Medical University of South Carolina researcher Andy Shih and his lab are taking advantage of the properties of light to visualize what happens in the brain during a stroke. Two-photon microscopy is a relatively new technology that is leading to exciting imaging discoveries, including the details of how a stroke affects individual blood vessels deep in the brain in real time.

Imaging studies of the brain have come a long way since the first visualization of blood vessels in the brain in 1927. Modern techniques include CT and MRI, but these methods can only reveal changes in large-scale brain structure. Other methods that reveal changes in cellular structure can only be done with dead tissue. The ability to visualize changes at the cellular level of a living organism would revolutionize the way that many neurological diseases are understood and treated. Now, new research from Shih's lab is working to advance the field of neuroimaging to do just that.

Shih is an imaging researcher at the Medical University of South Carolina who focuses on finding out exactly what happens in the brain during a stroke. South Carolina is part of the buckle of the “stroke belt,” or the portion of the US that has rates of stroke significantly higher than the national average.

“One of the things that we need to understand for stroke is how quickly the stroke is leading to bleeding in the brain, or leakage of blood vessels in the brain,” Shih says. To do that, Shih’s lab uses a form of neuroimaging that can visualize changes in the brain on the cellular level.

“My major interest is in seeing things,” Shih says. “I love pictures. I love visualizing what we are talking about all the time.”

To see how individual cells respond to stroke, Shih’s lab employs two-photon imaging. A photon is a packet of light energy, and the two-photon method relies on two photons hitting one molecule at the same time to generate a fluorescent glow.

“Fluorescence works like this: You shine a light of one wavelength into your tissue, and there’s a molecule in there that accepts the photon, absorbs it, and then releases a photon of a lower wavelength back out,” says Shih.

However, conventional fluorescent imaging can only be used with very thin sections of tissue. In Shih’s lab, they use in vivo imaging, which involves imaging of intact tissue in a live animal.

“There is a major problem with in vivo imaging,” Shih says. “The tissues are big and they’re thick. If you’re looking down at it from the surface, you never see anything below that.”

To see down through the tissue without cutting into it, two-photon imaging uses longer wavelengths of light, which can pass deeper into the brain. However, using these longer, lower energy wavelengths means that it is more difficult to excite a fluorescent molecule.

This is where the “two photon” part of “two-photon imaging” comes in, as two photons of a lower energy can deliver the same total energy as one photon of a higher energy. The effect of these two lower energy photons is that they excite the molecule enough so that it fluoresces, and structures deeper in the brain can be visualized.

Shih’s lab applies this technique to image cells important in stroke due to their location close to blood vessels. “There is a type of cell called a pericyte—not a parasite that infects the body, but a pericyte—that lives right next to the vessel wall,” says Shih. Pericytes release a type of enzyme called matrix metalloproteases (MMPs).

”Pericytes must be using the MMPs for something under normal conditions, because they probably haven’t evolved to damage the brain. Some people think that they use MMPs like a pair of scissors, cutting through the brain to help the vasculature grow in the way that it needs to,” Shih says.

“In a dense tissue like the brain, you have to be able to cut through carefully, so they use these MMPs like tightly regulated molecular shears to move around. But during stroke, those mechanisms go awry, and they release MMPs in an aberrant way that causes the blood vessels to start leaking.” However, pericytes are not always detrimental during stroke. Shih's upcoming paper in the journal Cell Reports focuses on key unanswered questions about pericyte biology.

His work focuses on the function of pericytes in an animal model of stroke. He says animal research can complement human research. “Animals are animals and humans are humans. The way I like to think of it is that it’s hard to translate exactly what’s happening during stroke, but you can imagine that some of the same things are happening in the human brain that are happening in our animals,” says Shih.

“We help to inform MRI, a powerful clinical tool, using animals as the common vehicle. We can image the animal with MRI, and then come back here and image at the cellular resolution with in vivo imaging to study the same structures. We can collect data from the same animals in the same locations within hours of each other.”

With the combined data from his lab’s studies using MRI and two-photon imaging, Shih’s group has also started to examine the MRI signals generated by microscopic strokes, called microinfarcts, which are surprisingly common in the aged brain, and may contribute to dementia

Another project in his lab focuses on the plasticity that occurs during stroke recovery. “We’re interested in how neurons rewire with each other after a stroke in the periphery of the stroke. This study uses high-resolution imaging of neuronal activity in order to understand how neurons are wired with each other,” Shih says.

Recovery of basic functions after stroke can take months or years, and is often incomplete. Understanding exactly how this recovery proceeds at the cellular level in the brain could improve therapy.

“If a person uses their arm to pick up a coffee, and then all of a sudden their ability to move that arm is lost after a stroke, they eventually learn how to pick up a coffee again. But how does this happen when that area of the brain is gone? The regions surrounding the stroke start to take on the functions of the brain tissue that was lost,” says Shih.

“How does recovery happen? What are the changes that are happening to allow these neurons to find new partners, connect with each other, and reinstate function? Let’s learn how they rewire, and then we can use some of the therapies that are clinically tested, like rehabilitation or trans-cranial magnetic stimulation, to see how these approaches are augmenting the rewiring process at the cellular level.”

Speaking of trans-cranial magnetic stimulation, or TMS, Shih has a project investigating that technology’s impact on neuronal function. One of his lab members, Manuel Levy, is collaborating with DeAnna Adkins, who has a very small TMS coil to use in an animal model of TMS therapy.

“The question is, when we stimulate, what are we doing? It’s a fundamental question of what type of neurons are we activating? How deep does it go? How long does it last for?” This work is funded by a mentored pilot project from the MUSC Stroke Recovery Research Center to Levy.

While these questions are still being researched, Shih maintains a big-picture view of the potential therapeutic impact of his lab’s work. He says applying the imaging methods that achieve the resolution of two-photon microscopy to human patients would be a game changer.

“If you could go in there and do this type of imaging and try to understand what’s happening to blood vessels when we have a stroke, you can answer some basic questions that we just don’t understand, like what is small vessel disease? What is it doing to the way that blood is flowing through capillaries, that we can’t visualize with any other method?”