Abstract
Abstract: This study was to investigate whether polymerized human placenta hemoglobin (PolyPHb) given before ischemia protects in vivo rat heart function against ischemia/reperfusion (I/R) injury. Forty-five male Sprague-Dawley rats were randomly divided (n = 15 per group) into a sham group, control group (pretreatment with Lactated Ringer's solution), or PolyPHb group (pretreatment with 0.1gHb/kg PolyPHb). Rat hearts were subjected to 30-min ischemia by occlusion of left anterior descending, followed by 2-hr reperfusion. As compared to the control group, PolyPHb preserved cardiac function and reduced cardiac troponin-I release and histopathological changes. Therefore, PolyPHb pretreatment provided a profound cardioprotective effect on the in vivo rat heart.
INTRODUCTION
Cardiac ischemia/reperfusion (I/R) injury is inevitable in patients with coronary heart disease, which is believed to be the leading cause of death worldwide. Numerous studies have reported that I/R injury may cause damage to the heart and lead to myocardial contractile dysfunction, cardiac arrhythmias, as well as myocardial infarction [Citation1,Citation2]. In addition, as the population ages, the problem of I/R injury is likely to increase given that elderly hearts are more susceptible to I/R injury [Citation3,Citation4]. Thus, strategies that provide protection of the heart are urgently required.
Polymerized human placenta hemoglobin (PolyPHb), one type of hemoglobin-based oxygen carrier (HBOC), was initially developed to treat patients with trauma and hemorrhagic shock [Citation5,Citation6]. In our previous studies, we have demonstrated that PolyPHb had a potentially protective effect on an isolated rat heart after cold storage and reperfusion, and the mechanism was due to attenuation of mitochondrial oxidative damage and restoration of myocardial nitroso-redox balance [Citation7–9]. As a promising HBOC, PolyPHb allows more oxygen transport to the hypoxia tissues owing to its higher oxygen affinity, lower viscosity, and smaller mean diameter than human red blood cells. It may thus have a role to play in restoring microcirculatory perfusion and alleviating myocardial I/R injury [Citation5,Citation10–12]. Therefore, this study was designed to investigate the effect of PolyPHb pretreatment on in vivo rat heart function after 30-min left ventricular ischemia and 2-hr reperfusion.
METHODS
The present study was performed in adherence with the Guidelines on the Use of Laboratory Animals published by the National Institutes of Health and approved by the Animal Care and Use Committees in Sichuan University. Forty-five adult male Sprague-Dawley rats, approximately 10 weeks old, weighing 200–250g, 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
PolyPHb was prepared as we previously described [Citation5,Citation6]. Briefly, hemoglobin from fresh human placenta blood (donated by Tianjin Union Stem Cell and Genetic Engineering Ltd, Tianjin, China) was purified and viral inactivation provided by heat treatment, then intra- and inter-molecular cross-linking were performed by using pyridoxal phosphate (PLP) and glutaraldehyde (GDA), respectively. After that, ultrafiltration and molecular sieve chromatography were performed to harvest PolyPHb with molecular weight range from 64 KD to 600 KD. The final concentration of PolyPHb solution was 8 gHb/dL.
General Preparation
Forty-five male Sprague-Dawley rats, weighing 250-300g, were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and heparin (500 IU). Anesthesia was maintained by injection of 15 mg/kg sodium pentobarbital into the tail vein during the experiment. Tracheal intubation was performed and then mechanical ventilation was achieved by connecting the tracheal tubing to a rodent ventilator (tidal volume was 8–10 mL, frequency was 70–80 times per minute, and expiration: inspiration was 2:1) (DH-150, Medical Instrument Company of Zhejiang University, Hangzhou, Zhejiang, China). Body temperature was maintained at 36–37°C using a warming blanket. The standard limb lead II configuration electrocardiographic system was attached subcutaneously by needle electrodes to confirm ST segment changes during ischemia. A polyethylene catheter was placed in the left femoral vein for pretreatment and vein blood sampling. Total occlusion of left anterior descending coronary artery (LAD) was performed. Briefly, a lateral leftsided thoracotomy was performed through the fourth intercostal space, followed by a pericardiotomy. A 6–0 silk suture was passed and tightened around LAD to form a reversible coronary artery occlusion. Successful myocardial ischemia was verified by the emergence of regional epicardial cyanosis and ST segment elevation ().
Figure 1. The experimental protocol of this study. Representative ECGs at baseline and LAD occlusion are shown at the bottom. ECG: electrocardiogram; LAD: left anterior descending coronary artery; L-RS: Lactated Ringer's solution; PolyPHb: polymerized human placenta hemoglobin.
Experimental Protocol
As shown in , after a 10-min stabilization period, 45 rats were randomly assigned to sham, control, and PolyPHb groups (n = 15/group). In the PolyPHb and control groups, rats were pretreated with 0.1 gHb/kg PolyPHb solution or isovolumetric Lactated Ringer's solution (L-RS) for 5 min, respectively. After 10-min washout, we tightened the suture around the LAD to induce 30-min ischemia, and then loosened it to allow 2-hr reperfusion. In the sham group, the suture was merely passed around the LAD without tightening.
Cardiac Function Measurement
A 24-G heparin-filled catheter (Spacelabs Medical, Inc., Redmond, WA, USA) was inserted from the right carotid artery to the left ventricle for measurement of cardiac functional parameters, including heart rate (HR), left ventricular systolic pressure (LVSP), rate of maximum LVDP increase (+ dp/dt) and decrease (-dp/dt), and LV end-diastolic pressure (LVEDP) (ADInstruments Pty Ltd., Bella Vista, NSW, AUS).
Myocardial Histopathological Analysis
After 2 hr of reperfusion, the LV tissues were immediately fixed for 24 hours in 4% paraformaldehyde in 0.1 M phosphate buffer saline (pH 7.4), and then dehydrated in a series of ethanol and embedded with paraffin. After that, 4-μm sections were prepared and stained with hematoxylin and eosin (HE). The result of HE staining was assessed in a blinded fashion by a pathologist for the following histological examination: acute myocardial necrosis, cellular swelling, and fatty changes.
Cardiac Enzyme Determination
The release of cardiac troponin-I (cTnI) was determined by use of a commercially available ELISA kit (Life Diagnostics, Inc., West Chester, PA) from blood samples collected at the following time points: 1) at the end of the 10-min baseline stabilization period; and 2) at the end of the 2-hr reperfusion period.
Statistical Analysis
All values in the text and figures are presented as mean ± SEM. The values of HR, LVSP, ± dp/dt, and LVEDP were analyzed by 2-factor ANOVA with repeated measures, and use of a post hoc t test with Bonferroni correction for multiple comparisons. The release of cTnI was analyzed by 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
PolyPHb Improved Cardiac Function after I/R Injury
Under basal conditions, no significant differences in cardiac functional parameters were observed among the groups (). LAD occlusion was accompanied by reductions in LVSP and ± dp/dt and an increase in LVEDP, which indicated the cardiac function was depressed when compared with the sham group. Even PolyPHb pretreatment did not totally reverse the damage caused by I/R injury; it still improved the recovery of HR (P < 0.01), LVSP (P < 0.01) and ± dp/dt (P < 0.05 and P < 0.01, respectively) to a greater degree as compared to the control group (). The LVEDP elevation during reperfusion was also significantly attenuated in the PolyPHb group (P < 0.05 vs. the control group, ).
Figure 2. Cardiac function during reperfusion. The recovery of HR (A), LVSP (B), ± dp/dt (C and D), and LVEDP (E) of the three group hearts. Values are presented as mean ± SEM (n = 15). *P < 0.05 and **P < 0.01 vs. the control group; ‡P < 0.001 vs. the sham group. HR: heart rate, LVSP: left ventricular systolic pressure; ± dp/dt: maximum left ventricular pressure increase and decrease rate; LVEDP: left ventricular end-diastolic pressure.
Table 1. The basal cardiac functional parameters of the three groups.
PolyPHb Limited Histopathological Changes after I/R Injury
The results of HE staining showed that the sham group exhibited minimal histopathological changes (). After LAD occlusion and reperfusion, the extents of acute myocardial necrosis, cellular swelling, and fatty changes (arrows) were greatly augmented in the control group, while these histopathological changes were attenuated by PolyPHb pretreatment.
Figure 3. Representative photomicrographs of HE-stained left ventricular tissue sections. Magnification × 400, scale bar: 100 μm. Arrows indicate the locations of acute myocardial necrosis, cellular swelling, or fatty changes (n = 5).
