New imaging techniques illuminate muscle and nerve, speeding research and aiding clinical trials
Researchers are very good at understanding what is going on with things they can’t see directly, whether it be the membranes of muscle cells or the motor neurons of the spinal cord. But what is true for the rest of us is equally true for scientists: Seeing something is often the quickest and most direct way to understand it.
For neuromuscular disease researchers, new developments in imaging muscles and nerves are bringing new understanding of both healthy tissue and disease processes. These new imaging techniques are allowing researchers for the first time to look deep within the body to track disease and to monitor the response to new therapies. They also have the potential to reduce the need for uncomfortable biopsies, to increase the speed and accuracy of diagnosis, and even to reduce the number of animals needed to conduct neuromuscular disease research.
In this article, we’ll take our own look at these techniques, and explore how they are helping researchers accelerate the hunt for cures in muscular dystrophies, amyotrophic lateral sclerosis (ALS) and other diseases.
“Exon skipping is one of the most interesting and promising approaches to treatment of Duchenne muscular dystrophy,” says neurologist and neurogeneticist Kenneth Fischbeck. “But the need in the field is to see whether this — or any — approach to muscular dystrophy is working.”
|Neurologist and neurogeneticist Kenneth Fischbeck heads the Neurogenetics Branch of the National Institute of Neurological Disorders and Stroke at the National Institutes of Health. His research group is studying the mechanisms of hereditary neurological and neuromuscular diseases with the goal of developing effective treatments for these disorders.|
Fischbeck, a distinguished investigator at the National Institute of Neurological Disorders and Stroke (NINDS) Neurogenetics Branch, is talking about an experimental strategy in which a molecule called an antisense oligonucleotide is used to circumvent certain mutations that cause Duchenne MD (DMD). (See Exon Skipping in DMD: What Is It and Whom Can It Help? Quest, October-December 2011.)
The antisense molecule is targeted to a particular section (exon) of the gene for dystrophin, the protein that’s missing in DMD. In patients with certain genetic mutations, exon skipping can change the way the cell “reads” genetic instructions and allow muscle fibers to produce functional dystrophin protein.
Early results suggest that exon-skipping therapy may have real promise as a disease-modifying treatment for DMD, and Fischbeck is leading an imaging study of boys receiving this experimental therapy.
The challenge, according to Fischbeck, is that the standard ways of assessing response to treatment in DMD are inadequate. Clinical trials in DMD, including the current exon-skipping trials, typically measure clinical improvement by determining how far a child can walk in six minutes.
“But the six-minute walk test is notoriously variable,” he says, “and not the kind of thing you ideally want to determine if the treatment is working.” That variability arises for multiple reasons, including that people, perhaps especially young children, vary in their energy level, or how they are feeling, or how much they want to push themselves on a given day.
The walking test results ultimately will be needed in order to know whether the treatment offers a benefit in daily life (and thus is “clinically meaningful,” in the language of drug testing). But before that, Fischbeck says, “the need is to get some kind of early indication to see if the treatment is having a biological effect.” Finding a reliable marker that will track response to treatment of DMD is a major goal as the field begins to test more and more disease-modifying therapies.
In the past, that role has fallen to muscle biopsy, in which a small sliver of muscle is removed, usually with a needle. In the short term, muscle biopsy will remain the “gold standard” for assessing treatment response, since it alone can show the presence of the dystrophin protein.
But Fischbeck and others are hoping that, in the long run, biopsy can be largely replaced by noninvasive magnetic resonance imaging (MRI) of the muscle. Studies currently under way are meant to collect the kind of information that may allow MRI and other forms of imaging to serve as reliable biological markers (biomarkers) for many kinds of muscle diseases. The images can provide a range of information, including the integrity of the muscle and the degree of deleterious changes that have taken place in it over time.
The potential advantages of MRI in studying muscle disease are multiple and significant.
First, in contrast to biopsy, it provides an image of the entire muscle. Muscle damage in DMD is “patchy,” explains Ami Mankodi, an assistant clinical investigator in the Neurogenetics Branch at NINDS, who is working with Fischbeck on the imaging study of boys with DMD who are receiving experimental antisense treatment. This means that while some muscle fibers are damaged, others are unaffected, and still others are healing. With a biopsy, there is no guarantee that the tiny portion of the muscle that’s sampled will be representative of the entire muscle.
Second, “imaging can be safely repeated as often as desired,” Mankodi says. This is especially important in a progressive disease such as DMD, in which multiple invasive procedures over time are not ideal, and raise concerns in the family.
Third, imaging may provide clinically important information early on, before there is measurable weakness. According to Krista Vandenborne, who is assessing the potential of muscle imaging to track the progress of DMD, “MRI can detect muscle changes even before we can see a change of function.” Vandenborne is a professor and chair of the Department of Physical Therapy at the University of Florida in Gainesville, and has received MDA support to develop noninvasive muscle imaging in DMD patients.
Even in her youngest subjects, boys who are 5 years old, she sees “consistently, across the board” that there are imaging changes indicative of damage and inflammation within the muscle “much higher than what we think is a normal range.”
Fourth, MRI makes it possible to look at multiple muscles simultaneously, Vandenborne says. Since the disease may progress at different rates in different muscles, the ability to capture change in more of them means a more accurate picture of the whole person, and a better chance of seeing if there is a response to therapy.
Finally, Fischbeck says, imaging may be the best way to test therapies tailored for small numbers of patients. While the current antisense therapy is meant to address the mutation in 13 percent of DMD patients, treatment for rarer mutations will require other antisense molecules, and these must be tested as well.
The hope is that imaging may provide a rapid and simple way to tell whether any new antisense molecule is beneficial without the burden of a full-scale clinical trial.
Vandenborne, whose work is independent of the exon-skipping trial, hopes to develop a long-term understanding of the DMD disease process by using MRI and a related technique called magnetic resonance spectroscopy (MRS). Both are done in the same machine and at the same time.
|MRI of DMD-affected thigh muscles: In this cross-section of the thigh muscles of a 6-and-a-half-year-old boy with DMD, the muscle is gray, and the fat is white. The white area around the perimeter of the thigh muscle is the normal fat layer between the skin and the muscle. The large, black-rimmed white spot is the thigh bone. (The white part is bone marrow, and the black part is hard bone.) Boys with DMD have more fat accumulation in their muscles than is normal. Fat in muscle can be measured using either MRI or MRS.|
“Spectroscopy is essentially a noninvasive biochemical assay. It provides additional information about the underlying composition or chemistry of the muscle,” such as how much fat is present, she says.
As DMD progresses, Vandenborne notes, there is a progressive increase in the amount of fat in the muscle. The amount of fat, therefore, is an indication of the severity of disease, and that’s a key measure in her studies.
“What we see is that, in general, when the boys are younger there is a slower increase. But then between the ages of 8 and 10, we really see it accelerate. We are trying to capture that, and trying to understand where the point is that it begins to progress more rapidly.” That information will lead to a better understanding of the best therapeutic options for the different stages of the disease.
“The ultimate goal,” Vandenborne says, “is to validate these imaging techniques so we can use them as outcome measures in clinical trials.”
Vandenborne sees an important role for MRI and MRS in the future of understanding and tracking DMD, and potentially other muscle diseases.
“These are powerful tools that really have a tremendous potential for muscular dystrophy. The fact that they are not invasive is really going to allow us to look at boys in a longitudinal way, very differently than we have been able to do. If we can take away the trauma of muscle biopsy and follow boys repeatedly in a noninvasive way, I think it could be a huge step forward.
But she cautions that the MRI approach that she is using is not the same as what is done in the average hospital with a scanner. “People assume that any site will do, because everybody does MRI.” But in her study, and that of Fischbeck and Mankodi, it is the raw data, not the final image, which is giving them the information they need to find the subtle changes in muscle structure they are studying. “We need to be able to take these measures and quantify them,” she says. “Not every hospital is going to be able to do that.”
Lighting up a transporter in ALS
For more than 15 years, ALS researchers have “had their eye” on glutamate transporters, proteins that carry a substance called glutamate in the nervous system. Soon, that may be more than just a metaphor, as scientists are finishing a five-year process of developing a tool that will help them look directly at these important proteins in ALS patients.
Glutamate helps neurons communicate with one another, but it is thought to harm motor neurons in large amounts. Keeping glutamate in check is the job of some glutamate transporters, specifically one called EAAT2, which carries glutamate away from neurons that could be damaged by it.
In 1995, neurologist and neurophysiologist Jeffrey Rothstein and colleagues showed that the level of EAAT2 was dramatically lowered in the central nervous systems of ALS patients, suggesting that too much glutamate might be accumulating and contributing to the disease. (Rothstein is a current and longtime MDA research grantee at Johns Hopkins University in Baltimore, where he also directs the MDA/ALS center.)
The glutamate finding also suggested that increasing EAAT2 might be therapeutic, setting off a search for drugs with this effect. That search turned up ceftriaxone, an antibiotic that is currently being tested in a clinical trial in ALS patients.
One challenge has been to find a way to monitor the effectiveness of the drug — the extent to which EAAT2 levels rise — during the trial. That has fallen to neuroscientist Rita Sattler, who co-directs Rothstein’s laboratory at Hopkins.
“It is obviously very difficult to look into the brain or spinal cord to see if there is more of this transporter,” she says, but the need for such a tool is great. So she set out to develop a molecule (called a ligand) that could stick to EAAT2 and could be seen with positron emission tomography, or PET, an imaging technique that can give an exquisitely detailed look inside tissues.
Any tissue in which the PET ligand has bound to EAAT2 will be detected and show up brightly, and the more transporter there is, the brighter the image will be.
|PET scan of a rat brain: The rat is facing the reader’s left. The more intense color in this image corresponds to the presence of higher levels of the glutamate transporter EAAT2. Orange indicates the highest EAAT2 level, followed by yellow and green for somewhat lower levels.|
“We are trying to get something that, within a couple weeks on the drug, should be able to tell us about any changes in the levels of the transporter protein,” Sattler says. She is working in close collaboration with John Gerdes and Richard Bridges from the University of Montana, who have been developing the PET ligand for their own research on glutamate transport, as well as Henry VanBrocklin from the University of California, San Francisco, an expert on PET imaging. Together, they are close to finally achieving their goal.
Unlike MRI, PET requires the use of small amounts of radioactivity, which is incorporated into the ligand. As the radioactivity decays, it emits small energetic particles called positrons, which can be picked up by sensitive detectors to create a three-dimensional image.
Sattler notes, “The difficulty we had in the beginning was to find a ligand to cross the blood-brain barrier,” a tight seal that prevents most types of molecules from entering the brain. They discovered the best strategy was to use a precursor compound whose chemical structure allows it to cross the barrier. Once in the brain, it is quickly broken down to the active ligand form that carries the radioactive tag and binds to EAAT2, thereby allowing the detection of the target glutamate transporter in the central nervous system.
The compound has been successfully tested in rodents and is advancing into primates. Assuming all goes well, the goal is to begin safety testing the PET ligand in humans later in 2012, and to begin evaluating it in ALS patients in 2013 for the ability to detect EAAT2. The researchers “are on the home stretch,” Sattler said.
Until the ceftriaxone trial is completed, it will not be clear whether increasing EAAT2 is in fact therapeutic in ALS, but Sattler notes that the PET ligand she and her team have developed may be useful for other purposes besides monitoring clinical trials. Most ALS researchers believe that there are likely multiple causes of ALS, and that low EAAT2 may be only one of them. If so, it may be possible to use the marker to identify a distinct subgroup of patients — those with especially low levels of the transporter — and develop treatments specifically for them.
The problems with biopsy and the advantages of imaging aren’t just seen in humans. There are benefits in animal research, too.
Kanneboyina Nagaraju, an immunologist and veterinarian at Children’s National Medical Center in Washington, D.C., works with mouse models of muscular dystrophies, studying the effects of potential therapies before they get to the clinic.
“Just like in the human, the muscle damage in the mdx [dystrophin-deficient, DMD-like] mouse is very patchy,” he says. “There will be an area of extensive muscle damage, but if you move a couple of fields under the microscope, suddenly you have an apparently normal area. This creates a big problem for studying muscle pathology in mice. Because of that, you need to study large numbers of mice, to compensate for the individual variation.”
