Using A Three-Way Regeneration Model

Posted by Sam Maddox in Research News on August 24, 2015 # Research

Clearing out the spinal cord scar using a bacterial roto-rooter; seeding the injury site with fragments of peripheral nerve; souping up inactive spinal cord nerve cells by modifying their genetic blueprints – these are three well-studied ideas that have each captured major mindshare in the spinal cord research community, and have over years of animal experiments and news stories become part of the inventory of hope for people with paralysis.

Now comes a new paper from the Veronica Tom lab at Drexel in Philadelphia. She simultaneously employs all three concepts to power up spinal cord regeneration – in a completely cut, transection model – by digesting the scar barrier with chondroitinase; by adding a growth support matrix using peripheral nerve grafts at the injury site; and by delivering a new gene code to trick axons into thinking they are young again.

And? Yes, this triple whammy seemed to work. This is from the paper, from the Journal of Neuroscience, “Expressing Constitutively Active Rheb in Adult Neurons after a Complete Spinal Cord Injury Enhances Axonal Regeneration beyond a Chondroitinase-Treated Glial Scar:”

We demonstrate, for the first time, that simultaneously addressing neuron-related, intrinsic deficits in axon regrowth and extrinsic, scar-associated impediments to regeneration results in significant regeneration after SCI.

OK, caveat alert: the regeneration was indeed significant but not the kind of growth that reanimates muscles; the injury was treated acutely; and the Tom trifecta is clinically challenging, especially the gene-mod, by way of a permanent code-swap delivered to spinal cord axons by way of a virus. But the work is significant because when therapies do become relevant to people with chronic injuries, and they do target the spinal cord bundles that really matter for function, the underlying scheme is going to tale a combination approach, probably a lot like this one.

I spoke with Dr. Tom to get some background and to learn more about the new paper. She grew up in the New York area and got the bug to study science pretty early on in high school. She went to New York University and was attracted to the brain and its response to injury. “We looked at the idea of nerve plasticity, and the response of nerves after injury. I studied neurodevelopment and began to look at regeneration after injury. I fell in love with this area.” After graduation Tom wanted to continue her work in nerve repair, specifically in the spinal cord. She approached Jerry Silver at Case Western in Cleveland, then and now a major investigator of SCI recovery.

Tom recalls that her first brush with the Silver lab was about the time the lab published a paper in Nature, Stephen Davies as lead author, showing that microtransplants of nerve chunks from the dorsal roots allowed regeneration along spinal cord nerve tracts that hadn’t been seen before. “I though what they were doing there was really cool,” said Tom. Readers here are quite familiar with Jerry Silver; he is as firm a believer in the capacity of spinal cord repair as there is in the field; Tom got her doctorate at Case and clearly a lot of Silver’s optimism rubbed off on her.

Tom got a post-doc position with John Houle, who was in the process of moving his lab from Arkansas to Drexel. Houle is a veteran spinal cord specialist whose work includes transplants and growth factors to promote recovery (he and Tom had previously worked with the Silver lab on an idea to bridge the broken gap of the injured cord, using micropumps to inject chondroitinase, plus PNS grafts, to promote regeneration).

Here’s the basis of Tom’s work:

“It’s clear,” she explains, “that the CNS [central nervous system] does retain some capacity for repair – we’ve known this since the days of [early 20th Century Spanish neuroscientist] Ramon y Cajal. The classic work of [Albert] Aguayo brought this idea back [in the 1980s]. Repair, however, is not really robust. A), there is a brick wall that forms at the site of injury, and B), the motorneurons do not have the capacity for growth that they did during development. I tell my students that the neurons are like people who get old. They can get cranky. And their priorities shift. Mature nerve cells have to maintain synapses [connections between nerve cells] and do not have the need for a robust growth response, as young neurons do.”

So, the strategy is to break the brick wall, the scar, using chondroitninase, AKA ch’ase, and then to revive nerve cells’ growth patterns by changing their gene coding. The third part of the study, using peripheral nerve grafts (PNS), was necessary, Tom says, because nerve axons require a growth substrate. “They do not grow in space,” she said.

Tom knows ch’ase can help with the scar; she and Houle published work showing regeneration and functional recovery in animals treated with ch’ase. Its promise hasn’t been realized because there is not yet a clinically relevant way to deliver the scar buster. In this latest research, she and her team used implanted osmotic minipumps to squirt the enzyme intrathecally, directly to the interface with the peripheral nerve grafts, for at least two weeks. Because ch’ase alone is not stable at body temperature, they mixed it with a sugar compound.

As for the gene mod, that’s complicated. Tom credits Zhigang He and his work with turning off the PTEN gene to restore growth capacity in spinal cord nerve cells. Turning off PTEN turns on mTOR, so Tom and her group decided to take different approach to do the same thing. Rheb (stands for Ras homolog enriched in brain) is a molecule that directly upregulates mTOR. It gets delivered to nerve cells using a virus-based vector.

Permanent viral-based gene manipulation, Tom admits, might make people “squeamish.” She reminds us that PTEN is a growth switch that was discovered by people looking for clues to cancer. They want if off; we want it to be on. But at some point, we too may want it off. The technology is not there yet, says Tom. But in her experiments, it all seems to hang together, a least in principal, using a cord injury model that is much more damaged that what would be found in human SCI.

Neurons in large numbers grew into the PNS grafts. These neurons were from the propriospinal area of the cord, all located fairly close to the region of injury. There was no evidence that more functionally important neurons from the brain – so called supraspinal neurons – regrew . From the paper:

To our knowledge, no study has definitively established that increasing mTOR activity after a complete SCI promotes axonal regeneration of injured fibers. Here we asked whether caRheb-mediated activation of mTOR in neurons following SCI enhances axon growth. Expressing caRheb alone was not sufficient to spur regrowth of axons out of a PNG. However, combining condroitinase treatment of the glial scar and neuronal caRheb expression significantly augmented the number of propriospinal axons capable of reinnervating distal spinal cord. Importantly, these axons formed putative synapses. The functional relevance of this regeneration will be assessed in future experiments.

And so the work continues. Tom and her group want to see if some form of the triple treatment can be useful in a chronic model; they also want to see if they can get brainstem nerve cells to regenerate and cross the scar on the PNS bridges.

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