Artificial Organs

36(2):185193, Wiley Periodicals, Inc.

2012, Copyright the Authors
Artificial Organs 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Liposome-Encapsulated Hemoglobin Improves Energy

Metabolism in Skeletal Muscle Ischemia and
Reperfusion in the Rat
*Daisuke Kurita, *Akira T. Kawaguchi, Kensuke Aso, Mariko Yamano,
Haruyuki Minamitani, and *Munetaka Haida
*Tokai University School of Medicine; Tokai University IT Education Center; Keio University Faculty of Science and
Technology; Tokai University Junior College of Nursing and Medical Technology, Kanagawa; and Osaka Prefecture
University, Habikino, Osaka, Japan

Abstract: The effect of liposome-encapsulated hemoglobin (LEH) was tested in a rodent model of limb ischemia
and reperfusioncausing local reperfusion injury and a
cascade of systemic responses. Intracellular pH (pHi) and
phosphocreatine (PCr)/inorganic phosphate (Pi) ratio were
serially monitored using 31P-nuclear magnetic resonance
spectroscopy with a 2-cm solenoid coil on a rodent hind
limb. After baseline measurements, the right hind limb
underwent ischemia for 70 min, followed 10 min later by
intravenous administration of LEH (10 mL/kg, n = 6),
homologous red blood cells (RBCs, n = 6), saline (n = 6), or
no treatment (n = 6). Reperfusion was then observed for an
additional 60 min. While pHi decreased precipitously after
the onset of ischemia and even following reperfusion,
LEH-treated rats had significantly milder intracellular acidosis compared with all other groups during ischemia, and
after reperfusion as well throughout the observation with

the saline-treated rats. In contrast, the PCr/Pi ratio

decreased regardless of treatment after ischemia until reperfusion, when the ratio returned toward normal or the
energy status improved only in the LEH-treated rats, while
the ratio remained depressed in the control animals receiving RBC, saline, or no treatment. Morphological studies
7 days later revealed a tendency toward suppressed mononuclear cell infiltration with preservation of muscular mass
and structure in the LEH-treated rats. LEH treatment after
early limb ischemia appeared to improve intracellular
energy metabolism and eventually preserve skeletal muscle
in a rodent model of limb ischemia and reperfusion.
Key Words: 31P-nuclear magnetic resonanceMyopathic
nephrotic metabolic syndromePhosphocreatineHighenergy phosphate complexInorganic phosphate
Intracellular
pHAnaerobic
metabolismAerobic
metabolism.

Skeletal muscle is reported to be one of the most

vulnerable tissues to ischemia (1). Depending on the
severity and duration of limb ischemia, clinical
impact varies widely from limited local damage to
systemic and multimodal consequences due to metabolic effluence from the affected limb, leading to
systemic acidosis, hyperkalemia, nephropathy, and
circulatory collapse (14). As liposome-encapsulated
hemoglobin (LEH) (5) has been reported to improve
circulation in tissues with ischemia or deranged

perfusion (611), we investigated its effects on limb

ischemia and early reperfusion using 31P-nuclear
magnetic resonance spectroscopy (31P-NMR) (12
17), following changes in intracellular pH (pHi) and
high-energy phosphate (phosphocreatine, PCr) to the
inorganic phosphate (Pi) ratio (PCr/Pi) in comparison with animals receiving homologous red blood
cells (RBCs), saline, or no treatment as control. 31PNMR was first used to detect energy metabolism in
excised muscle (14), whole body (15), skeletal muscle
of hind limb (1619), brain (2022), and myocardium
(23,24) because its noninvasiveness allows repeated
studies for longitudinal observations. Based on these
characteristics, we evaluated the hypothesis that
LEH might efficiently supply oxygen (O2), maintain
aerobic energy metabolism, and therefore ameliorate ischemia and reperfusion injury by means of

doi:10.1111/j.1525-1594.2011.01419.x
Received April 2011; revised September 2011.
Address correspondence and reprint requests to Dr. Akira T.
Kawaguchi, Tokai University School of Medicine, Shimokasuya
143, Isehara, Kanagawa 259-1193, Japan. E-mail akira@is.icc.utokai.ac.jp

185

aor_1419

185..193

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D. KURITA ET AL.

31

P-NMR spectroscopy (1419), a highly specific, sensitive, and noninvasive technique that allows serial
observation of the status of pHi and energy metabolism in rat hind-limb skeletal muscle.

