Mapping oxygen concentration in the awake mouse brain

Thank you for submitting your work entitled "Mapping oxygen concentration in the awake mouse brain" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by David Kleinfeld as the Reviewing Editor and Timothy Behrens as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. All reviewers are enthusiastic about this work. Each raised a number of questions. I ask you address each question by clarifying the manuscript. No new data is required. I have summarized the key questions:

1) The hematocrit is reported to be significantly higher in cortex than in olfactory bulb (subsection “Capillary flow and Po₂ values in the somatosensory cortex of the awake resting mouse”). This needs to be discussed – how is this difference produced? Is it somehow produced by plasma skimming at a series of junctions of the vessels supplying the olfactory bulb? As this is an acute preparation, one needs to be certain that this is not due to some small amount of tissue damage when making the imaging window.

2) Please show a raw trace of the Po₂ profile between RBCs, or state explicitly that the line in the inset to panel A IS raw data (and not a schematic); why is that line continuous to the left and dotted on the right hand side?

3) The authors repeatedly stress that previous measurements using two-photon phosphorescence only report Mean Po₂. Could the authors please include a comparison of their Mean Po₂ values against those made by other groups also using two-photon phosphorescence probe?

4) Please explain the bars in Figure 1B far better. State completely clearly that Po₂ is the mean (over vessels) of a single time averaged value obtained from each vessel, while SD is mean (over vessels) of the standard deviation of the Po₂ in each vessel over time (if that is what it is!).

5) The Po₂ seems (subsection “Laminar organisation of capillary blood flow and Po₂ values in the superficial layers of the somatosensory cortex”) to be determined by the local capillary density. Does this imply that local O₂ consumption is independent of laminar position (one might have expected vessel density to be proportional to local O₂ usage)? Is it possible to derive, from the laminar distribution of [O₂] and the blood flow reaching each lamina, how much O₂ is consumed by each cell layer?

6) A more thorough clarification of the argument of how isoflurane increases interstitial oxygen concentration would strengthen the manuscript. The current explanation links increased bloodstream oxygenation to the vasodilatory effect of isoflurane (subsection “Brain oxygenation is greatly enhanced by isoflurane anesthesia”). However, the increase in RBC flux (Figure 3A) resulting from vasodilation would lead to a decrease in oxygen unloading from RBCs, as evidenced by the increase in RBC Po₂ (Figure 3A). If less oxygen is unloaded from the red blood cells, then less oxygen diffuses through the vessel wall, leading to a decrease in tissue oxygenation. This is contradicted by the statement that cellular processes occur at a higher oxygen concentration during isoflurane anaesthesia. This matter is further confused by the delivery of isoflurane in a 21% oxygen gas mixture.

7) Theoretical models are cited as evidence that InterRBC Po₂ is in equilibrium with the surrounding tissue (subsection “Capillary flow and Po₂ values in the somatosensory cortex of the awake resting mouse”). The theoretical models rely on accurate hematocrit prediction to determine the distribution of oxygen to the surrounding tissue. The relationship between radial oxygen gradients around capillaries, hematocrit and RBC velocities have been discussed in (I. Gould, A. Linninger, "Hematocrit Distribution and Tissue Oxygenation in Large Microcirculatory Networks", Microcirculation, 22, p1-18, 2015). The authors may consider inclusion of this reference to support the argument.

8) The authors mention that they anaesthetise all mice before initiating even awake imaging experiments. Is it possible to head fix the mice without using anaesthesia? Is the anaesthesia necessary to do the retro-orbital injection of the fluorescein dextran and oxygen probe? It seems awake mice could be tail/vein injected. This might also affect the results since there could residual effects of anaesthesia. At the very least this confound should be acknowledged.

The reviewers have also identified some technical and typographic errors that you should attend to. See the full reviews attached below. We look forward to a revised submission together with a tally of brief answers to each of the seven points above. This is beautiful work and we hope to see it published soon.

Reviewer #1:

This manuscript provides important data (obtained using a relatively non-invasive technique) characterising the O₂ level at different parts of the vasculature in awake non-anesthetized animals. Although mainly descriptive, the data will be of great interest to all those working on the control of cerebral blood flow, energy supply to the brain, brain metabolism and BOLD fMRI signals, because all of these subjects depend critically on the local O₂ concentration, which is poorly defined in vivo. Previous work has only provided similar data on anesthetized animals (or with more invasive techniques in freely moving animals), and it turns out, importantly, that the values are very different in the unanesthetized animal. This paper is therefore a significant step forward, and I support its publication.

There are some areas where the discussion should be improved, as follows.

1) The hematocrit is reported to be significantly higher in cortex than in olfactory bulb (subsection “Capillary flow and Po₂ values in the somatosensory cortex of the awake resting mouse”). This needs to be discussed – how is this difference produced? Is it somehow produced by plasma skimming at a series of junctions of the vessels supplying the olfactory bulb? Why might that occur more for the supply to the olfactory bulb than for that to the cortex?

2) The Po₂ seems (subsection “Laminar organisation of capillary blood flow and Po₂ values in the superficial layers of the somatosensory cortex”) to be determined by the local capillary density. Does this imply that local O₂ consumption is independent of laminar position (one might have expected vessel density to be proportional to local O₂ usage)? Is it possible to derive, from the laminar distribution of [O₂] and the blood flow reaching each lamina, how much O₂ is consumed by each cell layer?

3) It is surprising that this study finds a lower Po₂ in the surface layers of the cortex, whereas Devor and Sakadzic found a decrease in [O₂] with depth. Is there any methodological difference that could account for this, e.g. O₂ penetration into the brain from the cortical window in the earlier studies)? Was the laminar profile of O2 affected by isoflurane?

4) Figure 1A. Please show a raw trace of the Po₂ profile between RBCs, or state explicitly that the line in the inset to panel A IS raw data (and not a schematic); why is that line continuous to the left and dotted on the right hand side?

5) Figure 1B. Please explain the bars far better. State completely clearly that Po₂ is the mean (over vessels) of a single time averaged value obtained from each vessel, while SD is mean (over vessels) of the standard deviation of the Po₂ in each vessel over time (if that is what it is!). Please try out your text on a naive reader so that it is comprehensible.

6) Figure 3 legend. Stress that the measurements in A are from the glomerular layer, unlike the vessel image on the right which is pial.

7) Figure 6. Add labels for Olfactory bulb and Cortex to the panels so the reader can grasp what is plotted at a glance.

8) In the first paragraph of the subsection “Laminar organisation of capillary blood flow and Po₂ values in the superficial layers of the somatosensory cortex” state explicitly how deep capillaries "deeper than 410 um" could be.

Reviewer #2:

The paper reports micron scale mapping of oxygen concentration, hematocrit, and RBC flux in both awake and anaesthetized mice using the two-photon phosphorescence probe PtP-C343. Additionally, these measurements are replicated in both the primary somatosensory cortex (n=3) as well as the olfactory bulb (n=5). Measurements of erythrocyte-associated transients were measured in individual capillaries, and oxygen tension was reported in RBCs (RBC Po₂), in between two red blood cells (InterRBC Po₂) and the mean between these two values (Mean Po₂). These measurements are report for each layer of the cortex.

This manuscript provides an impressively thorough analysis of the correlation between hematocrit, RBC flux and the three oxygen concentration measurements in the bloodstream. Additionally, the comparison between awake and anaesthetized mice clearly demonstrates the increased oxygen concentration in the bloodstream. Additionally, inclusion of a comparative study with previous measurements of bloodstream oxygen concentration in both the awake and anaesthetized mice would strengthen the argument that isoflurane increases interstitial Po₂. The explanation of how isoflurane increases interstitial Po₂ is left somewhat unclear.

The reviewers recommend that this manuscript be published with minor revisions.

Technical Comments:

1) A more thorough clarification of the argument of how isoflurane increases interstitial oxygen concentration would strengthen the manuscript. The current explanation links increased bloodstream oxygenation to the vasodilatory effect of isoflurane (subsection “Brain oxygenation is greatly enhanced by isoflurane anesthesia”). However, the increase in RBC flux (Figure 3A) resulting from vasodilation would lead to a decrease in oxygen unloading from RBCs, as evidenced by the increase in RBC Po₂ (Figure 3A). If less oxygen is unloaded from the red blood cells, then less oxygen diffuses through the vessel wall, leading to a decrease in tissue oxygenation. This is contradicted by the statement that cellular processes occur at a higher oxygen concentration during isoflurane anaesthesia. This matter is further confused by the delivery of isoflurane in a 21% oxygen gas mixture.

2) The authors repeatedly stress the importance of differentiating between RBC Po₂, InterRBC Po₂, and Mean Po₂ – stating that previous measurements using two-photon phosphorescence only report Mean Po₂. Could the authors please include a comparison of their Mean Po₂ values against those made by other groups also using two-photon phosphorescence probe?

3) Similarly, the authors cite previous low-resolution studies on the effects of isoflurane on brain tissue oxygenation, but the manuscript is lacking a side by side comparison with previous studies. An inclusion of the previous measurements would strengthen the conclusion made in this paper.

4) Theoretical models are cited as evidence that InterRBC Po₂ is in equilibrium with the surrounding tissue (subsection “Capillary flow and Po₂ values in the somatosensory cortex of the awake resting mouse”). The theoretical models rely on accurate hematocrit prediction to determine the distribution of oxygen to the surrounding tissue. The relationship between radial oxygen gradients around capillaries, hematocrit and RBC velocities have been discussed in (I. Gould, A. Linninger, "Hematocrit Distribution and Tissue Oxygenation in Large Microcirculatory Networks", Microcirculation, 22, p1-18, 2015). The authors may consider inclusion of this reference to support the argument.

Reviewer #3:

The authors describe methods and results using 2-photon phosphorescence lifetime mediated measurements of tissue oxygenation in vivo. The authors extend their exciting work demonstrating that local O₂ tension changes over the time course of each RBC passing (up to 100 pass per second, with each making a local O₂ transient). This work extends a proven strategy of using compounds with a phosphorescence lifetime that is oxygen sensitive. The approach enables probe-specific calibration of oxygen levels using the phosphorescence lifetime. The methods (O₂ probe) although not developed by Charpak`s lab, is applied quite effectively here. The authors extend previous work by David Boas and make measurements of oxygenation from awake animals. The work examines different cortical layers and contrasts with olfactory bulb. The analysis and presentation of the data is strong. Novel data includes capillary specific measurements and relationships between local oxygenation and red blood cell flux. The work also is significant for examining potentially confounding effects of well-used anaesthesia such as isoflurane. In this way the current work strikes an important cautionary tone with respect to anesthesia.

1) The authors’ analysis is impressive and I don`t have any real concerns with it, since I am familiar with this literature. However, for those which have seen these phosphorescence measurements for the first time, I think it would be important to show supplementary data illustrating how the measurements are obtained. This could be the calibration curve or some raw data showing how Po₂ and velocity/flux are calculated in a single experiment.

2) I would also like to see the authors give some measurements for arteriole and vein diameters with isoflurane anaesthesia vs. the control awake state, the example images point towards a robust finding.

3) The authors mention that they anaesthetise all mice before initiating even awake imaging experiments. Is it possible to head fix the mice without using anaesthesia? Is the anaesthesia necessary to do the retro-orbital injection of the fluorescein dextran and oxygen probe? It seems awake mice could be tail/vein injected. This might also affect the results since there could residual effects of anaesthesia. At the very least this confound should be acknowledged.

4) The authors should comment on how their results relate to recent work from David Boas lab which propose that significant exchange of oxygen with tissues can occur through veins.

5) The authors should also comment on whether there are changes on efficacy of O₂ signal overtime. From the protocol it sounds as though this probe must have an extremely long half-life. Why this probe would not be cleared by the liver is uncertain, its pharmacokinetics should be further investigated or at least if there is information this should be discussed so others can have a better feel for how long after injection can quantification of O₂ be done.

6) I thought the idea that some regions of cortex could have very low flow and oxygenation to be interesting, however this is also an acute preparation and one needs to be certain that this is not due to some small amount of tissue damage when making the imaging window.