Steven M. Strasberg

Cold preservation of rat sinusoidal endothelial cells causes actin disassembly, cell rounding, matrix metalloproteinase (MMP) secretion, and platelet adhesiveness. Studies in rats suggest that gelatinases MMP2 and MMP9 are the key mediators of the injury. We created a model of cold preservation injury in mouse sinusoidal endothelial cell (MSEC) to examine the effect of cold on MSEC, specifically on MSEC from genetically deleted mice (MMP9/KO) mice. MSEC were isolated from wild-type and MMP9/KO mice and cold preserved for up to 24 hours. MMP activity was measured in culture supernatants and in effluents from preserved whole mouse livers. Cellular and actin morphology were studied by light and fluorescence microscopy. A platelet-MSEC adhesion assay was performed. Yield, growth, and appearance of MSEC were similar in wild-type and MMP9/KO mice. Cold-preserved wild-type MSEC exhibited actin disassembly and cell rounding as in the rat but at a much slower rate. These morphologic cell changes were attenuated in MSEC from MMP9/KO mice. Both MMP2 and MMP9 were present in liver effluents of wild-type mice, but MMP9 was absent in effluents from MMP9/KO mice. Total MMP activity in culture supernatants was greater after preservation in wild-type than in MMP9/KO mice. There was significantly more platelet adhesion to wild-type MSEC than to MSEC from MMP9/KO mice. In conclusion, MSEC is an excellent model system for the study of cold preservation injury. Injury is similar to rat sinusoidal endothelial cells but delayed. MMP9 is a key mediator of the cold preservation injury. (Liver Transpl 2004;10:1041-1048.)

Summary Background Data: Saline-linked surface radiofrequency (RF) ablation is a new technique for applying RF energy to surfaces. The surface is cooled, which prevents charring and results in deeper coagulation. However, subsurface heating may lead to steam formation and a form of tissue disruption called steam popping. We determined parameters that predict steam popping and depth of tissue destruction under nonpopping conditions. A commercially available saline-linked surface RF cautery device (Floating Ball 3.0, TissueLink, Inc.) was used. Methods: One hundred eighty circular lesions were created varying in lesion diameter, duration, power, and inflow occlusion. Variables affecting popping were determined. Then factors influencing lesion depth were studied at fixed nonpopping diameter/power combinations (1 cm/10W, 2 cm/15W, 4 cm/60W). Tissue viability was determined in selected samples by staining of tissue NADH. Results: The probability of steam popping was directly related to power level and inflow occlusion, and indirectly related to lesion diameter. Depth of injury under safe nonpopping conditions was directly related to power, lesion size, and inflow occlusion. Maximum depth in excess of 20 mm was achieved using a 4 cm diameter at 60W with inflow occlusion. Microscopy of NADH-stained tissues showed a complete cell killing in the macroscopically visible coagulated area. Conclusions: Steam popping can be avoided by selecting power level/lesion diameter combinations. Tissue destruction to 20 mm can be safely achieved with short periods of inflow occlusion. The device has promise as a treatment of superficial tumors and close resection margins.

Leukocyte adhesion on reperfusion is a critical component of cold preservation injury, and involves increased intercellular adhesion molecule 1 (ICAM-1) expression on sinusoidal endothelial cells (SEC). This study determined whether ICAM-1 expression occurs during cold preservation and whether actin disassembly is necessary for ICAM-1 expression and leukocyte adhesion. ICAM-1 expression was measured in isolated rat SEC during 8 hours of cold preservation by immunofluorescence techniques. Leukocyte adhesion to cold-preserved SEC was measured in an assay using fluorescently labeled leukocytes. The calpain inhibitors N-acetyl-leu-leu-norleucinal/N-acetyl-leu-leu-methioninal and the actin stabilizer phalloidin were added in some studies to prevent actin disassembly. Cold-exposed SEC showed a rapid increase of surface ICAM-1 expression, reaching maximum values in 1 hour. Studies in permeabilized cells suggested that ICAM-1 moved from a perinuclear location to the cell surface. Actin stabilization had no effect on the time-dependent increase in ICAM-1 expression, but seemed to affect the distribution of ICAM-1 on the cell surface. Leukocyte adhesion to SEC correlated with ICAM-1 expression and was reduced to control levels by an anti-ICAM-1 antibody. Although actin stabilization did not reduce ICAM-1 expression, it did reduce leukocyte-SEC adhesion to control values. Increased ICAM-1 expression on cold-preserved SEC is a direct effect of cold. It is not related to actin disassembly, although it seems that actin disassembly affects the distribution of ICAM-1. Leukocyte adhesion to cold-preserved SEC requires both increased ICAM-1 and actin disassembly. Agents that inhibit actin disassembly can significantly decrease leukocyte adhesion regardless of increased ICAM-1 expression.

