Alzheimer’s Risk Linked to Brief, Localized Oxygen Deficits in Brain

This illustration conveys the key takeaway of our research. At the center, there's an image of a head with two faces, one looking left and the other right, inside of which a brain is depicted. The left side of the image symbolizes a person at rest, possibly daydreaming, in contrast to the right side, which shows a person in motion, actively running. The scenario on the left is characterized by a gloomier atmosphere, with dark clouds dominating the sky. Similarly, within the brain of the person on the left, there are more dark clouds compared to the right side. These dark clouds symbolize hypoxic pockets, indicating that they are more prevalent in individuals who are resting than in those who are active. The central message is that physical activity reduces the presence of hypoxic areas in the brain. [Dan Xue]
This illustration conveys the key takeaway of our research. At the center, there’s an image of a head with two faces, one looking left and the other right, inside of which a brain is depicted. The left side of the image symbolizes a person at rest, possibly daydreaming, in contrast to the right side, which shows a person in motion, actively running. The scenario on the left is characterized by a gloomier atmosphere, with dark clouds dominating the sky. Similarly, within the brain of the person on the left, there are more dark clouds compared to the right side. These dark clouds symbolize hypoxic pockets, indicating that they are more prevalent in individuals who are resting than in those who are active. The central message is that physical activity reduces the presence of hypoxic areas in the brain. [Dan Xue]

Researchers at the University of Rochester and at the University of Copenhagen have developed a bioluminescence imaging technique that creates highly detailed, and visually striking, images of the movement of oxygen in the brains of mice. The technique offers a way to learn more about brain oxygen tension (PO2), a measure of oxygen delivery and demand in brain tissue that changes dynamically, but is not well understood. The investigators suggest that the method, which can be easily replicated by other labs, will enable researchers to more precisely study forms of hypoxia in the brain, such as the denial of oxygen to the brain that occurs during a stroke or heart attack.

The team’s experiments, including monitoring oxygen in awake mice, have provided insight into why a sedentary lifestyle may increase 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, MD, co-director of the Center for Translational Neuromedicine (CTN), which is based at both the University of Rochester and the University of Copenhagen. “This provides us a 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.”

Nedergaard, and colleagues, reported on their work in Science, in a paper titled “Oxygen imaging of hypoxic pockets in the mouse cerebral cortex.”

The human brain consumes vast amounts of energy, which is almost exclusively generated from a form of metabolism that requires oxygen. “The human brain uses ∼20% of total body oxygen consumption at rest,” the authors wrote. “Delivery and demand of oxygen (O2) are so finely balanced that maintaining tissue oxygenation may be the most critical of all brain functions.” But while the efficient and timely delivery of oxygen is known to be critical to healthy brain function—“Consciousness is lost within seconds upon cessation of cerebral blood flow,” the team noted—the precise mechanics of this process have largely remained unknown.

Scientists’ understanding of the dynamics of brain tissue oxygen tension (PO2) under physiological conditions has been limited largely because of the lack of spatially precise measurement techniques for PO2 imaging, the team continued. “Currently, tissue PO2 can be measured by phosphorescence and by Clark-type electrodes. Neither approach provides sufficiently high spatiotemporal sensitivity to detect physiological changes in cortical PO2”.

To address these limitations the team developed a new technique, which employs a genetically encoded bioluminescent oxygen indicator protein, chemical cousin of the bioluminescent proteins found in fireflies. Such proteins, which have been used in cancer research, are generated using a virus that delivers instructions to cells to produce a luminescent protein in the form of an enzyme.  When the enzyme encounters a second chemical compound, a substrate called furimazine, the chemical reaction generates light. The authors explained, “Green enhanced Nano-lantern (GeNL) is a luminescent fusion protein consisting of the luciferase NanoLuc and the fluorescent protein mNeongreen. During the enzymatic conversion of its luminescent substrate furimazine to furimamide, energy is emitted in the form of light … The enzymeatic reaction of GeNL with furimazine depends on O2,  and the intensity of the bioluminescence signal is linearly correlated to the availability of O2 when O2 is the rate-limiting factor in the enzymatic reaction.”

These illustrations depict the capillary network within the brain's blood vessels. The left panel shows oxygen-rich blood, colored in red, flowing through the capillaries into blue arteries. During this flow, oxygen is released from red blood cells and absorbed by brain tissue, which is essential for producing energy to sustain neuronal activity. On the left side, the image displays the oxygenation levels in the brain of a resting mouse. We observed that resting mice exhibited a higher number of hypoxic pockets—areas with low oxygenation—compared to mice that were active, as shown on the left side. In summary, physical activity appears to reduce the occurrence of hypoxic pockets in the brain, thereby lessening the brain's hypoxic burden. [Dan Xue, Felix Beinlich]
These illustrations depict the capillary network within the brain’s blood vessels. The left panel shows oxygen-rich blood, colored in red, flowing through the capillaries into blue arteries. During this flow, oxygen is released from red blood cells and absorbed by brain tissue, which is essential for producing energy to sustain neuronal activity. On the left side, the image displays the oxygenation levels in the brain of a resting mouse. We observed that resting mice exhibited a higher number of hypoxic pockets—areas with low oxygenation—compared to mice that were active, as shown on the left side. In summary, physical activity appears to reduce the occurrence of hypoxic pockets in the brain, thereby lessening the brain’s hypoxic burden. [Dan Xue, Felix Beinlich]

Like many important scientific discoveries, employing this process to image oxygen in the brain was stumbled upon by accident. First author  Felix Beinlich, PhD, an assistant professor in the CTN at the University of Copenhagen, had originally intended to use the luminescent protein to measure calcium activity in the brain.  It became apparent that there was an error in the production of the proteins, causing a months-long delay in the research.

