New device crucial to study of newborn muscle development
Rather than removing tissue to study cells under a microscope, imagine placing a microscope inside living tissue – with no more trauma than getting a shot.
That’s precisely the type of new research tool that Roger Cornwall, MD, is using at Cincinnati Children’s to launch a novel clinical study of children with cerebral palsy and brachial plexus injuries.
Cornwall is the first scientist in the world to employ a device called the Zebrascope, an improved form of microendoscope with resolution powerful enough to capture images of the inner structure of muscle cells – without removing tissue samples. The device, created by researchers at Stanford University, is so tiny that its light-emitting and light-sensing fibers can fit inside a pair of needles.
“It’s less invasive than getting a flu shot,” says Cornwall, who also serves as co-director of the Hand and Upper Extremity Center at Cincinnati Children’s. “It’s the first device of its kind that allows looking inside a muscle cell, in vivo, in real time. This will allow us to confirm findings from research in the mouse about why permanent limitations to arm movement often occur when newborns sustain brachial plexus nerve damage during difficult deliveries. This also will allow new studies of muscle function in cerebral palsy, for which no animal model has been developed.”
How the device works
The development of the Zebrascope began with a 2008 paper in Nature. But the early version of the device involved a large, table-top laser scanning microscope that cannot be used easily in clinical settings. The hand-sized Zebrascope was developed in 2012 by Gabriel Sanchez, MS, then a doctoral student at Stanford working in the lab of Scott Delp, PhD. Since then Delp, Sanchez and other partners formed Zebra Medical Technologies to produce the device. Cincinnati Children’s received the company’s first product.
The probe features two needles that can span several muscle fibers. A 20-gauge needle houses one microendoscope that emits a laser signal through the fibers. A 30-gauge needle with another microendoscope picks up the signal and transmits it to a microscope, which displays the image real time on a computer screen.
The scope can capture still images and video within an 80-micron field of view with resolution capable of distinguishing the difference between 2.5 and 2.6 microns. This resolution is powerful enough to measure sarcomeres, the key building block of muscle fibers.
The Zebrascope derives its name from these sarcomeres appearing as a series of black and white stripes on the microscope.
Measuring a chain
Muscles, Cornwall says, are structured somewhat like chains, with sarcomeres being the individual links. Muscles contract by shrinking each sarcomere a tiny bit, which when multiplied by the large number of sarcomeres within a muscle fiber, results in significant contraction.
When a muscle fiber has too few sarcomeres, however, the tiny links become stretched and they lose the ability to contract a muscle across the normal range of movement. This is what Cornwall and a few other scientists believe occurs among newborns with brachial plexus injuries and with cerebral palsy.
Cerebral palsy (occurring in 3.6 per 1,000 live births in the US) and neonatal brachial plexus injury (occurring in 1.52 per 1000 live births) are the two most common causes of neurogenic muscle contractures in childhood.
In studies of mice using traditional methods of tissue removal, scientists found that the lack of nerve input caused by neonatal brachial plexus injuries prevents normal muscle cell development and results in permanent elbow flexion contractures.
“In terms of muscle development, there’s a ‘fourth trimester’ after a child is born during which muscle stem cells are still forming mature muscle fibers,” Cornwall says. “If you disturb the nerve inputs for muscle growth at birth, you alter muscle function forever. Even when surgical repairs or natural healing restores nerve input, the muscles cannot move normally because they were not formed normally.”
This type of muscle contracture does not occur among children or adults who sustain brachial plexus injuries after that fourth trimester. For them, if nerve function can be restored, arm movement returns to normal.
“We think this same kind of muscle growth problem also happens in cerebral palsy,” Cornwall says.
Measuring the length of sarcomeres allows scientists to distinguish normal from abnormal muscle tissue. However, although they can in mice, researchers cannot ethically remove tissues from healthy children to measure human sarcomeres.
Cornwall predicts the new device will help confirm that sarcomere lengths are longer than normal in diseased human tissue.
If that finding proves true, then several molecular pathways affected by lack of nerve input that Cornwall and colleagues have identified in mice may also affect human muscle development. And if that’s the case, there may be ways to preserve range-of-motion by developing drugs that mimic nerve input during that crucial fourth trimester, Cornwall says.
“Currently, a muscle is like a sealed bag of coins. You can weigh the bag, measure the volume of the bag, but you cannot know how much money there may be unless you know what kinds of coins are in there,” Cornwall says. “This new device lets us see inside the bag.”
Confirmation study begins
So far, Cornwall and colleagues have published three papers related to this line of research. Their initial work helped convince the Stanford team to produce the first device for Cincinnati Children’s.
“This technology has attracted the attention of many clinicians and researchers around the world, and we have received many requests for collaborative application of the technology,” Scott Delp states in a letter to a study review committee in October 2013. “(Dr. Cornwall’s) hypothesis, tying together muscle contracture pathophysiology in cerebral palsy and brachial plexus palsy could allow dramatic advances in our understanding of neurological control of muscle growth. For these reasons, Dr. Cornwall’s team will be the first to gain access to this technology.”
The new clinical trial is expected to last about a year as it recruits up to 30 participants. Children ages 6 to 18 with elbow flexion contractures caused by brachial plexus or cerebral palsy will be included.
The first patient to join the study, a 10-year-old girl with one arm damaged by an early brachial plexus injury, was tested with the new device on Jan. 13, 2015.
“We saw in her the same kinds of differences in the sarcomeres that we have seen in mice,” Cornwall says. “But these are early days. We need to gather information from more children, across a range of arm positions, to reach any conclusions.”
Ultimately, the researchers hope their findings with brachial plexus injuries will also apply to muscle development in children with cerebral palsy.
“About 90 percent of the surgeries performed on children with cerebral palsy are to treat secondary muscle contracture problems,” Cornwall says. “If we can trick muscles into thinking they have normal nerve input, we could prevent many of those secondary muscle problems and dramatically change the lives of kids.”