Correction of heat-induced susceptibility changes in respiratory-triggered 2D-PRF-based thermometry for monitoring of magnetic resonance-guided hepatic microwave ablation in a human-like in vivo porcine model.

PURPOSE: To develop and evaluate susceptibility corrected 2D proton resonance frequency (PRF)-based magnetic resonance (MR)-thermometry for the accurate assessment of the ablation zone of hepatic microwave ablation (MWA). METHODS AND MATERIALS: Twelve hepatic MWA were performed in five LEWE minipigs with human-like fissure-free liver. Temperature maps during ablation of PRF-based MR-thermometry were corrected by modeling heat induced susceptibility changes. Ablation zones were determined using cumulative equivalent minutes at 43 °C (CEM43) as tissue damage model. T1 weighted (w) post-ablation contrast-enhanced (CE) MR-imaging and manually segmented postmortem histology were used for validation. The agreement of uncorrected (raw) and susceptibility corrected (corr) MR-thermometry with T1w post-ablation CE MR-imaging and histology was evaluated. The Wilcoxon-signed rank test and Bland-Altman analysis were applied. RESULTS: With the susceptibility corrected MR-thermometry a significantly increased dice coefficient (raw: 77% vs. corr: 83%, p < 0.01) and sensitivity (raw: 72% vs. corr: 82%, p < 0.01) was found for the comparison to T1w-CE imaging as well as histopathology (dice coefficients: raw: 76% vs. corr: 79%, p < 0.001; sensitivity: raw: 72% vs. corr: 74%, p < 0.001). While major axis length was significantly increased (7.1 mm, p < 0.001) and minor axis length significantly decreased (2.2 mm, p < 0.001) in uncorrected MR-thermometry compared to T1w-CE MR-imaging, no significant bias was found after susceptibility correction. CONCLUSION: Using susceptibility corrected 2D PRF-based MR-thermometry to predict the ablation zones of hepatic MWA provided a good agreement in comparison to T1w post-ablation CE MR-imaging and histopathology.

Supportive Hepatocyte Transplantation after Partial Hepatectomy Enhances Liver Regeneration in a Preclinical Pig Model.

BACKGROUND: Hepatocyte transplantation (HTx) is regarded as a potential treatment modality for various liver diseases including acute liver failure. We developed a preclinical pig model to evaluate if HTx could safely support recovery from liver function impairment after partial hepatectomy. METHODS: Pigs underwent partial hepatectomy with reduction of the liver volume by 50% to induce a transient but significant impairment of liver function. Thereafter, 2 protocols for HTx were evaluated and compared to a control group receiving liver resection only (group 1, n = 5). Portal pressure-controlled HTx was performed either immediately after surgery (group 2, n = 6) or 3 days postoperatively (group 3, n = 5). In all cases, liver regeneration was monitored by conventional laboratory tests and the novel noninvasive maximum liver function capacity (LiMAx) test with a follow-up of 4 weeks. RESULTS: Partial hepatectomy significantly impaired liver function according to conventional liver function tests as well as LiMAx in all groups. A mean of 4.10 ± 1.1 × 108 and 3.82 ± 0.7 × 108 hepatocytes were transplanted in groups 2 and 3, respectively. All animals remained stable with respect to vital parameters during and after HTx. The animals in group 2 showed enhanced liver regeneration as observed by mean postoperative LiMAx values (621.5 vs. 331.3 µg/kg/h on postoperative day 7; p < 0.001) whereas HTx in group 3 led to a significant increase in mean liver-specific coagulation factor VII (112.2 vs. 54.0% on postoperative day 7; p = 0.003) compared to controls (group 1), respectively. In both experimental groups, thrombotic material was observed in the portal veins and pulmonary arteries on histology, despite the absence of clinical symptoms. CONCLUSION: HTx can be performed safely and effectively immediately after a partial (50%) hepatectomy as well as 3 days postoperatively, with comparable results regarding the enhancement of liver function and regeneration.

An Approach towards Motion-Tolerant PPG-Based Algorithm for Real-Time Heart Rate Monitoring of Moving Pigs.

Animal welfare remains a very important issue in the livestock sector, but monitoring animal welfare in an objective and continuous way remains a serious challenge. Monitoring animal welfare, based upon physiological measurements instead of the audio-visual scoring of behaviour, would be a step forward. One of the obvious physiological signals related to welfare and stress is heart rate. The objective of this research was to measure heart rate (beat per minutes) in pigs with technology that soon will be affordable. Affordable heart rate monitoring is done today at large scale on humans using the Photo Plethysmography (PPG) technology. We used PPG sensors on a pig's body to test whether it allows the retrieval of a reliable heart rate signal. A continuous wavelet transform (CWT)-based algorithm is developed to decouple the cardiac pulse waves from the pig. Three different wavelets, namely second, fourth and sixth order Derivative of Gaussian (DOG), are tested. We show the results of the developed PPG-based algorithm, against electrocardiograms (ECG) as a reference measure for heart rate, and this for an anaesthetised versus a non-anaesthetised animal. We tested three different anatomical body positions (ear, leg and tail) and give results for each body position of the sensor. In summary, it can be concluded that the agreement between the PPG-based heart rate technique and the reference sensor is between 91% and 95%. In this paper, we showed the potential of using the PPG-based technology to assess the pig's heart rate.

Long-term Functional Maintenance of Exteriorized Portal Venous Catheters in a Porcine Animal Model.

BACKGROUND: Portal venous access for blood sampling, infusion therapy, and measurement of portal venous pressure is of special interest for experimental studies in surgery, pharmacology, and hepatology. Chronic animal models with continuous portal venous access are rare and especially thrombosis or clotting of permanent catheters is a frequent complication. Aim of this study was to establish a preclinical pig model with a permanent portal venous catheter (PVC). MATERIALS AND METHODS: PVC implantation was performed in 21 LEWE mini pigs. The catheter was inserted in the distal part of the superior mesenteric vein and fixated with a tobacco-pouch suture. Animals were followed up for 4 wk, directly after implantation of the PVC. Blood gas analyses and portal venous pressures were recorded. Three different groups with continuous infusion via the catheters were defined: NaCl solution (2 mL/h) (group 1), NaCl solution (2 mL/h) + enoxaparin sodium injection (anti-Xa levels of 0.3-0.8 U/mL) (group 2) and heparinized NaCl (2 I.E./mL, 2 mL/h) (group 3). RESULTS: All 21 PVC implantations were performed without any complications. Application of continuous perfusion with heparinized NaCl (group 3) enabled portal venous access for the entire experiment in 8 of 10 cases (mean of 23.7 d) without any signs of dysfunction. However, for use of NaCl alone or in combination with enoxaparin sodium, catheters were only functional for 6.8 d and 6.9 d, respectively. CONCLUSIONS: Permanent portal venous access through PVC in mini pigs is achievable by continuous infusion of low-dose heparinized NaCl solution.

Alloresponses of Mixed Lymphocyte Hepatocyte Culture to Immunosuppressive Drugs as an In-Vitro Model of Hepatocyte Transplantation.

BACKGROUND Hepatocyte transplantation (HCTx) has the potential for the treatment of end-stage liver disease. However, failure of engraftment and the long-term acceptance of cellular allografts remain significant challenges for its clinical application. The aim of this study was to investigate the efficacy of the immunosuppressive agents, Cyclosporine, Everolimus, and Belatacept to suppress the alloresponse of primary human hepatocytes in a mixed lymphocyte-hepatocyte culture (MLHC) and their potential hepatotoxicity in vitro. MATERIAL AND METHODS Primary human hepatocytes were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an MLHC. Proliferative alloresponses were determined by flow cytometry, and cytokine secretion was measured using Luminex-based multiplex technology. Using an MLHC, the alloresponses of primary human hepatocytes were compared in the presence and absence of Cyclosporine, Everolimus, and Belatacept. Cultured primary human hepatocytes were assessed for the production of albumin, urea, aspartate transaminase (AST) and DNA content. Metabolic activity was determined with the MTT assay. RESULTS Immune responses induced by primary human hepatocytes were effectively suppressed by Cyclosporine, Everolimus, and Belatacept. Everolimus significantly reduced the metabolic activity of primary human hepatocytes in vitro, suggesting impairment of cell viability. However, further functional analysis showed no significant differences between treated and untreated controls. CONCLUSIONS Cyclosporine, Everolimus, and Belatacept suppressed the alloresponse of primary human hepatocytes in an MLHC without significant cytotoxicity or functional cell impairment.

Porcine model for the study of liver regeneration enhanced by non-invasive 13C-methacetin breath test (LiMAx test) and permanent portal venous access.

INTRODUCTION: Despite advances in perioperative management and surgical technique, postoperative liver failure remains a feared complication after hepatic resection. Various supportive treatment options are under current discussion, but lack of structured evaluation. We therefore established a porcine model of major liver resection to study regeneration after partial hepatectomy in a reliable and well-defined pre-clinical setting. METHODS: Major hepatectomy was performed on seven minipigs with the intention to set up a non-lethal but relevant transient impairment of liver function. For steady postoperative vascular access (e.g. for blood withdrawal, measurement of venous pressure), permanent catheters were implanted into the internal jugular and portal veins, respectively. Animals were followed up for 30 days; clinical and laboratory results were recorded in detail. Monitoring was enhanced by non-invasive determination of the maximum liver function capacity (LiMAx test). RESULTS AND CONCLUSIONS: The established porcine model appeared suitable for evaluation of postoperative liver regeneration. Clinical characteristics and progression of liver function impairment as well as subsequent recovery were comparable to courses known from surgery in humans. Laboratory parameters (e.g. liver enzymes, bilirubin, INR, coagulation factor II) showed relevant derangements during postoperative days (POD) 0 to 3 followed by normalization until POD 7. Application of the LiMAx test was feasible in minipigs, again showing values comparable to humans and kinetics in line with obtained laboratory parameters. The exteriorized portal vein catheters enabled intra- and postoperative monitoring of portal venous pressures as well as easy access for blood withdrawal without relevant risk of postoperative complications.

Silencing of HLA class I on primary human hepatocytes as a novel strategy for reduction in alloreactivity.

In contrast to the whole liver, primary hepatocytes are highly immunogenic. Thus, alternative strategies of immunomodulation after hepatocyte transplantation are of special interest. Silencing of HLA class I expression is expected to reduce the strength of allogeneic immune responses and to improve graft survival. In this study, primary human hepatocytes (PHH) were isolated using a two-step-collagenase perfusion-technique and co-cultured with allogeneic lymphocytes in terms of a mixed lymphocyte hepatocyte culture. Expression of HLA class I on PHH was silenced using lentiviral vectors encoding for ß2-microglobulin-specific short hairpin RNA (shß2m) or non-specific shRNA (shNS) as control. The delivery of shß2m into PHH caused a decrease by up to 96% in ß2m transcript levels and a down-regulation of HLA class I cell surface expression on PHH by up to 57%. Proliferative T cell alloresponses against HLA-silenced PHH were significantly lower than those observed form fully HLA-expressing PHH. In addition, significantly lower secretion of pro-inflammatory cytokines was observed. Levels of albumin, urea and aspartate-aminotransferase did not differ in supernatants of cultured PHH. In conclusion, silencing HLA class I expression on PHH might represent a promising approach for immunomodulation in the transplant setting without compromising metabolic function of silenced hepatocytes.