While Beinlich waited for a new batch from the manufacturer, he decided move forward with the experiments to test and optimize the monitoring systems. The virus was used to deliver enzyme-producing instructions to astrocytes, ubiquitous support cells in the brain that maintain the health and signaling functions of neurons, and the furimazine substrate was injected into the brain via a craniotomy.

The recordings revealed activity, identified by a fluctuating bioluminescence intensity (BLI), something that the researchers suspected, and would later confirm, reflected the presence and concentration of oxygen. “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 Beinlich. The authors further stated, “Changing the O2 concentration from 20% to 40% increased BLI by ~200%, whereas a reduction of O2 concentration in the inhaled air to 10% decreased BLI by ~50% from baseline.”

This figure demonstrates that hypoxic pockets result from capillary stalling. We introduced microspheres into the bloodstream of mice, selecting a size comparable to white blood cells, which are primarily responsible for capillary stalling. We monitored the fluorescence of these microspheres concurrently with tissue oxygenation measurements. The top panel uses a color scheme of yellow and lilac to depict oxygen levels, where yellow indicates high oxygen and lilac signifies low oxygen. The bottom panel visually represents the microspheres in white. A line plot on the right side illustrates the temporal changes in oxygen levels. The occlusion of a capillary by a microsphere, indicated by a rise in the blue line, leads to a decrease in oxygen concentration. During a hypoxic pocket, as highlighted by a grey shade, there is an elevation in microsphere fluorescence concurrent with a reduction in tissue oxygenation. [Felix Beinlich]
This figure demonstrates that hypoxic pockets result from capillary stalling. We introduced microspheres into the bloodstream of mice, selecting a size comparable to white blood cells, which are primarily responsible for capillary stalling. We monitored the fluorescence of these microspheres concurrently with tissue oxygenation measurements. The top panel uses a color scheme of yellow and lilac to depict oxygen levels, where yellow indicates high oxygen and lilac signifies low oxygen. The bottom panel visually represents the microspheres in white. A line plot on the right side illustrates the temporal changes in oxygen levels. The occlusion of a capillary by a microsphere, indicated by a rise in the blue line, leads to a decrease in oxygen concentration. During a hypoxic pocket, as highlighted by a grey shade, there is an elevation in microsphere fluorescence concurrent with a reduction in tissue oxygenation. [Felix Beinlich]

While existing oxygen monitoring techniques provide information about a very small area of the brain, the researchers were able to observe, in real time, a large section of the cortex of the mice. The intensity of the bioluminescence corresponded with the concentration of oxygen, which the researchers demonstrated by changing the amount of oxygen in the air the animals were breathing. Changes in light intensity also corresponded with sensory processing. 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.

The brain cannot survive long without oxygen, a concept demonstrated by the neurological damage that quickly follows a stroke or heart attack. But what happens when very small parts of the brain are denied oxygen for brief periods? This question was not even being asked by researchers until the team in the Nedergaard lab began to look closely at the new recordings. While monitoring the mice, the researchers observed that specific tiny areas of the brain would go dark, sometimes for minutes, meaning that the oxygen supply was being cut off. “Continuous imaging of BLI showed that PO2 under resting conditions was highly dynamic, exhibiting local transient dips in PO2. These local hypoxic events were spatially constricted, lasting several seconds up to minutes, and typically showed a sharp on- and offset in relative tissue PO2.”

This figure illustrates that altering the oxygen concentration in the air inhaled by mice affects the bioluminescence intensity of our biosensor within the brain tissue. Specifically, an increase in the intensity of bioluminescence indicates a rise in the oxygen concentration in the brain. The oxygen concentration in the inhaled air was modified at intervals of 60 seconds. [Dan Xue, Felix Beinlich]
This figure illustrates that altering the oxygen concentration in the air inhaled by mice affects the bioluminescence intensity of our biosensor within the brain tissue. Specifically, an increase in the intensity of bioluminescence indicates a rise in the oxygen concentration in the brain. The oxygen concentration in the inhaled air was modified at intervals of 60 seconds. [Dan Xue, Felix Beinlich]

Oxygen is circulated throughout the brain via a vast network of arteries and smaller capillaries—or microvessels—which permeate brain tissue. Through a series of experiments, the researchers were able to determine that oxygen was being denied due to capillary stalling, which occurs when white blood cells temporarily block microvessels and prevent the passage of oxygen carrying red blood cells. These areas, which the researchers named “hypoxic pockets,” were more prevalent in the brains of mice during a resting state, compared to when the animals were active. “Manipulations that either increased or blocked capillary flow showed that local interruption of the microcirculation is responsible for the occurrence of hypoxic pockets,” the team also pointed out.

Capillary stalling is believed to increase with age and has been observed in models of Alzheimer’s disease. “Increased capillary stalling has been observed in models of Alzheimer’s disease, raising questions about the long-term impact of capillary stalling and its potential role in long-term neuronal viability,” the scientists pointed out. “Hypoxia-induced increase in expression of hypoxia inducible factor 1a (HIF1a) impairs plasticity by disrupting synaptic physiology and spatial memory,”

“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,” said Nedergaard. “It also provides a tool to test different drugs and types of exercise that improve vascular health and slow down the road to dementia.”

The authors further concluded, “Our study predicts that physical inactivity has direct effects on tissue PO2 by favoring capillary occlusions and increasing the number of hypoxic pockets. Conversely, simply increasing sensory input or locomotion rapidly suppress the occurrence of hypoxic pockets, perhaps explaining the linkage between sedentary lifestyle and an increased risk of dementia.”