Case Western Reserve scientist Jerry Silver, Ph.D, a longtime member of the Science Advisory Council for the Reeve Foundation
By Sam Maddox
Trauma to the spinal cord destroys some nerves, damages others. The survivors try to recover. They can't; they become disoriented, mired in poisonous chaos and hemmed in by a fibrous net of scar. They are really stuck. One of the core issues in neuroscience is to get them unstuck and functional. But even if they can be teased away from the injury site, will they ever again find a proper target, or conduct a proper signal? Spinal cord regeneration is complicated beyond our comprehension.
Case Western Reserve scientist Jerry Silver, a longtime member of the Science Advisory Council for the Reeve Foundation, has been working on the stuck-nerve problem for better than 30 years. He's well aware how daunting the task is. But he got to thinking one day, what if, instead of trying to bulldoze through all the cellular mayhem at the site of injury, what if a different approach were used? Rather than attack the tortured infrastructure within the cord itself, how about trying to sneak around it?
More Progress in Research
Silver, a native Clevelander, likes traffic analogies. Start with the tip of a nerve, the growth cone: "Imagine a growth cone being like a car on a highway: The cone is lost, chugging along on crummy fuel with a lousy motor and bad tires. The conditions are icy and it's sliding around all over the place. Suddenly though the cone makes contact with a mat somebody has thrown out on the ice." Silver explains that the mat might be the equivalent of a cellular treatment, an implanted Schwann cell, for example. "There's now some traction: the cone can move a bit but macrophages [immune commando cells] threaten to attack it if it moves off the mat. So – what to do? You could throw in lots of other mats and get rid of the macrophages so the cone could more or less move from mat to mat. But on the periphery is a barbed wire fence – the scar."
So, mused Silver, maybe we can ramp up the motor or do something to civilize that nasty environment. There's still that barrier. But wait, he says. Looks like there may be a way out: Detour ahead.
That's the oversimplified concept behind an important study Silver and colleagues published recently. The work was partly sponsored by the Reeve Foundation.
Using a cervical injury rodent model, significant breathing function was restored using a surgical detour – by stitching in a half-inch piece of the host animal's peripheral nerve above and below the lesion site on the outside of the cord. In effect, Silver's team created an escape route for axons stuck in traffic above the injury to get around the problem area and resume their connection to the diaphragm. To cut open a pathway for the axons to exit the cord and merge into the graft, Silver's team used a chemical bolt cutter, the enzyme drug chondroitinase (ch'ase).
This study is important because two repair strategies were combined to enhance recovery in the complex circuitry that controls breathing; the results were published as an article in the top-tier journal Nature. The nerve implants restored normal or near-normal breathing in nine of 11 test animals. "It's pretty amazing," said Silver. "Our work is to-date one of the most convincing demonstrations of the return of robust function after paralysis."
It's also important to note that if the outside detour bridge is cut, the effect vanishes; the animals revert to their previous, single-lung activity. That shows that the repair of breathing was indeed due to regenerating nerves originating above the injury.
Silver emphasized that to this point, the detour protocol does not dramatically affect the animals' ability to walk, even though there is some locomotor improvement. "Although there are axons regenerated from above the lesion to below the lesion, there's no evidence of anything extraordinarily interesting in terms of their walking behavior. Walking is a different ballgame."
A major paper in the journal Science in 1996 by Henrich Cheng and Lars Olson reported restored walking after a peripheral nerve graft similar to that of Silver's group (without ch'ase). "Cheng and Olson had originally said that animals can walk. It made them very famous. It was big. Ours is basically the Cheng and Olson strategy with chondroitinase and some improvements. The animals don't walk. Walking involves the entire body. It's head balanced on the neck and it's alternating steps. It's walking over uneven surfaces with different compositions. It's so complicated."
Silver doesn't think a graft or even a series of grafts around the lesion is the most likely strategy, at least not without further modifications and additional intensive rehabilitation, for ambulation.
Silver and his team, pondering which other clinically relevant muscle group would have a major impact on people with paralysis, have now applied the detour strategy to the bladder. Said Silver, "From what I read, most spinal cord injured people put at the top of their list being able to control their urine."
In a recent presentation to the Society for Neuroscience, Silver's team (including Yu-Shang Lee of the Cleveland Clinic) demonstrated significant improvement in bladder function in a group of animals that had a peripheral nerve bridge (again with ch'ase and also a growth-promoting substance called FGF) around a complete thoracic spinal cord lesion. He calls the treatment the "full Monty."
"It's really remarkable. While the recovery is not perfect it is amazingly improved; you still get periods of abnormally increased bladder pressure and less than maximally efficient urination, but the activity of the sphincter and other urodynamic parameters are really nicely fixed. It's not normal, but it's patterned. We have not yet examined sensory function but this new graft strategy could, indeed, allow for sensory regeneration. I've got to tell you, I'm really psyched."
What about stitching in peripheral nerve bridges to affect hand function? Silver thinks this might work to a limited extent, but again, rehab will be an important component going forward. "I'm thinking maybe two bridges, one each to the major extensors and flexors could give you function; I'm not saying pick up a grain of rice, but maybe grab a cup, you know, or a doorknob or something crude."
Silver has focused his career on glial cells – once thought of as support cells for the "more important" neuron cells. But as glia become better understood, it is evident they are much more important in the function, non-function, and repair of the spinal cord. One very important glial response to injury is the formation of a barrier or scar that axons can't penetrate – Silver's barbed wire. His work with ch'ase, which in essence digests the scar, has opened up exciting possibilities in numerous labs in the U.S. and abroad for possible regeneration in the spinal cord. (See sidebar for other ch'ase work supported by the Reeve Foundation.)
Silver notes that his recent results are built on 30 years of work. "I'm one of the first persons ever to ask the question, 'Why don't axons grow where they don't?' Everybody else was asking, 'Why do axons grow where they do?'" He began to look at proteoglycans, barrier molecules known to be inhibitory in development. There was some literature about these inhibitors in cartilage, which because of proteoglycans is not innervated.
"But the most convincing paper that I read that proteoglycans are really inhibitory is one where they asked the question, 'Why doesn't the placenta eat the uterus?' Wow. That's a cool question. The placenta is a highly invasive tissue, but it only invades the surface of the uterus from the inside, not the whole thing. And the question is, why? Turns out in this paper proteoglycans are in high abundance in the stroma of the uterus. And if you get rid of the proteoglycans with certain enzymes, the placenta takes over the whole uterus, and will kill the mother. I mean, we owe our existence to proteogylcans. Wow. That's potently inhibitory. I thought, maybe I ought to look for them in the nervous system."
Silver said the problem studying proteoglycans was that until 1990 there were no antibodies available that could detect them. Three years later, Silver was first to confirm their presence in the glial scars of rats; he has since then been nearly obsessed with removing proteoglycans and thus promoting regeneration. He won both the Ameritec Award and the Reeve-Irvine Research Medal for this work.
Silver has for many years studied the enzyme chondroitinase, which breaks down proteoglycans. This led to the work with peripheral nerve detours, alongside John Houle's group at Drexel University. In 2006 they worked up an animal model for a nerve bypass. It worked; the animals had better use of a paw. "I thought that was one of the first and best demonstrations of long distance functional regeneration. But it wasn't walking. It wasn't full hand function. It was just really wrist extension. It looks kind of cool when they do it, but they can't use their toes. They can't groom."
Silver said that's when he decided "to tackle this whole regeneration problem at a more simple level. I asked the question, 'If we can restore the function of one important muscle really well, wouldn't that be a good thing? And I said, 'Let's fix the diaphragm.' I mean, that's a really important muscle; we need to breathe to live. The diaphragm is pretty simple, much like a big bag."
Silver conjured up the seminal nerve graft experiments of Santiago Ramón y Cajal from 100 years ago, and of Albert Aguayo from the 1980s – both showed that central nerve axons liked to grow into peripheral grafts. So, using a hemisection injury (only half of the cord is affected), Silver set up his model: "Only one lung is paralyzed. This way, the animals don't have to be on a respirator. But the animal can compensate by breathing faster and deeper on the other side. So it's a really nice model."
