dependent manner, but this can be overcome to produce new connections and increased recovery after stroke.”

Mapping New Connections

Dr. Carmichael and colleagues used sequential tracer injections to label neurons that are forming new connections in the brains of stroke-induced rats. The first tracer was injected at the time of the stroke and the second seven to 21 days later, a “clinically relevant time period for neurorehabilitative therapy,” explained Dr. Carmichael, Associate Professor, Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles. The tracers enabled the researchers to map pathways of molecular regeneration and the time frame in which it occurs.

Dr. Carmichael’s group then identified neurons that were double labeled and back labeled with only the second tracer in both young and aged animals, cut them out, and amplified the RNA to compare the gene expressions of neurons from the same animal and regions to determine the regeneration growth program.

“We found several interesting categories of molecules that differ between sprouting neurons in aged versus young adult animals after stroke. One major difference is that aged neurons paradoxically upregulate receptors for growth inhibitory molecules,” Dr. Carmichael explained. “One is EphA4, selectively induced in sprouting neurons of aged animals and not in young adults, and this is a receptor for the glial growth inhibitory molecule EphrinA5. So, paradoxically, aged neurons are activating the receptor for a major glial growth inhibitory protein, which itself is activated by stroke in the region of sprouting.”

A Novel Drug Delivery System

Using a quantitative brain mapping system to trace axonal output in mice, researchers compared connections in a normal mouse, a mouse with stroke, and mouse with stroke and different treatments. They performed a series of experiments to determine if EphrinA5 signaling after stroke blocks the formation of new connections, thereby inhibiting recovery. To conduct these experiments, the investigators developed a novel drug delivery system to target areas of the brain where axonal sprouting occurs.

“A major problem in stroke repair therapies is that the target of the therapy is a relatively small region of the CNS. Drugs that promote repair in this region usually have to be given systemically, where they can produce side effects in other tissue,” Dr. Carmichael explained. “We modified a biopolymer hydrogel that can be injected into the stroke cavity and then slowly releases the drug over one month. The stroke cavity is an ideal site, because it can accept a large volume of injection without damaging normal brain and is directly adjacent to the site of neural repair—the peri-infarct tissue.”

Blocking EphrinA5 signaling resulted in a statistically significant increase in axonal sprouting and improved behavioral therapy, while inducing EphrinA5 signaling blocked sprouting and subsequent recovery. Behavior models showed similar results, in which blocking EphrinA5 signaling resulted in gradual, but significant functional recovery.

Maximizing Behavioral Recovery

One of the only treatments that have been consistently shown to produce improved behavioral recovery after stroke is overuse of the affected limb, or constraint-induced therapy, according to the researchers. They modeled constraint-induced therapy in the mouse after stroke—by using onabotulinumtoxin A to force the mouse to use its affected limb—and found that it caused new connections to form in the brain. They then blocked EphrinA5 signaling along with the behavioral therapy and found a substantial increase in axonal sprouting.

“The dramatic thing that happens when you force a mouse to overuse its forelimb motor cortex and then block EphrinA5 signaling [is] this tremendous increase in axonal connections in the forelimb motor cortex, prefrontal cortex, and temporal areas,” Dr. Carmichael explained. “This is without precedent in the literature and prompted great alarm on my part, so we repeated the analysis and it was validated twice.”

EphA4 and EphrinA5 signaling are just one piece of the puzzle of brain remapping after stroke, according to Dr. Carmichael. GAP43, a growth-associated protein, and insulin-like growth factor 1 signaling are two other emerging areas of study, he noted.

—Rebecca K. Abma

Axonal Sprouting Boosts Motor Recovery in Stroke Patients