LIVER TRANSPLANTATION 20:1372–1382, 2014
ORIGINAL ARTICLE
Biomarkers Distinguish Apoptotic and Necrotic Cell Death During Hepatic Ischemia/ Reperfusion Injury in Mice Min Yang,1 Daniel J. Antoine,2 James L. Weemhoff,1 Rosalind E. Jenkins,2 Anwar Farhood,3 B. Kevin Park,2 and Hartmut Jaeschke1 1 Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 2MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, England; and 3Department of Pathology, St. David’s North Austin Medical Center, Austin, TX
Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin–stained and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)–stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury. Liver Transpl C 2014 AASLD. 20:1372-1382, 2014. V Received March 10, 2014; accepted July 12, 2014.
Abbreviations: AcHMGB1, hyperacetylated high-mobility group box 1; ALT, alanine aminotransferase; CK18, caspase-cleaved fragment of cytokeratin 18; FK18, full-length cytokeratin 18; G/E, galactosamine/endotoxin; H&E, hematoxylin and eosin; HMGB1, high-mobility group box 1; HPF, high-power field; IRP, ischemia/reperfusion; miR-122, micro-RNA 122; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. Potential conflict of interest: Nothing to report. This work was supported in part by the National Institutes of Health (grants R01 DK070195 and R01 AA12916), the National Center for Research Resources (5P20RR021940-07), and the National Institute of General Medical Sciences of the National Institutes of Health (8 P20 GM103549-07). Additional support came from an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health (P20 GM12345), the Training Program in Environmental Toxicology of the National Institute of Environmental Health Sciences (T32 ES007079-26A2), the Medical Research Council (G0700654 to Daniel J. Antoine, Rosalind E. Jenkins, and B. Kevin Park), and the Wellcome Trust (to Daniel J. Antoine). Address reprint requests to Hartmut Jaeschke, Ph.D., Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 1018, Kansas City, KS 66160. Telephone: 913-588-7969; FAX: 913-588-7501; E-mail: [email protected] DOI 10.1002/lt.23958 View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases
C 2014 American Association for the Study of Liver Diseases. V
LIVER TRANSPLANTATION, Vol. 20, No. 11, 2014
Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery using the Pringle maneuver, during liver transplantation, and during hemorrhagic shock. This clinical condition has been recognized for more than 30 years and has been extensively investigated with various animal models. The initial hypothesis that IRP injury is caused by an intracellular oxidant stress induced by xanthine oxidase1 was rapidly disputed.2,3 Subsequently, it was recognized that postischemic oxidant stress is derived largely from resident Kupffer cells4 and neutrophils,5,6 which are responsible for early and late injury phases, respectively. Although proinflammatory mediators such as tumor necrosis factor a (TNF-a),7 complement factors,8 and chemokines9,10 as activators of neutrophils were identified relatively early, the concept of sterile inflammation has emerged more recently.11,12 Currently, a major focus of research is the identification of damage-associated molecular patterns such as high-mobility group box 1 (HMGB1) protein and DNA fragments13 released by damaged cells and the involvement of toll-like receptors in triggering the formation of cytokines in macrophages.14 Views on the mechanisms of cell death during hepatic IRP injury have changed over time. Initially, it was thought that inflammatory cells kill hepatocytes by necrosis, and this conclusion was supported by extensive liver enzyme release.15 However, with the emerging recognition of apoptotic cell death during the late 1990s, a major shift occurred.16 With the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay as the test for apoptosis, it was concluded that up to 80% of hepatocytes and endothelial cells die by apoptosis during the first 3 hours of