Researchers at Boston Children's Hospital now provide insight into why nerve pathways of spinal cord remain quiet. They also show that a small-molecule compound, given systemically, can revive these circuits in paralyzed mice, restoring their ability to walk
"For this fairly severe type of spinal cord injury, this is most significant functional recovery we know of," says He. "We saw 80 percent of mice treated with this compound recover their stepping ability." The study, led by Zhigang He, Ph.D., in Boston Children's F.M. Kirby Neurobiology Center, was published online July 19 by the journal Cell.
Waking up dormant spinal circuits
Many animal studies looking to repair spinal cord damage have focused on getting nerve fibers, or axons, to regenerate, or to getting new axons to sprout from healthy ones.
While impressive axon regeneration and sprouting have been achieved, by His lab and others, their impacts on the animals' motor function after a severe injury are less clear. Some studies have tried using neuromodulators such as serotonergic drugs to simulate the spinal circuits, but have gotten only transient uncontrolled limb movement.
He and colleagues took another approach, inspired by the success of epidural electrical stimulation-based strategies, the only treatment known to be effective in patients with spinal cord injury. This treatment applies a current to the lower portion of the spinal cord; combined with rehabilitation training, it has enabled some patients to regain movement.
"Epidural stimulation seems to affect the excitability of neurons," says He. "However, in these studies, when you turn off the stimulation, the effect is gone. We tried to come up with a pharmacologic approach to mimic the stimulation and better understand how it works."
He, first author Bo Chen and colleagues selected a handful of compounds that are already known to alter the excitability of neurons, and can cross the blood-brain barrier. They gave each compound to paralyzed mice in groups of 10 via intraperitoneal injection. All mice had severe spinal cord injury, but with some nerves intact. Each group was treated for eight to ten weeks.
Inhibiting inhibition by re-activating KCC2
One compound, called CLP290, had the most potent effect, enabling paralyzed mice to regain stepping ability after four to five weeks of treatment. Electromyography recordings showed that the two relevant groups of hindlimb muscles were active. The animals' walking scores remained higher than the controls' up to two weeks after stopping treatment. Side effects were minimal.
CLP290 is known to activate a protein called KCC2, found in cell membranes, that transports chloride out of neurons. The new research shows that inhibitory neurons in the injured spinal cord are crucial to the recovery of motor function.
After spinal cord injury, these neurons produce dramatically less KCC2. As a result, He and colleagues found, they can't properly respond to signals from the brain. Unable to process inhibitory signals, they respond only to excitatory signals that tell them to keep firing. And since these neurons' signals are inhibitory, the result is too much inhibitory signaling in the overall spinal circuit. In effect, the brain's commands telling the limbs to move aren't relayed.
By restoring KCC2, with either CLP290 or genetic techniques, the inhibitory neurons can again receive inhibitory signals from the brain, so they fire less. This shifts the overall circuit back toward excitation, the researchers found, making it more responsive to input from the brain. This had the effect of reanimating spinal circuits disabled by the injury.