Hepatic ischemia/reperfusion injury (IRI) occurs primarily in the course of extended liver resection procedures necessitating the performance of a prolonged Pringle maneuver after liver transplantation as well as after cardiogenic or hemorrhagic shock. IRI may not only cause severe prolonged disturbances of liver function or liver failure, but also involve other organ systems, especially the lung and the kidneys (1-3) and is therefore of highest clinical importance.
The loss of mitochondrial adenosine triphosphate (ATP) production is thought to be closely related to ischemia. ATP depletion results in disrupted cellular volume control and disintegration of mitochondria. Some researchers have suggested that these phenomena may be responsible for cell death after longer periods of anoxia (4, 5). There is a broadly shared consensus that reactive oxygen species (ROS), which are produced under conditions of prolonged warm ischemia and subsequent reperfusion, play an important role in the induction of IRI (5, 6). Under these conditions, tumor necrosis factor α (TNF-α) plays a key role in signaling the pathway of IRI. TNF-α is primarily expressed by Kupffer cells, the resident macrophages of the liver, during inflammatory processes, such as IRI (7, 8). Despite its role as a mediator of cell damage and inflammation, TNF-α is also known to be one of the major growth factor of hepatic regeneration (9).
A number of substances capable of scavenging cytotoxic oxygen radicals have been studied in both animal models and humans, and a hepatoprotective effect has been shown for several substances, such as α-tocopherol, glycine, or glutathione (10-12). In addition, ischemic preconditioning (IPC) is one of the most effective strategies to decrease liver injury in both animals and human studies (13). Interestingly, however, in case of small liver remnants, the protective effects of IPC were lost (14), and in this context, IPC also seems to impair liver regeneration.
In view of the fact that preconditioning strategies in liver surgery needs to be rapid, readily performed, and without side effects, we aimed to characterize the potency of α-lipoic acid (LA) in pharmacological preconditioning of the liver. LA is a disulfide with coenzyme function in mitochondrial multienzyme complexes. The significant reduction potential of α-LA defines the substance as a radical scavenger with an antioxidant effect (15, 16). In addition to these antioxidant effects, LA has been shown to activate specific cytoprotective cell signaling pathways (17). The mechanism of action of LA in IRI of the liver in vivo are, as yet, completely unknown.
The current study should therefore clarify the mechanism of action of LA in ischemia/reperfusion of the liver in vivo and test the impact of LA on liver regeneration.
MATERIALS AND METHODS
Male Brown Norway rats weighing 175 to 200 g (Harlan, Paderborn, Germany) were used. All animals had access to water and rat chow ad libitum (Global Diet; Harlan); APS Guiding Principles in the Care and Use of Animals were adhered to. The animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). The study was registered with the Institutional Animal Care and Use Committee.
In a model for acute experiments, 90 min of ischemia of the left liver lobe was performed by clamping the corresponding branch of the portal vein, the hepatic artery, and bile duct proximal to its origin. Reperfusion was initiated by removal of the clamp. Thereafter, the abdominal cavity was closed with running suture, and animals were allowed to awake. After 1 h of reperfusion, anesthetized animals were killed, and liver tissue and plasma were stored for further examination. Three experimental groups were studied: (a) sham-operated animals (n = 3); (b) 90 min of ischemia with 1 mL of normal saline via the abdominal vena cava 15 min before ischemia (n = 5, no treatment [NT]), and (c) 90 min of ischemia with 500 μmol of LA (120 μg) via the abdominal vena cava 15 min before ischemia (n = 5, LA pretreatment [LA]). The body temperature was monitored and maintained at 37 ± 0.4°C by heating lamp.
Long-term outcome and animal survival were investigated by resecting nonischemic liver tissue at the time of reperfusion. After inhalation anesthesia with ether, the abdominal cavity was accessed through a midline incision. Inflow occlusion of the median lobe was performed by placing a microvascular clamp to the median branch of the portal vein, hepatic artery, and bile duct. Ischemia was maintained for 90 min. Immediately after ischemia, the nonischemic liver tissue (i.e., the caudate and the right and left lobes of the liver) was resected (70% liver resection). One group of animals was monitored for 30 days. Another group of animals was killed 32 h after operation for histology and immunohistochemistry.
