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Keywords:

  • Adenosine;
  • cardiopulmonary bypass;
  • ischemia-reperfusion;
  • nitric oxide;
  • total body cooling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

We have evaluated the involvement of hepatic preconditioning mediators (adenosine, adenosine A1 and A2 receptors) during normothermic recirculation (NR) in a model of liver transplantation from non-heart-beating donor (NHBD) pigs.

Application of NR after 20 min of warm ischemia (WI) reversed the lethal injury associated with transplantation of NHBD livers (achieving 5-day survival and diminishing glutathione S-transferase (GST), aspartate aminotransferase (AST) and hyaluronic acid (HA)).

Adenosine administration prior to WI simulated the effect of NR. Measuring adenosine, we found that during NR, hepatic adenosine levels increased and xanthine levels decreased. Then when we blocked A2 receptors the effect of NR was abolished, whereas the blocking of A1 receptors further protected the liver. Furthermore, A2 blocking improved hepatic perfusion during NR whereas A1 blocking reduced it.

The study suggests that NR has a preconditioning effect by maintaining adequate adenosine and xanthine levels. During NR, adenosine protects the liver through A2 activation and damages it through A1 activation although simultaneous stimulation of both receptors exerts a clear beneficial effect. The possible relation of NR mechanism with other preconditioning mediators such as cAMP and nitric oxide synthesis are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

Non-heart-beating donors (NHBD) have shown to be a good source of organs for transplantation (1–7). However, clinical experience has been limited to kidney transplantation where primary non-function is not a life-threatening problem. Whereas in the case of the liver, the absence of knowledge of the quality of the graft and its viability has maintained this type of transplant in an experimental level. This fact has motivated active research in the field mainly focused on the methodology of organ procurement.

In this context, we have demonstrated that organ procurement after cardiac arrest (warm ischemia) by using a cardiopulmonary bypass (CPB) at 37°C for 30 min (normothermic recirculation, NR) before total body cooling, clearly improves graft function (8–12). The use of the so-called normothermic recirculation improves the quality of renal grafts clinically (13), and liver grafts experimentally (8–12). However, the underlying cellular mechanisms are yet unknown. Nevertheless, the beneficial role of an NR during organ procurement/preservation has not been fully understood, since it has been accepted worldwide that in organ preservation ‘warm is our enermy’.

On the other hand, ischemic preconditioning has been defined as the protective effect conferred by transient and brief normothermic ischemia-reperfusion (I-R) periods over the subsequent injury caused by prolonged period of ischemia. The protective effect of ‘preconditioning’ was first described in the heart (14), and it is now well recognized in most of the organs, including the liver (15).

The use of NR after a period of warm ischemia, may change the overall situation and transform the period of cardiac arrest into a brief and transient period of ischemia. In other words, some of the underlying mechanisms suggested to take place during preconditioning may, in fact, have a major role during NR.

Hepatic preconditioning has been reported as being mediated by a transient increase in adenosine levels (15,16). In addition to Peralta et al. (15,16), other authors have outlined the key role of adenosine in the preconditioning effect. As it happens in the heart model, all these authors have shown that the activation of adenosine during the transient periods of ischemia, is responsible for the beneficial effect after reperfusion. Moreover, adenosine could exert its effect by counteracting its receptors. Until now, only A2 receptors have been clearly demonstrated to be involved in the preconditioning effect in the liver; however the presence of the other A1 subtype has been proven in hepatocytes as well (17).

Taking into account this possibility, we designed a study in order to evaluate the role of adenosine in our experimental model of liver procurement from non-heart-beating donors. The aim was to demonstrate whether NR had a similar role to adenosine after a given period of warm ischemia.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

Sixty outbred weanling pigs, weighing 25–30 kg, were transplanted with an allograft from an NHBD.

Experimental design

To evaluate the effect of adenosine and NR, the following experimental groups were formed and randomly allocated:

Control group (CG, n = 10). The donor animal was submitted to a period of 20 min of cardiac arrest. No drugs neither NR were applied in this group.

