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

Abstract

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. 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. 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. 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.