Three experimental groups were studied: (a) animals that were only treated by liver resection of the caudate and the right and left lobes of the liver (70% liver resection) without ischemia and with 1 mL of normal saline via the infrahepatic vena cava 15 min after laparotomy (n = 3, resection only), (b) 90 min of ischemia of the median liver lobe with 1 mL of normal saline via the abdominal vena cava 15 min before ischemia and resection of the caudate and right and left lobes of the liver (70% liver resection, n = 5 NT), and (c) 90 min of ischemia with 500 μmol LA (120 μg) via the abdominal vena cava 15 min before ischemia and resection of the caudate and the right and left lobes of the liver (70% liver resection, n = 5, LA). Animal survival was calculated using the Kaplan-Meier method.
In our previous study, we could demonstrate reduction of IRI by 50 μmol of LA in our in vitro model (17). Therefore, we tried to show the influence of 50 μmol of LA in our described animal survival model; however, we observed that after pretreatment with 50 μmol of LA, no animals survived (n = 3, data not shown). Next, we used 500 μmol of LA to reduce IRI of the liver. Here, we could observe a massive reduction of IRI of the liver in the same model.
Homogenization of liver tissue
The liver tissue was weighed before homogenization and diluted 1:5 with 0.01 mol/L phosphate-buffered saline. Homogenization was then carried out with a Potter tissue homogenizer. A homogeneous mixture was achieved at 800 rpm. Next, the mixture was centrifuged at 1000g for 10 min. The supernatant was pipetted into an Eppendorf cup and centrifuged at 10,000g for 20 min. The clear supernatant was pipetted into a fresh Eppendorf cup and stored frozen at −20°C until further measurement.
Blood samples for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assessment were obtained and analyzed using a serum analyzer (Olympus AU Connector 640; Olympus, Hamburg, Germany).
Determination of lipid peroxidation
Determination of lipid peroxidation (LPO) in homogenized liver tissue was carried out with a standardized test (Cayman Chemical, Grünberg, Germany) kit in accordance with the manufacturer's instructions.
Measurement of ATP
Measurement of ATP in liver tissue was carried out using a standardized method as described by Trautschold et al. (18). The liver slices were weighed before homogenization and diluted in 4% HClO4 (1:10). After homogenization, the supernatant was pipetted into an Eppendorf cup and neutralized with 150 μL of 2 mol/L Tris, 150 μL of KOH, and phenol red. Then 5 mol/L of KOH was added until a blue-lilac complex was observed. The mixture was again centrifuged at 10,000 U/min, 5 min, and 4°C. The supernatant was measured immediately by UV photometry and hexokinase/glucose-6-phosphate dehydrogenase.
Histology and immunohistochemistry
Formalin-fixed tissue samples were embedded in paraffin, and 5-μm sections were cut. Replicate sections were stained with hematoxylin-eosin (H&E) for the evaluation of oncosis and liver regeneration, stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and stained for Ki-67 protein. All histological evaluations were done in a blinded fashion.
Oncotic cell injury was evaluated in serial sections stained with H&E. Morphological criteria of oncotic cell injury such as increased swelling, vacuolization, and blebbing were used. The percentage of oncosis was estimated by evaluating the number of microscopic fields with oncosis compared with the entire histological section.
To determine hepatocyte replication, mitotic figures were counted in 10 high power fields (HPF, 400-fold magnification) and given as mitotic index (number of mitotic figures per HPF).
