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THROMBOCYTOSIS AND BONE MARROW CELL TRANSPLANTATION REDUCE CCL INDUCED LIVER FIBROSIS IN MICE
S88
POSTERS
12111 THROMBOPOlETlNlTHROMBOCYTOSlS AND BONE MARROW CELL TRANSPLANTATION REDUCE CCL4 INDUCED LIVER FIBROSIS IN MICE M. Watanabe, S. Murata, Y . Nakano, A. Myronovych, K. Hisakura, 0. Ikeda, R. Matsuo, N. Ohkohchi. Department of Surgerji, Uniuer~xit?,of Tsnkuha, Tsnkuhu, Iliumki, Jupun E-mail: [email protected] Background and Aims: Liver cirrhosis was considered irreversible, but recently several studies have reported that it could be ameliorated and bone marrow cell (BMC) transplantation had effect of improving fibrosis. We found the strong capability of platelet for promotion of liver regeneration. The aim of this study is to investigate the effect of thrombopoietin (TPO), thrombocytosis, and BMC transplantation, on suppression of fibrous change of liver. Materials and Methods: C57BL6 female mice were used. Iml/kg of carbon tetrachloride (CC14) by intraperitoneal injection was administered to mice twice a week to make liver fibrosis for 8 weeks. To estimate the effect of trombocytosis and BMC transplantation on liver fibrosis, mice were divided into four groups as follows; (i) control group, saline were administered for 8 weeks, (ii) CCl4 group, CCl4 was administered for 8 weeks, (iii) thrombocytosis group, TPO (PEG-rHuMGDF, Kirin, Japan) were administered by intraperitoneal injection once a week from 5 to 8 week in addition to CCl4 administration, (iv) BMC transplantation group, 1 x 106 of BMC from limbs of GFP mouse were administered by retroorbital venous injection at 5 week in addition to CC14 administration. Examination items: (i) platelet counts of peripheral blood, (ii) liver fibrosis calculated by Sirius red staining, (iii) hydroxyproline contents of liver tissue, (iv) expressions of TGF-beta, MMP-9 and TTMP-I that were analysed by semi quantitative RT-PCR. Results: The number of platelets in thrombocytosis group was higher than the other groups. Liver fibrosis in thrombocytosis and BMC transplantation groups were decreased significantly compared to CCl4 group (p < 0.01, p < 0.05). Hydroxyproline contents were decreased in thrombocytosis and BMC transplantation groups comparing with CCl4 group (p i 0.001, p 0.01). Expression of TGF-beta in thrombocytosis group was decreased rather than other groups, and expressions of MMP-9 in thrombocytosis and BMC transplantation group were higher than CCl4 group. Conclusions: Our results suggest that TPO/thrombocytosis and BMC transplantation suppress the chronic liver fibrosis.
12121 BRAIN SWELLING AND COMA IN CIRRHOTIC RATS ADMINISTERED ENDOTOXIN IS ASSOCIATED WITH INCREASED PROTEIN NITROSATION G. Wright’, N.A. Davies’, D.L. Shawcross’, S.J. Hodges’, C. Zwingmann’, A. Mani’, K.P. Moore’, D. Harry’, V Stadlbauer’, D.C. Davies3, H.F. Brooks3, Z. Z0u4, R. Williams’, R. Jalan’. ’Liver Fuilure Group, the UCL Institute of Hepatology, Division of Medicine, Uniuersih~College London, UK; ’Center of Rexearch, Department of Medicine, linioevsiiy of Montreal, Quebec, Canada; ”Dioision of’ Busic Medicul Sciences, St George :x Uniuersity of London, UK; 4Beijing 302 Hospital of PLA, Beijing Institute oflnf+ctious Diseases, Beijing, China E-mail: [email protected] Background: In patients, inflammation is known to decompensate cirrhosis, resulting in edema and coma; yet the underlying mechanisms are unknown. Recent evidence has suggested a possible association with systemic proinflammatory cytolcine release and effects on protein tyrosine nitrosation (PTN) and cytolcine actions within the brain. Aims: To explore the hypothesis that endotoxemia induces brain edema in cirrhotic rats and determine whether it is associated with hyperammonemia, proinflammatory cytokine milieu and/or PTN. Methods: Adult Sprague-Dawley rats 4 weeks following bile-duct ligation (BDL; n = 14) or Sham operation ( n = 14), and Naive rats fed a 1 week
hyperammonemic diet (HD; n = 14), were injected with Lipopolysaccharide (LPS: 0.5mg/kg, 1.P) or saline, and sacrificed 3 hours later. Plasma biochemistry, ammonia, cytokine and nitriteinitrate concentrations as well as brain water content, and nitrotyrosine were measured. Results: Plasma and brain ammonia levels were higher in HD and BDL rats than Sham-operated controls, and did not change significantly with administration of LPS in any group; routine biochemistry between groups were similar. LPS administration increased frontal cortex brain water in HD, BDL and Sham-operated groups (p=0.012, p=0.004 and p=0.038 respectively). Interestingly, coma only occurred in BDL rats administered LPS. Plasma Nitritehitrate (NOx) and Nitrite levels increased significantly in LPS-treated groups compared to the non-LPS treated controls (p 0.05, respectively), but frontal cortex Nitrotyrosine levels only increased in the cirrhotic rats injected with LPS compared to all other groups (p 0.05: ANOVA, figure 1).In BDL rats compared to Sham-operated rats the levels of plasma TL-6 and TNF-a (1.8 and 1.4 fold respectively) and brain TL-6 and TNF-a ( I .6 and I .5 fold respectively) were elevated and augmented by LPS (22-38 fold in plasma and 1-5 fold in brain). Conclusion: Injection of LPS into cirrhotic rats causes worsening edema and coma, due to the synergistic effect of hyperammonemia and endotoxaemia, possibly augmenting a ‘primed’ inflammatory state in cirrhotic rats. This phenomenon is associated with a proinflammatory state, and nitration of brain proteins which appears not to be directly correlated to NO production and therefore likely to be due to reactive oxygen intermediates previously demonstrated to be increased in LPS-administered BDL rats.
12131 LIPOPOLYSACCHARIDE EXACERBATES BRAIN EDEMA AND PRODUCES COMA IN CIRRHOTIC RATS THROUGH A CYTOTOXIC MECHANISM: AN ELECTRON-MICROSCOPIC STUDY G. Wright’, N.A. Davies’, D.C. Davies’, H.F. Brooks’, D.L. Shawcross’, S.J. Hodges’ , R. Jalan’ . ‘Liver Fuilure Group, the UCL Institute of Heputology, Division of Medicine, University College London, UK; 2Division of Basic Medical Sciences, St George’s University of London, UK E-mail: gavin.wright~ucl.ac.uk Background: Inflammation worsens brain edema and predisposes to coma in patients with cirrhosis; but the mechanisms related to the development of brain edema remain unclear. Aims: To explore the hypothesis that endotoxemia induces brain edema in cirrhotic rats and determine whether it is associated with anatomical disturbance of the blood-brain barrier. Methods: Adult Sprague-Dawley rats 4 weeks after bile-duct ligation (BDL; n = 8 ) or Sham operation (n=8), and Naive rats fed a 1 week hyperammonemic diet (HD; n = X), were injected with Lipopolysaccharide (LPS: 0.5mg/kg, 1.P) or saline, and sacrificed 3 hours later. Plasma biochemistry and ammonia was measured. During sacrifice, the rats were perfused (100-1 10 mmHg) with a heparinised saline rinse before administering gluteraldehyde/paraformaldehyde fixative containing 2% Lanthanum nitrate (a low molecular weight (MW 139.8) ionic tracer) to evaluate the barrier integrity. The brains were extracted and processed into modified Spur’s resin before morphology was examined (Hitachi 7100 transmission electron microscope at 75kV; using a Gatan column mounted CCD camera, resolution 1024 x 1024: scale-bar, 1 pm). Results: BDL was associated with biochemical evidence of cirrhosis. Plasma ammonia levels were higher in HD and BDL rats than Shamoperated controls, and did not change significantly with LPS administration in any group; routine biochemistry between groups were similar. Interestingly, coma only occurred in BDL rats administered LPS. Histology is shown in Figure. I a-f. Sham-operated rats showed normal microvessel architecture. BDL rats showed markedly collapsed microvessels with moderate perimicrovessel edema worsened by administration of LPS. HD rats showed reasonably retained microvessel architecture but, gross
POSTERS
12111 THROMBOPOlETlNlTHROMBOCYTOSlS AND BONE MARROW CELL TRANSPLANTATION REDUCE CCL4 INDUCED LIVER FIBROSIS IN MICE M. Watanabe, S. Murata, Y . Nakano, A. Myronovych, K. Hisakura, 0. Ikeda, R. Matsuo, N. Ohkohchi. Department of Surgerji, Uniuer~xit?,of Tsnkuha, Tsnkuhu, Iliumki, Jupun E-mail: [email protected] Background and Aims: Liver cirrhosis was considered irreversible, but recently several studies have reported that it could be ameliorated and bone marrow cell (BMC) transplantation had effect of improving fibrosis. We found the strong capability of platelet for promotion of liver regeneration. The aim of this study is to investigate the effect of thrombopoietin (TPO), thrombocytosis, and BMC transplantation, on suppression of fibrous change of liver. Materials and Methods: C57BL6 female mice were used. Iml/kg of carbon tetrachloride (CC14) by intraperitoneal injection was administered to mice twice a week to make liver fibrosis for 8 weeks. To estimate the effect of trombocytosis and BMC transplantation on liver fibrosis, mice were divided into four groups as follows; (i) control group, saline were administered for 8 weeks, (ii) CCl4 group, CCl4 was administered for 8 weeks, (iii) thrombocytosis group, TPO (PEG-rHuMGDF, Kirin, Japan) were administered by intraperitoneal injection once a week from 5 to 8 week in addition to CCl4 administration, (iv) BMC transplantation group, 1 x 106 of BMC from limbs of GFP mouse were administered by retroorbital venous injection at 5 week in addition to CC14 administration. Examination items: (i) platelet counts of peripheral blood, (ii) liver fibrosis calculated by Sirius red staining, (iii) hydroxyproline contents of liver tissue, (iv) expressions of TGF-beta, MMP-9 and TTMP-I that were analysed by semi quantitative RT-PCR. Results: The number of platelets in thrombocytosis group was higher than the other groups. Liver fibrosis in thrombocytosis and BMC transplantation groups were decreased significantly compared to CCl4 group (p < 0.01, p < 0.05). Hydroxyproline contents were decreased in thrombocytosis and BMC transplantation groups comparing with CCl4 group (p i 0.001, p 0.01). Expression of TGF-beta in thrombocytosis group was decreased rather than other groups, and expressions of MMP-9 in thrombocytosis and BMC transplantation group were higher than CCl4 group. Conclusions: Our results suggest that TPO/thrombocytosis and BMC transplantation suppress the chronic liver fibrosis.
12121 BRAIN SWELLING AND COMA IN CIRRHOTIC RATS ADMINISTERED ENDOTOXIN IS ASSOCIATED WITH INCREASED PROTEIN NITROSATION G. Wright’, N.A. Davies’, D.L. Shawcross’, S.J. Hodges’, C. Zwingmann’, A. Mani’, K.P. Moore’, D. Harry’, V Stadlbauer’, D.C. Davies3, H.F. Brooks3, Z. Z0u4, R. Williams’, R. Jalan’. ’Liver Fuilure Group, the UCL Institute of Hepatology, Division of Medicine, Uniuersih~College London, UK; ’Center of Rexearch, Department of Medicine, linioevsiiy of Montreal, Quebec, Canada; ”Dioision of’ Busic Medicul Sciences, St George :x Uniuersity of London, UK; 4Beijing 302 Hospital of PLA, Beijing Institute oflnf+ctious Diseases, Beijing, China E-mail: [email protected] Background: In patients, inflammation is known to decompensate cirrhosis, resulting in edema and coma; yet the underlying mechanisms are unknown. Recent evidence has suggested a possible association with systemic proinflammatory cytolcine release and effects on protein tyrosine nitrosation (PTN) and cytolcine actions within the brain. Aims: To explore the hypothesis that endotoxemia induces brain edema in cirrhotic rats and determine whether it is associated with hyperammonemia, proinflammatory cytokine milieu and/or PTN. Methods: Adult Sprague-Dawley rats 4 weeks following bile-duct ligation (BDL; n = 14) or Sham operation ( n = 14), and Naive rats fed a 1 week
hyperammonemic diet (HD; n = 14), were injected with Lipopolysaccharide (LPS: 0.5mg/kg, 1.P) or saline, and sacrificed 3 hours later. Plasma biochemistry, ammonia, cytokine and nitriteinitrate concentrations as well as brain water content, and nitrotyrosine were measured. Results: Plasma and brain ammonia levels were higher in HD and BDL rats than Sham-operated controls, and did not change significantly with administration of LPS in any group; routine biochemistry between groups were similar. LPS administration increased frontal cortex brain water in HD, BDL and Sham-operated groups (p=0.012, p=0.004 and p=0.038 respectively). Interestingly, coma only occurred in BDL rats administered LPS. Plasma Nitritehitrate (NOx) and Nitrite levels increased significantly in LPS-treated groups compared to the non-LPS treated controls (p 0.05, respectively), but frontal cortex Nitrotyrosine levels only increased in the cirrhotic rats injected with LPS compared to all other groups (p 0.05: ANOVA, figure 1).In BDL rats compared to Sham-operated rats the levels of plasma TL-6 and TNF-a (1.8 and 1.4 fold respectively) and brain TL-6 and TNF-a ( I .6 and I .5 fold respectively) were elevated and augmented by LPS (22-38 fold in plasma and 1-5 fold in brain). Conclusion: Injection of LPS into cirrhotic rats causes worsening edema and coma, due to the synergistic effect of hyperammonemia and endotoxaemia, possibly augmenting a ‘primed’ inflammatory state in cirrhotic rats. This phenomenon is associated with a proinflammatory state, and nitration of brain proteins which appears not to be directly correlated to NO production and therefore likely to be due to reactive oxygen intermediates previously demonstrated to be increased in LPS-administered BDL rats.
12131 LIPOPOLYSACCHARIDE EXACERBATES BRAIN EDEMA AND PRODUCES COMA IN CIRRHOTIC RATS THROUGH A CYTOTOXIC MECHANISM: AN ELECTRON-MICROSCOPIC STUDY G. Wright’, N.A. Davies’, D.C. Davies’, H.F. Brooks’, D.L. Shawcross’, S.J. Hodges’ , R. Jalan’ . ‘Liver Fuilure Group, the UCL Institute of Heputology, Division of Medicine, University College London, UK; 2Division of Basic Medical Sciences, St George’s University of London, UK E-mail: gavin.wright~ucl.ac.uk Background: Inflammation worsens brain edema and predisposes to coma in patients with cirrhosis; but the mechanisms related to the development of brain edema remain unclear. Aims: To explore the hypothesis that endotoxemia induces brain edema in cirrhotic rats and determine whether it is associated with anatomical disturbance of the blood-brain barrier. Methods: Adult Sprague-Dawley rats 4 weeks after bile-duct ligation (BDL; n = 8 ) or Sham operation (n=8), and Naive rats fed a 1 week hyperammonemic diet (HD; n = X), were injected with Lipopolysaccharide (LPS: 0.5mg/kg, 1.P) or saline, and sacrificed 3 hours later. Plasma biochemistry and ammonia was measured. During sacrifice, the rats were perfused (100-1 10 mmHg) with a heparinised saline rinse before administering gluteraldehyde/paraformaldehyde fixative containing 2% Lanthanum nitrate (a low molecular weight (MW 139.8) ionic tracer) to evaluate the barrier integrity. The brains were extracted and processed into modified Spur’s resin before morphology was examined (Hitachi 7100 transmission electron microscope at 75kV; using a Gatan column mounted CCD camera, resolution 1024 x 1024: scale-bar, 1 pm). Results: BDL was associated with biochemical evidence of cirrhosis. Plasma ammonia levels were higher in HD and BDL rats than Shamoperated controls, and did not change significantly with LPS administration in any group; routine biochemistry between groups were similar. Interestingly, coma only occurred in BDL rats administered LPS. Histology is shown in Figure. I a-f. Sham-operated rats showed normal microvessel architecture. BDL rats showed markedly collapsed microvessels with moderate perimicrovessel edema worsened by administration of LPS. HD rats showed reasonably retained microvessel architecture but, gross