31P-NMR

IV infusio

n line

Coil

MATERIALS AND METHODS

Halothane 2%

LEH
Relevant characteristics of LEH (Terumo Co.
Ltd., Tokyo, Japan) have been reported (5). Briefly,
it is a liposome capsule measuring approximately
250 nm in mean diameter, containing purified
hemoglobin eluted from human RBCs outdated
for transfusion. The liposome capsule is coated
with polyethylene glycol to reduce aggregation and
capture by the reticuloendothelial system, in order
to prolong the circulation half-life to approximately 13 h in rodents (5) and to 70 h in monkeys
(5,8). Inositol-hexaphosphate is included for 2,3diphosphoglycerate to adjust the O2 affinity to
P50O2 = 40 mm Hg in LEH with low O2 affinity
(l-LEH), lower than that of rodent RBCs
(P50O2 = 30 mm Hg, Fig. 1). l-LEH is considered to
be more efficient in O2 transport to tissues under
atmospheric respiration or even better with supplemental O2 than RBCs (Fig. 1). LEH is suspended in
saline to a hemoglobin concentration of 6 g/dL or
20% of volume at pH 7.4 (5). l-LEH is precipitated

O2 Dissociation Curve

100

SO2 (%)

80

RBC (P50O2=30 mm Hg)

40

60

l-LEH (P5002=40 mm Hg)

O2 transport
between
40 200 mm Hg

20

O2 transport
between
40 100 mm Hg

50

100
150
PO2 (mm Hg)

200

FIG. 1. O2 dissociation characteristics of LEH and rodent RBCs.

Whereas LEH with low O2 affinity (l-LEH, P50O2 = 40 mm Hg) is
considered to have a higher O2 delivery than RBCs (RBC,
P50O2 = 30 mm Hg) between 100 and 40 mm Hg under atmospheric respiration, l-LEH is even more efficient in O2 delivery
under respiration with supplemental O2 between 200 and
40 mm Hg. P50O2 (mm Hg), the definition of O2 affinity, is the
partial pressure of O2 under which half of the O2 carrier is bound
to O2.
Artif Organs, Vol. 36, No. 2, 2012

Oxygen 3 L/min

Weight 3 kg
3 m clear of metals

Heating Pad

FIG. 2. Experimental setting, illustrating animal positioning, and

equipment alignment is shown. The whole body of the animal
was placed in the gantry of the NMR system with the right hind
leg in a solenoid coil. All other equipment was made of either
nonmagnetic materials or was placed at least 3 m away from the
NMR system. The most proximal portion of the right hind leg was
occluded by the tourniquet, with the distal leg covered by a
solenoid coil with its axis angled 90 degrees from the main
magnetic axis of the NMR system.

between plasma and RBCs by centrifugation at

10 000 g for 10 h. A sibling rat donated homologous blood, which was separated from other
components, washed three times, and diluted with
saline to 20% hematocrit to serve as a control
solution (RBC solution) containing a comparable
amount of rat hemoglobin.
Animals
All experiments were approved by the institutional
review board of Tokai University School of Medicine.
Animals received humane care as required. The
experiments were performed on 24 male SpragueDawley rats (9 weeks old, 270300 g, mean 283 g).
Animals were anesthetized and maintained with 2%
halothane and O2, and were laid prone on a heating
pad to maintain body temperature during the NMR
study. The right hind limb was held in a solenoid coil
during NMR spectroscopy, with care being taken that
magnetic material and equipment were at least 3 m
apart. The right hind limb was occluded using a fluorocarbon loop with a weight of 3 kg (Fig. 2). Ten
minutes after onset of ischemia, rats were randomly
assigned to one of four groups, by intravenously
receiving 10 mL/kg of body weight of l-LEH (n = 6),
transfusion of the same amount of hemoglobin (RBC
solution, n = 6), saline (n = 6), or no treatment (n = 6)
via tail vein over 10 min at slow speed (2.7 to 3.0 mL/
10 min) to avoid acute volume load. Spectra were
continuously measured for 70 min, when the weight
was removed to relax the stricture, and the limb was
reperfused with spectra being recorded for an additional 60 min.
31

P-NMR spectroscopy
31
P-NMR spectra were obtained with a 2.0-Tesla,
31-cm bore superconducting magnet (BEM250/80,

