Comparative evaluation of PEGylated bovine hemoglobin as a perfusate in rat ex vivo lung perfusion

BACKGROUND: Ex vivo lung perfusion (EVLP) is pivotal for assessing and preserving marginal donor lungs. While red-blood-cell (RBC)-based perfusates enhance oxygen delivery, they increase pulmonary vascular resistance, thereby potentially exacerbating perfusion-induced injury. This study compared a novel acellular oxygen carrier, PEGylated bovine hemoglobin (PEG-bHb), against RBC-based and acellular perfusates in a rat EVLP-transplantation model. METHODS: A total of 49 rats were included in this study. Lungs underwent 4 h EVLP with acellular, RBC or PEG-bHb solutions, followed by left-lung transplantation and 2 h reperfusion; unperfused lungs served as controls. Respiratory mechanics, gas exchange, inflammatory cytokines, oxidative-stress markers, tight junction integrity (Occludin), and histology were evaluated. Bulk RNA-sequencing, qRT-PCR, and immunofluorescence for endoplasmic reticulum (ER) stress markers (ATF-6, ATF-4, CHOP and ubiquitin) were employed to explore underlying mechanisms. RESULTS: PEG-bHb demonstrated lower pulmonary vascular resistance and higher dynamic compliance than the RBC group. Inflammatory cytokine (IL-6, IL-1β, TNF-α) and Oxidative markers (MDA, HIF-1α) in lung tissue were reduced compared to acellular and RBC groups. Occludin expression indicated superior alveolar-capillary integrity versus control and acellular groups and parity with RBC group. Post-transplantation, the PEG-bHb group exhibited the least histological injury and the highest IL-10 expression, indicating attenuated early graft damage. Transcriptomic analysis suggested concurrent regulation of endoplasmic-reticulum stress and cytokine pathways; immunofluorescence demonstrated reduced ATF-6, increased ATF-4, and stable CHOP expression. CONCLUSION: PEG-bHb reduced early post-transplant injury by preserving lung compliance, sustaining barrier integrity, and mitigating inflammatory stress during EVLP. It represents a promising candidate for clinical EVLP application.