Volume 114, Issue 1, Pages 153-163 (January 1998) Hydrogen peroxide derived from hepatocytes induces sinusoidal endothelial cell apoptosis in perfused hypoxic rat liver  Satoru Motoyama, Yoshihiro Minamiya, Satoshi Saito, Reijiro Saito, Ikuo Matsuzaki, Shichisaburo Abo, Hideo Inaba, Katsuhiko Enomoto, Michihiko Kitamura  Gastroenterology  Volume 114, Issue 1, Pages 153-163 (January 1998) DOI: 10.1016/S0016-5085(98)70643-2 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Perfusion flow in each treatment group. Isolated rat liver was perfused with KHB containing 5% bovine serum albumin and 10% washed rat red blood cells. The perfusion flow was continuously measured by an electromagnetic flow transducer. We produced LFH by reducing the afferent pressure (10, 7.5, 5.0, and 2.5 cm H2O) and established a reliable constant low flow. (B) Oxygen uptake in each treatment group. ○, Control group (n = 5); ●, 75% LFH group (n = 4); 2, 50% LFH group (n = 4); ■, 25% LFH group (n = 5); ▵, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Perfusion flow in each treatment group. Isolated rat liver was perfused with KHB containing 5% bovine serum albumin and 10% washed rat red blood cells. The perfusion flow was continuously measured by an electromagnetic flow transducer. We produced LFH by reducing the afferent pressure (10, 7.5, 5.0, and 2.5 cm H2O) and established a reliable constant low flow. (B) Oxygen uptake in each treatment group. ○, Control group (n = 5); ●, 75% LFH group (n = 4); 2, 50% LFH group (n = 4); ■, 25% LFH group (n = 5); ▵, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 DCF fluorescence images of the rat liver microcirculation (typical images). To visualize the H2O2 production, 5 μmol/L of DCFH-DA (final concentration) was added to the perfusate at 80 minutes. After 120 minutes of perfusion, DCF fluorescence images were recorded with a silicon-intensified target TV camera. (A) DCF fluorescence was negligible in the control group. (B) The intensity of DCF fluorescence increased significantly in the 25% LFH group, especially in the midzone. (C) Pretreatment of (−)BOF 4272 attenuated the increase of LFH-induced DCF fluorescence. P, terminal portal venules; T, terminal hepatic venules. All panels were 4×. (D) The amount of DCF fluorescence was calibrated using reagent DCF. We measured the fluorescence intensities of the known concentrations of reagent DCF using the same system. The fluorescence intensities expressed as 256-gray level (linear) were plotted against the different DCF concentrations (logarithmic). The relationship between DCF concentration and fluorescence intensity was always linear, and the correlation was always significant (P < 0.05). y = 314.9 log × +31.7; r2 = 0.978. (E) Quantification of H2O2 production in the liver. Using a calibration line, we determined H2O2 in the three zones in each group (4-5 livers in each group). The amount of H2O2 is expressed as the DCF concentration. DCF fluorescence intensities in the 50% and 25% LFH groups were significantly higher than in the control group in all three zones (*P < 0.05). In the 25% LFH group, DCF fluorescence intensity in the M zone was significantly higher than that in the other two zones ($P < 0.05). Pretreatment of (−)BOF 4272 significantly attenuated the increase of LFH-induced DCF fluorescence (#P < 0.05). Control, control group (n = 4); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 DCF fluorescence images of the rat liver microcirculation (typical images). To visualize the H2O2 production, 5 μmol/L of DCFH-DA (final concentration) was added to the perfusate at 80 minutes. After 120 minutes of perfusion, DCF fluorescence images were recorded with a silicon-intensified target TV camera. (A) DCF fluorescence was negligible in the control group. (B) The intensity of DCF fluorescence increased significantly in the 25% LFH group, especially in the midzone. (C) Pretreatment of (−)BOF 4272 attenuated the increase of LFH-induced DCF fluorescence. P, terminal portal venules; T, terminal hepatic venules. All panels were 4×. (D) The amount of DCF fluorescence was calibrated using reagent DCF. We measured the fluorescence intensities of the known concentrations of reagent DCF using the same system. The fluorescence intensities expressed as 256-gray level (linear) were plotted against the different DCF concentrations (logarithmic). The relationship between DCF concentration and fluorescence intensity was always linear, and the correlation was always significant (P < 0.05). y = 314.9 log × +31.7; r2 = 0.978. (E) Quantification of H2O2 production in the liver. Using a calibration line, we determined H2O2 in the three zones in each group (4-5 livers in each group). The amount of H2O2 is expressed as the DCF concentration. DCF fluorescence intensities in the 50% and 25% LFH groups were significantly higher than in the control group