To Grasp the Cord

Spinal cord injury research in Canada seeks improved basic understanding and successful regenerative, repair and diagnostic methodologies

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By Deborah Komlos

It may send a chill down your spine to realize the complexity and sensitivity of the region that controls our essential functions. In fact, that region lies right within the spine itself.

Protected by the spinal column, the spinal cord is the information conduit linking the body and the brain. Via neuronal communication between the brain and spinal cord — the two regions that make up the central nervous system (CNS) — voluntary, involuntary and sensory functions can occur, such as walking, breathing and feeling pain, respectively. Trauma to the spinal cord affects body areas situated below the injury level, and an injury can be either complete or incomplete, depending on the extent of the nerve damage.

“The majority of the spinal cord injured patients are partially injured; that means there are some residual groups of nerve fibres that are preserved,” says Wolfram Tetzlaff, MD, PhD, associate director of Pre-clinical Discovery Science at ICORD, the International Collaboration On Repair Discoveries at the University of British Columbia (UBC) (Vancouver, BC). “The trick is to actually engage those spared fibre tracks in the spinal cord to actually become functionally useful.”

Also a professor with UBC’s department of Zoology, Tetzlaff is one of approximately 50 principal investigators associated with ICORD, an interdisciplinary research organization that is working to develop strategies for promoting functional recovery and improved quality of life following spinal cord injury. A new 10,000-square-metre facility to be located at the Vancouver General Hospital (Vancouver, BC) will house the researchers beneath one roof. Contingent on funding approval for ICORD, groundbreaking for the new centre is projected for early to mid 2005, says Chris McBride, PhD, ICORD’s managing director.

Key Concepts
Tetzlaff’s research involves studying the response of neurons to injury.

“You have to understand that neurons in the brain send their processes to the spinal cord to instruct the motor neurons and others in the spinal cord to, for instance, execute a movement,” Tetzlaff explains. “And these long, fine processes get interrupted after spinal cord injury. And our question is, first of all, do these neurons understand that their processes have been interrupted, and do they make the right efforts to regrow these processes?”

Neuronal regeneration after spinal cord injury is complicated, Tetzlaff says, by the presence of inhibitory scars and other factors, such as myelin-associated inhibitors.

“The wisdom was for a long time that many of these nerve cells actually die after spinal cord injury,” he says. “What we described . . . is that they actually shrink; they don’t die. They shrink beyond detection and sit there, and they sit there even for over a year in the rat and you can revive them. And then you can actually promote their regrowth if you provide them with a decent growth substrate to grow their axons on.”

Tetzlaff’s team has found that application of brain-derived neurotrophic factor to cell bodies in the midbrain of rats that had their rubrospinal neurons’ axons cut in the spinal cord one year previously resulted in regeneration of these axons. (Fig. 1) As the majority — more than 90 per cent — of human spinal cord injuries were sustained more than a year ago, Tetzlaff says research on chronic injury is important. “Most of the therapies that are aiming at neuroprotection are immediate regeneration and so on; these are therapies that work in the acute setting, but they don’t work in the chronic setting,” he says.

Other research includes Tetzlaff’s work with fellow UBC and ICORD researcher Jane Roskams, PhD, investigating the use of olfactory ensheathing glial cells derived from the nasal mucosa to promote regeneration of nerve processes after spinal cord injury. This approach is novel, Tetzlaff says, because other groups worldwide that are investigating these cells take them from the olfactory bulb in the brain, and this does not translate well into a clinical setting. The more practical source would also benefit autotransplantation, he notes. “It’s much more likely that your own cells stay alive. You can get them from your own body without necessity to immune suppress,” he says.

“The theory is that (the olfactory ensheathing glial cells) would guide the growth of axons,” Tetzlaff says. In both rat and mouse models, the researchers have found promising results with the transplanted cells, with numerous axonal populations sprouting into and across the spinal cord lesion sites.

