Isovolemic hemodilution with glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) attenuated rat liver ischemia/reperfusion injury

Abstract

This study was to investigate whether glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) could attenuate ischemia/reperfusion (I/R)-induced liver injury. Isovolemic hemodilution of SD rats was performed by exchanging 15% total blood volume with PolyPHb. I/R was induced by left liver lobes pedicle cross-clamping for 60 min and reperfusion for 2 h. Blood pressure moderately elevated after PolyPHb infusion and returned to basal level within 10 min. The hepatic histopathological damage and the activities of liver injury markers were reduced by PolyPHb. The TUNEL staining and caspase assay indicated hepatic apoptosis was also inhibited. Therefore, our findings suggest PolyPHb can reduce liver I/R injury.

Keywords::

• apoptosis
• hemoglobin-based oxygen carrier
• ischemia/reperfusion injury
• liver

Introduction

Ischemia/reperfusion (I/R) injury is a major mechanism which leads to end-organ damage. In liver transplantation and surgery, I/R injury is unavoidable for that the surgical procedure involves vascular occlusion and reopening (Tawadrous et al. 2002). Transient disruption of liver blood supply followed by reperfusion induces an acute generation of reactive oxygen (ROS) and nitrogen species. These changes initiate a chain of deleterious cellular responses leading to hepatic inflammation, apoptosis, and death (Horváth et al. 2012, Pacher et al. 2007). Even though the duration of warm ischemia in hepatic surgery minimizes the intraoperative blood loss, it may also result in postoperative liver dysfunction and failure (Garcea and Maddern 2009).

Glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) is a particularly promising hemoglobin-based oxygen carrier (HBOC) developed in China (Li et al. 2006a, 2006b). It allows transport of more oxygen (O₂) to hypoxic tissues owing to its higher O₂ affinity, lower viscosity, and smaller mean diameter than human red blood cells, which suggests that PolyPHb may be helpful in providing sufficient microcirculation perfusion and thereby alleviating organ I/R injury (Wu et al. 2011, 2009). Our previous studies have demonstrated that PolyPHb provided protections on various organs against I/R injury, including heart (Li et al. 2009a, 2010a, 2009b, 2010b, 2011), lung (Li et al. 2013), and kidney (Li et al. 2012). The underlying molecular mechanisms are implicated in the attenuation of apoptosis, quenching of oxidative stress, and restoration of nitroso–redox balance (Li et al. 2009a, 2009b, 2010b). Here, we tested the hypothesis that isovolemic hemodilution with PolyPHb could also protect liver from warm I/R injury.

Materials and methods

This study was approved by the Institutional Animal Care and Use Committee of Sichuan University, and all animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Thirty adult male Sprague-Dawley (SD) rats, approximately 10 weeks old, and weighing 200–250 g, were housed at constant temperature (22 ± 3°C) on a 12–12 h light/dark cycle and given free access to food and water.

Preparation of PolyPHb

The PolyPHb was prepared as previously described (Li et al. 2009a, 2006a, 2006b). Briefly, fresh human placenta blood (Tianjin Union stem cell genetic engineering Ltd, Tianjin, China) was washed and suspended in hypotonic phosphate buffer solutions (PBS) to get free hemoglobin. Then hemoglobin purification and viral inactivation were achieved by heat treatment (60 ± 1°C for 10–12 h). The resulting hemoglobin was modified with pyridoxal phosphate to obtain optimal O₂ affinity. After cross-linkage with glutaraldehyde, ultrafiltration and molecular sieve chromatography were employed to harvest PolyPHb with molecular weight of 64–600 kDa. In the final product, we did not add antioxidants such as superoxide dismutase and catalase.

Animal preparation and experimental protocol

Thirty male SD rats, weighing 200–250 g, were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and heparin (500 IU). After endotracheal intubation, all the rats were mechanically ventilated with 95% O₂ at respiratory rate (RR) of 30 bpm and tidal volume (Vt) of 6 ml/kg (DH-150, Medical Instrument Company of Zhejiang University, Hangzhou, Zhejiang, China). Anesthesia was maintained by injection of 15 mg/kg sodium pentobarbital via tail vein. Body temperature was maintained at 36–37°C during the experiment with a heating blanket. A polyethylene catheter was placed in the left femoral artery to withdraw blood and measure blood pressure using PowerLab data-acquisition system (ADInstruments Pty, Bella Vista, NSW, AUS). Another polyethylene catheter was placed in the left femoral vein for infusion of PolyPHb or blood.

Rats were randomly divided into three groups (n = 10 in each group): sham group, whole blood group, and PolyPHb group. Total blood volume was calculated from 7% of body weight. Fifteen percent of total blood volume was removed from femoral artery and equivalent volume of PolyPHb (PolyPHb group) or the shed blood (whole blood group) was infused via femoral vein. After 30 min of stabilization, rat model of liver I/R injury was established as reported previously with modifications (Fukai et al. 2005). Briefly, a transverse abdominal incision was performed and the left liver lobes were mobilized. Liver ischemia was induced by left liver lobes pedicle cross-clamping for 60 min. The Yasargil clip was then released to allow 2 h of reperfusion. After that, all the rats were sacrificed using a bolus of i.v. sodium pentobarbital (120 mg/kg). The experimental protocol is schematically shown in Figure 1.

Figure 1. A schematic representation of the experimental protocol.

Determination of activities of liver enzymes

Blood samples were collected before liver ischemia and 2 h after reperfusion. After isolation of plasma by centrifugation, the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by use of an Auto Clinical Chemistry Analyzer (BS-120, Mindray, China).

Histological examination

The left lateral lobe of the liver was placed in 10% neutral formalin and embedded in paraffin, then sectioned into 5-μm intervals and stained with Hematoxylin and Eosin (H&E). A pathologist blinded to the treatment was assigned to assess the histopathological changes of live tissue. The extent of hepatocyte apoptosis was determined using terminal dUTP nick labeling (TUNEL) by commercial kit (Promega, Madison, WI, USA).

Measurement of caspase-3/7 activity

Liver tissue was homogenized and centrifuged. The protein concentration of its supernatant was determined using the BCA method (Pierce, Rockford, IL, USA). Then, the activity of caspase-3/7 was determined by a Caspase-Glo 3/7 assay kit (Promega).

Statistical analysis

All values in the text and figures were presented as mean ± SD. The blood pressure was analyzed by the Student's t test for two group comparisons. Other data were subjected to one-way ANOVA followed by LSD correction for post hoc t test (SPSS 13.0 software). P values < 0.05 were considered statistically significant.

Results

Effect of isovolemic hemodilution with PolyPHb on blood pressure

The effect of isovolemic hemodilution with PolyPHb on blood pressure was evaluated. As shown in Figure 2, the blood pressure was quickly increased after PolyPHb infusion, which was higher than that after infusion of shed blood (P < 0.05). However, 10 min after infusion, the blood pressure of both groups recovered to basal levels.

Figure 2. The blood pressure at baseline and during the period of 30 min after whole blood or PolyPHb infusion. Values are presented as mean ± SD (n = 10). *P< 0.05 vs. the whole blood group.

PolyPHb attenuates activities of markers of hepatic I/R injury

To assess the liver damage after I/R injury, the plasma transaminase activities (AST and ALT) and markers of necrosis were measured. Before ischemia, the plasma levels of ALT and AST activities were similar among groups. While after liver I/R injury, a dramatic increase in liver enzyme activities were observed in the whole blood group (all P < 0.001 vs. the sham group, Figure 3), indicating a significant hepatocellular damage. In the PolyPHb group, the I/R-induced enzyme activity increases were greatly reduced (all P < 0.001 vs. the whole blood group).

Figure 3. The activities of ALT (A) and AST (B) in plasma before and after liver I/R injury. Values are presented as mean ± SD (n = 5). ***P< 0.001 vs. the whole blood group. ALT: alanine aminotransferase; AST: aspartate aminotransferase.

PolyPHb-inhibited, I/R-induced histopathological damage

H&E staining of liver sections showed that I/R-induced marked coagulation necrosis and inflammatory cell infiltration in the whole blood group (Figure 4). While in the PolyPHb group, these histological damages were dramatically attenuated. The sham animals had no obvious changes on liver histopathology.

Figure 4. Representative photomicrographs of HE-stained hepatic tissue sections (n = 5). Original magnification × 400; scale bar: 50 μm.

PolyPHb-reduced, I/R-induced hepatic apoptosis

After 60-min warm ischemia and 2-h reperfusion, significantly increased TUNEL-positive hepatic cells were observed in the whole blood group (31.05 ± 3.54% vs. 4.45 ± 1.42% in the sham group, P < 0.001, Figure 5A). However, in the PolyPHb group, the apoptotic hepatic cells were remarkably reduced when compared with the whole blood group (18.40 ± 4.52%, P < 0.01). Consistently, notable increase of the caspase-3/7 activity was observed in the whole blood group (Figure 5B). While in the PolyPHb group, it was remarkably down-regulated, suggesting I/R-induced hepatic apoptosis was inhibited by isovolemic hemodilution with PolyPHb.