in all three zones (*P < 0.05). In the 25% LFH group, DCF fluorescence intensity in the M zone was significantly higher than that in the other two zones ($P < 0.05). Pretreatment of (−)BOF 4272 significantly attenuated the increase of LFH-induced DCF fluorescence (#P < 0.05). Control, control group (n = 4); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 DCF fluorescence images of the rat liver microcirculation (typical images). To visualize the H2O2 production, 5 μmol/L of DCFH-DA (final concentration) was added to the perfusate at 80 minutes. After 120 minutes of perfusion, DCF fluorescence images were recorded with a silicon-intensified target TV camera. (A) DCF fluorescence was negligible in the control group. (B) The intensity of DCF fluorescence increased significantly in the 25% LFH group, especially in the midzone. (C) Pretreatment of (−)BOF 4272 attenuated the increase of LFH-induced DCF fluorescence. P, terminal portal venules; T, terminal hepatic venules. All panels were 4×. (D) The amount of DCF fluorescence was calibrated using reagent DCF. We measured the fluorescence intensities of the known concentrations of reagent DCF using the same system. The fluorescence intensities expressed as 256-gray level (linear) were plotted against the different DCF concentrations (logarithmic). The relationship between DCF concentration and fluorescence intensity was always linear, and the correlation was always significant (P < 0.05). y = 314.9 log × +31.7; r2 = 0.978. (E) Quantification of H2O2 production in the liver. Using a calibration line, we determined H2O2 in the three zones in each group (4-5 livers in each group). The amount of H2O2 is expressed as the DCF concentration. DCF fluorescence intensities in the 50% and 25% LFH groups were significantly higher than in the control group in all three zones (*P < 0.05). In the 25% LFH group, DCF fluorescence intensity in the M zone was significantly higher than that in the other two zones ($P < 0.05). Pretreatment of (−)BOF 4272 significantly attenuated the increase of LFH-induced DCF fluorescence (#P < 0.05). Control, control group (n = 4); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 DCF fluorescence images of the rat liver microcirculation (typical images). To visualize the H2O2 production, 5 μmol/L of DCFH-DA (final concentration) was added to the perfusate at 80 minutes. After 120 minutes of perfusion, DCF fluorescence images were recorded with a silicon-intensified target TV camera. (A) DCF fluorescence was negligible in the control group. (B) The intensity of DCF fluorescence increased significantly in the 25% LFH group, especially in the midzone. (C) Pretreatment of (−)BOF 4272 attenuated the increase of LFH-induced DCF fluorescence. P, terminal portal venules; T, terminal hepatic venules. All panels were 4×. (D) The amount of DCF fluorescence was calibrated using reagent DCF. We measured the fluorescence intensities of the known concentrations of reagent DCF using the same system. The fluorescence intensities expressed as 256-gray level (linear) were plotted against the different DCF concentrations (logarithmic). The relationship between DCF concentration and fluorescence intensity was always linear, and the correlation was always significant (P < 0.05). y = 314.9 log × +31.7; r2 = 0.978. (E) Quantification of H2O2 production in the liver. Using a calibration line, we determined H2O2 in the three zones in each group (4-5 livers in each group). The amount of H2O2 is expressed as the DCF concentration. DCF fluorescence intensities in the 50% and 25% LFH groups were significantly higher than in the control group in all three zones (*P < 0.05). In the 25% LFH group, DCF fluorescence intensity in the M zone was significantly higher than that in the other two zones ($P < 0.05). Pretreatment of (−)BOF 4272 significantly attenuated the increase of LFH-induced DCF fluorescence (#P < 0.05). Control, control group (n = 4); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 DCF fluorescence images of the rat liver microcirculation (typical images). To visualize the H2O2 production, 5 μmol/L of DCFH-DA (final concentration) was added to the perfusate at 80 minutes. After 120 minutes of perfusion, DCF fluorescence images were recorded with a silicon-intensified target TV camera. (A) DCF fluorescence was negligible in the control group. (B) The intensity of DCF fluorescence increased significantly in the 25% LFH group, especially in the midzone. (C) Pretreatment of (−)BOF 4272 attenuated the increase of LFH-induced DCF fluorescence. P, terminal portal venules; T, terminal hepatic venules. All panels were 4×. (D) The amount of DCF fluorescence was calibrated using reagent DCF. We measured the fluorescence intensities of the known concentrations of reagent DCF using the same system. The fluorescence intensities expressed as 256-gray level (linear) were plotted against the different DCF concentrations (logarithmic). The relationship between DCF concentration and fluorescence intensity was always linear, and the correlation was always significant (P < 0.05). y = 314.9 log × +31.7; r2 = 0.978. (E) Quantification of H2O2 production in the liver. Using a calibration line, we determined H2O2 in the three zones in each group (4-5 livers in each group). The amount of H2O2 is expressed as the DCF concentration. DCF fluorescence intensities in the 50% and 25% LFH groups were significantly higher than in the control group in all three zones (*P < 0.05). In the 25% LFH group, DCF fluorescence intensity in the M zone was significantly higher than that in the other two zones ($P < 0.05). Pretreatment of (−)BOF 4272 significantly attenuated the increase of LFH-induced DCF fluorescence (#P < 0.05). Control, control group (n = 4); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 CLA-dependent chemiluminescence. Neutrophils were isolated from peripheral blood samples taken from healthy humans. The neutrophil suspensions were prepared in bovine serum albumin–HEPES buffer at 1 × 106 cells/mL. In the presence of 1 μmol/L CLA, chemiluminescence activities were evoked by adding phorbol myristate acetate to a final concentration of 0.1 μg/mL. The maximum chemiluminescence activities for 5 minutes were monitored using the luminometer (n = 4). Treatment of (−)BOF 4272 did not affect the amount of neutrophil superoxide anion production even when its concentration was increasing to 10−4 mol/L. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 H2O2 detection by cerium electron-microscopic method. To visualize H2O2, the liver was perfused with a reaction medium containing CeCl3 at the end of the perfusion. Cerium deposition, which shows H2O2 production, was examined by electron microscopy. (A) The cerium deposition was marked on the surface of hepatocyte plasma membrane in the midzone in the 25% LFH. Few depositions were observed on the SEC and the Kupffer cell (original magnification 9000×). (B) The SEC was shrunken, and the nuclear chromatin was compacted into uniformly dense masses in 25% LFH (original magnification 11,400×). These changes indicate apoptosis in the SECs. (C) On the other hand, there were no changes in morphology in the SEC, and few cerium depositions were seen on the hepatocyte in the control group (11,400×). H, hepatocyte; E, SEC; K, Kupffer cell (bar = 1 μm). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 H2O2 detection by cerium electron-microscopic method. To visualize H2O2, the liver was perfused with a reaction medium containing CeCl3 at the end of the perfusion. Cerium deposition, which shows H2O2 production, was examined by electron microscopy. (A) The cerium deposition was marked on the surface of hepatocyte plasma membrane in the midzone in the 25% LFH. Few depositions were observed on the SEC and the Kupffer cell (original magnification 9000×). (B) The SEC was shrunken, and the nuclear chromatin was compacted into uniformly dense masses in 25% LFH (original magnification 11,400×). These changes indicate apoptosis in the SECs. (C) On the other hand, there were no changes in morphology in the SEC, and few cerium depositions were seen on the hepatocyte in the control group (11,400×). H, hepatocyte; E, SEC; K, Kupffer cell (bar = 1 μm). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 H2O2 detection by cerium electron-microscopic method. To visualize H2O2, the liver was perfused with a reaction medium containing CeCl3 at the end of the perfusion. Cerium deposition, which shows H2O2 production, was examined by electron microscopy. (A) The cerium deposition was marked on the surface of hepatocyte plasma membrane in the midzone in the 25% LFH. Few depositions were observed on the SEC and the Kupffer cell (original magnification 9000×). (B) The SEC was shrunken, and the nuclear chromatin was compacted into uniformly dense masses in 25% LFH (original magnification 11,400×). These changes indicate apoptosis in the SECs. (C) On the other hand, there were no changes in morphology in the SEC, and few cerium depositions were seen on the hepatocyte in the control group (11,400×). H, hepatocyte; E, SEC; K, Kupffer cell (bar = 1 μm). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 TUNEL staining of the rat livers. (A) Few cells were stained in the control livers (×10). (B) In contrast, many TUNEL-positive cells were observed mainly in the midzone in the 25% LFH liver. (C) In high-power views, they were classified as nonparenchymal cells but not as hepatocytes (×100) in the 25% LFH livers. (D) Pretreatment of (−)BOF 4272 attenuated the increase of TUNEL-positive cells (original magnification 10×). (E) The relationship between TUNEL-positive and trypan blue–positive nonparenchymal cells. Almost all of the trypan blue–positive cells were also TUNEL positive. ○, Control group; ●, 75% LFH group; 2, 50% LFH group; ■, 25% LFH group. y = 0.78× + 0.56; r = 0.98; P < 0.05. (F) The number of TUNEL-positive nonparenchymal cells in each group. The TUNEL-positive cells ratio was defined as (the number of stained cells)/(the number of stained and unstained cells). The number of TUNEL-positive cells in the 25% LFH group was significantly higher than in the control and (−)BOF 4272 groups (P < 0.05). Control, control group (n = 5); 75%, 75% LFH group (n = 4); 50%, 50% LFH group (n = 4); 25%, 25% LFH group (n = 4); 25% + (−)BOF 4272, (−)BOF 4272 group (n = 5). *P < 0.05. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 DNA fragmentation of the nonparenchymal cell and hepatocyte suspension in the control and 25% LFH groups. To obtain the nonparenchymal cell suspension, a modification of the method of Seglen was used.26 Briefly, the perfused rat livers in the 25% LFH (n = 4) and control groups (n = 2) were perfused with 30 mL of prewarmed 0.2% trypsin-ethylenediaminetetraacetic acid solution for 10 minutes. The perfusate was collected and centrifuged at 50g for 10 minutes. The supernatant was centrifuged at 300g for 10 minutes. (A) We examined the percentage of the SECs in the cell suspension by flow cytometry. Cells were indirectly immunostained with monoclonal antibody SE-1 followed by fluorescein isothiocyanate–labeled rabbit anti-mouse immunoglobulin G. This separation technique yielded a sample >30% SECs. (B) The pellet was lysed and electrophoretically separated on 2% agarose gel containing 0.1 μg/mL of ethidium bromide. DNA was visualized by a UV transilluminator. Lane 1, hepatocytes in the 25% LFH group; lane 2, nonparenchymal cells in the control group; lane 3, nonparenchymal cells in the 25% LFH group. The characteristic ladder pattern of apoptosis is seen in lane 3. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 DNA fragmentation of the nonparenchymal cell and hepatocyte suspension in the control and 25% LFH groups. To obtain the nonparenchymal cell suspension, a modification of the method of Seglen was used.26 Briefly, the perfused rat livers in the 25% LFH (n = 4) and control groups (n = 2) were perfused with 30 mL of prewarmed 0.2% trypsin-ethylenediaminetetraacetic acid solution for 10 minutes. The perfusate was collected and centrifuged at 50g for 10 minutes. The supernatant was centrifuged at 300g for 10 minutes. (A) We examined the percentage of the SECs in the cell suspension by flow cytometry. Cells were indirectly immunostained with monoclonal antibody SE-1 followed by fluorescein isothiocyanate–labeled rabbit anti-mouse immunoglobulin G. This separation technique yielded a sample >30% SECs. (B) The pellet was lysed and electrophoretically separated on 2% agarose gel containing 0.1 μg/mL of ethidium bromide. DNA was visualized by a UV transilluminator. Lane 1, hepatocytes in the 25% LFH group; lane 2, nonparenchymal cells in the control group; lane 3, nonparenchymal cells in the 25% LFH group. The characteristic ladder pattern of apoptosis is seen in lane 3. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Identification of TUNEL-positive nonparenchymal cells in the midzone of the 25% LFH group. (A) To identify the TUNEL-positive cells, tissues stained with the TUNEL method were immunohistochemically double-stained with monoclonal antibody SE-1. The SECs were stained pink (developed with new fuchsin). The nuclei of TUNEL-positive cells were stained dark brown (developed with 3,3'-diaminobenzidine tetrahydrochloride). Arrowhead, SE-1–positive and TUNEL-positive; arrow, SE-1–positive and TUNEL-negative. (B) The tissues that were stained with the TUNEL method were immunohistochemically double-stained with monoclonal antibody ED-2. Kupffer cells were stained dark brown (developed with 3,3'-diaminobenzidine tetrahydrochloride). TUNEL-positive cells were stained violet (developed with 3-amino-9-ethylcarbazole). Arrowhead, ED-2–positive and TUNEL-negative; arrow, TUNEL-positive and ED-2–negative. These photographs show that TUNEL-positive cells were stained with monoclonal SE-1, but not with ED-2 (original magnification 100×). Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Apoptosis induced by reagent H2O2. To determine the effect of H2O2, the small pieces of liver were exposed to reagent H2O2. The liver pieces (5 × 5 × 2 mm) were incubated in the cultured medium containing 0.5 mmol/L of reagent H2O2 at 37°C for 2 hours. Then the pieces were fixed with 2% glutaraldehyde and stained with TUNEL method. There were few apoptotic cells in the liver pieces incubated without H2O2. In the liver pieces incubated with a reagent H2O2, the SECs were TUNEL positive without zonality, whereas the hepatocytes were not. Gastroenterology 1998 114, 153-163DOI: (10.1016/S0016-5085(98)70643-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions