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

Background: 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.

Methods: 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.

Results: Acute hypoxia (10% O₂, 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.

Conclusions: The technological platform established in this study overcomes the limitations of traditional approaches, simultaneously monitoring pulmonary artery function and cellular CA²⁺ signaling mechanisms in HPV.