The monoclonal antibody MIB-5 (DAKO, Hamburg, Germany) was used for immunohistochemical analysis of hepatocellular regeneration. After 20 min of microwave pretreatment of dewaxed formalin-fixed paraffin sections in citrate buffer (pH 6.0), immunostaining was performed on an automated immunostainer (Techmate 500plus; DAKO) using an DAB peroxidase-based detection system (K5001; DAKO). The proliferative activity was assessed by counting the number of labeled hepatocytes nuclei per HPF (400-fold magnification) and given as proliferative index (number of Ki-67-positive cells per HPF).
To determine apoptotic cell injury, we perform TUNEL staining: livers were fixed with freshly prepared 4% paraformaldehyde in neutral phosphate-buffered saline. Frozen sections (5 μm) of the fixed tissue were prepared and stained with the TUNEL method using a commercial kit (ApoTag peroxidase apoptosis detection kit; Chemicon International, Hampshire, UK). The number of TUNEL-positive hepatocytes was counted in 10 HPF (400-fold magnification).
Before cutting the liver into slices and shock freezing, a small liver sample obtained from each liver was fixed using 0.1 mmol/L glutaraldehyde (Sigma, Hamburg, Germany). Fixation of the tissue was carried out in preparation for electron microscopy: the tissue was subsequently stained using methylene blue. All other procedures were done according to standardized techniques.
Phosphorylation of Akt
Phosphorylation of Akt was determined by Western blot as previously described (17).
Caspase activity in liver tissue
Caspase 3, 8, and 9 activity was measured using a colorimetric reaction (Caspase Colorimetric Assay; R&D Systems Germany, Wiesbaden, Germany) at a wavelength of 405 nm. All samples were prepared in pairs, one pair was measured with and one without substrate, according to the manufacturer's instructions. The results are expressed as fold increase in caspase activity in postischemic versus sham liver tissue (19).
Determination of TNF-α in liver tissue and serum
TNF-α determination in homogenized liver tissue and serum was carried out using a commercial enzyme-linked immunosorbent assay (R&D Systems Germany) kit in accordance with the manufacturer's instructions.
Polymerase chain reaction
The expression of TNF-α, Bax, and Bcl-2 was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The expression of 18 S was used as the housekeeping gene. Total RNA was isolated using Qiagen (Hilden, Germany) RNeasy Mini-Kits. After photometric determination of the amount of RNA, first-strand complementary DNA (cDNA) was synthesized from 4 μg of RNA from each liver, using the following procedure: the transcription mix contained 5 μL of oligo(dT) (Gibco-BRL, Paisley, UK), 3.6 μL dNTP mix (10 mmol/L) (Gibco-BRL), 8 μL first strain buffer, 4 μL DTT (0.1 mol/L), 1.5 μL reverse transcriptase (MLV-T, Roche Diagnostics), and 1.5 μL RNA free water (Qiagen). Controls were performed without the addition of reverse transcriptase. Cycle conditions were chosen as follows: 90 min at 37°C, 10 min at 94°C, and 30 s at 4°C. The cDNA was checked by amplification of 18 S. Primers were designed specifically for rats with the Primer 3 Software (Whitehead Institute, Boston, Mass), and synthesized by MWG Biotech (Eberberg, Germany). The following primers were used:
- Bcl-2 neu
- forward: 5′-TGC-AGA-GAT-GTC-CAG-TCA-GC-3′
- reverse: 5′-CAT-CCA-CAG-AGC-GAT-GTT-GT-3′ (expected product 200 bp);
- forward: 5′-ACA-GAT-CAT-GAA-GAC-AGG-GG-3′
- reverse: 5′-CAA-AGT-AGA-AGA-GGG-CAA-CC-3′ (expected product 203 bp).
All sequences were compared with the complete rat GenBank library to ensure that each primer was unique for its intended target and that areas of sequence polymorphisms were avoided.
Equivalent levels of starting cDNA were used for amplification of PCR products. The master reagent mix consisted of 4 μL dNTP mix (1.25 mmol/L), 1 μL specific primer (50 pmol/μL), 1 μL Taq polymerase (5 U/μL; Boehringer, Ingelheim, Germany), and 10 μL PCR buffer (1.5 mmol/L) were heated to 93°C, for 1 min at 59°C, 1 min at 72°C, and then cooled to 4°C. The resulting PCR products were visualized by gel electrophoresis stained with ethidium bromide.
Real-time quantitative RT-PCR analysis
Real-time quantitative RT-PCR analysis of transcripts for TNF-α and glyceraldehyde-3-phosphate dehydrogenase was performed with predesigned and optimized TaqMan Gene Expression Assays (Applied Biosystems, Foster City, Calif) on an Applied Biosystems 7500 Real-Time PCR System according to the manufacturer's instructions. Relative quantification was carried out using the delta-delta-CT method.
All data are expressed as means ± SE. Statistical differences between experimental groups were calculated by GraphPad Prism (GraphPad Software, San Diego, Calif) using one-way analysis of variance followed by the Mann-Whitney U test. Survival curves were calculated using Kaplan-Meier method. Proportions were calculated using Fishers exact test. Differences were considered statistically significant at P < 0.05.
No TUNEL staining was observed in the livers of the sham-operated animals (data not shown, Figs. 1A, 5). In the untreated group, the number of TUNEL-positive cells was increased after 90 min of ischemia and 1 h of reperfusion. The LA-treated group showed a significantly lower incidence of TUNEL-positive hepatocytes at the same time point.
Oncotic cell injury
After 90 min of ischemia and 1 h of reperfusion there was a marked increase in oncotic hepatocytes in the untreated group compared with that observed in the sham-operated animals (not detectable; Figs. 1B, 5). In the LA-pretreated group, we observed a massive decrease of oncotic hepatocytes after 1 h of reperfusion (Fig. 1B).
AST, LPO, and ATP
After 90 min of ischemia and 1 h of reperfusion, AST release increased markedly in the untreated group in comparison with the sham-operated group (Fig. 2), whereas AST increased only slightly after 1 h of reperfusion in the LA group. Serum ALT levels followed a similar pattern (data not shown).
LPO in liver tissue
LPO values obtained 1 h after reperfusion showed a substantial increase in the untreated livers compared with the sham-operated group. In the LA group, LPO was reduced by about 85% (Fig. 3).
ATP content in liver tissue
In comparison with the sham-operated animals, ATP levels in liver tissue after 90 min of ischemia and 1 h of reperfusion were decreased in the untreated group to about 10% of sham-treated animals. LA induced a marked increase in ATP levels (Fig. 4).
Significant differences were found for all of the described caspases after 1 h of reperfusion after 90 min of ischemia (Fig. 6). We observed a three-fold increase of caspase 3 activity in the untreated group in comparison with sham-operated animals (Fig. 6A). Caspase 8 activity in the untreated animals was doubled in comparison with that observed in the sham-operated animals (Fig. 6B), and a three-fold increase in caspase 9 activity was noted in the untreated group compared with sham-operated animals (Fig. 6C). In the LA group, caspase 3, 8, and 9 activities were significantly decreased in comparison with the untreated group (Fig. 6, A-C).
Messenger RNA expression of Bax and Bcl-2 in liver tissue
In sham-operated animals, we saw no expression of Bcl-2 in liver tissue, whereas messenger RNA (mRNA) of Bax was detectable (Fig. 7, A and D). In the untreated group, there was an expression of Bax and Bcl-2 after 90 min of ischemia and 1 h of reperfusion (Fig. 7, B and E). In the LA-pretreated rats, PCR product of Bcl-2 was detectable at the same time point (Fig. 7E). Interestingly, mRNA of Bax was absent in the LA group after 90 min of ischemia and 1 h of reperfusion (Fig. 7C). Phosphorylation of Akt was not influenced by ischemia/reperfusion or LA (data not shown).
TNF-α level in liver tissue and serum
TNF-α was measured in both serum and liver tissue. After 90 min of ischemia and 1 h of reperfusion, we could not detect any TNF-α in serum (data not shown).
After 90 min of ischemia and 1 h of reperfusion, we observed a substantial increase of the TNF-α content in liver tissue after LA pretreatment in comparison with the livers of the sham-operated and untreated group (Fig. 9). Accordingly, our real-time RT-PCR showed significant up-regulation of TNF-α in LA-pretreated livers in comparison with the untreated group (18.1 ± 3.9 vs. 2.6 ± 0.2; Fig. 9).
We performed electron micrograph investigations after 90min of ischemia and resection of the nonischemic liver tissue and 32 h of reperfusion. We could observe mitochondrial swelling and rupture of the mitochondrial membrane in the untreated group (Fig. 10B), whereas most of the mitochondria of the LA group were without severe features of injury (Fig. 10C).
We determined the parameters of liver regeneration after 90 min of ischemia and resection of the nonischemic liver tissue 32 h after ischemia. We observed a mild increase of mitosis and Ki-67 staining in animals that were only treated by liver resection (Fig. 8, 11, A and B). After 90 min of ischemia, levels of mitotic index increased in the untreated group in comparison with animals that underwent liver resection without ischemia. In the LA-pretreated group, the mitotic index was almost doubled in comparison with the untreated group.
The proliferative index of Ki-67 staining was increased in the untreated group in comparison with animals who were only treated by liver resection. After pretreatment with LA, the proliferative index was strongly increased.
We compared the survival rates of animals from the LA-treated group, the nontreated group, and the group undergoing liver resection as the only measure (Fig. 12). All animals of the untreated group died within the first 3 days (n = 5). In the 500-μmol-LA group, 4 of 5 animals survived the 90 min of ischemia and resection of the nonischemic lobes of the liver up to 30 days after surgery. All animals with liver resection and without ischemia survived the 30-day monitoring period (n = 3).
Results of the present study demonstrate a substantial reduction in hepatic IRI accompanied by increased liver regeneration and animal survival after preconditioning with α-LA.
LA is a naturally occurring compound present in the majority of prokaryotic and eukaryotic cells. For many years, LA has been used as a pharmacological agent against diabetic polyneuropathy (20). It has also gained considerable attention as a powerful lipophilic antioxidant (21, 22). After oral and intravenous application, LA is subjected to a high hepatic extraction (23). Moreover, studies have shown that the liver has a high capacity for uptake and accumulation of LA in carrier-mediated and diffusion-transport mechanisms. The detection of biliary excretion of LA metabolites confirms that LA is transported into and not only bound to the hepatocyte (24).
Interestingly, we found that both necrosis- and apoptosis-related cell deaths were decreased in the present model under preconditioning with LA. These data are in contrast to the previous study (17), where Akt phosphorylation but not protection from apoptotic cell death was observed under LA preconditioning in isolated buffer-perfused livers. This difference could be explained by the different models of liver ischemia: protective effects of Akt activation have been described in ex vivo and in vivo settings such as isolated perfused rat liver and liver transplantation (17, 25). Studies that describe Akt activation in a model of selective and warm liver ischemia with duration of 90 min are rare.
In the present model, the application of LA leads to an increase in the ATP level in liver tissue after ischemia and reperfusion. These findings corroborate the results obtained by our previous study, showing the identical capacity of LA in isolated perfused rat livers (17). The potency of LA to increase ATP levels in rat heart mitochondria has been reported by another group (26). This gives rise to the question as to how LA increases ATP levels in liver tissue after ischemia/reperfusion. One possible answer may be the known potential of LA to reform vicinal thiol groups in the ATP synthase. Dithiols have been shown to form disulfide bridges during ischemia/reperfusion. ATP synthesis occurs only if the dithiols between the F1 and the F0− part of the enzyme remain functional (27). The lipophilic character of LA allows it to reach the inner membrane of the mitochondrion and to enhance mobility and integrity of the enzyme (28). ATP synthesis may therefore also occur under conditions of ischemia/reperfusion. Accordingly, our electron microscopic investigations show decreased injury of the mitochondrial membrane after LA preconditioning.
