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reperfusion in fatty liver rats
Induction of Immediate Early Genes and Apoptosis After Ischemia/Reperfusion in Fatty Liver Rats T. Tsuchiya, T. Abe, T. Saito, M. Miyazawa, H. Kanno, S. Ishii, M. Suzuki, H. Inoue, and R. Motoki
I
SCHEMIA/reperfusion (I/R) injury is an important cause of primary nonfunction (PNF) in liver transplantation. It is well known that severe fatty liver graft is susceptible to increased hepatic I/R injury,1 although some papers reported that the rate of PNF in mild and moderately fatty liver graft did not increase in comparison with normal liver graft.2 The exact reasons for the increased I/R injury seen with the fatty livers are poorly understand. The immediate early genes (IEGs) (c-fos, c-jun, etc.) are rapidly and transiently induced in most tissues by a variety extracellular stimuli, including I/R injury.3 These genes encode proteins that are members of the leucine zipper family and relate to the process of cell repair. Recent studies indicate that the induction pattern and the level of c-fos and c-jun expression in transplanted livers with various conditions may reflect the differences in severity of the transplantrelated insults and the differences in subsequent graft survival upon transplantation.4 A continuous expression of c-fos and c-jun has been suggested to be associated with cell damage and programmed cell death, including apoptosis.5,6 To clarify the mechanisms of the fatty liver injury against I/R, we investigated the induction of the IEGs (c-fos, c-jun) and apoptosis in fatty and normal liver using I/R model in rats. MATERIALS AND METHODS Experimental Design We established four groups, normal liver (NL), fatty liver (FL), shunted normal liver (S-NL), and shunted fatty liver (S-FL). In these all groups, 8-week-old Wister rats were used. In the FL and S-FL groups, rats were fed chow deficient in choline for 4 weeks. In the S-NL and S-FL groups, portosystemic shunts were induced by subcutaneous transposition of the spleen, according to Benmark’s method, 4 weeks before the operation.
Total Hepatic Ischemia The rats were fasted for 12 hours before the experiment but allowed to drink water freely. They were anesthetized with ether. We induced total hepatic warm ischemia in vivo by clamping the hepatic artery, portal vein, and bile duct for 45 minutes and then initiated reperfusion. Before ischemia and at 0, 0.5, 1, 1.5, 2, 3, 6, 12, 24, 48 hours after reperfusion, liver tissues were sampled (n 5 6).
One-Week Survival Study Forty-five minutes of total hepatic ischemia was generated in 46 rats (n 5 15, NL and FL groups; n 5 8, S-NL and S-FL groups), for the preliminary 1-week survival study.
Histologic Examination Liver tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections (4 mm thick) were stained with hematoxilin and eosin (HE). In the FL group, occupational rate of fat drops was analyzed by NIH image before I/R.
Northern Blot Analysis of IEG mRNAs in Liver Tissue For the study of I/R, 6 animals in each group were sacrificed after 45 minutes of hepatic ischemia without reperfusion (time 0) and at 0.5, 1, 1.5, 2, 3, 6, 12, 24, 48 hours after reperfusion. The liver was processed immediately. The liver specimens, taken from the median lobe (2 to 3 g wet weight) were promptly frozen in liquid nitrogen and stored at 270°C. RNA preparation and Northern blot analysis were carried out as described previously.7 Total RNA (30 mg), extracted from liver tissue by a guanidinium thiocyanate method, was size-fractionated by 1% (w/v) agarose-formaldehyde gel electrophoresis, transferred onto Hybond-N nylon membranes (Amersham). The membranes were prehybridized for 30 minutes at 65°C in rapid hybridization solution (Amersham). DNA probes for c-fos and c-jun were labeled with [32P]-dCTP (Bresatec, Adelaide, Australia) by the random multiprime method (Pharmacia, Sydney, Australia) and used rediprime DNA labeling system, then added to the prehybridization solution. After hybridization at 65°C for 2 hours, the membranes were washed to a final stringency of 0.1% (w/v) SDS, 0.23 sodium chloride sodium phosphate EDTA (SSPE) (1 3 SSPE is 0.15 M NaCl, 10 mM NaH2PO4.H2O, 1 mM EDTA) at 65°C for 30 minutes and exposed to BAS-IIIs IP film (FUJIX) with intensifying screens for 8 hours. Loading and transferred RNAs were monitored by reprobing the membranes with an oligonucleotide probe for G3PDH mRNA. Intensity of autoradiographic bands was measured by BAS 1000 image analyzer and adjusted for variation in the intensity of G3PDH ribosomal RNA. From the First Department of Surgery, Fukushima Medical College (T.T., T.A., T.S., M.M., H.K., S.I., M.S., H.I., R.M.), Fukushima, Japan. This work was supported by a grant for scientific research from the Ministry of Education, Science and Culture of Japan and Fukushima Society for the Promotion of Medicane. Address reprint requests to Dr T. Tsuchiya, 1-Hikarigaoka, Fukushima, 960-12 Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00869-0
Transplantation Proceedings, 30, 2919–2922 (1998)
2919
2920
Fig 1. The expression of c-fos in livers of experimental groups after 45 min of ischemia and reperfusion. (A): nonshunted groups (h: normal liver rats, o: fatty liver rats). *P , .05, **P , .01 versus normal liver rats. (B): shunted groups (■: shunted normal liver rats, h: shunted fatty liver rats). *P , .05, **P , .01 versus shunted normal liver rats. Values are mean 1 SD (n 5 6) in all groups.
Detection of Apoptotic Cells Apoptosis in liver tissue was detected by the TUNEL method that used ApopTag Plus in situ apoptosis detection kit (Oncor).
RESULTS One-Week Survival Study
The 1-week survival rates for normal (NL) and fatty (FL) liver rats were 80% and 33.3%, respectively (P , .05); in the shunted (S-NL) and shunted fatty liver (S-FL) groups, all rats survived after 45 minutes I/R. Histologic Examination
TSUCHIYA, ABE, SAITU ET AL
Fig 2. The expression of c-jun in livers of experimental groups after 45 min of ischemia and reperfusion. (A): nonshunted groups (h: normal liver rats, o: fatty liver rats). *P , .05, **P , .01 versus normal liver rats. (B): shunted groups (■: shunted normal liver rats, h: shunted fatty liver rats). *P , .05 versus shunted normal liver rats. Values are mean 6 SD (n 5 6) in all groups.
peak value, c-fos and c-jun expressions of NL were significantly stronger than FL. In NL and S-FL, the expression of c-fos returned to basal levels by 3 hours after reperfusion. In contrast, the expression of c-fos in FL that the survival rate was worst returned to basal levels by 6 hours after reperfusion. In S-NL, the expression of c-fos returned to basal levels most quickly, by 2 hours. The induction pattern of c-jun expression was similar to that of c-fos. In FL, the expression of c-jun persisted up to 2 hours and disappeared 3 hours after reperfusion. In NL and S-FL, the expression of c-jun returned to basal levels by 2 hours after reperfusion. In S-NL, the expression of c-jun returned to basal levels most quickly, by 1.5 hours.
Liver specimens of FL and S-FL groups showed moderately fatty infiltration and sinusoidal lumens were narrow and irregular. In FL group, fat drops occupied 51.1 6 2.5% (n 5 5) of the liver. At 24 hours after I/R, mild congestion and slightly focal necrosis were observed in the NL and FL groups. In the S-NL and S-FL groups, the congestions were observed but focal necroses were not evident.
Expression of Apoptosis
Expression of c-fos and c-jun mRNA
DISCUSSION
In all groups, c-fos and c-jun mRNAs increased markedly with a peak value at 1 hour after I/R (Figs 1 and 2). At the
Previous papers have reported that the severe fatty infiltration of liver graft tends to exhibit PNF after transplantation.
