Vacuum-stabilized lung window enables real-time and simultaneous imaging of vascular and calcium responses to hypoxia in vivo

This study introduced a vacuum-stabilized lung window technique for intravital imaging, which for the first time enabled the simultaneous, high-resolution, real-time monitoring of both pulmonary arterial vascular reactivity and dynamic changes in intracellular calcium signaling smooth muscle cells during acute hypoxic pulmonary vasoconstriction (HPV) in spontaneously breathing mice. By integrating two-photon microscopy with intravascular fluorescent tracers (FITC, Evans Blue) and a smooth muscle-specific genetically encoded calcium indicator (GCaMP6f), this technique enabled stable imaging of pulmonary arteries within a diameter range of 13.95–80.16 μm and supported the systematic extraction and analysis of calcium signals from multiple regions of interest along the longitudinal axis of the vascular wall. Acute hypoxia (10% O 2 , 30 min) elicited a time-dependent vasoconstriction, with an average 8% reduction in diameter of 30–50 μm pulmonary arteries. Hypoxia triggered PASMC calcium signals characterized by a distinct latency period and marked spatial heterogeneity, with the earliest calcium responses observed in 30–40 μm arteries. Pharmacological intervention with dasatinib prolonged the latency of the calcium response and enhanced the amplitude of calcium oscillations, with effects preferentially targeting vessels in the 30–50 μm range. Simultaneous recordings confirmed that the rise in intracellular calcium consistently preceded vasoconstriction, and revealed that dasatinib treatment modulated calcium-contraction coupling in a vessel size-dependent manner. The technological platform established in this study overcomes the limitations of traditional approaches, simultaneously monitoring pulmonary artery function and cellular Ca 2+ signaling mechanisms in HPV.