reperfusion.17,18 Also, reports emerged that caspase inhibitors protected against hepatic IRP injury.19,20 Most of these studies were performed with rats. However, a detailed morphological study that considered hallmarks of apoptosis such as cell shrinkage, chromatin condensation, and apoptotic bodies together with hepatic caspase-3 activity could not find any relevant increase in apoptotic cell death in the postischemic liver tissue.21 Gujral et al.21 concluded that >95% of all cell death occurred by necrosis. Importantly, it was also demonstrated that most necrotic cells stained positively in the TUNEL assay.21 These findings supported the previously raised concern that this assay, which detects DNA strand breaks,22 may not be specific for apoptosis.23 Taken together, the preponderance of the experimental evidence suggested that oncotic necrosis was the dominant mode of cell death during hepatic IRP injury in rats.24 During the last decade, more and more IRP studies have been performed with mice. Using the TUNEL assay and occasionally additional parameters such as cleavage of procaspase-3 or B cell lymphoma 2–associated X protein expression, many reports still argue for a substantial role of apoptosis over necrosis.25-27 However, most of these studies did not directly com-
YANG ET AL. 1373
pare apoptotic and necrotic cell death. In addition, the fact that apoptotic cells, in contrast to necrotic cells, are visible for only a limited time in tissue sections can make a quantitative comparison difficult. Thus, the question remains: how relevant is apoptosis for overall postischemic cell injury? To address this important question, plasma biomarkers for necrotic and apoptotic cell death were used because these parameters are more reflective of the process in the entire liver. Previous studies have shown the utility of plasma levels of total HMGB1 protein, micro-RNA 122 (miR-122), and full-length cytokeratin 18 (FK18) as indicators for cell necrosis; hyperacetylated highmobility group box 1 (AcHMGB1) as a marker of inflammation; and caspase-cleaved fragment of cytokeratin 18 (CK18) and caspase-3 activities as measures of apoptotic cell death in acetaminophen hepatotoxicity and bile duct ligation.28-33
MATERIALS AND METHODS Animals and Experimental Procedures C57bl/6j mice (20-25 g of body weight) were purchased from Jackson Laboratories (Bar Harbor, ME). All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (publication 86-23, revised 1985). All experimental protocols were approved by the animal use committees of the University of Kansas Medical Center. With mice under ketamine anesthesia (a mixture of 75 mg/kg ketamine, 5.1 mg/kg xylazine, and 0.75 mg/kg acepromazine given intramuscularly), laparotomy was performed, and the blood supply to the median and left hepatic lobes was occluded with an atraumatic vascular clamp for 45 minutes as described elsewhere.34 Reperfusion was initiated by removal of the clamp. The body temperature was maintained in all animals with an infrared heating lamp. After reperfusion had been initiated, 4 mL/kg physiological saline was injected intraperitoneally, the abdominal incision was closed with 4-0 silk and wound clips, and the animals were allowed to recover. The sham operation included all procedures except for clamping of the blood supply. Groups of animals were sacrificed at the end of ischemia or after 1, 3, 6, 12, or 24 hours of reperfusion. Blood was drawn from the vena cava into a heparinized syringe. The liver was rapidly removed and rinsed in saline, and sections were placed in a phosphate-buffered 10% (vol/ vol) formalin solution and embedded in paraffin for histological evaluations or snap-frozen in liquid nitrogen. In addition to the time-course experiments, groups of animals were treated with the pancaspase inhibitor Z-VD-fmk (10 mg/kg intraperitoneally) 30 minutes before ischemia and at the time of reperfusion or with a vehicle (50 mM trishydroxymethylaminomethane buffer, pH 7.0; 10 mL/kg).35 Furthermore, as a