Normothermic Recirculation group (NR, n = 10). After 20 min of cardiac arrest, a period of normothermic recirculation was performed. No drugs were added to this group.

Adenosine group (ADE, n = 10). Adenosine was administered as a continuous infusion (350 μg/kg/min, i.v.) for 15 min prior to 20 min of cardiac arrest, and NR was not used.

The effect of A1 and A2 antagonist over NR:  In case of a positive adenosine effect, the study included the use of:

DPCPX group (n = 10): 8-cyclopentyl-1,3dipropylxanthine (DPCPX)—an A1 adenosine receptors antagonist—was administered as a continuous infusion (10 μg/kg/min, i.v.) during NR.

CSC group (n = 10): 8-(3-chlorostyryl) caffeine (CSC)—an A2 adenosine receptors antagonist—was administered (0.2 μg/kg, i.v.) as a single bolus at the beginning of NR period.

Surgical procedures

Donor Procedures:  Animals had fasted for 36 h prior to surgery. Anesthetic and monitoring procedures were performed as described elsewehere (8–12). After opening the abdomen, the hepatic hilium was exposed. A non-invasive flowmeter (Transonic Systems Inc. HT207. Ithaca, New York) was placed around the hepatic artery and portal vein. Baseline flow values were determined. At this point, heparin was given intravenously (3 mg/kg). Liver and esophagic temperatures were also monitored (Mon-A-Therm, Mallinckrodt Medical Inc®, St Louis, MO).

Cardiac arrest was then produced by intravenous injection of KCl (10–15 mL 2M) and maintained for 20 min in all the animals studied.

In the groups where NR was performed, the jugular vein, aorta and the inferior vena cava were cannulated (22 Fr, 16 Fr and 28 Fr respectively) and connected to a blood oxygenator (Bard Quantum Oxygenator and Venous reservoir, HF6000–H6770VR, CR Bard Inc®; Haverhill, MA), a heat exchanger (Módulo Normo-hipotermia Palex S.A., Barcelona, Spain) and a non-pulsatile roller pump (Stöckert-Shiley®, Munich, Germany). The circuit was primed with saline solution 500 mL, mannitol 0.5 g/kg and Haemoce® (saline solution of polygeline) 500 mL.

After 20 min of cardiac arrest, NR (extracorporeal circulation and tissue oxygenation at 37°C) was run and maintained for 30 min at the maximum pump flow rate permitted by the venous blood return, with a target of 2.2 L/min/m2 body surface. The pump flow rate was recorded, as an indirect estimate marker of circulatory function, during the whole NR period, and the mean pump flow rate was calculated. Portal and hepatic artery blood flows were continuously registered before cardiac arrest, during the 30 min period of NR and during total body cooling. Flow data are corrected to the animal body surface. The mean arterial pressure, liver and esophageal temperatures, and the temperature of the heat exchanger were recorded every 5 min.

Sodium bicarbonate was added to the circuit, to correct metabolic acidosis, but no other fluids were introduced into the circuit. Total body cooling by extracorporeal circulation was started and it continued until the animal progressively reached a liver temperature of 15°C.

Organ procurement:  Liver procurement was then performed in each donor in a standard manner, as described elsewhere (12), with University of Wisconsin solution perfusion through the aorta and portal vein. The liver was then cooled and preserved at 4°C for 6 h.

Recipient procedures:  The anesthetic management was similar to that of the donors. Once the abdomen was opened, a standard hepatectomy was performed as previously described (12). The donor liver was flushed in the back table with saline solution at room temperature. The allograft was placed in the recipient and anastomosis were performed in the following order: first the suprahepatic vena cava and portal vein, immediately revascularizing the liver, then the infrahepatic vena cava and the hepatic artery and finally the biliary tract, leaving the gallbladder in place. No veno-venous bypass was performed because the duration of the anhepatic stage lasted no longer than 20 min in any case. The hepatic arterial blood flow was reconstructed between the donor celiac axis and the recipient's hepatic artery by means of magnifying lenses. Biliary reconstruction was performed with an intraluminal stent externally secured with two silk stitches. The cold ischemia time, from the end of total body cooling to reperfusion in the recipient, ranged from 5 to 6 h. The abdominal wall was closed in two layers and the skin was closed with a running silk suture.

