The Long and Winding Path of Axon Regeneration
After spinal cord injury (SCI), nerve fibers (axons) have a hard time growing across the area of damage to reestablish functional connections, which often results in loss of sensation and movement below the level of injury. In research laboratories, many different models of SCI are utilized to better understand injury processes and develop therapies for improving function. In rodents, intraorbital optic nerve crush is commonly used as a model to study axon regeneration. The optic nerve transmits visual information from the retina to the brain and is composed of the axons of retinal ganglion cells (RGCs). After the optic nerve is crushed, axons degenerate and cell death occurs, similar to what happens in the spinal cord following injury.
Scientists have developed various strategies to improve optic nerve regrowth and have identified numerous genes that play a role in axon regeneration. By modifying those genes, researchers can make axons grow longer into, and sometimes through, the injury site. However, the paths of regenerated axons are often misguided and end up growing in the wrong direction.
Kevin Park, PhD, an Associate Professor at The Miami Project, and the researchers in his lab noticed that the paths of regenerating axons were particularly misguided in areas around the injury site. They figured that glial cells, which become activated immediately following injury, might be disrupting the path of regenerating axons. They wondered what would happen if axons were encouraged to regenerate at a later time point, after the glial cells return to a normal “un-activated” state.
Dr. Park and his colleagues introduced a gene (ciliary neurotropic factor, CNTF) using a virus (adeno-associated virus, AAV), which allows the genetic material to enter the cells. The CNTF gene encodes a protein that has been shown to promote axon regeneration in previous studies. In one group of animals, they injected AAV2-CNTF immediately following injury. In the second group of animals, they waited eight weeks, which is considered the “chronic phase” of injury in a mouse model, before injection the AAV2-CNTF. In both groups, they waited 3 weeks and then looked at how the axons were regenerating (Yungher et al, 2017).
They discovered that RGCs were able to regenerate long after injury, suggesting that there is not a critical time window for intervention. They saw enhanced growth rates in axons after AAV2-CNTF injection, even after treatments that was administered eight weeks following injury. However, they found that very few RGCs survived injury, with only 4% living eight weeks later. For this reason, they wanted to see whether a larger pool of cells could be triggered to regenerate their axons, as long as they are protected from cell death.
So, they brought in a special type of mouse that had been genetically engineered to undergo very little cell death following an injury to the nervous system. The Bax gene, which regulates cell death, was eliminated from the DNA of these mice, so they are called “Bax knockout (KO)” mice. Dr. Park and colleagues repeated their delayed intervention experiments with AAV2-CNTF and compared the response in Bax KO mice to the response in normal, wild-type mice.
In the Bax KO mice, they saw similar rates of axon regeneration, whether treatment was provided immediately or eight weeks later. In the animals that were treated immediately with the gene that promotes axon regeneration (CNTF), they actually found that a smaller portion of RGCs were capable of regeneration in the Bax KO mice, even though there were many more cells. Although it is unclear why the majority of RGCs failed to generate, it seems as though the surviving RGCs are resilient and able to generate axons long after injury.
When they compared the paths of regenerating axons, they found more linear growth in animals treated at the later time point. By then, the environment around the injury site may be more permissive, since many of the growth factors and cytokines present immediately after injury may be downregulated.
In order to more comprehensively evaluate the axon growth patterns near the injury site, Dr. Park and colleagues collaborated with other Miami Project researchers to label the axons, making individual axons easily visible (Bray et al, 2017). With the help of Dr. Pantelis Tsoulfas, along with their laboratory groups, they were able to see that, although the axons are capable of regenerating long distances, particularly after CNTF treatment, the growth is not in the right direction. The results of their work highlight the importance of chronic regenerative and protective strategies, as well as encouraging axon growth in the right direction.
Yungher BJ, Ribeiro M, Park KK. (2017). Regenerative responses and axon pathfinding of retinal ganglion cells in chronically injured mice. Investigative Ophthalmology Visual Science. 58: 1743-1750.
Bray ER, Noga M, Thakor K, Wang Y, Lemmon VP, Park KK, Tsoulfas P. (2017). 3D visualization of individual regenerating retinal ganglion cell axons reveals surprisingly complex growth paths. eNeuro. 4(4). pii: ENEURO.0093-17.2017.