Dynamic Detection: Real-Time Brain Hypoxia Imaging
By Dr. Noor Mehandi
Human life doesn’t exist without oxygen as it is required for energy production. With each breath we take, tiny oxygen molecules make their way from the air into the lungs. There they cross thin membranes into blood vessels that carry it all the way to every single tissue and cell. The brain, the center of consciousness, is one of the organs that consumes the most oxygen. Our grasp of the molecular dynamics of oxygen in the brain is evolving. The blood flow to the brain is tightly regulated to maintain a sufficient supply of oxygen and nutrients, given that our brain is functioning constantly. Even in a growing fetus, there is a brain-sparing effect in situations of compromised blood supply.
This study aims to understand the distribution of oxygen in the brain during physiological conditions using an innovative imaging technique in mice. GeNL – the Green enhanced Nano-lantern – is a one-of-a-kind specialized protein that detects oxygen through a chemical reaction that produces light in the presence of oxygen. This light is then detected by an ultra-sensitive camera, and the amount of illuminance helps estimate the oxygen content. This protein is expressed in a particular type of cell present in the brain (called astrocytes). With this, the team discovered the existence of sharply defined areas with decreased oxygen in the brain, which they named “hypoxic pockets.” Using spectroscopy, a technique to analyze material composition based on interaction with light, they detected the state of hemoglobin within the blood vessels to map the blood flow in the brain. Additionally, they noted that the hypoxic pockets were a result of a lack of blood flow to these areas and that methods that increased blood flow decreased the occurrence of these hypoxic pockets. The changes in oxygen levels in the brain were further tested with varying oxygen concentrations in the air, compared between anesthetized versus awake states, sensory stimulation, and exercise. They noted an overall reduction in hypoxic pockets in the awake state, with sensory stimulation and physical activity.
This paper enhances our understanding of oxygen distribution and utilization in the brain cortex during various phases of activity. We are yet to understand the clinical implications of these hypoxic pockets in physiological development and pathological disease and how we can extrapolate the data from the mouse brain to the human brain. It would also be interesting to note if the sites of hypoxic pockets are related to memory and skill formation. The correlation between physical activity and decreasing hypoxic pockets opens opportunities for advancing research on the effects of a sedentary lifestyle in progressing the aging of our brain. The increase in hypoxic pockets in anesthetized versus awake mice also supports hesitation in the use of sedatives in neonatology. Moreover, this paper establishes advancements in monitoring the dynamics of oxygen levels. The technique used can be incorporated into diagnosing, understanding neurological conditions, and studying the effect of interventions on the oxygen tension in the brain. Additionally, it would be interesting if we could have a similar GeNL protein expressed in other organs to understand the effect of normal oxygen distribution therein, as well as changes with hypoxia.
Publication here.
Q/A with Drs. Hirase and Beinlich
Q1. How did your team come about this study to look at oxygen content in the brain?
Dr. Hirase: Funnily enough, we were initially trying to record astrocytic Ca2+ fluctuations using bioluminescence. Still to date, fluorescence imaging is the method of choice for measuring astrocytic Ca2+. However, to get fluorescence signals, we need to excite with higher-energy light. Bioluminescence on the other hand does not require excitation light. Felix (Dr. Beinlich) expressed a control (i.e., Ca2+ insensitive) bioluminescent probes in astrocytes. Astrocytes glowed in green; that is exciting, but Felix’s very initial recording also showed rich dynamics in the recorded bioluminescent signal. Further, he showed such rich bioluminescence dynamics in all mice he recorded. Antonis’ (Dr. Asiminas) thorough analysis indicated that the fluctuations are not random. We were puzzled for a while (perhaps several months); but after a while, we came to know that the oxygen is the primary rate limiting factor for this bioluminescent molecule (Renilla luciferase) [cf. the firefly luciferin-luciferase reaction is dependent on ATP and O2]. So, we were inspired to use the Renilla luciferase as an in situ/in vivo oxygen sensor.
Dr. Beinlich: I only imaged the control probe at that time because we accidentally got the wrong plasmid from our collaborators and while I was waiting for the new virus with the Ca2+ sensing probe I tested our microscope system and observed the amazing – unexpected – dynamics of oxygen fluctuations in the cortex. Given that background, it was mostly luck and timing rather than planning to measure oxygen.
Q2: Have your team expanded this technique to detect the oxygen content in other organs? Q3: What future directions can we expect from your team in regards to this study?
Dr. Beinlich: No, we have not expanded the technique and most likely will not to other organs than the brain because our focus within the institute lies in the brain. However, I can imagine that lung and brain especially will give very interesting oxygen-derived bioluminescence intensity dynamics.
Dr. Hirase: I personally think the capillary networks of skeletal muscles, the liver, and the kidney are interesting because of their characteristic vascularization.
Q3: What future directions can we expect from your team in regards to this study?
Dr. Beinlich: We are currently in the process of making a comprehensive protocol for the method going beyond the details explained in the science publication.
Figure: An O2 Odyssey: hypoxia detection in the brain of living mice