Portal blood flow regulates volume recovery of the rat liver after partial hepatectomy: molecular evaluation.

BACKGROUND/AIM: Liver regeneration is a finely tuned process that is closely regulated by multiple cell cycle steps. Although the portal blood flow affects liver regeneration, the molecular mechanism by which the blood flow regulates gene expression and liver function is largely unknown. The aim of this study was to investigate the molecular effect of portal blood flow on hepatocyte proliferation and gene regulation during liver regeneration. MATERIALS AND METHODS: We developed a simple surgical rat model to investigate the relation between portal blood flow and liver regeneration by partially ligating the portal trunk with 8-0 Proline sutures under microscopy to reduce the blood flow by 40%. We investigated recovery of liver volume, DNA synthesis, and gene expression associated with cell cycle regulators, comparing partially hepatectomized (PH) rats without (PH group; n = 30) and with partial portal ligation (PHPL group; n = 30) for 7 days after the operation. RESULTS: The hepatic tissue blood flow and the recovery ratio between liver weight and body weight in the PHPL group were significantly lower than in the PH group after hepatectomy. The peak 5-bromo-2'-deoxyuridine labeling index in the PHPL group was delayed and weak compared with the PH group. The expression of CT-1 and cyclin D, E, and B mRNAs indicated that the liver regeneration in the PHPL group was delayed and weak. In addition, there was reciprocal expression of C/EBPalpha and C/EBPbeta mRNAs, an observation supported by their nuclear protein levels. Furthermore, the cytochrome P-450 protein level in the PHPL group was higher than that in the PH group 1 day after hepatectomy. CONCLUSION: The portal blood flow regulates the activity of liver regeneration and the gene expression associated with cell cycle regulators, while the functions are maintained.

Plasma level of a 5-fluorouracil metabolite, fluoro-beta-alanine correlates with dihydropyrimidine dehydrogenase activity of peripheral blood mononuclear cells in 5-fluorouracil treated patients.

5-fluorouracil (5-FU) is mostly metabolized after administration, and the metabolizing enzyme, dihydropyrimidine dehydrogenase (DPD), seems to be the rate-limiting factor. However, there are few reports on the final metabolite, fluoro-beta-alanine (FBAL). We report here the results of determination of the FBAL level in 5-FU treated patients and the correlation between the FBAL level and the DPD activity in peripheral blood mononuclear cells (PBMCs). Blood samples were collected from 20 patients, who had received continuous intravenous infusion (CIV) of 5-FU (320 mg/m2/24 hr) after resection of colorectal cancer, and the FBAL level was determined by high performance liquid chromatography (HPLC), after derivatizing into o-phthalaldehyde (OPA) and detecting fluorescence. DPD activity was measured in cytosol prepared from PBMCs using HPLC radioassay. The average FBAL plasma level during CIV of 5-FU was 911.0 ng/ml (521.0 to approximately 1834.6 ng/ml) and that of DPD activity in PBMCs was 282.6 pmol/min/mg-protein (145.0 to approximately 568.0 pmol/min/mg-protein). There was a significant correlation between the FBAL level and the DPD activity (r=0.805, p<0.0001). FBAL level in plasma may be useful in predicting the DPD activity in PBMCs, however, further studies are required considering the small number of cases in this study.

Successful ex vivo normothermic liver perfusion with purely artificial products using artificial blood.

We tried to make an ex vivo functioning liver with an artificial perfusate that consisted of artificial blood in the pig liver. A liver graft from a female pig weighing 20 kg was harvested in the usual manner. The perfusion solution consisted of artificial blood, L-15 medium, distilled water, bovine serum albumin, NaHCO3, NaOH, KCl, human regular insulin, 50% glucose solution, and dexamethasone. The isolated liver was perfused with this oxygenated perfusate through the portal vein at a rate of 300 ml/min for 9 hours. Seven livers were perfused for 9 hours in this system. Five of the livers showed mean oxygen consumption of over 8 ml-O2/min during perfusion. Histological findings showed that the hepatic architecture was almost completely preserved and numerous hepatocytes exhibited PAS-positive cytoplasmic glycogen deposits in these livers. These observations indicate that we have succeeded in developing an ex vivo functioning liver with an artificial perfusate employing artificial blood.

Adenosquamous carcinoma of the colorectum: report of two cases.

Adenosquamous carcinomas of the colorectum are rare neoplasms. Our experience with two cases is presented in this paper. One patient, who complained of bloody stool, was found to have adenocarcinoma in the sigmoid colon. He received a laparoscopy-assisted sigmoidectomy. The histological examination revealed that the tumor was adenosquamous carcinoma. To date, he has survived six months post operatively without evidence of recurrence. The other patient, who complained of anal bleeding, was found to have rectal adenocarcinoma and received a low anterior resection. Histological examination revealed that the tumor was an adenosquamous carcinoma. He remains alive, with no evidence of recurrence, nine years post operatively. Both cases showed paracolic lymph node metastasis. Because of its very low incidence, the histogenesis, malignancy and prognosis of this disease remain unclear. Thus, further clinical and histological study of this disease entity is required.

Sepsis and cholestasis: basic findings in the sinusoid and bile canaliculus.

It is well known that the liver plays a major role in the clearance of systemic toxemia and is postulated as a regulational organ in the host-defense system. The well-controlled interaction between hepatic parenchymal cells and sinusoidal lining cells including macrophages and Kupffer cells can systematically regulate even critical infections. However, when patients are under the overload condition caused by severe infection, rejection of a transplanted liver and other hapatic dysfunction often are experienced following surgery. Among various signs and symptoms of hepatic dysfunction, progressive cholestasis is recognized as a polarized representation of the irreversible changes in hepatic constitutional cellular functions, especially in hepatic parenchymal cells. Bile canaliculi, the smallest components of the biliary tree, lie between the apical surfaces of adjacent hepatocytes. Septic cholestasis might be a result of disturbance of the total bile canalicular system, i.e., bile secretion, canalicular contraction, and so on. Recently, the molecular biology of the hepatocellular transport system has become better understood, and the pathophysiological condition of cholestasis can be explained as a representation of the intracellular molecular transcriptional system. Cellular changes in surgical cholestasis and molecular findings concerning the bile canaliculus are introduced in this article.

[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents. Special references to bacteria isolated between April 1998 and March 1999].

The annual multicenter studies on isolated bacteria from infections in general surgery and their antimicrobial susceptibility have been conducted in Japan since July 1982. This paper describes the results obtained in fiscal 1998 (from April 1998 to March 1999). The number of cases investigated as objectives was 225 for one year. A total of 429 strains (121 strains from primary infections and 308 strains from postoperative infections) were isolated from 183 cases (81.3% of total cases). In primary infections, the isolation rates of anaerobes and Escherichia coli were higher than in postoperative infections, while in postoperative infections, those of Gram-positive aerobes and Pseudomonas aeruginosa were higher than in primary infections. On the whole, among Gram-positive aerobes, the isolation rate of Enterococcus faecalis was the highest, followed by Staphylococcus aureus with high frequency in isolation from postoperative infections. Among Gram-positive anaerobes, Peptostreptococcus spp. and Streptococcus spp. were predominantly isolated. Among Gram-negative aerobes, E. coli, P. aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae were frequently isolated. Among Gram-negative anaerobes, Bacteroides fragilis group was the majority of isolates. In primary infections, the percentage of Gram-negative aerobes has gradually increased since fiscal 1995 or 1996 with these years as the turning point, while those of Gram-positive and Gram-negative anaerobes have gradually declined. In postoperative infections, the percentage of Gram-negative anaerobes has increased continuously since the mid-1980s. The percentage of MRSA among S. aureus rose to 89.7%, which was the highest level since the beginning of this study. The susceptibilities of B. fragilis, which did not show apparent changes, were recognized to have decreased against cephems in fiscal 1998. Among other bacteria in B. fragilis group, development of resistance to cephems has continued on a long-term basis since the mid-1980s. E. coli and K. pneuminiae have obviously not changed in susceptibilities, however, the susceptibilities of isolated strains in fiscal 1998 against high-generation cephems, oxacephems and monobactams have declined. We found neither vancomycin-resistant nor teicoplanin-resistant strains of S. aureus and Enterococcus spp.

[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents. Special references to bacteria isolated between April 1997 and March 1998].

The annual multicenter studies on isolated bacteria from infections in general surgery and their antimicrobial susceptibility have been conducted in 19 facilities in Japan since July 1982. This paper describes the results obtained during the period from April 1997 to March 1998. The number of cases investigated as objectives was 215 for one year. A total of 420 strains (170 strains from primary infections and 250 strains from postoperative infections) were isolated from 174 cases (80.9% of total cases). In primary infections, the isolation rate of anaerobic bacteria was higher than in postoperative infections, while in postoperative infections, those of aerobic Gram-positive bacteria and Pseudomonas aeruginosa were higher than in primary infections. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was the highest, followed by Staphylococcus aureus, which was frequently isolated from postoperative infections. Among anaerobic Gram-positive bacteria, Peptostreptococcus spp. and Streptococcus spp. were commonly isolated from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was most predominantly isolated from primary infections, followed by P. aeruginosa, Klebsiella pneumoniae in this order, and from postoperative infections, P. aeruginosa was most predominantly isolated, followed by E. coli and K. pneumoniae. Among anaerobic Gram-negative bacteria, Bacteroides fragilis group was the majority of isolates from both types of infections. We found neither vancomycin nor arbekacin resistant strains of S. aureus, and found no vancomycin resistant strains of Enterococcus spp. The susceptibility of P. aeruginosa against carbapenems did not decline in the year 1997, while resistance of B. fragilis group against cephems advanced increasingly.

Changes in hepatic venous oxygen saturation in hepatic warm ischemia/reperfusion injury in pigs.

To clarify the changes that occur in hepatic venous oxygen saturation (ShVO2) during hepatic ischemia/reperfusion (I/R) injury, we examined the relationship between ShVO2, hepatic tissue blood flow (HTBF), and portal vein pressure (PVP) in a warm I/R model using pig livers. Female pigs weighing 18-23 kg were subjected to warm I/R under extracorporeal circulation between the superior mesentric vein and the left jugular vein to avoid portal congestion. The warm ischemic times were 120 min (n = 4), 180 min (n = 14), and 240 min (n = 4). ShVO2, HTBF, and PVP were measured after reperfusion. The survival rates of the pigs 3 days after reperfusion were 100% in the 120-min group, 57% in the 180-min group, and 25% in the 240-min group. In the 180-min group, the ShVO2 was lower in the pigs that died than in those that survived. There was a significant correlation between ShVO2 and both PVP and HTBF after reperfusion. Histological examination revealed findings of severe I/R injury in pigs with a low ShVO2, and mild I/R injury in pigs with a stable ShVO2. These observations suggest that the changes in ShVO2 could reflect the degree of hepatic I/R injury, especially that related to microcirculatory disturbances occurring at the sinusoid levels.

Power Doppler sonography: evaluation of hepatocellular carcinoma after treatment with transarterial embolization or percutaneous ethanol injection therapy.

OBJECTIVE: The aim of this study was to compare tumor detectability by assessing the vascularity on power and color Doppler sonography and CT after transarterial embolization or percutaneous ethanol injection therapy or both in hepatocellular carcinoma. SUBJECTS AND METHODS: Forty-seven nodules of hepatocellular carcinoma (size, 28 +/- 7 mm [mean +/- standard deviation]; range, 20-40 mm) in 38 patients were treated with transarterial embolization (n = 6), percutaneous ethanol injection therapy (n = 23), and transarterial embolization plus percutaneous ethanol injection therapy (n = 9). Power Doppler sonography, color Doppler sonography, and CT were performed before and 2 weeks, 3 months, and 6 months after the treatments. The existence of hepatocellular carcinoma was confirmed by positive findings for color signals on both Doppler sonography techniques and for tumor stains on CT. All the tumors were determined to be malignant by microscopic examination of biopsy specimens. RESULTS: Before the treatments, power Doppler sonography (100%) and CT (100%) were significantly more effective than color Doppler sonography (61.7%) (p < 0.001, for both). Six months after the treatments, the sensitivity of power Doppler sonography (87.5%) was significantly better than that of color Doppler sonography (12.5%) but was not significant in comparison with CT (66.6%). However, power Doppler sonography detected color signals in two of three tumors in which iodized oil was accumulated and no tumor stain appeared on CT, and the two lesions detected with power Doppler sonography were carcinomas. CONCLUSION: Power Doppler sonography can be considered the most sensitive technique in assessing the viability of hepatocellular carcinoma treated with transarterial embolization or percutaneous ethanol injection therapy or both.

[Estimation of 99mTc-GSA receptor amount by non-linear 3-compartment model: ligand-receptor binding model without blood sampling].

99mTc-GSA (galactosyl serum albumin) receptor amount (Ro) was estimated by a non-linear 3-compartment model of the ligand-receptor binding without blood sampling. Forward/reverse rate constants and receptor amount were assumed to be variable. Relationship between this parameter and other conventional parameters including ICG R15 (15-minutes retention rate of indocyanine green) was evaluated for 43 surgical candidates with liver tumors. Linear relationships between Ro and HH15 and LHL15 were R2 = 0.73 and 0.72, respectively. Linear relationship between Ro and Rmax, the maximum removal rate, is also good (R2 = 0.84), and the regression line (y = 0.038x + 0.066) was slightly over 0 at y-interception. Linear relationship between Ro and ICG R15, was poor (R2 = 0.39) and relationship was rather a concave shape. Linear relationship of reduction rate between Ro and non-tumor tissue volume of the liver, which assessed at the same day of two weeks after the operation, was y = 1.09x - 0.01 (R2 = 0.82). GSA receptor amount, Ro, seems to change proportional to non-tumor liver tissue volume changing before and after hepatectomy. Complementary to ICG R15, it may be an useful and intuitive parameter for hepatectomy.

[Mechanisms of cell death in hypoxic stress].

The present article attempts to summarize and introduce the mechanisms of hypoxia-induced cell death. Necrosis is associated with rapid metabolic collapse that leads to cell swelling, early loss of plasma membrane integrity, and ultimate cell rupture, in which cytosolic contents leak from necrotic cells causing injury to and inflammation of the surrounding tissue. In contrast, apoptosis is an energy-requiring, gene-directed process, which results in cell suicide without any injury to surrounding tissues. Although apoptosis and necrosis are conceptually distinct pathways of cell death, recent advances have revealed that hypoxic cell damage can induce both necrosis and apoptosis simultaneously. Loss of the mitochondrial membrane potential (MMP) precedes the morphological changes in cell death, and overexpression of Bcl-2 or Bcl-XL blocks apoptosis as well as hypoxia-induced necrosis by maintaining MMP. These findings indicate that apoptosis and some types of necrosis share common features in the death signaling pathway. The factors that determine whether cells undergo apoptosis or necrosis are still unclear, but intracellular ATP levels and/or their rate of decline are considered to be one possible determinant of the manifestation of cell death.

Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver.

