Oxygen-Derived Free Radicals Mediate Liver Damage in Rats Subjected to Tourniquet Shock

Abstract

The placement of rubber band tourniquets upon rat hind-limbs for 5 h followed by reperfusion of the extremities results in a severe form of circulatory shock characterized by hypotension and death within 24 h of tourniquet release. Oxidative damage to muscle tissue is an early consequence of hind-limb reperfusion on tourniquet release, yet this local damage does not explain the lethal hypotensive shock state which evolves within the next 24 h. Multiple system organ failure (MSOF), of as of yet unknown causes, is usually described in relation to several shock states. It has been suggested that injured or necrotic tissue may activate neutrophils, platelets, and the coagulation system leading to embolization in remote tissues. Effective decreases in hepatic blood flow have been observed in several forms of sepsis which precedes the biochemical evidence consistent with an ischemic insult of the liver. In support of our original hypothesis, that organ failure has its genesis in a primary perfusion abnormality with secondary ischemic organ injury, herein we have assessed the possibility that oxygen-derived free radicals are generated in the liver of rats after reperfusion of their hind-limbs on release of the tourniquets. We report on the protective effects of allopurinol (ALLO) and a mixture of superoxide dismutase (SOD) catalase (CAT) and dimethylfulfoxide (DMSO) on liver free sulfhydryl content (SH), thiobarbituric acid-reactive substances (TBARS), and on the release of aspartic acid (AsT) and alanine aminotransferase (AIT) activities, and of alkaline phosphatase during a 5 h tourniquet period and after 2 h of reperfusion of the hind-limbs. During the hind-limb ischemic period hepatis tissue SH levels remained essentially constant during the first hour (6.02 ± 0.36 to 5.65 ± 0.20 μmoles/g wet tissue), and decreased significantly, over and above the normal circadian decrease of liver glutathione levels, to 4.02 ± 0.69 μmoles/g wet tissue after the third hour and remained lowered until tourniquet release. A further significant decrease (3.11 ± 0.49 μmoles/g wet tissue) was observed after 2h of reperfusion. TBARS production remained constant during the 5 h hind-limb ischemic period (168.4 ± 37.3 μmoles/g wet tissue) and rose by 55+ to 261.7 ± 55.8 μmoles/g wet tissue after 2 h of tourniquet release. ALLO, but not the SOD-CAT-DMSO combination, protected hepatic SH loss during the hind-limb ischemic insult, yet both offered protection after 2 h of tournoquet release. With regard to TBARS production, ALLO and the SOD-CAT-DMSO mixture had no effect on basal levels during the ischemic period, but both significantly reduced liver TBARS production after the two hour reperfusion period of hind limb reperfusion. Plasma AsT levels rose 8-fold from 99.4 ± 7.2 to 193 ± 17.0 U/L after the 5-hour tourniquet period, and to 844.8 ± 75.1 U/L two hours after hind-limb reperfusion. The plasma levels of AsT were significantly lower in both the ALLO and SOD-CAT-DMSO pre-treated animals. This was not the case with plasma AIT levels which increased 3-fold during the reperfusion period, but which could not be protected with these same pre-treatment protocols. Alkaline phosphatase plasma levels increased 2-fold during the same period. It is concluded that oxidative stress to the liver, as a result of himd-limb ischemia followed by reperfusion, is partly responsible for the MSOF which leads to circulatory derangements and death of rats subjected to this tourniquet shock model.

Key Words:

• Oxidative stress
• oxyradicals
• liver
• tourniquet shock
• ischemia
• reperfusion