After 3 hours of I/R, diffuse apoptotic cells were observed widely in the FL and S-FL groups. In contrast, apoptotic cells in the NL and S-NL groups were localized at the pericentral lesion (Zone 1) of the liver (Fig 3). After 48 hours of I/R, apoptotic cells were detected in the FL group only.
IMMEDIATE EARLY GENES AND APTOSIS
2921
Fig 3. Microscopic findings at 3 hours after reperfusion by the TUNEL method (3200). A: NL group. B: FL group. C: S-NL group. D: S-FL group. TUNEL positive cells were observed at hepatic pericentral lesion in NL and S-NL groups. TUNEL positive cells were observed at widely in FL and S-FL groups.
Several reasons have been suggested to explain the increased I/R injury of fatty liver graft: (1) increased damage to the sinusoidal microcirculation, (2) increased blood cell adhesion, (3) increased Kupffer cell activation,8 (4) lipid peroxidation,2 and others. But mechanisms responsible for the increased I/R injury seen with the fatty livers are still not fully understood. Here we show the different expression of the IEGs and changes in the presence of apoptotic cells between normal and fatty liver after I/R. In FL group, the 1-week survival rates after I/R were worse than that of the NL group. However all the rats in the S-NL and S-FL groups survived after 45 minutes I/R. Light microscopy findings in FL group revealed narrow and irregular sinusoidal lumens, and most of the endothelial cells were swollen and peeled off at 24 hours after I/R.
Therefore, we thought that the change of the sinusoidal width increased damage of microcirculation after I/R. After I/R, fatty liver may be severely damaged by intestinal congestion. Exception of intestinal congestion reduced the liver injury after I/R in the shunted fatty liver group. The IEGs (c-fos and c-jun) are rapidly and transiently induced after hepatic I/R. These genes encode proteins or delayed early genes that related with the cell repair and cell proliferation. Goto et al.4 reported that the pattern of IEGs expression might represent the status of the liver graft and its likely long-term response to insult in liver transplantation. After long hepatic ischemia and reperfusion time, IEG expression in poor survival groups showed a protractive pattern. In our fatty liver group (poor survival group) c-fos and c-jun expressed longer after I/R than normal liver and
2922
shunted fatty liver. We thought about this reason, usually IEGs are controlled by negative feedback from encoded protein or delayed genes, but severely damaged liver cannot produce the proteins or delayed genes. Thus, we suggest that expression pattern of hepatic c-fos and c-jun might be correlated with the liver damage. On the other hand, the induction of IEG has been suggested to be associated with programmed cell death, including apoptosis.5,6 Colotta et al. reported that the expression of c-fos and c-jun may represent an important early event in the activation of the genetic program of cell death.9 Smeyne et al5 reported that continuous c-fos expression precedes programmed cell death. We observed the apoptotic cells in all groups at 3 hours after I/R, but could not detect them at 48 hours after I/R without the FL group that showed protractive expression of IEGs. Continuous expression of liver IEGs may be associated with programmed cell death. However, it will be necessary to perform more experiments about in situ hybridization, immunohistochemical study, gene targeting, and so on to confirm this theory.
TSUCHIYA, ABE, SAITU ET AL
In conclusion, we suggest that fatty liver is more vulnerable than healthy liver against warm ischemia in the rat. Expression of liver IEGs might be correlated with liver damage and induction of apoptosis after I/R. REFERENCES 1. Todo S, Demetris AJ, Makowka L, et al: Transplantation 47:903, 1989 2. D’Alessandro AM, Kalayaglu M, Sollinger HW, et al: Transplantation 51:157, 1991 3. Morgan JI, Curran T: Annu Rev Neurosci 14:421, 1991 4. Goto S, Matsumoto I, Kamada N, et al: Transplantation 58:840, 1994 5. Smeyne RJ, Yendrell M, Hayward M, et al: Nature 363:166, 1993 6. Dragnunow M, Young D, Hughes P, et al: Mol Brain Res 18:347, 1993 7. Le F, Wilce PA, Cassady I, et al: Neurosci Lett 120:271, 1990 8. Teramoto K, Bowers JL, Kruskal JB, et al: Transplantation 56:1076, 1993 9. Colotta F, Polentarutti N, Sironi M, et al: J Biol Chem 267:18278, 1992
I
SCHEMIA/reperfusion (I/R) injury is an important cause of primary nonfunction (PNF) in liver transplantation. It is well known that severe fatty liver graft is susceptible to increased hepatic I/R injury,1 although some papers reported that the rate of PNF in mild and moderately fatty liver graft did not increase in comparison with normal liver graft.2 The exact reasons for the increased I/R injury seen with the fatty livers are poorly understand. The immediate early genes (IEGs) (c-fos, c-jun, etc.) are rapidly and transiently induced in most tissues by a variety extracellular stimuli, including I/R injury.3 These genes encode proteins that are members of the leucine zipper family and relate to the process of cell repair. Recent studies indicate that the induction pattern and the level of c-fos and c-jun expression in transplanted livers with various conditions may reflect the differences in severity of the transplantrelated insults and the differences in subsequent graft survival upon transplantation.4 A continuous expression of c-fos and c-jun has been suggested to be associated with cell damage and programmed cell death, including apoptosis.5,6 To clarify the mechanisms of the fatty liver injury against I/R, we investigated the induction of the IEGs (c-fos, c-jun) and apoptosis in fatty and normal liver using I/R model in rats. MATERIALS AND METHODS Experimental Design We established four groups, normal liver (NL), fatty liver (FL), shunted normal liver (S-NL), and shunted fatty liver (S-FL). In these all groups, 8-week-old Wister rats were used. In the FL and S-FL groups, rats were fed chow deficient in choline for 4 weeks. In the S-NL and S-FL groups, portosystemic shunts were induced by subcutaneous transposition of the spleen, according to Benmark’s method, 4 weeks before the operation.
Total Hepatic Ischemia The rats were fasted for 12 hours before the experiment but allowed to drink water freely. They were anesthetized with ether. We induced total hepatic warm ischemia in vivo by clamping the hepatic artery, portal vein, and bile duct for 45 minutes and then initiated reperfusion. Before ischemia and at 0, 0.5, 1, 1.5, 2, 3, 6, 12, 24, 48 hours after reperfusion, liver tissues were sampled (n 5 6).
One-Week Survival Study Forty-five minutes of total hepatic ischemia was generated in 46 rats (n 5 15, NL and FL groups; n 5 8, S-NL and S-FL groups), for the preliminary 1-week survival study.
Histologic Examination Liver tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections (4 mm thick) were stained with hematoxilin and eosin (HE). In the FL group, occupational rate of fat drops was analyzed by NIH image before I/R.
Northern Blot Analysis of IEG mRNAs in Liver Tissue For the study of I/R, 6 animals in each group were sacrificed after 45 minutes of hepatic ischemia without reperfusion (time 0) and at 0.5, 1, 1.5, 2, 3, 6, 12, 24, 48 hours after reperfusion. The liver was processed immediately. The liver specimens, taken from the median lobe (2 to 3 g wet weight) were promptly frozen in liquid nitrogen and stored at 270°C. RNA preparation and Northern blot analysis were carried out as described previously.7 Total RNA (30 mg), extracted from liver tissue by a guanidinium thiocyanate method, was size-fractionated by 1% (w/v) agarose-formaldehyde gel electrophoresis, transferred onto Hybond-N nylon membranes (Amersham). The membranes were prehybridized for 30 minutes at 65°C in rapid