ARTIFICIAL O2 CARRIER ALLEVIATES MUSCULAR ISCHEMIA

Phospho-Energetic, Inc., Philadelphia, PA, USA)
operating at 34.53 MHz for 31P nuclei.A 2-cm doubleturn solenoid coil was used to acquire spectra from
the right hind limb. The coil was placed at the most
homogeneous portion of the magnetic field. Shimming of the magnetic field was performed by optimizing the shape and intensity of the spectrum of water
protons. Homogeneity was adjusted for at least 0.3
parts per million (ppm) for each rat. The 31P-NMR
spectra were acquired using a single 90-degree pulse
(18 ms) and collected with 2048 data points and
5000 Hz spectral width. Spectra were accumulated
for 3 min with a repetition time of 10 s at 3-min
intervals. We previously confirmed that a repetition
time of 10 s was optimal for the accumulation of
spectra over 3 min without affecting the PCr/Pi ratio
for the hind limb muscle. Prior to fast Fourier transform, 18 averaged free-induction decays were multiplied by an exponential window function of 10 Hz.
31

P-NMR spectra interpretation

The 31P-NMR spectrum during rest was obtained
for 3 min prior to limb ischemia.Then the time course
of any changes in the 31P-NMR spectra during limb
entrapment and reperfusion was measured. For spectral analysis, special software (OriginPro ver.7 Peak
Fitting Module, OriginLab Corporation, Northampton, MA, USA) was used for each spectrum. Each
peak area and center was obtained by Levenberg
Marquart nonlinear least squares curve fitting using
the Voigt function (15), which was the convolution
function of Gaussian and Lorentzian. The peak areas
and centers of a baseline, Pi, PCr, and g-, a-, and
b-adenosine triphosphate (ATP), were determined

187

by a minimum c2 value. In the normal skeletal muscle

(1214), peaks of Pi, PCr, and ATPs were observed
and identified by 31P-NMR (Fig. 3, left). The dynamics of PCr and Pi in intact tissues were measured.
When anaerobic metabolism prevailed due to O2
depletion (Fig. 3, right), PCr decreased and Pi
increased because of degradation of the high-energy
phosphate complex. Thus, their ratio, PCr/Pi, may
serve as a sensitive and reliable measure of the muscular energy metabolism and status (1217); changes
in PCr/Pi ratio were followed by its measured values
and presented as the changes from preischemic
values (%PCr/Pi) as well. Any changes in pHi are
given by the following equation (16): pHi = 6.
77 + log(d - 3.29)/(5.58 - d), where d is the chemical
shift of Pi from PCr. The area ratio of PCr/Pi can be
used as an index of muscular energy metabolism.

Other variables
Before and after acquisition of 31P-NMR spectra,
venous blood (<0.1 mL) was taken through the
tail vein for hematocrit and volumes of l-LEH
(LEHcrit). Systemic blood pressure was monitored in
the left femoral artery of some rats from each group.
In these animals, blood samples were analyzed within
30 min of the end of NMR spectra measurement for
plasma electrolytes and lactate using a portable clinical analyzer (i-stat, FUSO Pharmaceutical Industries,
Ltd., Tokyo, Japan). Then, the animals were reversed
from anesthesia and returned to cages with water and
fed ad libitum under room air. Seven days later, the
animals were anesthetized for final NMR spectroscopy recordings.

31P-NMR

Normal

Anoxia
PCr / Pi

PCr

=
Pi
PCr ATP

ATP
Pi

-20 -15 -10 -5

FIG. 3. Typical 31P-NMR spectrum of skeletal muscle under normal condition

(Normal) and ischemia (Anoxia), showing
increase in inorganic phosphate (Pi) and
decrease in phosphocreatine (PCr). As a
result, the PCr/Pi ratio decreased as the
cellular aerobic condition changed from
normal (left) to ischemia (right). In contrast, peaks for ATP showed no change
even under ischemia, suggesting that degradation of PCr supplied energy to maintain ATP levels for cellular survival.

0 5 10 15 20
ppm
Artif Organs, Vol. 36, No. 2, 2012

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D. KURITA ET AL.
(Fig. 4). While all the groups had similar O2 content
before, RBC-treated rats had the highest O2 content
in whole blood, and LEH-treated rats had the highest
O2 content in the plasma fraction (Fig. 4) because of
the presence of l-LEH. Systemic blood pressure
showed a temporary elevation in response to leg
ischemia followed by administration of solution, but
returned to baseline by 30 min after the onset of
ischemia was seen in all groups. Releasing the loop
for reperfusion did not alter the mean systemic pressure significantly in any group. Among the determinants in blood samples drawn after NMR recording,
there was no significant or consistent difference in
arterial blood gases, plasma electrolytes, glucose, or
lactate levels in rats that received LEH (1.63
0.5 mg/dL), saline (1.97 0.5 mg/dL), RBCs (1.87
0.5 mg/dL), or no treatment (1.57 0.1 mg/dL).