While these transplanted cells are not identical to the bulb-derived ones, “they have quite remarkable overlapping properties,” Tetzlaff notes. But more work is needed, he points out, because regeneration is one component, but seeing functional recovery is another.

“Often times in our research, it’s not possible to be 100 per cent sure whether a particular regenerated fibre was giving you the increased function,” he says. Any number of the hundreds of thousands of fibres may also be stimulated to grow, he says, and neuronal plasticity may contribute to functional recovery as well. His team is currently designing experiments to tease apart these possibilities.

Bridge Building
Molly S. Shoichet, PhD, a University of Toronto (Toronto, ON) associate professor in the department of Chemical Engineering and Applied Chemistry, emphasizes the importance of a combination of strategies to study spinal cord injury.

Shoichet’s lab has taken two approaches in the rat model, both involving a tissue engineering regenerative strategy. The first involves an intubulation strategy on a fully transected spinal cord, an injury that may result from a stab or gunshot wound. “So the idea is that you have two nerve ends and you put a tube in, and the tube serves as a nerve-guidance channel. So it provides a permissive environment for regeneration within the tube,” says Shoichet, the Canada Research Chair in Tissue Engineering. (Fig. 2)

The strategy uses hydrogel tubes filled with a cocktail of fibrin, fibroblast growth factor (FGF)-1 and peripheral nerve tissue. Over a 15-week period, significant treatment differences were noted, as measured by the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale that assesses functional recovery after spinal cord injury, and ranges from zero (complete paralysis) to 21 (complete mobility). With the tube and cocktail treatment, the best rat scored eight or nine, Shoichet says, with an average of five, versus an average of three with the other strategies. Her team is currently replicating the work and expects results over the summer.

A less-invasive approach involves injection into the spinal cord of a fast-gelling collagen containing epidermal growth factor and FGF-2. This treatment is for a compressed spinal cord, Shoichet says, and while the group has demonstrated that it is a safe delivery mode, further combination strategies are being explored.

Overall, much more work is needed, she says.

“The transection injury model is an excellent injury model for examining methods that will stimulate regeneration. But I think the bar is higher for us to demonstrate more regeneration before that would be tested in humans,” Shoichet says. The less-invasive strategy is closer to application, she says. “But again, we don’t have the magic bullet,” she adds. “And again, I don’t think there’s just one.”

To further advance the technology toward products, Shoichet founded matRegen Corp. (Toronto, ON) in 2002. The firm is currently developing its platform technology SpinFX for a series of applications, including neurosurgical.

Shoichet says clinical trial work is important to demonstrate activity in spinal cord research, even when the trials are not successful — a recent example being Acorda Therapeutics Inc.’s (Hawthorne, NY) announcement that its two Phase III chronic spinal cord injury trials failed to meet their primary end points. “If there are no clinical trials, then there’s less hope. And with clinical trials, it doesn’t mean they’re going to work, but at least there’s something that’s gotten to that stage,” she says.

Stimulating Results
Predicting when her work may get to clinic is something Vivian K. Mushahwar, PhD hesitates to do because much more experimentation is required over the next five years to show whether or not it has clinical feasibility, she says.

An assistant professor with the University of Alberta (Edmonton, AB) department of Biomedical Engineering, Mushahwar works with a technique called intraspinal microstimulation (ISMS), a procedure in which six to 15 microwires — about 25 to 30 microns in diameter — are implanted into each side of the spinal cord. Through these wires, 10 to 300 microamperes of electrical stimulus is delivered, Mushahwar says, to elicit functional limb movement.

In both a rat and cat model, the implanted wires are localized in a tiny region of the spinal cord called the lumbosacral enlargement. In adult humans, this region is about five centimetres in length and is located at the base of the back. The wires are linked to an external device where stimulation is applied.

“The more we understand about this region from the basic science, the better off we are because we can target these control centres and have them move the leg in more natural ways than what you would get by stimulating each muscle separately,” Mushahwar says. “The small implant would now be condensed in a five-centimetre region, as opposed to long wires implanted throughout the leg, for example, as is conventionally done right now.