hybridization solution (Amersham). DNA probes for c-fos and c-jun were labeled with [32P]-dCTP (Bresatec, Adelaide, Australia) by the random multiprime method (Pharmacia, Sydney, Australia) and used rediprime DNA labeling system, then added to the prehybridization solution. After hybridization at 65°C for 2 hours, the membranes were washed to a final stringency of 0.1% (w/v) SDS, 0.23 sodium chloride sodium phosphate EDTA (SSPE) (1 3 SSPE is 0.15 M NaCl, 10 mM NaH2PO4.H2O, 1 mM EDTA) at 65°C for 30 minutes and exposed to BAS-IIIs IP film (FUJIX) with intensifying screens for 8 hours. Loading and transferred RNAs were monitored by reprobing the membranes with an oligonucleotide probe for G3PDH mRNA. Intensity of autoradiographic bands was measured by BAS 1000 image analyzer and adjusted for variation in the intensity of G3PDH ribosomal RNA. From the First Department of Surgery, Fukushima Medical College (T.T., T.A., T.S., M.M., H.K., S.I., M.S., H.I., R.M.), Fukushima, Japan. This work was supported by a grant for scientific research from the Ministry of Education, Science and Culture of Japan and Fukushima Society for the Promotion of Medicane. Address reprint requests to Dr T. Tsuchiya, 1-Hikarigaoka, Fukushima, 960-12 Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00869-0
Transplantation Proceedings, 30, 2919–2922 (1998)
2919
2920
Fig 1. The expression of c-fos in livers of experimental groups after 45 min of ischemia and reperfusion. (A): nonshunted groups (h: normal liver rats, o: fatty liver rats). *P , .05, **P , .01 versus normal liver rats. (B): shunted groups (■: shunted normal liver rats, h: shunted fatty liver rats). *P , .05, **P , .01 versus shunted normal liver rats. Values are mean 1 SD (n 5 6) in all groups.
Detection of Apoptotic Cells Apoptosis in liver tissue was detected by the TUNEL method that used ApopTag Plus in situ apoptosis detection kit (Oncor).
RESULTS One-Week Survival Study
The 1-week survival rates for normal (NL) and fatty (FL) liver rats were 80% and 33.3%, respectively (P , .05); in the shunted (S-NL) and shunted fatty liver (S-FL) groups, all rats survived after 45 minutes I/R. Histologic Examination
TSUCHIYA, ABE, SAITU ET AL
Fig 2. The expression of c-jun in livers of experimental groups after 45 min of ischemia and reperfusion. (A): nonshunted groups (h: normal liver rats, o: fatty liver rats). *P , .05, **P , .01 versus normal liver rats. (B): shunted groups (■: shunted normal liver rats, h: shunted fatty liver rats). *P , .05 versus shunted normal liver rats. Values are mean 6 SD (n 5 6) in all groups.
peak value, c-fos and c-jun expressions of NL were significantly stronger than FL. In NL and S-FL, the expression of c-fos returned to basal levels by 3 hours after reperfusion. In contrast, the expression of c-fos in FL that the survival rate was worst returned to basal levels by 6 hours after reperfusion. In S-NL, the expression of c-fos returned to basal levels most quickly, by 2 hours. The induction pattern of c-jun expression was similar to that of c-fos. In FL, the expression of c-jun persisted up to 2 hours and disappeared 3 hours after reperfusion. In NL and S-FL, the expression of c-jun returned to basal levels by 2 hours after reperfusion. In S-NL, the expression of c-jun returned to basal levels most quickly, by 1.5 hours.
Liver specimens of FL and S-FL groups showed moderately fatty infiltration and sinusoidal lumens were narrow and irregular. In FL group, fat drops occupied 51.1 6 2.5% (n 5 5) of the liver. At 24 hours after I/R, mild congestion and slightly focal necrosis were observed in the NL and FL groups. In the S-NL and S-FL groups, the congestions were observed but focal necroses were not evident.
Expression of Apoptosis
Expression of c-fos and c-jun mRNA
DISCUSSION
In all groups, c-fos and c-jun mRNAs increased markedly with a peak value at 1 hour after I/R (Figs 1 and 2). At the
Previous papers have reported that the severe fatty infiltration of liver graft tends to exhibit PNF after transplantation.
After 3 hours of I/R, diffuse apoptotic cells were observed widely in the FL and S-FL groups. In contrast, apoptotic cells in the NL and S-NL groups were localized at the pericentral lesion (Zone 1) of the liver (Fig 3). After 48 hours of I/R, apoptotic cells were detected in the FL group only.
