Abstract
Dysfunction of the graft after liver transplantation caused by ischaemia-/reperfusion (I/R) injury is a serious clinical problem. The aim of this study was to evaluate the influence of different kinds of reperfusion on I/R injury in a rat model. Arterialized orthoptic rat liver treatment was performed on male LEWIS-(RT1)-rats. Three groups (n = 7) were formed. Group I: antegrade reperfusion with a 6-min delayed reperfusion via the hepatic artery. Group II: Antegrade reperfusion, simultaneously, via the portal vein and the hepatic artery. Group III: Retrograde reperfusion via the vena cava. Serum parameters were determined one, 24 and 48 h after operation. Furthermore, after 48 h, the liver was taken for histological assessment. After 48 h, rats of group III showed significantly lower aspartate amino transferase and alanine amino transferase serum levels compared with group I and group II rats. Forty-eight hours after transplantation, glutamate dehydrogenase serum level was significantly lower in group III than in group II. In histology, group III livers showed significantly less necrotic spots than group I and group II livers. Maximum size of the necrotic spots was significantly lower in group III than in group I. Also, significantly more necrotic spots were seen in the ‘Rappaport′s zone’ 1 and 2 of group I than in group III. Our data suggested that the expression of I/R-injury correlates with the type of reperfusion. Furthermore, under standard conditions, this study was able to demonstrate that in a rat model, the retrograde reperfusion leads to a lower expression of I/R-injury than the antegrade reperfusion.
Keywords: I-/R injury, rat liver transplantation, retrograde reperfusion
One of the most serious complications after liver transplantation (LTx) is the primary non-function (PNF). The frequency of the appearance of an organ failure is not exactly indicated in the literature (4.2–8.4%) (Ploeg et al. 1993; Porte et al. 1998; Moreno Sanz et al. 1999; Bennett-Guerrero et al. 2001). The influence of I/R-injury as a significant risk factor for PNF is widely unquestioned. The I/R-injury is responsible for approximately 10% of PNF after LTx (Fondevila et al. 2003). There is a high correlation between pre-existing damage of the liver and the I/R-injury (Briceno et al. 2002; Caraceni et al. 2005). Because of the increasing organ shortage, it is necessary to transplant every available organ and to develop new strategies to minimize the damage caused by conservation, ischaemia and reperfusion. Especially fatty livers are prone to this kind of damage and, accordingly, are more predisposed to PNF after LTx (Sun et al. 2001; Perez-Daga et al. 2006). The type of reperfusion during liver transplantation has an influence on I/R-injury (Brockmann et al. 2005; Polak & Porte 2006).
In a number of studies which compare different kinds of antegrade reperfusion, no agreement could be reached concerning the question which type of reperfusion will lead to the lowest expression of I/R injury, though most studies have found the most positive effect in the simultaneous arterial and portal venous reperfusion (Massarollo et al. 1998; Brockmann et al. 2005). The primary retrograde reperfusion via the V. cava with the following reperfusion via the portal vein in the ‘Piggy Back technique’ is a new method in liver transplantation (Kneipeiss et al. 2003). So far, it has only been evaluated in two retrospective trials (Kniepeiss et al. 2003; Heidenhain et al. 2006). The aim of this trial was to compare the method of retrograde reperfusion with the common antegrade reperfusion methods in an animal model under standard conditions to evaluate if there is an advantage to this method.
Materials and methods
Animals
Male LEWIS-(RT1) rats with a weight of 220–240 g were used in this study.
Groups
Group I: Antegrade reperfusion via portal vein with a 6-min delayed reperfusion via hepatic artery (n = 7).
Group II: Antegrade reperfusion, simultaneously via portal vein and hepatic artery (n = 7).
Group III: Retrograde reperfusion via infra-/suprahepatic inferior V. cava (n = 7).
Operation
The operation was performed as an arterialized orthotopic rat liver transplantation with micro-surgical suture technique of the venous blood vessels. The bile duct and the hepatic artery were reconstructed by splint-technique. The operation, the blood sampling and the dissection were performed under Isoflurane anaesthesia. At the beginning of the explantation, the graft liver was mobilized. After that, a Polyethylene-splint was inserted into the bile duct before it was cut off (22-G, Vasculon® Plus; Ohmeda AB, Helsingborg, Sweden). The perfusion was performed via the abdominal aorta with 0.9% NaCl solution at a temperature of 4 °C. The supra- and infra-hepatic V. cava, as well as the portal vein, were cut off leaving a maximum stump length at the liver. The hepatic artery together with the truncus coeliacus and an aortal patch were left on the liver. The graft liver was preserved in 0.9% NaCl solution for 2 h at a temperature of 4 °C. We choose this short time interval to prevent the liver from extensive damage caused by preservation.
To start with the host operation in group I and group II, the original liver was removed before the graft liver was orthotopically placed into the situs. Then, the anastomosis of the suprahepatic inferior V. cava and the portal vein was performed by microsurgical suture technique. The next step was the anastomosis of the artery with a heparinized plastic splint (24-G, Vasculon® Plus, Ohmeda AB). In group I, the blood flow was opened via portal vein and suprahepatic inferior V. cava, which ended the anhepatic period. After 6 min, the blood flow of the liver artery was released too. In group II, the reperfusion via the portal vein and liver artery was performed simultaneously. After reperfusion, the infrahepatic inferior V. cava anastomosis was performed by microsurgical suture technique and the bile duct was reconstructed by splint technique in both groups. In group III, the anastomosis of the suprahepatic inferior V. cava was performed first, as in groups I and II. After that, the anastomosis of the infrahepatic inferior V. cava was performed by microsurgical suture technique. Then, the blood flow was opened via the inferior V. cava, which led to a retrograde perfusion of the liver with bleeding out from the portal vessel stump. After loss of 0.1 ml blood, it was occluded by a vessel clamp. Now, the anastomosis of the portal vein by microsurgical suture technique and the anastomosis of the liver artery by splint technique were performed. The liver artery was opened 6 min after the portal vein. The bile duct was reconstructed by splint technique. It took 22.0 (±3.0) min for placing the graft liver into the host situs to reperfusion in all groups.
Measurement of the serum parameters
Blood was sampled 1, 24 and 48 h after oRLTx under Isoflurane anaesthesia and the aspartate amino transferase (AST), alanine amino transferase (ALT) and glutamate dehydrogenase (GLDH) levels of the host serum were measured.
Histology
Liver tissue was taken 48 h after transplantation and fixed in buffered formalin. After embedding in paraffin, 5-μm-thick sections were stained with haematoxylin and eosin. In all groups, the right half of the Lobus medianus, the Lobus lateralis dexter and the Lobus lateralis sinister were examined. The histological analysis was performed by two pathologists who were unaware of the treatment groups.
Statistical analysis
Data were expressed as mean ± SD. Statistical differences between the control and the experimental groups were analysed using one-way anova. A P-value of <0.05 was considered significant. The statistical analysis of the histological parameters was carried out using the ‘Fisher-Test’.
Results
Serum parameters
Table 1 shows the values of AST, ALT and GLDH in the serum of animals 1, 24 and 48 h after oRLTx.
Table 1.
Serum AST, ALT and GLDH levels
| U/l | Group I | Group II | Group III |
|---|---|---|---|
| AST after 1 h | 1893.4 (±402.4) | 2026.4 (±350.7) | 1756.0 (±367.3) |
| AST after 24 h | 1723.7 (±235.5) | 2613.3 (±343.9) | 1186.4 (±252.9) |
| AST after 48 h | 425 (±86.9) | 588.3 (±67.9) | 213.7 (±44.1) |
| ALT after 1 h | 1688.86 (±442.80) | 1677.71 (±285.85) | 1462.14 (±357.52) |
| ALT after 24 h | 1089.71 (±171.02) | 1598.43 (±159.36) | 662.57 (±160.7) |
| ALT after 48 h | 214.57 (±46.1) | 332.0 (±43.9) | 106.29 (±31.99) |
| GLDH after 1 h | 138.89 (±52.55) | 180.97 (±63.51) | 237.63 (±103.84) |
| GLDH after 24 h | 975.71 (±426.84) | 1500.57 (±323.75) | 610.0 (±211.47) |
| GLDH after 48 h | 489.0 ±145.96) | 921.97 (±178.19) | 267.81 (±66.51) |
AST, aspartate amino transferase; ALT, alanine amino transferase; GLDH, glutamate dehydrogenase.
Significant differences of AST levels after 24 h could be seen between group I and group II (P= 0.008) and between group II and group III (P < 0.001). In comparison, after 48 h, there was a significant difference between all groups (group I vs. group II: P = 0.039; group I vs. group III: P = 0.01; group II vs. group III: P < 0.001.
Significant differences of ALT levels were seen after 24 h (group I vs. group II: P = 0.009, group I vs. group III: P = 0.025, group II vs. group III: P < 0.001) and 48 h (group I vs. group II: P = 0.016, group I vs. group III: P = 0.024, group II vs. group III: P = 0.024) between all groups.
After 24 h, the mean GLDH value of group III was significantly lower than the mean value of group II (P = 0.002). After 48 h, the GLDH values of group I and group III were significantly lower than that of group II (group I vs. group II: P = 0.009; group III vs. group II: P < 0.001).
Histology
Type of necrosis
We differentiated four kinds of necrosis as follows: No necrosis, single-cell necrosis, isolated necrosis and confluent necrosis. The distribution of the necrosis types per group is shown in Table 2. In a statistical comparison, significant differences can be seen between group I and group II (P = 0.029).
Table 2.
Frequency of type of necrosis in per cent
| Type of necrosis | Group I (%) | Group II (%) | Group III (%) |
|---|---|---|---|
| No necrosis | 0 | 14.30 | 42.90 |
| Single cell | 0 | 14.30 | 0 |
| Isolated | 14.30 | 14.30 | 42.90 |
| Confluent | 85.70 | 57.10 | 14.30 |
Number of necrotic spots
The number of necrotic spots was counted per image area. A significant lower number was found in group III compared with groups I and II. The mean number of necrotic spots was 11.57(±4.97) in group I, 9.0(±2.8) in group II and 1.57(±1.1) in group III (Figure 1).
Figure 1.
Number of necrotic spots per image area. Significance between group I and group III: P = 0.007 and between group II and group III: P = 0.026.
Minimum size of necrotic spots
There were no significant differences between the minimum size necrotic spots in groups I, II and III.
Maximum size of necrotic spots
As an essential part of the histological analysis, the mean maximum size of the necrotic spots was evaluated. The mean maximum size was 3 (±0.65) mm in group I, 2.4 (±1.55) mm in group II and 0.37(±0.31) mm in group III. There were significant differences between groups I and III (P = 0.001) (Figure 2).
Figure 2.
Maximum size of necrotic fields. Detectable significance between group I and group III (P = 0.001).
Zone division of necrosis
In addition, the distribution of the necrosis between the different zones of a liver azinus, according to Rappaport, was evaluated in the histological analysis of the graft-liver tissue (Schoniger-Hekele et al. 2002; Snover et al. 1987). In Zone 1, the frequency of necrosis was 71.4% in group I, 42.9% in group II and 0% in group III. The differences showed a significance between groups I and III (P = 0.021). In Zone 2, the frequency of necrosis was 100% in group I, 71.4% in group II and 14.3% in group III. The differences showed a significance between groups I and III (P = 0.005). The highest number of necrosis could be seen in Zone 3 of all groups. Group I showed a necrosis occurrence of 100%, group II of 85.7% and group III of 57.1%. No significant differences were found between groups.
Discussion
The major finding of this study on the ischaemia-/reperfusion (I/R) injury after liver transplantation in the rat is that the liver damage after oRLTx with retrograde reperfusion is clearly less than after oRLTx with antegrade reperfusion.
As the parameters for liver damage, we chose serum transaminases and serum GLDH, which are also used as liver-damage parameters in routine clinical diagnosis (Sezer et al. 2001). Furthermore, we performed an extensive histological examination of the liver tissue, 48 h after liver transplantation. At this point, liver damage caused by other reasons than I-/R injury in a syngen oRLTx seems unlikely. We could show that AST and ALT levels were significantly lower in the retrograde reperfused graft livers than in the antegrade reperfused livers after 48 h. Similarly, ALT levels were significantly lower in the retrograde reperfused livers after 24 h. After 24 and 48 h, the GLDH levels in the retrograde reperfused group were significantly lower than in Group II. These subjective parameters, which indicate less cell damage in Group III livers, are supported by the histological findings, which show less and smaller necrotic spots in the retrograde reperfused livers. Furthermore, the most extensive collection of necrosis was seen in ‘Rappaport′s zone’ 3. The reason for this is, in our opinion, the microcirculatory disturbances because of I-/R injury (Vollmar et al. 1994). After retrograde reperfusion, we observed the lowest number of necrosis here but without the proof of significance.
Altogether, our data suggest that retrograde reperfusion in liver transplantation decreases the occurrence of an I/R injury in the rat. These data are also supported by the findings of two retrospective clinical trials (Kniepeiss et al. 2003; Heidenhain et al. 2006). However, Heidenhain et al. found a significantly higher occurrence of bile duct complications after liver transplantation with retrograde reperfusion (Heidenhain et al. 2006). Nevertheless, retrograde reperfusion in liver transplantation should be investigated in further clinical trials because of its obvious beneficial effect on I-/R injury.
Acknowledgments
We thank Dr Peter Luppa (MD) and Ms Ingrid Cepeha from Department of Clinical Chemie (Technical University Munich) for their help in measuring the serum parameters. We acknowledge the efforts of Ms Elisabeth Kern and Mrs Lloyd-Wiedemann in reviewing and carefully reading the manuscript.
Author contribution
Kern, H., Matevossian, E., Stangl, M. and Thorban, S. designed research. Kern, H., Bald, C., von Weihern, C., Fend, F. and Brill, T. conducted research. Kriner, M., Hüser, N. Kern, H., Matevossian, E. and Stangl, M. analysed data. Kern, H., Matevossian, E.and Bald C wrote the paper.
Conflict of interest
We, the authors, declare that we have no commercial association that might pose a conflict of interest in connection with the submitted article. We hereby disclose any financial and personal relationship with other people and organizations that could have influenced our work.
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