Mechanisms of hypothermic protection against ischemic liver
injury in mice
Atsushi
Kato1,
Saurabh
Singh2,
Kenneth R.
McLeish2,
Michael J.
Edwards1, and
Alex B.
Lentsch1
Departments of 1 Surgery and
2 Medicine, University of Louisville School of
Medicine, Louisville, Kentucky 40292
 |
ABSTRACT |
Hepatic hypothermia can safely
prolong the duration of hepatic inflow occlusion during complex liver
resectional surgeries. The mechanism(s) by which hypothermia protects
against this form of liver ischemia-reperfusion injury are not
completely understood. In this study, we sought to determine whether
hypothermia protects against ischemia-reperfusion injury by altering
the hepatic inflammatory response. Mice undergoing 90 min of partial
hepatic ischemia followed by up to 8 h of reperfusion had their
body temperatures regulated at 35-37°C (normothermic) or
unregulated, in which rectal temperature dropped as low as 25°C by
the end of ischemia (hypothermic). Hypothermic mice had less liver
injury vs. normothermic mice, as assessed histologically, by serum
transaminase levels (89% decrease), and by liver wet-to-dry weight
ratios (91% decrease). Neutrophil accumulation was absent in
hypothermic mice (99% reduction vs. normothermic mice). Production of
the proinflammatory cytokines tumor necrosis factor-
,
interleukin-1
, and macrophage inflammatory protein-2 were reduced by
up to 92%. Activation of the transcription factor nuclear factor-
B
was not reduced in hypothermic mice, but activation of c-Jun
NH2-terminal kinase (JNK) and the transcription factor activator protein (AP)-1 were greatly diminished. These data suggest that hypothermia suppresses the hepatic inflammatory response through
selective inhibition of JNK and AP-1.
inflammation; cytokines; neutrophils
 |
INTRODUCTION |
SURGICAL MANAGEMENT OF
HEPATIC resectional surgery often requires hepatic inflow
occlusion (Pringle maneuver) to reduce intraoperative bleeding. The
resultant hepatic ischemia-reperfusion leads to an acute inflammatory
response that may cause significant hepatocellular damage and organ
dysfunction (13, 19). This inflammatory response is
characterized by early production of the proinflammatory cytokines tumor necrosis factor (TNF)-
and interleukin (IL)-1
, which are released by activated Kupffer cells immediately after hepatic ischemia-reperfusion (28, 30). These cytokines appear to
propagate the inflammatory response by upregulating adhesion molecules
on the hepatic vascular endothelium and increasing the production of
CXC chemokines (4-6, 18). The cooperative
effects of adhesion molecules and CXC chemokines results in the
recruitment of neutrophils from the vascular space into the hepatic
parenchyma (15). These activated neutrophils then cause
hepatocellular damage by obstructing the microcirculation and releasing
toxic substances such as reactive oxygen species and proteases
(15, 16, 29). In experimental models, blockade of
inflammatory mediators has proven effective against liver injury after
hepatic ischemia-reperfusion (5, 6, 9, 26), suggesting
that interventional therapies designed to suppress the inflammatory
response during hepatic ischemia-reperfusion may reduce the
morbidity of hepatic resectional surgery.
Hepatic hypothermia has been shown to have a protective effect on
surgical resection of the liver (31). In 1953, Raffucci et
al. (25) noted that dogs rendered hypothermic by total
body cooling could survive 1 h of hepatic ischemia, whereas
normothermic dogs all died within the same period of time. Since then,
numerous methods of hypothermia have been developed and adapted for
clinical application (11, 20, 31). Subsequently,
hypothermic techniques are now performed in selected patients with
large metastatic liver tumors and hepatocellular carcinoma associated
with cirrhotic liver disease (12, 14, 32). However, the
physiological mechanisms by which hypothermia confers protection
against hepatic ischemia-reperfusion injury remain largely unknown. In
the current studies, we investigated whether hypothermia protects
against ischemia-reperfusion injury by altering the hepatic
inflammatory response.
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MATERIALS AND METHODS |
Hepatic ischemia-reperfusion injury model.
Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) weighing
23-27 g were used in all experiments. This project was approved by
the University of Louisville Animal Care and Use Committee and was in
compliance with the National Institutes of Health guidelines. The
animals were divided into three groups: sham control, normothermic hepatic ischemia-reperfusion, and hypothermic hepatic
ischemia-reperfusion. Partial hepatic ischemia was induced as
previously described (18). Briefly, mice were anesthetized
with sodium pentobarbital (60 mg/kg im). A midline laparotomy was
performed, and an atraumatic clip was used to interrupt blood supply to
the left lateral and median lobes of the liver. The caudal lobes
retained intact portal and arterial inflow and venous outflow,
preventing intestinal venous congestion. After 90 min of partial
hepatic ischemia, the clip was removed, initiating hepatic reperfusion.
The animals received 0.2 ml of sterile saline subcutaneously, the wound
was closed in layers with 4-0 silk, and the animal was allowed to recover. Sham control mice underwent the same protocol without vascular
occlusion. Rectal temperature was measured by using an electronic
thermometer with a probe (Fisher Scientific, Pittsburgh, PA) every 15 min after injection of anesthesia. In sham and normothermic groups,
body temperature was maintained at 37°C by using a heating pad. In
the hypothermic group, body temperature was not regulated. For
assessment of hepatic reperfusion, 20 mg/kg Evans blue dye was injected
intravenously 1 min before reperfusion. At the indicated times,
postischemic livers were excised, weighed, and then homogenized in 2 ml
PBS. The homogenate was diluted with 2 vol of formamide and incubated
for 2 h at 60°C, followed by centrifugation for 30 min at 5,000 g. The absorbance of the supernatant was measured at 620 nm
and 740 nm, and Evans blue concentration was determined by a standard
curve. Correction for contaminating heme pigments was calculated by the
formula E620
(1.426 × E740 + 0.03). Final Evans blue dye concentration was expressed as nanograms
Evans blue dye per gram liver tissue.
RT-PCR.
Liver tissue was removed after 90 min of ischemia and 1 h
reperfusion. Total RNA from liver tissue was extracted with TRIzol reagent (GIBCO-BRL, Rockville, MD). RNA (1 µg) was reverse
transcribed to cDNA using random hexamers (Perkin-Elmer, Foster City,
CA). cDNA products were coamplified by PCR (30 cycles: 95°C for
60 s, 59°C for 90 s, and 72°C for 10 s). Primers for
TNF-
, IL-1
, macrophage inflammatory protein (MIP)-2, and
-actin have been described elsewhere (1, 35). PCR
products were electrophoresed in a 3.5% agarose gel, stained with
ethidium bromide, and photographed.
Liver neutrophil accumulation.
Liver myeloperoxidase (MPO) assay was performed on animals 8 h
after reperfusion. Liver MPO content was assessed by methods described
elsewhere (18). Briefly, liver tissue (100 mg) was homogenized in 2 ml of buffer A (3.4 mmol/l
KH2HPO4 and 16 mmol/l Na2HPO4, pH 7.4). After being centrifuged for
20 min at 10.000 g, the pellet was resuspended in 10 vol of
buffer B (43.2 mmol/l KH2HPO4, 6.5 mmol/l Na2HPO4, 10 mmol/l EDTA, and 0.5%
hexadecyltrimethylammonium, pH 6.0) and sonicated for 10 s. After
being heated for 2 h at 60°C, the supernatant was reacted with
3,3',3,5'-tetramethylbenzidine and the optical density was read at 655 nm.
Blood and tissue analysis.
Blood was obtained by cardiac puncture 8 h after reperfusion for
analysis of serum alanine aminotransferase (ALT) as an index of
hepatocellular injury. Measurements of serum ALT were made using a
diagnostic kit (Sigma, St. Louis, MO). Serum levels of TNF-
,
IL-1
, and MIP-2 were measured by sandwich ELISA according to the
manufacturer's instructions (R&D Systems, Minneapolis, MN). Ischemic
lobes (or corresponding lobes in the sham group) were excised for
tissue analysis. Liver edema was determined by organ wet-to-dry weight
ratios. Tissues were fixed in 10% formalin and then embedded in
paraffin for light microscopy. Sections were stained with hematoxylin
and eosin for histological examination.
Western blot.
Liver homogenates were sonicated on ice three times for 10 s and
then centrifuged at 14,000 rpm for 30 min at 4°C. Supernatants were
removed, and protein content was estimated using the Bradford method.
Tyrosine phosphorylation of c-Jun NH2-terminal kinase (JNK)
was determined by Western blot with a phosphospecific antibody (Cell
Signaling Technology, Beverly, MA). Samples were separated by 10%
SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked with
5% milk in Tween-20 Tris-buffered saline (TTBS) for 1 h at room
temperature. Blots were probed with the anti-phospho JNK in 5%
BSA/TTBS overnight at 4°C, and antibodies were detected by using a
peroxidase-conjugated secondary antibody in 5% milk/TTBS for 1 h
at room temperature. Products were visualized by enhanced chemiluminescence. To verify equal loading of proteins in each lane,
the blots were stripped and restained using an antibody that stains
total JNK (Cell Signaling Technology). Western blot films were
digitized, and activation of JNK (phosphorylated/total) was determined
by image analysis with software from Adobe Systems (San Jose, CA).
Electrophoretic mobility shift assay.
Nuclear extracts of liver tissue were prepared by the method of
Deryckere and Gannon (7) and analyzed by electrophoretic mobility shift assay. Briefly, double-stranded nuclear factor (NF)-
B
consensus oligonucleotide (Promega, Madison, WI) was end labeled with
[
-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; Amersham,
Arlington Heights, IL). Binding reactions containing equal amounts of
nuclear protein extract (20 µg) and 35 fmol (~50,000 cpm, Cherenkov
counting) of oligonucleotide were incubated at room temperature for 30 min. Binding reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography.
Statistical analysis.
All data are expressed as means ± SE. Data were analyzed with a
one-way ANOVA with subsequent Student-Newman-Keuls test. Differences were considered significant when P < 0.05. To
calculate percentage change, negative control values were subtracted
from positive control and treatment group values.
 |
RESULTS |
Regulation of body temperature.
Rectal temperature was monitored every 15 min during hepatic
ischemia-reperfusion. Just before induction of anesthesia, the average
rectal temperature of mice was 37.1 ± 0.05°C (Fig.
1). After injection of 60 mg/kg of
pentobarbital, body temperature decreased in all mice. In mice
regulated with a heating pad, body temperature was 33.8 ± 0.1°C
at the initiation of hepatic ischemia. In mice without any
thermoregulatory support, body temperature was 30.6 ± 1.3°C on
hepatic ischemia. By the end of the ischemic period, regulated mice had
a rectal temperature of 36.7 ± 0.4°C, whereas those with no
thermoregulatory support had a rectal temperature of 25.9 ± 0.5°C. Mice in which body temperature was controlled maintained
rectal temperatures in the range of 35-37°C for the duration of
hepatic reperfusion. In mice in which body temperature was not
controlled, rectal temperature was 24.9 ± 0.4°C 15 min after
reperfusion and returned to temperatures in the range of 35-37°C
within 4 h of reperfusion. Thus mice not receiving
thermoregulatory support were significantly hypothermic for nearly
4 h after induction of hepatic ischemia.

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Fig. 1.
Rectal temperatures during hepatic ischemia-reperfusion
injury in mice without thermoregulatory support ( ) and
mice in which body temperature was regulated with a heating pad
( ). Values are means ± SE; n = 5/group. * P < 0.05 vs. mice with regulated
temperature.
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Effects of hypothermia on hepatic reperfusion.
Because mice in which body temperature was not controlled were
hypothermic for an extended time during reperfusion, we sought to
determine if this reduction in body temperature affected the rate at
which the liver reperfused. To address this issue, normothermic and
hypothermic mice were injected intravenously with Evans blue dye just
before reperfusion. The ischemic lobes were resected after 1, 5, 10, 20, and 30 min of reperfusion. The liver content of Evans blue dye
increased at similar rates in normothermic and hypothermic mice and
reached a maximum in both groups by 20 min (Fig.
2A). Macroscopically,
reperfusion appeared to be more uniform in hypothermic mice compared
with normothermic mice (Fig. 2B). These data suggest that
the reperfusion rates were similar in normothermic and hypothermic mice
but that hypothermia facilitates more uniform reperfusion.

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Fig. 2.
Rate of hepatic reperfusion in normothermic and
hypothermic mice as measured by Evans blue dye content. Mice were
injected with 20 mg/kg Evans blue dye 1 min before reperfusion.
A: at times indicated, livers were excised and weighed and
the Evans blue dye content was determined. B: visual
observation of the livers after 5 min of reperfusion showed that in
normothermic mice reperfusion was patchy, whereas in hypothermic mice
reperfusion was uniform.
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Effects of hypothermia on inflammatory liver injury.
To assess the effects of hypothermia on liver injury induced by
ischemia-reperfusion, we measured serum levels of ALT and liver
wet-to-dry weight ratios. After hepatic ischemia and 8 h reperfusion in normothermic mice, there was a significant increase in
hepatocellular injury compared with the sham controls (Fig. 3A). Hypothermia reduced
hepatocellular injury by 89% (P < 0.001). Similarly,
normothermic ischemia-reperfusion induced a significant increase in
liver wet-to-dry weight ratios compared with sham controls (Fig.
3B). Hypothermic mice had far less hepatic swelling, with a
91% reduction in liver wet-to-dry weight ratios compared with
normothermic mice (P < 0.001). To further assess the
effects of hypothermia on hepatic ischemia-reperfusion injury, sections of liver obtained from the ischemic lobe after ischemia and 8 h of
reperfusion were evaluated for histopathological changes. In
normothermic mice undergoing ischemia-reperfusion, liver
sections were characterized by patchy areas of coagulative necrosis,
especially in zones 2 and 3 of hepatic lobules
(Fig. 4A). Neutrophils and red
blood cells were numerous in the necrotic areas, and there was a marked
destruction of sinusoidal structure consistent with the no-reflow
phenomenon (21, 29). In contrast, hepatic architecture of
hypothermic mice appeared normal; there was no evidence of hepatocyte
necrosis and no inflammatory cell infiltrates (Fig. 4B).
These data demonstrate that hypothermic mice are greatly protected from
ischemia-reperfusion-induced hepatocellular injury.

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Fig. 3.
Effects of hypothermia on ischemia-reperfusion-induced
liver injury (I/R). Serum alanine aminotransferase (ALT) and liver
wet-to-dry weight ratios were measured after ischemia and 8 h of
reperfusion as indices of liver injury. Values are means ± SE;
n = 5/group.
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Fig. 4.
Histological analysis of livers from normothermic mice
(A) and hypothermic mice (B) after hepatic
ischemia and 8 h of reperfusion. Liver sections from the ischemic
lobes of normothermic mice showed areas of hepatocyte necrosis
associated with leukocyte infiltrates, erythrocyte accumulation, and
sinusoidal collapse. Liver sections from the ischemic lobes of
hypothermic mice appeared virtually normal.
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The liver injury induced by ischemia-reperfusion is known to be
mediated by the secretory products of activated neutrophils (16). To determine whether hypothermia reduced the hepatic
recruitment of neutrophils, we measured the content of MPO in liver
samples obtained after ischemia and 8 h of reperfusion. In
normothermic mice, ischemia-reperfusion caused a significant increase
in liver MPO content compared with sham controls (Fig.
5). In hypothermic mice this effect was
completely abrogated, with liver MPO content reduced 99% from that of
normothermic mice (P < 0.001), demonstrating that
hypothermia prevents the recruitment of neutrophils into the liver.

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Fig. 5.
Effects of hypothermia on liver neutrophil recruitment
induced by hepatic ischemia and 8 h of reperfusion. Liver
neutrophil accumulation was assessed by liver content of
myeloperoxidase (MPO). Values are means ± SE; n = 5/group.
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Effects of hypothermia on the production of proinflammatory
cytokines and chemokines.
The hepatic inflammatory response to ischemia-reperfusion is
characterized by increased production of proinflammatory cytokines and
the subsequent production of neutrophil-attracting chemokines (5,
6, 18). In an attempt to identify the mechanism by which
hypothermia conferred its protective effects, we first assessed whether
hypothermia altered the expression of mRNA for the cytokines TNF-
and IL-1
and the chemokine MIP-2 in liver tissues obtained after
ischemia and 1 h of reperfusion. As shown in Fig.
6, expression of TNF-
, IL-1
, and
MIP-2 mRNA was increased in normothermic mice compared with sham
controls. TNF-
and IL-1
mRNA expression in hypothermic mice was
similar to sham controls, whereas MIP-2 mRNA in hypothermic mice was
higher than in sham controls (Fig. 6). We next measured serum protein
levels of TNF-
, IL-1
, and MIP-2. In normothermic mice, serum
levels of TNF-
, IL-1
, and MIP-2 after ischemia-reperfusion were
all significantly greater than serum levels in sham controls (Fig.
7). Hypothermia resulted in an 80%
reduction in TNF-
(P < 0.001), a 76% decrease in
IL-1
(P = 0.003), and a 92% reduction in MIP-2
(P < 0.001) compared with normothermic mice. Thus
hypothermia appears to confer its protective effects by greatly
suppressing the production of proinflammatory cytokines and chemokines.

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Fig. 6.
Effects of hypothermia on hepatic mRNA expression of
tumor necrosis factor (TNF)- , interleukin (IL)-1 , and macrophage
inflammatory protein (MIP)-2. Liver RNA extracts were analyzed by
RT-PCR. Coamplification of the cDNA for the housekeeping gene -actin
was performed as an internal control. Results are representative of 2 independent experiments.
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Fig. 7.
Effects of hypothermia on serum levels of TNF- ,
IL-1 , and MIP-2 after hepatic ischemia and 8 h of reperfusion.
Serum samples were analyzed by ELISA. Values are means ± SE;
n = 5/group.
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Effects of hypothermia on activation of NF-
B, JNK, and activator
protein-1.
The expression of proinflammatory cytokines is controlled by a number
of different signal transduction pathways. The transcription factor
NF-
B is a primary regulator of a large number of cytokines and
chemokines (24). We investigated whether the decreased
cytokine and chemokine generation in hypothermic mice might be caused
by suppressed activation of NF-
B. However, we found that NF-
B
activation in hypothermic mice was not decreased compared with
normothermic mice (data not shown). Because the expression of cytokines
and chemokines is also controlled in part by c-Jun (33,
34), and since other studies have suggested a role for JNK in
the hepatic response to ischemia-reperfusion (3, 23), we
investigated whether the activity of JNK was altered by hypothermia. As
shown in Fig. 8, no activated
(phosphorylated) JNK was detected in livers from sham controls. In
livers from normothermic mice, both JNK isoforms 1 and 2 were activated
after 1 and 4 h of ischemia but were increased minimally after
8 h of reperfusion (Fig. 8). In hypothermic mice, there was no
activation of the JNK2 at any time point (Fig. 8). There did appear to
be slight activation of JNK1 after 1 h of reperfusion, but there
was no activation of this form at either 4 or 8 h after
reperfusion (Fig. 8). To determine if this effect was specific for JNK
or was a generalized effect on mitogen-activated protein kinases, we
assessed the activation of two other mitogen-activated protein kinases,
p38 and extracellular signal-regulated protein kinase (ERK). No
phosphorylated p38 could be detected in any group (data not shown).
Phosphorylated ERK was detected in both normothermic and hypothermic
livers, but there was no difference between groups (data not shown).
This suggests that hypothermia selectively suppresses the activation of
JNK but not other mitogen-activated protein kinases.

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Fig. 8.
Effects of hypothermia on the activation of c-Jun
NH2-terminal kinase (JNK) in liver during
ischemia-reperfusion injury. Lysates of liver tissues from independent
mice were analyzed by Western blot and stained with phospho-specific
antibodies to JNK (A, top). The blot was then
stripped and restained for total JNK proteins (A,
bottom). JNK1 (B) and JNK2 (C)
activation was assessed by image analysis of digitized Western blots.
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Because JNK activation can lead to transcriptional activation of
activator protein (AP)-1 (17), and since AP-1 is known to
contribute to the gene expression of TNF-
and other proinflammatory mediators (33, 34), we assessed the activation of AP-1 in normothermic and hypothermic mice after hepatic ischemia-reperfusion. In normothermic mice, AP-1 activation was greatly increased after ischemia and 1 h of reperfusion compared with normothermic sham controls (Fig. 9A).
Hypothermia completely abrogated this increase in hepatic AP-1
activation. There was a faster-migrating band observed in the sham and
hypothermia groups. To determine the composition of this band, as well
as that of the expected AP-1 band, supershift assays were performed.
The faster-migrating band observed in the hypothermia group did not
supershift on addition of antibodies to FosB, JunB, c-Jun, or JunD
(Fig. 9B). The composition of the AP-1 complex in
normothermic mice appeared to be composed of JunB, c-Jun, and JunD.
These data suggest that suppressed activation of JNK and the resulting
reduction in AP-1 activation may contribute to the protective effects
of hypothermia on hepatic ischemia-reperfusion.

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Fig. 9.
Effects of hypothermia on liver activator protein (AP)-1
activation. A: liver nuclear extracts from hypothermic (HT)
mice and normothermic (NT) mice were obtained after ischemia and 1 h of reperfusion and were analyzed by electrophoretic mobility shift
assay. Results are representative of 3 independent experiments.
B: to identify the components of the AP-1 complex,
supershift assays were performed. Liver nuclear extracts from HT and NT
mice were incubated in the presence or absence of antibodies for FosB,
JunB, c-Jun, and JunD. The solid arrowheads indicate the position of
the AP-1 complex, the open arrowheads indicate the position of the
supershifts, and the open circles indicate nonspecific bands.
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 |
DISCUSSION |
Hypothermia has been used to safely prolong hepatic ischemia
resulting from inflow occlusion during resectional surgery
(31). Furthermore, hypothermia has been shown to reduce
ischemia-reperfusion injury to the liver (11, 12, 14, 20, 25,
32). To date, the mechanism for the protective effects of
hepatic hypothermia has been attributed to reduced metabolism and
oxygen consumption. Early studies showed that total body cooling to a
rectal temperature as low as 23°C for up to 12 h does not
adversely affect liver function (10). Subsequently, it was
shown that extracorporeal cooling of the liver to 25°C resulted in a
36% reduction in the oxygen consumption by the liver
(22). More recent reports suggest that hepatic hypothermia
reduces liver ATP levels, resulting in a prolongation of hepatocellular
viability in ischemic tissues (8). Transcriptional
activation and protein synthesis demand a great deal of energy, and
thus hypothermia may attenuate transcriptional activation and protein
synthesis by suppressing the cellular metabolic activity and oxygen
demand. Kupffer cells are known to be activated by liver ischemia and
are thought to initiate the hepatic inflammatory response through their
production of proinflammatory cytokines and chemokines. However,
hepatocytes are also a significant source of these mediators, and it is
likely that hypothermia reduces the metabolic rate and energy
requirement of Kupffer cells and hepatocytes during the initial phase
of hepatic ischemia-reperfusion, thereby decreasing proinflammatory
cytokine and chemokine production and suppressing the subsequent
inflammatory response.
To the best of our knowledge, this is the first study to demonstrate
that hypothermia suppresses the hepatic inflammatory response to
ischemia-reperfusion. We found that mice that were hypothermic during
the ischemic and early reperfusion periods had dramatically reduced
production of the proinflammatory cytokines TNF-
and IL-1
and the
CXC chemokine MIP-2. This hypothermia-induced suppression of these
mediators resulted in almost complete abrogation of hepatic neutrophil
accumulation. Furthermore, liver histology of hypothermic mice appeared
almost completely normal, whereas normothermic livers displayed the
typical signs of hepatocellular necrosis, sinusoidal collapse, and
leukocyte accumulation. Correspondingly, hypothermia almost completely
ameliorated the hepatocellular injury induced by ischemia-reperfusion.
The liver injury induced by ischemia-reperfusion is greatly dependent
on neutrophils (15, 16). Numerous studies have shown that
prevention of neutrophil recruitment to liver by blockade of cytokines,
chemokines, or adhesion molecules suppresses the development of tissue
injury (5, 6, 9, 26). Our data suggest that hypothermia
prevents the initiation of the inflammatory response in the liver by
preventing the transcription of proinflammatory mediators.
The gene expression of the mediators studied here is controlled largely
by the transcription factor NF-
B (24). However, hypothermia did not cause a reduction in NF-
B activation in liver, suggesting that hypothermia may affect other transcriptional regulators involved in the expression of TNF-
, IL-1
, and MIP-2. A likely candidate is AP-1, which is known to contribute to the transcription of
proinflammatory mediators (33, 34). Hypothermia greatly suppressed the activation of JNK1 and JNK2. Both of these isoforms are
involved in the activation of AP-1, but JNK2 has been shown to have
10-fold greater activity for c-Jun phosphorylation (27). Interestingly, JNK2 phosphorylation was completely inhibited in hypothermic mice. Correspondingly, there was almost complete inhibition of AP-1 activation in the liver after ischemia-reperfusion. Thus it
seems that reduced hepatic temperature somehow differentially affects
NF-
B and AP-1. The activation of these two transcription factors has
been documented in hepatic ischemia-reperfusion injury, and both share
similar activation kinetics (38). Previous studies by us
and others have suggested a central role of NF-
B in the hepatic
inflammatory response to ischemia-reperfusion (35, 38). However, NF-
B activation is known to be necessary for hepatocyte regeneration (2), and thus a potential role of NF-
B in
hepatoprotection has also been suggested. The role of AP-1 in the
hepatic inflammatory response has not been rigorously investigated.
Optimal gene expression of multiple cytokines and chemokines requires
cooperative activation of both NF-
B and AP-1, suggesting that the
reduced production observed during hypothermia may be due to the
selective effect on AP-1. Furthermore, our studies suggest that
selective inhibition of AP-1 may provide a potential therapeutic
benefit for the hepatic inflammatory response induced by
ischemia-reperfusion.
There is evidence that hypothermia reduces the production of reactive
oxygen species induced by hepatic ischemia (36, 37). AP-1
is activated by oxidant stress, and it is possible that the hypothermia-induced reduction of reactive oxygen species may result in
decreased AP-1 activation. However, NF-
B is also redox sensitive, and therefore reduced oxidant stress in hypothermic mice would be
expected to reduce the activation of NF-
B. We did not observe any
reduction in NF-
B activation in livers from hypothermic mice. Furthermore, adenoviral expression of mitochondrial superoxide dismutase in liver greatly reduces hepatic ischemia-reperfusion injury in association with reduced activation of both AP-1 and NF-
B
(39). Thus it appears unlikely that reduced generation of
reactive oxygen species by hypothermia results in selective inhibition
of AP-1 activation. The mechanism by which hypothermia suppresses JNK
activation and subsequent activation of AP-1 warrants further investigation.
In summary, our data demonstrate that hypothermia during hepatic
ischemia-reperfusion prevents the activation of the transcription factor AP-1 and subsequent production of proinflammatory cytokines and
chemokines. These effects result in abrogation of liver neutrophil accumulation and hepatocellular damage. Our findings offer a new explanation for the beneficial effects of hypothermia on hepatic ischemia-reperfusion injury. Furthermore, they support the use of
hypothermic therapy in the management of complex liver resectional surgeries that require extended occlusion times.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases (DK-56029) and the Department of Veterans Affairs.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
A. B. Lentsch, Dept. of Surgery, Univ. of Louisville School
of Medicine, James Graham Brown Cancer Center, Rm. 426, 529 South
Jackson St., Louisville, KY 40202 (E-mail:
alentsch{at}louisville.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00454.2001
Received 25 October 2001; accepted in final form 11 December 2001.
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