BACKGROUND: Although inhibition of endothelial nitric oxide synthase (eNOS) has been reported to aggravate hepatic ischemia-reperfusion (I/R) injury, the role of inducible nitric oxide synthase (iNOS) has been still unknown. We investigated the role of NO produced by iNOS, and evaluated the effect of an iNOS inhibitor on prolonged warm I/R injury in the pig liver. METHODS: Pigs were subjected to 120 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in serum and hepatic microdialysate NO2- + NO3- (NOx) and the cellular distribution of eNOS and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of iNOS inhibitor was also investigated. RESULTS: Hepatic I/R induced new nitric oxide production in serum and hepatic microdialysate NOx after reperfusion and severe hepatic damage in the centrilobular region where nitrotyrosine was strongly expressed. Diffuse eNOS expression in sinusoidal endothelium did not differ before and after reperfusion. In contrast, strong iNOS expression in Kupffer cells and neutrophils appeared strongly in the centrilobular region after reperfusion. Pigs with intraportal administration of N(G)-nitro-L-arginine (10 mg/kg) died during the period of ischemia or early in the period of reperfusion with a high mortality rate (80.0%). Intraportal administration of aminoguanidine hemisulfate (10 mg/kg) significantly suppressed nitric oxide production and serum aspartate aminotransferase after reperfusion, inhibited nitrotyrosine expression, and attenuated hepatic damage. CONCLUSIONS: These results indicate that hepatic I/R injury is triggered by centrilobular iNOS expression; and attenuated by inhibition of iNOS.

A case of partial autotransplantation of the liver in advanced hepatocellular carcinoma.

Hepatocellular carcinoma in Japan is frequently complicated by chronic hepatic disease such as chronic hepatitis and liver cirrhosis, and it is often impossible to decide the range to be resected only based on clinical stage and other tumor factors. We experienced a case with advanced hepatocellular carcinoma complicated by liver cirrhosis that directly infiltrated into the right and middle hepatic vein. Right trisegmentectomy was performed, the tumor site was extracorporeally removed and the hepatic posterior segment was autotransplanted. An anastomosis of the right hepatic vein and the inferior vena cava was performed with a vascular prosthesis. The patencies of the anastomosed vessels in the vascular reconstructions were confirmed by Doppler sonography, which was very useful, providing an easy and exact evaluation of hepatic blood flow at the patient's bedside. Throughout the post-operative course before the patient's discharge, no abnormal hepatic function was found. Though cases for which partial hepatic autotransplantation is appropriate may be few, this operation procedure, which applies hepatic transplantation techniques, is significant in that it increases the resectability and achieves curative resection of hepatocellular carcinoma.

Beta-glucan reflects liver injury after preservation and transplantation in dogs.

Graft failure and extrahepatic organ complications, which frequently develop after transplantation, may be related to inflammatory mediators stimulated by endotoxin (ET). The role of endotoxemia after liver transplantation is controversial and may depend upon differences in the ET assay method used in the various contradicting studies. While the standard Limulus amebocyte lysate (LAL) is reactive for ET and beta-glucan, a novel turbidimetric assay method enables separate determinations of ET and beta-glucan. Beagle dogs undergoing orthotopic liver transplantation were divided into two groups. In Group I (n = 6) the grafts were transplanted immediately and in Group II (n = 6) grafts were preserved for 48 h in University of Wisconsin (UW) solution. Animals received cyclosporine immunosuppression and were followed for 14 days. Daily measurements of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were performed. Samples for ET and beta-glucan measurement were collected serially and processed using the turbidimetric assay method. While no graft failure was seen in Group I, three of six Group II animals died from graft failure within 1 day after transplantation. Preservation and reperfusion injury was much more severe in the Group II grafts than in Group I grafts. While endotoxemia could not be detected, postoperative beta-glucan levels (undetectable pretransplant) were seen in both groups. Beta-glucan levels were much higher in Group II grafts than in Group I grafts, and correlated with the severity of liver damage. In conclusion, this study shows that beta-glucan, instead of ET, appears during the early posttransplant period. We believe that posttransplant elevation of beta-glucan is related to liver damage, especially endothelial damage by preservation and reperfusion.