7

of nitroso-redox balance [710]. However, the inuence

of deoxygenated HBOC on the I/R heart has never been
reported, and whether deoxygenated HBOC pretreatment
provides the same cardioprotection as ischemia precondi-
tioning is poorly understood. Therefore we designed this
study to investigate the inuence of deoxygenated HBOC
pretreatment on the isolated rat heart after warm ischemia
and reperfusion, and compared with the effect of ischemia
preconditioning.
MATERIALS AND METHODS
The present study was performed in adherence with
the Guidelines on the Use of Laboratory Animals pub-
lished by the National Institutes of Health and approved
by the Animal Care and Use Committees in Sichuan
University.
Comparison of the Cardioprotective Effect of Deoxygenated Hemoglobin-based
Oxygen Carrier (HBOC) Pretreatment and Ischemia Preconditioning
Zhen You
Department of Hepato-Bilio-Pancreatology, West China Hospital, Sichuan University, Chengdu, China
Tao Li
Laboratory of Anesthesiology and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, China
Chengmin Yang
Institute of Tianjin Union Biotechnology Development, Tianjin, China
Hong Wu and Yong Zeng
Department of Hepato-Bilio-Pancreatology, West China Hospital, Sichuan University, Chengdu, China
Abstract: This study was designed to compare the inuence of deoxygenated hemoglobin-based oxygen carrier (HBOC) pretreatment
and ischemia preconditioning on cardiac ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts were pretreated with
0.1 gHb/dL deoxygenated HBOC or ischemia, then subjected to 50-min warm ischemia and 2-hr reperfusion. The results indicated
that deoxygenated HBOC pretreatment and ischemia preconditioning both equally improved the recovery of cardiac function, and
reduced the cardiac enzyme release and myocardial histopathological changes as compared to the control group. Therefore our study
demonstrated that deoxygenated HBOC pretreatment and ischemia precondition provided equivalent protection to the isolated heart
against I/R injury.
Keywords: hemoglobin-based oxygen carrier, pretreatment, ischemia preconditioning, ischemia/reperfusion injury
INTRODUCTION
Ischemia/reperfusion (I/R) injury is harmful to the cardio-
vascular system and responsible for cardiac infarction and
ischemic heart disease [13]. Reduction of cardiac I/R
injury improves the survival and prognosis of patients,
which is urgently required in clinic settings. As we know,
ischemia preconditioning is commonly recognized as
a promising method of cardioprotection [46]. In other
words, repetitive brief episodes of I/R are benecial to
decreasing the following I/R injury.
Our previous studies have demonstrated that
oxygen-saturated polymerized human placenta hemoglo-
bin (Poly PHb), one type of hemoglobin-based oxygen
carrier (HBOC), can protect the isolated rat heart from
I/R injury, and the underlying mechanisms are impli-
cated in attenuation of myocardial apoptosis, quenching
cardiac mitochondrial oxidative stress and restoration
Zhen You and Tao Li contributed equally to this study.
This study was supported by grants from the National Nature Science Foundation of China (30828030 and 30801083) and the
Postdoctoral Science Foundation of China (20090461338).
Address correspondence to Yong Zeng, MD, Department of Hepato-Bilio-Pancreatology, West China Hospital, 37 Wainan Guoxue
Road, Chengdu 610041, China. E-mail: zengyong@medmail.com.cn
Articial Cells, Blood Substitutes, and Biotechnology, 39: 711
Copyright 2011 Informa Healthcare USA, Inc.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.3109/10731199.2010.495037
8 Z. You et al.
HBOC Solution Preparation
HBOC in this study was PolyPHb, which was prepared
as we previously described [11,12]. Before use, the
HBOC solution was added into a Krebs-Henseleit buffer
(KHB: 120.0 mM NaCl, 4.5 mM KCl, 20.0 mM NaHCO
3
,
1.2 mM KH
2
PO
4
, 1.2 mM MgCl
2
, 2.5 mM CaCl
2
and 10.0
mM glucose, pH 7.4, 37 C) to a nal concentration of
0.1 gHb/dL, and bubbled with 95% nitrogen gas 5%
carbon dioxide for 10 mins for deoxygenation. As a
result, the oxygen tension of the above HBOC solution
was 42 15 mmHg, which was signicantly lower than
that of the KHB alone (532 25 mmHg).
Experimental Proposal
Forty male Sprague-Dawley rats, weighing 250300 g,
were anesthetized with an intraperitoneal injection of
sodium pentobarbital (50 mg/kg) and heparin (500
IU). The hearts were quickly excised and perfused on a
Langendorff apparatus with KHB at a constant pressure
of 100 cmH
2
O. A thin-wall latex balloon was inserted into
the left ventricle (LV) through the left atrium to continu-
ously monitor the LV function, including left ventricular
developed pressure (LVDP), maximum LVDP increase
( dp/dt) and decrease rate (dp/dt), and LV end-diastolic
pressure (LVEDP) (AD Instruments Pty Ltd., Bella Vista,
NSW, Australia). Coronary ow rate (CF) was calculated
from the coronary efuent, sampling time, and cardiac
wet weight.
As shown in Figure 1, after 10 mins of basal
perfusion, hearts were randomly assigned to sham,
control (without pretreatment), IPC (ischemia pre-
conditioning), and deoxy-HBOC groups (pretreatment
with deoxygenated HBOC). In the IPC and deoxy-
HBOC groups, a sequence of three 5-min deoxygen-
ated HBOC perfusion or ischemia and 5-min KHB
perfusion was performed, respectively. The control
group hearts were basal perfused for 40 mins without
pretreatment. Then the hearts were subjected to 50-min
ischemia and 2-hr reperfusion. The sham group hearts
were perfused by KHB for 160 mins without pretreat-
ment and I/R injury.
Cardiac Enzyme Release Measurement
The coronary efuents at the end of 10-min baseline and
2-hr reperfusion were collected; the release of cardiac
enzyme, including creatine kinase-MB (CK-MB) and
lactate dehydrogenase (LDH), was measured by an
Olympus AU5400 autoanalyser (Olympus Diagnostics,
Melville, NY) within 2 hrs.
Myocardial Histopathological Analysis
After 2 hrs of reperfusion, the LV tissues were immedi-
ately xed 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 in parafn. After that,
4- m sections were prepared and stained with hematoxy-
lin 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.
Statistical Analysis
All values in the text and gures were presented as mean
SEM. The values of LVDP, dp/dt, LVEDP and CF
were analyzed by 2-factor ANOVA with repeated mea-
sures. The releases of CK-MB and LDH 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 signicant.
RESULTS
Deoxygenated HBOC Pretreatment Increased LV
Function
There were no signicant differences in LVDP, dp/dt,
LVEDP and CF among the 4 groups at basal perfusion,
but during reperfusion the LVDP was greatly improved
in the IPC and deoxy-HBOC groups as compared with
the control group ( P 0.01 and P 0.01, respectively,
Figure 2A). Both the dp/dt and dp/dt were also greatly
increased in the IPC and deoxy-HBOC groups when
Figure 1. The experimental protocol of this study. In the IPC
and deoxy-HBOC groups hearts, a sequence of three 5-min
deoxygenated HBOC perfusion or ischemia and 5-min KHB
perfusion were performed, then subjected to 45 mins of ischemia
at 37

C and 2 hrs reperfusion. Hearts suffered from I/R injury
without pretreatment were used as the control group. Hearts
perfused with KHB for 160 mins without pretreatment and I/R
injury were used as the sham group. KHB: Krebs-Henseleit
buffer.
The Cardioprotection of HBOCs 9
compared to the control group ( P 0.01 and P 0.01, re-
spectively, Figure 2B and 2C). In addition, the LVEDP of
the control group was greatly elevated during reperfusion;
however, ischemia preconditioning and deoxygenated
PolyPHb pretreatment signicantly reduced its eleva-
tion ( P 0.05 and P 0.05, respectively, Figure 2D).
There was no signicant difference in the CF among
the 4 groups during reperfusion (data not shown). When
we compared the cardiac function of the IPC group and
deoxy-HBOC group, we found there were no signicant
differences.
Deoxygenated HBOC Pretreatment Reduced
CK-MB and LDH Release
At baseline, the levels of CK-MB and LDH release
were low and showed no signicant difference
among the 4 groups. After 2-hr reperfusion, both of
them were up-regulated in the control group (62.34
6.33 IU/L/g wet heart for CK-MB release and 120.51
12.37 IU/L/g wet heart for LDH release). Ischemia
preconditioning and deoxygenated HBOC pretreatment
exhibited equally inhibitory effects on the reduction of
CK-MB and LDH release (CK-MB release: P 0.01
and P 0.01 vs. the control group, respectively; LDH
release: P 0.01 and P 0.01 vs. the control group,
respectively, Figure 3).
Deoxygenated HBOC Pretreatment Limited
Myocardial Histopathological Changes
As shown in Figure 4, after I/R injury, the extents of acute
myocardial necrosis, cellular swelling and fatty changes
(arrows) were greatly augmented in the control group as
compared to the sham group. However, these histopatho-
logical changes were largely lessened in the IPC and
deoxy-HBOC groups.
DISCUSSION
HBOC was initially developed as an oxygen-carrying
agent for emergency use when traditional blood trans-
fusion is unavailable or unsafe [1316]. Besides, more
and more studies have demonstrated its cardioprotective
effect against I/R injury, which revealed an alternative

Figure 2. The cardiac function recovery of LVDP (A), dp/dt (B and C) and LVEDP (D) of the 4 group hearts. Values were presented as
mean SEM (n 10). * P 0.05 and * * P 0.01 vs. the control group. LVDP: left ventricular development pressure, dp/dt: maximum
LVDP increase and decrease rate, LVEDP: left ventricular end-diastolic pressure.
10 Z. You et al.
clinical use of HBOC [710,17,18]. As a promising oxy-
gen carrier, HBOC is able to freely diffuse in microcir-
culation and transport oxygen to hypoxia tissues owing
to its high oxygen afnity, low viscosity and small mean
diameter. However, the effect of deoxygenated HBOC
on cardiac I/R injury has never been investigated. In the
present study, we compared the inuence of deoxygen-
ated HBOC pretreatment and ischemia preconditioning
on the I/R heart, and the results demonstrated that there
were no signicant differences of cardiac functional re-
covery, enzyme release, and histopathological changes
between these two groups. Both deoxygenated HBOC
pretreatment and ischemia preconditioning greatly
improved the cardiac contractile performance recovery,
as evidenced by the elevated LVEDP and dp/dt and
reduced LVEDP during reperfusion. In addition, the
CK-MB and LDH release and histopathological changes
were also equally inhibited in the IPC and deoxy-HBOC
groups.
In my opinion, just like ischemia precondition-
ing, the cardioprotective effect of deoxygenated HBOC
pretreatment can also be attributed to repetitive brief
episodes of I/R, because deoxygenated HBOC pretreat-
ment induced a sequence of hypoxia and reperfusion to
myocardium too. As we know, deoxygenated HBOC can
take away the oxygen preloaded to myocardium during

Figure 3. The myocardial CK-MB (A) and LDH (B) releases at the end of baseline and reperfusion of the 4 group hearts. Values were
presented as mean SD (n 58). * * P 0.01 vs. the control group. CK-MB: creatine kinase-MB; LDH: lactate dehydrogenase.
Figure 4. Representative photomicrographs of HE-stained left ventricular tissue sections. Magnication 400, scale bar: 100 m.
Arrows indicate the locations of acute myocardial necrosis, cellular swelling or fatty changes (n 5).
The Cardioprotection of HBOCs 11
basal perfusion instead of providing oxygen. Therefore, in
theory, deoxygenated HBOC would induce a higher level
of hypoxia as compared to ischemia, thus deoxygenated
HBOC pretreatment might provide more protection to the
I/R heart than ischemia preconditioning. Unfortunately,
our results found that there was no signicant difference
between these 2 groups. Another important nding of
this study is that hypoxia may be the key factor medi-
ated the cardioprotection of ischemia preconditioning
and hypoxia preconditioning, because hypoxia precondi-
tioning produced by deoxygenated HBOC pretreatment
had similar cardioprotective effect as ischemia precon-
ditioning in the present study. We believe the method of
deoxygenated HBOC pretreatment can be widely used in
clinical situations; for example, it can be performed on
the patient before cardiopulmonary bypass to protect the
myocardium.
There were some limitations of this study. First,
in the clinic situation, ischemia preconditioning is
not an ideal method of cadioprotection; we cannot
block the aorta to induce cardiac ischemia. However,
according to regulation of the fraction of inspired
oxygen, hypoxia preconditioning is easy to perform. We
probably should employ hypoxia preconditioning as a
control instead of ischemia preconditioning. Second,
even though we demonstrate that deoxygenated HBOC
pretreatment and ischemia preconditioning had similar
cardioprotective effects by the results of cardiac func-
tion recovery, enzyme release, and histopathological
changes. However, these results were far from enough
to support that deoxygenated HBOC pretreatment and
ischemia preconditioning shared similar mechanisms
of cardioprotection.
In conclusion, the present study provided distinct evi-
dence that deoxygenated HBOC pretreatment provided
protection to the isolated heart against I/R injury and this
protection was equivalent to the ischemia precondition-
ing, which revealed the alternative clinic uses of HBOC in
cardiac-related surgery.
Declaration of interest: The authors report no conicts of
interest. The authors alone are responsible for the content
and writing of the paper.
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