EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: We aimed to determine the biochemical and histopathologic effects of direct oxygen supply to the preservation fluid of static cold storage system with a simple method on rat livers.

Materials and Methods: Sixteen rats were randomly divided into 2 groups: the control group, which contained Ringer’s lactate as preservation fluid; and the oxygen group, which contained oxygen and Ringer’s lactate for preservation. Each liver was placed in a bag containing 50 mL Ringer’s lactate and placed in ice-filled storage containers. One hundred percent oxygen supplies were given via a simple, inexpensive system created in our laboratory, to the livers in oxygen group. We obtained samples for histopathologic evaluation in the 12th hour. In addition, 3 mL of preservation fluid was subjected to biochemical analysis at 0, sixth, and twelfth hours. Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and pH levels were measured from the preservation fluid.

Results: In oxygen-supplemented group, the acceleration speed of increase in alanine amino-transferase and lactate dehydrogenase levels at sixth hour and lactate dehydrogenase, alanine aminotransferase, and lactate dehydrogenase levels at 12th hour were statistically significantly reduced. In histopathologic examination, all parameters except ballooning were statistically significantly better in the oxygen-supplemented group.

Conclusions: This simple system for oxygenation of liver tissues during static cold storage was shown to be effective with good results in biochemical and histopathologic assessments. Because this is a simple, inexpensive, and easily available method, larger studies are warranted to evaluate its effects (especially in humans).

Key words : Liver, Preservation, Oxygen, Supply

Availability of donor organs has gained more importance with the increased demand for liver transplant and rising mortality in patients awaiting transplant.¹ Ischemia, preservation, and reperfusion injury (IPRI) affects long-term prognosis of graft viability and patient outcome because of early graft dysfunction. Ischemia, preservation, and reperfusion injury are characterized by intracellular energy depletion leading to activation of the innate immune system.²

The preservation injury is associated with the alteration of energy metabolism. In that respect, hypothermia is important to preserve organs by reducing the cellular metabolic activity about 90% to 95%, but metabolism still requires oxygen. Mitochondria take up the oxygen to synthesize adenosine triphosphate (ATP). In the absence of oxygen, ATP can only be generated through the anaerobic glycolysis and once the cellular glycogen stores are consumed, ATP depletion rapidly arises leading to irreversible cell injury and death.³ In studies, therapies targeting specific aspects of IPRI have been determined to be especially effective when applied before the onset of IPRI.^4,5 The early recovery of cellular metabolism after transplant is accomplished with the prevention of IPRI. The livers are stored in preservation solutions on melting ice at 0ºC to 4ºC, allowing a safe preservation up to 12 hours; however, a cold ischemia longer than 12 hours also is associated with a greater risk of graft dysfunction.⁶

The preservation period is an important factor determining donor organ viability. Currently, there are 2 modes of preservation methods for livers: static and dynamic. Static storage is the simple cold storage (SCS), while dynamic preservation includes hypo-thermic machine perfusion (HMP), normothermic machine perfusion, and oxygen persufflation methods. Stegemen and associates determined that viability of long preserved liver grafts can be augmented by transient hypothermic reconditioning using either machine perfusion or gaseous oxygen persufflation, both prevent initial mitochondrial dysfunction and subsequent tissue injury in their study with rat livers.⁷

It is generally believed that improving preservation techniques contributes to the improved maintenance of organ quality and minimizing ischemia-reperfusion injury; thus, resulting in more successful transplant outcomes. In the last decade, experimental studies have shown that continuous perfusion of the liver during preservation can improve graft viability and challenge the limits of the current static storage.^8,9 However, the mode of oxygen supply remains unclear. Optimization of oxygen delivery during hypothermic preservation is essential. The oxygen supplied during cold ischemia should guarantee the ATP synthesis for the residual metabolic activity, but at the same time, it may favor generation of reactive oxygen species (ROS), leading to the exacerbation of cellular injury.^10,11

It has been shown that conventional SCS tech-niques may not provide sufficient oxygen to the core of the organs.¹² Regarding this, HMP, normothermic machine perfusion, or oxygen persufflation may provide additional advantages.^13,14 Hypothermic or normothermic machine perfusion systems use a device that generally enables dual perfusion via both the hepatic artery and the portal vein in a closed circuit. Similarly, persufflation also requires specific and expensive systems that are not widely available. We aimed to determine the biochemical and histopathologic effects of direct oxygen supply to the preservation fluid of static cold storage system with an unpretentious method on the rat livers.

Materials and Methods

Study procedures
This study was conducted in Çukurova University Medical Experiments and Investigation Laboratories. Ethical approval was obtained before the study (09.2014-6/2 Çukurova University). Sixteen mature Wistar Albino male rats weighing 250 to 300 grams were used, which was in accordance with the rules of the National Institute of Health Guide for the Care and Use of Laboratory Animals. All rats were obtained from the same center, and they were housed in a temperature- and humidity-controlled room under a constant 12-hour light/dark cycle. Animals had free access to water, but their food consumption was restricted 12 hours before the surgery.

