For people with paralyzing neck injuries, the greatest loss is the ability to breathe on their own. A repair technique, published in the July 14 Nature, could someday sever the tether between them and the perpetual hum of the respirator. Researchers at Case Western Reserve University in Cleveland, Ohio, restored diaphragm function to injured rats with a two-pronged approach: They combined nerve grafts with an enzyme to break up proteins blocking regeneration. Although it took three months, the rats eventually regained diaphragm function—in some cases, the muscle activity was even stronger than an uninjured diaphragm. First author Warren Alilain, who is now moving on to a job at Metropolitan General Hospital in Cleveland, led the study in the Case Western laboratory of senior author Jerry Silver.
“It is one of the first, if not the first, paper to show convincing long-term functional regeneration, especially with regard to respiration,” commented Phillip Popovich of Ohio State University in Columbus.
For severed nerves to regenerate, their axons must make it through the extracellular matrix, which is chockfull of sticky proteoglycans. Normally, these molecules cement nerves in place after development, keeping synapse structure rigid. But the same proteoglycans flood tissue after spinal cord injury, preventing new axons from getting through.
To punch through the goo, scientists have taken advantage of an enzyme produced by the bacterium Proteus vulgaris. This parasite is a potent invader of animal tissues precisely because it possesses chondroitinase ABC, which breaks down the proteoglycans. Adding chondroitinase to the site of spinal cord injury improves regeneration (Bradbury et al., 2002), but in a limited fashion. Similarly, performing a peripheral nerve graft promotes regeneration by providing a bridge conducive to axon growth (David and Aguayo, 1981). But when the new axons hit the proteoglycan barrier at the end of the bridge, they simply turn around, Silver said.
Many spinal injury researchers are expanding their experiments beyond one single treatment, said Harry Goshgarian of Wayne State University in Detroit, Michigan. “People are realizing that there is no magic bullet,” he said. “It might be that the best way to go is with a combination of therapies.”
Silver’s group first tried chondroitinase plus a nerve graft in a 2006 study, in which they were able to restore some function to paralyzed forelimbs in rats (Houle et al., 2006). The rats could flex their wrists a bit, but could not move individual digits or unite the various muscles involved in walking. The problem, Silver said, was that all of the new axons stopped extending as soon as they reached the end of the nerve graft. It is as if every driver crossing the Golden Gate Bridge took the first exit on the other side, with no one going any farther.
Silver decided that if he could only restore function to one muscle, he would make it an important one: the diaphragm. More than half of spinal cord injuries occur in the neck, Goshgarian noted, and loss of respiratory function is the most common cause of death in those cases. Losing breathing is “pretty much the last vestige of independence; you cannot even yell by yourself,” added Oswald Steward of the University of California in Irvine. A treatment “could be a huge positive impact on people’s lives,” he said.
The diaphragm provided the researchers with an ideal model system to compare damaged and whole nerve networks. Alilain performed hemisections such that the nerves leading to one side of the muscle were severed, leaving the other side intact. The rats could still breathe with half a diaphragm.
Alilain inserted peripheral nerve grafts at the injury site, injecting chondroitinase above and below the graft. A week later, he cut the axons again. This actually promotes sprouting, Silver said, rather like pruning a rose bush to improve growth and branching.
Then they waited for three, six, eight weeks—but saw no change. “It was very depressing,” Silver said. The researchers gave up and put the rats away.
Fortunately, Alilain happened to come back to those rats when he needed to test out some electrodes. Soon, he was running to Silver’s office to report that the paralyzed sides of the diaphragms had started moving again. “Between 10 and 12 weeks, the activity in the diaphragm just blooms,” Silver said.
Hundreds of nerve fibers emerged from the end of the graft, and with the proteoglycans out of the way, they were able to find their targets. Using electromyography (EMG), Alilain found that the animals that received the combo treatment had 80-90 percent of normal activity in the affected side of the diaphragm. In some animals, the peak amplitude on the EMG surpassed that of the other, unaffected side of the muscle. In comparison, a couple of untreated animals recovered perhaps 10 percent of function, Silver said, while the graft alone only provides an improvement of 10-20 percent.
The authors were particularly surprised, and pleased, to observe that, after axon regeneration, the diaphragm moved in the correct pattern of breathe in, breathe out. There were few twitches in between or in the middle of breaths, for example. Silver suspects that interneurons in the spinal cord, left over from before the injury, filter the signals so only the appropriate ones reach the muscle. Selective filtering and pruning of the new connections could be one reason the repair took so long, he suggested; re-myelinating the new axons could also take a while.
“If it is as good as it looks—and it looks pretty good—it is not something that would require a huge amount of extra work to get it to the clinic,” Steward suggested. One key hurdle would be to show that chondroitinase is safe for people. Silver has not seen major side effects in animals, he told ARF. Popovich also cautioned that Alilain created a clean hemisection in the rats; people may have larger or more complicated lesions.
Silver has a couple of ideas in mind to make the therapy work even better. For one, he has noticed that the new nerve fibers are straight like straws instead of branched like trees. He suspects that there are not enough neurotrophins in an adult to support arborization; thus, added neurotrophin might improve the results.
In addition, Zhigang He and colleagues at Children’s Hospital in Boston have found that knocking out phosphatase and tensin homolog (PTEN) improves regeneration (see ARF related news story on Liu et al., 2010). PTEN normally suppresses mTOR, a growth-enhancing factor. Silver suspects that getting rid of PTEN will also enhance regeneration in his model.
Restoring complex functions such as walking could be difficult, but Silver thinks another simple circuit could be amenable to his technique: bladder control. The group has already started testing on this function, the most important concern for people with lower spinal cord injury.
Could this treatment be useful for neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS)? Opinions differ on that point. “ALS would not be a target that I would think of,” Steward said. “The problem is loss of the motor neurons that supply the diaphragm, so regenerating the connection from the brain is not going to help with that.”
However, Popovich suggested that chondroitinase treatment might open up the spinal cord for remaining motor neurons to form new connections, perhaps slowing symptoms. A nerve graft based on fetal tissue or stem cells might also help repair some degeneration, Goshgarian speculated.
Part of Alilain’s success relied on Schwann cells to guide and support the new axon growth. A July 13 paper in the Journal of Neuroscience offers a better understanding of how Schwann cells contribute to peripheral neuropathy—it comes down to mitochondria. Researchers knew these organelles were key to the metabolism and function of neurons, but they are just as crucial in Schwann cells, report a research team led by Jeffrey Millbrandt of Washington University in St. Louis, Missouri.
The authors engineered mice with a floxed version of the gene for mitochondrial transcription factor A (Tfam), which is essential for mitochondrial division and maintenance. They activated Cre recombinase to cut out this gene specifically in Schwann cells. Surprisingly, the Schwann cells were able to survive without Tfam. The neurons fared worse, with demyelination and loss of first small-, then large-caliber axons. The animals developed progressive peripheral neuropathy as they aged.
Alilain WJ, Horn KP, Hu H, Dick TE, Silver J. Functional regeneration of respiratory pathways after spinal cord injury. Nature. 2011 Jul 14;475:196-200. Abstract
Zukor K, He Z. Drawing breath after spinal injury. Nature. 2011 Jul 14;475:178-9. Abstract
Viader A, Golden JP, Baloh RH, Schmidt RE, Hunter DA, Milbrandt J. Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function. J Neurosci. 2011 Jul 13;31(28):10128-40. Abstract