Shailendra Jain

Survival transplantation of preserved non???heart-beating donor rat livers: preservation by hypothermic machine perfusion1

Background. Non–heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. Methods. This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. Results. Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07±1.42 and 2.02±0.69 U (mean±SE), respectively, compared with SCS, 15.54±0.81 and 3.41.3±0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17±0.49 mg/dL) were comparable to controls (0.91±0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. Conclusions. These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Improved Preservation of Warm Ischemic Livers by Hypothermic Machine Perfusion with Supplemented University of Wisconsin Solution

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs–Henseleit solution. Livers preserved with SUW showed significantly (p <. 001) improved recovery of tissue ATP levels (μ mol/g liver), 2.06 ± 0.10 (mean ± SE), as compared to the UW group, 0.70 ± 0.10, and the level was 80% of that of fresh control livers (2.60 ± 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p <. 05) lower in the SUW group (429 ± 58) as compared to ischemia–reperfusion (IR) (781 ± 12) and the UW group (1151 ± 83). Bile production (μ g/min/g liver) was significantly (p <. 05) higher in the SUW group (280 ± 13) as compared to the IR (224 ± 24) and the UW group (114 ± 14). The tissue edema formation assessed by tissue wet–dry ratio was significantly (p <. 05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Recovery of Energy Stores of Non-Heart-Beating Donor Livers Following Preservation by Hypothermic Machine Perfusion

The shortage of donor organs for liver transplantation requires investigations into expanding the donor pool. Non-heart beating donor (NHBD) livers are an untapped source of organs. NHBD organs are those that have had a period of warm ischemia prior to cold preservation. A main concern of NHBD livers is that there is a depletion of energy stores during the warm ischemia and subsequent cold storage. This study is to investigate the use of hypothermic machine perfusion to regenerate energy stores in the liver during 5hr storage and after 1hr rewarming in a rat transplantation model. Results will be compared with control. (no warm ischemia) and simple cold stored tissues.Copyright

The Effects of Shear Stress on Endothelial Cells at Hypothermic Temperatures

Hypothermic machine perfusion preservation (MPP) has proven to be a successful technique for hypothermic kidney storage, however this technology has not successfully been applied to the liver. Recent research has indicated that the endothelial cells lining the liver sinusoids display rounding phenomena during MPP that is not fully understood. In order to gain a better understanding of endothelial cell shear stress response and the factors that induce rounding, a temperature-controlled micro-shear chamber has been designed and fabricated. The micro-shear chamber has been used to apply shear stresses, corresponding to those imposed during MPP, to rat liver primary endothelial cell cultures in order to form an understanding of how these stresses affect endothelial cell morphology. The chamber allows for the application of shear stresses ranging from 0.2 ± .01 dynes/cm2 to 2.3 ± 0.3 dynes/cm2 , corresponding to what occurs during MPP.] Twenty-four hour in vitro experiments with shear stresses ranging from 0 to 1.49 dynes/cm2 at 4 °C were conducted in order to replicate in vivo conditions of the liver during hypothermic MPP. It has been demonstrated that endothelial cell rounding increases with increasing shear and can be prevented by utilizing low flow rates.Copyright

Visualization of Endothelial Cell Structure During Machine Perfusion Preservation of Livers

The development of machine perfusion preservation (MPP) of kidney has led to significant improvements and greater success rates in kidney transplantation by providing superior preserved tissue and viable non-heart-beating donor tissue. However, machine perfusion of livers has not been successful in improving preservation. Currently, the major cause of damage associated with MPP of livers remains unclear. Previous studies showed increased vascular resistance and blockages during and after 24hrs MPP but no direct evidence existed. Utilizing a novel two colors fluorophores labeling, an intravital microscopic study was conducted to obtain real time images and confocal microscopy to get detailed images in order to correlate fluorescent-tagged endothelial cells (ECs) with red cell stasis. Fluorescein isothiocynate (FITC) was used to label red blood cells (RBCs) and DiI acetylated low-density lipoprotein (DiI acLDL) was used to mark ECs. Structure of ECs was recorded and assessed during 24hrs MPP with University of Wisconsin (UW) solution at 4°C with a flow rate of 4ml/min. Images recorded from intravital microscopy and confocal microscopy show ECs rounding over a period of 24 hrs and subsequent red blood cells stasis after 24hrs MPP and during rewarming.Copyright