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If You Give a Mouse a CONCUSSION

At an NIH lab in Maryland, a brilliant intern stumbled on a novel way to observe the brains of concussed mice in real time. The resulting research is transforming ideas of how the brain reacts to mild traumatic injury and pointing the way to possible treatment

ONE OF the most important recent developments in the treatment of brain trauma—and by extension, the future of football—may have been discovered by a clumsy intern.

Theo Roth, 22, is a St. Louis--born, Alabama-raised Stanford graduate who finagled his way into a National Institutes of Health internship in the summer of 2010, after his senior year at Vestavia Hills High near Birmingham. With a recommendation from a mentor of his parents (both doctors), Roth appealed directly to Dr. Dorian McGavern at the NIH's Bethesda, Md., research campus. That helped Roth sidestep more than 10,000 college kids who were gunning for 1,000 spots. McGavern's team was using a new research technique, pioneered in 2007 at New York University, which involved shaving down a small portion of a mouse's skull to shine light into the brain and record its processes. McGavern wanted to study the effects of meningitis, but his new intern couldn't handle the tiny ballpoint saw without concussing the mice and muddling the results. "He was really bad," McGavern says of Roth. "Just couldn't get the hang of it."

Roth's ineptitude was—and remains—difficult to accept for a kid who scored 35 out of 36 on his ACTs and 2350 out of 2400 on his SATs. "It's really hard to do, and they only gave me a week to learn," he says. "They have neurosurgeons come in and get it wrong."

But in the subsequent images of the rodents' concussed brains, Roth and McGavern observed a flurry of action: leakage from the blood vessels that line the skull seeping down and damaging brain tissue. Toward the end of the summer the two started talking about what they had seen. Roth, a former high school wrestler and a St. Louis Rams fan, began to grasp the larger significance of their findings. "We saw the [concussed] brain operating in ways no one had ever recorded before, and right around the same time traumatic brain injury was becoming a hot topic," Roth says. "People were starting to realize how detrimental it was in the NFL and for guys coming back from Iraq and Afghanistan."

Roth and McGavern were chatting one morning in the cramped computer room in McGavern's lab when something clicked. "It was like, Wait a minute, we're actually re-creating what happens in a mild traumatic brain injury," Roth says. "This may actually be very important."

It became Roth's personal project to study concussed mice—after all, he was the best in the lab at concussing them. Roth shaved the bone of the fully anesthetized mouse from a density of one millimeter to 30 microns, about the width of a human hair. The mouse was then strapped under a two-photon microscope, a machine about four feet tall connected to a computer that allows for imaging of living tissue. Over the rest of that summer and the summer of 2011 the bright blue, green and purple images on the computer screens gave McGavern and his team an outline for the mechanisms of damage from minor head trauma. Here's a thumbnail of what happens:

• A mild brain injury occurs—in this case, when skull is pressed into brain.

• The impact damages blood vessels lining the skull, causing some to burst or leak.

• The body responds, in part, by producing molecules called reactive oxygen species (ROS), which mistake the injury for the intrusion of a foreign body.

• Useful in fighting bacterial infections such as E. coli, the ROS swarm around the injury and instead of healing cause damage by tearing up the glial limitans, the thin membrane separating the brain from the fluids around it.

• Fluids carrying ROS from the damaged blood vessels leak through the new holes in the membrane and come into contact with brain tissue, destroying it.

McGavern believes this process could play a fundamental role in the development of chronic traumatic encephalopathy (CTE), the disease that has been found in the autopsied brains of deceased football players, including Bears safety Dave Duerson and Chargers linebacker Junior Seau, who both committed suicide. It's also listed as evidence in the case made against the NFL by thousands of former players who sued over the alleged mistreatment of concussions for decades by NFL doctors. CTE and the concussion epidemic are the reason the NFL agreed to pay $765 million to those players, and to allocate $30 million to the NIH to fund studies on the biggest problem confronting football at all levels of the game.

But the NFL wasn't funding this NIH project. This one was led by a researcher whose main interest was viral infections and an intern with shaky hands and a special mind. Roth became obsessed. He went to Palo Alto with mice on his mind. He joined the Stanford marching band, which became his distraction, but as soon as winter and spring breaks began, he was on a plane east. "I was much more excited to be doing the research than I was to be taking classes," he says. "It was a little rough to be studying for a midterm when I was thinking about the next experiment we could do. I was doing work that was brand new, as opposed to learning things from a book."

HAVING OBSERVED the process of concussions in real time, the researchers brainstormed ways to treat them. McGavern recalled that in grad school at the Mayo Clinic his wife had worked on ROS and ways to block them, using an antioxidant called glutathione. McGavern's team obtained the readily available organic chemical, and Roth tried it out on mice. He concussed the mouse, placed a small quantity of the drug on top of its skull and observed it under the microscope. "You think of the skull as a bone that keeps everything out, but it is a porous filter," McGavern says.

The day after their first experiment with glutathione, McGavern arrived at 9 a.m. and found Roth grinning, having slept at the computer. "I saw him, and I already knew that we had it," McGavern says. "He showed me the result, and the cells look like they are totally naive. It looks like there's no injury that's happened whatsoever. It looks like a normal brain.

"After that Theo lived in the laboratory, 16, 18, 20 hours at a time. He laid his pillow out on the keyboard and would just sleep between experiments. He was a man possessed."

McGavern reached out to Dr. Lawrence Latour working one floor below, who had been studying concussions in human patients for years. At two local hospitals concussion patients were invited to participate in an NIH study for which they were injected with dye and an MRI was taken of the brain. In half of the patients with mild brain injuries the dye showed up in the brain tissue, which meant the same process observed in mice was happening in humans.

"You can't get down to the resolution we can see with the mice," McGavern says, "but if you look at the human brain, you saw the leakage."

Roth became the lead researcher on a paper published in the journal Nature that made national headlines last fall. He reported, among other findings, that passing glutathione through the mouse skull immediately after a concussion reduced brain tissue damage by an average of nearly 70%.

(The anesthetized mice endure no pain during the procedure, McGavern says, and are able to live normally with their thinned skulls, but typically they're killed and dissected to further examine their brain tissue. McGavern has no qualms about using mice in his studies. "When you consider the possible benefit for our species," he says, "it's an easy call.")

Roth was elated. All those spring breaks spent in a lab devoid of daylight, staring at a computer—they meant something now. This was not a cure for brain injury, but it might lead the way to a concussion treatment.

"I don't know if I've ever had a person better than him in my lab in 10 years of doing this," McGavern says. "He's just brilliant. The amount of information he can collect is ridiculous. And the dedication—think of all the spring break destinations; Cabo, Florida—and instead he was at the lab."

"An incredible feeling," Roth says.

Next up for Roth is grad school. He applied to 20 M.D.-Ph.D programs and got into most, choosing UC San Francisco. As for the research, McGavern and Roth's work spawned two more studies. McGavern's lab will look into the long-term effects of multiple brain injuries, with and without antioxidant treatment. Within the next several weeks another researcher at NIH, with McGavern's help, will study the treatment on pigs, whose skull thickness is very similar to humans'. While more work needs to be done to see whether such therapy would be effective on humans, someday, McGavern says, a person could be treated with a dose of antioxidant pressed to the scalp immediately after brain trauma. There would be no clear telling if it worked, short of a dye MRI and, for football players, a decrease over time in the number of players who suffer loss of brain function.

No doubt Roth would like to get in on subsequent research, though he's not sure he's capable of devoting 16 hours at a time to the cause. "I was young and had plenty of energy then," says Roth. "It feels like I'm getting old."

Roth's paper reported, among other findings, that passing an antioxidant through the skull immediately after a concussion reduced brain tissue damage by nearly 70%.



A LOOK INSIDE The two-photon microscope can peer through the mouse's skull to provide images of live tissue.



POWER OF CIRCUMSTANCE Roth's original difficulty in preparing subject mice for a meningitis study turned into an apparent breakthrough in concussion science.



LOOKING AHEAD Roth (left) spent his college summers and time off working with McGavern (above) on the groundbreaking research. Their findings have spawned further studies on glutathione treatment for concussions.



[See caption above]