Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats.
To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed.
Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (
Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.
Hepatic dysfunction due to ischemic injury by hemorrhage or prolonged interruptions of blood supply during liver resection and liver transplantation have been continuing problems in the past few years.
One method reported to reduce injury is ischemic preconditioning (IP), in which blood flow is blocked for a short time initially with recovery of blood flow by reperfusion, subsequently allowing blood flow to be blocked for a relatively long period.
Therefore, this study examined the difference in liver regeneration capacity after ischemic injury using the liver cirrhosis animal model established previously.
Experimental animals were maintained in a pyogen-free (SPF) animal facility at the Clinical Research Center, Yonsei University College of Medicine, under constant room temperature (22℃) and humidity (55%), with a supply of sterile drinking water and sterile animal feed according to the standards of the Association of Assessment and Accreditation of Laboratory Animal Care International. In addition, animal experiments were conducted after approval of the experiment procedures by the ethical animal experiment guideline established by the Department of Experimental Animals, Clinical Medical Research Center, Yonsei University College of Medicine.
Liver cirrhosis was induced by injecting 300 mg/kg thioacetamide (Sigma, St. Louis, USA) intraperitoneally into 8 week-old Sprague-Dawley (SD) white rats, twice a week, for 16 weeks.
To compare the extent of liver damage according to the ischemic period, the left portal pedicle including hepatic artery and portal vein was clamped for 30, 60, 90, or 120 min, and liver tissue was obtained from the ischemic area. The degree of apoptosis was assessed by the ApopTag Peroxidase
Liver cirrhosis-induced Sprague-Dawley white rats (N=12) were equally (N=3) divided into the non-clamping (NC), total clamping (TC), IP, and the intermittent clamping (IC) groups (
Zoletil (30 mg/kg, Virbac, Carros, France) was intraperitoneally injected and after the animal was fully sedated, the abdomen was opened through a midline skin incision and the liver hilum was exposed. The left portal pedicle was identified and ischemia was induced by blocking blood flow through clipping the portal vein and hepatic artery running into the left hepatic lobe using aneurysmal clips. Immediately after the induction of ischemic injury or 90 min after laparotomy in the NC group, the total injured liver, approximately 75% of the area of the total liver was resected.
Immediately prior to ischemic injury (day 0) as well as 1, 3, 5, and 7 days after injury, rats were anesthetized and blood was collected from the tail vein. The blood was centrifuged at 3,000 rpm for 15 min, and using the supernatant, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a biochemical analyzer at the Korean Animal Clinical Research Center.
In each experimental group, the presence or absence of liver cirrhosis was evaluated by trichrome staining (HT15 Trichrome Stain [Masson] Kit, Sigma, St. Louis, USA) as follows. The deparaffinized slides were hydrated, incubated with heated Bouin's solution (56℃) for 15 min, and cooled by immersing in water. They were washed until the yellow color disappeared from the tissues, and stained with Weigert's Iron Hematoxylin solution for 5 min. Slides were washed with running water for 5 min and immersed in distilled water for a while followed by staining with Biebrich Scalet-Acid fuchsin for 5 min, and incubation in phosphotungstic/phosphomolibdic acid solution for 5 min. They we then incubated in aniline blue solution for 5 min, incubated with 1% acetic acid for 2 min, washed, and dehydrated with alcohol, and then collagen fibrosis was examined.
To evaluate the level of hepatocyte injury caused by liver ischemia in the experimental groups, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay were performed as follows.
Liver tissues were fixed with 10% formaldehyde and prepared as paraffin slides. After the deparaffinization procedure of treating sequentially with xylene, and then 100%, 75%, and 50% ethanol; hematoxylin staining, washing, eosin staining, washing, dehydration with lower concentration of alcohol up to high concentration, and xylene treatment were performed sequentially, and the injury level of liver tissues was assessed by hyalinization.
The level of liver injury in the groups was compared by extracting the liver immediately after ischemic injury induction and performing TUNEL assay. In brief, after sequential deparaffinization and washing with PBS, the slides were incubated with proteinase K for 15 min, treated with 3% hydrogen peroxide for 15 min, and washed with PBS. The slides were pretreated with the equilibration buffer (19054; QIAGEN, USA) for 20 min and reacted with the TdT mixture for 1 hour in a 37℃ humidified chamber. Samples were then treated with the stop/wash buffer for 10 min, and washed with PBS. Antidigoxigenin conjugated solution at room temperature was added for 30 min, washed with PBS, followed by reaction with diethyl aminobenzidine (00-2014; Invitrogen, San Francisco, CA, USA) for 5 min. Subsequently, apoptosis was examined.
To evaluate the cell division capacity, proliferating cell nuclear antigen (PCNA) staining was performed as follows: Liver extracted at the time of sacrifice of cirrhosis rat model was added to 10% formalin solution, and embedded in paraffin by conventional methods. After the hydration procedure with Xylene and alcohol, the slides were incubated with anti-PCNA antibody diluted to 1:50 at 4℃ for 24 hours, washed with PBS, and then incubated with anti-mouse immunoglobulin G antibody (Sigma M9902) diluted to 1:100 at room temperature for 40 min. They were then incubated with the PAP Complex (Sigma P2416, in mouse) diluted to 1:100 at room temperature for 40 min, and stained with 0.025% 3,3-diaminobenzidine in PBS and 0.045 % hydrogen peroxide at room temperature for 3 min, and counterstained with hematoxylin. PCNA showing immune response was extracted by the hydrogen peroxide method, and examined under a light microscope. After counting 25 fields, the averages were obtained, and statistical analysis was performed. After PCNA staining, PCNA-positive cells were counted in a 10-µm × 10-µm area. At 400× magnification, 25 areas were selected randomly, and the number of stained cells according to each staining method was counted (cells/field).
Using the statistics program, SPSS 15.0 (SPSS, Chicago, IL, USA), differences in apoptosis and cell division capacity among the experimental groups were assessed by analysis of variance. The results were presented as mean±standard error of the mean, and cases with
After 60 min of ischemia, the degree of injury was not significant as evaluated by assessing for apoptotic cells in areas of Zone I. After 90 min, the extent of the injured area in Zone I had expanded; and after 120 min, most areas of the liver tissue were severely injured (
On day 1, ALT and AST were markedly increased in the TC and IP groups with the highest level of both AST and ALT in the IP group on day 1, and returned to basal levels on day 3 and 5 after ischemic liver injury (
Following the induction of ischemia according to the various methods, liver tissue obtained from the ischemic injury area was stained with H&E, and the degree of tissue injury was examined (
To assess the effect of ischemia on liver generation capacity in each group, rats were sacrificed 7 days after hepatectomy, and the residual liver was stained with H&E (
With accumulated experience with hepatectomy and liver transplantation, as well as the development of new techniques, indications for the procedures have been increasing. However, apoptosis of liver cells occurring during hepatectomy and liver transplant may be detrimental for patients, thus prompting recent active studies on the apoptosis occurring during liver ischemia and reperfusion.
Ischemic injury was induced by clipping the portal vein and hepatic artery entering the left lobe of the liver. In the total ischemic model, severe injury to the liver tissue and hepatocyte apoptosis was observed. During ischemia, leukocytes increase, macrophages are activated, and the injury of liver tissues and hepatocytes is mediated by the interaction of the inflammatory reaction, free radical production, and apoptosis. These mechanisms lead to injury at the time of reperfusion following ischemia, and the disruption mitochondrial ion homeostasis by hepatocyte ischemia causes cellular calcium influx resulting in protein and DNA damage. Xanthine oxidase synthesis, which generates free radicals, is also accelerated.
It has been reported that IP can be applied to prevent liver injury, and that the activation of Kupffer cells was lower than TC, with the major effect of protecting hepatocytes and sinusoidal cells from apoptosis.
In total ischemic treatment that blocks blood flow completely, the level of liver injury is severe, and liver regeneration is difficult after surgical treatments. In intermittent treatment, repeated ischemia and reperfusion is burdensome, and during reperfusion, hemorrhage may be induced at the resected surface. In comparison, IP treatment induces relatively less liver injury, and may result in more effective liver regeneration after surgery. Ischemic treatment during liver transplant or liver resection may cause fatal injury to liver tissue, and thus when surgical treatments are performed, more effective methods should be developed to minimize liver damage, and promote regeneration.
Cytokines and growth factors expressed during liver regeneration are associated with expression of their receptors. It has been reported that during liver resection or injury, epithelial-cell growth factor receptor and the hepatocyte growth factor receptor c-Met are activated.
Our study performed the IP procedure that has been used to reduce ischemic injury, and the level of liver injury and regeneration capacity were compared to other procedures blocking blood flow. Particularly, since most liver transplants are associated with liver cirrhosis, animal models should also have similar clinical features. Generally, for the induction of liver cirrhosis in rats, thioacetamide, carbon tetrachloride, dimethylnitrosamine, and other drugs are used. Carbon tetrachloride needs to be diluted in oil, and thus intraperitoneal administration is difficult. In addition, it may induce pain in rats, and thus is not efficient.
First, to determine the ischemic period, the level of apoptosis according to various periods were compared. If the injury is too small, it would be difficult to distinguish the level of injury from the regeneration capacity. On the other hand, if injury is too severe, the regeneration capacity would noticeably deteriorate. Hence, in our study, 90 min was selected as the optimal ischemic period. After ischemia, the level of liver-cell damage was most severe in TC group. The IP group also developed injury; nonetheless, comparison of the regeneration of hepatocytes shows that the regeneration capacity of IP group was good. This could be explained by the fact that in liver damage, cell proliferation is an indicator of liver regeneration, and in IP treatment, increased resistance by the suppression of c-fos gene during ischemia and reperfusion could contribute to reduced injury.
This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084120).
alanine aminotransferase
aspartate aminotransferase
hematoxylin and eosin
intermittent clamping
ischemic preconditioning
non-clamping
phosphate buffered saline
proliferating cell nuclear antigen
total clamping
terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling
Experimental protocols. Eachcolumn is 5 min. Blue columns indicate ischemic clamping time. Data in all panels are representative of at least three independent experiments (N=3 rats per group).
Degree of apoptosis of the liver tussue from the rats after ischemic injury assessed by ApopTag peroxidase in situ apoptosis detection kit (×40). The brownish spots indicated by arrows represent apoptotic cells. (A) 60-min ischemic injury, (B) 90-min ischemic injury, and (C) 120-min ischemic injury.
Level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 0, 1, 3, 5, and 7 days after ischemic injury (N=3 rats per group).
NC, non-clamp; TC, total clamp; IP, ischemic precondition; IC, intermittent clamp.
H&E (A-D), trichrome (E-H), and TUNEL (I-L) stains of the liver after various ischemic injuries (×40). The arrow identifies the thick blue bundles of separating collagen (A, E, and I: non-clamp [NC]; B, F, and J: total clamp [TC]; C, G, and K: ischemic precondition [IP]; D, H, and L: intermittent clamp [IC]).
H&E (A-D), trichrome (E-H), and proliferating cell nuclear antigen (PCNA) stains (I-L), (×100) at 7 days after various kinds of ischemic injury (A, E, and I: non clamp [NC]; B, F, and J: total clamp [TC]; C, G, and K: ischemic preconditioning [IP]; D, H, and L: intermittent clamp [IC]).
The numbers of proliferating cells were measured by proliferating cell nuclear antigen (PCNA) stain (×100) on cirrhotic liver 7 days after various kinds of liver ischemic injury (*
NC, non-clamp; TC, total clamp; IP, ischemic precondition; IC, intermittent clamp.