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Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8  wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O 2 + 5% CO 2 + 3.8% H 2 and N 2 as the base gas to verify the response of primary hepatocytes in a high concentration of hydrogen gas in vitro. Mice in the CSAA + HRW group had lower serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and milder histological damage. The inflammatory cytokines were expressed at lower levels in the HRW group than in the CSAA group. Importantly, HRW reversed hepatocyte fatty acid oxidation and lipogenesis as well as hepatic inflammation and fibrosis in preexisting hepatic fibrosis specimens. Molecular hydrogen inhibits the lipopolysaccharide-induced production of inflammation cytokines through increasing heme oxygenase-1 (HO-1) expression. Furthermore, HRW improved hepatic steatosis in the CSAA + HRW group. Sirtuin 1 (Sirt1) induction by molecular hydrogen via the HO-1/adenosine monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway suppresses palmitate-mediated abnormal fat metabolism. Orally administered HRW suppressed steatosis induced by CSAA and attenuated fibrosis induced by CDAA, possibly by reducing oxidative stress and the inflammation response. NEW & NOTEWORTHY The mRNA expression of inflammatory cytokines in the HRW group was lower than in the CSAA group. HRW reversed hepatocyte apoptosis as well as hepatic inflammation and fibrosis in NASH specimens. Molecular hydrogen inhibits LPS-induced inflammation via an HO-1/interleukin 10 (IL-10)-independent pathway. HRW improved hepatic steatosis in the CSAA + HRW group. Sirt1 induction by molecular hydrogen via the HO-1/AMPK/PPARα/PPARγ pathway suppresses palmitate-mediated abnormal fat metabolism.

Background: Pure laparoscopic donor hepatectomy (PLDH) is being increasingly performed at centers with experienced surgeons [1-6]. This procedure is still developing and is associated with several challenges owing to its technical difficulty [7-9]. Conversely, the anterior approach is sometimes applied to both laparoscopic and open right hepatectomy for management of tumors in the liver [10, 11]. However, there are no reports regarding the use of the anterior approach for PLDH. We found this method to be useful; therefore, we aimed to introduce the novel procedure using a video clip., Methods: The donor was placed in the supine position. First, the right side of the inferior vena cava was dissected instead of performing the liver hanging maneuver. The right Glissonean pedicle was encircled and controlled, and the liver parenchyma was completely transected. Thereafter, the ligaments around the liver were dissected, and the graft was mobilized. The hilar vessels were respectively separated. Finally, the right hepatic duct, right hepatic artery, right portal vein, and right hepatic vein were divided, and the graft liver was retrieved. This study was approved by institutional ethics board (MH2019-119), and informed consent was taken from the patient., Results: The overall surgical time was 400 min, the volume of blood loss was 31 mL, the warm ischemic time was 7 min, and no complications were seen., Conclusion: The advantages of the anterior approach for right-sided PLDH might be attribute to reduction of compression injury and incidence of subcapsular hematoma, as liver mobilization is easily performed because of increased liver mobility. However, PLDH is a highly-skilled procedure, and indications for PLDH should be extended in a step-wise manner. Further, the procedure should be performed only by highly proficient surgeons having extensive experience in both laparoscopic liver resection and living donor liver transplantation.

Ischemia-reperfusion (IR) injury is a kind of injury resulting from the restoration of the blood supply after blood vessel closure during liver transplantation and is the main cause of graft failure. The pathophysiological mechanisms of hepatic IR include a variety of oxidative stress responses. Hepatic IR is characterized by ischemia and hypoxia inducing oxidative stress, immune response and apoptosis. Fat-denatured livers are also used as donors due to the lack of liver donors. Fatty liver is less tolerant to IR than normal liver. Heme oxygenase (HO) is an enzyme that breaks down hemoglobin to bilirubin, ferrous iron and carbon monoxide (CO). Inducible HO subtype HO-1 is an important protective molecule in mammalian cells used to improve acute and chronic liver injury owing to its characteristic anti-inflammatory and anti-apoptotic qualities. HO-1 degrades heme, and its reaction product CO has been shown to reduce hepatic IR injury and increase the survival rate of grafts. As an induced form of HO, HO-1 also exerts a protective effect against liver IR injury and may be useful as a new strategy of ameliorating this kind of damage. This review summarizes the protective effects of HO-1 in liver IR injury, especially in fatty liver.

