A new publication from University of Pittsburgh researchers announces the use of advanced three-dimensional imaging techniques to reveal the architecture of nerves inside knee joint tissue, according to a July 7 report. The study, led by Alejandro Almarza, professor of oral and craniofacial sciences at the School of Dental Medicine with a secondary appointment in bioengineering, explores how these nerve patterns may relate to pain in densely innervated joints such as the temporomandibular joint (TMJ).
Traditional methods for studying joint nerves involve slicing tissue into thin sections and staining them with dyes for microscopic analysis. However, this process destroys the three-dimensional structure necessary for understanding how nerves branch throughout a joint. To address this limitation, Almarza collaborated with Simon Watkins and Alan Watson from Pitt's Center for Biologic Imaging (CBI) to employ light sheet fluorescence microscopy alongside an imaging technique known as tissue clearing.
"Tissue clearing makes an entire piece of tissue transparent for 3D imaging so you can visualize the nerves inside, and the microscope we used works like a wall of light sweeping through the volume of tissue all at once, making it faster than a traditional microscope while still achieving near-confocal resolution with minimal tissue damage," Almarza said. "Some of the best of these systems in the world are custom-built here at Pitt by Simon Watkins, and the clearing methods have been developed by Alan Watson."
The team compared two methods: PEGASOS—a previously established protocol—and c-Clear, which was developed in-house at CBI. While PEGASOS left behind autofluorescent protein that interfered with imaging quality, c-Clear introduced a photobleaching step that improved visualization by allowing fluorescent antibodies to bind more effectively to neurofilament proteins. "The c-Clear method takes about six to eight weeks to obtain an image, making it far more labor and time-intensive than normal histological methods, but the result is an extremely powerful and clear representation of how these nerves branch," Almarza said.
The research generated large volumes of data—one terabyte per nerve map—with total project data reaching approximately sixteen terabytes. The CBI’s computing infrastructure supported this effort by providing seven petabytes of storage and high-performance computing resources needed for data processing and public sharing on the National Institute of Health's SPARC Portal.
Almarza is part of ReJoin Consortium under NIH HEAL Initiative—a $50 million project focused on mapping nerve architecture across various joints—to expand understanding around pain signaling. "There are a lot of people whose radiographs look like they should have pain in their TMJ, but they're actually talking just fine," Almarza said. "Is it because of the type of nerves in there? And why is it different from people with pain? That's the type of question this research is hoping to answer."
