Endoplasmic and Vascular Surface Activation During Organ Preservation: Refining Upon the Benefits of Machine Perfusion

Abstract

The endoplasmic reticulum (ER) represents a subcellular target reactive to various cytosolic impairments. The involvement of ER-stress in organ preservation was investigated, comparing machine preservation, cold storage (CS) and a novel concept of only temporary perfusion after procurement. Rat livers were retrieved 30 min after cardiac arrest and preserved for 18 h by CS, oxygenated machine perfusion for 18 h (18 h MP) or for 2 h with subsequent CS for 16 h (2 h MP + 16 h CS). Upon reperfusion, 18 h MP significantly improved enzyme leakage (ALT, LDH) and promoted a 2-fold increase of metabolic recovery compared to CS. However, vascular stress, evaluated by endothelinrelease, was significantly elevated after 18 h MP. Interestingly, better viability was obtained using the shortterm perfusion protocol (2 h MP + 16 h CS), which further reduced enzyme leakage, maintained energetic recovery and mitigated endothelin-release compared to 18 h MP. Caspase 12-mRNA was upregulated in the 18 h MP-group but unchanged after CS or 2 h MP + 16 h CS. Activation/cleavage of caspase 12 protein was significantly enhanced after 18 h MP and very low in the 2 h MP + 16 h CS-group. Correspondingly, electron microscopy showed ultrastructural alterations of ER after CS and especially after 18 h MP but not after 2 h MP + 16 h CS. At this time mitochondrial appearance was unaffected in all groups, suggesting the ER to be an early subcellular target of preservation injury. In our model, ER and vascular endothelium were best protected by only temporary machine perfusion, which also maintained overall graft viability.

Role of perfusion medium, oxygen and rheology for endoplasmic reticulum stress-induced cell death after hypothermic machine preservation of the liver

“…However, despite the fact that restoration of tissue aerobiosis is conjectured to be the most valuable means for preventing progression of tissue alterations during preservation of the NHBD graft [10], this technique is rather cumbersome, needs an attentive surveillance during transportation and may be prone to induce alterations at the vascular endothelial site, if performed for a long time or under suboptimal conditions [11,12].…”

“…Accumulating evidence suggests perturbations at the endoplasmic reticulum (ER) as a novel subcellular effector, possibly involved in promotion of cell death in various pathologies [13] including the pathophysiology of organ preservation [12,14].…”

“…Both mechanisms have been found operative in experimental or clinical liver preservation [12,14] and the first data suggest that ER-stress activation is especially favoured by long term HMP, where ultrastructural alterations of the ER, as well as particular ER-stress-associated gene responses, were significantly more prominent after 18 h of machine preservation compared with cold storage (CS) [12]. In this study, however, machine perfusion was performed with histidine-tryptophan-ketoglutarate (HTK), being a noncolloid containing solution, primarily designed for static preservation and not yet fully validated for long-term graft perfusion.…”

Protective effect of tanshinone IIA on rat kidneys during hypothermic preservation

“…It has been reported that induction of CHOP could be detected between 4 and 8 h of exposure to ischemia, and pro-caspase-12 was also detectable in the same time frame (17). Minor et al showed significant ultrastructural alterations of ER and marked up-regulation of caspase-12 mRNA following cold storage or oxygenated machine perfusion of rat liver for 18 h, while mitochondrial appearance was unaffected, suggesting the ER to be an early subcellular target of preservation injury (18). Our previous study reported that ER stress was triggered during the hypothermic preservation, and reducing the levels of CHOP and caspase-12 may preserve the donor organ viability (19).…”

Raman spectral signatures of human liver perfusates correlate with oxidation reduction potential

“…This was the rationale for initiating a research program to develop whole human livers (ethically donated for transplantation and research but rejected for transplantation) to determine whether drug-induced hepatic toxicity can be predicted in real-time after dosing into the blood. Thus far, ex-vivo organ perfusion experiments have relied on physical parameter measurements and offline chemical assays to assess the viability of each organ throughout the normothermic perfusion period (10,11). These include the release of a host of potential biomarkers, including liver enzymes (AST, ALT, LDH and GGT) into the perfusate.…”