Steatosis has a low tolerance against ischemia-reperfusion injury (IRI). To prevent IRI in the steatotic liver, we attempted to elucidate the protective effect of astaxanthin (ASTX) in the steatotic liver model by giving mice a methionine and choline-deficient high fat (MCDHF) diet. Levels of lipid peroxidation and apoptosis, the expression of inflammatory cytokines and heme oxygenase (HO)-1, in the liver were assessed. Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver. ASTX decreased serum ALT and AST levels, the amount of TUNEL, F4/80, or 4HNE-positive cells and the mRNA levels of inflammatory cytokines in MCDHF mice by IRI. Moreover, HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepatocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX. Pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.

Purpose: We compared the image quality of free-breathing diffusion-weighted imaging (FB-DWI) to that of respiratory-triggered DWI (RT-DWI) after proper optimization., Materials and Methods: Three healthy subjects were scanned to optimize magnetic resonance (MR) parameters of FB-DWI to improve image quality, including spatial resolution, image noise, and chemical shift artifacts. After this optimization, we scanned 32 patients with liver disease to assess the clinical feasibility of the optimized FB-DWI. Of the 32 patients, 14 had a total of 28 hepatocellular carcinomas (HCCs), four had a total of 15 metastatic liver tumors, and the other 14 had no tumor. Qualitatively, we compared the image quality scores of FB-DWI with those of RT-DWI with the Wilcoxon signed-rank test. Quantitatively, we compared the signal-to-noise ratios (SNRs) of the liver parenchyma, lesion-to-nonlesion contrast-to-noise ratios (CNRs) and apparent diffusion coefficient (ADC) values of the liver parenchyma and liver tumor by the paired t-test., Results: The average scores of image quality for sharpness of liver contour, image noise, and chemical shift artifacts were significantly higher for FB-DWI than RT-DWI (P < 0.05). SNRs, CNRs, and ADC values of the liver parenchyma and tumors did not differ significantly between the 2 DWI methods., Conclusion: Compared with RT-DWI, the optimized FB-DWI provided better spatial resolution, fewer artifacts, and comparable SNRs, lesion-to-nonlesion CNRs, and ADC values.

Background: In order to analyze postoperative liver regeneration following hepatic resection after chemotherapy, we retrospectively investigated the differences in liver regeneration by comparing changes of residual liver volume in three groups: a living liver donor group and two groups of patients with colorectal liver metastases who did and did not undergo preoperative chemotherapy., Methods: This study included 32 patients who had at least segmental anatomical hepatic resection. Residual liver volume, early postoperative liver volume, and late postoperative liver volume were calculated to study the changes over time. From the histopathological analysis of chemotherapy-induced liver disorders, the effect on liver regeneration according to the histopathology of noncancerous liver tissue was also compared between the two colorectal cancer groups using Kleiner's score for steatohepatitis grading {Hepatology, 41(6):1313-1321, 2005} and sinusoidal obstruction syndrome (SOS) grading for sinusoidal obstructions {Ann Oncol, 15(3):460-466, 2004}., Results: Assuming a preoperative liver volume of 100%, mean late postoperative liver volumes in the three groups (the living liver donor group and the colorectal cancer groups with or without chemotherapy) were 91.1%, 80.8%, and 81.3%, respectively, with about the same rate of liver regeneration among the three groups. Histopathological analysis revealed no correlation between either the Kleiner's scores or the SOS grading and liver regeneration., Conclusions: As estimated by liver volume, the level of liver regeneration was the same in normal livers, tumor-bearing livers, and post-chemotherapy tumor-bearing livers. Liver regeneration was not adversely affected by the extent to which steatosis or sinusoidal dilatation was induced in noncancerous tissue by chemotherapy in patients scheduled for surgery.