“The idea there is to see if we can target control centres, like control centres for the leg in the spinal cord, and take advantage of that complicated circuitry that’s already available for us in the spinal cord and basically provide it with this artificial stimulus that would have been provided by the brain had the brain still been connected to there,” Mushahwar says.

Her team has administered ISMS for up to six months, a duration that she says has been used by many experimentalists going from the animal model to human because it is long enough to establish stability of any approach. “If the implant is going to move a bit out of its place for whatever reason, it happens in those early days of the implantation; it won’t happen later,” she says.

Mushahwar says her team has been repeatedly successful in the animals, but many questions remain to be answered. For one, can the microwires stay implanted for a long period of time and not harm the spinal cord? Second, can the same limb movements be elicited over and over again?

The ultimate goal, she says, is to establish functional leg movements that would permit a daily total of at least four hours of standing and walking. “I’m talking walking at best with crutches because balance is a difficult issue, and what we’re targeting is leg movements without consideration to trunk balance right now,” she says.

The fact that not all injuries are sensory complete is an important consideration, Mushahwar emphasizes, because individuals may still have sensation even though they lack voluntary movement. “So if you’re going to stimulate in the spinal cord, you really want to make sure that you’re not evoking responses . . . there are regions that we completely avoid. We avoid them because we know that these are the regions where sensory information comes in — pain and skin sensation and things like that that could cause discomfort,” she says.

“We really shouldn’t be forgetting about the bladder and sexual function,” Mushahwar adds, mentioning that there are research groups addressing these concerns. “People want that back, and individuals with paraplegia rank standing, walking, bladder, sexual function at just about the same level. They want them all back. They do put sexual function first, though.”

Visual Displays
Developing effective spinal cord injury treatments remains a top priority, but applications to better assess injury complement those efforts.

Krisztina L. Malisza, PhD, a scientific officer with the MR Technology Group, Institute for Biodiagnostics, National Research Council of Canada (Winnipeg, MB), is exploring this area through the use of functional magnetic resonance imaging (fMRI) in a rat model.

“What we’re trying to do is to develop a tool that can be used by researchers and clinicians to demonstrate where there is neuronal function that’s maintained in the spinal cord, either in healthy individuals — how that function shows up on the functional images — or once there’s been an injury, and what the results are of the injury and where the neuronal function is, how it changes over time and things like that,” Malisza says.

Malisza, who is also a member of the Spinal Cord Research Centre at the Faculty of Medicine, University of Manitoba (Winnipeg, MB), says the current goal is to thoroughly evaluate the tool in order to prove that it is showing neuronal function. “So that we can actually prove that, OK, I’ve got functional activation here in the grey matter in the cord, and show that it actually does correlate to where there is actual neuronal activation,” she says. This involves correlating results of the functional imaging with direct measurements by gold standards such as histology, she says.

Malisza explains that while functional imaging was developed in the early ’90s, its application was mainly for the brain, and shifting to the spinal cord — which began about six years ago — has been a challenge.

For one, bone affects the quality of the image, and motion of the spinal column will give rise to artifacts that, in turn, produce false positive regions or false negative regions, Malisza says.

The technique of fMRI uses a slight modification of BOLD (blood oxygen- level dependent) fMRI, which is used for the brain. The variation is termed SEEP (signal enhancement by extravascular protons), and enables better images because it avoids the difficulties related to fMRI of the spinal cord, Malisza says.

Rather than using X-rays, which are harmful, or having to inject a contrast agent to properly visualize details, “fMRI is a non-invasive technique in that it’s only looking at what’s naturally within your body, within the animal,” she says. “You’re only looking basically at water, and you’re exciting the spins of the protons in the water and you’re detecting that when you’re getting an MR image.”

The team has yet to enter an animal injury model, but that is a goal, Malisza says. Currently, electrical stimulation of the rat forepaw is used to detect regions of functional activation in the spinal cord.