But whole-body imaging in live mice avoids that problem, Nagaraju says. “We can see the entire muscular system,” meaning many fewer animals are needed for each study.
And, best of all, imaging can be done without sacrificing the animal, which often has to be done to examine muscles by other methods. “If we treat with a therapeutic agent, we don’t have to sacrifice the mouse to look at its muscles at a specific time point,” Nagaraju says. The same mouse can be imaged at different points in a study to see, for instance, how a treatment is affecting muscles over time.
Nagaraju is beginning to use MRI in his studies (the mouse must be anesthetized for the imaging procedure), but also has been extensively using optical imaging, in which the inflammation in mouse tissue is stained with a harmless fluorescent dye that emits skin-penetrating light upon activation of an enzyme in the inflamed area. While not practical for imaging deeper muscles and tissues at this stage, it has been very effective for those closest to the surface.
“Even 10 years ago, the technology was pretty primitive,” Nagaraju says. But it is advancing yearly. “We currently use between 50 and 90 mice per drug that we test, but if the technology evolves to where I can see all the muscles, even the deeper ones, then I can cut down the numbers at least by half. That is a clear possibility in the next 10 years.”
These new imaging technologies are helping neuromuscular disease researchers see more of what’s going on inside muscles and nerves. And they’ll be using those images to better understand disease, develop better treatments, and take better care of all people with neuromuscular diseases in the future.
MRI — magnetic resonance imaging — is so familiar to so many people that it is easy to forget it has been in routine medical use only since the 1980s.
The MRI machine houses a very powerful, doughnut-shaped magnet. When a person is placed into the machine and the magnet is turned on, it alters the magnetic “spin” of atomic nuclei in some molecules in the tissues. This change in spin is harmless and can’t be felt. Different tissues and different molecules react slightly differently to the magnet, and by detecting and manipulating the properties of those spinning nuclei, imaging specialists can develop “pictures” of the tissues. It is important to realize that the MRI machine uses no radiation. Thus, there is no increased risk of cancer or other harmful effects, as is seen with repeated and prolonged exposure to X-rays.
It’s also important to note that, unlike a camera, the machine itself doesn’t make the pictures. Instead, it simply records the responses of the atomic nuclei to the magnet, which are stored as a dense series of numbers. Researchers then use this raw data to create fine-scale images of the tissues, highlighting the features in which they are interested. This ability to draw out several different features from a single MRI scan is one of the reasons it is such a powerful research tool.
Undergoing imaging in an MRI scanner is painless, uses no sedation, and requires little more than lying still and enjoying a good video. If you are going in for a scan (or are accompanying a child who is going to get one), you’ll be asked to remove your belt, jewelry and any other metal object you may have. “The magnet is extremely powerful, and we don’t want any flying objects!” Krista Vandenborne says.
Boys in her study can bring in their favorite DVD for entertainment during the 90-minute procedure. They lie on their backs wearing headphones to block out the noise of the machine (similar to a jackhammer at close range), and are asked to remain very still. Sandbags may be placed against the legs to help keep them from moving.
A parent remains in the room with the child along with a member of the imaging staff. “One of the things that people underestimate is the importance of the personnel in the room. When you’re dealing with children, you want to have a positive and comforting attitude,” says Vandenborne. “Having a kid-friendly environment is extremely important.”
Ami Mankodi has nothing but praise for the young boys in her study. “They show so much perseverance! I love being in the MRI suite with them.” If the procedure goes on too long for them, she says, “they can come out if they want to. They are the boss.”
These optical images compare inflammation in leg muscles from normal mice and leg muscles from mdx mice, which lack the muscle protein dystrophin. Dystrophin deficiency is the underlying problem in DMD.
The more intense color corresponds to the activity of a protein called cathepsin, which is involved in muscle repair and is located primarily in areas where there is inflammation of the muscle tissue.
The highest cathepsin and inflammation levels are orange, and the lowest are purple. Yellow, green and blue indicate levels between orange and purple. The highest inflammation levels are in the back leg of the mdx mouse.
Note: Click on photo to expand; rollover photo for cutline.
Richard Robinson is a freelance medical and science writer based in Sherborn, Mass.