Morphological studies
After recording the final NMR spectrum 7 days
after ischemia/reperfusion, all animals were sacrificed under deep anesthesia. The bilateral soleus
muscles were excised for pathological studies by an
author, who was blinded to the study protocol (M.Y.).
The specimens were fixed and stained with
hematoxylin-eosin (H&E) and Massons trichrome
for microscopic observation.
Statistical analysis
The physiologic determinants and other values of
the rats were averaged for each group, presented as
mean standard deviation, and compared among
groups by least significant difference method unless
otherwise defined. A P value <0.05 was considered
significant.

pHi
Whereas pHi declined precipitously in all animals
(Fig. 5A), progression of intracellular acidosis was
slower in the l-LEH-treated rats, with a significant
difference (*P < 0.05) being observed in the salinetreated rats starting even during infusion (18 min)
and throughout the observation (127 min). In contrast, pHi was least different in rats with no treatment, which showed a significant difference in the
progression of acidosis starting from 18 min until reperfusion, as in the rats treated with RBCs (63 min,
#P < 0.05). Thus, the level of pHi at the end of
ischemia varied according to the treatment:
6.54 0.04 in l-LEH, 6.39 0.04 in RBC, 6.32 0.13
in saline (P < 0.05 vs. LEH), 6.47 0.09 in the non-

RESULTS
Hematocrit, LEHcrit, O2 content, and blood
pressure changes
Before and after ischemia/reperfusion, hematocrit
increased significantly only in the RBC transfusion
group (45.5 1.8% to 47.9 1.2%, P < 0.05), while
there were no changes in the group receiving l-LEH
(46.6 2.0% to 45.9 1.1%), saline (45.4 1.7% to
44.0 1.0%), or no treatment (47.1 2.0% to
47.3 3.1%). Furthermore, 10 mL/kg of l-LEH infusion yielded LEHcrit of 1.8 0.6%. These volumes
of RBCs and l-LEH allowed calculation of the O2
content in whole blood and plasma fraction (including LEH) before ischemia and after reperfusion

20

Plasma

O2 Content (O2 mL/dL)

Pre

O2 Content (O2 mL/dL)

Whole Blood

20

Post
Saline group

10

10

0
1

0
1

Pre

Post
LEH group

0.5

0.5

20

40

60

80

PO2 mm Hg
Artif Organs, Vol. 36, No. 2, 2012

100

20

40

60

PO2 mm Hg

80

100

FIG. 4. Oxygen contents in whole blood

and plasma fraction. Volume changes in
RBC (hematocrit) and LEH (LEHcrit)
allowed O2 content calculation in whole
blood (upper panels) and plasma fraction
(lower panels) before (left panels) and
after infusion (right panels) in each treatment group; l-LEH (heavy solid line), RBC
(dotted line), saline (masked line), and no
treatment (thin solid line). While the preischemic condition was the same among the
groups, O2 content in whole blood became
highest in the RBC-transfused and nontreated groups, and O2 content in the
plasma fraction was elevated only in LEHtreated animals. Note that the amount of
O2 contained in the plasma fraction with
l-LEH was less than 1/20 of the O2 content
in whole blood. Some lines were superimposed on each other.

ARTIFICIAL O2 CARRIER ALLEVIATES MUSCULAR ISCHEMIA

189

LEH
Saline
RBC
None

6.6

Infusion

Intracellular pH

7.1

6.1

Ischemia
5.6
-1

18

27

36

45

54

63

72

82

91 100 109 118 127

Time after Occlusion (min)

B

FIG. 5. NMR spectroscopy changes.

Intracellular pH (A) decreased precipitously in each group even following
reperfusion. The animals treated with
l-LEH had significantly higher pHi, starting
during infusion up to reperfusion, compared to RBC-treated rats and animals
without treatment (#P < 0.05), and
throughout the observation in rats treated
with saline (*P < 0.05). The PCr/Pi ratio
(B) compared to self-preischemic value
(%PCr/Pi) decreased after the onset of
ischemia in all groups until reperfusion,
when the ratio reversed to increase (normalize) only in the LEH-treated animals
(#P < 0.05 vs. nontreated control), while
the ratio varied or remained unchanged
in the other groups. The %PCr/Pi ratio
7 days later (C) showed a tendency
toward better preservation in the LEHtreated rats, with no significant difference
from the other treatment groups.

