Key words : Cytokines, Liver transplantation, Static cold storage

Introduction

The liver is the most frequently transplanted solid organ worldwide after kidneys. The important advantages of transplant from living donors are that living donor transplant allows the recipient’s general health status to be stabilized, functional allograft can be obtained, and there is no need for long-term organ preservation before transplant.¹ The Global Observatory on Donation and Transplantation estima-ted that 37 436 liver transplants were performed worldwide in 2022 and that approximately 24% of these were from living donors.² Although allografts from deceased donors are more susceptible to injuries and have lower success rates of transplant than living donors, deceased donors continue to constitute a large part of the donor pool as a result of organ shortages and the increasing number of patients who need organ transplant.

Each tissue that is procured from the living body to an ex vivo environment may be subjected to ischemia. Hypoxia, which develops after cessation of blood flow, triggers the cell death characterized by apoptosis or necrosis, possibly causing ATP depletion, release of various proinflammatory cytokines, and activation of proteolytic cascades.^3-6 The severity of these changes is directly related to allograft function posttransplant.^4,7 This is especially important for organs donated from deceased donors, who are often far from the recipient,^8,9 because death causes a cytokine storm that results in inflammation, leukocyte infiltration, complement system activation, and oxidative stress.¹⁰

Static cold storage (SCS) is an organ preservation method used for all organs and involves cooling to 4 ºC and the use of organ preservation solutions. The time that an organ spends in SCS until it is procured from the donor, perfused with a preservative solution, and transplanted to the recipient is named “cold ischemia time.”^3,4 Cooling slows down the deve-lopment of the above-mentioned changes by reducing the metabolism rate,¹¹ but metabolism does not completely stop⁴ and ATP production is still required to maintain basal metabolism. Although cold ischemia time may vary according to the existing risk factors, the general recommendation is for cold ischemia time of the liver in SCS to not exceed 12 hours to preserve liver allograft function.^3,11 Otherwise, prolonged SCS may result in the discard of a large proportion of donor organs, where the supply is already low. Therefore, new strategies are needed to save time in the perioperative period by reducing cold ischemia injury.

Hyperbaric oxygen therapy (HBO2T) is admi-nistered in hypoxia-related pathologic disorders.¹² It is known that HBO₂T has antioxidant, antiapoptotic, and immunomodulatory effects. In an organ preserved in SCS after procurement, HBO₂T has the potential to support ATP production by providing the oxygen required for oxidative phosphorylation¹³ and to slow the transition to anaerobic metabolism associated with ischemia.¹⁴ Increased evidence shows that HBO₂T attenuates ischemia-reperfusion injury by modulating both the humoral and cellular immune response.¹⁵ However, the effects of HBO₂T on inflammatory mediators in terms of preserving allograft viability and increasing the duration of cold ischemia remain unclear.

In this study, we aimed to investigate the effects of HBO₂T administered to rat livers for 60 and 120 minutes during SCS on the cytokine-mediated immune response, allograft viability, and histopatho-logic changes and to evaluate the potential of HBO₂T to prolong cold ischemia time.

Materials and Methods

The study protocol was approved by the Çanakkale Onsekiz Mart University Animal Studies Local Ethics Committee (approval No. 2019-1900068039) and performed according to the Guide for the Care and Use of Laboratory Animals approved by the National Research Council Committee.¹⁶

Animals
Female Wistar rats (n = 24; 3-4 weeks old) were supplied by the Çanakkale Onsekiz Mart University, Experimental Research Application and Research Center. We housed rats in individual cages under a standard 12:12-hour light/dark cycle at 22 ± 2 ºC and in a room with standard humidity (45%-50%). Rats were fed ad libitum with food and water. We stopped feeding 12 hours before the perfusion process, but they were allowed to drink water.

Liver tissue procurement
We performed the surgical procedure as described previously.^17,18 Briefly, the abdominal cavity of rats under ketamine (Ketalar, Pfizer, Turkey)/xylazine (Rompun, Bayer, Canada) (50/10 mg/kg, intraperitoneal) anesthesia was opened with a mid-abdominal incision. After a cannula was inserted into the portal vein, the liver was perfused with perfusion solution (cold storage solution BEL-GEN, Institut Georgez Lopez) at 4 ºC. The hepatic vein was dissected, and the procedure was continued until the perfusion solution flowed lucidly through it. Rats were sacrificed by cervical dislocation, and livers were removed and placed into Falcon tubes containing 15 mL of preservation solution.