The rats were randomly divided into 2 groups, each including 8 rats: the control group (group C), which contained Ringer’s lactate (RL) as preservation fluid; in the oxygen group (group O), which contained oxygen and RL for preservation.

Surgical methods
General anesthesia was provided by 60 mg/kg of ketamine hydrochloride (Ketalar/Eczacýbasý, Ýstanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Rompun/Bayer, Leverkusen, Germany). A midline incision was made from the xiphoid process to the pelvic region after skin antisepsis with povidone iodine. The small intestines were taken from the body after the laparotomy, to identify the portal pedicle. For the continuation of intrahepatic circulation 500 IU/0.1 mL heparin (Nevparin/heparin sodium 25.000 IU/5 mL-Mustafa Nevzat, Ýstanbul, Turkey) was injected from the portal vein. The portal vein was cannulated (22 gauge) and after distal attachment pedicle, the liver was perfused with RL at 4°C, until the fluid ran clear; coming from the superior vena cava, the hepatectomy was performed thereafter. After the hepatectomy, the rats were killed using high-dose anesthetics (intracardiac ketamine, 300 mg/kg).

Each liver was placed into a bag containing 50 mL RL to be placed in ice-filled storage containers. We obtained samples for histopathologic evaluation in the twelfth hour. In addition, 3 mL of preservation fluid was subjected to biochemical analysis at the zero, sixth, and twelfth hours.

Oxygen supplementation
Two sterile silicon cannulas with a diameter of 4 mm that would be inserted from the openings of the bags containing livers preserved in ice bags were prepared. One of those cannulas carried oxygen to the preservation fluid (with a length of 40 cm from the oxygen tube to the fluid); while the other cannula maintained the way for oxygen produced in preservation fluid to go out (15 cm in length). During the experiments, the bags containing livers were all conserved in ice-filled bags to produce cold transport model. The oxygen system was inserted in the bag used in cold transport (Figure 1). The oxygen supply (100% O2) was established in a circular manner with 1 hour supply (flow rate: 2 L/min) and 15-minute interruption periods for 12 hours. The total load added to the carrying bag with this system together with 5 L oxygen tube was 2300 ± 50 grams.

Biochemical analysis
Samples obtained from the preservation fluid were taken to laboratory for subsequent measurements of aspartate aminotransferase (AST), alanine amino-transferase (ALT), lactate dehydrogenase (LDH) levels. Moreover, the pH value of this fluid also was evaluated by pH meter (ADWA pH meter) during the sixth and twelfth hours.

Pathological evaluation
Each liver sample was stored in a separate box containing 10% formaldehyde until histopathologic evaluation. Tissue samples were embedded in paraffin blocks and slices (0.4 microns) were obtained. The slices were deparaffinized in incubator at 75ºC for 45 minutes. The pathologist did not know the groups of rats. Evaluation of histopathologic injury was performed in hematoxylin-eosin–stained sections using some parameters. Sinusoidal dilatation (around central vein), acinar transformation, sinusoidal inflammation, congestion (around central vein and sinusoids), hydropic degeneration (around central vein), ballooning, and steatosis were evaluated. All parameters were assessed as 0, absent; 1, mild; 2, moderate; and 3, severe. Steatosis was evaluated as absent, 1% to 30% present, 31% to 60%, or 61% to 100% present (Figure 2).

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). Results are expressed as the mean ± standard deviation. An independent samples t test was used to analyze the biochemical and histopathologic differences between the groups. A value of P < .05 was considered to be statistically significant.

Biochemical results
The biochemical results of preservation fluid of both groups are summarized in Table 1. The acceleration speed of increase in AST and LDH levels at sixth hour, and ALT, AST, and LDH levels in the twelfth hour were statistically significantly reduced. Moreover, in the control group, increased acidity on pH values of preservation fluid at sixth and twelfth hours were statistically significant (Table 1).

Histopathologic results
In histopathologic examination performed from the biopsy specimens taken at the twelfth hour. All histologic parameters except ballooning were statistically significantly better in the oxygen-supplemented group (Table 2). In the control group, sinusoidal dilatation (around the central vein) (P = .004), acinar transformation (P = .014), sinusoidal inflammation (P = .005), congestion (both around the central vein and sinusoids) (P = .001), hydropic degeneration (around the central vein) (P = .002), and steatosis (P = .018) were worse than those of the oxygen-supplemented group.

We evaluated the effects of direct oxygen supplementation with a simple, inexpensive model to the static cold preservation fluid of rat livers and determined that direct oxygen supplementation improved the biochemical and histopathologic parameters in this animal model. To the best of our knowledge, this is the first report evaluating the effects of direct oxygen supply to the SCS.