The therapeutic approach to treating patients with hepatocellular carcinoma (HCC) has changed radically over the past few decades and continues to evolve as the biology and natural history of the disease become better understood and new effective treatment modalities are developed. Modern cancer therapy has evolved so that it can be specifically tailored in the best interests of an individual patient to avoid too much or too little therapy and to provide reasonably predictable outcomes and expectations. Such individualized therapy requires an accurate estimation of prognosis. Accurate prognostication, in turn, depends upon understanding the natural history of the disease for a given tumor stage, as well as the probability of survival in response to a specific therapy. Appropriate selection of individualized cancer therapy for patients with HCC is more complex than for most cancers because prognosis depends not just on the anatomic extent of the disease but also on liver function [ 1 x 1 Okuda, K., Ohtsuki, T., and Obata, H. Natural history of hepatocellular cancer and prognosis in relation to treatment. Study of 850 patients. Cancer. 1985; 56: 918–928 Crossref | PubMed | Scopus (1576) | Google Scholar See all References 1 ]. In fact, liver function is a more powerful predictor of survival as well as response to specific therapy than tumor stage for many patients with HCC.

This study was performed to determine the role of intracellular calcium concentration and calpain activity on the cellular events that occur in rat sinusoidal endothelial cells (SEC) in the cold. Intracellular calcium concentrations were measured in isolated cold preserved rat SEC. Dantrolene or 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was added in some studies. In other studies, calpain activity and m-calpain and μ-calpain expression were measured during cold preservation in the presence or absence of calpain inhibitors. The effect of addition of dantrolene to preservation solutions on function of whole livers after preservation was determined. Cold preservation caused an increase in intracellular calcium concentration first detected at 1 hour of preservation. This was associated with cell rounding and actin disassembly. Dantrolene and BAPTA-AM prevented the increase in intracellular calcium concentration and reduced cell rounding and actin disassembly. Cold preservation also resulted in increased calpain activity and expression on SEC. Calpain expression was reduced by dantrolene. Calpain inhibitors N-acetyl-leu-leu-norleucinal (ALLN) and N-acetyl-leu-leu-methioninal (ALLM) reduced calpain activity and expression and restored SEC cell shape and actin morphology. Dantrolene improved function of livers preserved in Eurocollins (EC) solution when tested on the isolated perfused rat liver (IPRL). In conclusion, exposure of SEC to cold results sequentially in elevated intracellular calcium concentration, increased calpain activity, and actin disassembly.

To present the survival results for patients with colorectal carcinoma metastases who have undergone liver resection after being staged by [18F] fluoro-2-deoxy-D-glucose–positron emission tomography (FDG-PET). Hepatic resection is standard therapy for colorectal metastases confined to the liver, but recurrence is common because of the presence of undetected cancer at the time of surgery. FDG-PET is a sensitive diagnostic tool that identifies tumors based on the increased uptake of glucose by tumor cells. To date, no survival results have been reported for patients who have actually had liver resection after being staged by FDG-PET. Forty-three patients with metastatic colorectal cancer were referred for hepatic resection after conventional tumor staging with computed tomography. FDG-PET was performed on all patients. Laparotomy was performed on patients not staged out by PET. Resection was performed at the time of laparotomy unless extrahepatic disease or unresectable hepatic tumors were found. Patients were examined at intervals in the preoperative period. FDG-PET identified additional cancer not seen on computed tomography in 10 patients. Surgery was contraindicated in six of these patients because of the findings on FDG-PET. Laparotomy was performed in 37 patients. In all but two, liver resection was performed. Median follow-up in the 35 patients undergoing resection was 24 months. The Kaplan-Meier estimate of overall survival at 3 years was 77% and the lower 95% confidence limit of this estimate of survival was 60%. This figure is higher than 3-year estimate of survival found in previously published series. The 3-year disease-free survival rate was 40%. Preoperative FDG-PET lessens the recurrence rate in patients undergoing hepatic resection for colorectal metastases to the liver by detection of disease not found on conventional imaging.

UW solution and HTK solution are both used for cold preservation of liver allografts. Although they are about equally effective, their compositions are very different, and they were formulated using different rationales. The authors recently showed an important role for MMPs in liver preservation injury and consequently postulated that these preservation solutions contain cryptic inhibitors of MMP activity. To determine this possibility, the ability of these solutions to inhibit MMP activity was studied. The source of MMP2 and MMP9 was human liver effluents obtained at the time of liver transplantation or commercially available human recombinant MMP2 and MMP9. MMP2 and MMP9 showed gelatinolytic activity at 37°C and also at 4°C, although activity at 4°C was reduced. Activity was inhibited by University of Wisconsin (UW) and Histidine/Tryptophan/ Ketoglutarate (HTK) solutions. Examination of individual ingredients disclosed that reduced glutathione (GSH) and lactobionate in UW solution and histidine in HTK solution were the cryptic inhibitors. HTK solution was a more effective inhibitor than UW solution. GSH inhibited the activity of both enzymes, but was a much more effective inhibitor of MMP9 than MMP2. Oxidized glutathione(GSSG) was a much less effective inhibitor of the enzymes. The inhibitor constants (K_i) of GSH for MMP2 and MMP9 were 34 μmol/L and 3 μmol/L, respectively. The authors conclude that MMP inhibition is a cryptic property of both commonly used liver preservation solutions and contributes importantly to their action. Furthermore, GSH appears to be an effective inhibitor of gelatinases at concentrations at which it is normally present in extracellular fluid.