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Interact CardioVasc Thorac Surg 2008;7:18-22. doi:10.1510/icvts.2006.150979 © 2008 European Association of Cardio-Thoracic Surgery
Hepatic injury in a rat cardiopulmonary bypass modelDepartment of Cardiothoracic Surgery, Jinling Hospital, Nanjing Clinical Medicine School of the Secondary Military Medical University, 305 East Zhongshan Road, Nanjing 210002, China Received 25 December 2006; received in revised form 2 October 2007; accepted 4 October 2007
*Corresponding author. Tel.: +86-25-80860075; fax: +86-25-84819984.
An increasing number of patients were undergoing cardiac surgery with cardiopulmonary bypass (CPB) and more attention had been paid to hepatic injury after CPB. This study was designed to study how CPB could induce and aggravate the hepatic injury in a rat model. Male Sprague-Dawley rats were randomly divided into two groups (n=12): sham and CPB groups. Blood samples were collected at the beginning, at the cessation of CPB, and at 0.5, 1, 2, 3 and 24 h post-operation. Liver samples were harvested at 24 h after operation. In CPB group, the levels of serum liver enzymes and tumor necrosis factor-  , activities of inducible nitric oxide synthase, malondialdehyde and myeloperoxidase in liver tissue were significantly increased. In addition, swollen hepatocytes, vacuolization and congestion in sinusoids were observed. On the contrary, the activities of liver antioxidative enzymes and the concentration of glutathione (GSH) decreased remarkably. All results indicated that CPB would induce and aggravate hepatic injury by facilitating oxidative stress and the systemic inflammatory response.
Key Words: Hepatic injury; Cardiopulmonary bypass; Rat; Systemic inflammation response; Oxidative stress
An increasing number of patients are undergoing cardiac surgery [1] and in most cases a cardiopulmonary bypass (CPB) is required. This technique is aggressive and the patients are subjected to a high degree of organ injury [1–3]. Today more and more studies have being focused on organ protection after CPB; however, these studies usually study organs such as heart, lung and kidney. In a clinical situation, about 10% of cardiac surgical patients with CPB, especially high-risk patients, experience hepatic injury, which directly influences their cure rate and mortality [1, 3]. There are mainly two pathophysiologic mechanisms involved in hepatic injury, the systemic inflammatory response syndrome (SIRS) and oxidative stress. However, most of them were based on liver ischemia and reperfusion (IR), toxic drug induced hepatic injury or acute inflammation models [2–6]. The need for studies concentrating on mechanisms of hepatic injury induced by CPB is essential. The aim of this study is to determine whether CPB induced and aggravated hepatic injury in a rat model by measurements of serum liver enzymes, hepatic inflammatory and antioxidant activity and histologic injury.
2.1. Animals care Male Sprague-Dawley rats (450–600 g) were used for the experiments. All animals received humane care in compliance with ‘The Principles of Laboratory Animal Care’ formulated by the National Society of Medical Research and ‘The Guide for the Care and Use of Laboratory Animals’ published by the US National Institutes of Health (Publication No. 85–23, revised 1996). The following experimental protocol was approved by the local Ethical Committee. Rats were randomly assigned to one of two groups (n=12 for each group): sham; CPB. The rat model of CPB was built as previously described in our laboratory [7] with some modifications. Rats were anesthetized with intraperitoneal administration of butaylone (50 mg/kg), thus providing stable anesthesia while maintaining spontaneous ventilation during the entire operative procedure. All subsequent procedures were performed under aseptic conditions. After surgical-level anethesia was achieved, the left femoral artery was cannulated with a 24-gauge Teflon heparinized catheter to monitor arterial pressure (AP) and to collect arterial blood for arterial blood gas analysis (blood gas analyzer, GEM Premier 3000, USA). Following administration of heparin (250 U/kg), a 16-gauge catheter, modified to a multiside-orifices cannula in the forepart, was inserted into the right jugular vein and advanced to the right atrium. A 22-gauge catheter was cannulated to the tail artery, which served as the arterial infusion line for the CPB circuit. The mini-CPB circuit comprised a venous reservoir, a roller pump, a specially designed membrane oxygenator, and sterile tubing with an inner diameter of 4 mm for the venous line and of 1.6 mm for the arterial line. We primed the CPB circuit with a total volume of 10 ml synthetic colloid (HAES-steril 9.5 ml, 5% NaHCO3 0.5 ml) solution with heparin (250 U/kg). At the initiation of perfusion, the flow-rate was gradually adjusted to sustain the mean arterial pressure (MAP) at about 80 mmHg. Gas flow (95% O2 and 5% CO2) was initiated at around 50–75 ml/min and adjusted to maintain blood gas analysis results in the physiological range. When the flow rate reached 100 ml/kg/min, it was maintained for 1 h. At the end of the CPB, the flow rate was turned down step by step in order to keep hemodynamic stabilization. When the rat was eased off CPB, the right jugular vein catheter was removed and the remaining priming solution was infused gradually through the tail artery catheter when MAP was <70 mmHg. After three hours of intensive postoperative care, the tail artery catheter and the femoral artery catheter were removed. Then the neck, tail and groin incisions were sutured. Throughout the experiment, body central temperature was monitored with a rectal probe and kept at 36.5–38.3 °C by a heat lamp placed above the animal and the CPB equipment; and MAP was maintained about 70–90 mmHg. The rats were given water and food 6 h after the operation, and they were monitored for 24 h postoperatively. Blood samples were collected from the femoral artery immediately after heparinization (T0), at the end of CPB (T1), 0.5 h (T2), 1 h (T3), 2 h (T4), 3 h (T5) and 24 h (T6) after the operation. Approximately 0.6 ml of blood was collected at each time point. The serum was stored at –70 °C in a refrigerator, which was used for later determination of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and tumor necrosis factor- (TNF-). At 24 h post operation, the rats were anesthetized again. The liver was harvested and divided into three parts. One was reserved in 10% formalin solution for microscopic examination and apoptosis detection, one was pre-fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C for electron microscopic observations, and the other was stored in liquid nitrogen until biochemical analysis. 2.5. Biochemical analysis of liver function AST, ALT and LDH were determined by an automated analyzer (Hitachi-917, Hitachi Ltd, Tokyo, Japan).
2.6. Assays of oxidative stress markers and TNF-
TNF- 2.8. Detection of apoptosis in hepatic sections Specimens obtained at 24 h after CPB were stained for DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), using a commercial kit (Roche, SUI). Ten random high-power fields were examined for each TUNEL-stained tissue sample. The number of apoptotic cells was counted and expressed as a percentage of the total number of cells (apoptotic index). All slides were examined by an independent pathologist who was unaware of the treatment groups.2.9. Electron microscopic observations Liver tissues were pre-fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 2 h and post-fixed in 1.5% osmium tetraoxide at 4 °C for 2 h. The samples were dehydrated and embedded in epoxy resin. Ultra-thin sections were cut using an Ultracut-OmU4 ultramicrotome, stained with uranyl acetate and lead citrate, and observed with an electron microscope (JEM-1200EX, JEOL Ltd, Tokyo, Japan). An experienced histologist who was unaware of the treatment conditions was invited to observe and make histological assessments. All values were expressed as mean±S.D. Data were analyzed using a commercially available statistics software package (SPSS for Windows v. 13.0, Chicago, IL, USA). One or two-way ANOVA was used for comparisons between the groups where appropriate. Post hoc comparisons were performed using LSD test or Dunnett's T3 test. P<0.05 was considered as statistically significant.
3.1. Physiological data All the rats survived from CPB procedures, and their physiologic data pre- and post-operation are summarized in Table 1.
3.2. Results of liver function and serum TNF- level
All the results are shown in Fig. 1.
3.3. Results of oxidative stress markers The effects of CPB on liver oxidative stress markers are shown in Table 2.
3.4. Histological results (Fig. 2) In sham group, hepatocytes and sinusoids presented normal morphology reflecting a well preserved liver parenchyma. No congestion was noticed in the sinusoids. However, in CPB group, hepatocytes were prominently swollen with marked vacuolization. Congestion was noticed in enlarged sinusoids (I).
TUNEL-stained sections of liver from rats in sham group revealed only scattered evidence of apoptotic cells. When liver sections from CPB group were TUNEL-stained, there was a clear increase in the number of positive cells (apoptotic index 27.4±11.7%) (II). Most of the mitochondria from the sham-operated animals were in a highly condensed form. The cristae were tightly packed and rather heavily stained. However, the mitochondria from CPB group were in a swollen state with an apparent disintegration of the cristae. Also, there were significant differences between the two groups (III).
The present study demonstrates that CPB induces and aggravates hepatic injury in a rat model of CPB, evidenced by biochemical and histopathological results. All the rats survived from CPB procedures, in the sham group the activities of ALT, AST and LDH retain a low level. An increase of ALT, AST and LDH levels is noted in the group treated with CPB. Increased levels of hepatic enzyme activity were considered as an indication of hepatocyte injury, which had been reported in previous studies [9, 10, 13]. Such results indicate that there is a severe hepatocyte injury in the liver after CPB. In CPB group, we observe significant increases in MDA, which is a product of lipid peroxidation. This finding is in agreement with a previous study that levels of lipid peroxidation (LPO) products were increased from 40 to 80% above basal value in patients undergoing CPB [15]. Furthermore, elevated levels of LPO would cause membrane pump activity reduction, such as Ca2+-ATPase, which is confirmed by the decrease of Ca2+-ATPase activity in CPB group. Nitric oxide (NO) was an important molecule mediator in hepatic injury, and mainly produced by iNOS in pathophysiological station. Compared to sham group, a remarkable elevation in activity of iNOS is observed in CPB group, which is in agreement with a previous study [11]. MPO activity, as an index to define the severity of neutrophil accumulation, is also determined in this study. Only about five-fold increase is observed in CPB group compared with sham group, which is not so much as the 18-fold enhancement of MPO activity documented in the previous study [13]. Nevertheless, the results of MPO activity still demonstrated that accumulation of a large number of neutrophils was induced by CPB supported by the remarkable increase of MPO activity in CPB group compared with sham group. A significant decrease in CAT and SOD activities and depletion of the hepatic GSH store in CPB group reveals that CPB not only dramatically elevates the severity of LPO and neutrophil accumulation but also lowers the level of antioxidants.
Increased TNF- Histological damage ranges from normal (sham group) to severe (CPB group). In sham group, hepatocytes and sinusoids are arranged around the central vein reflecting no signs of degeneration and most of the mitochondria from the sham-operated animals are in a highly condensed form. However, the tissues from CPB group show that hepatocytes are prominently swollen with marked vacuolization and the mitochondria are also in a swollen state with an apparent disintegration of the cristae. When liver sections from CPB group are TUNEL-stained, there is a clear increase in the number of positive cells, but only scattered evidence of apoptotic cells are revealed in sham group. Our experiment confirms hepatic injury in a rat CPB model in different aspects, which is evidenced by both biochemical indices and morphology examinations. And the exact mechanisms also need be studied further.
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Abstract
1. Introduction
