CLEVELAND, Ohio — Using tools familiar to any gardener -- a chemical fertilizer and a sort of high-tech trellis -- Cleveland researchers have coaxed skittish nerve fibers to bridge a gap in rats' damaged spinal cords and forge new connections.

The repairs, though experimental, revived the rats' partially paralyzed diaphragm muscles, restoring normal or near-normal breathing in nine of 11 test animals, the Case Western Reserve University scientists report Thursday in the journal Nature.

"It's pretty amazing," said CWRU neuroscientist Jerry Silver, who led the research project and described it as the culmination of 30 years of effort. "Our work is one of the most convincing demonstrations [to date] of the return of robust function" after paralysis.

The CWRU approach blends two nerve-regrowth methods, leveraging the power of each.

"The experiments are significant in demonstrating that this combination of two repair strategies can work together to enhance recovery in the complex circuitry that controls breathing," spinal cord injury researcher James Fawcett, who heads Britain's Cambridge Centre for Brain Repair at the University of Cambridge, said via email. "This paper from the Silver laboratory shows that a combination treatment can be very successful." Fawcett was not involved in the CWRU project.

Researchers have tried many techniques over the years to overcome the devastation of spinal cord injuries, which affect 12,000 Americans annually. Progress has been slow, a testament to the human nervous system's complexity.

Much of that experimental work has focused on re-establishing the ability to walk, a goal that remains unmet. Silver's lab has concentrated instead on the challenges of regaining breathing and bladder control. "Our goal was to target one critical muscle that [paralyzed] people would like back," he said.

Those two bodily functions are far less complicated than walking in terms of making neurological fixes. The shortest stroll requires a suite of highly coordinated limb and trunk movements involving dozens of muscles; by contrast, breathing and urination basically involve flexing only the diaphragm or the sphincter. Restoring them could boost paralyzed patients' long-term survival and dramatically improve their day-to-day lives.

Patients with high neck-level spinal injuries -- the most common type -- usually are tethered to breathing machines. They can't smell or taste food, must speak haltingly between breaths, and are vulnerable to infections such as pneumonia. On average, a paralyzed 20-year-old on a ventilator won't live to see his 45th birthday.

The vigorous resumption of breathing in the paralyzed CWRU lab rats suggests "strong hope that this [repair method] could be translated to people," Silver said. But he cautions that it must be verified first in bigger animals, such as cats, dogs or pigs, whose spinal cords are more similar in size to humans.

In a larger, longer cord, nerve fibers would have to cover more distance to skirt the injury site and form new connections downstream. In the rats, the splices were about half an inch. Achieving greater lengths is "a really critical first hurdle," Silver said.

An information conduit

The thick, rubbery spinal cord is the body's information superhighway, a data linkage more sophisticated than any fiber-optic cable or FireWire hookup. It's a two-way conduit connecting brain and body.

The dense bundles of nerve fibers running the cord's length relay commands -- Scratch your ear! Take a deep breath! -- outward from the brain, while simultaneously returning sensations -- Ow! That radiator is hot! -- and other updates from the body.

The signals rocket electro-chemically along lengthy strands called axons, which join a neuron to its neighbors. Neurons in the brain's various control centers send axons downward into the spinal cord, where they link to nerves that have connections throughout the body.

A sharp blow or a violent twist can sever those vital axons in the spinal cord, disrupting the brain-body data exchange below the injury site.

Nerve fibers elsewhere in the body are capable of re-growing and re-connecting -- though sometimes imperfectly -- after being damaged. But central nervous system axons in the brain and spinal cord don't regenerate on their own. When cut, their spidery filaments retract as if scalded, the tips ballooning into angry-looking bulbs.

Over the years, Silver and other researchers have worked to unravel the axons' strange behavior and the mystery of why they don't re-connect. They've discovered that the nerve fibers are reacting to powerful chemical and physical roadblocks that the body throws up as part of its attempts to prevent further damage after a spinal cord injury.

The wound site -- which scientists call the glial scar -- fills with cleanup enzymes that chomp whatever's in their path, whether cellular debris or healthy axons. Potent molecular "keep out" signals warn exploratory neural fibers away; trespassers are met by proteins that short-circuit the nerves' ability to re-wire. Long, chainlike structures called proteoglycans link up to create a protective straitjacket around existing nerve tendrils, hugging them like a burlap sack around a plant's roots.

In short, the wound becomes a toxic, walled-off dead zone.

All this activity probably is meant to be helpful -- to block invading bacteria, preserve whatever intact nerve fibers are left, and curb random, mis-wired connections that could confuse brain and body. In that sense, the glial scar is beneficial, like a bandage. But it also bars nerve regrowth, and the return of sensation and muscle control.

A hostile environment

Though the mechanisms of the scar are complex, the problem it poses is straightforward: how to get something to grow in a hostile setting. It's reminiscent of the challenges of gardening, which happens to be one of Silver's favorite pastimes.

"I think simply, in terms of what it would take if I were a nerve fiber, stuck in the scar, to get where I need to go," the CWRU researcher said. "You need fundamental tools. You need a bridge to get to where you're going, a bridge that will get you over long distances. One that will keep you alive, nurture you, like fertilizer. It'll give you directionality so you don't grow haphazardly, and not let you branch very far."

Silver knew those tools existed.

Since the early 1900s, researchers have experimented with neural bridges, snippets of peripheral nerves borrowed from rats' hind legs. Crushing the peripheral nerve kills its own fibers but preserves a network of revved-up support cells that encourage nearby axons to enter the graft, like a vine snaking down a rain gutter.

A seminal 1981 study by Canadian scientists using these makeshift scaffolds proved that neurons in rats' brains and spinal cords could be coaxed to bud and grow axons more than an inch into the grafts.

Getting them to come out the other side, and more importantly, to form working connections capable of relaying nerve signals, has been difficult.

Silver's own work has dealt with proteoglycans, those net-like proteins that physically ensnare nerve fibers after a spinal cord injury. Since first confirming their presence in the glial scars of rats in 1993, Silver has considered vanquishing the proteoglycans a key to unleashing axons' potential to regenerate.

To clip the proteoglycan net, Silver and other researchers have borrowed a trick that certain bacteria use when they're trying to breach a scar and invade the body. The marauding bacteria churn out a chemical scissors -- an enzyme called chondroitinase ABC, or "chase," for short. Chase slices through the links of the proteoglycan fence, allowing bacteria to slip inside.

Scientists injecting chase into the glial scars of spinal cord-injured rats have shown that it has the same effect, shearing the nets around axons. That makes it possible for the nerve fibers to sprout and grow again. But the results have been disappointing, with little or no recovery of meaningful limb movement.

Two methods better than one

Silver believed that combining the two approaches -- the neural graft and the chase enzyme -- would overcome the limitations of each individual procedure. Chase would unshackle the severed axons so they could start to grow, and the neural trellis would nurture and guide them around the scar, directing them to reconnect with the appropriate nerve node in the spinal cord.

Silver's post-doctoral assistant, Warren Alilain, who is now a researcher at MetroHealth Medical Center and is the study's lead author, mastered the tricky surgical techniques of installing the half-inch-long grafts in anesthetized rats. First, he nicked their spinal cords to paralyze half of the breathing muscle, stilling one lung. He carefully stitched one end of the graft in place on the cord's surface, upstream from the injury, and added a shot of chase. After a week to allow axons to enter the bridge, Alilain anchored the graft's other end to the cord, below the wound site, injecting a dose of chase there, too.

Then, Silver and Alilain waited. And ... nothing.

Days passed, then weeks, with scarcely a flicker of movement in the deadened portions of the rats' diaphragms. Their lungs remained paralyzed on one side. "Warren's got a lot of patience," Silver said. But "we were not seeing recovery in any animal at three weeks, and barely any activity coming back at six weeks."

At two months, the scientists pulled the plug on the experiment. "We were pretty depressed," Silver said. "I questioned my whole [professional] life. Am I done? Because at that time, it was about three years' worth of work."

A lucky break

That's when luck saved the day.

Shortly after calling it quits, Alilain needed to do a routine test of the electrodes the lab uses to gauge muscle activity. The scientist happened to try them on one of the rats from the graft/chase experiment.

"He said, 'Jerry, I see some activity! It's not bad -- better,' " Silver recalls. "I said let's wait a little longer. Between 10 and 12 weeks, activity bloomed in the diaphragm. It just took nearly three months. But when the activity does come back, it just explodes."

Nine of the 11 rats who got the graft/chase treatment regained a "robust" ability to breathe with their previously paralyzed lung, Silver said. (The neural bridges may have pulled loose in the two animals that didn't recover.)

Tests showed the nine recovered rats were able to inhale even more forcefully than normal animals do, though their breaths weren't as deep.

Silver speculates the months-long delay between treatment and the return of regular breathing may be because the regenerating axons need time to build up the necessary insulation. Or it might take a while for the nerve fibers to make proper connections.

Cutting the neural bridge re-paralyzed the rats' revived lung. The CWRU scientists say the shutdown is proof that the nerve fibers that had spread into and through the graft -- not some other mechanism -- were responsible for restoring breathing.

The source of those sprouting axons is a major surprise. Only about 10 percent of the nerve fibers that ventured across the bridge and re-linked to the spinal cord originated in the breathing-control center of the rats' brains.

The rest -- the vast majority of reconnected nerves from the graft -- had nothing to do with respiration. And yet the rats were able to resume breathing almost normally.

That suggests two things, Silver said. First, a small number of neurons can spark the diaphragm to work again. Second, the spinal cord is somehow able to filter out extraneous signals from all those nonrespiratory nerves, while letting the few important breathing signals through. It's comparable to standing in a noisy room and managing to hear a single conversation above the din.

Researchers have worried for decades that reconnecting a severed spinal cord might produce a torrent of confusing nerve messages, like a burst of static, that would throw muscles into a frenzy. In this case, at least, that didn't happen.

"It's remarkable," Silver said. "I think one of the most important parts of this paper is [demonstrating] what the spinal cord is capable of doing."

Future research and obstacles

Future targets for Silver's lab are to lure greater numbers of respiratory axons to cross the bridge around the glial scar, and to grow the nerve fibers for longer distances. That's essential if the technique is to be tried in humans, whose spinal cords are considerably longer than rats'.

Among the hurdles to human testing is what Silver describes as American neurosurgeons' concerns about any procedure that might further harm an extensively paralyzed person.

Those worries didn't dissuade Taiwanese neurosurgeon Heinrich Cheng, who in 2004 treated a single paralyzed patient with a neural bridge and a drug to encourage axon re-growth. Cheng reported that the patient, who was paralyzed below the waist, regained some feeling and eventually was able to switch from a wheelchair to a walker.

"There's a reluctance" in the United States to do such experimental operations, Silver said. "However, if we can show the technique is tolerated in larger animals . . . [and] that a surgeon really could do this subtly, without causing more damage, they might even do it for a quadriplegic."

Whoever advances the CWRU study, its core message is the resiliency of the nervous system – no surprise to an inveterate gardener.

"The nervous system is like a plant," Silver said. "It doesn't want to die. You can take a plant that's just about dead, and if you give it the right soil and some water and a little fertilizer, some magic can occur. All life wants to survive."