Assessing and developing treatment for spinal cord injuries has long proved difficult for scientists hampered by a lack of available tools and imaging techniques. Now however, a team of Cornell researchers has developed a method to potentially circumvent this problem by surgically implanting a window into the spinal cord of a mouse, allowing for dynamic and long term imaging at a cellular level.
In an article published in the January issue of Nature Methods, Prof. Chris Schaffer, biomedical engineering, and Matthew Farrar grad designed and inserted a chamber into the backs of mice that enables researchers to view of the cellular interactions in spinal cord injury sites. Working alongside Prof. Joseph Fetcho, neurobiology and behavior, Schaffer and Farrar aimed at identifying ways that researchers could advance the quality of spinal cord injury treatment by developing an improved imaging procedure.
The previous method for observing cells after spinal cord injury was to perform multiple surgeries to image the damaged site. However, according to Farrar, multiple surgeries are harmful to the mice because researchers must repeatedly reopen the skin and risk causing inflammation, increasing the risk of infection and the growth of fibrotic tissue. This makes carrying out multiple surgeries a less than ideal practice.
“The goal here is to be able to gain a better understanding of disease dynamics and to create a platform for the robust evaluation of therapeutic strategies,” Schaffer said. “People have been limited in terms of the tools they had available to study spinal cord injury and to develop strategies that could help injured axons regrow and regain function.”
Axons are like the wires of the nervous system, carrying information between the brain and the rest of the body. The axons in the central nervous system do not spontaneously regenerate in adults. After spinal cord trauma, the damaged ends of the axons degenerate from the injury site.
“In treating spinal cord injury, the first thing you have to do is to get the axons to stop dying back and start growing forward,” Farrar said.
Currently, researchers have two ways of assessing the efficacy of drug therapies designed to repair axonal regrowth after spinal cord injury, Schaffer said. The first is to evaluate behavioral differences between animals treated for spinal cord trauma and uninjured ones. The problem, according to Schaffer, is that the animals used in research respond well to drug therapy in general, so scientists can only observe minute differences.
The other technique is to use histology, or the study of microscopic anatomy of cells. Scientists take tissue samples from animals at different time points after a spinal cord injury. There are two problems with this technique, according to Schaffer: One, researchers cannot watch the same animal over time and examine its dynamic cellular behavior at the injury site. Second, researchers cannot watch for when the axons are growing forward.
“If you consider a bundle of axons in an injured spinal cord, some of them might be dying back, some of them might be growing forward, and some of them might not be injured at all,” Farrar said. “If all I show you is a single picture in time then you can’t know which ones are which and this makes it hard to pick out the overall effect of the drug.”
To solve this issue, Schaffer and Farrar developed a tool that enables imaging of the same axons in the same mouse over time, which allows researchers to determine if an axon is getting shorter or longer. This is crucial because some axons may at first, in response to a drug therapy, begin to grow forward, but with time recede back again, according to Farrar. If the axon is not monitored over time, then one might conclude that the drug was more effective than it really was Farrar said.
After designing the chamber, the team was to induced a very small spinal cord injury in the mice and tagged axons and blood vessels with fluorescent markers. This allowed Farrar and Schaffer to observe the superficially severed axons and their growth behavior.
“The idea that you are going to grow back every axon in someone that has spinal cord injury is probably a long way off. And you probably do not have to. The axons that are most likely to regenerate, I would think, are the ones that sort of most robustly remain in the lesion site. And now we have a way to find them using our chamber,” Schaffer said.
The research team also studied microglia, or the inflammatory cells, of the nervous system while studying of axon regrowth. Schaffer said this part of the experiment confirmed findings by other scientists that microglia can interfere with the ability of axons to grow through an injured region because microglia form what are known as glial scars. After cellular trauma, large numbers of inflammatory cells travel to the injury site, clean up debris and recruit other cells to lay down scar tissue. Some research suggests that scar tissue is not something that axons are able to grow through.
According to Schaffer, scientists disagree about how much inflammatory activity is good. While getting rid of cellular debris is crucial to cell functioning, too much of the microglia to clear the debris can form scar tissue through which axons cannot grow. Schaffer and Farrar’s chamber will allow scientists to better determine how to modulate the invasion of these inflammatory cells.
Schaffer and Farrar said that the next steps for scientists using the chamber they developed will be crucial. “This research was aimed at developing a procedure, an implant and an imaging strategy to be able to see spinal cord injury sites over an extended period,” Schaffer said. “But really that is just the beginning, because now we can begin to look how the milieu of things that are released in a spinal cord injury interacts with each other in a way that seems to favor axonal degeneration.”