Researchers at the University of Rochester and University of Copenhagen’s Center for Translational Neuromedicine developed a bioluminescence imaging technique that tracks the movement of oxygen in mice brains to reveal the level of oxygen present in the mice’s cortical tissue under different physiological conditions. As more oxygen enters the brain, the luminescence grows brighter. The method can be easily replicated by other labs and is expected to enable the forms of brain hypoxia, such as oxygen deprivation to the brain during a stroke or heart attack or in patients with dementia, to be more precisely studied. The researchers used a genetically encoded, bioluminescent oxygen indicator for partial oxygen tension imaging. Oxygen tension represents the balance between local oxygen delivery and consumption at any given time. They examined oxygen partial pressure in different parts of the mouse brain at high spatial and temporal resolution. Luminescent protein, substrate, and oxygen combine to create a chemical chain reaction that produces light. The images show the cortex of a mouse recorded over several minutes, as the oxygen concentration in the mouse brain fluctuates between 20%, 30%, and 40%. Courtesy of the University of Copenhagen. Instructions were delivered to the brain cells, via a virus, to produce a luminescent protein in the form of an enzyme. The researchers injected a substrate into the brain via a craniotomy. When the enzyme encountered the substrate, a chemical reaction occurred that generated light. “The chemical reaction in this instance was oxygen dependent, so when there is the enzyme, the substrate, and oxygen, the system starts to glow,” said Felix Beinlich, a professor at the University of Copenhagen. The fluctuating intensity in bioluminescence reflected the presence and concentration of oxygen. When the researchers altered the amount of oxygen available to the animals, they found that the intensity of the bioluminescence increased or decreased according to the concentration of oxygen. Changes in light intensity also corresponded with sensory processing in the mice. For example, when the mice’s whiskers were stimulated with a puff of air, the researchers could see the corresponding region of the brain light up. It is understood that the brain cannot survive long without oxygen. But what happens when very small parts of the brain are deprived of oxygen for brief periods? The bioluminescence imaging technique enabled the researchers to view a large section of the mouse cortex in real time. While monitoring the mice, they observed that specific, tiny areas of the brain would go dark, sometimes for minutes, which meant that the oxygen supply to that part of brain was being cut off. Through a series of experiments, the researchers determined that oxygen was being denied due to capillary stalling, a phenomenon that occurs when white blood cells temporarily block microvessels, preventing the passage of oxygen-carrying red blood cells. These temporary, spatially restricted periods of hypoxia, which the researchers named hypoxic pockets, occurred spontaneously and were more prevalent in the brains of mice during a resting state, compared to when the animals were active. Physical activity, like running, reduced the occurrence of hypoxic regions by 52%, compared with rest. In addition to offering a view of cortical oxygen dynamics in awake, behaving animals, the bioluminescence imaging tool provides a means to explore the importance of oxygen tension in physiological processes and neurological diseases in humans. Capillary stalling is believed to increase with age and has been observed in models of Alzheimer’s disease. By detecting incidents of hypoxia related to capillary stalling, the imaging tool could contribute to our understanding of why a sedentary lifestyle may increase the risk for diseases like Alzheimer’s. “This research demonstrates that we can monitor changes in oxygen concentration continuously and in a wide area of the brain,” said Maiken Nedergaard, a professor at the University of Rochester. “This provides us with a more detailed picture of what is occurring in the brain in real time, allowing us to identify previously undetected areas of temporary hypoxia, which reflect changes in blood flow that can trigger neurological deficits." “The door is now open to study a range of diseases associated with hypoxia in the brain, including Alzheimer’s, vascular dementia, and long COVID, and how a sedentary lifestyle, aging, hypertension, and other factors contribute to these diseases,” Nedergaard said. “It also provides a tool to test different drugs and types of exercise that improve vascular health.” The research was published in Science (www.doi.org/10.1126/science.adn1011).