To the Clinic
For BioAxone Therapeutic Inc. (Montreal, QC), moving to the clinic is just around the corner. The firm is currently completing its preclinical phase for Cethrin®, its therapeutic protein for the treatment of acute spinal cord injury, and plans to move into clinical trials by the second quarter of this year, says BioAxone’s CSO Lisa McKerracher, PhD. The firm submitted its IND for Cethrin in April.

Cethrin is a fusion protein that is an antagonist of Rho, a GTPase that regulates intracellular motility. By inactivating Rho — which becomes abnormally activated after spinal cord injury — the researchers have demonstrated axon regeneration as well as neuroprotection, by protecting cells from apoptosis. (Fig. 3)

“What we have found is that you do have an abnormal activation. It occurs within the earliest time point we’ve looked at, which is an hour and a half,” McKerracher says. That activation is reversed, she says, when Cethrin is administered.

“It’s a local application during a decompression or a fixation surgery that’s normally done afterwards, and about 80 per cent of the patients will have that surgery done,” she explains. “And it’s actually applied on top of the cord and has the ability to diffuse right into the cord. So we don’t have to inject needles. We don’t have to put in catheters or those kinds of things. So it’s fairly simple delivery.”

McKerracher agrees with the common sentiment that in the future, effective treatments for spinal cord injury will likely involve a combination of therapies. But for now, progress can be made gradually through examination of the acute spinal cord injury model, she says.

“We’re not going to have miraculous treatments and recoveries. I think in most medical fields, the improvements are done in small steps,” McKerracher says. “But in the case of spinal cord injury, those small steps can make a huge difference in quality of life for a patient.” SEEP (signal enhancement by extravascular protons) fMRI, and enables better images because it avoids the difficulties related to functional imaging of the spinal cord, Malisza says.

Rather than using X-rays, which are harmful, or having to inject a contrast agent to properly visualize details, SEEP fMRI “is a non-invasive technique in that it’s only looking at what’s naturally within your body, within the animal,” she says. “You’re only looking basically at water, and you’re exciting the spins of the protons in the water and you’re detecting that when you’re getting an MR image.”

The team has yet to enter an animal injury model, but that is a goal, Malisza says. Currently, electrical stimulation of the rat forepaw is used to detect regions of functional activation in the spinal cord.

To the Clinic
For BioAxone Therapeutic Inc. (Montreal, QC), moving to the clinic is just around the corner. The firm is currently completing its preclinical phase for Cethrin®, its therapeutic protein for the treatment of acute spinal cord injury, and plans to move into clinical trials by the second quarter of this year, says BioAxone’s CSO Lisa McKerracher, PhD. The firm submitted its IND for Cethrin in April.

Cethrin is a fusion protein that is an antagonist of Rho, a GTPase that regulates intracellular motility. By inactivating Rho — which becomes abnormally activated after spinal cord injury — the researchers have demonstrated axon regeneration as well as neuroprotection, by protecting cells from apoptosis. (Fig. 3)

“What we have found is that you do have an abnormal activation. It occurs within the earliest time point we’ve looked at, which is an hour and a half,” McKerracher says. That activation is reversed, she says, when Cethrin is administered.

“It’s a local application during a decompression or a fixation surgery that’s normally done afterwards, and about 80 per cent of the patients will have that surgery done,” she explains. “And it’s actually applied on top of the cord and has the ability to diffuse right into the cord. So we don’t have to inject needles. We don’t have to put in catheters or those kinds of things. So it’s fairly simple delivery.”

McKerracher agrees with the common sentiment that in the future, effective treatments for spinal cord injury will likely involve a combination of therapies. But for now, progress can be made gradually through examination of the acute spinal cord injury model, she says.

“We’re not going to have miraculous treatments and recoveries. I think in most medical fields, the improvements are done in small steps,” McKerracher says. “But in the case of spinal cord injury, those small steps can make a huge difference in quality of life for a patient.”