100

LEH
NS

LEH RBC
SalineNO
RBC
None

Infusion

%PCr / Pi

80

60

Ischemia

40

20

0
-1

18

27

36

45

54

63

72

82

91

100

109

118

127

Time after Occlusion (min)

C

%
50

%PCr/Pi

40
30
20
10
0

LEH

Saline

RBC

None

Artif Organs, Vol. 36, No. 2, 2012

190

D. KURITA ET AL.

treated group. The tendency of intracellular acidosis

remained progressive even after releasing the tourniquet, further reducing pHi at the end of observation,
or 60 min after reperfusion, when pHi reduction
appeared to have leveled off (6.21 0.14 in LEH,
6.14 0.13 in RBC, 5.96 0.32 in saline, 6.16 0.13
in the nontreated group), with less significant differences among the groups because of a large variation
after reperfusion.
PCr/Pi
While ATP did not change much in response to
ischemia (Fig. 3), PCr decreased and Pi increased
at the same time, rendering their ratio, PCr/Pi, to
decrease precipitously after the onset of ischemia,
with no difference among groups during ischemia
(Fig. 5B). After release of the tourniquet, however,
the PCr/Pi ratio reversed to increase in all animals in
the LEH-treated group significantly (2.9 2.8% to
13.7 8.4%, P < 0.05) significantly (#P < 0.05) compared to the nontreated group (1.3 1.1% to
1.8 1.9%). The values of the other control groups
were between these two groups, having significant
difference with LEH-treated rats only at 72 and
82 min (*P < 0.05), with no significant timedependent change on average (3.7 7.2% to
5.6 8.4% in saline-treated group, 2.3 3.0% to
7.3 8.4% in RBC-treated group). The %PCr/Pi
ratio 7 days after reperfusion showed variable results
(Fig. 5C), ranging from 20 to 30% of the values
obtained before ischemia. The rats treated with
l-LEH tended to have a higher ratio, although only
31 8%, than the other treatment groups (2023%
on average).
Morphological changes
In a typical H&E staining of the core of the soleus
muscle 7 days after reperfusion (Fig. 6A), there was
diffuse muscular necrosis determined by widened
intercellular space, muscular atrophy, anisocytosis,
and eosinophilic cytoplasm with karyolysis. In the
periphery of the soleus muscle (Fig. 6B), diffuse
mononuclear cell infiltration, or inflammation, was
persistent in all groups, suggesting that a replacement
process was underway. These findings appeared to be
less severe in animals treated with l-LEH.
DISCUSSION
Treatment for and outcome of limb ischemia and
reperfusion failed to be improved even during recent
years when various techniques were developed for
other organs to suppress reperfusion injury and
resultant organ dysfunction. It became clear that
Artif Organs, Vol. 36, No. 2, 2012

minimizing the ischemic damage and reperfusion

injury is crucial for suppressing skeletal muscle
damage to the affected limb as well as a subsequent
systemic inflammatory response (14). Using 31PNMR during ischemia and early reperfusion, we
tested the effects of LEH, which has been reported
to be beneficial in various experimental cases of
focal as well as global ischemia and reperfusion (5)
highly protective against brain edema after permanent occlusion of the middle cerebral artery (6),
preserving the cortex after reperfusion in the rat (7)
as well as in the monkey (8), protective in cochlear
ischemia/reperfusion (9), accelerated gastric (in
preparation) as well as skin wound healing (10), and
enhanced cancer radiotherapy (11). Moreover, LEH
has been manufactured to have a lower oxygen affinity (l-LEH; P50 = 40 mm Hg [5]) than RBC to transfer O2 efficiently under atmospheric or supplemental
O2 respiration (Fig. 1). Thus, we tested l-LEH
with O2 respiration in an early phase of skeletal
muscle ischemia induced by tourniquet occlusion,
finding that l-LEH-treated rats had significantly
milder intracellular acidosis, while the PCr/Pi ratio
decreased regardless of treatment after onset of
ischemia until reperfusion 70 min later, when the
PCr/Pi ratio returned toward normal only in the
LEH-treated rats.
Intracellular energy metabolism was followed by
31
P-NMR spectroscopy to detect subtle intracellular
changes in the rodent hind limb undergoing abrupt
ischemia followed by reperfusion. In preliminary
studies, we tested various methods to induce leg
ischemia that would be severe enough to be associated with a steady increase in Pi by 31P-NMR.
Arterial inflow occlusion by ligation, thrombotic
occlusion, or stripping of the femoral artery yields
only a temporary increase in Pi, which returns to
baseline, suggesting that a compensatory mechanism(s) quickly reinstates aerobic metabolism in the
rodent hind limb. Since we learned that circumferential heavy ligation induced skeletal muscle ischemia
(25), we developed the current tourniquet system to
allow remote control of tightening for ischemia and
releasing for reperfusion while keeping the animal in
place for continued NMR observation. Then, occlusion pressure was increased by changing the weight
from 2.0 to 2.5 kg and then to 3.0 kg to induce steady
and serious ischemia. Finally, the duration of
ischemia was adjusted to 70 min, not long enough to
induce muscular necrosis (1,16), in order to allow
observation of the reperfusion response in the
skeletal muscle, but not of the systemic consequences, which were undetectable by the current
protocol.

ARTIFICIAL O2 CARRIER ALLEVIATES MUSCULAR ISCHEMIA

A

191

H&E stainin
ng 1 week later (x 10)
LEH

Saline

RBC

None

H&E staining
g 1 week later (x 10)
LEH

Saline

RBC
C

None

Although the occlusion pressure was higher than

those reported in mice (25) or in rats (26), significant
deceleration in intracellular acidosis starting soon
after infusion suggested penetration of l-LEH across
the high occlusion pressure. As pHi largely depends
on the levels of intracellular lactate in a dynamic
balance between its production and washout (16
19,23), LEH treatment was considered to be associated with aerobic metabolism, reducing lactate
production and thereby decelerating the progression

FIG. 6. Morphological study. In a typical

H&E staining of the core of the soleus
muscle 7 days after reperfusion (A), there
was diffuse muscular necrosis determined
by widened intercellular space, muscular
atrophy, anisocytosis, and eosinophilic cytoplasm with karyolysis. In the periphery of the
soleus muscle (B), diffuse mononuclear cell
infiltration was persistent in all groups, suggesting a prevailing replacement process.
These findings appeared to be less severe
in animals treated with l-LEH.

of intracellular acidosis. This may be the first direct

evidence that LEH is effective during ischemia, as all
the previous studies but one (7) showed benefits of
LEH only after reperfusion (6,811), leaving the
inevitable possibility that LEH is effective not as an
O2 carrier, but as an agent that reduces oxidative
stress after reperfusion. During brain ischemia,
plasma flow has been reported to be persistent,
although decreased, even to the core of the ischemia
(27). There might be a similar effect of LEH under
Artif Organs, Vol. 36, No. 2, 2012

192

D. KURITA ET AL.

the permanent occlusion of the middle cerebral

artery (7), where reduction in brain edema was highly
significant in various parts of the brain despite the
absence of reperfusion. Thus, the benefits of LEH
may be described as its perfusion with O2 delivery,
supporting aerobic energy metabolism and preserving pHi during ischemia, which ameliorates the reinstatement of microcirculation early after reperfusion.
In contrast to pHi, the PCr/Pi ratio decreased
abruptly during ischemia without any differences
among treatment groups, and there was also not
much difference in the ATP levels among the groups,
similar to a previous observation by Morikawa et al.
(16), who reported that changes occurred in the order
of PCr reduction, Pi increase, and pHi decrease, followed by beta-ATP reduction after 3 h. This might
suggest that O2 delivery with LEH is obviously not
enough, and that PCr degradation and Pi production
occurred at a similar rate regardless of the treatment,
as it is the in-common step for muscular energy
storage and metabolism (1417) to keep the ATP
level constant, the final energy source for cellular
survival (1,16,23). These phenomena are compatible
with the observations by Hayes et al. (12), who
reported that myocyte glycogen and PCr were
depleted initially, in preference to ATP, in skeletal
muscle (13), as well as in the myocardium (23). After
a longer ischemic interval, diminishing ATP levels
correlated closely with worsening muscle necrosis
(13), suggesting that the current ischemia for only
70 min is not severe enough to deplete ATP or to
cause muscular necrosis (13,16). After reperfusion,
however, only LEH-treated rats had significantly
improved PCr/Pi ratio, while the nontreated animals
failed to show any improvement in energy status (Fig.
5B). This may be due to the effect of intravenous
infusion (10 mL/kg) during ischemia in the LEH,
RBC, and saline treatment groups, which may help to
reduce viscosity and retain plasma flow in capillaries
during ischemia and facilitate recovery of microcirculation after reperfusion. These speculations are
compatible with the report by Hammersen (13), who
precisely followed the morphological changes in capillaries during ischemia and reperfusion. In contrast,
nontreatment might have led to increased blood viscosity, which would likely be associated with thrombotic occlusion of the vessels (14), further delaying
the recovery of intracellular acidosis and energy
status.
This discrepancy, improving energy status (PCr/Pi)
under worsening intracellular acidosis, may be due to
the delay in recovery of pHi (14,18), being related to
prolonged anaerobic metabolism (14,19), consuming
intracellular buffering materials, accumulated lactate
Artif Organs, Vol. 36, No. 2, 2012