Purpose: To introduce and assess the TRacking Only Navigator echo (TRON) technique for diffusion-weighted magnetic resonance imaging (DWI) of the liver., Subjects and Methods: A total of 10 volunteers underwent TRON, respiratory triggered (RT), and free breathing (FB) DWI of the liver. Scan times of TRON and RT DWI were measured, and image sharpness in TRON, RT, and FB DWI was assessed and compared using nonparametric tests. Furthermore, 14 patients with liver metastasis who had undergone TRON and RT DWI of the liver were retrospectively assessed. Relative contrast ratios (RCRs) and apparent diffusion coefficients (ADCs) of the largest hepatic metastasis in TRON and RT DWI were measured. RCRs were compared using a parametric test and agreement in ADCs was assessed using the Bland-Altman method., Results: In the volunteers, mean scan times of TRON and RT relative to FB DWI were 110% to 112% and 261% to 290%, respectively. On axial images, there were no significant differences in images sharpness among TRON, RT, and FB DWI, but on coronal images image sharpness in TRON was nearly always significantly better (P < 0.05) than in RT and FB DWI. In the patients, mean RCRs between TRON and RT DWI were not significantly different (P = 0.9091). Mean difference in ADC +/- limits of agreement (in 10 mm/s) between TRON and RT DWI was -0.16 +/- 0.79., Conclusion: TRON offers sharp diffusion-weighted images of the liver using an efficient scan time, making it an excellent alternative to RT and FB DWI. The moderate to poor agreement in ADCs of liver metastases between TRON and RT DWI requires further investigation.

Purpose: To assess the feasibility of TRacking Only Navigator echo (TRON) for diffusion-weighted magnetic resonance imaging (DWI) of the liver at 3.0T., Materials and Methods: Ten volunteers underwent TRON, respiratory triggered, and free breathing DWI of the liver at 3.0 Tesla (T). Scan times were measured. Image sharpness, degree of stair-step and stripe artifacts for the three methods were assessed by two observers., Results: Mean scan times of TRON and respiratory triggered DWI relative to free breathing DWI were 34% and 145% longer respectively. In four of eight comparisons (two observers, two b-values, two slice orientations), TRON DWI image sharpness was significantly better than free breathing DWI, but inferior to respiratory triggered DWI. In two of four comparisons (two observers, two b-values), degree of stair-step artifacts in TRON DWI was significantly lower than in respiratory triggered DWI. Degree of stripe artifacts between the three methods was not significantly different., Conclusion: DWI of the liver at 3.0T using TRON is feasible. Image sharpness in TRON DWI is superior to that in free breathing DWI. Although image sharpness of respiratory triggered DWI is still better, TRON DWI requires less scan time and reduces stair-step artifacts.

Purpose: To assess cardiac motion-induced signal loss in diffusion-weighted magnetic resonance imaging (DWI) of the liver using dynamic DWI., Materials and Methods: Three volunteers underwent dynamic coronal DWI of the liver under breathholding, in the diastolic (DWI(diast)) or systolic (DWI(syst)) cardiac phase, and with motion probing gradients (MPGs) in phase encoding (P, left-right), frequency encoding (M, superior-inferior), or slice select (S, anterior-posterior) direction. Liver-to-background contrasts (LBCs) of DWI(syst) were compared to those of DWI(diast), for both the left and right liver lobes, using nonparametric tests. Signal decrease ratios (SDRs) were calculated as (1-(LBCDWI(syst)/LBCDWI(diast))) x 100%. DWI(syst) was further analyzed to determine which direction of MPGs was most affected by cardiac motion., Results: In the left liver lobe, LBCs of DWI(syst) (median 3.35) were significantly lower (P < 0.0001) than those of DWI(diast) (median 4.84). In the right liver lobe, LBCs of DWI(syst) (median 4.17) were also significantly lower (P < 0.0001) than those of DWI(diast) (median 5.35 ). SDRs of the left and right liver lobes were 25.5% and 17.3%, respectively. In DWI(syst), the significantly lowest (P < 0.05) LBCs were observed in the M direction (left liver lobe) and P direction (right liver lobe) of MPGs., Conclusion: Signal intensity of both liver lobes are affected by cardiac motion in DWI. In the left liver lobe, signal loss especially occurs in the superior-inferior direction of MPGs, whereas in the right lobe, signal loss especially occurs in the left-right direction of MPGs.