Experimental groups and hyperbaric oxygen therapy administration
We divided extracted livers into 3 groups: (1) livers stored at 4 ºC for 24 hours (control group); (2) livers placed in a pressure tank box with 2.5 atmospheres absolute (ATA) hyperbaric oxygen (HBO₂) kept constant at 4 ºC for 60 minutes and stored at 4 ºC for 23 hours (HBO₂-60 group); and (3) livers placed in a pressure tank box with 2.5 ATA HBO₂ kept constant at 4 ºC for 120 minutes and stored at 4 ºC for 22 hours (HBO₂-120 group). We monitored temperature with a digital thermometer (Berlin Fridge Tag Memory Digital Thermometer). We fixed stored liver samples in 10% neutral buffered formalin for 48 hours. After fixation, a routine tissue processing procedure was followed, and samples were embedded in paraffin blocks. We obtained 4-μm-thick sections from paraffin-embedded tissues using a microtome device (Leica RM2125 RTS; Leica Microsystems Inc) and placed sections on poly-L-lysine-coated slides; we deparaffinized for use in histological and immuno-histochemical procedures and for terminal deoxy-nucleotidyl transferase dUTP nick end-labeling (TUNEL).

Histopathological examination
We stained sections with a routine hematoxylin and eosin (H&E) staining protocol.¹⁹ Two histologists blinded to the groups scored hydropic degeneration and sinusoidal dilatation according to histopat-hological changes under a light microscope (Olympus CX43) as follows: 0 = no injury, 1 = mild injury, 2 = moderate injury, and 3 = severe injury.

Immunohistochemical examination
We immunostained sections with primary antibodies against tumor necrosis factor-α (TNF-α) (1:100, Biobryt Inc Firm) and interleukin 10 (IL-10) (1:150, Santa Cruz Biotechnology) by using the antigen retrieval method²⁰ and according to the manufacturer’s instructions. Briefly, the sections were left at 65 ºC for 1 hour and deparaffinized in xylene before we passed sections through an alcohol series for rehydration. We then incubated the sections with 10 mM EDTA in a 200-watt microwave oven for 20 minutes and cooled at room temperature for 20 minutes. We later surrounded each tissue by a tissue boundary pen, with incubation with 3% hydrogen peroxide and blocking with phosphate buffered saline (pH 7.4). We then incubated the sections with primary antibodies overnight at 4 ºC and then with secondary antibodies compatible with the primary antibodies for 2 hours at room temperature. We used 3,3-diaminobenzidine tetrahy-drochloride (Thermo Fisher Scientific) as a chromogen and counterstained with Harris hematoxylin. We then coverslipped sections by using 90% glycerol. Two histologists blinded to the groups evaluated immuno-staining of cells under a light microscope as follows:21 <10% = grade 0, 10% to 25% = grade 1, 25% to 50% = grade 2, and >50% = grade 3.

Apoptosis detection with the terminal deoxynucleotidyl transferase dUTP nick end-labeling method
We used the TUNEL method to detect apoptosis of liver tissue. We obtained 4-µm-thick sections from each paraffin block, which were then were immersed in changes of xylene and reducing alcohol before staining. After dewaxing, hydration, and serum blocking, we used the ApopTag Peroxidase in situ Apoptosis Detection kit (S7100, Merck Millipore) according to the manufacturer’s protocol. Two histologists who were blinded to the groups examined sections under a light microscope (Olympus CX43) with ×400 magnification. Cells with brown-stained nuclei were accepted as TUNEL-positive apoptotic cells. We calculated the apoptotic index according to the following formula: apoptotic index = (number of TUNEL-positive cells/total number of cells counted) × 100.¹⁸

Reverse transcriptase-polymerase chain reaction
We evaluated IL-6 gene expression by using reverse transcriptabe-polymerase chain reaction (RT-PCR). At the end of the experimental period, we homo-genized approximately 10 to 30 mg of liver tissue in a homogenizer (Mixer Mill MM 400, RETSCH). We determined total RNA from obtained homogenates by using a RNA mini kit (Ambion PureLink RNA Mini Kit, Thermo Fisher Scientific). We equalized concentrations of samples by measuring the RNA concentrations and purity by a NanoDrop photo spectrometer. We used the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) to determine cDNA synthesis from the obtained RNA samples. The PCR conditions were as follows: step 1 = 25 ºC for10 minutes, step 2 = 37 ºC for 120 minutes, and step 3 = 85 ºC for 5 minutes. The obtained cDNAs were amplified using the StepOne real-time PCR system (Thermo Fisher Scientific) in accordance with the TaqMan qPCR Master Mix protocol. We used β-actin as the housekeeping gene. We determined gene expression levels by using cycle threshold (Ct) values and evaluated fold changes by the 2^-ΔΔCT method (Ct target gene - Ct reference gene).

