See also Part 1.
Paralyzing spinal cord injuries are often no longer than an inch. They might as well be a mile. Repairing severed or severely damaged spinal cords is an intractable medical problem, and progress in solving it has been slow. Using rodent models, researchers are trying various means of coaxing neurons to extend their axons across spinal cord lesions, and several new methodologies were explored at this year’s annual meeting of the Society for Neuroscience, held 13-17 November in San Diego, California. Strategies ranged from rejuvenating neurons by reinitiating developmental pathways to breaking down physical barriers to axon growth in the damaged spine.
Researchers led by Zhigang He at Children’s Hospital, Boston, Massachusetts, achieved some success in rodents by knocking out the phosphatase and tensin homolog (PTEN), which is a negative regulator of mammalian target of rapamycin (mTOR) and a major driving force in neurodevelopment (see Part 1 of this series). At SfN, other researchers reported similar rejuvenation attempts. Paul Lu and Mark Tuszynski, both of the University of California in San Diego, simply grafted young nerves. They transplanted spinal cord neurons from embryonic day 14 (E14) rats into adult cords that are severed halfway through at the neck. Transplantation is a well-known method to enhance nerve repair, Lu told ARF in an interview, but until now, researchers were unsure how far axons from the transplanted neurons spread. It is very hard to track these cells, he said. To get an accurate picture of the transplants, Lu used E14 rat neurons that express green fluorescent protein. In addition, Lu improved upon previous graft techniques by including a fibrin matrix to support the transplanted cells at the injury site.
Lu observed glowing green axons spreading out from the graft site. He estimated that 1,000 axons were growing in a 30-micron section of spinal cord. These grew as far as 30 millimeters when he examined the tissues at three months post-treatment. Moreover, he observed synapse formation, and the treated rats were able to move their hind limbs.
Ryan Williams and Mary Bunge of the University of Miami Miller School of Medicine in Florida used cell transplants of a different sort, combined with a transcription factor, to make adult neurons act like young’uns again. Bunge’s laboratory has long been interested in using Schwann cell transplants to provide fertile ground for axon regeneration (see ARF related news story; Xu et al., 1997; Takami et al., 2002). The Schwann cells prevent secondary damage to remaining neurons and myelinate any remaining axons as well as new, regenerated ones. However, they do not allow for complete recovery, and Bunge believes a multifactorial approach combining Schwann cell transplants plus other treatments will be most effective (reviewed in Bunge, 2008).
In their study, the researchers combined the Schwann cell treatment with a transcription factor they hoped would nudge the adult neurons toward a regeneration-capable phenotype. Mammalian achaete-scute homolog-1 (Mash-1) is expressed during development, when brain stem neurons are sending processes toward the spinal cord. What we’re trying to do, basically, is make the nerve cell bodies younger, Bunge said.
Williams injected AAV vectors carrying the Mash-1 transgene into rats’ brainstems up to six weeks before completely transecting the thoracic spinal cord. He examined the animals six weeks after injury, when he stained tissues for dopamine-beta hydroxylase (DBH), a marker for neurons that reside in the brainstem, far from the lesion site, and send axons down the spinal cord. Those axons from these neurons were cut at the time of injury, but some then grew across the lesion. In the Mash-1-treated animals, there were 2.5-fold more DBH-positive axons, which grew at least 0.25 millimeters into the Schwann cell bridge, than in animals that received Schwann cell transplants alone. A handful of axons grew as far as 2.5 millimeters into the bridge. Moreover, Mash-1-treated animals moved their hind limbs better than the transplant-only rats.
Bunge was cautious about overinterpreting the results. It is not a really strong improvement, she told ARF. We probably need to change a few other genes, as well.
Another challenge in regrowing axons is to knock down the barriers in their way. Scar tissue, composed of proteoglycans, provides both a physical and biochemical barrier to regeneration, Bunge said, because the tissue produces growth inhibitors. One method to dissolve the scar is to use chondroitinase, an enzyme that breaks down proteoglycans. Barbara Grimpe and colleagues at Heinrich Heine University in D?orf, Germany, took a different approach to attack scar tissue. As described by coauthor Martin Oudega of the University of Pittsburgh, the researchers blocked scar tissue formation by targeting the enzyme xylosyltransferase-1 (XT-1), which adds glycosamino glycan side chains to proteoaminoglycans.
Grimpe and colleagues used a deoxyribozyme to knock down XT-1 transcription. These so-called DNA enzymes, widely used in cancer and virus research, are single-stranded DNA molecules that bind and digest a specific target mRNA. Compared to other RNA interference technology, the authors say, DNA enzymes are easier to administer, and cells, including neurons, naturally import the single-stranded DNA by endocytosis. This treatment blocked proteoglycan formation and, in combination with peripheral nerve grafts, boosted descending axon growth nearly 10-fold (Hurtado et al., 2008). Moreover, the scientists reported at SfN, the treatment was safe, and injured rats on the XT-1 treatment were better able to traverse a horizontal ladder than were untreated animals.
It is important that many laboratories investigate many different repair approaches, Grimpe and Oudega wrote in an e-mail to ARF. Bunge agreed: There is great promise in the combination of cell transplantation, administration of growth factors, manipulation of genes, and the application of the enzyme chondroitinase, which will attack the scar, she said.
This is Part 2 of a two-part series. See also Part 1.
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