PolyPHb Reduced cTnI Release after I/R injury
As shown in , the basal release of cTnI was low and showed no significant difference among the three groups. LAD occlusion and reperfusion greatly elevated the cTnI release in the control group (12.42 ± 2.21 ng/mL vs. 5.05 ± 1.29 ng/mL in the sham group, P < 0.001). The PolyPHb group showed a decrease in the release of cTnI (8.65 ± 2.57 ng/mL, P < 0.05 vs. the control group), but it was still higher than that of the sham group (P < 0.01).
Figure 4. The release of cTnI in the three group hearts at 10-min baseline and 2-hr reperfusion. Values are expressed as mean ± SEM (n = 8). *P < 0.05 vs. the control group; †P < 0.01 and ‡P < 0.001 vs. the sham group. cTnI: cardiac troponin-I.
DISCUSSION
In the present study, we pretreated the rat with 0.1gHb/kg PolyPHb to investigate the potential effect of PolyPHb on I/R heart after 30-min LAD occlusion and 2-hr reperfusion. The results demonstrated that PolyPHb pretreatment improved cardiac functional recovery, as evidenced by the elevated HR and systolic/diastolic performance during reperfusion when compared to the control group. In addition, the myocardial histopathological changes and enzyme release were also significantly reduced in the PolyPHb group, which further proved the protective effect of PolyPHb pretreatment on injury in the rat heart.
PolyPHb, a polymer of purified and viral-inactivated human placenta hemoglobin, is a promising HBOC developed in China. Like most HBOC products under investigation, PolyPHb can freely diffuse within microcirculation and transport oxygen to hypoxic tissues owing to its high oxygen affinity, low viscosity and small mean diameter [Citation14,Citation15]. There are three reasons why we employed human placenta hemoglobin as the raw material for production of this blood substitute: (1) The availability of fresh blood is limited in most clinical situations due to the ongoing shortage of donated blood. As a source of HBOCs, outdated blood will become increasingly difficult to obtain as a source of raw material; (2) Animal source blood is thought to be unsafe for various animal borne diseases, such as aftosa and mad cow disease; (3) Compared to adult hemoglobin, placenta hemoglobin has higher oxygen affinity, which means it may provide more oxygen supply to ischemic tissues.
Recently, the cardioprotective effect of HBOC has been reported in many animal studies [Citation7,Citation8,Citation10,Citation15–17]. Our previous study indicated that PolyPHb protected isolated rat heart from I/R injury, the mechanism of which was in part due to attenuation of myocardial apoptosis and restoration of the myocardial redox signaling. The results of this study provided additional evidence that PolyPHb could protect in vivo rat heart from I/R injury. However, according to some recently published clinical trials, there are several complications associated with systemic administration of HBOCs, such as coronary and cerebral vasospasm, gastrointestinal side-effects, chest and abdominal pain [Citation18–21]. But in the present study, we did not observe any of these complications, probably because the dosage used in this study was lower than that reported in the clinical trials. As far as we know, PolyPHb is far from perfect, like most blood substitutes around the world. A lot of work is still required to improve its quality and explore its alternative clinical uses.
In conclusion, our findings suggest that, using an in vivo rat heart I/R model, PolyPHb pretreatment before LAD occlusion exerts profound cardioprotective effect against the following I/R injury. These results reveal that PolyPHb may be a promising candidate for heart protection in the future clinical applications.
Declaration of interest: This study was supported by grants from the National Nature Science Foundation of China (81100180, 30801083 and 81070117), the China Postdoctoral Specialized Science Foundation (201003700), the Specialized Research Fund for the Doctoral Program of Higher Education (20100181120090) and the Major Program of the clinical high and new technology of PLA (2010gxjs039).
REFERENCES
- Yellon, D.M., Hausenloy, D.J. (2007). Myocardial Reperfusion Injury. N Engl J Med 357:1121–1135.
- McCord, J.M. (1985). Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med 312:159–163.
- Jahangir, A., Sagar, S., Terzic A. (2007). Aging and cardioprotection. J Appl Physiol 103:2120–2128.
- Boengler, K., Schulz, R., Heusch, G. (2009). Loss of cardioprotection with ageing. Cardiovasc Res:cvp033.
- Li, T., Yu, R., Zhang, H.H., Wang, H., Liang, W.G., Yang, X.M., Yang, C.M. (2006). A method for purification and viral inactivation of human placenta hemoglobin. Artif Cells Blood Substit Immobil Biotechnol 34:175–188.
- Li, T., Zhang, H.H., Wang, H., Yu, R., Yang, C.M. (2006). Purification and viral inactivation of hemoglobin from human placenta blood. Journal of Biomedical Engineering 23:640–644.
- Li, T., Li, J., Liu, J., Zhang, P., Wu, W., Zhou, R., Li, G., Zhang, W., Yi, M., Huang, H. (2009). Polymerized placenta hemoglobin attenuates ischemia/reperfusion injury and restores the nitroso-redox balance in isolated rat heart. Free Radical Biology and Medicine 46:397–405.
- Li, T., Liu, J., Yang, Q., Wu, W., Zhang, P., Yang, J., Li, J., Zhang, W., Yang, C. (2009). Polymerized Placenta Hemoglobin Improves Cardiac Functional Recovery and Reduces Infarction Size of Isolated Rat Heart. Artificial Cells, Blood Substitutes, and Biotechnology: An International Journal 37:48–52.
- Li, T., Zhang, P., Liu, J., Zhou, R., Li, Q., You, Z., Dian, K. (2010). Protective effects of hemoglobin-based oxygen carrier given to isolated heart during ischemia via attenuation of mitochondrial oxidative damage. Free Radical Biology and Medicine 48:1079–1089.
- Wu, W., Yang, Q., Li, T., Zhang, P., Zhou, R., Yang, C. (2009). Hemoglobin-based Oxygen Carriers Combined with Anticancer Drugs May Enhance Sensitivity of Radiotherapy and Chemotherapy to Solid Tumors. Artificial Cells, Blood Substitutes and Biotechnology 37:163–165.
- Standl, T., Freitag, M., Burmeister, M.A., Horn, E.P., Wilhelm, S., Am Esch, JS. (2003). Hemoglobin-based oxygen carrier HBOC-201 provides higher and faster increase in oxygen tension in skeletal muscle of anemic dogs than do stored red blood cells. J Vasc Surg 37:859–865.
- Klein, HG. (2000). The Prospects for Red-Cell Substitutes. N Engl J Med 342:1666–1668.
- Wu, W., Li, T., Liu, J., Yang, C. (2011). Pretreatment Before Ischemia Induction with Polymerized Human Placenta Hemoglobin (PolyPHb) Attenuates Ischemia/Reperfusion Injury-induced Myocardial Apoptosis. Artificial Cells, Blood Substitutes and Biotechnology 39:3–6.
- D’Agnillo, F., Chang, T.M. (1998). Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties. Nat Biotechnol 16:667–671.
- Burmeister, M.A., Rempf, C., Standl, T.G., Rehberg, S., Bartsch-Zwemke, S., Krause, T., Tuszynski, S., Gottschalk, A., Schulte am Esch, J. (2005). Effects of prophylactic or therapeutic application of bovine haemoglobin HBOC-200 on ischaemia-reperfusion injury following acute coronary ligature in rats. Br J Anaesth 95:737–745.
- Caswell, J.E., Strange, M.B., Rimmer, D.M. 3rd, Gibson MF, Cole P, Lefer DJ. (2005). A novel hemoglobin-based blood substitute protects against myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 288:1796–1801.
- George, I., Yi, G.H., Schulman, A.R., Morrow, B.T., Cheng, Y., Gu, A., Zhang, G., Oz, M.C., Burkhoff, D., Wang, J. (2006). A polymerized bovine hemoglobin oxygen carrier preserves regional myocardial function and reduces infarct size after acute myocardial ischemia. Am J Physiol Heart Circ Physiol 291:1126–1137.
- Jahr, J.S., Nesargi, S.B., Lewis, K., Johnson, C. (2002). Blood Substitutes and Oxygen Therapeutics: An Overview and Current Status. American Journal of Therapeutics 9: 437–443.
- Winslow, R.M. (2003). Current status of blood substitute research: towards a new paradigm. J Intern Med 253:508–517.
- Chen, J.Y., Scerbo, M., Kramer, G. (2009). A review of blood substitutes: examining the history, clinical trial results, and ethics of hemoglobin-based oxygen carriers. Clinics 64:803–813.
- Natanson, C., Kern, S.J., Lurie, P., Banks, S.M., Wolfe, S.M. Cell-Free Hemoglobin-Based Blood Substitutes and Risk of Myocardial Infarction and Death: A Meta-analysis. JAMA 2008;299:2304–2312.