Design and In Vivo Validation of a Constant-Flow Ventilation Circuit for Tissue Stabilization During Intravital Imaging of the Lungs

Purpose: Intravital lung microscopy requires lung stabilization, which has been accomplished by vacuum fixation or imaging during ventilatory plateau phases. We introduce a constant-flow ventilation (CFV) circuit for stabilizing the lungs during intravital microscopy while supporting oxygenation. In CFV, a cannula is placed in the trachea with its tip near the carina. Fresh gas enters the catheter at a steady, high flow rate. Outflow exits around the outside of the catheter at the same rate. Gas exchange occurs by diffusion. Constant-flow ventilation has previously been tested in large animals and humans. Methods: We implement CFV in rats, with a custom circuit and orotracheal cannula that enable smooth switching between CFV and conventional mechanical ventilation (CMV). We initiate lung injury with zero end-expiratory pressure/excessive tidal volume CMV. Then we ventilate with protective CMV; 5 min of CFV (100% oxygen, 1.2 L/min/kg flow rate and cannula tip at the carina) during which we surgically open a window in an intercostal space and image the lungs; and, again, protective CMV. Results: Throughout CFV, peripheral arterial oxygen saturation (93%) and heart rate (316 min⁻¹) are constant. Our CFV circuit enables sufficient lung stability, despite cardiac motion, for imaging by brightfield and longer-duration confocal microscopy. Airway pressure is stable during CMV-CFV switching. Conclusion: This technique could enable new research investigations, e.g., of in vivo microvascular/alveolar mechanics without vacuum artifacts or cardiopulmonary coupling, and could potentially have clinical applications, e.g., protection against ventilation-induced lung injury.