Background/aims: Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are the hepatic manifestation of metabolic syndrome. However, its therapeutic strategy has not been established. Recently, an angiotensin II type 1 receptor blocker, telmisartan (Tel), has received a great deal of attention as a therapeutic tool for metabolic syndrome. The aim of this study was to investigate the efficacy and mechanisms of Tel on a murine NASH model., Methods: C57BL/6 mice were fed a methionine- and choline-deficient high-fat diet (MCDHF) or a standard diet with/without the administration of Tel (10 mg/kg/day) for 8 weeks., Results: MCDHF feeding induced marked steatohepatitis with macrophage infiltration. Tel attenuated liver steatosis with decreased hepatic triglycerides (P<0.05) and fibrogenesis with decreased type I collagen and transforming growth factor-beta1 mRNA expressions (P<0.05). Tel also suppressed the infiltration of macrophages into the liver and decreased hepatic monocyte chemoattractant protein-1 and its receptor (CC-chemokine receptor 2; CCR2) mRNA expressions, especially CCR2. In vitro, Tel suppressed CCR2 expression, which was induced by low-density lipoprotein. The size of adipocyte in visceral fat tissue was reduced with an increased serum adiponectin concentration in the Tel group., Conclusions: In this study, we revealed that Tel attenuated steatohepatitis progression by suppressing the macrophage infiltration into the liver. Tel also affected the reduction of adipocyte size and elevation of serum adiponectin. Tel might serve as a new therapeutic strategy for NASH.

Purpose: To compare and determine the reproducibility of apparent diffusion coefficient (ADC) measurements of the normal liver parenchyma in breathhold, respiratory triggered, and free-breathing diffusion-weighted magnetic resonance imaging (DWI)., Materials and Methods: Eleven healthy volunteers underwent three series of DWI. Each DWI series consisted of one breathhold, one respiratory triggered, and two free-breathing (thick and thin slice acquisition) scans of the liver, at b-values of 0 and 500 s/mm2. ADCs of the liver parenchyma were compared by using nonparametric tests. Reproducibility was assessed by the Bland-Altman method., Results: Mean ADCs (in 10(-3) mm2/sec) in respiratory triggered DWI (2.07-2.27) were significantly higher than mean ADCs in breathhold DWI (1.57-1.62), thick slice free-breathing DWI (1.62-1.65), and thin slice free-breathing DWI (1.57-1.66) (P<0.005). Ranges of mean difference in ADC measurement+/-limits of agreement between two scans were -0.02-0.05+/-0.16-0.24 in breathhold DWI, -0.14-0.20+/-0.59-0.60 in respiratory triggered DWI, -0.03-0.03+/-0.20-0.29 in thick slice free-breathing DWI, and -0.01-0.09+/-0.21-0.29 in thin slice free-breathing DWI., Conclusion: ADC measurements of the normal liver parenchyma in respiratory triggered DWI are significantly higher and less reproducible than in breathhold and free-breathing DWI., (Copyright (c) 2008 Wiley-Liss, Inc.)

Recent reports have raised concerns over the feasibility of differentiating bone marrow cells (BMCs) into functional hepatocytes. Such augmentation is considered necessary for potential clinical use of these cells in liver diseases. The present investigation was designed to determine the kinetics of transplanted BMCs and evaluate the effects of repeated bone marrow transplantation (BMT) in rat models of CCl(4)-induced liver damage. The early kinetics of transplanted BMCs was evaluated with a charge-coupled-device (CCD) camera using BMCs obtained from green fluorescent protein (GFP) transgenic (Tg) rats and followed up with in vivo imaging system (IVIS) using BMCs obtained from firefly luciferase (luc) Tg rats. We used a portal infusion system for repeated BMT. BMCs were transplanted via a peripheral vein or the portal vein (PV) once or repeatedly using this system. The results revealed that BMCs accumulated more in the damaged liver than in the intact liver. In the experimental group receiving repeated BMT via the PV, the liver fibrosis was milder than that in the group not receiving BMT, and large clusters of albumin-producing cells were detected by albumin staining. The injected BMCs were shown to accumulate in the damaged liver. This strategy of repeated BMT has potential clinical use in enhancing the number of albumin-producing cells and suppressing liver fibrosis. This combination of beneficial effects may contribute to the benefits of cell transplantation therapy. Demonstration of the benefits of BMT in this study may be expected to have great significance for clinical trials.