(18), and failure in immediate reperfusion in some

animals that may even deter the recovery of microcirculation and lactate washout (13,28), as in the nontreated control rats. The important difference may be
the source of energy regeneration and transfer to
reproduce PCr, either aerobic or anaerobic; the latter
is much less efficient, with lactate production as
the end metabolite (2325,28). Thus, pHi changes
became similar and leveled off after reperfusion,
when O2 became available for aerobic energy production in viable cells. The presence of LEH in the
plasma was associated with increased O2 availability,
which had ameliorated the PCr/Pi ratio without
improving pHi (14) after reperfusion. Such persisting
metabolic failure may result in the observations
7 days later, when cell shrinkage and necrosis in the
core of ischemia and diffuse neutrophil infiltration in
the periphery were noted. These findings were in
accordance with the 31P-NMR observation that the
PCr/Pi ratio tended to be higher (albeit only 31%) in
LEH-treated rats than in the other animals, with 20 to
23% of preischemic control (24).
These precise and sensitive observations of pHi
and energy status were afforded by 31P-NMR, which
in turn made it difficult to obtain systemic and physiologic determinants, such as blood pressure, systemic
acid-base balance, and electrolyte levels. While the
observed changes in pHi and energy status appeared
to be irrelevant to reperfusion pressure, hematocrit,
whole blood O2 content, pH, or buffering capacity of
infused solutions, these physiologic parameters are
important as the systemic symptoms after reperfusion, and they will need to be followed in future
studies.
CONCLUSION
l-LEH (10 mL/kg) early after onset of ischemia
appeared to hamper progressive intracellular acidosis of skeletal muscle and improve energy regeneration after reperfusion, suggesting exertion of a
protective effect on energy metabolism during
ischemia and early reperfusion. A tendency toward
less severe neutrophil infiltration, and preservation of
PCr/Pi, muscular structure, and muscle mass 7 days
later may suggest improved muscular viability in rats
treated with l-LEH. While the current results are
compatible with the hypothesis, the mechanism(s)
remains speculative, and successive systemic influences need to be determined in future studies.
Acknowledgments: We gratefully acknowledge
the assistance and technical support of the Tokai University Education and Research Support Center.

ARTIFICIAL O2 CARRIER ALLEVIATES MUSCULAR ISCHEMIA

Funding: This study was supported in part by:
A) Grant-in-Aid for Scientific Research
(A16209037 and A20249072) from the Ministry of
Education, Culture, Science and Technology, Tokyo,
Japan. This was granted to A. Kawaguchi.
B) New Energy Development Organization
(NEDO), Tokyo, Japan. This was granted to Terumo
Co. Ltd. and Tokai University.
C) Japan Science and Technology Agency (JST),
Japan. This was granted to A. Kawaguchi.
Conflicts of interest and author contribution: D.
Kurita, A. Kawaguchi, and M. Haida are clinicians/
scientists responsible for the organization and summarization of this study. At the time of the
experiments, they were attached to Tokai University
School of Medicine, where all animal experiments
were carried out. K. Aso and H. Minamitani were
researchers belonging to Keio University School of
Science and Technology at the time of this work. M.
Yamano was doing research at Osaka Prefecture
University and did the anatomical studies. As these
authors had individual research funds (specified previously), there are no monetary dependencies or conflicts of interest.
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Artif Organs, Vol. 36, No. 2, 2012