TNF-α plays a central role in inducing apoptosis in hepatocytes (29). A prevention of cytochrome c release, activation of caspase 3, and DNA fragmentation was observed in TNF receptor 1-deficient mice, as well as following inhibition of TNF-α release with pentoxifylline. A survival rate of 60% was noted in a model of 75-minute total hepatic ischemia in the murine liver after the application of pentoxifylline (29). In the current study, a survival rate of up to 80% was observed after preconditioning with 500 μmol of LA accompanied by reduction of necrotic and apoptotic cell death. Interestingly, increased levels of TNF-α in liver tissue paralleled the reduction of apoptosis and necrosis. The improved regeneration and animal survival in our model suggests that LA-induced TNF-α preferably acts as a growth factor and not so much as a mediator of cell death and inflammation. In contrast to the induction of TNF-α in our LA-treated animals, LA has been demonstrated to significantly attenuate the lipopolysaccharide-induced production of TNF-α in isolated Kupffer cells (30). It therefore seems that the mechanism of action of LA, either increased or decreased TNF-α expression, depends on the stimulus applied, and might also differ between isolated cells and in vivo conditions.
After pretreatment with LA, we observed decreased content of mRNA of the proapoptotic protein Bax in postischemic liver tissue, whereas Bcl-2 expression increased in the untreated and LA groups. Decreased Bax by LA might represent a crucial event in attenuating IRI of the liver. Bax is localized in the cytosol of cells and translocates to mitochondria in response to death stimuli (31). Cleavage of Bax then stimulates cytochrome c release and apoptotic cell death in hepatocytes. It seems that LA regulates Bax and Bcl-2 balance of the rat liver. Because such modulation of proapoptotic and antiapoptotic proteins is described in rat brain to reduce IRI (32), our data strongly suggest that LA protects from apoptosis because of its action on proapoptotic and antiapoptotic proteins.
Generation of ROS could lead to the activation of caspase 8 and Bid (33-36). We could demonstrate decreased LPO and decreased activation of caspase 8 after LA pretreatment. Moreover, reduction of caspase 9 and 3 activation was observed at the same time point accompanied by reduced TUNEL staining after pretreatment with LA. Our data support the hypothesis that LA also reduces apoptosis by its antioxidative potential. This conclusion is further supported when comparing the hepatoprotection potential of LA, glycine, and IP; we observed that LA and IP decrease LPO in liver tissue; however, we also observed that LA is much more effective in reducing other parameters of hepatic injury in comparison with IP (in data preparation). As expected, glycine did not affect LPO in liver tissue and other parameters of warm hepatic injury and is therefore not as effective as LA in reducing IRI of the liver (37).
Previous studies have highlighted TNF-α as major and initial growth factor involved in liver regeneration (38). After LA pretreatment, we could demonstrate significantly higher levels of TNF-α in liver tissue compared with untreated animals. Our real-time RT-PCR results underline these findings. Moreover, we could demonstrate increased features of liver regeneration by improvement of the mitotic index and Ki-67 staining, while animal survival was highly increased by LA pretreatment. These findings are completely new and underline the therapeutic potential of LA in pharmacological preconditioning. In comparison with established methods of liver preconditioning, such as IPC, this might be a powerful advantage of LA (14).
In summary, we could demonstrate impressive results about the influence of LA on IRI of the liver and liver regeneration in vivo. The potency of LA to protect from IRI and improve liver regeneration leading to dramatically improved animal survival makes it an attractive candidate for clinical application (39). In comparison with other drugs that were only used in experimental settings, this is an important difference and improvement of LA.
The authors Marianne Müller and Karin Molter from the Institute of Pathology, Johannes Gutenberg University Mainz, for excellent technical support.
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Liver surgery; ischemic tolerance; liver failure; preconditioning of the liver; apoptosis