Gastroenterology
Volume 132, Issue 1 , Pages 372-383, January 2007

Mild Hypothermia Attenuates Liver Injury and Improves Survival in Mice With Acetaminophen Toxicity

  • Javier Vaquero

      Affiliations

    • Neuroscience Research Unit, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • ,
  • Mireille Bélanger

      Affiliations

    • Neuroscience Research Unit, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • ,
  • Laura James

      Affiliations

    • Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas
    • ,
  • Raquel Herrero

      Affiliations

    • Neuroscience Research Unit, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • ,
  • Paul Desjardins

      Affiliations

    • Neuroscience Research Unit, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • ,
  • Jean Côté

      Affiliations

    • Department of Pathology, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • ,
  • Andres T. Blei

      Affiliations

    • Northwestern University Feinberg School of Medicine, Chicago, Illinois
    • ,
  • Roger F. Butterworth

      Affiliations

    • Neuroscience Research Unit, Hôpital Saint-Luc (CHUM), Université de Montréal, Montréal, Quebec, Canada
    • Corresponding Author InformationAddress requests for reprints to: Roger F. Butterworth, PhD, DSc, Neuroscience Research Unit, Hôpital Saint-Luc (C.H.U.M.), 1058 St. Denis Street, Montréal, Quebec, H2X 3J4, Canada. fax: (514) 412-7314.

Received 7 July 2006; accepted 11 October 2006. published online 23 November 2006.

Article Outline

Background & Aims: Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice. Methods: Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5–37.5°C) by external warming or were allowed to develop mild hypothermia (32.0–35.0°C) after 2 hours from APAP administration. Results: Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8–12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis. Conclusions: Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.

Abbreviations used in this paper: ALF, acute liver failure, APAP, acetaminophen, BrdU, 5-bromo-2-deoxyuridine, HT, hypothermic, NAPQI, N-acetyl-p-benzoquinone imine, NT, normothermic, PARP, poly(ADP-ribose) polymerase, Sal, saline, TdT, terminal deoxynucleotidyl transferase, TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling

 

Acetaminophen (N-acetyl-p-aminophenol, APAP) hepatotoxicity, intentional and unintentional, has become a major cause of acute liver failure (ALF) and a serious public-health concern in many countries.1, 2 Knowledge of the mechanisms of APAP hepatotoxicity derives to a large extent from studies performed in mice treated with APAP. Under normal conditions, APAP is detoxified in the liver by glucuronidation and sulfation.3 A small fraction of APAP undergoes metabolism through cytochrome P-450, forming highly reactive compounds such as N-acetyl-p-benzoquinone imine (NAPQI) that are readily detoxified by conjugation with glutathione. Administration of high doses of APAP results in saturation of detoxification pathways, depletion of hepatic glutathione, and excessive production of NAPQI, which freely binds to cellular molecules in mice4 and human beings.5 In mice, covalent binding of APAP metabolites to liver proteins starts within 15 minutes of the overdose, concurrently with the beginning of glutathione depletion, and peaks within 1–2 hours.4, 6 This is followed by other pathogenetic events such as disturbance of intracellular calcium homeostasis,7 oxidative and nitrosative stress,8, 9, 10, 11 massive hepatic congestion,12 and activation of the innate immunity, including natural-killer and natural-killer cells with T-cell receptors,13 macrophages, and neutrophils.14, 15, 16 Oncotic necrosis is the main mode of hepatocyte cell death,17 but a variable role for apoptosis also has been suggested.13, 18 Despite a large amount of experimental literature, many aspects of APAP hepatotoxicity remain controversial,19 partly because of the variability of the APAP mouse model of liver injury.20 The body temperature of APAP-treated mice is known to fall spontaneously as a result of several factors that include the pharmacological action of APAP,21 the overnight fast (frequently used in the model),22 the development of ALF and hypovolemia,12 and the release of cytokines.23 Ambient temperature and the mode of drug administration also influence thermoregulatory responses in the mouse.24 Even though temperature is a major determinant of all (bio)-chemical reactions, the assessment of body temperature in APAP-treated mice has generally been omitted.25 Because the development of hypothermia may critically affect mechanisms studied in this model, the present study was designed to investigate the effect of body temperature on the progression of toxic liver injury in mice treated with APAP. These studies were also prompted by the evolving use of mild hypothermia for controlling intracranial hypertension in patients with ALF,26 together with the lack of experimental data on the effects of hypothermia on the injured liver itself, particularly on its capacity to regenerate.27

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Materials and Methods 

Products and Reagents 

The mouse monoclonal antibody against 5-bromo-2-deoxyuridine (BrdU), terminal deoxynucleotidyl transferase (TdT), proteinase K, anti–digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl-phosphate, and digoxigenin-1-2′-deoxy-uridine-5′-triphosphate were from Roche Diagnostics (Indianapolis, IN). The antibody against poly(ADP-ribose) polymerase (PARP) and the biotinylated goat anti-mouse immunoglobulin G were from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals and reagents were obtained from Sigma (St. Louis, MO).

Animals, Treatments, and Temperature Monitoring 

Male 8- to 10-week-old C57BL/6 mice (Charles River, St. Constant, Quebec, Canada) were acclimatized for 1 week in individual cages with free access to chow and water and 12-hour light–dark cycles. After an overnight fast, mice were administered APAP (300 mg/kg intraperitoneally) or saline at 8:00 am. The APAP was freshly prepared, dissolved in warmed saline (2% solution), filtered (0.2 μm), and maintained at 37°C in a water bath. After APAP injection, the cages were placed within heating pad–lamp assemblies that were periodically adjusted to maintain the body temperature of the mice within the desired range. In some 24-hour and 48-hour experiments, the cages were placed in an infant incubator (Model C-86; Air-Shields Inc, Hatboro, PA) to facilitate body temperature maintenance. Body temperature was measured with a rectal thermometer (introduced ∼2 cm) for mice (CMA150/Microdialysis, Solna, Sweden). All mice were maintained at normothermia (35.5–37.5°C) during the first 2 hours after APAP injection. After 2 hours, one group was maintained at normothermia (NT group), and the other group was allowed to develop mild hypothermia by decreasing active warming (mild-HT group, 32.0–35.0°C). Access to regular diet was resumed (after 12 hours) only in the 24-hour and 48-hour groups. Mice were decapitated at indicated time points (from 1 hour to 48 hours). Neck blood was collected in heparinized tubes, processed by centrifuge, and used to measure alanine aminotransferase (ALT) activity with an automated analyzer. The liver was rapidly dissected and rinsed in cold saline. Two portions of the left and medial lobes were fixed in 10% formalin-buffered solution for histological processing. The remaining liver was flash-frozen and stored at –70°C. To assess the integrity of the bowel walls, saline was injected through the anus of all mice with a PE-50 catheter. Bowel perforations detected by saline leakage and macroscopic examination were only observed in mice that were followed for more than 12 hours, in whom the perforation rate was 28%. Only animals with intact bowels were analyzed. Experiments were performed according to the Guidelines of Canadian Council of Animal Care and were approved by the Animal Research Committee at Saint-Luc Hospital (C.H.U.M.).

Histological Assessment of Liver Necrosis and Glycogen 

The extent of hepatocyte necrosis was evaluated in paraffin-embedded liver sections stained with hematoxylin-phloxine-saffron. Digital images of 6 low-power fields were obtained from each slide in a random and blinded fashion. The area of necrosis was delineated and quantified by using the National Institutes of Health Image J software (NIH, Bethesda, MD), and the average area of necrosis from each mouse was used for subsequent analysis. Histological assessment of hepatic glycogen was performed using the periodic acid–Schiff stain according to standard procedures.

Determination of Hepatic Hemoglobin Content 

The hemoglobin content of liver tissue was measured spectrophotometrically by using Drabkin’s reagent, as described elsewhere.28 Briefly, liver tissue (∼50 mg) was homogenized in distilled water (∼300 μL) containing protease inhibitors. Homogenates were sonicated (30 seconds) and processed by centrifuge (10,000g for 30 minutes at 4°C). To avoid the formation of turbidity and interference of spectrophotometric readings, diethyl-ether (1 mL) was added to the supernatant and processed by centrifuge (10,000×g for 10 minutes at 4°C). After discarding the organic phase, 53 μL of sample or standard were mixed with 1 mL of Drabkin’s reagent and allowed to react for 1 hour before measuring the absorbance of cyanmethemoglobin at 540 nm using a spectrophotometer. The hemoglobin content of liver tissue (milligrams of hemoglobin per gram of wet tissue) was calculated using standards prepared with hemoglobin from bovine blood.

Determination of APAP-Protein Adducts in Liver Tissue 

Approximately 100 mg of liver tissue was homogenized on ice in sterile phosphate-buffered saline (PBS). Homogenates were then processed by centrifuge (10,000×g for 10 minutes at 4°C). Supernatants were removed for analysis of APAP-protein adducts by using high-performance liquid chromatography with electrochemical detection, as described elsewhere.5, 6 Adducts were normalized to the protein content of the sample, as determined by the Bradford method.5

Determination of Total Hepatic Glutathione Content 

To measure total hepatic glutathione content (GSH + GSSG), liver tissue (∼40 mg) was homogenized in 5 volumes of PBS buffer (pH 6.2) with 1 mmol/L ethylenediaminetetraacetic acid and was processed by centrifuge (10,000×g for 15 minutes at 4°C). Supernatants were mixed with an equal volume of 10% metaphosphoric acid and were processed by centrifuge (10,000×g for 5 minutes at 4°C). Resulting supernatants were mixed with 4 mol/L triethanolamine (50 μL/mL sample) to increase the pH before assaying. Glutathione concentration was measured spectrophotometrically by using 5,5′-dithio-bis(2-nitrobenzoic acid), as described by Tietze.29

Determination of Hepatic Glycogen Content 

Hepatic glycogen content was determined as described by Huang and Veech.30 Briefly, liver tissue (∼150 mg) was homogenized in 2 mL of 20% KOH and 65% ethanol, incubated at 85°C for 15 minutes, and processed by centrifuge (800g for 20 minutes). The pellets were washed twice with 1 mL of chloroform–methanol (1:1). After evaporating residual organic solvents, glycogen was hydrolyzed by incubating the pellet with 350 μL of 1 N HCl at 100°C for 1 hour. The samples were then neutralized with 1 N NaOH and were processed by centrifuge (5 minutes at 800g). Glucosyl units in the glycogen hydrolysates were measured spectrophotometrically by using the hexokinase method as described by Bergmeyer.31 The reaction mixture contained the following final concentrations (final volume, 2.02 mL): 100 mmol/L Tris-HCl buffer (pH 7.8), 2 mmol/L magnesium acetate, 5 mmol/L nicotinamide adenine dinucleotide+, 0.5 mmol/L adenosine triphosphate, 2 U/mL of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 0.7 U/mL of hexokinase (EC 2.7.1.1), and 20 μL of sample or standard. Standards were prepared by dissolving known amounts of d-glucose in distilled water. The glycogen content (μmol of glucosyl units per gram of wet tissue) was calculated from the change in absorbance at 340 nm.

Western Blot Analysis 

Liver tissue was homogenized in buffer (50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L sodium chloride; 1 mmol/L ethylenediaminetetraacetic acid; 1% Triton X-100; 0.5% sodium deoxycholate; and 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail, and processed by centrifuge (12,000g for 1 hour at 4°C). Protein content was measured according to Lowry et al.32 Whole protein extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred overnight to polyvinylidene difluoride membranes. Membranes were blocked for 1 hour in Tris-buffered saline containing 5% dry milk and 0.1% Tween 20 and were probed for 1 hour with rabbit polyclonal antibodies against PARP (1:1000) or β-actin (1:150,000). Blots then were exposed to peroxidase-conjugated secondary antibodies (Promega, Madison, WI), and revealed by an enhanced chemiluminescence detection system (Amersham Bioscience, Arlington Heights, IL).

Assay by TUNEL 

Paraffin-embedded liver sections were placed in an oven at 60°C for 30 minutes, dewaxed in xylene, and rehydrated in decreasing ethanol solutions. An incubation step in 4% diethyl pyrocarbonate ethanolic solution for 30 minutes at 4°C was included before rehydration to reduce false-positive staining.33 Sections were exposed to proteinase K (20 μg/mL) for 10 minutes, washed extensively in TN buffer (30 mmol/L Tris-HCl, 300 mmol/L NaCl, pH 7.4) and distilled water, and incubated for 1 hour at 37°C in buffer (30 mmol/L Tris-HCl, 1 mmol/L CoCl2, 0.25 mg/mL BSA, 200 mmol/L sodium cacodylate) containing 10 nmol/mL digoxigenin-1-2′-deoxy-uridine-5′-triphosphate and 200 U/mL TdT. After extensive washing, the sections were incubated for 30 minutes in AP buffer (50 mmol/L Tris-HCl, 150 mM NaCl, pH 7.4) containing anti–digoxigenin-alkaline phosphatase conjugate (3 μL/mL). Labelled nick ends of DNA strands were visualized by incubation in NB buffer (100 mmol/L Tris-HCl, 100 mmol/L NaCl, 50 mmol/L MgCl2, pH 9.4) containing 3.4 μL/mL nitroblue tetrazolium chloride, 2.6 μL/mL 5-bromo-4-chloro-3-indolyl-phosphate, and 1 mmol/L levamisole. Incubation with buffer lacking TdT and liver sections from mice treated with Fas-agonistic antibody (BD Biosciences, Mississauga, Ontario) were included as negative and positive controls, respectively. Digital images of 5 low-power fields from each liver were obtained in a random and blinded fashion, and the number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)–positive hepatocytes were counted. The average number of TUNEL-positive hepatocytes in each animal was used for subsequent analysis.

BrdU Immunohistochemistry 

To evaluate liver regeneration, mice from the 24-hour and 48-hour groups were administered BrdU (50 mg/kg intraperitoneally, 0.4% solution in sterile PBS) 2 hours before they were killed. Paraffin-embedded liver sections were dewaxed by heating at 60°C and were rehydrated. The slides were incubated in 0.1 N HCl with pepsin (EC 3.4.23.1; 0.05 mg/mL) for 4 minutes at 37°C. After rinsing, they were incubated at 37°C in 2 N HCl for 20 minutes, and twice in 0.1 mol/L borate buffer (pH 8.5) for 5 minutes. Endogenous peroxidase activity was quenched by using 0.3% H2O2 in PBS. Nonspecific binding sites were blocked with PBS, 10% goat serum, and 0.5% Triton X-100 for 1 hour. The slides were incubated with an anti-BrdU mouse monoclonal antibody (1:200) for 1 hour in PBS, 2% goat serum. After washing, sections were exposed for 1 hour to biotinylated goat anti-mouse immunoglobulin G antibody (1:200) in PBS, 1.5% goat serum, followed by incubations with Vectastain ABC reagent (Vector Laboratories, Burlingame, CA), and 3,3′-diaminobenzidine containing urea-H2O2. Slides were finally counterstained with Mayer’s hematoxylin. Positive controls were performed using intestine from BrdU-injected mice, and negative controls included tissues from non–BrdU-injected animals and omission of primary antibody in BrdU-injected mice. Digital images of 5 low-power fields (∼1500 hepatocytes per field) from each liver were obtained in a random and blinded fashion, and the number of BrdU-labeled hepatocyte nuclei were counted. The average number of BrdU-positive hepatocytes in each animal was used for subsequent analysis.

Statistical Analysis 

Values are expressed as mean ± SEM. Unpaired t-test or 1-way ANOVA, followed by the Newman Keuls test, were used to assess differences between groups when only 1 time point was evaluated. Two-way ANOVA followed by Bonferroni posttest was used when the evaluation included two time points. The natural logarithm of variables was used for statistical purposes. A P value of <.05 was considered significant. Data were analyzed by using Prism 4.0 software (Prism 4.0, San Diego, CA).

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Results 

Body temperature was maintained at 37°C in all animals for the first 2 hours (Figure 1A). Maintenance of normothermia in APAP-treated mice required immediate active warming and intensive monitoring to avoid unwanted fluctuations of body temperature. Hypothermia spontaneously developed in mice in which active warming was reduced at 2 hours (mild-HT group), but most animals still required some degree of heating to avoid decreases of body temperature below 32°C. The group maintained at normothermia (NT group) required active warming during the entire course of experiments. Despite intensive monitoring, 13% of the mice followed for more than 8 hours presented uncontrollable episodes of hyperthermia or deep hypothermia and were excluded from the main study. This issue, together with the detection of bowel perforations in animals followed for long periods of time (see Methods), encourages the investigation of better methods for controlling body temperature in mice.

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  • Figure 1. 

    Effect of body temperature on survival in mice treated with acetaminophen (APAP; 300 mg/kg intraperitoneally). (A) Rectal temperature in control mice (Saline), APAP-treated mice maintained at normothermia (NT), and APAP-treated mice allowed to become mildly hypothermic (mild HT). The body temperature of both NT and mild-HT mice was maintained within normal values in the first 2 hours after APAP challenge. The mean ± SEM of each group is plotted at each time point. (B) Survival rates of NT and mild-HT mice (n = 9–10 per group) in the first 24 hours after APAP challenge. Log-rank test was used for statistical analysis.

Effect of Body Temperature on Survival and Hepatocellular Damage in Mice With APAP-Induced Liver Injury 

After APAP treatment, the 24-hour survival was drastically reduced in mice maintained at normothermia compared with those allowed to develop mild hypothermia (40% vs 100% at 24 hours; log-rank test, P < .01; Figure 1B).

Manipulation of body temperature significantly influenced the degree of APAP-induced hepatocellular damage. Macroscopically, the most conspicuous feature was the extreme hepatic enlargement and congestion in NT compared with mild-HT mice. On light microscopy, APAP-induced hepatocellular damage in NT animals consisted of centrolobular necrosis with varying degrees of confluent necrosis and hemorrhagic congestion. These features were more severe in NT mice and resulted in larger areas of hepatocyte necrosis compared with mild-HT mice (54.0% ± 6.1% vs 32.3% ± 4.1%, P < .01; Figure 2A, C, and D). The hepatoprotective effect of hypothermia was confirmed by the lower levels of plasma ALT in the mild-HT group at 12 hours and 24 hours (at 24 hours: saline [Sal]: 52 ± 8 U/dL vs NT: 10,394 ± 1908 U/dL vs mild HT: 3815 ± 1311 U/dL; P < .001 for Sal vs the rest and P < .01 for NT vs mild HT; Figure 2B).

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  • Figure 2. 

    Effect of body temperature on the progression of liver injury in mice after an acetaminophen (APAP) challenge (300 mg/kg intraperitoneally). (A) Percentage area of hepatocyte necrosis in APAP-treated normothermic (NT; open circles) and mildly hypothermic (mild-HT; filled circles) mice. Each point represents the mean percentage area of hepatocyte necrosis in a single mouse, calculated from 6 low-power fields. Horizontal lines represent the mean percentage area of necrosis in each group. (B) Plasma ALT values in saline-treated mice (Sal) and APAP-treated NT (hatched bars) and Mild-HT (filled bars) mice. The number of mice analyzed for each time point is indicated in the abscissa axis. Bars represent mean ± SEM. *P < .001 vs Sal; #P < .05 NT vs mild HT; ##P < .01 NT vs mild HT. (C, D) Representative histological liver sections from mice followed for 24 hours showing hemorrhagic congestion and extensive centrilobular necrosis in NT mice (C) and smaller areas of necrosis in mild-HT mice (D). Hematoxylin-phloxine-saffron. Calibration marks in the lower right are 100 μm.

Effect of Body Temperature on the Formation of APAP-Protein Adducts and Hepatic Glutathione 

Metabolism to the highly reactive metabolite NAPQI, leading to depletion of hepatic glutathione and binding to cellular macromolecules, is a key step in APAP hepatotoxicity. Therefore, we assessed whether these mechanisms were influenced by body temperature. As expected, the hepatic concentration of APAP-protein adducts significantly increased in the first 2 hours after the administration of APAP (Figure 3A). Mild hypothermia did not significantly reduce the hepatic concentration of APAP-protein adducts; they remained elevated to a similar extent in both NT and mild-HT mice at 4 hours (0.763 ± 0.065 nmol APAP-Cys per milligram of protein vs 0.637 ± 0.059 nmol APAP-Cys per milligram of protein, NS). Consistent with previous studies,4 administration of APAP also resulted in a drastic depletion of hepatic glutathione stores in the first 2 hours (Figure 3B). The subsequent rebound in hepatic glutathione at 4 hours was lower in mild-HT mice (Figure 3B). To further assess potential effects of hypothermia on APAP bioactivation, we performed additional experiments in which hypothermia was allowed to develop immediately after the administration of APAP. In these latter experiments, hypothermic mice presented similar hepatic concentrations of APAP-protein adducts at 1 hour and 2 hours compared with mice maintained at normothermia (Figure 3C); concentrations of hepatic glutathione were dramatically depleted by APAP treatment in both groups (Figure 3D).

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  • Figure 3. 

    Effect of body temperature on the formation of acetaminophen (APAP)-protein adducts and on the depletion of hepatic glutathione stores in mice after an APAP challenge (300 mg/kg intraperitoneally). (A, B) Concentration of (A) APAP-protein adducts and (B) total glutathione (reduced GSH plus oxidized GSSG) in liver tissue of saline-treated mice (Sal), APAP-treated mice maintained at normothermia (NT; hatched bars), and APAP-treated mice allowed to develop mild hypothermia after the first 2 hours of APAP injection (mild HT, filled bars). (C, D) Concentration of APAP-protein adducts (C) and total glutathione (D) in liver tissue when mild hypothermia was allowed to develop immediately after the APAP injection (filled bars). The number of mice analyzed for each time point is indicated in the abscissa axes. Bars represent mean ± SEM. *P < .05 vs Sal; **P < .001 vs Sal; #P < .05 NT vs mild HT.

Effect of Body Temperature on Hepatic Hemoglobin Content 

The degree of hemorrhagic congestion was assessed by measuring the concentration of hemoglobin in liver tissue. Maintenance of normal body temperature after APAP challenge resulted in severe hepatic hemorrhagic congestion, which was significantly reduced in mild-HT mice (milligrams of hemoglobin per gram of tissue at 12 hours: Sal, 12.2 ± 1.2 vs NT, 54.4 ± 9.5 vs mild HT, 18.8 ± 4.1; P < .01 for Sal vs NT and P < .001 for NT vs mild HT; Figure 4A). This was an early effect of hypothermia.

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  • Figure 4. 

    Effect of body temperature on hepatic hemorrhagic congestion and hepatic glycogen content in mice after an acetaminophen (APAP) challenge (300 mg/kg intraperitoneally). (A, B) Hepatic hemoglobin (Hb; A) and glycogen (B) content in liver tissue of saline-treated mice (Sal), and APAP-treated normothermic (NT; hatched bars) and hypothermic (mild HT; filled bars) mice. The number of mice analyzed for each time point is indicated in the abscissa axes. Bars represent mean ± SEM. *P < .01 NT vs Sal; **P < .001 vs Sal; #P < .001 NT vs mild HT.

Effect of Body Temperature on Hepatic Glycogen Content 

Hepatic glycogen content was evaluated both quantitatively by using an enzymatic assay (Figure 4B) as well as histologically in periodic acid–Schiff-stained liver sections (Figure 5A–D). Administration of APAP resulted in significant depletion of hepatic glycogen beginning at 2 hours. In NT mice, hepatic glycogen remained low at 4 hours and increased at 8 hours and 12 hours without reaching normal values. In contrast, recovery of hepatic glycogen stores in mild-HT mice started at 4 hours and overshot basal values at the 8-hour and 12-hour time points.

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  • Figure 5. 

    Representative periodic acid–Schiff-stained liver sections for histological assessment of hepatic glycogen stores in mice after an acetaminophen (APAP) challenge (300 mg/kg intraperitoneally). In fasted mice treated with saline, glycogen stores were observed predominantly around central veins (A). In mice treated with APAP that were maintained at normothermia (NT), periodic acid–Schiff staining was strongly reduced at 2 hours (B) and 4 hours (not shown) and subsequently was increased at 8 hours around portal areas (C). In APAP-treated mice that were allowed to develop mild hypothermia (mild HT), glycogen started to increase around portal areas at 4 hours (not shown) and was strongly increased over basal levels at 8 hours (D). Calibration marks in the lower right are 100 μm.

Effect of Body Temperature on Hepatocyte Cell Death by Apoptosis 

Oncotic necrosis is the primary mode of cell death in APAP hepatotoxicity,17 but a variable relevance and activity of apoptotic pathways have been reported.13, 34, 35, 36 It is important to note that temperature has been shown to affect Fas-mediated hepatocyte apoptosis in vitro.37 In the present study, cleavage of PARP, a marker of caspase activity, was detected by Western blot only in liver extracts from NT mice at 8 hours and, particularly, at 12 hours after APAP challenge (Figure 6A). In contrast, no cleavage of PARP was detected at any time point in APAP-treated mice that were maintained at mild hypothermia. The presence of apoptotic cell death was also investigated by using the TUNEL technique. In mice that were administered saline, the presence of TUNEL-positive hepatocytes was negligible (Figure 6B). Administration of APAP was associated with an increase in the number of TUNEL-positive hepatocytes at 12 hours in both NT and mild-HT mice; such increases, however, were significantly attenuated in the later group (Figure 6B). In both groups, TUNEL staining included hepatocytes with isolated staining of the nucleus as well as hepatocytes with staining of both the nucleus and the cytosol (Figure 6C), consistent with findings in a previous study.10

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  • Figure 6. 

    Evaluation of apoptosis in mice after an acetaminophen (APAP) challenge (300 mg/kg intraperitoneally). (A) Western blot analysis of poly(ADP-ribose) polymerase (PARP) expression at 8 hours and 12 hours in liver from saline-treated mice (Sal), and APAP-treated normothermic (NT) and hypothermic (mild HT) mice. Whole protein liver extracts (50 μg per lane) were resolved on 8% sodium dodecyl sulfate–polyacrylamide gels. Liver protein from a mouse treated with Fas-agonistic antibody was used as positive control. The 85-kDa band corresponding to the proteolytic cleavage of PARP was visible only in NT mice. No cleavage of PARP was observed at 4 hours in any group (not shown). Actin is shown as loading control. (B) Quantification of hepatocytes positive for terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in liver sections from saline-treated mice and from APAP-treated NT and mild-HT mice at 12 hours after APAP challenge. Graph shows the average number of TUNEL-positive hepatocytes per low-power field (LPF) in each group (n = 6–9 per group). Bars represent mean ± SEM. *P < .05 vs Sal and mild HT; #P < .05 vs Sal and NT. (C) High-power field of a representative TUNEL-stained liver section from an APAP-treated mouse. Notice the presence of hepatocytes with distinct nuclear staining and others with staining of both nucleus and cytosol. Calibration mark in the lower right is 20 μm.

Effect of Body Temperature on Hepatocyte Proliferation (DNA Synthesis) 

Because the extent of liver damage and, therefore, the stimulus for liver regeneration, was different in NT and mild-HT mice, a precise assessment of the effect of body temperature on liver regeneration was not possible. Consequently, we investigated whether hypothermia abrogated hepatocyte proliferation in APAP-treated mice by assessing the degree of DNA synthesis by using BrdU immunohistochemistry. At 24 hours, only occasional hepatocyte nuclei labeled for BrdU were seen in both the NT and mild-HT groups (Figure 7A). At 48 hours, the number of labeled nuclei was significantly increased in both groups, although to a lesser extent in mild-HT mice (Figure 7A, C, and D). The extent of hepatocyte DNA synthesis, however, depended mainly on the extent of damage, because it was significantly correlated with the area of hepatocyte necrosis for each mouse, independent of body temperature (Figure 7B).

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  • Figure 7. 

    Hepatocyte DNA synthesis evaluated by BrdU immunohistochemistry in mice after an acetaminophen (APAP) challenge (300 mg/kg intraperitoneally). (A) Quantification of hepatocyte DNA synthesis in liver sections from saline-treated mice and APAP-treated mice maintained at normothermia (NT; hatched bars) or allowed to develop mild hypothermia (mild HT; filled bars). Graph shows the average number of BrdU positively stained hepatocyte nuclei per low-power field (LPF) in each group (n = 5–9 per group). Bars represent mean ± SEM. *P < .01 vs Sal; **P < .001 vs Sal; #P < .001 NT vs mild HT. (B) Relationship between body temperature, extent of liver injury, and liver regeneration in NT (open circles) and mild HT (filled circles) mice surviving 48 hours after the APAP challenge. Data points represent the percentage area of hepatocyte necrosis in individual mice (assessed in hematoxylin-phloxine-saffron stained liver sections), plotted against the average number of BrdU–labeled hepatocyte nuclei per low-power field of BrdU immunohistochemistry liver sections. The dotted line represents the linear regression line. (C, D) Representative photomicrographs of BrdU immunohistochemistry in an NT and a mild-HT mouse killed 48 hours after the injection of APAP. Arrows indicate BrdU–labeled hepatocyte nuclei. Calibration mark in the lower right is 100 μm.

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Discussion 

Preservation of basic animal physiology is important to adequately interpret in vivo models of disease. In particular, body temperature is a major physiological variable influencing systemic, cellular, and molecular processes in living organisms. Even though hypothermia is known to develop in the APAP-treated mouse,23, 38 the control of body temperature has generally been omitted in previous studies.25 In the present study, APAP-treated mice spontaneously developed a profound decrease of body temperature under standard experimental conditions (overnight fast, 300 mg/kg APAP dose, intraperitoneally administration, and ambient temperature ∼24°C). A mild decrease of body temperature afforded complete protection against APAP-induced mortality under normothermic conditions and a significant reduction of APAP-induced hepatocellular necrosis, assessed both histologically and by measurement of plasma ALT. Mild hypothermia also attenuated hepatic hemorrhagic congestion, improved the recovery of hepatic glycogen stores, and led to reduced expression of markers of apoptotic cell death. These results suggest that mild hypothermia is a useful tool to modulate APAP-induced liver damage and indicate that the control of body temperature is essential for an adequate interpretation of findings in the APAP-treated mouse, particularly when survival is an end point.

Earlier studies of carbon tetrachloride exposure of spinal cord–transected rats, which become poikilothermic, suggested that hypothermia protects the liver by reducing the bioactivation of compounds.39 In the present study, the hepatoprotective effect of mild hypothermia was not a result of decreased APAP bioactivation. All mice were maintained at normothermia for the first 2 hours after APAP challenge, the time frame within which most of its reactive metabolites are produced.4 Indeed, the highest concentrations of covalent adducts of APAP metabolites with liver proteins and the depletion of hepatic glutathione occurred in the first 2 hours, and they showed similar subsequent kinetics independent of body temperature. Even with an immediate development of hypothermia after APAP challenge, the changes of APAP-protein adducts and glutathione were similar to normothermic controls. The hepatoprotective effect of hypothermia appeared to be the result of interference with mechanisms downstream of the initial bioactivation of APAP.

Mild hypothermia led to an early attenuation of APAP-induced hepatic hemorrhagic congestion, as assessed by the hemoglobin content of liver tissue, and improved recovery of glycogen concentrations. Hepatic hemorrhagic congestion is an early pathological feature of APAP hepatotoxicity12, 40, 41 and sinusoidal endothelial cells are targets of APAP hepatotoxicity in both animals11, 42, 43 and human beings.44 In mice, as much as 50% of circulatory red blood cell volume is trapped in the liver early (∼4 hours) after an APAP challenge, resulting both in severe hypovolemia and secondary hypoxic liver damage as a result of the obstruction of the hepatic microcirculation.12, 40, 43 By reducing hepatic congestion, mild hypothermia likely improved systemic and hepatic hemodynamics and oxygenation, as well as ameliorated ischemic areas of the liver, given its established protective effects against ischemic liver injury.45, 46 Hepatic glycogen was not depleted until the 2nd hour after APAP challenge, and the development of mild hypothermia was associated with a faster and more complete return to normal values. Such effect may be pathogenetically relevant, because the availability of glucose for anaerobic glycolysis influences the mode of cell death (necrosis vs apoptosis) in APAP hepatotoxicity.47 Furthermore, glycogen is a source of uridine 5′-diphosphate–glucose for glucuronidation reactions, the major route of APAP detoxification. It is important to note that uridine 5′-diphosphate–glucose may be directed toward the synthesis of ascorbate in conditions of glutathione depletion,48 thereby enhancing the antioxidant defenses of hepatocytes against APAP-induced oxidative stress. Results of the present study emphasize that in addition to their relevance as markers of liver damage, hepatic hemorrhagic congestion and glycogen depletion are key mechanisms in the development of APAP hepatotoxicity.

Oncotic necrosis is the principal mode of hepatocyte cell death in APAP hepatotoxicity.17 However, the activity of apoptotic pathways, including the Fas–FasL system, is inconsistent in previous studies of APAP-treated animals.17, 34, 35, 36 Several groups have shown that inhibition of apoptosis after the administration of inhibitors36 or via genetic manipulation13, 18 attenuates APAP-induced liver damage in vivo. It is important to note that the attenuation of Fas-mediated hepatocyte apoptosis by hypothermia described in vitro37 suggests that body temperature may influence the mode of cell death. In the present study, the APAP-induced increase of the number of TUNEL-positive hepatocytes was significantly attenuated by mild hypothermia. Despite including an incubation step in the TUNEL procedure that is reported to reduce false positives,33 TUNEL staining occurred in both nucleus and cytosol of many hepatocytes, in contrast to the distinct nuclear staining that is induced by genuine apoptotic stimuli.10 The presence of other hepatocytes with isolated TUNEL staining of the nucleus as well as the detection only in normothermic mice of PARP cleavage (a less sensitive but more specific marker of caspase activity) strongly suggests that mild hypothermia attenuates the activity of apoptotic pathways in hepatocytes of APAP-treated mice. Uncontrolled body temperature, therefore, may have influenced the assessment of apoptosis in previous studies. The mechanisms by which hypothermia affects apoptotic events and whether controlling body temperature results in changes in the ultimate mode of cell death in APAP hepatotoxicity merits further investigation.

Concerns regarding the clinical use of hypothermia for treating intracranial hypertension in patients with ALF, particularly in nontransplant candidates, have been centered on the potential impairment of liver regeneration.27 In the present study, the data from BrdU immunohistochemistry suggest that mild hypothermia does not delay or impair hepatocyte DNA synthesis, because the latter was in accordance with the degree of hepatocyte necrosis in all animals, independent of body temperature. Although the evaluation of DNA synthesis could have been biased (more than 50% of normothermic mice could not be evaluated because of early mortality), this survivor bias has probably resulted in a global overestimation of DNA synthesis in the normothermic group. Such a situation would strengthen the notion that mild hypothermia does not impair liver regeneration. Further studies are required to completely resolve this issue.

In the context of the promising clinical experiences reported with the use of mild hypothermia for treating intracranial hypertension in human ALF,26 the present study provides novel insight into the potential effect of hypothermia on the liver itself. Mild hypothermia did not appear to have major detrimental effects on the injured liver, and its hepatoprotective effect against APAP hepatotoxicity adds to the beneficial effects described against the hepatotoxicity of carbon tetrachloride39 and azoxymethane,49 ischemia–reperfusion liver injury,45, 46 and hepatocyte apoptosis.37 Improved liver function may underlie the decrease of arterial ammonia observed in ALF mice49 and patients26 treated with mild hypothermia. Issues such as active versus passive cooling and the differences between animal and human physiology, however, should be taken into account when considering human ALF. Globally, the results of the present study emphasize the need to control body temperature in future studies in animal models of ALF and suggest that the benefits of mild hypothermia in ALF extend beyond those related to reduced cerebral complications.

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 Supported by grants from the Canadian Institutes of Health Research (CIHR; R. F. B.) and from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK-DK067999; L. J.). J. V. was supported by a fellowship from Fondo de Investigacion Sanitaria (Instituto de Salud Carlos III, Spain), and M. B., by a CIHR Doctoral Research Award.

PII: S0016-5085(06)02480-2

doi:10.1053/j.gastro.2006.11.025

Gastroenterology
Volume 132, Issue 1 , Pages 372-383, January 2007

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