Effect of diaspirin cross-linked hemoglobin on normal and
postischemic microcirculation of the rat
pancreas
Ernst
von Dobschuetz1,
Tomas
Hoffmann2,
Clemens
Engelschalk3, and
Konrad
Messmer1
1 Institute for Surgical
Research and 3 Institute for
Clinical Chemistry, Klinikum Grosshadern, Ludwig-Maximilians
University, D-81366 Munich; and
2 Maria-Theresia
Klinik, D-80336 Munich, Germany
 |
ABSTRACT |
Microcirculatory alterations
with reduced nutritive supply to the pancreas could be the cause of
hyperamylasemia, which occurs in some patients receiving the vasoactive
oxygen carrier diaspirin cross-linked hemoglobin (DCLHb) in clinical
studies. Therefore, the effects of DCLHb on rat pancreas
microcirculation were evaluated. Anesthetized Sprague-Dawley rats
received one of the following treatments during baseline conditions
(n = 7 rats/group): 10% hydroxyethyl starch (HAES) (0.4 ml/kg), DCLHb (400 mg/kg), or DCLHb
(1,400 mg/kg). After 1 h of complete, reversible pancreatic ischemia, other animals received 10% HAES (0.4 ml/kg) or DCLHb (400 mg/kg) during the onset of reperfusion. The number of
red blood cell-perfused capillaries (functional capillary density, FCD)
and the level of leukocyte adherence in postcapillary venules in the
pancreas were assessed by means of intravital microscopy during 2 h
after treatment. In the nonischemic groups, FCD was 18% greater after
DCLHb (1,400 mg/kg) than after 10% HAES treatment without any increase
in leukocyte adherence. In the inschemia-reperfusion (I/R) 10% HAES
group, FCD was significantly (P < 0.05) lowered, leukocyte adherence enhanced, and mean arterial pressure
(MAP) reduced by 31% compared with nonischemic animals. DCLHb
treatment in the I/R group resulted in a slight increase in FCD, a
significant (P < 0.05) reduction of
leukocyte adherence, and a complete restoration of MAP compared with
the animals of the I/R control group. Thus our data provide no evidence
for a detrimental effect on the pancreatic microcirculation or an
enhanced risk of postischemic pancreatitis by DCLHb.
hemoglobin solutions; blood substitute; endothelin; nitric oxide; shock treatment
 |
INTRODUCTION |
DIASPIRIN CROSS-LINKED hemoglobin (DCLHb, HemAssist)
was designed as an oxygen carrier for the treatment of shock,
hypovolemia, and anemia. DCLHb, a modified stroma-free human
hemoglobin, has undergone multicenter clinical trials in the United
States and Europe for the treatment of shock and stroke and for use in
elective surgery (19). In the phase I
study, an increase of blood pressure and a slight decrease of heart
rate were seen in healthy volunteers after administration of DCLHb,
without other serious side effects (23). DCLHb does not show
immunogenicity in humans, has lowered infectious risks compared with
blood transfusions, and is immediately available in an emergency
situation, since there is no need for cross-matching and blood typing.
The beneficial oxygen-carrying properties of DCLHb have been
demonstrated in several in vitro and in vivo situations in animals
subjected to controlled and uncontrolled hemorrhagic shock (19).
Probably due to the nitric oxide (NO)-scavenging mechanism of free
hemoglobin, DCLHb also appears to be advantageous in septic shock
patients (24) suffering from hyporeactivity to vasoconstrictive drugs
and mediators. Endothelium-derived relaxing factor (NO) (28),
endothelin, and the potentiation of adrenoreceptors (19) by DCLHb are
considered to be involved in the vasoreactive reactions observed after
DCLHb injection. DCLHb shows an alteration of blood flow and regional
vascular resistance to many organs (28). The pancreas is reported to be
a susceptible organ to ischemia-reperfusion (I/R) in patients with hemorrhagic shock. Hypoperfusion of the pancreas is possible during cardiac surgery due to the extracorporal circulation, and I/R
reactions would be anticipated following pancreas transplantation and
during surgery of the thoracic and thoracoabdominal aorta due to the
clamping of the blood supply to the pancreas (7, 8). Experimental acute
pancreatitis induced by different means shows microcirculatory
perfusion failure with a reduction of the nutritive supply to the
pancreatic tissue (13). Vasoactive changes that are seen after DCLHb
administration could result in an alteration of tissue blood and oxygen
supply to the pancreas. The role of enhanced endothelin-1 and lowered
NO concentrations, as seen after DCLHb administration, in experimental
acute pancreatitis is controversially discussed. Some studies show
beneficial and some detrimental effects of these factors in the course
of acute pancreatitis (14, 15, 34). To date, the hamster skin muscle is
the only organ in which microcirculatory alterations after DCLHb
injection have been investigated utilizing intravital microscopy
techniques. In muscle, these experiments showed an increase of venular
red blood cell velocity under nonischemic conditions after DCLHb
treatment, without changes of functional capillary density (FCD) or
leukocyte adherence in postcapillary venules (20). After 4 h of
pressure ischemia in the dorsal skinfold chamber, there was a
reduction of leukocyte adherence in postcapillary venules after DCLHb
treatment during reperfusion time (21). In 4 of 14 septic patients who
received DCLHb in a phase II trial, sepsis was caused by acute pancreatitis (24). When considering treatment of such patients with DCLHb, it is important to exclude possible disturbances of pancreatic microvascular perfusion after induction of acute pancreatitis. In clinical studies, some patients showed hyperamylasemia after DCLHb administration. Microcirculatory alterations of DCLHb could be the trigger mechanism of such enzyme enhancement. Thus the purpose of this study was to investigate microcirculatory reactions and possible side effects after DCLHb administration under normal conditions and after an inflammatory stress
induced by 1 h of ischemia-induced acute pancreatitis.
 |
MATERIALS AND METHODS |
Anesthesia and monitoring.
Male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) weighing
180-260 g were anesthetized by ether and pentobarbital sodium (50 mg/kg body wt ip) after an overnight fast with free access to tap
water. After tracheotomy, the rat respiration was volume controlled by
a ventilator (frequency: 57-65 breaths/min, tidal volume:
2-2.5 ml, inspired oxygen fraction: 0.25-0.40; rodent ventilator 683, Harvard Apparatus, South Natick, MA). The right carotid
artery and the right jugular vein were cannulated by a polyethylene
catheter (PE-50, 0.58 mm ID, Portex, Hythe, Kent, UK) for continuous
monitoring of mean arterial pressure (MAP), heart rate, and continuous
volume replacement (3-5 ml/h iv 0.9% NaCl) by a syringe pump.
Rectal temperature between 36.5 and 37.5°C was kept constant by
means of a heating pad (Fa. Effenberger, Pfaffing, Germany). Adequate
anesthesia was maintained by intravenous injection of pentobarbital
sodium (12 mg · kg body
wt
1 · h
1)
and N2O admixture
(0.65-0.85). Arterial blood gases were measured intermittently (ABL 300, Radiometer, Copenhagen, Denmark) and were
adjusted to the following values for baseline conditions by means of
breathing adjustment and 8.4% sodium bicarbonate injection: PO2 = 100-120 mmHg,
PCO2 = 30-40 mmHg, pH = 7.39 ± 0.02, and base excess = 0 ± 2. Hematocrit values in arterial blood were measured by Coultercounter
T540 (Coulter Electronics, Hialeah, FL). The experiments were performed
in accordance with German legislation for the protection of animals.
Animal model and experimental protocol.
After a transverse laparatomy, complete ischemia of the
pancreas was induced by means of microvascular clips (closing force of
70 g; Aesculap, Tuttlingen, Germany) on the four arteries supplying blood to the pancreas (left gastric artery, gastroduodenal artery, splenic artery, and caudal pancreaticoduodenal artery). The
microsurgical procedure has been described elsewhere (9). Sham-operated
animals underwent the same preparation but without induction of
ischemia. After a stabilization period of 15 min, animals were
randomly assigned to five groups (n = 7 animals/group): 1) sham-operated group without ischemia that received 4 ml/kg body wt of 10%
hydroxyethyl starch (HAES) (200/0.5) after operation procedures (sham
control), 2) sham-operated group
without ischemia that received 400 mg/kg (4 ml/kg) body wt
DCLHb (sham DCLHb), 3) sham-operated
group without ischemia receiving 1,400 mg (14 ml/kg) body wt
DCLHb (sham high-dosage DCLHb), 4)
1-h ischemia group that received 4 ml/kg body wt of 10% HAES
(200/0.5) at the onset of reperfusion (ischemia control), 5) 1-h ischemia group that
received 400 mg/kg (4 ml/kg) body wt DCLHb during the onset of
reperfusion (ischemia DCLHb). ["HAES (200/0.5)"
means the artificial colloid HAES (macromolecular polymer, which is
manufactured from amylopectin and consists of hydroxyethylated glucose
molecules linked by
-1.4 bonds) with a molecular weight of 200,000. The degree of hydroxyethylation is 0.5, which means that 5 of 10 glucose molecules are substituted by hydroxyethyl groups.] HAES
was chosen as a control solution for DCLHb to show that possible
microcirculatory or macrocirculatory effects of DCLHb are not caused
just by its volume-expanding property. An ischemia control
group receiving no HAES infusion was not considered necessary, since
there was no significant difference in microvascular parameters between
these two groups in experiments performed to establish this model (9).
The circulatory response of DCLHb is well characterized in rats and
pigs and is dose dependent up to a dosage of 400-500 mg/kg body
wt. At higher doses of DCLHb, there is no further increase of MAP (3).
We assumed that the major microcirculatory effect of DCLHb on the
pancreas should occur at the doses chosen. Because other experiments
that investigated the circulatory effects of DCLHb by microsphere
technique also used a dose of 400 mg/kg body wt, we used the same dose
to make our results comparable to these studies (5, 6). The high dose
(1,400 mg/kg body wt) injected during baseline conditions allowed us to
investigate whether a high, top-load dose of DCLHb would elicit greater
microcirculatory effects compared with the 400 mg/kg dose.
Samples of arterial blood (1 ml) were taken before administration of
the solutions, 15 min after administration, and at the end of the
experiment. The withdrawn blood volume was immediately replaced by a
0.9% NaCl solution (1:1). Fifteen minutes after administration of the
solution, the pancreas and spleen were exteriorized for intravital
microscopy on an adjustable stage and covered by a thin Teflon membrane
to prevent drying. MAP was continuously registered on a recorder (XT
Kompensograph, Siemens, Munich, Germany). Heart rate was obtained from
the phasic blood pressure curves. Heart rate, arterial blood gases, and
hematocrit were measured at baseline conditions and 15, 60, and 120 min
after administration of the solutions. The experiments were terminated
by injection of an overdose of pentobarbital sodium.
Drugs.
HemAssist (DCLHb) was provided in 4.5-ml containers by Baxter
Healthcare (lot no. 97J10AD11-111997, Round Lake, IL). The
physiochemical and pharmacokinetic characteristics of HemAssist given
by manufacturer were as follows: 10 g/dl hemoglobin crosfumaril
content, methemoglobin concentration of <5%, oxygen half-saturation
value (P50) = 32 mmHg (normal
blood P50 = 26 mmHg),
right-shifted dissociation curve compared with that of red blood cells
for transfusion, pH adjusted to 7.4 at 37°C, half-life time = 13 h
in healthy men and women, and oncotic pressure = 42 mmHg (human plasma = 25 mmHg). Containers were stored in a 70°C freezer and thawed 0.5 h before use. The 10% HAES (200/0.5) was purchased from Fresenius (Bad Homburg, Germany).
Intravital microscopy and quantification of microcirculation.
After intravenous injection of 0.15 ml of 0.75% HAES (molecular weight
of 200,000) labeled with FITC (FITC-HAES; Laevosan, Linz, Austria) for
contrast enhancement of microvessels and 0.1 ml of 0.2% rhodamine 6G
(molecular weight of 497; Sigma, St. Louis, MO) for in vivo staining of
cytochrome c-containing cells
(leukocytes), intravital microscopy of the pancreas was performed using
a modified Leitz-Orthoplan microscope (Leitz, Wetzlar, Germany) with a
100 W mercury vapor lamp attached to a Ploemo-Pak illuminator with I2/3 (excitation 450-490 nm,
emission >515 nm) and N2
(excitation 530-560 nm, emission >580 nm) filter blocks (Leitz)
for epi-illumination. A saltwater immersion objective (×25/0.6;
Leitz) allowed magnification of approximately ×800. The
observations were recorded by means of a charge-coupled device video
camera (FK 6990, Cohu, Prospective Measurements, San Diego, CA) and
stored on video tape (Panasonic video recorder, Munich, Germany) for
off-line evaluation. Quantitative assessment of the microcirculation
included determination of the FCD and the number of adherent leukocytes
in the postcapillary venules. These parameters were measured at three
time points: 45, 90, and 120 min after injection of the solutions. FCD
is defined as the number of red blood cell-perfused capillaries (cm)
per observation area (cm2). The
FCD was determined by analysis of the video tapes according to
Schmid-Schönbein et al. (26) by means of superimposing a grid
system (squared-type) on the video screen (square side = 50 µm). The
number of intersections of red blood cell-perfused capillaries with the
grid system were counted, and FCD was calculated as described earlier
(9). Ten randomly selected tissue surface areas (400 × 300 µm)
of the pancreas were evaluated at each time point. For quantification
of leukocyte-endothelial interaction, at least three identical
postcapillary venules (diameter of >40 µm and length of <150
µm) per animal were recorded for 30 s at each time point. Adherent
leukocytes were defined as cells remaining stationary on the surface of
the endothelium for the whole 30-s observation time. The surface area
of the vessel segments was calculated on the basis of diameter
measurements, assuming a cylindrical geometry of the vessels. Adherent
leukocytes are given as cells per area
(mm2).
Measurement of serum values of
-amylase and
interleukin-6.
Blood samples were withdrawn from the carotid artery and centrifuged at
3,000 g for 10 min at 4°C. Serum
samples were stored in a
70°C freezer until assay. Serum
-amylase activity was measured by a random-excess analyzer (COBAS
INTEGRA, Roche, Basel, Switzerland). The interleukin-6 (IL-6) serum
concentration was measured by means of a rat blood IL-6 ELISA kit (no.
I051502A, Laboserv, Staufenberg, Germany). Interference of DCLHb with
the two performed tests was investigated by spiking plasma with
increasing concentrations of DCLHb. Because the amylase activity test
yielded valid data up to a DCLHb concentration of 200 mg/dl, samples
were diluted for measurement. No interference of the IL-6 ELISA test
was encountered up to a DCLHb concentration of 1,500 mg/dl. Tests were
not performed in the high-dosage group due to technical problems with
these two tests.
Light microscopy.
Tissue samples from the corpus of the pancreas were taken at the end of
the experiment from four animals per group and immediately fixed in
10% neutral buffered Formalin. The samples were dehydrated, embedded
in paraffin, cut at ~3 µm (Microtome, Leica, Munich, Germany), and
stained with polymorphonuclear esterase staining (Sigma). In 10 regions
of interest (diameter of 250 × 250 µm) per animal, granulocytes
were counted (cells/mm2) by a
blind observer.
Statistics.
All data are presented as mean values ± SE. After normal
distribution testing was performed, data were subjected to one-way ANOVA and pair-wise Student-Newman-Keuls test between the groups. Non-normally distributed data were tested by one-way ANOVA on ranks
followed by a pair-wise Dunn's method comparison procedure. Within
each individual group, one-way repeated-measures ANOVA (normally
distributed data) followed by Dunnett's method, or a Friedman
repeated-measures ANOVA on ranks (non-normally distributed data)
followed by Dunnett's method was performed. The amylase values were
tested by paired t-test (normally
distributed data) or Wilcoxon's signed rank test (non-normally
distributed data) (SigmaStat 2.0, Jandel, San Rafael, CA).
P < 0.05 was considered to be
statistically significant.
 |
RESULTS |
Effects of DCLHb on normal pancreas.
To investigate the influence of DCLHb on pancreatic microcirculatory
perfusion changes, FCD (Fig.
1A)
was assessed by intravital microscopy. DCLHb (400 mg/kg) administered
during baseline conditions resulted in no significant difference of FCD
compared with 10% HAES treatment. A higher dosage of DCLHb (1,400 mg/kg) increased microcirculatory perfusion by 18% compared with the
HAES-treated group at the end of the experiments. In all three
sham-operated groups, there was a significant increase of leukocyte
adherence during the observation time compared with the 45-min values
(Fig. 2A). No
differences in degree of leukocyte adherence in postcapillary venules,
amylase serum activity (Fig. 3), and IL-6
serum concentration (Fig. 4) could be
detected between the two sham-operated groups. In groups receiving 400 mg/kg and 1,400 mg/kg DCLHb, respectively (Fig.
5),
MAP was elevated by 18% and 21%, respectively, compared with the 10%
HAES-treated control group. Hypertension persisted in the 1,400 mg/kg
DCLHb group over the whole observation period. In the 400 mg/kg DCLHb
group, the MAP returned to baseline values between 15 and 120 min after
injection. DCLHb resulted in a significant amelioration of base excess
compared with the 10% HAES control group at 60 and 120 min after
solution injection (Table 1).


View larger version (72K):
[in this window]
[in a new window]
|
Fig. 1.
Functional capillary density given as the length of red blood
cell-perfused capillaries per observation area
(cm/cm2).
A: diaspirin cross-linked hemoglobin
(DCLHb; 400 mg/kg) in nonischemic animals yields no significant
differences compared with the hydroxyethyl starch (HAES) control group.
At the higher dose (1,400 mg/kg), FCD was significantly increased.
B: ischemia-reperfusion injury
induced significant capillary "no-reflow" compared with the
nonischemic control group. This effect was reduced by DCLHb treatment
at the end of the experiment. Values are means ± SE.
# P < 0.05 vs. sham control, + P < 0.05 vs.
ischemia control group.
|
|


View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
Number of adherent leukocytes in at least 3 postcapillary venules.
A: there were no significant
differences in nonischemic animals between the groups. Values are means ± SE. + P < 0.05 vs. 45 min values. B: 1-h ischemia of
the pancreas resulted in a 3-fold increase in the number of adherent
leukocytes compared with nonischemia (sham control). Leukocyte
adhesion was significantly (P < 0.05) reduced by DCLHb treatment at the end of observation
time. Values are means ± SE.
# P < 0.05 vs. sham-HAES
control, + P < 0.05 vs. ischemia-HAES control.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
Serum amylase concentrations at baseline conditions and at the end of
the experiment. Ischemia-reperfusion resulted in a significant
(P < 0.05) increase in amylase
concentration, which was completely absent in the group receiving
DCLHb. Values are means ± SE. + P < 0.05 vs. baseline.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Interleukin-6 (IL-6) serum concentration at baseline and after
treatment. No changes were detected in the nonischemic animals.
Significant elevation of IL-6 was found after 2 h of reperfusion in the
10% HAES-treated ischemia-reperfusion group, which was absent
in the DCLHb-treated ischemia-reperfusion group. Values are
means ± SE. + P < 0.05 vs.
baseline and DCLHb-sham. p. inf., Postinfusion.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of mean arterial pressure (MAP). Injection of DCLHb into
nonischemic animals resulted in a significant increase in MAP, which
lasted 1 h in the 400 mg/kg sham and 2 h in the 1,400 mg/kg sham group.
A significant decrease of MAP can be seen during the reperfusion time
in the 10% HAES-treated ischemia-reperfusion group, whereas
MAP was preserved in DCLHb-treated animals. Values are means ± SE.
# P < 0.05 vs. sham HAES
control, + P < 0.05 vs. before
injection (b inj).
|
|
Effects of DCLHb on postischemic pancreas.
I/R resulted in a significant (P < 0.05) reduction of FCD during the reperfusion time (from 266 ± 15 to 188 ± 12 cm
1)
compared with the sham control group (from 362 ± 10 to
318 ± 10 cm
1) (Fig.
1B). This impairment of capillary
perfusion was attenuated by DCLHb treatment at the end of the
reperfusion time (220 ± 12 cm
1)
(P < 0.05). I/R significantly
enhanced leukocyte adherence (P < 0.05) at all time points. This effect was less pronounced in DCLHb-treated animals (P < 0.05)
compared with the ischemia control group (Fig.
2B). A significant increase of both
amylase activity (Fig. 3) and IL-6 concentration (Fig. 4) during the
experiment was only observed in the 10% HAES-treated I/R group but was
completely absent in the DCLHb-treated animals. When reperfusion
occurred, MAP fell immediately by ~30%. DCLHb treatment thereafter
was followed by an immediate and significant
(P < 0.05) increase in blood
pressure back to control values, which was sustained to the end of the experiment. In contrast, 10% HAES treatment in the I/R group resulted in only a short-lasting (5 min) increase in MAP, with the animals remaining hypotensive until the end of the experiment.
Effects of DCLHb on granulocyte infiltration in the pancreatic
tissue.
Extravasated leukocytes were seen only in close proximity to the
endothelium of the vessels. There were no significant differences when
the number of extravasated leukocytes between the experimental groups
was compared (sham control: 0.8 ± 0.5/mm2, sham DCLHb: 1.2 ± 1.2/mm2, sham
high-dosage DCLHb: 1.6 ± 0.7, ischemia control: 39.1 ± 20.2, ischemia DCLHb: 6.1 ± 3.6).
 |
DISCUSSION |
Effects of DCLHb on normal pancreas.
In this study, we have observed a significant enhancement of pancreatic
FCD after top-load infusion of a very high dosage of DCLHb into healthy
animals. These results are similar to data gained by microsphere and
laser-Doppler methods, indicating that, when DCLHb is infused,
perfusion of the pancreas is increased in healthy animals (5). As
expected from other studies (19), MAP increased significantly after
DCLHb injection in a dose-dependent manner. However, no adverse
microcirculatory effect of DCLHb, for example a fall of FCD or enhanced
leukocyte adherence, could be detected in the normal pancreas; it is
therefore unlikely that hyperamylasemia seen after DCLHb injection into
patients in clinical studies is due to a disturbance of pancreatic
microvascular perfusion. The decrease of FCD and the considerable
increase in the number of adherent leukocytes observed after infusion
of 10% HAES in the sham group may be explained by an inflammatory
reaction due to the exteriorization of the pancreas. These changes
appear to be counteracted by DCLHb treatment, as suggested by the lower increase of amylase (Fig. 3) and IL-6 (Fig. 4) levels, as well as
stable FCD values (Fig. 1A) in the
DCLHb-treated sham groups.
Effects of DCLHb on postischemic pancreas.
This is the first study on microvascular perfusion after reversible
normothermic ischemia to the pancreas. As we have shown previously, 1 h of complete pancreatic ischemia provokes mild pancreatic injury (9). We assumed that both harmful and beneficial effects of DCLHb should be detectable in this preparation. In the
postischemic pancreas, DCLHb resulted in a slight improvement of FCD,
reduction of leukocyte adherence, and a stable arterial blood pressure;
this appears important for the treatment of the inflamed gland, since
systemic hypotension is a major complicating factor in the early phase
of acute pancreatitis in humans. The concentrations of IL-6 and amylase
were slightly reduced in the DCLHb I/R group, a finding in agreement
with the microcirculatory data showing that DCLHb does not exacerbate
the inflammatory reaction. IL-6 is a valuable prognostic parameter to
assess the course and severity of disease during the early onset of
acute pancreatitis (27). As in our previous study (9), I/R resulted in
a comparable reduction of FCD, enhancement of leukocyte adherence, and
elevation of amylase, even though the control group was receiving 10%
HAES. This group was included to exclude a major volume effect being responsible for changes observed after infusions of the hyperoncotic HAES solution, since DCLHb has itself plasma-expanding properties (oncotic pressure = 42 mmHg).
I/R in the pancreas.
Post-I/R injury is characterized by a microcirculatory perfusion
failure, called capillary no-reflow, which causes continuing impediment
of oxygen supply to the tissue, thereby exacerbating the primary
ischemic damage to tissue. The causes for capillary no-reflow are up to
now not completely understood. Among other mechanisms,
hemoconcentration in the capillaries due to the enhanced plasma
leakage, disequilibrium of endothelin-1 and NO balance controlling
microvascular flow, leukocyte plugging of capillaries, and endothelial
cell swelling are discussed as possibilities (18). Further hallmarks of
reperfusion injury are the activation of leukocytes and the enhancement
of leukocyte-endothelium interactions mediated through upregulation of
adhesion molecules on the surface of endothelial cells of postcapillary
venules (8, 18). Adherent leukocytes are considered to play a pivotal
role in I/R tissue damage due to their release of proteinases and
generation of superoxide radicals (respiratory burst) (8). These
phenomena have been described for striated muscle (18), heart, liver,
lung, and many other organs (8). Our group was the first to describe these phenomena in I/R-induced experimental pancreatitis, and we have
demonstrated that the high susceptibility of the pancreas to I/R
results in histological changes resembling those seen in acute
pancreatitis (8, 9). I/R in pancreas causes an inflammatory reaction
similar to acute pancreatitis and is considered a major determinant in
progression of pancreatitis caused by other factors. For example,
ethanol ingestion, next to gallstones, is a major associated factor for
acute pancreatitis and causes a significant increase of endothelin-1
levels and a drastic reduction (up to 50%) of pancreas perfusion (33),
indicating that reduced perfusion of the pancreas, as well as
consecutive low-flow ischemia, can contribute decisively to
pancreas tissue damage. To evaluate if there are potentially harmful
microcirculatory effects of DCLHb in patients with acute pancreatitis,
we have studied postischemic pancreatitis, particularly since elevated
serum amylase and lipase values have in fact been observed after
infusion of DCLHb to patients. To date, DCLHb has proven its usefulness
in both systemic (20) and organ ischemia in animal in vivo
models of kidney (22), heart (17), skin muscle (21), and brain (1). In
our study, there is only a slight improvement of pancreatic FCD failure
after I/R by DCLHb. There is a significant reduction of leukocyte
adherence in the DCLHb-treated I/R group. These results let us conclude that the beneficial effect of DCLHb on postischemic pancreatitis is, in
contrast to other organs, very low. The mechanisms by which DCLHb,
injected as a topload infusion, beneficially influences I/R damage
could be the following. 1) Treatment
of I/R-damaged muscle of the rat and hamster (29) with hyperbaric
oxygen enhanced oxygen supply to the tissue and showed a beneficial
effect. Thus treatment of I/R damage with the oxygen carrier DCLHb,
which obviously enhances the
PO2 of
postischemic tissue (21), is probably beneficial in I/R injury. In
fact, infusion of DCLHb after I/R injury of the kidney did not increase
radical generation occurring after ischemia (22).
2) After normothermic I/R of the
pancreas, the animals suffered from hypotension. Reduction of the MAP
induced by tourniquet ischemia of the rat hindlimb resulted in
a reduction of FCD in the pancreas of 40% (13). Similar results were
obtained in our model, indicating that reduction of MAP by bradykinin
resulted in a tremendous decrease in FCD (11). Because DCLHb stabilizes the macrohemodynamic parameters, this could be another reason for the
protection of the microcirculation by DCLHb in our experimental model.
Vasoactive effects of DCLHb and possible influence on pancreas
microcirculation and I/R damage.
DCLHb induces changes of blood flow and regional resistance to flow in
various organs by virtue of its vasoactive properties. Several studies,
using the radioactive microsphere technique to investigate the normal
perfusion of pancreas and mesentery after DCLHb administration, have
shown results similar to those obtained in our experiments. In these
studies, DCLHb (400 mg/kg body wt) caused an increase in blood flow
velocity to the pancreas of almost 100% from baseline (after 15 min);
baseline values were reached again within 60 min (5).
These effects of DCLHb on the blood supply of the pancreas were nearly
abolished when the endothelin receptor antagonist BQ-123 or the
substrate for NO synthase,
L-arginine, was present,
indicating that endothelin release and scavenging of NO are involved in
the regional blood flow changes. There is considerable controversy in
the literature concerning NO and endothelin in normal and inflamed
tissues. In contrast to red blood cell-encapsulated hemoglobin, which
does not cross the endothelial cell barrier, the
-
cross-linked
tetramer is considered able to penetrate the endothelial and smooth
muscle layer (4). Endothelin (the endothelium-derived constriction
factor) and NO (endothelium-derived relaxing factor) regulate the
vascular tone (25). Thus extravasating DCLHb is capable of increasing
vascular tone by NO scavenging. The scavenging of NO by free hemoglobin
is a well-known fact (19). Two known mechanisms are
1) the reaction between
oxyhemoglobin and NO to give methemoglobin and nitrite and
2) binding of NO at the Fe center of
deoxyhemoglobin. On the other hand, endothelin-1 is markedly increased
after DCLHb injection (6). Furthermore, endothelin-1 has been shown to
convert acute edematous pancreatitis into hemorrhagic pancreatitis (15)
in rats. In dogs, endothelin reduced blood flow to the pancreas by
~50% (31). In contrast, one experimental study on acute pancreatitis
reported a tissue-protective effect of endothelin-1 (14). Other
contradictory results exist concerning pancreatic perfusion and
influence on acute pancreatitis with regard to the effects of NO and NO
scavenging by DCLHb. The administration of NO donors resulted in an
amelioration and the administration of NO inhibitors in an aggravation
of experimental acute pancreatitis (2, 34). In an intravital microscopy
study, administration of a NO synthase inhibitor caused an increase of leukocyte adherence in postcapillary venules of cerulein-induced acute
pancreatitis (12). Another animal study showed a significant increase
of NO production after I/R of the pancreas (32). In contrast to the
above-mentioned studies, a beneficial effect of inhibiting NO synthase
by maintaining normotension was observed in severe experimental
pancreatitis (100% mortality) in untreated rats (16). This study is in
agreement with our results, namely, a preservation of MAP when animals
with pancreatic I/R were treated with DCLHb. Several studies describe
that inhibition of NO either by NO scavenging or by NO synthase
inhibition might have ambivalent effects on both I/R and acute
pancreatitis. Formation of peroxynitrite by rapid reaction of NO (32)
and potentiation of superoxides in the reperfusion phase (30) are
widely accepted findings. Thus the removal of NO is considered
beneficial to the target tissues. Bradykinin has been investigated by
our group (10) as a potentially harmful mediator of pancreatic I/R
injury. Bradykinin synthetized during acute pancreatitis is reported to
have vasodilating effects due to a NO-releasing mechanism by intact
endothelium (8). The hypotension, a typical feature of pancreatic I/R, may be caused by systemic bradykinin release, whereas the scavenging of
NO by DCLHb would allow the preservation of arterial pressure as
observed in the I/R group treated with DCLHb.
In conclusion, our results show that in the normal pancreas DCLHb
increases microcirculatory perfusion in a dose-dependent manner without
adverse microcirculatory side effects. We did not encounter signs of
acute pancreatitis. There is a slight reversal of capillary no-reflow
as well as a reduction of leukocyte adherence, serum amylase activity,
and IL-6 concentration when DCLHb is administered after induction of
postischemic pancreatitis. DCLHb does not exacerbate the inflammatory
and microcirculatory pancreatic damage of postischemic pancreatitis.
DCLHb is able to maintain the MAP during the time of pancreatic reperfusion.
 |
ACKNOWLEDGEMENTS |
We are indebted to Steffen Massberg, Tadashi Kondo, David Stobbe, and
Elke Schütze for helpful advice; without their assistance, the
project could not have been completed.
 |
FOOTNOTES |
This work was supported in part by Baxter Healthcare (Round Lake, IL)
and Institute for Surgical Research, Ludwig-Maximilians University
(Munich, Germany).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. von
Dobschuetz, Institute for Surgical Research, Klinikum Grosshadern,
Marchioninistr. 15, D-81366 Munich, Germany (E-mail: ernst.dobschuetzvon{at}stud.tu-muenchen.de).
Received 3 December 1998; accepted in final form 22 February 1999.
 |
REFERENCES |
1.
Cole, D. J.,
J. Nary,
L. W. Reynolds,
P. Patel,
and
J. C. Drummond.
Experimental subarachnoid hemorrhage in rats: effect of intravenous
-
diaspirin crosslinked hemoglobin on hypoperfusion and neuronal death.
Anesthesiology
87:
1486-1493,
1997[Medline].
2.
Dobosz, M.,
S. Hac,
and
Z. Wajda.
Does nitric oxide protect from microcirculatory disturbances in experimental acute pancreatitis in rats?
Int. J. Microcirc. Clin. Exp.
16:
221-226,
1996[Medline].
3.
Farmer, M.,
R. J. Przybelski,
J. E. McKenzie,
and
K. Burhop.
Preclinical data and clinical trials with diaspirin cross-linked hemoglobin.
In: Artificial Red Cells, edited by E. Tschuida. New York: Wiley, 1995, p. 177-185.
4.
Gould, S. A.,
L. R. Sehgal,
H. L. Sehgal,
R. DeWoskin,
and
G. S. Moss.
The clinical development of human polymerized hemoglobin.
In: Blood Substitutes: Principles, Methods, Products and Clinical Trials, edited by T. M. S. Chang. Basel: Karger, 1997, p. 12-38.
5.
Gulati, A.,
A. C. Sharma,
and
G. Singh.
Role of endothelin in the cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin.
Crit. Care Med.
24:
137-147,
1996[Medline].
6.
Gulati, A.,
G. Singh,
S. Rebello,
and
A. C. Sharma.
Effect of diaspirin crosslinked and stroma-reduced hemoglobin on mean arterial pressure and endothelin-1 concentration in rats.
Life Sci.
56:
1433-1442,
1995[Medline].
7.
Gullo, L.,
L. Cavicchi,
P. Tomassetti,
C. Spagnolo,
A. Freyrie,
and
M. D'Addato.
Effects of ischemia on the human pancreas.
Gastroenterology
111:
1033-1038,
1996[Medline].
8.
Hoffmann, T. F.,
R. Leiderer,
A. G. Harris,
and
K. Messmer.
Ischemia and reperfusion in pancreas.
Microsc. Res. Tech.
37:
557-571,
1997[Medline].
9.
Hoffmann, T. F.,
R. Leiderer,
H. Waldner,
S. Arbogast,
and
K. Messmer.
Ischemia reperfusion of the pancreas: a new in vivo model for acute pancreatitis in rats.
Res. Exp. Med. (Berl.)
195:
125-144,
1995[Medline].
10.
Hoffmann, T. F.,
R. Leiderer,
H. Waldner,
and
K. Messmer.
Bradykinin antagonists HOE-140 and CP-0597 diminish microcirculatory injury after ischaemia-reperfusion of the pancreas in rats.
Br. J. Surg.
83:
189-195,
1996[Medline].
11.
Hoffmann, T. F.,
M. Steinbauer,
H. Waldner,
and
K. Messmer.
Exogenous bradykinin enhances ischemia/reperfusion injury of pancreas in rats.
J. Surg. Res.
62:
144-151,
1996[Medline].
12.
Inagaki, H.,
A. Nakao,
T. Kurokawa,
T. Nonami,
A. Harada,
and
H. Takagi.
Neutrophil behavior in pancreas and liver and the role of nitric oxide in rat acute pancreatitis.
Pancreas
15:
304-309,
1997[Medline].
13.
Kerner, T.,
B. Vollmar,
M. D. Menger,
H. Waldner,
and
K. Messmer.
Determinants of pancreatic microcirculation in acute pancreatitis in rats.
J. Surg. Res.
62:
165-171,
1996[Medline].
14.
Kogire, M.,
K. Inoue,
S. Higashide,
K. Takaori,
Y. Echigo,
Y. J. Gu,
S. Sumi,
K. Uchida,
and
M. Imamura.
Protective effects of endothelin-1 on acute pancreatitis in rats.
Dig. Dis. Sci.
40:
1207-1212,
1995[Medline].
15.
Liu, X. H.,
T. Kimura,
H. Ishikawa,
H. Yamaguchi,
M. Furukawa,
I. Nakano,
M. Kinjoh,
and
H. Nawata.
Effect of endothelin-1 on the development of hemorrhagic pancreatitis in rats.
Scand. J. Gastroenterol.
30:
276-282,
1995[Medline].
16.
Lomis, T. J.,
C. W. Siffring,
S. Chalasani,
D. W. Ziegler,
K. E. Lentz,
K. E. Stauffer,
A. McMillan,
N. Agarwal,
C. J. Lowenstein,
and
J. E. Rhoads.
Nitric oxide synthase inhibitors N-monomethylarginine and aminoguanidine prevent the progressive and severe hypotension associated with a rat model of pancreatitis.
Am. Surg.
61:
7-10,
1995[Medline].
17.
McKenzie, J. E.,
E. Cost,
D. M. Scandling,
N. W. Ahle,
and
R. W. Savage.
Effects of diaspirin crosslinked haemoglobin during coronary angioplasty in the swine.
Cardiovasc. Res.
28:
1188-1192,
1994[Medline].
18.
Menger, M. D.,
M. Rücker,
and
B. Vollmar.
Capillary dysfunction in striated muscle ischemia/reperfusion: on the mechanisms of capillary "no-reflow."
Shock
8:
2-7,
1997[Medline].
19.
Nelson, D.
Blood and HemAsisst (DCLHb): potentially a complementary therapeutic team.
In: Blood Substitutes: Principals, Methods, Products and Clinical Trials, edited by T. M. S. Chang. Basel: Karger, 1997, p. 39-61.
20.
Nolte, D.,
A. Botzlar,
S. Pickelmann,
E. Bouskela,
and
K. Messmer.
Effects of diaspirin-cross-linked hemoglobin (DCLHb) on the microcirculation of striated skin muscle in the hamster: a study on safety and toxicity.
J. Lab. Clin. Med.
130:
314-327,
1997[Medline].
21.
Pickelmann, S.,
D. Nolte,
R. Leiderer,
E. Schütze,
and
K. Messmer.
Attenuation of postischemic reperfusion injury in striated skin muscle by diaspirin-cross-linked Hb.
Am. J. Physiol.
275 (Heart Circ. Physiol. 44):
H361-H368,
1998[Abstract/Free Full Text].
22.
Pincemail, J.,
O. Detry,
C. Philippart,
J. O. Defraigne,
C. Franssen,
K. Burhop,
C. Deby,
M. Meurisse,
and
M. Lamy.
Diaspirin crosslinked hemoglobin (DCLHb): absence of increased free radical generation following administration in a rabbit model of renal ischemia and reperfusion.
Free Radic. Biol. Med.
19:
1-9,
1995[Medline].
23.
Przybelski, R. J.,
E. K. Daily,
J. C. Kisicki,
C. Mattia-Goldberg,
M. J. Bounds,
and
W. A. Colburn.
Phase I study of the safety and pharmacologic effects of diaspirin cross-linked hemoglobin solution.
Crit. Care Med.
24:
1993-2000,
1996[Medline].
24.
Reah, G.,
A. R. Bodenham,
A. Mallick,
E. K. Daily,
and
R. J. Przybelski.
Initial evaluation of diaspirin cross-linked hemoglobin (DCLHb) as a vasopressor in critically ill patients.
Crit. Care Med.
25:
1480-1488,
1997[Medline].
25.
Rubanyi, G. M.,
and
M. A. Polokoff.
Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology.
Pharmacol. Rev.
46:
325-415,
1994[Medline].
26.
Schmid-Schönbein, G. W.,
B. W. Zweifach,
and
S. Kovalcheck.
The application of stereological principles to morphometry of the microcirculation in different tissues.
Microvasc. Res.
14:
303-317,
1977[Medline].
27.
Schölmerich, J.
Interleukins in acute pancreatitis.
Scand. J. Gastroenterol. Suppl.
219:
37-42,
1996[Medline].
28.
Sharma, A. C.,
G. Singh,
and
A. Gulati.
Role of NO mechanism in cardiovascular effects of diaspirin cross-linked hemoglobin in anesthetized rats.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H1379-H1388,
1995[Abstract/Free Full Text].
29.
Sirsjö, A.,
H. A. Lehr,
D. Nolte,
T. Haapaniemi,
D. H. Lewis,
G. Nylander,
and
K. Messmer.
Hyperbaric oxygen treatment enhances the recovery of blood flow and functional capillary density in postischemic striated muscle.
Circ. Shock
40:
9-13,
1993[Medline].
30.
Szabo, C.
The pathophysiological role of peroxynitrite in shock, inflammation, and ischemia-reperfusion injury.
Shock
6:
79-88,
1996[Medline].
31.
Takaori, K.,
K. Inoue,
M. Kogire,
S. Higashide,
T. Tun,
T. Aung,
R. Doi,
N. Fujii,
and
T. Tobe.
Effects of endothelin on microcirculation of the pancreas.
Life Sci.
51:
615-622,
1992[Medline].
32.
Tanaka, S.,
W. Kamiike,
H. Kosaka,
T. Ito,
E. Kumura,
T. Shiga,
and
H. Matsuda.
Detection of nitric oxide production and its role in pancreatic ischemia-reperfusion in rats.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G405-G409,
1996[Abstract/Free Full Text].
33.
Toyama, M. T.,
M. P. Lewis,
A. M. Kusske,
P. U. Reber,
S. W. Ashley,
and
H. A. Reber.
Ischaemia-reperfusion mechanisms in acute pancreatitis.
Scand. J. Gastroenterol. Suppl.
219:
20-23,
1996[Medline].
34.
Werner, J.,
J. Rivera,
C. Fernandez del Castillo,
K. Lewandrowski,
C. Adrie,
D. W. Rattner,
and
A. L. Warshaw.
Differing roles of nitric oxide in the pathogenesis of acute edematous versus necrotizing pancreatitis.
Surgery
121:
23-30,
1997[Medline].
Am J Physiol Gastroint Liver Physiol 276(6):G1507-G1514
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society