Effect of polymerized human placenta hemoglobin on hemodynamic parameter and cardiac function in a rat hemorrhagic shock model

Abstract

To investigate whether polymerized human placenta hemoglobin (PolyPHb) improved hemodynamic parameter recovery and cardiac function after hemorrhagic shock, a mean arterial pressure (MAP) of 30 mmHg was maintained for 60min. Then, all the rats were randomly resuscitated with hetastarch (HES), shed blood (Whole Blood), or PolyPHb (PolyPHb). The MAP was greatly improved by PolyPHb, but it was still lower than that of the Whole Blood group. Meanwhile, the cardiac function and enzyme releases in the PolyPHb group were similar to the HES group. Therefore, our findings suggest that PolyPHb moderately improved hemodynamic recovery and provided little cardioprotective effect in hemorrhagic shock.

Keywords::

• Hemoglobin-based oxygen carrier
• hemorrhagic shock
• resuscitation
• cardiac function

Introduction

Despite the fact that fluid resuscitation markedly improved short-term survival of patients with hemorrhagic shock, multiple organ failure still remains the leading cause of morbidity and mortality of these patients (Cohn et al. 2007, Yu et al. 2011). Hemorrhagic shock results in excessive productions of proinflammatory mediators and reactive oxygen species, which plays a significant role in the development of multiple organ dysfunction (Fink 2002, Maier et al. 2007). Thus, agents that can both resuscitate patients from hemorrhagic shock and preserve organ function are of great clinical benefit.

Polymerized human placenta hemoglobin (PolyPHb) is a novel oxygen therapeutic agent developed in China (Li et al. 2006a, 2006b). The primary application of PolyPHb is to treat patients suspected of suffering from ischemia, such as in trauma and hemorrhagic shock. While considering the excellent oxygen-transporting capacity of PolyPHb, we have designed a series of experiments to investigate its cardioprotective effect. Fortunately, our previous studies demonstrated that PolyPHb did protect the heart against ischemia/reperfusion (I/R) injury both in vivo and in vitro (Wu et al. 2009, Standl et al. 2003, McNeil et al. 2001, Li et al. 2011). The underlying mechanisms include attenuation of NO-mediated myocardial apoptosis, restoration of nitroso-redox balance, and preservation of mitochondria function (Li et al. 2009a, 2009b). Therefore, PolyPHb seems to be a prospective candidate in shock therapy. Accordingly, this study was designed to investigate the effect of PolyPHb on hemodynamic parameter recovery and cardiac function.

Materials and 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 of Sichuan University.

PolyPHb preparation

PolyPHb was prepared as we previously described (Li et al. 2006a, 2006b). Briefly, hemoglobin from fresh human placenta blood (donated by Tianjin Union Stem Cell and Genetic Engineering Ltd, Tianjin, China) was intra- and inter-molecularly cross-linked by pyridoxal phosphate and glutaraldehyde. Then, ultrafiltration and molecular sieve chromatography were performed to harvest PolyPHb with molecular weight range from 64 kD to 600 kD. The parameters of PolyPHb solution are described in Table I.

Table I. The parameters of PolyPHb solution

Parameters	Results or Range
Placenta hemoglobin (gHb/L)	10
Methemoglobin (%)	∼8 %
P50 *(mmHg)	21–24
Colloid Osmotic Pressure (mmHg)	20–25
Osmolality (mosm/kg)	290–310
Molecular Weight (kDa)	64–600
Unpolymerized hemoglobin (%)	∼3 %

* P₅₀: the PO₂ level at that hemoglobin is half-saturated with O₂.

Animal preparation

Sixty male Sprague-Dawley rats, weighing 200 250g, 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 min, 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 hot blanket. A polyethylene catheter was placed in the left femoral artery to withdraw blood and measure mean arterial pressure (MAP). Another polyethylene catheter was placed in the left femoral vein for infusion of blood or solutions.