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.

Background/aims: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats., Methods: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1., Results: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1., Conclusion: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

Intrahepatic lymphoid follicle (ILF) formation is one of the most characteristic and commonly observed histological features in patients with chronic hepatitis C. However, little is known regarding whether follicles in the liver belong to functional lymphoid tissues, where B cells are activated, differentiated, and proliferated, or if the lymphocytes are merely infiltrated after recruitment from the secondary lymphoid organs. To ascertain this possibility, we examined the expression of markers for B-cell activation, differentiation, and proliferation in ILFs in patients with chronic hepatitis C using surgically resected specimens, and compared them with specimens of perihepatic lymph nodes by an immunohistochemical technique. Germinal center (GC) formation in the ILFs was frequently found in HCV-positive cases. The distribution of immunoglobulin M (IgM)-, IgD-, and IgG-positive cells and the expression patterns of Ki-67, CD23, or bcl-2 and bcl-6 gene products in the follicles with GC formation in the liver of patients with chronic hepatitis C were similar to those of lymph nodes, indicating that B cells are activated, proliferated, and differentiated in the ILFs with GC formation in patients with chronic hepatitis C. Oligoclonal expansion of B cells in the livers with ILFs was confirmed by an analysis of immunoglobulin heavy chain (IgH) gene rearrangement determined by polymerase chain reaction (PCR). These data strongly suggest that ILFs with GC formation, which are frequently found in patients with chronic hepatitis C, may functionally be the same as those found in lymph nodes with respect to B-cell expansion and maturation.

The purpose of this study was to elucidate the mechanism behind low efficacy of interferon therapy to hepatitis C virus infected patients by using primary monkey hepatic parenchymal cells as a surrogate of primary human liver cells. The effects of various cytokines on the antiviral activity of IFNs in the monkey hepatic cells were studied to search for physiological inhibitors. Interleukin-1 alpha, EGF, and HGF showed suppressive effects on the antiviral activity of IFN-alpha, -beta in primary monkey hepatic cells when examined by the yield reduction method using vesicular stomatitis virus (VSV). In contrast, 50 ng/ml of TNF and IL-6 had no suppressive effect on the IFN-induced antiviral state in the hepatic cells.

Background/aims: Matrix metalloproteinase-13, one of the principal neutral proteinases capable of cleaving native fibrillar collagens, is important in the degradation and remodeling of extracellular matrix. However, its precise expression in liver injury has not been characterized. We examined the kinetics of the expression of matrix metalloproteinase-13 and one of its specific inhibitors, tissue inhibitor of metalloproteinase-1, in acute liver injury in rats., Methods: Acute liver injury was induced by administration of carbon tetrachloride or two different doses of D-galactosamine hydrochloride in Wistar rats. Hepatic matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 mRNA levels were then examined by Northern blotting., Results: All rats survived after liver injury induced by carbon tetrachloride or low doses of D-galactosamine hydrochloride. However, rats died 5 days after induction of liver injury by high doses of D-galactosamine hydrochloride. In carbon tetrachloride-induced liver injury, matrix metalloproteinase-13 mRNA was transiently increased between 6 h and 1 day after injury. Tissue inhibitor of metalloproteinase-1 mRNA expression was increased between 6 h and 3 days after the peak of matrix metalloproteinase-13 expression. Similar patterns of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 expression were observed in low-dose D-galactosamine hydrochloride-induced liver injury. In contrast, in high-dose D-galactosamine hydrochloride-induced liver injury, tissue inhibitor of metalloproteinase-1 expression peaked before matrix metalloproteinase-13 expression, which was increased 2 days after injury. Both mRNA levels continued to increase until death., Conclusions: Transient expression of matrix metalloproteinase-13, followed by that of tissue inhibitor of metalloproteinase-1, was observed during recovery from acute liver injury induced by carbon tetrachloride and low-dose D-galactosamine hydrochloride. In contrast, disordered expression of matrix metalloproteinase-13 was observed in fatal liver injury caused by high-dose D-galactosamine hydrochloride. These results indicate that matrix metalloproteinase13 plays an important role in the early phase of recovery from liver injury.