IMMEDIATE EARLY GENES AND APTOSIS
2921
Fig 3. Microscopic findings at 3 hours after reperfusion by the TUNEL method (3200). A: NL group. B: FL group. C: S-NL group. D: S-FL group. TUNEL positive cells were observed at hepatic pericentral lesion in NL and S-NL groups. TUNEL positive cells were observed at widely in FL and S-FL groups.
Several reasons have been suggested to explain the increased I/R injury of fatty liver graft: (1) increased damage to the sinusoidal microcirculation, (2) increased blood cell adhesion, (3) increased Kupffer cell activation,8 (4) lipid peroxidation,2 and others. But mechanisms responsible for the increased I/R injury seen with the fatty livers are still not fully understood. Here we show the different expression of the IEGs and changes in the presence of apoptotic cells between normal and fatty liver after I/R. In FL group, the 1-week survival rates after I/R were worse than that of the NL group. However all the rats in the S-NL and S-FL groups survived after 45 minutes I/R. Light microscopy findings in FL group revealed narrow and irregular sinusoidal lumens, and most of the endothelial cells were swollen and peeled off at 24 hours after I/R.
Therefore, we thought that the change of the sinusoidal width increased damage of microcirculation after I/R. After I/R, fatty liver may be severely damaged by intestinal congestion. Exception of intestinal congestion reduced the liver injury after I/R in the shunted fatty liver group. The IEGs (c-fos and c-jun) are rapidly and transiently induced after hepatic I/R. These genes encode proteins or delayed early genes that related with the cell repair and cell proliferation. Goto et al.4 reported that the pattern of IEGs expression might represent the status of the liver graft and its likely long-term response to insult in liver transplantation. After long hepatic ischemia and reperfusion time, IEG expression in poor survival groups showed a protractive pattern. In our fatty liver group (poor survival group) c-fos and c-jun expressed longer after I/R than normal liver and
2922
shunted fatty liver. We thought about this reason, usually IEGs are controlled by negative feedback from encoded protein or delayed genes, but severely damaged liver cannot produce the proteins or delayed genes. Thus, we suggest that expression pattern of hepatic c-fos and c-jun might be correlated with the liver damage. On the other hand, the induction of IEG has been suggested to be associated with programmed cell death, including apoptosis.5,6 Colotta et al. reported that the expression of c-fos and c-jun may represent an important early event in the activation of the genetic program of cell death.9 Smeyne et al5 reported that continuous c-fos expression precedes programmed cell death. We observed the apoptotic cells in all groups at 3 hours after I/R, but could not detect them at 48 hours after I/R without the FL group that showed protractive expression of IEGs. Continuous expression of liver IEGs may be associated with programmed cell death. However, it will be necessary to perform more experiments about in situ hybridization, immunohistochemical study, gene targeting, and so on to confirm this theory.
TSUCHIYA, ABE, SAITU ET AL
In conclusion, we suggest that fatty liver is more vulnerable than healthy liver against warm ischemia in the rat. Expression of liver IEGs might be correlated with liver damage and induction of apoptosis after I/R. REFERENCES 1. Todo S, Demetris AJ, Makowka L, et al: Transplantation 47:903, 1989 2. D’Alessandro AM, Kalayaglu M, Sollinger HW, et al: Transplantation 51:157, 1991 3. Morgan JI, Curran T: Annu Rev Neurosci 14:421, 1991 4. Goto S, Matsumoto I, Kamada N, et al: Transplantation 58:840, 1994 5. Smeyne RJ, Yendrell M, Hayward M, et al: Nature 363:166, 1993 6. Dragnunow M, Young D, Hughes P, et al: Mol Brain Res 18:347, 1993 7. Le F, Wilce PA, Cassady I, et al: Neurosci Lett 120:271, 1990 8. Teramoto K, Bowers JL, Kruskal JB, et al: Transplantation 56:1076, 1993 9. Colotta F, Polentarutti N, Sironi M, et al: J Biol Chem 267:18278, 1992