Ichiro Ohsawa

The Cytoprotective Effects of Addition of Activated Protein C into Preservation Solution on Small-for-Size Grafts in Rats

Small‐for‐size liver grafts are a serious obstacle for partial orthotopic liver transplantation. Activated protein C (APC), a potent anticoagulant serine protease, is known to have cell‐protective properties due to its anti‐inflammatory and antiapoptotic activities. This study was designed to examine the cytoprotective effects of a preservation solution containing APC on small‐for‐size liver grafts, with special attention paid to ischemia‐reperfusion injury and shear stress in rats. APC exerted cytoprotective effects, as evidenced by (1) increased 7‐day graft survival; (2) decreased initial portal pressure and improved hepatic microcirculation; (3) decreased levels of aminotransferase and improved histological features of hepatic ischemia‐reperfusion injury; (4) suppressed infiltration of neutrophils and monocytes/macrophages; (5) reduced hepatic expression of tumor necrosis factor α and interleukin 6; (6) decreased serum levels of hyaluronic acid, which indicated attenuation of sinusoidal endothelial cell injury; (7) increased hepatic levels of nitric oxide via up‐regulated hepatic endothelial nitric oxide synthesis expression together with down‐regulated hepatic inducible nitric oxide synthase expression; (8) decreased hepatic levels of endothelin 1; and (9) reduced hepatocellular apoptosis by down‐regulated caspase‐8 and caspase‐3 activities. These results suggest that a preservation solution containing APC is a potential novel and safe product for small‐for‐size liver transplantation, alleviating graft injury via anti‐inflammatory and antiapoptotic effects and vasorelaxing conditions. Liver Transpl 16:1–11, 2010.

Selective Stimulation of C Fibers by an Intra-Epidermal Needle Electrode in Humans

We recorded evoked potentials (EPs) induced by intra-epidermal electrical stimulation using a needle electrode with specific parameters. We identified the fibers activated by this specific stimulation by assessing the conduction velocity (CV) of the peripheral nerve. The EPs were recorded from the Cz electrode (vertex) of the International 10-20 system in ten healthy male subjects. The dorsum of the left hand and forearm were stimulated with an intensity of 0.01 mA above the sensory threshold. The mean P1 latency of EPs for the hand and forearm were 1007 ± 88 and 783 ± 80 ms, respectively, and the CV estimated from the latency of P1 was 1.5 ± 0.7 m/s. The CV indicated that the fibers activated by the stimulation were C fibers. Since the method of stimulation is convenient and non-invasive, it should be useful for investigating the functions of small fibers.

In vivo luminescent imaging of cyclosporin A-mediated cancer progression in rats

Background. Immunosuppressed individuals undergoing organ transplantation are at increased risk of recurrences of initial cancers, although how immunosuppressive therapy increases early cancer metastasis remains unclear. Methods. The metastatic fate of luciferase-expressing rat metastatic colon cancer cells (luc-RCN-H4) injected intravenously into the liver of syngeneic and allogeneic rats was examined in the presence of the immunosuppressant cyclosporin A (CsA) by in vivo luminescent technique. With respect to potential tumor-progressing factors, contribution of chemokine receptors and transforming growth factor (TGF)–β1 to early metastasis was evaluated using their specific signaling inhibitors. Results. F344 rats injected in the liver with luc-RCN-H4 cells did not always exhibit the formation of tumors and showed a dormant state as long as 60 days after inoculation without CsA. However, CsA released early luc-RCN-H4 cells from dormancy within 2 weeks at nearly 100% in liver and preferentially promoted metastasis to the lymph nodes (approximately 40%). A similar dissemination occurred even in minor histocompatibility complex–disparate hosts. As a tumor-progressing factor, RCN-H4 cells aberrantly expressed chemokine receptors CXCR4 and CCR7. The chemokine receptor (CXC) R4–specific antagonist AMD3100 decreased early metastasis of luc-RCN-H4 cells in rats with ischemic liver conditions (P<0.05), but CsA treatment did not enhance early adhesion. Use of CsA was able to facilitate TGF-β1 expression and the subsequent TGF-β–mediated random migration was blocked by the use of the specific signaling inhibitor SB431542 in vitro. Conclusions. Whereas the chemokine receptor expression by cancer cells is implicated with early organotropic dissemination even under CsA-mediated immune suppression, rather, CsA enhances the late-phase progression after tumor adhesion through TGF-β1 expression.

Dual cytoprotective effects of splenectomy for small-for-size liver transplantation in rats.

The problems associated with small‐for‐size liver grafts (ie, high mortality rates, postoperative complications, and acute rejection) remain critical issues in partial orthotopic liver transplantation (OLT). In association with partial OLT, splenectomy (SP) is a procedure used to reduce the portal pressure. However, the precise effects of SP on partial OLT have been unclear. In this study, using small‐for‐size liver grafts in rats, we examined the cytoprotective effects of SP on OLT. Liver grafts were assigned to 2 groups: a control group (OLT alone) and an SP group (OLT after SP). SP significantly increased animal survival and decreased liver damage. SP exerted the following cytoprotective effects: (1) it improved hepatic microcirculation and prevented increases in the portal pressure after OLT, (2) it suppressed the hepatic infiltration of neutrophils and macrophages through the direct elimination of splenic inflammatory cells before OLT, (3) it decreased the hepatic expression of tumor necrosis factor α and interleukin‐6, (4) it attenuated sinusoidal endothelial injury, (5) it decreased plasma endothelin 1 levels and increased hepatic heme oxygenase 1 expression, (6) it suppressed hepatocellular apoptosis through the down‐regulation of hepatic caspase‐3 and caspase‐8 activity, and (7) it increased hepatic regeneration. In conclusion, SP for small‐for‐size grafts exerts dual cytoprotective effects by preventing excessive portal vein hepatic inflow and eliminating splenic inflammatory cell recruitment into the liver; this in turn inhibits hepatocellular apoptosis and improves liver regeneration. Liver Transpl, 2012.

Activated protein C prevents hepatic ischaemia–reperfusion injury in rats

Background: Hepatic ischaemia–reperfusion injury (IRI) is a serious complication of liver surgery, especially extended hepatectomy and liver transplantation. Activated protein C (APC), a potent anticoagulant serine protease, has been shown to have cell‐protective properties by virtue of its anti‐inflammatory and anti‐apoptotic activities.

Assessment of liver graft function and regeneration by galactosyl‐human serum albumin (99mTc‐GSA) liver scintigraphy in adult living‐donor liver transplantation

Abstract:  Background:  In adult living‐donor liver transplantation (LDLT), the assessment of the graft functional reserve is very important. We evaluated the graft functional reserve by technetium‐99m‐diethylenetriaminepenta‐acetic acid‐galactosyl‐human serum albumin (99mTc‐GSA) liver scintigraphy.

Thrombomodulin Administration Attenuates Ischemia-Reperfusion Injury of the Remnant Liver After 70% Hepatectomy in Rats: Simulated Model of Small-for-size Graft in Living Donor Liver Transplantation

BACKGROUND Hepatic ischemia-reperfusion injury (IRI) is a serious complication affecting liver function and postoperative course after liver transplantation. Thrombomodulin (TM) has been known to have anticoagulant and anti-inflammatory activities exerting a cytoprotective effect. We evaluated the cytoprotective effect of recombinant human soluble TM (rhsTM) on the remnant liver exposed to IRI after 70% hepatectomy in rats, which was the simulated model of small-for-size graft in living donor liver transplantation. MATERIALS AND METHODS A Wistar rat underwent 70% hepatectomy followed by 20-minute IRI for the remnant liver. rhsTM (1 mg/kg) (TM group) or saline (control group) was intravenously administered 30 minutes before operation. RESULTS Alanine aminotransaminase levels were more significantly decreased during the 24 hours after operation in the TM group than in control group, especially at 6 hours. Intrahepatic infiltration of macrophages/monocytes (ED-1 immunohistochemical staining) at 6 hours was significantly decreased in the TM group compared to the control group. The number of proliferating cell nuclear antigen-positive cells at 12 hours (hepatocyte proliferation) was significantly higher in the TM group than in the control group; although liver weight 7 days after operation did not differ between the two groups. Hepatocyte apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, also known as TUNEL assay) at 24 hours was more significantly diminished in the TM group than in the control group. CONCLUSION These results suggest that rshTM attenuates hepatocyte injury through its anti-inflammatory effect, and promotes hepatocyte proliferation in the reduced-size liver exposed to hepatic IRI.

Immunological aspects in late phase of living donor liver transplant patients: usefulness of monitoring peripheral blood CD4+ adenosine triphosphate activity.

Aim. To evaluate whether the combination of the peripheral blood CD4+ adenosine triphosphate activity (ATP) assay (ImmuKnow assay: IMK assay) and cytochrome P450 3A5 (CYP3A5) genotype assay is useful for monitoring of immunological aspects in the patient followup of more than one year after living donor liver transplantation (LDLT). Methods. Forty-nine patients, who underwent LDLT more than one year ago, were randomly screened by using IMK assay from January 2010 to December 2011, and the complete medical records of each patient were obtained. The CYP3A5 genotypes were examined in thirty-nine patients of them. Results. The mean ATP level of the IMK assay was significantly lower in the patients with infection including recurrence of hepatitis C (HCV) (n = 10) than in those without infection (n = 39): 185 versus 350 ng/mL (P < 0.001), while it was significantly higher in the patients with rejection (n = 4) than in those without rejection (n = 45): 663 versus 306 ng/mL (P < 0.001). The IMK assay showed favorable sensitivity/specificity for infection (0.909/0.842) as well as acute rejection (1.0/0.911). CYP3A5 genotypes in both recipient and donor did not affect incidence of infectious complications. Conclusions. In the late phase of LDLT patients, the IMK assay is very useful for monitoring immunological aspects including bacterial infection, recurrence of HCV, and rejection.

Combination assays for evaluation of immune function and CYP3A5 genotype to identify the risk of infectious complications and mortality in living donor liver transplant patients.

BACKGROUND The aim of this study is to evaluate whether the combination of the ImmuKnow assay (IMK) and cytochrome P450 3A5 (CYP3A5) genotype assay is useful for identifying the risk of morbidity and mortality in living donor liver transplantation (LDLT) patients, and also to investigate the optimal cutoff value of IMK level of immune status. MATERIAL AND METHODS Sixty six LDLT patients, who were randomly screened by using IMK between March 2002 and June 2011, were divided into 2 groups: patients in whom at least 1 IMK value was <175 ng/mL (Group A, n=16) and patients in whom all IMK values were >175 ng/mL (Group B, n=50). Both donors and recipients were evaluated for the CYP3A5 genotype. RESULTS The frequencies of cytomegalovirus and bacterial infection in Group A were significantly higher than those in Group B (P<0.001). The short term mortality was 4 (25.0%) in Group A and none in Group B, and the IMK level in all four cases became <100 ng/mL at least one time before death. The rate of CYP3A5*1 allele (expressors) among recipients was significantly higher in Group A than in Group B (63.6% vs. 22.2%, P=0.0147). The rates of CMV infection and bacterial infection, and the IMK levels was significantly higher in recipients with expressors (p=0.0216, p=0.0332, and p=0.0433, respectively). CONCLUSIONS The combination of the IMK and CYP3A5 genotype assay is useful for monitoring immune status after LDLT, and the IMK level <100 ng/ml might be the critical level to make a recovery from the severe immunesupressive condition.

Postoperative Liver Dysfunction in Living Donors After Left-Sided Graft Hepatectomy: Portal Venous Occlusion of the Medial Segment After Lateral Segmentectomy and Hepatic Venous Congestion After Left Lobe Hepatectomy

BACKGROUND The aim of this study was to evaluate how sacrifice of the portal vein and/or hepatic vein affects remnant liver dysfunction after lateral segmentectomy or left lobe hepatectomy. MATERIALS AND METHODS Among 130 patients who underwent donor hepatectomy between March 2002 and July 2011, we enrolled lateral segment (n=15) and left lobe donors (n=40). We evaluated the postoperative courses and the territory of venous obstruction or congestion based on the sacrificed portal vein or hepatic vein after the donor operation: lateral segment grafts (P4a, P4b, LV4) and left lobe grafts (MV5, MV8) according to the results analyzed by MeVis Distant Service. RESULTS Among lateral segment donors, the predicted sacrificed territory of portal vein and hepatic vein was 14.3% (7.3%-19.4%) in P4a+4b: (P4a: 8.6%, P4b: 5.8%) and 2.9% (0%-8.4%) in LV4, respectively. On the other hand, in left lobe donors, the predicted congestive territory of the hepatic vein was 17.6% (2.8%-33.0%) in MV5+8 (7.8% in MV5 and 9.8% in MV8, respectively). The incidence of patients whose postoperative peak aspartate aminotransferase (AST) or alanine aminotransferase levels were higher than 500 IU/L was 20% in the lateral segment donors and 5% in the left lobe donors. The peak postoperative AST levels and territory of MV5+8 showed a significant positive correlation (R=0.569, P<.05) among left lobe donors. CONCLUSION Territories of P4 in lateral segment donors and MV5+8 in left lobe donors impacted postoperative liver dysfunction. It is important to recognize the precise territory of the portal vein and the hepatic vein before the donor operation.