Aim: Investigating spatio-temporal relationship between regional metabolic changes and microvascular responses in hypoxic brain is critical for unravelling local O2-sensing mechanisms. However, no reliable method to examine the relationship has been available because of inherent disadvantages associated with use of a conventional cranial window preparation. We aimed to devise a method to solve the problem. Methods: Anaesthetized mice were equipped with either a conventional cranial window with craniotomy or a thinned-skull preparation. Mice were mechanically ventilated to avoid hypercapnia and exposed to systemic isobaric hypoxia for 30min. Using two-photon laser scanning microscopy, nicotinamide adenine dinucleotide, reduced form (NADH) autofluorescence and diameter changes in penetrating and pre-capillary arterioles within the parenchyma were visualized to examine their temporal alterations. Results: With the conventional cranial window preparation, marked vertical displacement of the tissue occurred through oedema within 30s after inducing hypoxia. With a thinned-skull preparation, however, such hypoxia-induced displacement was diminished, enabling us to examine acute spatio-temporal changes in diameters of penetrating and pre-capillary arterioles and NADH autofluorescence. Vasodilatation of these microvessels was evoked within 1min after hypoxia, and sustained during the entire observation period despite the absence of hypercapnia. This event coincided with parenchymal NADH elevation, but the onset and peak dilatory responses of the penetrating arterioles preceded the local metabolic response of the parenchyma. Conclusion: Observation of hypoxia-exposed brain by the thinned-skull preparation combined with two-photon intra-vital microscopy revealed rapid vasodilatory responses in penetrating arterioles preceding parenchymal NADH elevation, suggesting the presence of acute hypoxia-sensing mechanisms involving specific segments of cortical arterioles within the neurovascular unit.
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