Departments of 1 Anesthesia, 3 Surgery, and 4 Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283; and 2 Department of Anesthesiology and Critical Care Medicine, Hadassah University Hospital, Jerusalem, Israel
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ABSTRACT |
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Sepsis is the leading cause of
death in surgical intensive care units. Although both mild sepsis
secondary to cecal ligation and single puncture (CLP) and fulminant,
double puncture CLP (2CLP) may provoke hepatocyte death, we hypothesize
that regeneration compensates for cell death after CLP but not 2CLP. In
male Sprague-Dawley rats, hepatic necrosis, as determined by serum
-glutathione S-transferase (
-GST) levels, was
significantly but equally elevated over time after both CLP and 2CLP.
Apoptosis, evaluated using both terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling and morphological
examination, was minimal after both CLP and 2CLP. Regeneration, assayed
by staining tissue for incorporation of exogenously administered
bromodeoxyuridine, was present after CLP but not after 2CLP. To further
substantiate impaired regeneration, steady-state levels of mRNAs
encoding JunB, LRF-1, and cyclin D1 were determined. After 2CLP, the
absence of JunB, LRF-1, and cyclin D1 mRNAs confirmed failed activation
of the mitogen-activated protein kinase-linked proliferative pathway
and progression through the cell cycle. Therefore, failed hepatocyte
regeneration may be a manifestation of hepatic dysfunction in fulminant sepsis.
necrosis; apoptosis; liver; hepatocyte; cytokines; tumor
necrosis factor; interleukin-1; interleukin-6; bromodeoxyuridine; tunel; -glutathione S-transferase; cyclin D1; LRF-1; JunB
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INTRODUCTION |
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SEPSIS AND THE RELATED SYSTEMIC inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) are the leading causes of death in critically ill surgical patients (3, 5). Both involve disordered metabolism (2, 16, 29, 35). Because the liver plays an essential role in the orchestration of complex metabolic responses, hepatic dysfunction is an important component of these syndromes. The liver is the second most commonly affected organ in sepsis/SIRS/MODS, and, as hepatic processes fail, liver cells can be lost (5, 13, 38). The pathophysiological mechanisms leading to hepatocyte dropout in late sepsis remain unknown.
Hepatocyte death can occur via either necrosis or programmed cell death (apoptosis) and has been described in a number of clinically relevant settings such as ischemia-reperfusion, organ transplantation, or toxic injury (17, 22, 26, 27, 39). However, the liver is unique in that it retains the capacity to regenerate in adult life (11, 19, 27, 33). In most settings in which cell loss is accelerated, compensatory mechanisms that include regeneration are activated so that overt hepatic failure is unusual. The role of hepatic regeneration in sepsis, SIRS, and MODS, however, has not been investigated.
In previous studies, we have investigated a murine model of sepsis,
cecal ligation and puncture (CLP), with specific reference to hepatic
dysfunction. When this model involves a single cecal puncture,
resulting in mild sepsis, we have shown that transcription of a number
of liver-specific genes is decreased transiently and mortality is
essentially nil (2, 2, 14, 15). In addition, we have shown
enhanced expression of the interleukin (IL)-6-dependent acute phase
reactant 2- macroglobulin and the IL-6-linked
transcription factor Stat-3 (1). However, when fulminant,
highly fatal sepsis is induced via cecal ligation and double puncture
(2CLP), decreased transcription is persistent, and Stat-3
activation/
2-macroglobulin expression abruptly
decrease. In addition to its effect on acute phase gene
expression, IL-6 also is an essential activator of hepatocyte
proliferation/regeneration (11, 50). Recent studies indicate that this process is IL-6 dependent but Stat-3 independent and
involves activation of mitogen-activated protein (MAP) kinase-linked transcription of genes such as those encoding JunB and LRF-1
(24). Therefore, in this study, we investigated hepatocyte
death and regeneration after CLP and 2CLP. Specifically, we tested the
hypothesis that CLP-induced hepatocyte necrosis and apoptosis
are compensated for by regeneration. In contrast, after 2CLP, we
propose that regeneration fails. Furthermore, we hypothesize that the
mechanism underlying this difference involves a decrease in the
expression of the MAP kinase-linked immediate-early genes junB
and lrf-1 and the gene encoding the cell cycle
modulator protein cyclin D1.
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MATERIALS AND METHODS |
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Induction of sepsis.
All animal studies were approved by the University Laboratory Animals
Resources committee of the University of Pennsylvania and conformed to
National Institute of Health standards for laboratory animals. Rats
were injected subcutaneously with 50 mg/kg of bromodeoxyuridine (BrdU;
2 mg/ml in saline) at the time of the initial operation. Under
methoxyflurane anesthesia, reversible sepsis was induced in male,
adolescent (250-275 g) Sprague-Dawley rats (Charles River, Boston,
MA) via cecal ligation and single 18-gauge puncture (CLP) as described
previously (1, 2, 14, 15). Fulminant sepsis was induced
with 2CLP (1, 2, 14, 25). Sham-operated (SO) animals
served as controls. After the procedure, animals were fluid
resuscitated with 50 ml/kg sterile saline injected subcutaneously,
awakened, and allowed free access to water and food. At time
0 (unoperated control) and at 3, 6, 16, 24, 48, and 72 h
after surgery, rats were reanesthetized with a 40 mg/kg intraperitoneal
injection of pentobarbital sodium. Inferior vena cava blood was
obtained to determine -glutathione S-transferase (
-GST) levels, and animals were killed via exanguination. In one set
of rats, liver tissue was perfusion fixed with 2% paraformaldehyde and
harvested for immunohistochemical detection of BrdU incorporation. In a
second set of animals, the liver was fixed with 10% formalin for
apoptosis staining using the terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. In a
third set, total RNA was isolated as previously described (1, 2, 14, 15, 25).
Determination of -GST levels in vena caval blood.
Blood was collected from the inferior vena cava, placed in a serum
separator tube (Becton-Dickenson, Franklin Lakes, NJ), and allowed to
coagulate. Samples were spun at 13,000 rpm for 5 min, and the aqueous
layer was isolated and stored at
70°C.
-GST levels were
determined in duplicate from serum using a quantitative enzyme
immunoassay (GST EIA; Biotrin International, Newton, MA; see Refs.
6, 37, and 44). Samples from six
animals at each time point were averaged.
Apoptosis detection. In situ cell death was evaluated using TUNEL (Boehringer Mannheim; see Refs. 21, 22, and 32). Paraffin-embedded tissue was fixed in 10% formalin, dewaxed, rehydrated in an ethanol gradient, treated with proteinase K, blocked, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The fluorescein-containing TUNEL reaction mixture, diluted 1:1 with PBS, was incubated on sections for 1 h. TdT was used to enzymatically label the 3'-OH DNA breaks with dUTP. Sheep anti-fluorescein antibody conjugated with horseradish peroxidase was added and further bound to metal-enhanced diaminobenzidine (DAB; Pierce, Rockford, CA) for light microscopic evaluation. The presence of apoptotic hepatocytes was evaluated by an independent pathologist (E. E. Furth), and apoptosis was designated as present only if cells contained both condensed chromatin and positive TUNEL staining.
Detection of cell division/regeneration. Paraffin-embedded, paraformaldehyde-fixed tissue was dewaxed, rehydrated, quenched for endogenous peroxidase activity, and blocked with horse serum/PBS and avidin-biotin blocking solution. Sections were then incubated with a mouse monoclonal primary antibody to BrdU (Sigma, St. Louis, MO) for 45 min, followed by a horse anti-mouse secondary antibody for 30 min (11, 18, 30). Chromogenic staining was performed with DAB as previously described (18).
Northern blot hybridization analysis.
Northern blot hybridization analysis was performed on total RNA samples
isolated from liver harvested 0, 3, 6, 16, 24, 48, and 72 h after
SO, CLP, or 2CLP as previously described (1, 2, 14, 25).
cDNAs complementary to the mRNAs encoding the MAP kinase-activated
immediate-early genes JunB and LRF-1, the cell cycle protein cyclin D1,
and the constitutively expressed 2-macroglobulin were
32P labeled, and blots were hybridized for 16 h.
Membranes were washed, and autoradiography and densitometry were
performed. Densitometric data for each gene of interest were divided by
the density for
2-macroglobulin at the same time point.
Studies were performed on RNA obtained from three rats at each time
point, and means and SD were calculated.
Data analysis. Cells incorporating BrdU were counted by two independent observers examining six different low-power fields. Apoptosis was evaluated by an independent pathologist. The highest and lowest counts were excluded. Statistical significance (P < 0.05) for cell counts and densitometric data were determined using ANOVA with the Bonferroni test of post hoc significance.
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RESULTS |
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Outcome. As in previous studies (1, 2, 14, 25), SO, CLP, and 2CLP provoked different clinical responses. SO animals recovered uneventfully. CLP was followed by signs of mild sepsis, including lethargy, decreased spontaneous movement, and poor grooming. All resolved within 48 h. Six to sixteen hours after 2CLP, rats exhibited decreases in food intake, spontaneous movement, and grooming. Diarrhea, piloerection, tachypnea, and extreme lethargy were evident by 16 h.
Outcome data paralleled previous studies (1). Six unoperated controls, the 36 SO animals, and the 36 CLP animals all survived. In contrast, mortality was high after 2CLP. Between 16 and 24 h, mortality was 50%, between 24 and 48 h 75%, and between 48 to 72 h 90%.Hepatic necrosis after CLP and 2CLP.
Figure 1 depicts -GST serum levels
after SO, CLP, and 2CLP. Relative to SO and time 0,
-GST
levels were significantly elevated 16, 24, 48, and 72 h after both
CLP and 2CLP.
-GST levels were significantly higher in the 2CLP
group than in CLP animals at 24, 48, and 72 h.
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Hepatic apoptosis induced by CLP and 2CLP.
A small number of TUNEL and morphologically positive apoptotic
cells was detected between 6 and 48 h after both CLP and 2CLP (Fig. 2). Apoptosis was evident
in both hepatocytes and nonparenchymal cells. There was no difference
in the number of apoptotic cells per high-powered field between CLP
and 2CLP. A few apoptotic cells were detected after SO.
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Hepatic regeneration after CLP and 2CLP.
BrdU uptake was evident 24 h after single-puncture CLP. At this
time point, 20 ± 4 cells/low-powered field were BrdU positive. This increased to 28 ± 6 cells/low-powered field and 34 ± 7 cells/low-powered field at 48 and 72 h after CLP, respectively
(Fig. 3). Fewer than five BrdU-positive
cells per low-powered field were evident at any time point in 2CLP
animals.
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DISCUSSION |
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In the present study, hepatocyte loss in both CLP and 2CLP occurs
via necrosis, reflected in elevated -GST levels. Low levels of
apoptosis were detected also. Most interestingly, hepatic
regeneration, as measured by BrdU uptake, was present after CLP but not
after 2CLP.
In previously published data from our laboratory, we have demonstrated
the divergence of outcome between CLP and 2CLP (1, 2, 14,
25). Furthermore, we found that CLP induced a transient but
profound downregulation of several liver-specific genes
(2). This decrease was persistent after 2CLP (14,
25). Pathological changes in the liver were more profound after
2CLP (1, 2, 14, 25). In the present study, hepatic
necrosis appears to correlate with this divergence in outcome and
transcription. Low levels of -GST release, however, make it unlikely
that this correlation has pathological significance. In contrast,
regeneration inversely parallels mortality and the previously described
persistent alterations in transcription (1, 14, 25).
Additionally, hepatocyte regeneration was evident at later time points
than necrosis and apoptosis, suggesting that it is a
compensatory process. The decreased expression of JunB, LRF-1, and
cyclin D1 is also consistent with our finding of an inverse correlation
between cellular regeneration and a selective but irreversible loss of
transcription 24 h after 2CLP (1, 14, 25).
Hepatocyte necrosis was evaluated by -GST serum levels.
-GST is
unique to hepatocytes, is rapidly released in the circulation, and
therefore is a more sensitive indicator of hepatocellular injury than
standard transaminase levels (6, 36, 37, 44, 47).
Although necrosis is a well-known component of late sepsis-associated liver failure, the presence of apoptosis has not been documented universally. Apoptosis is well described after direct hepatotoxic injury, such as that associated with carbon tetrachloride poisoning or after ischemia-reperfusion (10, 41). CLP-induced parenchymal cell apoptosis has been previously described in the lung, gut, muscle, kidney, and lymphoid tissue (20, 21). However, most studies show no evidence of apoptosis in the liver, brain, and heart. Indeed, others report that a second insult, such as galactosamine administration or ischemia, is required for apoptosis to be clearly evident in the liver after inflammatory insults (22, 23, 31, 42, 43, 45). Our studies confirm this, because only low levels of apoptosis could be appreciated. Apoptosis seen in CLP and 2CLP rats indicates that sepsis/MODS may be associated with some upregulation of programmed cell death, but it is likely that this is a less important process than necrosis.
Cytokines, such as tumor necrosis factor- (TNF-
), IL-1
, and
IL-6, have been implicated in the modulation of intrahepatic sepsis-associated responses (1, 4, 9, 13, 28, 34, 49).
These cytokines may play a role in cell death and regeneration as well.
It is known, for example, that TNF-
and IL-1
have been implicated
in inducing cell death via both apoptosis and necrosis (28). However, both exert anti-apoptotic effects via
activation of the transcription factor nuclear factor-
B (7,
28, 46, 48). Given the pleiotropic nature of TNF-
/IL-1
mediated responses, it is not surprising that there was only a minor
difference in necrosis and apoptosis after CLP and 2CLP.
TNF-
and IL-6 are also important components in the early signaling
pathways leading to regeneration (33). Cressman et al.
(12) found impaired regeneration and increased mortality
after 75% hepatectomy in IL-6-deficient mice. Similarly, Yamada et al.
(50) documented failed regeneration after hepatectomy in
mice deficient in the 55-kDa TNF-
receptor. In both studies,
regeneration was restored after a single dose of IL-6. In a previous
study, we demonstrated that CLP and 2CLP evoked different IL-6-linked
responses (1). Specifically, activation of the IL-6-linked
transcription factor Stat-3 and expression of the IL-6-activated
acute-phase reactant
2-macroglobulin decreased to nearly
undetectable levels between 16 and 24 h after 2CLP but not after
CLP. This is clearly consistent with, and might well explain, failed
regeneration in the liver after 2CLP but not CLP.
In conclusion, the present study indicates that failed hepatocyte regeneration may be an additional manifestation of hepatic dysfunction in fulminant sepsis. These findings suggest that further studies will clarify the role of liver regeneration in sepsis and may identify an important therapeutic approach.
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ACKNOWLEDGEMENTS |
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We thank L. Greenbaum and A. DeMaio for critically reviewing the manuscript. We gratefully acknowledge the support of Dr. David Longnecker and the faculty and staff of the Dept. of Anesthesia, University of Pennsylvania, whose hard work and dedication make academic opportunity possible.
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FOOTNOTES |
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This work was presented in part at the 20th annual symposium on Shock in Indian Wells, CA, in June 1997.
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants K08-DK-02179 and DK-50306.
Address for reprint requests and other correspondence: C. S. Deutschman, Dept. of Anesthesia, Dulles 773/HUP, 3400 Spruce St., Philadelphia, PA 19104-4283 (E-mail: csd{at}mail.med.upenn.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.
Received 12 July 2000; accepted in final form 5 December 2000.
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