The Role of Free Oxygen Radicals in Liver Damage Following Ischaemia and Reperfusion
Clinic of Surgery, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki
The importance of the oxygen free radicals (OFRs) has recently been recognized especially in human medicine. During the last two decades the main area of concern has been the deleterious effect of OFRs following ischemia and reperfusion in various organs. These studies have proved that OFRs are involved in more than 100 pathological processes, such as AIDS, arthritis, cardiovascular diseases and neoplasia. The presence of OFRs in blood is detected with the aid of specific instrumentation (electron paramagnetic resonance or electron spin resonance).
Any atom or molecule with an unconjugated electron in its outer layer is called free radical. Some free radicals contain oxygen and they are called oxygen free radicals. The peroxide anion, hydroperoxyl radical, hydroxyl radicals and lipid peroxyl radicals, hydrogen peroxide and solitary oxygen could potentially act as cytotoxic free radicals. Following every ischemic episode, the oxygenized blood carries substances like the ones mentioned earlier. In cases of a short period ischemia, the intra-cellular defense mechanisms are capable to neutralize the OFRs. In ischemic episodes of longer duration the defense mechanisms are exhausted and the cells are irreversibly damaged.
Although, these mechanisms have been reported in humans, it seems that similar processes are taking place in other species as well. Especially for dogs, OFRs seem to be related with ischemic episodes, autoimmune diseases, exposure to radiation, lower respiratory diseases, renal disease, multi-systemic diseases, ocular diseases, gastrointestinal diseases and integument diseases. More specifically, experimental studies have incriminated OFRs for causing more damage after reperfusion in a series of pathological processes. For example, in canine gastric dilatation-volvulus or diaphragmatic hernia, the epithelial hepatic cells could be irreversibly damaged not only due to ischemia but also to the large quantities of OFRs that are produced. In acute pancreatitis, pre-medication with OFRs scavengers prevented the destruction of that organ. Moreover, tissue ischemia was considered to be the main causative factor of post-operative adhesions, which represent vascular bridging between healthy tissue and ischemic area. It has been proven that OFRs have a detrimental effect on proteins, carbohydrates, fat acids and DNA and that they inhibit ATP production. These effects could be avoided or decreased by the act of OFRs scavengers, such as ascorbic acid (vitamin C) and mannitol.
In this study hepatic ischemia and reperfusion were created in dogs in order to evaluate: a) the hepatic injury caused by OFRs, and b) the effectiveness of ascorbic acid and mannitol in neutralizing the deleterious effects of OFRs as far as lipid peroxidation, hepatic function, hepatic structure and ultrastructure are concerned.
For that purpose 21 mongrel dogs were used. Prior to the beginning of the experiment all dogs were examined clinically and a series of biochemical values (ALP, ALT, SGPT, SGOT, LDH, total bilirubin, bile acids, glucose) were estimated. Only healthy dogs were allowed to participate in the study. In order to create hepatic ischemia, each dog was anesthetized and anesthesia was maintained with a mixture of halothane in oxygen. Through midline laparotomy the left hepatic artery was occluded temporarily with Bulldog artery forceps in order to create left lobe ischemia. The 21 dogs were separated in groups of seven. In the first group hepatic ischemia lasted for 90 minutes; in the second group for 120 minutes; in the third 10 minutes prior to hepatic artery occlusion a combination of vitamin C (25 IU/kg iv) and mannitol (0,2 mg/kg iv) was administered and ischemia lasted for 120 minutes. Just before and 10 minutes after the artery forceps were removed, samples of hepatic tissue from the left lobe were removed for histopathological examination and OFRs determination. For the latter examination the malonyldialdehyde (MDA) method was used. Blood sample collection was performed in all groups: 30 minutes before ischemia, immediately after reperfusion and every 48 hours for 14 days, for determination of ALP, ALT, AST, LDH activities and total bilirubin concentration.
For the statistical analysis of data, a Student t-test was used at 5% level of significance. As far as liver MDA concentration is concerned, there was a statistically significant increase for groups A and B. On the contrary, the difference in MDA concentrations for dogs in group C was not statistically significant. Moreover, it seems that the increase in the duration of occlusion results in a relative increase in MDA concentrations as proven from the comparison between groups A, B. As far as histopathology is concerned, tissue samples were examined with the light and the electron microscope. In our samples, as found in other studies, extensive necrosis and diffuse hemorrhage was noted. These lesions were accompanied by fatty and hydropic degeneration of hepatic cells. Most notably, the fatty degeneration presented as numeric fat vesicles of small size (microvesicular) or as a single or dual drop of fat. In the last case, the fatty drops compressed the nucleus of the epithelial cells, causing destruction of their normal appearance. On top of these findings, electron microscopic evaluation revealed: 1) lysis of mitochondrial cristae with density and local disappearance of their matrix; 2) the presence of crystalloids in the matrix of mitochondria; and 3) the presence of membranic cysts of different size into the hepatic cells. These lesions were moderate for dogs of group A, quite severe for dogs in group B but only mild or even absent for dogs in group C, which have been pre-medicated with oxy radical scavengers. Moreover, these lesions were aggravated in samples taken immediately after reperfusion and this was put down to hepatic injury due to oxygen free radical production.
Analysis of data of the biochemical profile did not give any statistically significant results.
It is obvious that the production of oxygen free radicals is closely related to the duration of ischemia. This is the reason why MDA concentration is markedly elevated in groups A and B. On the contrary, no statistically significant elevation was found for group C, where dogs have been pre-medicated with vitamin C and mannitol. It was decided that both substances should be used as they are not toxic, inexpensive and act through different mechanisms. Based on the results of this study it can be concluded that the concomitant preoperative administration of these scavengers could significantly inhibit the OFRs production. Furthermore, the hepatic lesions noted in groups A and B were similar in character and their only difference was in severity. As expected, the severity of the lesions increased in relation to the duration of ischemia. This could be attributed to both the higher MDA concentration and the prolonged influence of the OFRs to the hepatic cell. Once again, pre-medication with radical scavengers prevented the extend and the severity of the OFRs toxic effect.
Finally, it is worth noting that enzyme activity was not proven to be a reliable method of estimating hepatic damage due to OFRs activity, as their normal range and deviation is rather wide, their half-life short and false positives (increase) have been found in healthy dogs.
In conclusion, based on the results of this study, it can be assumed that OFRs production could cause a problem in numerous surgical or other procedures in dogs where hepatic ischemia is anticipated, their toxic effect on the hepatic cell is related to the duration of ischemia, changes in the hepatic cells are characterized mainly by fatty and hydropic degeneration and, more importantly, the premedication of the canine patient with vitamin C and mannitol could easily prevent that toxicity.
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