Protocol

Blood was withdrawn from the femoral artery over a period of 5 min until a MAP of 30 mmHg was achieved. This level of hypotension was maintained for 60 min by blood withdrawal or by infusion of shed blood. The rats were then randomly resuscitated with a 10 min infusion of equivalent volume of hetastarch (HES group, n =–20), shed blood (Whole Blood group, n =–20), or PolyPHb solution (PolyPHb group, n =–20). Rats were observed for 2 h after fluid resuscitation (Figure 1). Five rats in the HES group, two rats in the Whole Blood group, and three rats in the PolyPHb group were removed from this protocol for sudden cardiac arrest or uncontrolled blood loss during operation.

Figure 1. The experimental protocol of this study. HES: hetastarch; MAP: mean arterial pressure.

Measurement of cardiac function

A 24-G heparin-filled catheter connected to PowerLab data-acquisition system (ADInstruments Pty Ltd., Bella Vista, NSW, AUS) was inserted from the right carotid artery to the left ventricle for measurement of cardiac function, including heart rate (HR), left ventricular systolic pressure (LVSP), maximum left ventricular developed pressure increase (+ dp/dt) and decrease rate (−dp/dt), and left ventricular end-diastolic pressure (LVEDP).

Determination of cardiac enzyme release

Cardiac enzyme releases, including creatine kinase-MB (CK-MB) and cardiac troponin-I (cTnI), were determined by use of a commercial ELISA kit (Life Diagnostics, Inc., West Chester, PA) from plasma of vein blood samples collected before hemorrhagic shock and 2 h after resuscitation.

Statistical analysis

All values in the text and figures were presented as mean ± SD. The values of MAP, 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 results of cardiac enzyme releases were 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 MAP recovery after hemorrhagic shock

As shown in Figure 2, PolyPHb resuscitation significantly improved the recovery of MAP when compared with the control group (P <–0.001). But it was not as good as the Whole Blood group. In particular, after 60-min resuscitation, the MAP of the PolyPHb group was remarkably lower than that of the Whole Blood group (P <–0.05).

Figure 2. The MAP recovery after hemorrhagic shock and resuscitation of the three groups. Values were presented as mean ±SD (n = 15 – 18). ^***P < 0.001 vs. the HES group. ^†P < 0.05 vs. the Whole Blood group. MAP: mean arterial pressure.

Whole blood improved cardiac function, but PolyPHb did not

At basal conditions, there were no significant differences in HR, LVDP, ± dp/dt, and LVEDP among the three groups. However, during the period of resuscitation, whole blood infusion greatly improved HR and LVDP when compared to the HES and PolyPHb groups (Figures 3A and 3B). The ± dp/dt was also greatly increased in the Whole Blood group as compared with the HES and PolyPHb groups (Figures 3C and 3D). Consistently, the LVEDP was significantly attenuated by whole blood infusion (Figure 3E). There were no significant differences in cardiac functional recovery between the HES and PolyPHb group.

Figure 3. The HR (A), LVSP (B), ± dp/dt (C and D), and LVEDP (E) of the three groups. Values were presented as mean ± SD (n = 15 – 18). ^{* * *}P < 0.001 vs. the HES group. HR: heart rate; LVSP: left ventricular systolic pressure, ±dp/dt: maximum left ventricular developed pressure increase and decrease rate, LVEDP: left ventricular end-diastolic pressure; N.S.: nonsignificant.

Whole blood reduced cardiac enzyme release, but PolyPHb did not

Before shock, the releases of cardiac enzyme, including CK-MB and cTnI, were similar in the three groups. After 2 h resuscitation, these enzyme releases were largely increased in the HES group. Whole blood resuscitation significantly inhibited this shock-induced cardiac enzyme release (CK-MB: P <–0.05 vs. the HES group; cTnI: P <–0.05 vs. the HES group), while PolyPHb infusion could not limit cardiac enzyme release (Figure 4).

Figure 4. The CK-MB and cTnI releases of the three groups before shock and 2 h after resuscitation. Values were expressed as mean ± SD (n = 15 – 18). ^*P ± 0.05 vs. the HES group. CK-MB: creatine kinase-MB; cTnI: cardiac troponin-I; N.S.: nonsignificant.

Discussion

The present study provided distinct evidence that PolyPHb infusion can help to improve the MAP recovery after hemorrhagic shock, even though this effect was not as good as the whole blood. Also, our study clearly indicated that PolyPHb used in this way cannot reverse the cardiac dysfunction induced by hemorrhagic shock.

PolyPHb is a type of hemoglobin-based oxygen carrier (HBOC) developed by the research group of Professor Yang (Li et al. 2006a). As with most HBOCs, this product is initially used for treatment of patients in hemorrhagic shock and trauma (D’Agnillo and Chang 1998, Burmeister et al. 2005). In addition, we have designed a series of studies to investigate clinical alternative uses from several years ago. PolyPHb is able to freely diffuse in microcirculation and transport oxygen to hypoxia tissues owing to its high oxygen affinity, low viscosity, and small mean diameter. These abilities make PolyPHb an attractive agent for treatment of organ I/R injury. Our previous studies have proven the promising protective effect of PolyPHb on in vivo and in vitro hearts (Wu et al. 2009, Standl et al. 2003, McNeil et al. 2001, Li et al. 2011). However, in contrast to our hypothesis, the result of this study indicated no positive cardiac effect provided by PolyPHb in hemorrhagic shock. We believe there are two main reasons for this phenomenon. First, in the present study, the final PolyPHb concentration in the body after resuscitation reached 4–5 gHb/dL, while the PolyPHb level for optimal cardioprotective effect was only 0.1–0.5 gHb/dL, as we previously reported. Second, nitric oxide (NO) is important in maintaining vascular tone in the endothelium. Under physiologic conditions, hemoglobin is mostly insulated from the vascular endothelium. However, when free hemoglobin releases from red blood cells, it will form potent NO scavengers and cause systemic and pulmonary vasoconstriction (Levy 2011). Despite development of preparation technology, the remaining unpolymerized hemoglobin in the PolyPHb solution is still up to 3%. Thus, vasoconstriction is unavoidable when the amount of free hemoglobin reached the threshold. Consistent with our speculation, during the early 60 min of resuscitation, the MAP was greatly elevated by PolyPHb, even slightly higher than that of the Whole Blood group, suggesting that free hemoglobin in PolyPHb did influence the vascular tone. Although PolyPHb improved oxygen carrying in blood, the free hemoglobin induced vasoconstriction actually will reduce microcirculation perfusion and limit oxygen delivery to myocardium.

To overcome these disadvantages, further improvement of PolyPHb itself is necessary, such as increasing its structural stability and reducing its vasoactivity. Also, pretreatment of patient with agents that can relieve vasoconstriction and hypertension, like inhaled nitric oxide or nitroglycerin, is reported to be capable of excluding these side-effects, and ultimately protecting the heart against possible I/R injury during hemorrhagic shock (Yu et al. 2008, Yu et al. 2010, Katz et al. 2010). Last but not least, the main cause of the cardiac dysfunction is believed to be the burst of superoxide and reactive oxygen species during hemorrhagic shock and resuscitation. Therefore, HBOC with antioxidant property, such as PolyHb-SOD-CAT (D’Agnillo and Chang 1998), or alternative use of HBOC and these antioxidants, will be beneficial in improving cardiac function of the patient with sustained and more severe shock.

In conclusion, our findings suggest that PolyPHb moderately improved the outcome of hemodynamic recovery and provided little protective effect against cardiac dysfunction in a rat hemorrhagic shock model.

Acknowledgments

This study was supported by grants from the National Nature Science Foundation of China (81100180 and 81070117), the 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).

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