Ivo C.J.H. Post

Real-time assessment of renal cortical microvascular perfusion heterogeneities using near-infrared laser speckle imaging

Laser speckle imaging (LSI) is able to provide full-field perfusion maps of the renal cortex and allows quantification of the average LSI perfusion within an arbitrarily set region of interest and the recovery of LSI perfusion histograms within this region. The aim of the present study was to evaluate the use of LSI for mapping renal cortical microvascular perfusion and to demonstrate the capability of LSI to assess renal perfusion heterogeneities. The main findings were that: 1) full-field LSI measurements of renal microvascular perfusion were highly correlated to single-point LDV measurements; 2) LSI is able to detect differences in reperfusion dynamics following different durations of ischemia; and 3) renal microvascular perfusion heterogeneities can be quantitatively assessed by recovering LSI perfusion histograms.

Laser speckle contrast imaging for assessment of liver microcirculation

OBJECTIVE Laser speckle contrast imaging (LSCI) is a novel technique for microcirculation imaging not previously used in the liver. The aim of the present experimental study was to evaluate the use of LSCI for assessing liver microcirculation. MATERIALS AND METHODS In six male Wistar rats, the median liver lobe was exposed through a midline laparotomy. Liver blood perfusion was measured simultaneously with LSCI and sidestream dark-field (SDF) imaging at baseline and during sequential temporary occlusions of the portal vein, hepatic artery, and total blood inflow occlusion. Both the inter-individual variability associated with perfusion sampling area and comparisons in perfusion measurements between both imaging techniques were investigated and validated for the application of LSCI in the liver. RESULTS Occlusion of the hepatic artery, portal vein, and total inflow occlusion resulted in a significant decrease in LSCI signal to 74.7±6.4%, 15.0±2.3%, and 10.4±0.5% respectively (p<0.005 vs. baseline). The LSCI perfusion units correlated with sinusoidal blood flow velocity as measured with SDF imaging (Pearsons r=0.94, p<0.001). In a 10 mm diameter region of interest, as measured with LSCI, baseline inter-individual variability measured by the coefficient of variability was 13%. CONCLUSION Alterations in LSCI signal during sequential inflow occlusions were in accordance with previously published results on hepatic hemodynamics in the rat and correlated well with our SDF imaging-derived sinusoidal blood flow velocity measurements. We found that LSCI was able to produce reproducible real-time blood perfusion measurements of hepatic microcirculation. Compared to established techniques for liver blood perfusion measurements LSCI holds the advantages of non-contact measurements over large surfaces with a high speed of data acquisition.

A novel oxygenated machine perfusion system for preservation of the liver.

Machine perfusion (MP) is a potential method to increase the donor pool for organ transplantation. However, MP systems for liver grafts remain difficult to use because of organ-specific demands. Our aim was to test a novel, portable MP system for hypothermic preservation of the liver. A portable, pressure-regulated, oxygenated MP system designed for kidney preservation was adapted to perfuse liver grafts via the portal vein (PV). Three porcine livers underwent 20 h of hypothermic perfusion using Belzer MP solution. The MP system was assessed for perfusate flow, temperature, venous pressure, and pO2 /pCO2 during the preservation period. Biochemical and histological parameters were analyzed to determine postpreservation organ damage. Perfusate flow through the PV increased over time from 157 ± 25 mL/min at start to 177 ± 25 mL/min after 20 h. PV pressure remained stable at 13 ± 1 mm Hg. Perfusate temperature increased from 9.7 ± 0.6°C at the start to 11.0 ± 0.0°C after 20 h. Aspartate aminotransferase and lactate dehydrogenase increased from 281 ± 158 and 308 ± 171 U/L after 1 h to 524 ± 163 and 537 ± 168 U/L after 20 h, respectively. Blood gas analysis showed a stable pO2 of 338 ± 20 mm Hg before perfusion of the liver and 125 ± 14 mm Hg after 1 h perfusion. The pCO2 increased from 15 ± 5 mm Hg after 1 h to 53 ± 4 mm Hg after 20 h. No histological changes were found after 20 h of MP. This study demonstrated the feasibility of a portable MP system for preservation of the liver and showed that continuous perfusion via the PV can be maintained with an oxygen-driven pump system without notable preservation damage of the organ.

Optimal flow and pressure management in machine perfusion systems for organ preservation.

Intra-organ flow is the most critical parameter in machine-perfused organ preservation systems (MPS). Ultrasonic flow sensors (UFS) are commonly employed in MPS. However, UFS are sensitive to changes in fluid composition and temperature and require recalibration. Novel Coriolis-type mass flow sensors (CFS) may be more suitable for MPS because the measurement technique is not amenable to these factors. The effect of viscosity, colloids, temperature, pressure, and preservation solution on flow measurement accuracy of UFS and CFS was therefore investigated. A CFS-based MPS was built and validated for setpoint stability using porcine kidneys and the ability to reproduce different pressure and flow waveforms. The UFS exhibited a temperature- and preservation solution-dependent overestimation of flow rate compared to the CFS. The CFS deviated minimally from the actual flow rate and did not require recalibration. The CFS-based MPS conformed to the preprogrammed temperature, flow, pressure, and vascular resistance settings during 6-h kidney preservation. The system was also able to accurately reproduce different pressure and flow waveforms. Conclusively, CFS-based MPS are more suitable for organ preservation than UFS-based MPS. Our CFS-based MPS provides a versatile yet robust experimental platform for testing and validating different types of clinical and experimental MPS.

Antibiotic prophylaxis in (sub)normothermic organ preservation: in vitro efficacy and toxicity of cephalosporins.

Background Bacterial contamination during cold organ preservation occurs without major complications. However, with organ preservation steering toward (sub)normothermic temperatures, bacterial contamination may be detrimental with limited evidence to support the choice of antibiotic. Methods This study aimed to determine the effective antibiotic prophylaxis for (sub)normothermic preservation by investigating whether Staphylococcus epidermidis was capable of growing in a subnormothermia-compatible preservation solution Polysol (PS) and in solutions designed for cold preservation (University of Wisconsin solution, histidine-tryptophan-ketoglutarate solution, and Belzer-machine perfusion solution). Various S. epidermidis and Staphylococcus aureus strains were exposed to ceftriaxone and cefazolin at concentrations from 0 to 1000 &mgr;g/mL under subnormothermic and normothermic conditions in PS. To mimic procedural conditions, the effect of cefazolin was determined after exposure of bacteria to 20-hr incubation at 28°C in the presence of cefazolin and subsequent incubation at 37°C in the absence of cefazolin. The toxicity of cefazolin was assessed by cell viability and caspase activation assays in porcine kidney endothelial cells. Results Without antibiotics, PS sustained bacterial growth under sub(normothermic) conditions, whereas growth was absent in cold preservation solutions. Cefazolin exhibited greater bactericidal effect on S. epidermidis than ceftriaxone. However, after inoculating PS with 106 colony-forming units/mL, only a cefazolin concentration of 1000 &mgr;g/mL was able to exert a complete bactericidal effect on S. epidermidis and S. aureus strains and maintain sterility after removal of cefazolin. Finally, 1000 &mgr;g/mL cefazolin showed no adverse effects on porcine kidney endothelial cells. Conclusions Based on these findings, we recommend that high-dose cefazolin be used for prophylaxis in (sub)normothermic organ preservation with PS.

Efficacy of liver graft washout as a function of the perfusate, pressure, and temperature.

Donor graft washout can be impaired by colloids in organ preservation solutions that increase the viscosity and agglutinative propensity of red blood cells (RBCs) and potentially decrease organ function. The colloid‐induced agglutinative effects on RBCs and RBC retention after liver washout with Ringers lactate (RL), histidine tryptophan ketoglutarate solution, University of Wisconsin solution, and Polysol were determined as a function of the washout pressure (15 or 100 mm Hg) and temperature (4 or 37°C) in a rat liver washout model with 99mTc‐pertechnetate–labeled RBCs. Colloids (polyethylene glycol in Polysol and hydroxyethyl starch in University of Wisconsin) induced RBC agglutination, regardless of the solutions composition. RL was associated with the lowest degree of 99mTc‐pertechnetate–labeled RBC retention after simultaneous arterial and portal washout at 37°C and 100 mm Hg. RL washout was also associated with the shortest washout time. A single portal washout with any of the solutions did not result in differences in the degree of RBC retention, regardless of the temperature or pressure. In conclusion, no differences were found in portal washout efficacy between colloidal solutions, histidine tryptophan ketoglutarate, and RL. Simultaneous arterial and portal washout with RL at 37°C and 100 mm Hg resulted in the least RBC retention and the shortest washout time. Liver Transpl 19:843‐851, 2013.

Appraisal of the porcine kidney autotransplantation model.

Animal models are extensively used for transplantation related research, especially kidney transplantation. Porcine autotransplantation models are considered to be favorable regarding translatability to the human setting. The key determinants for translatability of the model are discussed, comprising animal age, development, anatomy, anesthesia and surgical protocols, and perioperative care. With the detailed discussion of these determinants and the pitfalls in diagnosing animal discomfort, an attempt is made to provide a uniform porcine kidney autotransplantation model with tools to improve currently used models.

Characterization and quantification of porcine circulating endothelial cells

Endothelial damage is a critical step in the development of (xeno) transplantation‐related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker.