| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 7, 2002 as doi:10.1096/fj.01-1034fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
University Department of Surgery and Liver Transplantation Unit, Royal Free and University College Medical School, University College London, Royal Free Hospital, London, UK
3Correspondence: Professor of Surgery and Liver Transplantation, University Department of Surgery and Liver Transplantation Unit, Royal Free and University College Medical School, Royal Free Hospital, Pond St., London NW3 2QG, UK. E-mail: b.davidson{at}rfc.ucl.ac.uk
SPECIFIC AIM
The aim of this study was to investigate the effect of ischemic preconditioning on tissue oxygenation and how it relates to nitric oxide (NO) metabolism in a rat model of lobar ischemia reperfusion (IR) injury of the liver.
PRINCIPAL FINDINGS
1. IR results in decreased tissue oxygenation and significant hepatocellular injury
To determine the extent of liver injury with ischemia and reperfusion, rats were subjected to 45 min of lobar ischemia, followed by 2 h of IR. At the end of 2 h of reperfusion, ischemia reperfusion resulted in significantly decreased oxygenated hemoglobin, increased deoxygenated hemoglobin (extracellular oxygenation), and decreased cytochrome oxidase CuA redox state (intracellular oxygenation) (Fig. 1
). IR also resulted in significantly increased plasma ALT [5173±215 vs. 414±268 (sham), P<0.001] and decreased NOx [10.4±2.3 vs. 16.9±3.7 (sham), P<0.01] (Fig. 2
).
|
|
2. Ischemic preconditioning (IPC) results in increased intracellular oxygenation and decreased hepatocellular injury
To determine the effect of preconditioning on IR injury, rats had 5 min lobar ischemia and 10 min reperfusion (IPC) before 45 min ischemia and 2 h of reperfusion. Extracellular oxygenation at the end of the reperfusion phase was not significantly different from the IR group. However, IPC produced a significant increase in intracellular oxygenation (cytochrome oxidase CuA redox state) during 45 min ischemia [1.2±0.6 vs. -12.4±5.8 (IR), P<0.05] and at the end of the reperfusion phase [9.9±7.6 vs. -14.5±9.9 (IR), P<0.05] (Fig. 1)
. At the end of the preconditioning period, plasma ALT levels were not significantly different but NOx levels were significantly increased [38.3±3.6 vs. 16.2±2.1 (sham), P<0.05]. At the end of 2 h reperfusion, plasma ALT was significantly decreased [1380±320 vs. 5173±215 (IR), P<0.05] and NOx significantly increased [93.8±4.3 vs. 10.4±2.3 (IR), P<0.05] (Fig. 2)
.
3. Nitric oxide modulates tissue oxygenation and hepatocellular injury in ischemic preconditioning
To determine the role of NO, L-arginine was administered before IR and N
-nitro-L-arginine methyl ester (L-NAME) was administered before IPC + IR. Nitric oxide stimulation with L-arginine treatment did not attenuate the impairment in hepatic oxygenation after IR (P>0.05 vs. IR) (Fig. 1)
. In contrast, inhibition of NO synthesis blocked the effect of IPC and further decreased tissue oxygenation [cytochrome oxidase CuA redox state, -23.4±2.8 vs. -14.5±9.9 (IR), P<0.05; deoxyhemoglobin, 253.9±132.7 (IR), P<0.05], suggesting an exacerbation of IRI (Fig. 1)
. Preconditioning and L-arginine treatment both increased NOx [93.8±4.3 and 84.0±4.7, respectively, vs. 10.4±2.3 (IR), both P<0.05] and decreased plasma ALT [1380±320 and 1257±290, respectively, vs. 5173±215 (IR), both P<0.05]. In contrast, L-NAME with IPC, decreased NOx [3.3±1.7 vs. 10.4±2.3 (IR), P<0.05] and increased plasma ALT [6962±317 vs. 5173±215 (IR), P<0.05]. Nitric oxide activity and hepatocellular injury correlated significantly with Hepatic oxygenation. At the end of reperfusion NOx had a positive correlation with oxyhemoglobin and cytochrome oxidase CuA redox state (r=0.9 and 0.9, respectively, P<0.001) and ALT a negative correlation with oxyhemoglobin and cytochrome oxidase CuA redox state (r=0.8 and 0.9, respectively, P<0.001).
CONCLUSIONS AND SIGNIFICANCE
Ischemic preconditioning has the potential to increase the tolerance of the liver to ischemic insults of liver surgery and transplantation. This study has investigated the relationship between hepatic tissue oxygenation and NO metabolism in IPC of the liver. The use of near-infrared spectroscopy to measure liver oxygenation has been investigated extensively and was recently applied to severity grading of IR injury.
Forty-five minutes of ischemia, followed by 2 h of IR, resulted in substantial liver injury as assessed by elevation in plasma ALT. Cellular enzymes are released into the circulation after rupture of plasma membrane due to cell injury and serum enzyme levels correlate with the degree of cell injury. IR was associated with decreased NOx. A large body of evidence indicates that NO maintains perfusion of the hepatic microcirculation and modulates liver injury through its vasodilatory and anti-inflammatory effects. IPC and NO stimulation reduced IR-induced liver injury, and both were associated with increased NOx levels. In contrast, NO inhibition not only inhibited the IPC-associated reduction in liver injury and increased NOx, but also increased ALT levels and decreased NOx, indicating an exacerbation in IR-associated liver injury. These changes in ALT and NOx in the experimental groups suggest an association of NO with liver injury in relation to IPC. Other researchers have reported an aggravation of liver injury by 90% after inhibition of NO synthesis with a nonselective NOS inhibitor in an experimental model of liver ischemia and endotoxemia.
There were changes in ischemic and postischemic (reperfusion) hepatic tissue oxygenation in all groups when compared with sham operated control values. Near-infrared spectroscopy measurement of hepatic tissue oxygenation correlates with hepatic venous blood oxygenation. With 45 min ischemia, there was a decrease in deoxygenated hemoglobin and an increase in oxygenated hemoglobin in all groups, reflecting the dissociation of oxygen from hemoglobin as oxygen is extracted by the hepatic tissue. These changes reflect reduced blood and oxygen supply to the tissue. The reduction in cytochrome oxidase CuA redox state reflects severe cellular hypoxia. On reperfusion, in the IR group the failure of recovery of hepatic extra- and intracellular oxygenation indicates persistent tissue and cellular hypoxia due to low liver blood volume and an inability to fulfill the oxygen demand with the reperfusion injury events. Other mechanisms responsible could be an increase in hepatic arterial resistance due to vasoconstriction or hypocapnia and alkalosis, all of which accompany tissue hypoxia.
After ischemic preconditioning, a significantly elevated cytochrome oxidase CuA redox state was maintained during 45 min of sustained ischemia. This implies preservation of intracellular oxygenation and is likely due to reduced energy consumption during sustained ischemia in the preconditioned livers. It is unlikely there would be any increased collateral flow to the ischemic lobes due to the peculiar anatomy of the rat liver, which is multilobed with individual blood inflow to each lobe. On reperfusion in preconditioned livers, there was a significant increase in cytochrome oxidase that persisted throughout the 2 h reperfusion period. There was no significant change in extracellular oxygenation despite recovery of recovery of blood volume during the reperfusion period in preconditioned livers. Taken together, these findings indicate a limitation or delay of hepatocellular injury as well as reduced cellular metabolism in the preconditioned livers. Studies of the myocardium have demonstrated that features contributing to the anti-infarct effect of preconditioned myocardium are a slower rate of energy metabolism, with decreased utilization of high-energy phosphates and delay of cellular ultrastructural damage.
NO regulates sinusoidal perfusion and thus may modulate liver injury. In this study, when using the amino acid substrate for NO biosynthesis L-arginine before IR, hepatic oxygenation values did not show statistically significant differences when compared with the IR group. Treatment with L-arginine did, however, increase NOx and significantly reduce plasma ALT, comparable to the IPC group. Increased NO production may have reduced hepatocellular injury by a direct cytoprotective effect. Use of NO synthesis inhibitor L-NAME with IPC, however, significantly decreased hepatic oxygenation and increased ALT levels, showing statistically significant differences when compared with the IR group. Overall, the impairment of tissue oxygenation was greater with NO inhibition than that observed with IR and would suggest it may have increased cellular sensitivity to ischemia. These data suggest that NO may be sufficient to limit the progression of liver injury; however, inhibition of NO synthesis blocks IPC and aggravates IR-induced liver injury. The significant increase in NOx levels at the end of the preconditioning period would further support that NO is important in the protective effect of IPC. Therefore, endogenous NO may influence the preconditioning effect.
Significant correlation was found between hepatic tissue oxygenation parameters measured by near-infrared spectroscopy and plasma ALT and NOx in the experimental groups. These data further emphasize that hepatic tissue oxygenation monitored by near-infrared spectroscopy is an indicator of hepatocellular injury and that NO production influences liver oxygenation and liver cell injury. The former observation may have important implications in clinical application of near-infrared spectroscopy to monitor hepatic ischemia.
In conclusion, the data in this study suggest that IPC has an important role in limiting or down-regulating IR-induced liver injury and may thereby increase ischemic tolerance of the liver. Furthermore, NO production is associated with the hepatic preconditioning effect.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1034fje; to cite this article, use FASEB J. (August 7, 2002) 10.1096/fj.01-1034fje 
2 Presented in part as poster of distinction at 8th United European Gastroenterology Week, Brussels, Belgium, November 2000 [Gut 2000, Vol. 47 (Suppl. III): A158 (abstr.)].
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