Figure 5. The rate of TUNEL-positive hepatic cells/total hepatocytes (A) and caspase-3/7 activity for each group (B). Values are presented as mean ± SD (n = 5). *P< 0.05 and **P< 0.01 vs. the whole blood group. TUNEL: terminal dUTP nick labeling.

Discussion

In the current study, we demonstrated that isovolemic hemodilution with PolyPHb significantly reduced rat liver warm I/R injury, as evidenced by greatly reduced activities of markers of liver injury (ALT and AST) and ameliorated hepatic histopathological damage. The TUNEL staining and caspase assay showed hepatic apoptosis was also inhibited after PolyPHb treatment, which provided additional evidence that the liver I/R injury was attenuated.

To prolong clinical effectiveness and to reduce the risk of bacterial contamination, human red blood cells have to be stored in exacting environment, which substantially limits its availability in emergency, such as at disaster sites or on the battlefield. Thus, developing blood substitutes, especially HBOC, as emergency alternatives to blood transfusion are necessary and of great clinical importance (Squires 2002). Ideal HBOC should have less stringent storage requirements, as well as be easy to sterilize and free of blood group antigens. In addition, the properties of high oxygen affinity, low viscosity, and small mean diameter of HBOC make it possible to freely diffuse in microcirculation and transport oxygen to hypoxia tissues. Thus, HBOC is a prospective agent for treatment of organ I/R injury (Alayash 2004, Chang 2007, Chang D’Agnillo and Chang 1998, Fergusson and McIntyre 2008, Winslow 2003). In our previous studies, we have proven that PolyPHb, a HBOC developed in China, could attenuate I/R injury in major solid organs including heart (Li et al. 2009a, 2009b, 2010a, 2010b, 2011), lung (Li et al. 2013), and kidney (Li et al. 2012). Our studies also suggested that the organ protective effect of PolyPHb should not be totally attributed to its oxygen transporting capacity, because PolyPHb could provide more profound protection to I/R heart than whole blood, which could transport oxygen too (Li et al. 2009a, 2010a, 2011). The possible mechanisms for its protective effect against organ I/R injury involved restoration of nitroso–redox balance and attenuation of mitochondrial oxidative damage (Li et al. 2009a, 2009b, 2010b).

The aim of the present study was to test our hypothesis that PolyPHb was also protective in liver I/R injury. The results confirmed our hypothesis and demonstrated that the hepatic apoptosis was greatly attenuated by PolyPHb. Apoptosis is defined by characteristic morphological changes of the hepatocytes. A hepatic cell that undergoes apoptosis shrinks, shows condensation, and marginizes chromatin in the nucleus, and eventually breaks down in apoptotic bodies. These morphological changes are associated with the activation of caspase cascade, which induce the breakdown of structural proteins, repair enzymes, and DNA (Jaeschke and Lemasters 2003). Since activation of the caspase cascade in hepatocytes involves amplification through the mitochondria and feedback activation of upstream caspases by effector caspases, depressing its activity prevents the progression of this cascade and effectively inhibits hepatocyte apoptosis (Malhi et al. 2006). In our study, we showed that PolyPHb significantly reduced the TUNEL-positive hepatocytes and the activity of caspase 3/7 in liver tissue. Thus, the antiapoptotic effect of PolyPHb on hepatocyte is clear.

Another finding of this study is that we observed an elevation of blood pressure after exchange of 15% total blood volume with PolyPHb, and this elevation reduced to basal level within 10 min. So we believe that the vasoactive effect of current PolyPHb product is moderate but still remained. As reported previously, vasoactive effect of HBOC was probably related to nitric oxide scavenging by free hemoglobin (Natanson et al. 2008). The HBOC autoxidation-induced ROS generation could also cause vasoconstriction (Hai 2012). In terms of PolyPHb, in addition to reduction of remaining free hemoglobin, we attempt to add some antioxidants in our following product so as to inhibit ROS generation. We hope these treatments could improve the quality of PolyPHb and avoid above complication.

In summary, by using a rat liver warm I/R injury model, we demonstrated that isovolemic hemodilution with PolyPHb (15% of total blood volume) provided protective effect on liver.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

This study was supported by the Applied Basic Research Funding of Sichuan Science and Technology Bureau (2012JY0005), National Nature Science Foundation of China (81100180), China Postdoctoral Specialized Science Foundation (201003700), the Specialized Research Fund for the Doctoral Program of Higher Education (20100181120090), the Major Program of the Clinical High and New Technology of PLA (2010gxjs039) and the Scientific Research Staring Foundation for Young Teachers of Sichuan University (2010SCU11022).