Statistical analyses
We used SPSS software version 22 (IBM) for statistical analyses. We evaluated data obtained from histological and immunohistochemical studies by using the Kruskal-Wallis test followed by the Mann-Whitney U test. We analyzed data obtained from IL-6 gene expression and TUNEL test with one-way analysis of variance followed by a post hoc Tukey test. Significance was accepted as P < .05. Data are shown as mean ± SEM, and graphs were made using GraphPad Prism 5.0 software (GraphPad Software Inc).

Results

Hematoxylin- and eosin-stained liver sections were analyzed in terms of hydropic degeneration and sinusoidal dilatation parameters. Representative images of H&E-stained sections are shown in (Figure 1A). No difference was detected between the hydropic degeneration grades of the groups. Although sinusoidal dilatation decreased in both HBO₂ groups compared with the control group (P ≤ .001), the decrease was greater in the HBO₂-120 group than in the HBO₂-60 group (P < .05; (Figure 1B)).

Representative images of liver sections stained with immunohistochemical primary antibodies of TNF-α and anti-IL-10 are shown in (Figure 2A and B), . Expression levels of both TNF-α and IL-10 decreased in both of the HBO₂ groups compared with the control (P < .01 and P < .001 for TNF-α; P < .05 and P < .001 for IL-10 for HBO₂-60 and HBO₂-120 groups, respectively). Expression levels for both proteins were less in the HBO₂-120 group compared with the HBO₂-60 group (P = .05 for TNF-α and P < .001 for IL-10; (Figure 2C)). Expression of IL-6 mRNA was lower in both HBO₂ groups than in the control group (P < .01); however, differences were not significant between the HBO₂ groups (Figure 2D).

(Figure 3A) shows representative TUNEL-positive cells. Data obtained from the TUNEL method showed that the apoptotic index of the HBO₂ groups was significantly reduced compared with the control group (P < .001). Furthermore, the apoptotic index of the HBO₂-120 group was lower than the HBO₂-60 group (P < .001; (Figure 3B)).

Discussion

Any injury to the allograft during procurement, transport, or transplant not only reduces the success rate of the transplant but may also damage other vital organs such as the heart and lungs. Allografts from deceased donors are more susceptible to injuries and have lower success rates of transplant than living donors. However, deceased donors continue to constitute a large part of the donor pool because of organ shortages and the increasing number of patients who are wait for organ transplants. Therefore, it is particularly important to preserve organs from deceased donors, who are often far from the recipient, from procurement to transplant.^8,9

The main objective of organ preservation is to allow resumption of normal cellular metabolism after transplant and to avoid potential morbidity and mortality associated with poor graft function, by reducing the severity of ischemic injury, which is a consequence of anaerobic metabolism and hypoxia.²² The metabolic rates of harvested tissues are halved with every 10 ºC decrease in temperature. In an environment of 4 ºC, metabolic rate drops to 10% of normal. Although SCS reduces tissue damage and therefore the rate of organ deterioration by reducing cellular metabolism, which includes mitochondrial enzyme activity and oxygen requirement,¹¹ SCS cannot completely stop metabolism.⁴ Organ preservation solutions are used to minimize cellular changes during SCS that lead to the loss of cell membrane functions and thus cell death.^8,9 However, even the most effective organ preservation solution cannot completely prevent extracorporeal ischemia in case of prolonged cold ischemia.¹ Therefore, the procured organ can be preserved only for a limited period by SCS. The general recommendation is for cold ischemia time for the liver in SCS not to exceed 12 hours to preserve liver allograft function.^3,11 However, injuries related to SCS have occurred when time exceeded 8 hours in Euro-Collins solution and 16 hours in University of Wisconsin solution, which were irreversible injuries after reperfusion of the liver.²³ Therefore, new techniques are needed that will save time in the perioperative period by reducing cold ischemia injury.

HBO₂ is 100% oxygen administered at a pressure higher than atmospheric pressure and has the potential to improve hypoxia-related pathologic disorders. In an organ preserved in SCS after procurement, HBO₂T has the potential to support ATP production by providing the oxygen required for oxidative phosphorylation,¹³ slow down the transition to anaerobic metabolism associated with ischemia, and allow extension of acceptable ischemic time by contributing to preservation of allograft vitality.¹⁴ In rat liver transplant models, administration of HBO₂ during SCS for 24 hours prevented ischemic injury by attenuating oxidant stress and ATP depletion without ischemia²⁴ and HBO₂ administered before and after transplant protected hepatocytes from necrosis and apoptosis.²⁵

Structural changes may develop during both SCS and reperfusion processes. Apoptotic cell, connective tissue, and sinusoidal dilatation are increased and glycogen level are decreased over time in the liver preserved by SCS.²⁶ The severity of these changes is directly related to allograft functions after transplant.^4,7 In addition, reperfusion may aggravate preexisting injuries.¹¹ In our study, we evaluated tissue injury by evaluating the development of hydropic degeneration and sinusoidal dilatation as structural changes and tissue viability by the apoptotic index. Hydropic degeneration resulting from disruption of ionic and fluid balance is characterized by cells that are swollen and often enlarged with cytoplasmic vacuolization. This characteristic is nonspecific, common, and usually reversible after reperfusion.²⁷ Sinusoidal dilatation is a histopathological finding that is considered an indicator of venous flow impairment²⁶ and centrilobular necrosis.²⁸

Our results showed that hydropic degeneration and sinusoidal dilatation developed in the liver stored in University of Wisconsin solution for 24 hours, as reported also by Sundberg and colleagues.²⁹ We administrated HBO₂ to cold-stored liver tissues for the first 1 to 2 hours. We found that HBO₂ administered for 60 minutes did not affect hydropic degeneration but reduced sinusoidal dilatation and apoptotic index compared with cold-stored tissues alone (control group). In addition, HBO₂ administered for 120 minutes further reduced sinusoidal dilation and apoptotic index. These results suggested that even limited-term administration of HBO₂ to liver tissue in SCS has the potential to reduce the risk of cold ischemia injury. The lack of reperfusion is a limitation of our study. Therefore, it cannot be known with certainty whether the histopathological changes after reperfusion will be reversible. However, preventing or reducing the severity of injuries before reperfusion contributes to allograft survival and reduction of transplant-related complications.

Immune activation is also an important factor causing organ ischemia in SCS. HBO₂ also has important effects on immune function and cytokine production.³⁰ Although evidence continues to increase that HBO₂T attenuates ischemia-reperfusion injury by modulating both the humoral and cellular immune response,¹⁵ its effects on the immunity and especially on inflammatory mediators in terms of preserving allograft viability and increasing the duration of cold ischemia remain unclear.

Kupffer cells are approximately 35% of nonpa-renchymal liver cells and 80% to 90% of all body macrophages.³¹ Active Kupffer cells release various cytokines such as TNF-α, IL-1, IL-6, IL-10, chemokines, prostaglandins, leukotrienes, and complement factors and cause the generation of free oxygen radicals and promote the migration of recipient inflammatory cells to the site of injury posttransplant.^4,32-35 Proinflam-matory cytokines such as TNF-α, IL-1, and IL-6 may cause exacerbation and deepen the inflammation process by affecting the production, release, or activation of each other. The proinflammatory response could be a major obstacle to allograft tolerance and the most important factor for ischemia-reperfusion injury.^10,36 The addition of anti-inflammatory drugs to ex vivo perfusion may have additional protective effects on the graft besides the avoidance of cold ischemic injury.³⁷ Kupffer cells are mainly activated after perfusion of the liver; however, Kupffer cells also may activate in response to hypoxia and prolonged cold ischemia.²³ An SCS period equivalent to the production of free oxygen radicals from Kupffer cells coincides with the time when cytokines begin to be released (>18 hours).³⁸

In this study, we assessed immune response by expressions of TNF-α and IL-6, which are important proinflammatory cytokines, and expression of IL-10, an anti-inflammatory cytokine. Our results showed that hyperbaric oxygen therapy for 1 hour in the liver placed in SCS after organ removal reduced expression levels of TNF-α, IL-10, and IL-6 mRNA in rat liver in SCS at hour 24. Although the decrease in IL-10 expression seems to be an unfavorable situation in this study, the suppression of proinflammatory cytokines, which are critical for triggering and maintaining inflammation, indicates that HBO₂ has a significant anti-inflammatory effect, and its effect is nonspecific. In addition, further reduction of TNF-α expression, level of sinusoidal dilation, and apoptotic index with the HBO₂T for 120 minutes demonstrates that the anti-inflammatory and antiapoptotic effects of HBO₂T are directly proportional to the exposure duration.

Conclusions

Our study showed that HBO₂T reduced histopat-hological changes and exhibited antiapoptotic and anti-inflammatory effects in rat liver preserved in SCS. Therefore, administration of HBO₂T with SCS, even for a limited period, may contribute to expanding the tolerable organ preservation time and increasing allograft survival.

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