Ischemia, preservation, and reperfusion injury are complex processes associated with both innate and adaptive immune systems. Several targeted therapies have demonstrated improvements in different steps of IPRI including inhibition of Kupffer cells, neutrophil depletion, cell surface receptor blockade, antioxidant effects, delivery of exogenous or endog-enous adenosine, and alteration of the endothelial nitric oxide balance.^5,15 In the pathophysiology of IPRI, intracellular energy depletion leading to activation of the innate immune system plays a central role.² In the absence of oxygen during preservation, ATP can only be generated through the anaerobic glycolysis. In our study, the more acidity in the preservation fluid of the control group is thought to be because of the increased lactic acid secretion from the anaerobic metabolism of liver cells as a sign of ischemia.

Although the effects of preservation injury are mainly present after reperfusion and they are difficult to determine the protective effects of liver preservation in a model without reperfusion, the prevention of IPRI is a significant aspect in the early recovery of cellular metabolism after transplant. The improvements in graft preservation represent a valuable advance in the chance of having viable donor organs. Currently, there are ways for oxygenation of the preservation fluid. The safety and feasibility of liver preservation with hypothermic machine perfusion (HMP) has been shown in liver transplanted patients. Although active oxygenation of the preservation solution could not be provided by the HMP in some studies, the PO₂ levels in the effluent perfusate remained relatively high and stable during preservation as a result of ambient air interchange at the organ chamber.¹⁶ In a recent study, Dirkes and associates reported a portable machine perfusion system for hypothermic preservation of the liver. In that study they adapted a portable, pressure-regulated, oxygenated machine perfusion system designed for kidney preservation to perfuse liver grafts via the portal vein. This study showed that continuous perfusion via the portal vein can be maintained with an oxygen-driven pump system without notable preservation damage of the organ.¹⁷ In a porcine liver transplant model, Vekemans and associates studied SCS-discarded human liver grafts to either 4 hours of HMP or an additional 4 hours of SCS. All livers were then warm reperfused to mimic ischemia-reperfusion injury ex vivo. They determined that during warm reperfusion, HMP versus SCS livers showed reduced AST and LDH release, but no morphologic difference. Further optimization of liver HMP may require different timing/duration of perfusion and/or a higher perfusion temperature.¹⁸

Another alternative of oxygenation during pre-servation is the venous systemic oxygen per-sufflation method.¹⁹ Vascular oxygen persufflation method, maintains cell and organ integrity and function, enables the repair of some damaged structures and restoration of cellular ion and signal homeostasis and most importantly, it restores the regenerative capacity of cells.²⁰

In an animal study by Minor and associates, control livers were cold stored for 10 hours at 4°C, while the livers of the treatment group were subjected additionally to hypothermic recon-ditioning (HR) by gaseous oxygen persufflation via the caval vein for 2 hours before transplant. It has been determined that HR significantly improved pretransplant energy charge and initial graft function after transplant, and molecular analyses revealed the prevention of ischemia-induced decline of cellular autophagy and mitigation of innate immune machinery as operative mechanisms among the protective effects provided by HR.²¹ Similarly, Koetting and associates also reported that hypothermic reconditioning results in a 40% to 50% reduction of serum levels of aspartate aminotransferase, lactate dehydrogenase, and tumor necrosis factor-α with a maximal effect after 2 hours of HR. They concluded that HR effectively ameliorated graft dysfunction after extended preservation of porcine livers.²² Although HMP and oxygen persufflation methods have been reportedly effective in oxygenating donor tissue, both of these are expensive and not widely available. Conversely, in none of these studies oxygen was supplied directly to the preservation fluid; instead, it was given directly to the liver, which makes our study unique.

About the oxygen supplementation in liver preservation, it should always be kept in mind that although oxygen supply is mandatory during cold preservation to maintain ATP source for the hepatocytes, it also can increase the formation of toxic ROS. Significant oxidative damage has been reported to occur after 24 hours of static or dynamic normobaric preservation, as indicated by the increased oxidized glutathione.²³ Moreover; the enhanced production of ROS during cold ischemia also has been described as a paradox.²⁴

One of the limitations of this study is the small sample size of the groups, which does not to damage more rats Secondly, we could not measure the direct oxygen content of preservation fluids that may be helpful to enlighten the difference between fluids directly. However, this also may be confusing, because in the presence of oxygen, the cells will consume the oxygen and its levels also will decrease. Moreover, we could not asses the ROS or glutathione levels as an indicator of oxidative stress. Because the main handicap with oxygen treatment during preservation period is the formation of reactive oxygen species, it would be valuable to determine the oxidant-antioxidant status in this study; nevertheless, in overall with its advantages and disadvantages, the histopathologic results of oxygen supplied group were statistically significantly better. It can be suggested that direct oxygenation of preservation fluid instead of the preserved organ may diminish the production of ROS, which makes the outcomes better. However, this hypothesis should be evaluated in future studies.

This simple system for oxygenation of liver tissues during SCS has been shown to be effective with better results in biochemical and histopathologic assessments. Because this is a simple, inexpensive, and easily available method, larger studies are warranted to evaluate its effects especially in humans.

Volume: 14 Issue: 6 December 2016

FULL TEXT