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* Department of Anesthesiology and Critical Care Medicine, University of the Saarland, D-66421 Homburg, Germany;
# Neurosurgical Research Laboratory, University of the Saarland, D-66421 Homburg, Germany;
Department of Pharmacology and Toxicology, University of Arkansas for Medical Science, Little Rock, Arkansas, USA; and
Department of Pathology, University Hospital Zürich, Zürich, Switzerland
1Correspondence: Department of Anesthesiology and Critical Care Medicine, University of the Saarland, D-66421 Homburg, Germany. E-mail: aimbau{at}uniklinik-saarland.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: necrosis • apoptosis • necrapoptosis • liver • ATP • oxygen free radicals
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The decrease of cardiac output after hemorrhage is accompanied by an even disproportionally greater decrease in hepatic blood flow, resulting in hepatocellular ischemia. Although hemorrhagic hypotension leads to an overall decrease in oxygen delivery to the sinusoids, ischemic injury is most pronounced in downstream hepatocytes located in the pericentral or centrilobular region of the acinus. Thus, the hallmark of hepatocellular injury in low flow states is characterized by pericentral damage, also referred to as pericentral necrosis (2)
. Apoptosis or programmed cell death is a morphologically distinct form of cell death that has been shown to contribute to hepatocellular injury, e.g., in models of Fas antibody- or tumor necrosis factor (TNF) -induced liver damage (3
4
5)
. Its contribution to liver injury in models of complete cold or warm ischemia/reperfusion is, however, highly controversial (6
, 7)
. Induction of either necrotic or apoptotic pathways of hepatocellular injury may depend on the degree of ischemic injury and the adequacy of restoration of oxygen supply to the cells since oxidative metabolism is of outstanding significance for the regulation of programmed cell death (8)
. Moreover, many agents that induce apoptosis are either oxidants or stimulators of oxidative metabolism (9
, 10)
, suggesting that the aggravation of hepatocellular injury associated with resuscitation from hemorrhage (also referred to as oxygen paradox) may depend on generation of oxygen free radicals (11)
and subsequent induction of apoptotic pathways (12)
. Thus, characterization of the mechanisms underlying hepatocellular injury is of particular importance since treatment strategies such as inhibition of caspase activity may offer novel pharmacological tools to prevent hepatocellular dysfunction and ultimately multiple organ failure (MOF) in critically ill patients (4
, 7
, 13)
. In the present study we provide evidence that prolonged ischemia results in necrotic injury to the pericentral region, whereas reoxygenation after shorter periods of ischemia produces oxygen free radical (OFR) -dependent apoptotic injury to pericentral hepatocytes in vivo. These data may help to explain the controversy regarding molecular mechanisms of ischemia/reperfusion-induced injury to the liver.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental design
Rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg b.w.). Anesthesia was maintained by intermittent intravenous pentobarbital application (1–3 mg/kg b.w.). Subsequently, a tracheotomy was performed to facilitate spontaneous breathing. The right jugular vein was cannulated for drug administration and fluid resuscitation. A continuous infusion of Ringer’s solution [10 mL/(kg·h)] was supplied to compensate for losses during preparative surgery. The left carotid artery was cannulated to allow blood withdrawal and measurement of systemic arterial blood pressure with a standard pressure transducer (Medex Medical, Ratingen, Germany).
Animals (n=6–9/group) were subjected to hemorrhagic shock with a mean arterial blood pressure (MAP) of 35 ± 5 mmHg for 1, 2, or 3 h in a modified Wiggers model, followed by fluid resuscitation. In the first group, MAP was lowered to 35 ± 5 mmHg for 1 h, followed by 2 h of resuscitation. In the second group, hemorrhagic hypotension was maintained for 2 h, followed by 1 h of fluid resuscitation; in the third group, MAP was lowered for 3 h without resuscitation. This model was used to produce varying grades of ischemic liver injury. Another series of experiments was undertaken to assess the effect of resuscitation/reoxygenation. In these experiments, rats were subjected to a MAP of 35 ± 5 mmHg at a variable of 1, 2, or 3 h, followed by resuscitation for 2 h irrespective of the duration of hemorrhagic hypotension. Shed blood was collected in syringes containing citrate-phosphate-dextrose solution (0.14 mL/mL shed blood). Animals were resuscitated if appropriate with 60% of the shed blood withdrawn infused during the first 10 min of resuscitation and twice the shed blood volume as Ringer’s solution during the first hour of resuscitation. The infusion rate of Ringer solution was lowered to a volume equaling the maximal bleed-out volume for the second hour of resuscitation.
Based on results obtained with the above specified model of graded shock suggesting occurrence of apoptotic cells in the pericentral region with short periods of hemorrhage, in another series of experiments hemorrhagic hypotension was induced for 1 h, followed by 2 h of resuscitation to assess the influence of either the membrane-permeable radical scavenger tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) or the pancaspase inhibitor z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone) on indicators of apoptosis after hemorrhagic shock. Tempol was administered as bolus injection (30 mg/kg b.w.), followed by a continuous infusion of 30 mg/kg b.w./h during resuscitation; z-VAD-fmk was injected twice as a bolus (3 mg/kg b.w. each) 30 min before hemorrhage and along with resuscitation fluids. To investigate the influence of antiapoptotic strategies on liver function in a further series of experiments, animals were pretreated with vehicle, tempol, or z-VAD-fmk and subjected to hemorrhagic hypotension for 1 h, followed by 5 h of resuscitation. Indocyanine clearance was determined before and 2 and 5 h after onset of fluid resuscitation, as described in detail below.
Sham-operated animals that did not undergo hemorrhage received a constant infusion of 10 mL/(kg·h) Ringer’s solution over the entire period of the experiment and served as controls.
Measurement of indocyanine green (ICG) clearance
ICG (Pulsion, München, Germany) was freshly prepared by dissolving in distilled water at a concentration of 2 mg/mL. ICG (0.8 mg/kg b.w.) was rapidly administered as a bolus into the right jugular vein; 10 s after injection, 0.1 mL of blood was withdrawn for determination of baseline ICG plasma concentration. Blood samples (0.1 mL) were taken at 1, 2, 4, and 8 min after administration. After centrifugation, ICG plasma concentrations were determined spectrophotometrically at 800 nm using preinjection blank serum as zero reference and the specific concentration read against a standard curve. The ICG clearance (CL) was calculated as: CL = D/AUC (D = dose administered, µg/kg b.w.; AUC = area under the curve).
Quantitative determination of liver enzymes
Blood samples were taken at baseline and at the end of the experiment. Serum was prepared and aliquots thereof were stored at –70°C until analysis. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GLDH) were analyzed with commercially available kits (Roche Diagnostics, Mannheim, Germany).
Determination of hepatic ATP content
To quantify the ATP content in the liver, the tissue was freeze-clamped at the end of the experiment and stored in liquid nitrogen until analysis. Frozen liver samples were rapidly homogenized in 3% sulfosalicylic acid with a Powergen 125 tissue homogenizer (Fisher Scientific, Pittsburgh, PA, USA) and ATP levels were determined enzymatically in the supernatant with Sigma test kit 366-UV. A standard curve with purified ATP was used to calculate tissue concentrations.
To semiquantitatively assess the regional ATP content in liver sections, a bioluminescence method was used. At the end of the experiment the left liver lobe was frozen immediately in liquid nitrogen and stored at –70°C until analysis. Frozen liver lobes were cryosectioned in a cryostat at 20 µm. Sections were freeze-dried for 24 h at –20°C and the sections subsequently were heated to 95°C to inactivate endogenous enzymes. Bioluminescence imaging of ATP was performed as described previously (14)
. A solution was prepared for the substrate-specific bioluminescence reaction in the absence of ATP containing 12 mL basic buffer (200 mM hydroxypiperizino-ethanesulfonic acid [HEPES] + 100 mM arsenate, pH 7.6). Pulverized dried light organs (260 mg) from fireflies (Phontinus pyralis) were added. After homogenization and centrifugation, the supernatant was mixed with 25 µL 1 M MgCl2. The solution was frozen and cut into 60 µm slices in a cryostat at –20°C. A freeze-dried and heat-inactivated liver section was then covered with a 60 µm section of frozen enzyme block and was placed onto a photographic film (Agfaplan, 100 ASA, Agfa, Köln, Germany) to record bioluminescent light emitted from the section after warming to room temperature. Exposure time was 30 s. Quantification of the signal was performed by computer-assisted densitometry using an image analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA).
In situ assessment of caspase activity, DNA fragmentation, and apoptotic cell death
Cleavage of genomic DNA during apoptosis resulting in DNA strand breaks was identified by labeling free 3'OH termini with modified nucleotides in an enzymatic reaction involving terminal deoxynucleotidyl transferase (TdT). For this determination a commercially available kit (in situ Cell Death Detection Kit; Roche Molecular Biochemicals) was used. Frozen liver samples were cryosectioned (7 µm) and stained by the TUNEL (Tdt-mediated dUTP nick end labeling) method according to the manufacturer’s instructions. TUNEL-positive hepatocytes were counted in 20 random high-power fields per tissue sample (400x) in a blinded fashion by fluorescence microscopy. Only TUNEL-positive hepatocytes with additional morphological characteristics of apoptosis such as cell shrinkage, chromatin condensation, and margination were counted as apoptotic cells. In addition, the caspase-mediated cleavage of intracellular proteins that occurs during apoptosis was detected in sections based on the staining for a neo-epitope of cytokeratin 18 that becomes available at an early caspase cleavage event using the monoclonal antibody M30 (M30 CytoDEATH, Roche Molecular Biochemicals).
Hematoxylin-eosin (HE) staining
Formalin-fixed, paraffin-embedded, dewaxed liver sections (5 µm) were used for HE staining. After deparaffinization, liver sections were stained in acid hematoxylin for 5 min and rinsed under running water for 10 min. Sections were stained in diluted eosin solution (1:10) an additional 5 min and rinsed with distilled water. After dehydration, the stained sections were coated with Roti-HistokittTM (Roth, Karlsruhe, Germany).
Transmission electron microscopy
Liver samples were stored in 2.5% glutaraldehyde in phosphate buffer (0.1 mol/L, pH 7.4) for <1 wk before further processing. Specimen were postfixed with osmium tetroxide, dehydrated in graded alcohol, and embedded in Epon. Ultrathin liver sections (60–80 nm) were cut on an ultramicrotome (Leica, Glattbrugg, Switzerland) and contrasted with uranyl acetate and lead citrate. Stained sections were viewed in a Phillips CM 10 electron microscope (Phillips, Dietikon, Switzerland) operating at 60 KV.
Statistical analysis
Data are presented as means ± standard error of the mean (SE). Differences were evaluated by analysis of variance (ANOVA), followed by post hoc multiple comparison according to the Student-Newman-Keuls method. According to the study design, either a one-way ANOVA or a repeated measure ANOVA was performed using the SigmaStat software package (Jandel Scientific, San Rafael, CA, USA). When criteria for parametric testing were violated, the appropriate nonparametric test, i.e., Kruskal Wallis ANOVA on ranks and Friedman test, were used. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 16)
. Consistent with these results, macrohemodynamics recovered in all animals subjected to 1 h of hemorrhage and 2 h of resuscitation, reflecting a fully reversible model of compensated hemorrhagic shock (data not shown). In animals subjected to 2 or 3 h of hemorrhagic hypotension, repeated injections of Ringer’s solution were required in the second and third hour to maintain a MAP of 35 ± 5 mmHg reflecting decompensation. Although all animals subjected to 2 h of hemorrhagic hypotension could be successfully resuscitated for up to 2 h, 
1/3 of the animals subjected to 3 h of hemorrhage failed to recover despite volume replacement (irreversible shock) and thus were not used for analysis.
Effect of graded hemorrhagic shock on hepatic ATP levels
The effect of graded hemorrhagic shock on tissue ATP levels is summarized in Fig. 1
. The upper panel shows regional distribution of ATP in a cross section of a representative left liver lobe. The lower panel reflects the ATP content measured in homogenates (n=6 livers for each condition). Resuscitation using autologous fresh shed blood and Ringer’s solution after 1 h of hemorrhagic hypotension (Fig. 1B
) restored ATP concentrations in homogenates virtually to the content of sham-operated controls with no locally persistent impairments of ATP content (Fig. 1A
). In contrast, prolonged hemorrhagic hypotension was paralleled by a gradual depletion of hepatocellular ATP concentrations. Restoration of ATP was heterogeneous in the specimen obtained after resuscitation, most notably after 2 h of hemorrhagic hypotension, when some areas presented an almost complete restoration and ATP concentrations in other areas within the same liver lobe remained severely depressed (Fig. 1C
). After 3 h of hemorrhagic hypotension without fluid resuscitation, almost no ATP was detectable in the whole liver lobe (Fig. 1D
).
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Histological evidence of hepatocellular injury after different periods of hemorrhagic shock
Histologic examination of hematoxylin-eosin (HE) -stained liver sections obtained from animals subjected to 1 h of hemorrhagic hypotension, followed by 2 h of resuscitation (Fig. 2
B), only rarely displayed scattered foci of eosinophilic or swollen hepatocytes and areas of early necrosis. However, condensated nuclei were frequently observed in these samples. Sections revealed hepatocellular coagulative necrosis in pericentral regions after 2 h of hemorrhage, followed by 1 h of resuscitation (Fig. 2C
). After 3 h of hemorrhagic hypotension, areas of pericentral necrosis were observed frequently extending into midzonal regions of the liver acinus (Fig. 2D
). Sinusoids were congested with red cells indicative of sinusoidal perfusion failure in particular after prolonged hemorrhagic hypotension. Evidence of apoptotic and necrotic hepatocytes as observed in HE-stained slides with the different shock models was further confirmed by transmission electron microscopy. In contrast to sham-operated controls (Fig. 3
A, B), apoptotic hepatocytes were frequently detected in sections of livers subjected to hemorrhagic shock for 1 h, followed by 2 h of fluid resuscitation (Fig. 3C, D
). These hepatocytes show typical morphological signs of apoptotic cell death, i.e., condensation of nucleus and chromatin. In contrast, in livers subjected to hemorrhagic hypotension for 3 h without resuscitation areas of extensive necrosis were observed over the whole liver lobe (Fig. 3E, F
).
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Apoptotic hepatocytes after different periods of hemorrhagic shock and resuscitation
An increase in DNA fragmentation/condensation as identified by morphological analysis of TUNEL-positive nuclei was observed in pericentral and midzonal hepatocytes after different periods of hemorrhagic hypotension (Fig. 4
B–D) compared with sham-operated controls (Fig. 4A
). The upper panel demonstrates representative liver sections. The lower panel represents the mean of apoptotic hepatocytes of 20 liver acini per individual experiment assessed by TUNEL assay in combination with morphological criteria. A maximum of TUNEL-positive cells displaying additional signs of apoptotic cell death was detected after 1 h of hemorrhage, followed by 2 h of resuscitation (Fig. 4B
). In contrast, apoptotic hepatocytes in pericentral and midzonal regions of the liver acinus were observed less frequently after prolonged periods of hemorrhagic hypotension. Two hours of hemorrhage, followed by 1 h of fluid resuscitation (Fig. 4C
) and 3 h of hemorrhagic shock without resuscitation (Fig. 4D
), resulted in an only moderate increase in the number of TUNEL-positive hepatocytes compared with sham-treated controls.
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Similarly, when animals were allowed to recover for 2 h after various durations of hemorrhagic hypotension (1, 2, or 3 h) apoptotic injury was detectable after 1 or 2 h of low flow, followed by adequate resuscitation; after 3 h of hemorrhage, signs of apoptotic injury were negligible despite resuscitation. Whereas volume resuscitation was sufficient to restore hepatocellular ATP after 1 or 2 h of hemorrhagic hypotension, it failed to normalize hepatocellular ATP after 3 h of shock (Fig. 5
).
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Effects of tempol and z-VAD-fmk on hepatocellular injury after short periods of low flow ischemia
Regulatory mechanisms controlling apoptotic cell death were studied in rats subjected to 1 h of hemorrhage and 2 h of resuscitation based on the results obtained for graded hemorrhage. Rats were pretreated in these experiments with the antioxidant tempol or the pancaspase inhibitor z-VAD-fmk before hemorrhage. The caspase-mediated cleavage of intracellular proteins was confirmed in sections based on the staining for a neo-epitope of cytokeratin 18 using the monoclonal antibody M30 (Fig. 6
,left panel). In sham-operated controls no cytokeratin 18 neo-epitope was detectable (Fig. 6A
). In contrast to untreated animals subjected to hemorrhagic shock, which represent high binding activity of the M30 antibody (Fig. 6C
), treatment with tempol (Fig. 6E
) or z-VAD-fmk (Fig. 6G
) resulted in a marked decrease in M30-positive hepatocytes. Similarly, the marked increase in DNA fragmentation detected by TUNEL immunohistochemistry (right panel) after hemorrhagic shock and resuscitation (Fig. 6D
; Fig. 7
) vs. controls (Fig. 6B
; Fig. 7
) was significantly attenuated after pretreatment with tempol (Fig. 6F
; Fig. 7
) or z-VAD-fmk (Fig. 6H
; Fig. 7
).
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Hepatocellular injury was further assessed by measurement of serum enzyme levels of AST, ALT, and GLDH. Serum activities of AST, ALT, and GLDH were significantly increased after hemorrhage with a comparable rise after 1 h or 2 h of hemorrhagic hypotension, followed by 2 h or 1 h of resuscitation, respectively. Enzyme leakage tended, however, consistent with a lack of washout from the liver, to be lower after 3 h of hemorrhagic hypotension without subsequent resuscitation despite substantial evidence of histologic injury (Table 1a
). The highest enzyme levels were observed in serum from animals after 3 h of shock, followed by 2 h of resuscitation indicating reperfusion injury. Pretreatment with the antioxidant tempol or the pancaspase inhibitor z-VAD-fmk in animals subjected to 1 h hemorrhage, followed by 2 h of resuscitation, attenuated serum enzyme levels of AST, ALT, and GLDH without reaching statistical significance (Table 1b)
.
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Hepatocellular function after hemorrhagic shock as determined by ICG clearance
A significant decrease (40%) in the clearance of indocyanine green after 1 h of hemorrhagic hypotension compared with baseline values was observed at 2 and 5 h of resuscitation in vehicle-treated animals (Fig. 8
). In contrast, in animals pretreated with the pancaspase inhibitor z-VAD-fmk and those pretreated with the antioxidant tempol, ICG clearance after hemorrhagic shock, followed by 2 h or 5 h of resuscitation, was well preserved and not significantly different from the respective baseline or from time-matched sham-operated controls (Fig. 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although hemorrhagic hypotension leads to an overall decrease in oxygen delivery to the sinusoids, ischemic injury is most pronounced in downstream hepatocytes located in the pericentral region since extraction of oxygen by upstream hepatocytes in the periportal region leads to a substantial decrease in oxygen availability to downstream hepatocytes. Thus, the hallmark of hepatocellular injury in low flow shock is characterized by pericentral damage, also referred to as "pericentral necrosis" (2)
. However, the protective effect of various antiapoptotic strategies in the present study suggests that, under appropriate conditions, the molecular mechanism of pericentral injury associated with low flow ischemia is rather apoptotic in nature.
Apoptosis and necrosis are two different forms of cell death with distinct morphological and biochemical characteristics, although there is evidence that a common death signal may culminate in either necrosis or apoptosis after initial progression of cell death via shared pathways ("necrapoptosis") (17
, 18)
. A contribution of apoptotic cell death to shock-induced hepatocellular dysfunction after shorter periods of hemorrhage in the present study is supported by a variety of morphological and biochemical (cytokeratin cleavage and TUNEL assay) markers as well as on the protective effect of a pancaspase inhibitor. Although evidence suggests that TUNEL staining might occur after necrotic cell death and may fail to discriminate between apoptotic, necrotic, and autolytic cell death (19)
, it is an early event in apoptosis but a late event in necrosis. The time-dependent fashion of occurrence of DNA fragmentation thus may explain that TUNEL-positive cells were observed frequently after short periods of hemorrhage but rarely after prolonged hemorrhage despite histological evidence of profound necrotic damage in the present study.
In vitro evidence suggests that the intensity of the insult and modifying factors such as ATP determine whether apoptosis or necrosis occurs, but in vivo data regarding molecular mechanisms of hepatocellular injury as a function of insult severity are scarce or controversial. In the present study, maximum apoptotic cell death was observed after shorter periods of hemorrhage (1–2 h) with adequate resuscitation leading to an almost complete restoration of hepatocellular ATP content. Conversely, after prolonged ischemia with or without resuscitation, extended necrotic areas were observed whereas only individual apoptotic hepatocytes were detected. Leist et al. demonstrated in Jurkat T cells that the decision between apoptosis and necrosis is highly dependent on intracellular ATP concentration (8)
. In these studies induction of ATP depletion for up to 90 min after administration of drugs provoking apoptosis shifted the pattern of cell death from apoptosis to necrosis. In addition, the necrotic cell death observed in ATP-depleted Jurkat T cells after pretreatment with apoptotic stimuli directed the cell toward apoptosis after restoration of intracellular ATP content for up to 2 h after drug challenge (8)
. Thus, death signals as they result from low flow hypoxia in vivo may initially progress via shared pathways and culminate either in cell lysis/necrosis or programmed cell death depending on the degree of resuscitation/reoxygenation. Consistent with a recent proposal by Lemasters et al. (17)
, cell death after hemorrhage and resuscitation may be viewed as an example of necrapoptosis where hypoxia reflects the common stress event that initiates shared pathways for propagation of cell death whereas restoration of ATP serves as a modifying factor deciding on necrotic vs. apoptotic demise.
As mentioned above, oxygen supply in the liver after hemorrhagic hypotension is lowest in pericentral regions due to the unidirectional blood flow in sinusoids. Thus, restoration of high-energy metabolites during resuscitation after hemorrhagic shock likely takes place first in periportal and midzonal regions of the liver acinus. In the present study we observed areas of coagulative necrosis in the pericentral region and a moderate number of apoptotic hepatocytes over the whole acinus, especially periportal and midzonal after longer periods of hemorrhagic hypotension (2 h), followed by resuscitation. This observation would explain the coexistence of both types of cell death after hemorrhagic shock in vivo, where individual cell death within the tissue would be determined by the energy supply. In addition to zonal heterogeneity, longer periods of hemorrhagic hypotension (2 h) produced substantial heterogeneity of local ATP content of larger vascular units within the liver lobes. This was paralleled by the occurrence of both types of cell death similarly scattered over the lobe, further supporting the concept of dependence of apoptotic cell death on restoration of hepatocellular ATP content in the liver in vivo.
Low flow ischemia associated with hemorrhagic hypotension and subsequent resuscitation may lead to the generation of OFR (15)
from intracellular (e.g., mitochondria) (11)
or extracellular sources (e.g., Kupffer cells) (20)
, which have been suggested as regulators of apoptotic injury. Similarly, other stimuli like TNF-
, which is also released by activated macrophages and neutrophils after hemorrhagic shock (21)
, may synergize to induce apoptotic injury in hepatocytes. In vitro studies with TNF- and ceramide have shown that these agents activate OFR production by mitochondria (22)
. Consistent with the concept of induction of apoptotic cell death after hemorrhagic hypotension by OFR, the extent of apoptosis was substantially reduced by administration of the antioxidant tempol before hemorrhage and along with resuscitation fluids in the present model. In contrast to these results, Gujral and co-workers demonstrated in a model of isolated liver ischemia that there is no evidence for apoptotic cell death after warm ischemia and subsequent reperfusion (6)
. However, in contrast to ischemia/reperfusion, hemorrhagic shock leads to a liver injury that is further aggravated specifically by a gut-derived endotoxemia (23)
. Therefore, ischemia and reperfusion of gut and liver as well as subsequent endotoxemia may activate macrophages and neutrophils to release a variety of cytotoxic mediators (24)
that may initiate in concert the apoptotic process through formation of OFR. A similar decrease in the number of apoptotic hepatocytes after hemorrhagic hypotension and pretreatment as observed with the antioxidant tempol was also noted after administration of the pancaspase inhibitor z-VAD-fmk. This is in line with recent reports that demonstrate an inhibition of hepatocyte apoptosis by blocking "effector" caspases after Fas- or TNF-induced apoptosis in mouse liver (3
, 4)
. Furthermore, the contribution of caspases in ischemia/reperfusion-induced apoptosis was shown in a model of myocardial injury by coronary occlusion, where z-VAD-fmk was shown to reduce infarct size upon ischemia/reperfusion (25)
. The mechanisms underlying ischemia/reperfusion-induced apoptosis are poorly understood. Apoptosis is mainly regulated by the apoptotic protease-activating factors (Apaf-1, -2, -3), the Bcl-2 family, and caspases (26
, 27)
. Recent evidence suggests that mitochondria are important in the apoptotic process and that alterations in the function of the mitochondrial membrane are pivotal for the initiation of cell death. Loss of the mitochondrial transmembrane electrical potential, part of a process described as mitochondrial permeability transition (MPT), precedes the gross morphological changes associated with apoptosis. These alterations appear to be regulated at least in part by the redox status of the cell. Onset of this MPT is caused by opening of high-conductance permeability transition pores. Pore opening leads to mitochondrial swelling, membrane depolarization, and uncoupling of oxidative phosphorylation (12
, 28)
. The permeability transition pore remains closed in unstressed cells. In contrast, during oxidative stress the pore opens suddenly (29)
. The immunosuppressive cyclic endecapeptide cyclosporin A specifically blocks onset of MPT and prevents apoptosis (30)
. This is consistent with our results observing a significant reduction in the number of apoptotic hepatocytes after short periods of hemorrhage/resuscitation and pretreatment with either the membrane-permeable antioxidant tempol or CSA (data not shown).
To further assess the functional significance of apoptosis for liver dysfunction, we studied the effect of tempol, an SOD mimetic known to confer protection in a variety of shock models (31
, 32)
, and z-VAD-fmk on depression of hepatocellular clearance capacity that accompanies hemorrhagic shock despite adequate resuscitation. Inhibition of the apoptotic pathway after hemorrhagic low flow ischemia and subsequent resuscitation by either the antioxidant tempol or the caspase inhibitor z-VAD-fmk was accompanied by significantly improved liver function, as assessed by indocyanine green clearance. Pretreatment with tempol or z-VAD-fmk tended to attenuate enzyme leakage from the liver compared with vehicle-treated animals. Thus, apoptosis seems to contribute significantly to liver dysfunction and injury in this model of compensated and reversible hemorrhagic shock that closely mimics the clinical scenario, whereas necrotic injury seems to be a major mechanism upon more severe ischemic insults in the liver.
In summary, we demonstrate that short periods of hemorrhagic hypotension with subsequent resuscitation accompanied by sufficient restoration of high energy phosphates lead to a profound induction of pericentral apoptotic cell death that mediates impaired hepatocellular clearance capacity in the liver in vivo. In contrast, prolonged hemorrhagic hypotension initiates typical pericentral necrosis. Pharmacological different agents that block apoptosis like the pancaspase inhibitor z-VAD-fmk or the antioxidant tempol may be useful in treating ischemic insults upon reperfusion by blocking the suicide response in cells that have suffered sublethal anoxia. Oxygen paradox and successful restoration of hepatocellular energy metabolism may thus, paradoxically, induce pericentral injury through pericentral apoptosis.
Received for publication June 27, 2002. Accepted for publication February 12, 2003.
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