Volume 41, Issue 5, Pages 706-713 (November 2004) Isoflurane pretreatment lowers portal venous resistance by increasing hepatic heme oxygenase activity in the rat liver in vivo  Rene Schmidt, Alexander Hoetzel, Tilo Baechle, Torsten Loop, Matjaz Humar, Michael Bauer, Heike L. Pahl, Klaus K. Geiger, Benedikt H.J. Pannen  Journal of Hepatology  Volume 41, Issue 5, Pages 706-713 (November 2004) DOI: 10.1016/j.jhep.2004.07.004 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Experimental protocol. After inhalational induction of anesthesia the animals were randomized into four groups. Groups 1 and 3 received pentobarbital sodium (PEN; 40mg/kg/h) during the entire experiment. Groups 2 and 4 were pretreated with isoflurane (ISO; 3.5vol%) for 5h followed by 1h of PEN anesthesia to avoid direct hemodynamic effects of ISO at the times of data acquisition. Six hours after onset, baseline hemodynamic measurements were obtained and vehicle (0.5ml sodium bicarbonate 8.4%; groups 1 and 2) or SnPP IX (50μmol/kg; groups 3 and 4) was applied. Five and ten minutes after the intervention additional data was collected and the livers were subsequently harvested for molecular analyses. Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Northern blot analysis of hepatic HO-1 messenger RNA (A) and 18S ribosomal RNA (B) expression in two representative animals subjected to 6h of pentobarbital anesthesia (PEN; lanes 1+2) or 5h of isoflurane pretreatment (ISO; lanes 3+4) followed by 1h of PEN anesthesia. Each group received either vehicle (lanes 1–4) or SnPP IX (50μmol/kg; lanes 5–8) 10min before organ harvesting to characterize the HO dependent effects. A profound induction of HO-1 transcripts was observed in the isoflurane pretreated groups ((A), lanes 3+4: ISO+vehicle; lanes 7+8: ISO+SnPP IX). (C) Quantification of hepatic HO-1 mRNA expression by densitometric analysis for the different groups. Relative densitometric units were calculated as dividends of the background-corrected densitometric values of HO-1/18S rRNA. Data is presented as mean±SEM relative densitometric units for n=6 animals per group. *P<0.05 versus PEN+vehicle; #P<0.05 versus PEN+SnPP IX. Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 (A) Western blot analysis of hepatic HO-1 protein in isoflurane pretreated animals and pentobarbital anesthetized controls. Isoflurane pretreatment led to a strong upregulation of hepatic HO-1 protein (lanes 3+4: ISO+vehicle; lanes 7+8: ISO+SnPP IX). In contrast, HO-1 protein in controls was only barely detectable (lanes 1+2: PEN+vehicle; lanes 5+6: PEN+SnPP IX). Proteins were isolated from the two representative rat livers shown in Fig. 2. (B) Measurements of HO enzyme activity in rat liver tissue presented as median (box: 25th and 75th percentiles; error bars: 10th and 90th percentiles) for n=6 animals per group. *P<0.05 versus PEN+vehicle; #P<0.05 versus ISO+vehicle. Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Time course of HO-1 mRNA (A) and HO-1 protein (C) expression was assessed in liver samples after 0.5h (lanes 1+2), 1h (lanes 3+4), 2h (lanes 5+6), 3h (lanes 7+8), 4h (lanes 9+10) and 5h (lanes 11+12) of isoflurane anesthesia. Equal loading of RNA samples was verified by 18S rRNA labeling (B). Isoflurane led to an induction of HO-1 mRNA and protein starting after 4h. A further increase of expression could be observed after 5h of isoflurane anesthesia. Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Expression of hepatic heat shock protein (hsp)-27 mRNA (A), hsp-70 mRNA (B), or hsp-90 mRNA (C) of two representative animals subjected to pentobarbital (PEN; lanes 3+4: PEN+vehicle; lanes 7+8: PEN+SnPP IX) or isoflurane (ISO; lanes 5+6: ISO+vehicle; lanes 9+10: ISO+SnPP IX) anesthesia. The animal which served as a positive control (lane 2) was subjected to 1h of hemorrhagic shock and 5h of resuscitation. The negative control (lane 1) is represented by an animal that was sacrificed without any treatment. Equal loading was verified by 18S rRNA labeling. No differences in hsp-27, hsp-70, or hsp-90 mRNA expression could be seen between the groups. Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Effects of in vivo blockade of the HO pathway in isoflurane pretreated and pentobarbital anesthetized controls on changes in (A) portal pressure, (B) portal blood flow and (C) hepatic arterial blood flow. Post-treatment measurements were performed 5 and 10min after administration of vehicle or SnPP IX (50μmol/kg). Inhibition of the HO metabolism led to a slight increase in portal pressure at 5min after intervention followed by a decrease in portal blood flow in control animals. Administration of SnPP IX in ISO pretreated rats caused a massive increase in portal pressure associated with a drop in portal blood flow at both times. In contrast, hepatic arterial blood flow remained unchanged in all groups. Data is presented as mean±SEM percent changes from baseline values. *P<0.05 versus respective baseline value; #P<0.05 versus all other groups at the respective time; §P<0.05 versus respective data measured at 10min (n=6 animals per group; ANOVA). Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Effects of in vivo blockade of the HO pathway in isoflurane pretreated animals and pentobarbital anesthetized controls on changes in (A) portal venous resistance, (B) hepatic arterial resistance and (C) systemic vascular resistance. Post-treatment measurements were performed 5 and 10min after application of vehicle or SnPP IX (50μmol/kg). Inhibition of the HO metabolism increased portal venous resistance up to 200% of baseline values in ISO pretreated rats whereas PEN controls showed no changes after blockade of the enzyme. Hepatic arterial resistance and systemic vascular resistance were not influenced by the application of SnPP IX. Administration of vehicle had no effect in any of the groups. Data is presented as mean±SEM percent changes from baseline values. *P<0.05 versus respective baseline value; #P<0.05 versus all other groups at the respective time (n=6 animals per group; ANOVA). Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Effects of HO blockade (SnPP IX; 50μmol/kg) on changes in hepatic microvascular flux in isoflurane (ISO) pretreated rats and pentobarbital (PEN) anesthetized controls. Data was assessed before application of vehicle or SnPP IX (baseline) with additional post-treatment measurements at 5 and 10min after pharmacological intervention. Inhibition of the HO pathway led to a significant decrease of microvascular flux in ISO pretreated animals. In contrast, no differences in flux values were observed after application of SnPP IX in controls. Administration of vehicle had no effect in both groups. Data is presented as mean±SEM percent changes of baseline. *P<0.05 versus respective baseline value (n=6 animals per group; ANOVA). Journal of Hepatology 2004 41, 706-713DOI: (10.1016/j.jhep.2004.07.004) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions