Two-photon microscopy measurement of cerebral metabolic rate of oxygen using periarteriolar oxygen concentration gradients

Abstract

The cerebral metabolic rate of oxygen ([Formula: see text]) is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying [Formula: see text] rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of [Formula: see text] based on a single measurement modality-two-photon imaging of the partial pressure of oxygen ([Formula: see text]) in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue [Formula: see text] distribution around cortical penetrating arterioles. [Formula: see text] is subsequently estimated by fitting the changes of tissue [Formula: see text] around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline [Formula: see text] in anesthetized rats and modulated tissue [Formula: see text] levels by manipulating the depth of anesthesia. This method provides [Formula: see text] measurements localized within [Formula: see text] and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.

Keywords: cerebral cortex; oxygen metabolism; oxygen partial pressure; phosphorescence; two-photon microscopy.

Fig. 1

The Krogh cylinder, cortical vascular morphology, and tissue ${\mathrm{PO}}_2$. (a) The Krogh cylinder model of oxygen diffusion from a vessel. An infinitely long tissue cylinder with radius $R_{\mathrm{t}}$ is supplied by an infinitely long arteriole with radius $R_{\text{art}}$. ${\mathrm{PO}}_2$ map on the right hand side is computed based on Krogh–Erlang equation [Eq. (2)]. The zero oxygen flux boundary condition ($d{\mathrm{PO}}_2/dr=0$ at $r=R_{\mathrm{t}}$) is satisfied at the external tissue boundary. (b) Maximum intensity projection of a $500\text{-}\mu \mathrm{m}$-thick microvascular stack. Pial artery and adjacent diving arterioles are colored red. Yellow circles emphasize capillary-free spaces around diving arterioles. Scale bar, $100\mu \mathrm{m}$. (c) Baseline tissue ${\mathrm{PO}}_2$ map (color coded), overlaid on a FITC image of the microvasculature $100\mu \mathrm{m}$ below the brain surface. Inset in upper left corner shows $200\text{-}\mu \mathrm{m}$-thick MIP of FITC-labeled microvasculature. Arterioles are colored red. White rectangle in inset outlines the position of the panel (c). Scale bar, $100\mu \mathrm{m}$.

Fig. 2

Baseline ${\mathrm{CMRO}}_2$ measurements. (a–d) Tissue ${\mathrm{PO}}_2$ maps (color coded) around penetrating arterioles overlaid on the corresponding FITC images of microvasculature in different animals (rats I to IV). Insets show MIPs of FITC-labeled microvasculature. Arterioles in insets are colored red. The white rectangle in each insert outlines the position of the corresponding panel with the ${\mathrm{PO}}_2$ data. Yellow lines outline regions of interest with ${\mathrm{PO}}_2$ data included in the fitting procedure. Imaging depths below brain surface are $100\mu \mathrm{m}$ (rat I), $150\mu \mathrm{m}$ (rat II), $124\mu \mathrm{m}$ (rat III), and $160\mu \mathrm{m}$ (rat IV). Scale bars, $100\mu \mathrm{m}$. (e–h) Tissue ${\mathrm{PO}}_2$ from the corresponding upper panels as a function of the radial distance from the penetrating arteriole with ${\mathrm{PO}}_2$ fit indicated by solid line.

Fig. 3

${\mathrm{CMRO}}_2$ estimation at different baseline tissue ${\mathrm{PO}}_2$ and two cortical depths along the same penetrating arteriole. (a, b) Tissue ${\mathrm{PO}}_2$ maps (color coded) overlaid on FITC images of microvasculature at depths of (a) $150\mu \mathrm{m}$ and (b) $130\mu \mathrm{m}$ below the brain surface. Inset in the lower left corner shows $160\text{-}\mu \mathrm{m}$-thick MIPs of FITC-labeled microvasculature. Arterioles are colored red. White rectangles in insets outline the position of the panels (a) and (b). Regions of interest with ${\mathrm{PO}}_2$ data included in the fitting procedure are the same as in rat II (Fig. 2). Scale bars, $100\mu \mathrm{m}$. (c) Tissue ${\mathrm{PO}}_2$ dependence on the radial distance from the arteriole with ${\mathrm{PO}}_2$ fits indicated by solid lines.

Fig. 4

${\mathrm{CMRO}}_2$ estimation at two anesthesia depths. (a, b) Tissue ${\mathrm{PO}}_2$ maps (color coded) at two different anesthesia levels, overlaid on FITC image of microvasculature $160\mu \mathrm{m}$ below brain surface. (a) Alpha-chloralose anesthesia; (b) combined alpha-chloralose and isoflurane anesthesia. Inset in lower right corner shows $312\text{-}\mu \mathrm{m}$-thick MIP of FITC-labeled microvasculature. Arterioles are colored red. White rectangle in inset outlines the positions of the panels (a) and (b). Regions of interest with ${\mathrm{PO}}_2$ data included in the fitting procedure are the same as in rat IV (Fig. 2). Scale bars, $100\mu \mathrm{m}$. (c) Tissue ${\mathrm{PO}}_2$ dependence on the radial distance from the arteriole with ${\mathrm{PO}}_2$ fits indicated by solid lines.