Hepatic perisinusoidal cell population consists of hepatic stellate cells, Kupffer cells, endothelial cells, and Pit cells. These cells are isolated by enzymic digestion and purified by density gradient centrifugation. With isolation of stellate cells, conventional method is unable to eliminate the contamination of Kupffer cells because the densities of these two cells are similar. We report here an improved method for isolation of highly purified hepatic stellate cells, using dichloromethylene diphosphate (CL2MDP), which has selective cytotoxicity of Kupffer cells. Three days after the single intravenous administration of liposome-encapsulated CL2MDP, the Kupffer cells disappeared almost completely from the liver. Following Percoll density gradient centrifugation, the purity of the hepatic stellate cells exceeded 98% without any contamination of the Kupffer cells. Kupffer cells are reported to affect the physiological functions of stellate cells. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.

In order to safely and reliably perform laparoscopic cholecystectomy in severe inflammatory cases (e.g., acute or chronic cholecystitis), we have designed a method of suturing the liver bed to the diaphragm, lifting it cephalad so as to maintain a good operative field. Initially, we dissect the gallbladder fundus, fully dissecting the neck of the gallbladder from the liver and finally dissecting the cystic duct (laparoscopic retrograde cholecystectomy facilitated by lifting the liver bed up to the diaphragm; Lap-RC). This method is different from laparoscopic standard cholecystectomy (Lap-SC), in which dissection of the cystic duct is done first. One hundred and twenty-nine consecutive laparoscopic cholecystectomies for various gallbladder diseases were carried out at Nerima General Hospital between August 1991 and June 1994. Fifteen cases of Lap-RC and six cases of Lap-SC in a severe inflammatory group were comparatively evaluated. Thirteen cases of Lap-RC and 92 cases of Lap-SC in a noninflammatory group were also comparatively evaluated. The rates of conversion to laparotomy were 0% in Lap-RC cases (0/15) and 33% in Lap-SC cases (3/9) in the severe inflammatory group. The incidences of major postoperative complications were 0% in Lap-RC cases (0/15) and 17% in Lap-SC cases (1/6) in the severe inflammatory group. In conclusion, Lap-RC showed satisfactory results in terms of both safety and reliability in patients with severe inflammatory disease.

Type VI collagen is a minor but essential matrix component in the liver. In this study, we utilized an acute and a chronic injury model to clarify the process of liver fibrosis in rats by administration of carbon tetrachloride. Collagen gene expression, with particular emphasis on type VI collagen, was studied by molecular hybridization techniques. The alpha 2(VI) collagen mRNA levels were markedly elevated on day 3 of acute injury and were approximately at the same high level at 7 and 14 weeks of chronic injury, as determined by Northern hybridizations and slot-blot analyses. Marked enhancement of type I collagen gene expression was similarly noted at these time points. The activation of collagen gene expression in acute injury, as determined by in situ hybridization, was particularly prominent in the vicinity of the central veins. Indirect immunofluorescence demonstrated marked accumulation of type VI collagen protein as early as day 3 of acute injury, and the reaction appeared to be initiated in the proximity of central veins. These results indicate that type VI collagen gene expression, together with other connective tissue components, including type I collagen, is activated in the early stages of the fibrotic process. Type VI collagen accumulation may contribute to the distorted architecture and functional impairment of the liver in hepatic fibrosis.

Immunolocalization of Type I, Type III and Type IV collagens, laminin and prolyl hydroxylase (PH), a key enzyme in collagen synthesis, was examined to clarify the fibrotic process in chronic, active liver disease. In piecemeal necrosis of chronic, active hepatitis (CAH) and active liver cirrhosis (LC), fat-storing cells (FSCs) and transitional cells (TSCs), containing abundant rough endoplasmic reticulum (RER), were increased in number and stained intensely for PH. Immunodeposits of extracellular matrix (ECM) components were found in the RER, Golgi apparatus (GA) and vesicles of these cells, especially in cases with marked inflammation. On the other hand, in the periportal areas of chronic, persistent hepatitis (CPH) or inactive LC, immunoreaction of ECM components was seldom found in the RER of FSCs and TSCs. In the portal tract, immunodeposits of ECM components were seldom found in the organelles of fibroblasts, although ECM was increased there. These findings indicate that FSCs and TSCs in piecemeal necrosis might play a role in the production of ECM components in the progression of fibrosis during the development of chronic active liver disease. In addition, ECM component production by FSCs and TSCs is associated with marked inflammation.

Northern analysis using total RNAs from the component cells of normal rat liver indicated that COL1A(I) mRNA is present in fat-storing cells (Ito cells) and sinusoidal endothelial cells. A fraction for Kupffer cells also contained this mRNA. When CCl4 was given, COL1A(I) mRNA was increased in a factor of 1.5 in the fractions of these component cells. After 48 h of the drug administration, hepatocytes appeared to possess over 60% of liver COL1A(I) mRNA, although in normal hepatocytes its level was below the range detectable by our procedures. Under this injured condition of liver, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was elevated, while activity of this enzyme was lowered by 50% of the control value. All the changes were obviously suppressed by the simultaneous administration of prostacyclin.

Monospecific antibodies, directed against type IV collagen and laminin, were used to clarify the process of sinusoidal capillarization in rats after carbon tetrachloride (CCl4) intoxication by the direct immunoperoxidase method. After acute intoxication, both type IV collagen and laminin were increased in the area of hepatic necrosis, adjacent to the central veins; however, sinusoidal capillarization was not found. During chronic intoxication, deposition of laminin was co-distributed with that of type IV collagen, but deposition proceeded more slowly than that of the type IV collagen. Deposition of laminin was increased in the Disse space. Sinusoidal capillarization was noted as thick deposition of both antigens by light microscopy. Immunoelectron microscopy showed that both components were continuously present in the Disse space. Intracellularly, both antigens were found in the rough endoplasmic reticulum (RER) of fat-storing cells (FSC) and endothelial cells, and these cells showed morphological changes, becoming slender and flattened. In contrast, few immunoreactive products of the two components were observed in the hepatocytes. These findings suggest that type IV collagen and laminin are indispensable for the establishment of sinusoidal capillarization, and that FSC play an important role in the production of both components.

Monospecific antibodies directed against type I, type III collagens, and prolyl hydroxylase were used to clarify the process of liver fibrosis after CCl4 intoxication in rats by the direct immunoperoxidase method. In acute CCl4 intoxication, fat-storing cells (FSCs) were increased in number in the areas of necrosis around the central veins. These FSCs exhibited intense positive stainings for type I, type III collagens, and prolyl hydroxylase in well-developed rough endoplasmic reticula and Golgi apparatus. This was the direct evidence that the collagens formed after CCl4 intoxication are produced by FSCs. In chronic CCl4 intoxication, increased FSCs in and around the fibers also contained strong immunoreactive materials of both collagens and prolyl hydroxylase mainly in the rough endoplasmic reticula. These collagens were also present in the Golgi apparatus and vesicles close to the cytoplasmic membrane, demonstrating the exocytic process of collagen formation of FSCs. In contrast, faint immunoreactions of both collagens were found in the rough endoplasmic reticula and Golgi apparatus of hepatocytes during the process of fibrosis. These findings indicate that FSCs play an important role in fibrogenesis after acute and chronic CCl4 intoxication in the rat.