A new imaging technique called coherent anti-Stokes Raman scattering, or CARS, has lead to the discovery that calcium ions could play a crucial role in multiple sclerosis (MS) by activating enzymes that degrade myelin, the fatty sheath that insulates nerve fibers.This unprecedented feat of looking real-time at the actual progress of demyelination will advance understanding of and perhaps promote early detection of conditions such as MS in which the myelin sheath has been found to degrade. Learning exactly how the myelin sheath is degraded might enable scientists to determine how to halt disease progress and reverse damage by growing new myelin, said Ji-Xin Cheng, an assistant professor in Purdue University's Weldon School of Biomedical Engineering and Department of Chemistry. The photo on the left was taken with two microscopic imaging techniques combined in the same platform, enabling researchers to conduct more specific and precise molecular analyses for research regarding multiple sclerosis and other conditions. Combining the techniques offers the hope of understanding the molecular mechanisms responsible for the impairment of the myelin sheath and overproduction of "astroglial filaments," which form bundles between critical nerve fibers and interfere with proper spinal cord functioning. Purdue researchers have shown how to combine three imaging techniques in one platform to analyze living tissues. The image above (left) combines pictures taken with two of those imaging techniques. It shows the myelin sheath in red, taken with a technique called coherent anti-Stokes Raman scattering, and astroglial filaments in green, taken with another imaging technique, sum frequency generation. (Courtesy Weldon School of Biomedical Engineering, Purdue University). Above right: In vivo epidetected CARS imaging of lyso-PtdCho-induced myelin swelling in a mouse sciatic nerve. Below: 3-D CARS imaging of intact myelin sheath (Images: Ji-Xin Cheng, courtesy Purdue University) "Although multiple sclerosis has been studied for many years, nobody knows exactly how the disease initially begins," he said. "The pathway is not clear." The scientists injected a compound called lysophosphatidylcholine (LPC) into the myelin of a mouse. Then, using CARS, they observed an influx of calcium ions into the myelin. This influx is now believed to start the process of myelin degradation. The LPC does not cause multiple sclerosis, but it is used extensively in laboratory research to study the deterioration of myelin, which insulates nerve fibers and enables them to properly conduct impulses in the spinal cord, brain and peripheral nervous system throughout the body. The increased concentration of calcium ions then activates two enzymes -- calpain and cytosolic phospholipase A2 -- which break down proteins and molecules in the myelin called lipids. "It is possible that the same pathway causes myelin degradation in people suffering from multiple sclerosis and spinal-cord injuries," Cheng said.The research demonstrates that CARS microscopy is a valuable research tool and could become a future clinical method to diagnose multiple sclerosis and detect damage to the spinal cord from accident trauma, which also causes the myelin to degrade, he said.Research findings are detailed in a paper appearing online this month in the Journal of Neuroscience Research. The paper was written by biomedical engineering doctoral student Yan Fu and postdoctoral research associate Haifeng Wang; Terry B. Huff, a graduate teaching assistant in the Department of Chemistry; Riyi Shi, an associate professor of basic medical sciences in Purdue's School of Veterinary Medicine and an associate professor of biomedical engineering; and Cheng.Characterization of lyso-PtdCho-induced myelin swelling by CARS microscopy. Laser beams were focused into the equatorial plane of axons. (A) CARS image of normal myelin sheath wrapping two parallel axons acquired at a speed of 1.13 seconds/frame. The same speed was used for other images. (B) CARS image of partially swollen myelin sheath acquired at 5 min after injecting 2 ?L of 10 mg/mL lyso-PtdCho into the tissue. (C) CARS intensity profiles of normal and swollen myelin fibers. Green: taken along the green line in (A). Red: taken along the red line in (B). Note the decrease of CARS intensity in the swollen region. (D) CARS images of normal myelin sheath with vertical (?) and horizontal (↔) excitation polarization. (E) CARS images of totally swollen myelin sheath with vertical (?) and horizontal (↔) excitation polarization. For all images, bar = 10 µm. (Images: Ji-Xin Cheng, courtesy Purdue University) "The findings of this study will help us to identify key steps in the progression of the demyelination, which is a hallmark of multiple sclerosis," said Shi, a researcher at Purdue's Institute for Applied Neurology and Center for Paralysis Research. "This information will also facilitate the design of pharmaceutical interventions that slow down or even reverse the development of the debilitating disease."The researchers used CARS to study and take images of healthy and diseased myelin. The researchers showed that an enzyme called cytosolic phospholipase A2 contributes to myelin degradation by snipping off one of the two tails that make up lipid molecules contained in the myelin. Cutting off one of the tails turns the lipid molecules into LPC, amplifying the effect and further degrading the myelin. The research was carried out in spinal cord tissues extracted from animals and in the sciatic nerves of living mice. Findings were confirmed by comparing CARS results with electron microscope images and measurements of electrical impulses in spinal cord tissue that distinguish between normal and diseased myelin. CARS imaging takes advantage of the fact that molecules vibrate at specific frequencies. In a CARS microscope, two laser beams are overlapped to produce a single beam having a new frequency representing the difference between the original two beams. This new frequency then drives specific molecules to vibrate together "in phase," amplifying the signals from those molecules.The research has been funded by the National Science Foundation and the National Institute of Biomedical Imaging and Bioengineering, with support from the state of Indiana and the Bindley Bioscience Center at Purdue's Discovery Park. Future work will include a collaboration with researchers at Northwestern University to study how to regrow the myelin sheath in animals. For more information, visit: www.purdue.edu