Thermal Preconditioning During <em>Ex-vivo</em> Lung Perfusion for the Rehabilitation of Damaged Lung Grafts before Transplantation

Ex-vivo lung perfusion (EVLP) has emerged as a breakthrough in lung transplantation, enabling both functional assessment of donor lungs and therapeutic interventions to rehabilitate marginal grafts. Reconditioning strategies include pharmacological and non-pharmacological approaches. Among the latter, we demonstrated that transient, controlled heat stress ("Thermal Preconditioning," TP) is an effective means of restoring damaged lungs by inducing an endogenous protective heat shock response. Here, we present a step-by-step protocol for a rat EVLP model that incorporates TP to recondition lungs subjected to warm ischemia, simulating donation after circulatory death (DCD). The protocol details lung preparation, cannulation, perfusion, and induction of the heat shock response. TP consists of a 30 min increase in perfusate temperature to 41.5 °C early in EVLP, followed by rapid return to 37 °C for the remainder of perfusion. In a 6 h EVLP model, TP-treated lungs (n = 30) showed increased heat shock protein 70 expression, improved static pulmonary compliance, reduced edema, lower release of von Willebrand factor (an endothelial injury biomarker), and attenuated histological damage compared to controls (n = 30). The protocol enables real-time monitoring of vascular resistance, airway pressures, pulmonary compliance, and oxygenation, and supports molecular and morphological analyses in both perfusate and tissue. This reproducible, scalable protocol is adaptable to larger animal or human EVLP systems, providing a practical translational platform for studying non-pharmacological interventions aimed at improving transplant outcomes.