1. Volume 44, Issue 5, Pages 894-901 (May 2006)
Role of pH in protection by low sodium against hypoxic injury in isolated perfused rat livers 
Mariapia Vairetti, Plinio Richelmi, Francantonio Bertè, Robert T. Currin, John J. Lemasters, Roberto Imberti 
Journal of Hepatology 
Volume 44, Issue 5, Pages (May 2006)
DOI: /j.jhep
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Protection of isolated rat livers by low-Na+ KHB during hypoxic perfusion. In normoxic control experiments, rat livers were perfused with KHB (●) or low-Na+ KHB (▪) equilibrated with 95% O2 and 5% CO2. During hypoxia, livers were perfused with KHB (○) or with low-Na+ KHB (▪) equilibrated with 95% N2 and 5% CO2. At 10-min intervals, LDH activity of the effluent perfusate was determined. During hypoxia, LDH release was higher with KHB than with low-Na+ KHB (P< 0.05, 4–6 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Accelerated injury after replacement of low-Na+ KHB with KHB during hypoxic perfusion. Isolated livers were perfused with KHB (○) or low-Na+ KHB (□) equilibrated with 95% N2 and 5% CO2. After 40min (arrow), low-Na+ KHB was replaced with KHB (σ). At 10-min intervals, LDH activity of the effluent perfusate was determined (4–6 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Effects of hypoxic liver perfusion on bile flow. Rat livers were perfused with the KHB (●,○) or low-Na+ KHB (▪,□), and bile was collected at 10-min intervals. During normoxia (●,▪), bile production (area under the curve) was higher with KHB than low-Na+ KHB, whereas after longer than 20min of hypoxia (○,□), bile production was greater in low-Na+ KHB (P<0.05, 4–6 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Effects of hypoxic perfusion on liver tissue ATP. Rat livers were perfused with low-Na+ KHB (black bars) and KHB (grey bars) and tissue ATP measured at indicated time points (*P<0.05, 4–5 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Effects of mannitol and sucrose on LDH release during hypoxic liver perfusion. Isolated livers were perfused with KHB (○), with KHB containing 150mM mannitol (▿) or with KHB containing 150 mM sucrose (▾). At 10-min intervals, LDH activity of the effluent perfusate was determined. LDH release (area under the curve) was lower during KHB plus mannitol than KHB plus sucrose or KHB alone (P<0.05, 4–6 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Liver weight after hypoxic liver perfusion. Livers were weighed at the end of perfusions and compared with their initial weight estimated from body weight, as described in Section 2. *P<0.05 versus KHB perfusion (4–6 perfusions per group). N.S.=non-significant.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

8. Fig. 7
Reversal by monensin of protection by low-Na+ KHB during hypoxic perfusion of isolated rat livers. Livers were perfused under hypoxic conditions with KHB for 120min (○), low-Na+ KHB at pH 7.4 for 120min (□), low-Na+ KHB at pH 7.4 with the addition of monensin (0.2μM, arrow) after 40min (♢), or low-Na+ KHB at pH 6.8 with the addition of monensin (0.2μM, arrow) after 40min (♦). At 10-min intervals, LDH activity of the effluent perfusate was determined. LDH release (area under the curve) was significantly higher with low-Na+ KHB plus monensin at pH 7.4 than with low-Na+ KHB plus monensin at pH 6.8 (P<0.01, 4–6 perfusions per group).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

9. Fig. 8
Effects of monensin on intracellular pH during hypoxic liver perfusion with low-Na+ KHB at normal and acidotic pH. Isolated livers were first perfused for 20min with normoxic KHB at pH 7.4. At time 0, the perfusate was changed to hypoxic low-Na+ KHB at pH 7.4 (●) or 6.8 (○). Intracellular pH was measured by confocal ratio imaging of BCECF fluorescence, as described in Section 2. Monensin (5μM) was added to the perfusate after 40min. Before monensin, intracellular pH (area under the curve) was greater at pH 7.4 than pH 6.8 (P<0.05). After monensin, intracellular pH was greater at an extracellular of pH 7.4 than pH 6.8 (P<0.01).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

10. Fig. 9
Trypan blue uptake after perfusion with KHB and low-Na+ KHB. After 40min of perfusion, livers were infused with trypan blue, fixed and prepared for histology, as described in Section 2. No trypan blue uptake occurred after aerobic perfusion with standard KHB (A) or low-Na+ KHB (B). After 40min of hypoxic perfusion with standard KHB, both parenchymal and non-parenchymal cells took up trypan blue (C). Perfusion with low-Na+ KHB protected parenchymal cells against hypoxia, but did not spare non-parenchymal cells (D). Eosin, magnification ×400.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

11. Fig. 10
Proposed mechanism of prevention of cell death by low-Na+ KHB during hypoxic liver injury. Low-Na+ KHB inhibits Na/H+ exchange and Na+/HCO3- co-transport, thus preventing intracellular alkalinization and pH-dependent cell death. Arrows labeled “-”indicate inhibitory effects.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions