Objectives: Ischemia is defined as the inability of the tissue to provide oxygen and other metabolites by the circulation and the removal of residual products. The University of Wisconsin solution is widely used to preserve ischemia and to preserve organs for transplant. Ozone is used in various areas of ischemia damage due to its antioxidant properties. The aim of our study was to investigate the effects of ozone added to University of Wisconsin solution on perfused liver preservation injury.
Materials and Methods: Our study included 24 Sprague Dawley rats with an average weight of 300 to 350 g. Animals were divided into 4 groups: group 1 (Ringer lactate), group 2 (Ringer lactate + ozone), group 3 (University of Wisconsin solution), and group 4 (University of Wisconsin + ozone). Solutions were perfused from the liver portal vein and aorta. After perfusion, rats were killed and liver biopsies were taken at 0, 6, and 12 hours of storage for pathological examination. For biochemical analysis, samples were collected from liver specimen storage solutions at 0, 6, and 12 hours.
Results: Mean alanine aminotransferase/aspartate aminotransferase levels in group 3 were 77/82 U/L at hour 0, 680/461 U/L at hour 6, and 1027/682 U/L at hour 12. In group 4, these levels were 35/31 U/L at hour 0, 415/295 U/L at hour 6, and 546/372 U/L at hour 12.
Conclusions: In terms of liver function values, we observed favorable result with University of Wisconsin solution with added ozone. Therefore, we suggest that the addition of ozone to the University of Wisconsin solution may be effective in preventing liver preservation damage.
Key words : Ischemia, Liver transplantation, Organ preservation
Organ preservation covers the period from the decision to take the tissue to be transplanted until it is transplanted to the recipient and its function begins. The aim is to preserve the functions and cell integrity of the organ taken from the donor until it is transplanted.1
Ischemia is defined as the inability of the tissue to meet its need for oxygen and other metabolites by the circulation and the inability to remove the formed residual products by the circulation.2 Reperfusion is the restoration of blood circulation in the ischemic tissue. Although reperfusion of an ischemic tissue meets the oxygen and other metabolic needs of the tissue, it paradoxically causes damage to the tissue.3 Hypothermia is the most important factor in slowing cell metabolism. Although hypothermia is necessary for organ preservation in transplantation, it cannot prevent negative events, such as oxidative stress, sodium pump inactivation, intracellular calcium accumulation, and iron ion release that occur during reperfusion. For this reason, there is a continued need to develop an ideal solution to prevent reperfusion damage.
The University of Wisconsin (UW) solution is a preservative solution containing glutathione and allopurinol as free radical inhibitors and xanthine oxidase inhibitors.4-6 It is primarily preferred in liver preservation and provides protection for up to 24 hours. Ozone, which has antioxidant effects, has been reported to reduce organ stress.5
The main reason for primary nonfunction after liver transplant is insufficient cold storage and the lack of a perfect preservation solution. For this reason, trying to develop new preservation solutions has been a continuing goal. With UW solution being a primary preferred preservation solution in liver transplant, we aimed to determine whether the liver could be more effectively protected with the addition of ozone, which has been shown to have positive effects in hepatic ischemia, to UW solution.
Materials and Methods
This study was approved by the Baskent University Animal Experiments Ethics Committee (project no. DA 19/08). Our study included 24 adult male Sprague Dawley rats with an average weight of 300 to 350 g. Rats were divided into 4 groups: group 1 (Ringer lactate only), group 2 (Ringer lactate + ozone), group 3, (UW solution only), and group 4 (UW solution + ozone). Solutions were perfused from the liver portal vein and aorta.
During the surgical procedure, rats were anesthetized by intraperitoneal administration of 50 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride under aseptic conditions. After anesthesia was provided, an incision area was shaved, skin antisepsis was provided with Povidine iodine, and rats were fixed in a 30-degree inclined surgical apparatus. After sterile draping, laparotomy was performed with a midline incision, the small intestines were removed, and the portal pedicle was defined. After portal vein cannulation, the distal portion was ligated and the suprahepatic vena cava was cut, after which perfusion was started (Figure 1). Rats were killed after perfusion, and standard hepatectomy was performed. Specimens from each of the 4 groups (Ringer lactate, Ringer lactate + ozone, UW solution, and UW solution + ozone) were stored in their solutions at -4 °C. Liver biopsies were taken at 6 hours for histological evaluation; at 0, 6, and 12 hours for pathological examination; and at 0, 6, and 12 hours for biochemical analysis. Samples were taken from the solutions in which the liver specimens were stored.
Evaluation of hepatocyte damage by hematoxylin-eosin staining
With regard to ballooning, 0 represented no damage, 1 represented mild damage, 2 represented moderate damage, and 3 represented severe damage. We evaluated fatty livers semi-quantitatively, dividing levels as 1% to 30%, 31% to 60%, and 61% to 100%. Pericentral hydropic change was graded as absent, focal, or diffuse, and sinusoidal dilatation was graded as absent, focal, or diffuse.
Samples were taken after 0, 6, and 12 hours of storage at -22 °C from the preservation solutions where liver specimens were stored. Alanine aminotransferase (ALT) was analyzed by ultraviolet test adapted to a standardized method, and aspartate aminotransferase (AST) was analyzed by the colorimetric method. Values are expressed in units per liter (U/L).
We used the SPSS version 24 package program for statistical analyses. Statistical power analysis was not performed because similar models of ischemia-reperfusion have been used previously. We determined that the number of animals would be sufficient for the statistical evaluation of our study results. We used t test, chi-square test, and analysis of variance multiple comparison analysis for comparisons between groups. P < .05 was considered to be statistically significant.
