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Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20928011 for Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation by İkbal Şencan, Tatiana Esipova, Kıvılcım Kılıç, Baoqiang Li, Michèle Desjardins, Mohammad A Yaseen, Hui Wang, Jason E Porter, Sreekanth Kura, Buyin Fu, Timothy W Secomb, David A Boas, Sergei A Vinogradov, Anna Devor and Sava Sakadžić in Journal of Cerebral Blood Flow & Metabolism

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20928011 for Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation by İkbal Şencan, Tatiana Esipova, Kıvılcım Kılıç, Baoqiang Li, Michèle Desjardins, Mohammad A Yaseen, Hui Wang, Jason E Porter, Sreekanth Kura, Buyin Fu, Timothy W Secomb, David A Boas, Sergei A Vinogradov, Anna Devor and Sava Sakadžić in Journal of Cerebral Blood Flow & Metabolism

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.

© 2014 Macmillan Publishers Limited. All rights reserved. What is the organization of cerebral microvascular oxygenation and morphology that allows adequate tissue oxygenation at different activity levels? We address this question in the mouse cerebral cortex using microscopic imaging of intravascular O2partial pressure and blood flow combined with numerical modelling. Here we show that parenchymal arterioles are responsible for 50% of the extracted O2at baseline activity, and the majority of the remaining O2exchange takes place within the first few capillary branches. Most capillaries release little O2at baseline acting as an O2reserve that is recruited during increased neuronal activity or decreased blood flow. Our results challenge the common perception that capillaries are the major site of O2delivery to cerebral tissue. The understanding of oxygenation distribution along arterio-capillary paths may have profound implications for the interpretation of blood-oxygen-level dependent (BOLD) contrast in functional magnetic resonance imaging and for evaluating microvascular O2delivery capacity to support cerebral tissue in disease.

Measurements of oxygen partial pressure (pO(2)) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO(2) measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here we report to our knowledge the first practical in vivo two-photon high-resolution pO(2) measurements in small rodents' cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 microm, sub-second temporal resolution and requires low probe concentration. The properties of the probe allowed for direct high-resolution measurement of cortical extravascular (tissue) pO(2), opening many possibilities for functional metabolic brain studies.

The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has focused scientific attention on the necessary tools to understand the human brain and mind. Here, we outline our collective vision for what we can achieve within a decade with properly targeted efforts and discuss likely technological deliverables and neuroscience progress.