Capitalizing on the flexibility of tiny cells inside the body's smallest blood vessels may be a powerful spinal cord repair strategy, new research suggests.
In mouse experiments, scientists introduced a specific type of recombinant protein to the site of a spinal cord injury where these cells, called pericytes, had flooded the lesion zone. Once exposed to this protein, results showed, pericytes change shape and inhibit the production of some molecules while secreting others, creating "cellular bridges" that support regeneration of axons - the long, slender extensions of nerve cell bodies that transmit messages.
Researchers observed axon regrowth in injured mice that received a single treatment injection of the growth-factor protein, and the animals also regained movement in their hind limbs. An experiment involving human cells suggests the results are not restricted to mice.
"There's a lot more that can be learned and a lot that can be expanded, but the more we worked on this, the more stunned we really were by the potency of this single treatment and how effective it was," said senior study author Andrea Tedeschi, associate professor of neuroscience in The Ohio State University College of Medicine. "This finding goes beyond spinal cord injury - it has implications in brain injury and stroke, and neurodegenerative diseases as well."
The work underscores how important blood vessel restoration is to recovery of neurological function after a spinal cord injury, researchers said.
"Spinal cord injuries are severe not only because they prevent transmission of information across the site of the injury, but because all of the vasculature structure and function is also compromised," said first study author Wenjing Sun, assistant professor of neuroscience at Ohio State. "Even if you are able to reestablish neuronal connectivity from one end to the other, the overall effect will still not be maximized unless you take care of everything else that falls apart."
The study was published April 18 in the journal Molecular Therapy.
Previous research suggesting pericytes interfere with spinal cord injury recovery had led some scientists to recommend clearing them from the lesion site to aid repair. But cancer research has indicated pericytes' properties change when they're exposed to a protein called platelet-derived growth factor BB (PDGF-BB) - which is one way tumors generate their own blood supply. In cancer, the aim is to block PDGF-BB signaling.
Earlier neuroscience research also indicated that pericytes are highly "plastic," meaning they are very responsive to changes in the microenvironment - including the presence of PDGF-BB. Tedeschi and colleagues saw potential to harness that cell-protein relationship to stabilize the vasculature surrounding a spinal cord injury. In the process, they found the newly sprouted blood vessels established a pathway for regenerated axons to follow.
Starting with imaging studies, the team showed that when a spinal cord is severed, pericytes migrate into the injury site over time but don't promote growth of functional blood vessels that are needed to support axon regeneration.
In cell-culture experiments, the researchers established a "carpet" of pericytes, added PDGF-BB, and then placed a layer of adult mouse sensory neurons on top and evaluated how much axons grew in 24 hours. The treated axons grew nearly as much as healthy axons extend under normal conditions.
PDGF-BB alone did not produce this result. Instead, experiments showed that pericytes combined with the growth factor rearranged fibronectin, a multifunctional adhesive glycoprotein that plays a critical role in tissue repair, cell attachment and motility. The cells themselves also change shape, becoming more elongated.
"We know these cells are going to infiltrate and deposit at the lesion epicenter. These elongated fiber structures that they become are far more permissive in promoting axons to regenerate from one end to the other and bypass the injury," Tedeschi said.
"To extend the clinical relevance of our findings, we cultured mouse neurons on top of human pericytes that were exposed to PDGF-BB, and that was sufficient to trigger a growth-promoting effect, suggesting that this might really be a generalized phenomenon that is not restricted to mice."
Turning to experiments in animals with spinal cord injury, researchers waited for seven days after the injury - the equivalent of about nine months in a human adult - before injecting a single dose of PDGF-BB at the injury site. Analysis of tissue four weeks after the injury showed that the PDGF-BB injection produced robust axon regenerative growth compared to the axon response in injured control mice.
"When we looked at formation of these pericyte structures that crossed the injury site, we saw the treatment promoted the growth of these bridges. And most if not all of these regenerating axons were able to escape the injury site by riding these cellular bridges that have formed in response to PDGF-BB administration," Sun said.
Electrophysiological and movement assessments of injured animals treated with PDGF-BB detected sensory activity beyond the lesion site and showed the mice regained better control of their hind limbs compared to control mice. The animals also were less sensitive to a non-painful stimulus, suggesting they did not experience the neuropathic pain that is often triggered by a spinal cord injury.
Analysis of the presence of inflammatory proteins during the repair process suggested that PDGF-BB administration not only promotes axon regeneration, but also reduces inflammation. RNA sequencing showed that spinal cord injury led to decreased gene expression by pericytes, but that the cells retained their core properties and did not convert into a different kind of cell - for example, a cell type that could end up being destructive to the injury environment.
"There was a decrease in some classical pericyte markers, but a gain of some additional function linked to the attempt to rebuild cellular bridges and functional vessels," Sun said. "From the overall gene signature in our data, they're still classified as a pericyte."
Because Tedeschi, Sun and colleagues have previously shown in mice that gabapentin promotes regeneration of neural circuits after spinal cord injury, there's potential to consider a multipronged approach to therapy, Sun said.
"We could combine both - modulating intrinsic properties of adult neurons with a drug, and what we are doing here, modulating the non-neuronal environment to produce cellular interactions that provide a more permissive substrate for the neuron to grow on," she said.
More work is planned to determine the precise timing for administration of PDGF-BB - with the presumption that pericytes take some time to migrate to the injury - as well as the ideal concentration of the treatment and a potential time-released delivery system.
This research was supported by the National Institute of Neurological Disorders and Stroke and Ohio State's Chronic Brain Injury Program.
Additional co-authors were Elliot Dion, Fabio Laredo, Allyson Okonak, Jesse Sepeda, Esraa Haykal, Min Zhou, Heithem El-Hodiri, Andy Fischer, Juan Peng and Andrew Sas, all of Ohio State, and Jerry Silver of Case Western Reserve University.