Silver hired respiratory specialist Warren Alilain from the Harry Goshgarian lab at Wayne State University in Detroit to perform the delicate detour surgeries. They had discouraging news early on. "Nothing was happening," said Silver. "In the 2006 paper, some projections came back within about six weeks. So we figured if we went eight weeks, there should be plenty of time to see something. But Warren saw nothing. So in the animals that he was recording from, from the diaphragm, all we were seeing was flatline. Six weeks and eight weeks, they start showing a tiny bit of activity, which was no more than spontaneous recovery. So that's eight weeks in a bunch of animals – three years of work – and Warren gave up; we quit. And, you know, we were really depressed."
Fortunately, said Silver, Alilain kept some of the animals to test electrodes for another experiment. Two weeks later, Warren came running into Silver's office. "He said, 'I see some activity and it's not bad.' And so I said, 'Let's wait a little longer.' So between 10 weeks and 12 weeks activity in many of the animals just bloomed. It took that long, which is pretty good evidence of regeneration. When the activity came back in some of the animals, breathing function was essentially restored to normal."
Silver said the duration of the breathing cycle is less than they'd like. "It's best in the chondroitinase treated animals, but still around 60 percent. We'd like to get more." He says he has strategies in mind on how to get more axons to travel the new highway. One would be to manipulate the PTEN gene, which switches cells into a robust growth mode. "They'll be like locomotives, you know? Blast their way out. Or we might try a neurotrophin [growth additive]. Those strategies, if we were to try them together, might really help."
Jerry Silver, Ph.D.
So what's the mechanism here? Where is the traffic in the grafts coming from? "It turns out," said Silver, "that the axons in the graft come from lots of places. This is a highway – back to the highway analogy – that any car can travel, as long as it can enter. But there are no more access or exodus restrictions anymore because the chondroitinase got rid of them." Silver said the enzyme drug acts to remove "toll booths" at the ends of the peripheral nerve roadway.
But because any cellular vehicle can merge onto the road, there is potential for some bad drivers. "People have thought for a millennium that if you got regeneration you could be worse off than with no regeneration at all because of misconnections. You could conceivably develop Huntington's disease-like motor movements or pain, horrible pain. And that's why we don't see spontaneous regeneration in the adult in the first place, because of the possibility of these weird connections. Well, I don't think that's true. As Reggie Edgerton says, a spinal cord is really smart. It's so smart, it can sort this out."
Silver recorded from the graft itself. "It is one mess of firing activity; total epilepsy." There are the good respiratory-related axons traveling along the nerve graft and along with them, it's "mostly garbage," said Silver. "What comes out the other side? It's garbage and some gold, but out to the phrenic nerve, right in that little segment of the cord, is only gold. All the junk that goes in gets filtered out. It's really remarkable – 90 percent of those axons have nothing to do with breathing and all their activity is weeded out." The spinal cord somehow filters out extraneous signals while letting the few breathing signals through.
What cell types are telling the regenerating axons, the ones related to breathing, what to do? "The axons have no innate respiratory rhythms themselves," said Silver. "We think it's interneurons – the relatively short axons that lie between supraspinal projection neurons and the motor neurons that send axons out to the muscles. They are the 'between' neurons and are really important but we don't know much about them. I don't know how the hell they do it. They just figure it out."
Silver has an eye on clinical application. The respiratory work is problematic, he said, since few neurosurgeons would risk surgery on the spinal cord so close to the brainstem. The bladder study involves lower risk surgery and might move more quickly to patients but there is much more to work out experimentally. His group is moving up to a larger animal model to test the surgery and bridge motif in both the breathing and bladder models.
Silver continues to ask the difficult questions that drive solid research. The detour is a solution but not the end of the road. Silver isn't ready to completely abandon the chaos at the injury site. "You probably know that many labs throughout the world now have almost abandoned long distance regeneration as a goal," he said. "I have not."