Postoperative care:  The animals were tracheally extubated for 20–35 min after the operation. They were placed in metabolic cages with heat lamps. Blood gas was monitored postoperatively for several hours. Analgesia was given by means of intramuscular injection of meperidin (100 mg) 1 h after tracheal extubation. The immunosuppressive regimen consisted of metilprednisolone 500 mg and azathioprine 1.5 mg/kg before liver reperfusion, and oral cyclosporine 25 mg/kg daily after the first postoperative day. Fluids were permitted orally on the first postoperative day and the animals were fed with commercial pig food and yogurt after the second day. The animals were killed on the 5th postoperative day by an i.v. overdose of sodium pentobarbital.

The study was approved by the Investigation and Ethics Committee of the Hospital Clínic. The animal experiments have followed the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication No. 86-23, 1985).

Blood, Tissue Samples and Measurements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

Blood samples from portal, suprahepatic vein and carotid artery were taken for blood gas, electrolytes and hemoglobin analysis (288 Blood Gas System, Ciba-Corning Diagnostics Corp. Medfield, MA) at baseline, 5 and 15 min after the beginning of NR and at the end of NR. With these data the following parameters were calculated: Oxygen content in hepatic artery (CaO2), portal vein (CpO2) and suprahepatic vein (CshO2) blood = pO2× 0.0023 + (hemoglobin × (O2 saturation/100) × 1.31); the values are expressed as mL O2 dL−1.Oxygen delivery (HDO2) to the liver = (CaO2× arterial flow) + (CpO2× portal flow).

In the donor, blood samples for biochemical assays were taken at the beginning of the procedure and after 30 min of recirculation at 37°C. In the recipient, samples were taken 1 h after reperfusion. Blood was immediately centrifuged and the plasma was distributed in aliquots, frozen and stored at –20°C for future testing. Hepatocellular and endothelial damage, were estimated by the plasma concentration, 1 h after reperfusion, of α-glutathione-S-transferase (α-GST) (18) and AST, and by hyaluronic acid (HA) (19–21). AST was also determined on the 5th postoperative day to asses the hepatocellular lesion just before animals were killed. Enzyme analyses were carried out using the standard laboratory methods (α-GST: Biotrin HEPKIT™α-porcine GST; Biotrin LTD. Dublin, Ireland). HA was measured by a radiometric assay (Pharmacia Diagnostics, Uppsala, Sweden).

Adenine nucleotide tissue content:  To study the effect of NR on the adenine nucleotides and their breakdown products, their tissue content was determined in liver biopsies obtained in the donor at the beginning of the procedure, after 20 min of cardiac arrest and at the end of the NR period. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, hypoxanthine and xanthine were determined by HPLC as described elsewhere (15,21). Pure standards of nucleotides were obtained from Sigma (St. Louis, MO). ATP, ADP, AMP, adenosine, hypoxanthine and xanthine levels were expressed as percentage of the total nucleotides (ATP + ADP + AMP + Adenosine + hypoxanthine + xanthine) present in each sample, to correct for tissue amount homogenised.

Histological evaluation of liver damage:  After the animal was killed on the 5th postoperative day, a large sample, including a whole hepatic segment, was kept in formaline. Serial sections perpendicular to the hepatic capsule were carried out every 0.5 cm. A careful macroscopic inspection was performed and two different randomly selected samples (1.5 cm2) were submitted for histologic evaluation. A third sample was chosen from the hilar area, including large hilar portal tracts. In case of macroscopically evident infarcted areas, a fourth sample was taken. All the specimens for histology were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections, 4-m thick, were routinely stained with hematoxylin and eosin. Sinusoidal congestion (SC), sinusoidal infiltration by polymorphonuclear cells and lymphocytes (SIPCL), ischemic cholangitis (IC) and centrilobular necrosis (CN) were evaluated in all specimens. All the histological parameters were semi-quantitatively evaluated according to the following criteria: absence (0), mild or focal (1), moderate (2) and severe (3). The pathologist was not aware of the times at which biopsies had been carried out or the treatment animals had been submitted. The reproducibility of the histological analysis, assessed by repeated blinded examination of 25% of the biopsies obtained at each time, showed 89% of agreement.

Quantitative morphometric analysis of the necrotic area was performed with the digital image processing software MIP (Microm, Barcelona, Spain), using an Olympus BH2 microscope (Olympus, Tokyo, Japan) at a magnification of 40×, with an eyepiece with a field of view of 26.5, with an intermediate device, each field resulting in the area of 15.45 mm2. Three randomly selected areas representing a total area of 46.35 mm2 were measured in each case. Necrotic areas were semi-automatically evaluated, manually drawing the necrotic areas with a clear camera. The results were expressed as percentage (mm2 of necrotic parenchyma per mm2 of total area).

Statistical analysis

The statistical analysis of the results was performed using the Fisher exact test, unpaired Student's t-test, Multiple analysis of variance test and Bonferroni test. Values of p < 0.05 were considered to be statistically significant. Results are expressed as mean + SE.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

Survival

Adenosine effect:  The absence of a period of NR after 20 min of cardiac arrest showed a survival rate of 20%. However, the addition, after those 20 min of CA, of a 30 min period of NR increased survival significantly (100%). Furthermore, when adenosine was added before cardiac arrest, survival was 90%, in spite of the absence of NR.

Adenosine antagonists effect:  The addition of specific A2-blocking agent such as CSC reduced survival to 30% in spite of the use of NR, whereas DPCPX treatment maintained a survival of 100%.

Hepatic adenosine and xanthine:  After 20 min of WI, adenosine did not change significantly either in the control group or in the groups where NR was used, although it obviously increased in the animals where adenosine was administered (p < 0.05). Furthermore, adenosine levels increased significantly at the end of NR in the groups where NR was used (NR, CSC, DPCPX) (p < 0.05) (Figure 1A).

image

Figure 1. Hepatic tissue adenosine (A) and xanthine (B), in the control, NR and adenosine groups, at baseline (basal), after 20 min of warm ischemia (WI), and after 30 min of normothermic recirculation (NR). Data are expressed as mean ± S.E. *p < 0.05 vs. basal, #p < 0.05 vs. preceding phase.

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Xanthine accumulates after 20 min of WI in the three groups (p < 0.05) and significantly decreases after NR running (p < 0.05), (Figure 1B).

Hepatocellular lesion

Adenosine effect:  No differences were found between basal plasma levels of α-GST (control: 96 ± 57 μg/L vs. NR: 94 ± 48 μg/L, ADE: 132 ± 69 μg/L) in the different groups (Figure 2A), nor in the plasma levels of AST (control: 55 ± 8 IU/L vs. NR: 75 ± 9 IU/L, ADE: 79 ± 11 IU/L), (Figure 2B).

image

Figure 2. (A) Plasma levels of α-Glutathione-S-transferase in the experimental groups at baseline (basal) and 1 h after reperfusion (RP). (B) Plasma levels of AST in the experimental groups at baseline (basal), 1 h after reperfusion (RP) and on the 5th postoperative day. Data are expressed as mean ± S.E. *p < 0.05 vs. the other groups except CSC group, #p < 0.05 vs. the other groups, p < 0.05 vs. the other groups. Numbers above columns at 5 days represent the number of surviving animals in each group.

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Plasma levels of α-GST increased throughout the procedure in all the groups reaching the peak value 1 h after reperfusion in the recipient. However, after reperfusion, there was a strikingly sharp increase in α-GST values in the control group, a feature not observed in the NR and ADE groups (p < 0.05).

The plasma levels of AST, increased throughout the procedure. Although the values were similar in all the groups before reperfusion, 1 h after reperfusion, the values were again significantly lower in treated animals (ADE and NR), when compared to the control group (Figure 2B).

Adenosine antagonists effect:  Similar baseline levels were found among the three study groups, with respect to α-GST (NR: 94 ± 48 μg/L, DPCPX: 51 ± 48 μg/L and CSC: 152 ± 68 μg/L) (Figure 2A), and AST (NR: 75 ± 9 IU/L, DPCPX: 59 ± 16 IU/L and CSC: 119 ± 52 IU/L) (Figure 2B).

α-GST and AST levels behave similarly in all three groups. After reperfusion, CSC-treated animals tended to have higher levels of α-GST and AST, the differences not being statistically significant.

Five days after transplantation CSC-treated animals showed the highest AST levels (p < 0.05), while DPCPX-treated animals, the lowest ones (p < 0.05), (Figure 2B).

Sinusoidal endothelial lesion

Adenosine effect:  There were no differences between the HA plasma levels in the groups at baseline (control: 220 ± 37 μg/L vs. NR: 181 ± 25 μg/L, ADE: 322 ± 57 μg/L), (Figure 3). HA progressively increased, reaching a peak value after reperfusion (p < 0.05). NR and ADE groups had lower levels of HA than control group, despite the fact that no statistical difference was achieved (Figure 3).

image

Figure 3. Plasma levels of hyaluronic acid in the experimental groups at baseline (B) and at the end of normothermic recirculation (NR). Data are expressed as mean ± S.E. *p < 0.05 vs. the other groups.

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Adenosine antagonists effect:  HA plasma levels were similar in the three groups at baseline (NR: 181 ± 25 μg/L, DPCPX: 138 ± 53 μg/L and CSC: 184 ± 26 μg/L) (Figure 3). HA progressively increased to reach a peak value after reperfusion (p < 0.05). At this time period the DPCPX group showed the lowest level of HA (p < 0.05). NR tended to have lower levels of HA than CSC, but the difference was not significant. HA levels in DPCPX-treated animals were significantly lower than in the CSC-treated animals (p < 0.05). Since HA uptake is mainly performed by the liver endothelial cells, these results indicate a significantly better endothelial cell function in the DPCPX group. (Figure 3).

Blood flows during NR

Hepatic blood flow rapidly recovered during NR in the NR, CSC, and DPCPX groups. However, higher flows were recorded throughout the study period in the CSC animals as compared to the DPCPX group (Figure 4A) (F = 4.3, p < 0.05). Pump blood flow increased quickly during NR in the three groups. Flows in CSC-treated animals tended to be higher than in NR and DPCPX groups. The NR group showed intermediate flows between the CSC and DPCPX groups (Figure 4B).

image

Figure 4. Hepatic (A) and extracorporeal pump blood flows (B), in the experimental groups, during NR at 5, 15 and 30 min. Data are expressed as mean ± S.E. *p < 0.05 between CSC and DPCPX groups.

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Histologic changes

Liver transplants from the 34 five-day surviving animals were examined (Figure 5). Sinusoidal congestion (SC), centrilobular necrosis (CN) and ischemic cholangitis (IC) tended to be worse in the control group than in the other groups. Among the study groups, the CSC animals demonstrated the higher intensity for SIPCL and CN. The animals in DPCPX group tended to be the ones with less intensity changes for all the variables. ADE and NR animals showed similar changes.

image

Figure 5. Histological findings evaluated on 5th postoperative day in the experimental groups. The intensity of these changes was evaluated by a semi-quantitative scoring system as absent (0), mild or focal (1), moderate (2) and severe (3). (SC: sinusoidal congestion; SIPCL: sinusoidal infiltration by polymorphonuclear cells and lymphocytes; CN: centrilobular necrosis and IC: Ischemic cholangitis). Data expressed as the median value ± S.E.

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Morphometric analysis of liver specimens revealed a greater percentage of the of necrotic parenchyma in control and CSC groups. Necrotic areas of 40 and 20% respectively were only observed in these two groups, the ones with less survival. Mean necrotic areas in ADE, NR and DPCPX groups were 4, 6 and 4%, respectively.

It must be taken into consideration that the number of samples was significantly reduced in the control and CSC groups because of low survival in these groups. Therefore, results in these groups were not so representative as in the others making the histological study difficult.

However, overall histological results seemed to be in agreement with the other lesion markers studied.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

Ischemic preconditioning (IP) is a phenomenon in which brief ischemia followed by reperfusion reduces the lesion associated with subsequent prolonged ischemia. Within the field of transplantation it may blunt the I-R damage, enhancing graft viability.

As a consequence of cardiac arrest immediately occurring prior to organ procurement, the organs from NHBD are additionally damaged by the unavoidable warm ischemia period. Recently, the use of cardiopulmonary bypass as a period of time of warm perfusion before graft procurement (the so-called ‘normothermic recirculation’) has been demonstrated to improve the quality of these grafts, decreasing the I-R associated to the procedure of transplantation (8,12). The first ideas of NR technique were introduced by Hoshino et al. (23) and then its usefulness was further demonstrated by our group and others (24,25). Thus, in NHBD procurement, NR seems to have a similar effect to that of ischemic preconditioning. The underlying cellular mechanisms leading to the beneficial effect of NR are still unknown.

The role of adenosine, in the process of IP, has long been known in the literature mainly in relation to the heart. However, recent data have shown the role of adenosine also in the process of IP within the liver (26). These results have proved clearly that adenosine increases after brief periods of ischemia, producing vasodilation by interacting with A2 receptors (27,28). Contrary to what happens in the heart, where A1 are the predominant type of receptors, it seems that A2 types are the predominant receptors within the liver.

We have clearly shown that the addition of adenosine before cardiac arrest has a similar effect to that of NR. Moreover, the use of NR increases the levels of adenosine within the liver. This is confirmed by the fact that the hepatocellular and sinusoidal endothelial cell (SEC) damage was attenuated in NR as well as in adenosine-treated livers when compared to controls. These results are in accordance with those by Peralta et al. in an ischemia-reperfusion model where they found that IP produces a significant increase in adenosine and a significant decrease in I-R injury after a long period of ischemia (27,28). The key role of adenosine in the cold preservation solution, in preventing reperfusion injury after liver transplantation in rats has been directly proven (29).

During ischemia, ATP degradation leads to the accumulation of xanthine, which has been established as an important source of superoxide radical. Harvey et al. demonstrated that non-viable livers develop higher levels of xanthine after WI than viable ones, and the same occurred with the levels of malondialdehyde indicating more lipid peroxidation of non-viable livers. Moreover these authors described how during cold preservation ATP is degraded to AMP, but ATP levels are not correlated with graft viability. It is suggested that graft viability does not depend on the availability of AMP to be rephosphorylated to ATP (30). In agreement with this idea, another protective effect of NR arises from the fact that NR significantly reduces the levels of hepatic xanthine (Figure 1B) (15,22,31).

Since it has been suggested that, in hepatic preconditioning, adenosine exerts its protective effect through the adenosine receptors stimulation, such receptors were selectively blocked during NR in our study (27,28). Considering that A2 receptors are predominant in the liver, we have found that CSC-mediated A2 blocking could abolish the beneficial effect of NR. And this is true not only for survival rate but also for hepatocellular and endothelial cell damage.

On the other hand, the use of specific A1 blocking agent deserves further comments. The preconditioning effect in the liver has been mainly described as being mediated through the A2 receptors, although recent studies report some presence of A1 receptors in the liver (17). If we carefully study the sinusoidal cell damage at the time reperfusion, and the hepatocellular damage at five days, we can surprisingly see a certain beneficial effect despite this selective blocking of the A1-type receptors. Therefore a partial role of A1 receptors during NR seems to exist.

This unexpected beneficial effect could be very well explained if we understand that by the use of DPCPX we block all A1 receptors in such a way that the activated adenosine produced by NR may significantly affect A2 receptors (the predominant type). Moreover, it has been recently shown that cyclic AMP (cAMP) may also play a role in liver IP (32,33). The stimulation of A2-type receptors may in fact stimulate this cyclic AMP production, while A1 stimulation decreases cyclic AMP producing a deleterious effect (34).

The specific effects on liver tissue at two different ends (endothelial and liver cell) may effectively explain the overall results obtained in our study. The effect of NR by stimulating both A1 (deleterious) and A2 (beneficial) receptors shows intermediate effect and this is true not only for survival rate but also for endothelial and hepatocellular damage.

This effect is consistent even when dealing with blood flows. We have previously demonstrated that survival was closely related to persistence of a good hepatic blood flow (12,35,36). The better the blood flow obtained during NR the better the survival, suggesting that preservation of the vascular bed is a key point in organ viability.

Furthermore, cAMP also regulates nitric oxide synthase (NOS)-mediated production of NO. Several studies show that this regulation is different on endothelial NOS (eNOS) and inducible NOS (iNOS). eNOS is expressed in endothelial cells and produces physiological levels of NO, and iNOS, mainly expressed in hepatocytes and kupffer cells, is strongly up-regulated under certain conditions producing large amounts of NO, which increase ischemia-reperfusion damage. Moreover, depending on the cell type and experimental conditions, cAMP may up-regulate eNOS-mediated NO production (36,37) and down-regulate iNOS-mediated NO production at different levels (39–41).

In this context, it is interesting to note that the blocking of A2 receptors may produce a significant decrease in clyclic AMP and thus producing at the endothelial level a marked decrease in NO (the beneficial one produced by eNOS), and at the hepatocellular level a significant increase in NO (the deleterious one, produced by iNOS). All these effects may in turn result in the prevalence of the iNOS-derived NO, which could explain the good blood flows but the maximum hepatocellular damage. On the other hand, A1 receptors blocking could be producing the opposite effect, favoring the prevalence of eNOS-derived NO during the process with good but lower blood flows and, what is more important, with minimum liver cell damage. During NR, we are stimulating both receptors (A1 and A2), producing an intermediate effect either over the endothelial or the hepatocellular damage. Further studies are needed blocking specifically the iNOS or the eNOS production in order to confirm these data. Nevertheless, experimental studies with other ischemia-reperfusion models have shown that eNOS inhibition increases cellular damage, on the contrary the inhibition of iNOS reduces reperfusion injury (42,43).

Because of the high mortality rate of the control and the CSC groups, we have not been able to show significant histological changes among groups. However, it seems that specimens at five days from controls and CSC, were more affected. On the other hand, those from NR and ADE had similar changes with the lowest mean necrotic areas. In accordance with these results, animals blocked by the use of an A1 antagonist such as DCPCX, showed minimal histological lesions suggesting that A1 blocking may indeed have an overall beneficial effect, in relation with the above-mentioned massive A2 receptors stimulation and/or the previously suggested effect on cyclic AMP.

In summary, the effect of NR seems to be partially related to adenosine. This adenosine-mediated effect, which is involved in ischemic preconditioning, predominantly acts at the level of A2 receptors. The results herein suggest that the blocking of A1 receptors favors the massive activation of A2 receptors ending in an increased overall beneficial effect.

Our experimental model was not specifically designed to understand the mechanisms of ischemic preconditioning, but to explain the beneficial effect of re-warming of an organ after cardiac arrest. It is clear that the mechanism of NR must be much more complex than what it may have been suggested in the study.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References

This study has partially been supported by ‘F.I.S.’ grant number 98/1247.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Blood, Tissue Samples and Measurements
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
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