Senolytic treatment during normothermic machine perfusion dampens ischemia reperfusion injury in old livers

Introduction: Donor organ shortage remains the leading cause of death among patients with end-stage organ failure awaiting transplantation. Older donor organs represent an underutilized opportunity to narrow the gap between demand and supply. However, their increased susceptibility to ischemia–reperfusion injury (IRI) has been associated with poorer outcomes. We have previously identified the accumulation of senescent cells and senescence-associated secretory phenotype (SASP) signaling as key drivers of exacerbated age-related IRI. Senolytic therapies, which selectively eliminate senescent cells, can mitigate these effects and improve graft survival in preclinical models. Nevertheless, clinical translation has been limited by the narrow therapeutic window between graft procurement and transplantation, as well as concerns regarding potential systemic adverse effects in transplant recipients. We therefore propose the application of senolytics during normothermic machine perfusion (NMP) as a targeted strategy to reduce IRI and improve the quality of older donor organs without recipient exposure.

Methods: Isolated livers from old male Sprague Dawley rats (12 months) were treated with a perfusate solution supplemented with a combination of Dasatinib (2 µM) and Quercetin (20 µM) (D&Q) during 6 hours of NMP. A modified version of a small NMP system previously developed by our group was used. Perfusate was collected at baseline, 2, 4 and 6 hours of perfusion and analyzed with Multi-ELISA, qPCR and liver function tests. After perfusion, tissue samples were analyzed with qPCR and stained for senescence-associated-β-galactosidase (sa-β-gal) to assess the amount of senescent cells.

Results: Compared to controls, the perfusate of D&Q-treated livers showed a consistent reduction in multiple key SASP-factors over 6 hours of perfusion, including cell-free mitochondrial DNA and IL-17 as major drivers of age-dependent IRI and rejection. This effect was accompanied by lower ALT and AST release and preserved bile production, suggesting reduced hepatocellular injury. Importantly, qPCR analysis of liver tissue collected at the end of perfusion revealed a prominent downregulation of key senescence markers, including p21 and p16, as well as pro-inflammatory SASP factors (e.g. TNF-α, IL-6, MMP3) in D&Q-treated livers. This observation was parralleled by a significantly lower number of sa-β-gal positive cells providing first evidence for a successful clearance of senescent cells during NMP.

Conclusion: Together, these data demonstrate for the first time that senolytic treatment during NMP is feasible and effective in dampening age-specific IRI which may be applicable not only for livers but older organ transplantation in general.