"Overshoot" of O₂ is required to maintain baseline tissue oxygenation at locations distal to blood vessels

Abstract

In vivo imaging of cerebral tissue oxygenation is important in defining healthy physiology and pathological departures associated with cerebral disease. We used a recently developed two-photon microscopy method, based on a novel phosphorescent nanoprobe, to image tissue oxygenation in the rat primary sensory cortex in response to sensory stimulation. Our measurements showed that a stimulus-evoked increase in tissue pO₂ depended on the baseline pO₂ level. In particular, during sustained stimulation, the steady-state pO₂ at low-baseline locations remained at the baseline, despite large pO₂ increases elsewhere. In contrast to the steady state, where pO₂ never decreased below the baseline, transient decreases occurred during the "initial dip" and "poststimulus undershoot." These results suggest that the increase in blood oxygenation during the hemodynamic response, which has been perceived as a paradox, may serve to prevent a sustained oxygenation drop at tissue locations that are remote from the vascular feeding sources.

Figure 1.

Baseline pO₂ relative to diving arterioles and surfacing venules. A, An image of the surface vasculature calculated as a maximum intensity projection of an image stack 0–300 μm in depth obtained using a 4× objective. Individual images were acquired every 10 μm. The fluorescent contrast is due to intravascular FITC. The image on the right shows a zoomed-in view of the region within the red square on the left at the depth plane of pO₂ measurements, 200 μm below the surface. A, Diving arteriole; V, surfacing venule. B, A grid of measured pO₂ values superimposed on the vascular reference image. C, pO₂ as a function of the radial distance from the center of a blood vessel: diving arterioles (left) or surfacing venules (right). The pO₂ values were obtained from grid measurements, as with the example in A and B. Data from multiple vessels from multiple animals are overlaid on each plot.

Figure 2.

Distribution of the measured pO₂ values as a function of cortical depth. Categories and numbers of samples are indicated above the histograms.

Figure 3.

Tissue pO₂ changes in response to transient stimulation. A, B, An example set of measurements from a plane 300 μm below the cortical surface. A, Points used to measure stimulus-evoked responses, color-coded according to the prestimulus (baseline) pO₂, are superimposed on the vascular reference image. A corresponding FITC image of the surface vasculature is shown on the left. B, Time courses of pO₂ change extracted from each point shown in A. The thick black curve shows the average. The red arrow in this and other figures indicates stimulus onset. C, Averaged time courses, grouped according to the baseline pO₂. The groups are indicated on the right. The inset shows mean peak pO₂ increase from the baseline (ΔpO₂) for each baseline category. The error bars represent SE across subjects. D, The same data as in C. The averaged time courses are grouped according to the baseline, above and below 14 mmHg. E, The same time courses as in D, baseline-subtracted and normalized to the peak amplitude.

Figure 4.

Tissue pO₂ changes in response to sustained stimulation. A, Averaged pO₂ time courses in response to the 20 s stimulus. The data were grouped according to the baseline pO₂. The groups are indicated on the right. The red line shows the average. The inset shows mean pO₂ increase from the baseline (ΔpO₂) during the plateau response for each baseline category. The error bars represent SE across subjects. B, A zoomed-in view of the first 10 s during the sustained stimulus (dashed line). The response to 2 s stimulus, group-averaged using the same baseline bins, is overlaid in solid black.