Mean ALT/AST levels in group 1 (Ringer lactate) were 118/136 U/L at hour 0, 986/1024 U/L at hour 6, and 1125/1205 at hour 12. In group 2 (Ringer lactate + ozone), mean ALT/AST levels were 105/112 U/L at hour 0, 856/929 U/L at hour 6, and 1050/1102 U/L at hour 12. In group 3 (UW solution only), ALT/AST levels were 77/82 U/L at hour 0, 680/461 U/L at hour 6, and 1027/682 U/L at hour 12. Finally, in group 4 (UW solution + ozone), ALT/AST levels were 35/31 U/L at hour 0, 415/295 U/L at hour 6, and 546/372 U/L at hour 12 (Table 1). In group 3 and group 4, the ALT/AST levels were similar, but these levels were lower than shown in group 1 and group 2. Statistical analyses showed similar results at each time period.
Liver biopsy results showed similar ischemic damage in both groups. Both groups had similar ballooning damage, fatty changes, pericentral hydropic changes, and sinusoidal dilatation results, with no statistical differences.
Structural changes occur in the mitochondria, nucleus, endoplasmic reticulum, lysosome, and cytoplasmic membrane of cells as a result of ischemia damage. Ischemia disrupts oxidative phosphorylation, leading to a decrease in intracellular adenosine triphosphate synthesis. As a result, there is accumulation of calcium, sodium, and water in the cell. Although it is difficult to determine whether these changes are reversible, it is known that damage to the mitochondria and cell membrane is irreversible.
If metabolism can be slowed in the ischemic process that develops through interruption of organ blood supply, cell damage development will also slow in direct proportion. The main factor in slowing cell metabolism is hypothermia. Metabolism decreases by a factor of 1.5 to 2.5 times with every 10 °C decrease in organ temperature, therefore slowing approximately 10 times from 37 °C to 0 °C.4,6 Although hypothermia is necessary for organ preservation in transplant procedures, it cannot prevent harmful events such as oxidative stress, sodium pump inactivation, intracellular calcium accumulation, and iron ion release that occur during reperfusion. For this reason, the need to develop an ideal protection solution to prevent reperfusion damage continues.
After reperfusion in liver transplant, the reperfu-sion process is dependent on the activation of inactive leukocytes and platelets to adhere to the endothelium and the release of inflammatory mediators. Experimental studies have shown that Kupffer cells are activated during extended clamping of the portal vein, secreting tumor necrosis factor-α and interleukin 1 and creating a systemic shock-like result.5,7
During the ischemic period, the amount of adenosine, xanthine, and hypoxanthine increases in cells of the organ. Xanthine oxidase enzyme, on the other hand, activates during the cold preservation period, causing a combination of xanthine and hypoxanthine with oxygen, thereby eliminating the formation of free oxygen radicals.7,8 The most important of these are superoxide, hydrogen peroxide, hydroxyl ions, and nitric oxide. These radicals are important factors in the pathogenesis of ischemia-reperfusion injury.8,9
The UW solution is a preservative solution containing glutathione and allopurinol as free radical inhibitors and xanthine oxidase inhibitors.4,6 It contains adenosine for ATP requirement, hydroxy-ethyl-starch to prevent interstitial edema, and phosphate ion for pH stabilization. Raffinose instead of glucose and sodium lactobionate can also be added. It is primarily preferred in liver preservation and provides protection for up to 24 hours.
The use of ozone, which has antioxidant effects, has been reported to reduce organ stress.5,9 Ozone therapy has been shown to have protective properties in experimental liver ischemia models.9 It has been shown that ozone regulates ATP levels as a result of its oxidative effect and may be protective against ischemia-reperfusion injury.10 Therefore, it is thought that ozone therapy contributes positively to ATP levels and adenosine, xanthine, and hypoxanthine metabolism in hepatic ischemia.11
In our study, in the animal group with UW solution plus ozone, ALT/AST levels measured from liver tissues perfused with this combined solution were lower than shown in the other 3 groups. However, this difference was not statistically significant. The fact that these measurements were taken from the solution where liver tissue was preserved may be an important limiting step of our study. In addition, showing differences between groups by immunohistochemical evaluations could be useful to demonstrate the effectiveness of the UW solution plus ozone combination.
For patients with chronic liver disease, the wait period for a liver donor has been increasing worldwide, and there are not enough donors to meet the number of patients waiting on the deceased organ donor wait list. Thus, providing the most ideal conditions for the preservation of donated organs remains crucial. In our study, we aimed to contribute to the finding of an ideal preservation solution in order to minimize the cold ischemic damage in a donated organ and to allow preservation for as long as possible. We believe that the UW solution plus ozone combination could be used for this purpose and could have a significant positive effect on organ preservation.
DOI : 10.6002/ect.2021.0453
From the 1Department of General Surgery, Medical School, Baskent University, Ankara, Turkey; and the 2Department of General Surgery, Baskent University, Adana Training and Research Center, Adana, Turkey
Acknowledgements: This study was supported by Baskent University Research Fund. The authors have no declarations of potential conflicts of interest. This manuscript was originally presented as part of the International Symposium on Benign and Malignant Tumors in Liver With or Without Cirrhosis held in Ankara, Turkey on June 24 and 25, 2021.
Corresponding author: Hüseyin Onur Aydın, Başkent University Faculty of Medicine, Department of General Surgery, Ankara, Turkey
Phone: +90 312 2036868
Figure 1. Perfusion Via Aorta and Portal Vein
Table 1. Biochemical Analyses of Preservation Solutions