1374 YANG ET AL.
positive control for apoptosis, some animals were treated intraperitoneally with 700 mg/kg Dgalactosamine (Sigma, St. Louis, MO) and 100 mg/kg Salmonella abortus equi endotoxin [Sigma; galactosamine/endotoxin (G/E)] dissolved in sterile phosphatebuffered saline (pH 7.0).35
Alanine Aminotransferase (ALT) Activities and Caspase Activation Plasma ALT activities were determined with the Pointe Scientific ALT kit (Pointe Scientific, Canton, MI) according to the manufacturer’s instructions. Caspase-3 activity in the liver or plasma was determined through the measurement of the z-VAD-FMK– inhibitable cleavage of the fluorescent caspase-3 substrate Ac-DEVD-AMC (Sigma-Aldrich St. Louis, MO), as described elsewhere.32 In addition, caspase-3 processing in liver and plasma was evaluated by western blotting with a caspase-3 antibody (Cell Signaling, Danvers, MA) as described in detail elsewhere.33
Measurement of Mechanism-Based Biomarkers of Liver Injury in Plasma FK18 and CK18 were identified and quantified by mass spectrometry as previously described in detail.28-30 miR-122 was measured by quantitative reverse-transcription polymerase chain reaction,31 let7d provided biological standardization, and lin-4 served as an internal standard for the assay. Total HMGB1 was quantified by immunoassay and mass spectrometry.28-30 The absolute quantification of AcHMGB1 was determined by mass spectrometry as previously described as a biomarker of immune cell activation.28 The investigators measuring the plasma biomarkers were blinded to the treatment groups of the animals.
Histology Sections of formalin-fixed paraffin-embedded liver samples were stained with hematoxylin and eosin (H&E) for the evaluation of liver cell death by a boardcertified pathologist (A.F.) as described in detail elsewhere.21,36 Additional liver sections were stained with the TUNEL in situ cell death assay (Roche) for visualization of DNA strand breaks as previously described.36 The quantitation of TUNEL-positive cells was performed by manual counting of the number of TUNEL-positive cells per 10 randomly selected highpower fields (HPFs) per section. The percentage of cells showing TUNEL-positive nuclei and cytoplasm was similarly quantified. All TUNEL-positive cells were included in the results, but hepatocytes and nonparenchymal cells were individually identified.
Statistical Analysis Data are given as means and standard errors. Statistical analysis was performed with SigmaPlot 11.0
LIVER TRANSPLANTATION, November 2014
(Systat Software, Chicago, IL). Data were assessed with a 1-way ANOVA followed by the StudentNewman-Keuls post hoc test for comparisons between means or Dunnett’s post hoc test for comparisons with a control. For data not normally distributed, we used the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. P < 0.05 was considered significant.
RESULTS C57Bl/6 mice were subjected to 45 minutes of hepatic ischemia followed by up to 24 hours of reperfusion. Progressive liver injury, as indicated by the increase in plasma ALT activities, was observed during reperfusion between 1 and 24 hours (Fig. 1A). No change in plasma ALT activities was observed in sham-operated animals. H&E-stained tissue sections showed tissue damage consistent with extensive necrosis (Fig. 1B). The area of necrosis was approximately 10% at 6 hours and progressed to >50% at 24 hours of reperfusion (Fig. 1A). No morphological evidence of apoptosis (apoptotic bodies) was detectable at any time during reperfusion. Staining sections with the TUNEL assay, which detects DNA strand breaks, showed very few individual positive cells (95% of all TUNEL-positive cells observed were hepatocytes. The progression of necrosis based on TUNEL staining was further illustrated after quantitation of both total TUNEL-positive cells (Fig. 2E) and the percentage of those exhibiting nuclear and cytoplasmic TUNEL staining (Fig. 2F). Although the number of total TUNEL-positive cells increased steadily during reperfusion, the staining of the cytosol developed between 3 and 6 hours and increased steeply afterward (Fig. 2F). These data are consistent with initial nuclear DNA fragmentation, which progressed to karyorrhexis and karyolysis. This TUNEL-staining pattern during IRP was fundamentally different from a positive control for apoptosis. In G/E-treated animals, numerous individual TUNEL-positive cells or apoptotic bodies were readily observed in which nuclei were stained but not the cytosol.33,36,37 It is well established that caspase-3 is an executioner caspase that is critical for apoptotic cell death
LIVER TRANSPLANTATION, Vol. 20, No. 11, 2014
YANG ET AL. 1375
Figure 1. Liver injury caused by IRP. Mice either were untreated controls (C) or were subjected to a sham operation (S) or 45 minutes of liver ischemia followed by reperfusion for various times (0, 1, 3, 6, 12, or 24 hours). (A) Liver injury was assessed by plasma ALT activities or quantitation of the areas of necrosis. Data are expressed as means and standard errors (n 5 3-6 animals per time point). *P < 0.05 versus controls. (B) Representative H&E-stained sections are shown for ischemia (45/0) and various times of reperfusion (45/X). The original magnification was 350.
in all liver cells.38 The activation of caspase-3 can be measured with an enzyme activity assay and with documentation of the formation of an active (eg, 17-kD) fragment by western blotting. Compared with very low levels of caspase-3 activities in controls, G/E treatment induced an extensive increase in enzyme activity in the liver and in plasma (Fig. 3A,B). This was confirmed by the reduction of procaspase-3 and the appearance of the active 17-kD fragment in the liver (Fig. 3C). In striking contrast to these observations in a liver with extensive apoptosis, livers from postischemic animals did not show any relevant increase of caspase3 activity in the liver or plasma (Fig. 3A,B) or evidence of caspase-3 processing (Fig. 3C) at any time point between 1 and 24 hours of reperfusion. The fact that no caspase-3 activity and no active caspase-3 fragments were detectable in the postischemic liver and in plasma suggest that this is a primary necrotic process and not secondary necrosis.39 miR-122 has been shown to be a highly sensitive marker for necrotic cell death.31,40 Although there
was no relevant increase in sham-operated animals compared with untreated controls, miR-122 levels increased dramatically during IRP (Fig. 4A) following the enhanced plasma ALT activities during reperfusion (Fig. 1A). However, peak miR-122 levels at 12 to 24 hours of reperfusion were 36,000- to 43,500-fold greater than the baseline in comparison with ALT levels, which were elevated 70- to 90-fold at this time. FK18 is a filamentous protein that can be passively released by necrotic cells. In contrast, CK18 reflects apoptotic cell death.41 During IRP, a time-dependent, massive release of FK18 was observed, with peak levels at 12 to 24 hours (Fig. 4B). Compared with baseline levels in controls (218 6 42 pmol/mL), FK18 concentrations were 220- and 440-fold higher between 3 and 24 hours of reperfusion, respectively. In contrast, the CK18 levels did not change significantly except for a
ORIGINAL ARTICLE
Biomarkers Distinguish Apoptotic and Necrotic Cell Death During Hepatic Ischemia/ Reperfusion Injury in Mice Min Yang,1 Daniel J. Antoine,2 James L. Weemhoff,1 Rosalind E. Jenkins,2 Anwar Farhood,3 B. Kevin Park,2 and Hartmut Jaeschke1 1 Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 2MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, England; and 3Department of Pathology, St. David’s North Austin Medical Center, Austin, TX
Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin–stained and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)–stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury. Liver Transpl C 2014 AASLD. 20:1372-1382, 2014. V Received March 10, 2014; accepted July 12, 2014.
Abbreviations: AcHMGB1, hyperacetylated high-mobility group box 1; ALT, alanine aminotransferase; CK18, caspase-cleaved fragment of cytokeratin 18; FK18, full-length cytokeratin 18; G/E, galactosamine/endotoxin; H&E, hematoxylin and eosin; HMGB1, high-mobility group box 1; HPF, high-power field; IRP, ischemia/reperfusion; miR-122, micro-RNA 122; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. Potential conflict of interest: Nothing to report. This work was supported in part by the National Institutes of Health (grants R01 DK070195 and R01 AA12916), the National Center for Research Resources (5P20RR021940-07), and the National Institute of General Medical Sciences of the National Institutes of Health (8 P20 GM103549-07). Additional support came from an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health (P20 GM12345), the Training Program in Environmental Toxicology of the National Institute of Environmental Health Sciences (T32 ES007079-26A2), the Medical Research Council (G0700654 to Daniel J. Antoine, Rosalind E. Jenkins, and B. Kevin Park), and the Wellcome Trust (to Daniel J. Antoine). Address reprint requests to Hartmut Jaeschke, Ph.D., Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 1018, Kansas City, KS 66160. Telephone: 913-588-7969; FAX: 913-588-7501; E-mail: [email protected] DOI 10.1002/lt.23958 View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases
C 2014 American Association for the Study of Liver Diseases. V
LIVER TRANSPLANTATION, Vol. 20, No. 11, 2014
Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery using the Pringle maneuver, during liver transplantation, and during hemorrhagic shock. This clinical condition has been recognized for more than 30 years and has been extensively investigated with various animal models. The initial hypothesis that IRP injury is caused by an intracellular oxidant stress induced by xanthine oxidase1 was rapidly disputed.2,3 Subsequently, it was recognized that postischemic oxidant stress is derived largely from resident Kupffer cells4 and neutrophils,5,6 which are responsible for early and late injury phases, respectively. Although proinflammatory mediators such as tumor necrosis factor a (TNF-a),7 complement factors,8 and chemokines9,10 as activators of neutrophils were identified relatively early, the concept of sterile inflammation has emerged more recently.11,12 Currently, a major focus of research is the identification of damage-associated molecular patterns such as high-mobility group box 1 (HMGB1) protein and DNA fragments13 released by damaged cells and the involvement of toll-like receptors in triggering the formation of cytokines in macrophages.14 Views on the mechanisms of cell death during hepatic IRP injury have changed over time. Initially, it was thought that inflammatory cells kill hepatocytes by necrosis, and this conclusion was supported by extensive liver enzyme release.15 However, with the emerging recognition of apoptotic cell death during the late 1990s, a major shift occurred.16 With the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay as the test for apoptosis, it was concluded that up to 80% of hepatocytes and endothelial cells die by apoptosis during the first 3 hours of reperfusion.17,18 Also, reports emerged that caspase inhibitors protected against hepatic IRP injury.19,20 Most of these studies were performed with rats. However, a detailed morphological study that considered hallmarks of apoptosis such as cell shrinkage, chromatin condensation, and apoptotic bodies together with hepatic caspase-3 activity could not find any relevant increase in apoptotic cell death in the postischemic liver tissue.21 Gujral et al.21 concluded that >95% of all cell death occurred by necrosis. Importantly, it was also demonstrated that most necrotic cells stained positively in the TUNEL assay.21 These findings supported the previously raised concern that this assay, which detects DNA strand breaks,22 may not be specific for apoptosis.23 Taken together, the preponderance of the experimental evidence suggested that oncotic necrosis was the dominant mode of cell death during hepatic IRP injury in rats.24 During the last decade, more and more IRP studies have been performed with mice. Using the TUNEL assay and occasionally additional parameters such as cleavage of procaspase-3 or B cell lymphoma 2–associated X protein expression, many reports still argue for a substantial role of apoptosis over necrosis.25-27 However, most of these studies did not directly com-
YANG ET AL. 1373
pare apoptotic and necrotic cell death. In addition, the fact that apoptotic cells, in contrast to necrotic cells, are visible for only a limited time in tissue sections can make a quantitative comparison difficult. Thus, the question remains: how relevant is apoptosis for overall postischemic cell injury? To address this important question, plasma biomarkers for necrotic and apoptotic cell death were used because these parameters are more reflective of the process in the entire liver. Previous studies have shown the utility of plasma levels of total HMGB1 protein, micro-RNA 122 (miR-122), and full-length cytokeratin 18 (FK18) as indicators for cell necrosis; hyperacetylated highmobility group box 1 (AcHMGB1) as a marker of inflammation; and caspase-cleaved fragment of cytokeratin 18 (CK18) and caspase-3 activities as measures of apoptotic cell death in acetaminophen hepatotoxicity and bile duct ligation.28-33
MATERIALS AND METHODS Animals and Experimental Procedures C57bl/6j mice (20-25 g of body weight) were purchased from Jackson Laboratories (Bar Harbor, ME). All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (publication 86-23, revised 1985). All experimental protocols were approved by the animal use committees of the University of Kansas Medical Center. With mice under ketamine anesthesia (a mixture of 75 mg/kg ketamine, 5.1 mg/kg xylazine, and 0.75 mg/kg acepromazine given intramuscularly), laparotomy was performed, and the blood supply to the median and left hepatic lobes was occluded with an atraumatic vascular clamp for 45 minutes as described elsewhere.34 Reperfusion was initiated by removal of the clamp. The body temperature was maintained in all animals with an infrared heating lamp. After reperfusion had been initiated, 4 mL/kg physiological saline was injected intraperitoneally, the abdominal incision was closed with 4-0 silk and wound clips, and the animals were allowed to recover. The sham operation included all procedures except for clamping of the blood supply. Groups of animals were sacrificed at the end of ischemia or after 1, 3, 6, 12, or 24 hours of reperfusion. Blood was drawn from the vena cava into a heparinized syringe. The liver was rapidly removed and rinsed in saline, and sections were placed in a phosphate-buffered 10% (vol/ vol) formalin solution and embedded in paraffin for histological evaluations or snap-frozen in liquid nitrogen. In addition to the time-course experiments, groups of animals were treated with the pancaspase inhibitor Z-VD-fmk (10 mg/kg intraperitoneally) 30 minutes before ischemia and at the time of reperfusion or with a vehicle (50 mM trishydroxymethylaminomethane buffer, pH 7.0; 10 mL/kg).35 Furthermore, as a
1374 YANG ET AL.
positive control for apoptosis, some animals were treated intraperitoneally with 700 mg/kg Dgalactosamine (Sigma, St. Louis, MO) and 100 mg/kg Salmonella abortus equi endotoxin [Sigma; galactosamine/endotoxin (G/E)] dissolved in sterile phosphatebuffered saline (pH 7.0).35
Alanine Aminotransferase (ALT) Activities and Caspase Activation Plasma ALT activities were determined with the Pointe Scientific ALT kit (Pointe Scientific, Canton, MI) according to the manufacturer’s instructions. Caspase-3 activity in the liver or plasma was determined through the measurement of the z-VAD-FMK– inhibitable cleavage of the fluorescent caspase-3 substrate Ac-DEVD-AMC (Sigma-Aldrich St. Louis, MO), as described elsewhere.32 In addition, caspase-3 processing in liver and plasma was evaluated by western blotting with a caspase-3 antibody (Cell Signaling, Danvers, MA) as described in detail elsewhere.33
Measurement of Mechanism-Based Biomarkers of Liver Injury in Plasma FK18 and CK18 were identified and quantified by mass spectrometry as previously described in detail.28-30 miR-122 was measured by quantitative reverse-transcription polymerase chain reaction,31 let7d provided biological standardization, and lin-4 served as an internal standard for the assay. Total HMGB1 was quantified by immunoassay and mass spectrometry.28-30 The absolute quantification of AcHMGB1 was determined by mass spectrometry as previously described as a biomarker of immune cell activation.28 The investigators measuring the plasma biomarkers were blinded to the treatment groups of the animals.
Histology Sections of formalin-fixed paraffin-embedded liver samples were stained with hematoxylin and eosin (H&E) for the evaluation of liver cell death by a boardcertified pathologist (A.F.) as described in detail elsewhere.21,36 Additional liver sections were stained with the TUNEL in situ cell death assay (Roche) for visualization of DNA strand breaks as previously described.36 The quantitation of TUNEL-positive cells was performed by manual counting of the number of TUNEL-positive cells per 10 randomly selected highpower fields (HPFs) per section. The percentage of cells showing TUNEL-positive nuclei and cytoplasm was similarly quantified. All TUNEL-positive cells were included in the results, but hepatocytes and nonparenchymal cells were individually identified.
Statistical Analysis Data are given as means and standard errors. Statistical analysis was performed with SigmaPlot 11.0
LIVER TRANSPLANTATION, November 2014
(Systat Software, Chicago, IL). Data were assessed with a 1-way ANOVA followed by the StudentNewman-Keuls post hoc test for comparisons between means or Dunnett’s post hoc test for comparisons with a control. For data not normally distributed, we used the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. P < 0.05 was considered significant.
RESULTS C57Bl/6 mice were subjected to 45 minutes of hepatic ischemia followed by up to 24 hours of reperfusion. Progressive liver injury, as indicated by the increase in plasma ALT activities, was observed during reperfusion between 1 and 24 hours (Fig. 1A). No change in plasma ALT activities was observed in sham-operated animals. H&E-stained tissue sections showed tissue damage consistent with extensive necrosis (Fig. 1B). The area of necrosis was approximately 10% at 6 hours and progressed to >50% at 24 hours of reperfusion (Fig. 1A). No morphological evidence of apoptosis (apoptotic bodies) was detectable at any time during reperfusion. Staining sections with the TUNEL assay, which detects DNA strand breaks, showed very few individual positive cells (95% of all TUNEL-positive cells observed were hepatocytes. The progression of necrosis based on TUNEL staining was further illustrated after quantitation of both total TUNEL-positive cells (Fig. 2E) and the percentage of those exhibiting nuclear and cytoplasmic TUNEL staining (Fig. 2F). Although the number of total TUNEL-positive cells increased steadily during reperfusion, the staining of the cytosol developed between 3 and 6 hours and increased steeply afterward (Fig. 2F). These data are consistent with initial nuclear DNA fragmentation, which progressed to karyorrhexis and karyolysis. This TUNEL-staining pattern during IRP was fundamentally different from a positive control for apoptosis. In G/E-treated animals, numerous individual TUNEL-positive cells or apoptotic bodies were readily observed in which nuclei were stained but not the cytosol.33,36,37 It is well established that caspase-3 is an executioner caspase that is critical for apoptotic cell death
LIVER TRANSPLANTATION, Vol. 20, No. 11, 2014
YANG ET AL. 1375
Figure 1. Liver injury caused by IRP. Mice either were untreated controls (C) or were subjected to a sham operation (S) or 45 minutes of liver ischemia followed by reperfusion for various times (0, 1, 3, 6, 12, or 24 hours). (A) Liver injury was assessed by plasma ALT activities or quantitation of the areas of necrosis. Data are expressed as means and standard errors (n 5 3-6 animals per time point). *P < 0.05 versus controls. (B) Representative H&E-stained sections are shown for ischemia (45/0) and various times of reperfusion (45/X). The original magnification was 350.
in all liver cells.38 The activation of caspase-3 can be measured with an enzyme activity assay and with documentation of the formation of an active (eg, 17-kD) fragment by western blotting. Compared with very low levels of caspase-3 activities in controls, G/E treatment induced an extensive increase in enzyme activity in the liver and in plasma (Fig. 3A,B). This was confirmed by the reduction of procaspase-3 and the appearance of the active 17-kD fragment in the liver (Fig. 3C). In striking contrast to these observations in a liver with extensive apoptosis, livers from postischemic animals did not show any relevant increase of caspase3 activity in the liver or plasma (Fig. 3A,B) or evidence of caspase-3 processing (Fig. 3C) at any time point between 1 and 24 hours of reperfusion. The fact that no caspase-3 activity and no active caspase-3 fragments were detectable in the postischemic liver and in plasma suggest that this is a primary necrotic process and not secondary necrosis.39 miR-122 has been shown to be a highly sensitive marker for necrotic cell death.31,40 Although there
was no relevant increase in sham-operated animals compared with untreated controls, miR-122 levels increased dramatically during IRP (Fig. 4A) following the enhanced plasma ALT activities during reperfusion (Fig. 1A). However, peak miR-122 levels at 12 to 24 hours of reperfusion were 36,000- to 43,500-fold greater than the baseline in comparison with ALT levels, which were elevated 70- to 90-fold at this time. FK18 is a filamentous protein that can be passively released by necrotic cells. In contrast, CK18 reflects apoptotic cell death.41 During IRP, a time-dependent, massive release of FK18 was observed, with peak levels at 12 to 24 hours (Fig. 4B). Compared with baseline levels in controls (218 6 42 pmol/mL), FK18 concentrations were 220- and 440-fold higher between 3 and 24 hours of reperfusion, respectively. In contrast, the CK18 levels did not change significantly except for a
