Developing cryo on-grid lamella workflows for the JEOL JIB 4700F

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death.Since many cytoprotective roles have been describe for H2S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs.Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H2S delivery effects on graft hypothermic preservation, were the main objectives of this investigation.Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K+). Livers were surgically removed and subsequently stored in HTK Solution (Histidine–tryptophan–ketoglutarate) at 0–4 °C. After 24 h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I – Livers procured before cardiac death and cold stored 24 h in HTK (BCD); II – Livers procured after cardiac death (45 min) and cold stored 24 h in HTK (ACD); III – Livers procured after cardiac death (45 min) and cold stored 24 h in HTK + 10 μM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45 min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-α and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia.On the other hand, warm ischemic livers exposed to H2S during cold storage, improved microcirculation, morphology and viability parameters during ex vivo reperfusion and showed significant modulation of HO-1 mRNA expression.In conclusion, HTK supplementation with Na2S arose as a potential treatment to recover non-heart beating harvested organs. Furthermore, an appropriate model of cardiac dead liver donors was successfully developed.