Acute ethanol causes hepatic mitochondrial depolarization in mice: role of ethanol metabolism.

BACKGROUND/AIMS: An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. METHODS: Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1-6 g/kg). RESULTS: Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼ 70% and ∼ 20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. CONCLUSIONS: Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde.

New method of stent-facilitated arterial reconstruction for orthotopic mouse liver transplantation.

BACKGROUND: Arterialized orthotopic liver transplantation (OLT) in the mouse mimics human liver transplantation physiologically and clinically. The present method of sutured anastomosis for reconstruction of the hepatic artery is complex and is associated with high incidence of complications and failure. This makes the endpoint assessment of using this complex model difficult because of the many variables of the technical aspect. METHODS: A total of 14 pairs of donors and recipients from syngeneic male mice were used for arterialized OLT. The grafts were stored in University of Wisconsin solution at 4°C for less than 4 h, and the recipients underwent OLT using a two-cuff technique. The arterial reconstruction was facilitated by the use of a single stent connecting the donor liver artery segment to the recipient common hepatic artery. RESULTS: All 14 recipients survived with the time for arterial reconstruction ranging from 4-10 min. Patency of the artery was confirmed by transecting the artery near the graft 2 and 14 d after transplantation. At day 2, five of the six arteries transected were patent and at day 14, seven of the remaining eight were patent for an overall patency rate of 85.7%. CONCLUSIONS: The stent-facilitated arterial reconstruction can be done quickly with a high patency rate. This model expands the translational research efforts to address marginal livers such as steatotic livers.

Icam-1 upregulation in ethanol-induced Fatty murine livers promotes injury and sinusoidal leukocyte adherence after transplantation.

Background. Transplantation of ethanol-induced steatotic livers causes increased graft injury. We hypothesized that upregulation of hepatic ICAM-1 after ethanol produces increased leukocyte adherence, resulting in increased generation of reactive oxygen species (ROS) and injury after liver transplantation (LT). Methods. C57BL/6 wildtype (WT) and ICAM-1 knockout (KO) mice were gavaged with ethanol (6 g/kg) or water. LT was then performed into WT recipients. Necrosis and apoptosis, 4-hydroxynonenal (4-HNE) immunostaining, and sinusoidal leukocyte movement by intravital microscopy were assessed. Results. Ethanol gavage of WT mice increased hepatic triglycerides 10-fold compared to water treatment (P < 0.05). ICAM-1 also increased, but ALT was normal. At 8 h after LT of WT grafts, ALT increased 2-fold more with ethanol than water treatment (P < 0.05). Compared to ethanol-treated WT grafts, ALT from ethanol-treated KO grafts was 78% less (P < 0.05). Apoptosis also decreased by 75% (P < 0.05), and 4-HNE staining after LT was also decreased in ethanol-treated KO grafts compared to WT. Intravital microscopy demonstrated a 2-fold decrease in leukocyte adhesion in KO grafts compared to WT grafts. Conclusions. Increased ICAM-1 expression in ethanol-treated fatty livers predisposes to leukocyte adherence after LT, which leads to a disturbed microcirculation, oxidative stress and graft injury.

C-Jun N-Terminal Kinase 2 Promotes Liver Injury via the Mitochondrial Permeability Transition after Hemorrhage and Resuscitation.

Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK) like c-Jun N-terminal kinase (JNK) 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R) was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT) and JNK2 deficient (KO) mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P < 0.05). Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123), indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT). By contrast, KO mice displayed less depolarization after H/R (23%, P < 0.05). In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R.

Minocycline decreases liver injury after hemorrhagic shock and resuscitation in mice.

Patients that survive hemorrhage and resuscitation (H/R) may develop a systemic inflammatory response syndrome (SIRS) that leads to dysfunction of vital organs (multiple organ dysfunction syndrome, MODS). SIRS and MODS may involve mitochondrial dysfunction. Under pentobarbital anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution containing minocycline, tetracycline (both 10 mg/kg body weight) or vehicle. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R with vehicle or tetracycline, ALT increased to 4538 U/L and 3999 U/L, respectively, which minocycline decreased to 1763 U/L (P < 0.01). Necrosis and TUNEL also decreased from 24.5% and 17.7 cells/field, respectively, after vehicle to 8.3% and 8.7 cells/field after minocycline. Tetracycline failed to decrease necrosis (23.3%) but decreased apoptosis to 9 cells/field (P < 0.05). Minocycline and tetracycline also decreased caspase-3 activity in liver homogenates. Minocycline but not tetracycline decreased lipid peroxidation after resuscitation by 70% (P < 0.05). Intravital microscopy showed that minocycline preserved mitochondrial polarization after H/R (P < 0.05). In conclusion, minocycline decreases liver injury and oxidative stress after H/R by preventing mitochondrial dysfunction.

Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers.

This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20 h after gavaging with ethanol (5 g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5 µM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.

Matrix metalloproteinases and descending aortic aneurysms: parity, disparity, and switch.

Central to the pathologic changes in developing aortic aneurysms are alterations in the abundance and activity of proteases, of which the most important for aneurysm production comprise the matrix metalloproteinase (MMP) family. In this review, literature demonstrating the role of MMPs in the development of aortic aneurysms is presented, with emphasis on the parity and disparity between the thoracic and abdominal aorta. Furthermore, the role of embryologic cellular origins and evidence of phenotypic switch will be addressed in terms of how this process alters MMP production during aneurysm development.

Mucinous cystic neoplasms of the pancreas: how much preoperative evaluation is needed?

Cystic lesions of the pancreas are identified with increasing frequency by modern imaging. The mucinous cystic neoplasm (MCN) is treated with resection for its malignant potential. How much preoperative evaluation is needed before undertaking operation is frequently a diagnostic dilemma. A retrospective review of 32 patients who underwent resection of a MCN between 1994 and 2007 was performed to define the preoperative evaluation and operative treatment of MCN patients. Thirty-two patients (30 women; mean age 49) had histology-proven MCN. Twenty-seven patients had symptomatic cysts (84%). Five had a history of gallstones and/or acute pancreatitis. All patients were worked up with CT and/or MRI. Endoscopic ultrasound was performed in 14 (44%) and endoscopic retrograde cholangiopancreatography in six (18%). Cytology was obtained in 13 (40%). Pathology revealed 22 benign MCNs (68%), five malignant MCNs (16%), and five MCNs with borderline pathology. Preoperative workup including CT or MRI imaging and cytology suggested MCN as the lesion in 15 patients (46%). CT features by itself predicted MCN in three patients (9%). Cytology revealed another six patients (19%) with possible MCN. In this series, preoperative workup did not identify three of five patients with MCN malignancy. A preoperative diagnosis cannot be made in most patients with MCN. Operative treatment can be based on clinical presentation and CT imaging because endoscopic ultrasound and fine needle aspiration for evaluation may be misleading. Middle-aged women with cystic lesions in the tail of the pancreas without prior gallstone or pancreatitis history most typically fit the profile of the MCN patient.

Inhibition of transforming growth factor-beta/Smad signaling improves regeneration of small-for-size rat liver grafts.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of cell proliferation. This study investigated whether overexpression of Smad7, which blocks TGF-beta-induced activation of Smad2/3, could prevent the suppression of regeneration of small-for-size liver grafts. Rats were intravenously given adenoviruses (2 x 10(10) pfu/rat) carrying the LacZ gene or the Smad7 gene (Ad-Smad7) 3 days prior to liver harvesting. Half-size livers were implanted into recipients of the same weight or twice the donor weight, and this resulted in half-size or quarter-size liver grafts. Cell proliferation, detected by 5-bromo-2'-deoxyuridine (BrdU) incorporation, increased to 23% in half-size grafts at 38 hours after implantation but was only 4% in quarter-size grafts. Graft weight did not increase after 38 hours in full-size and quarter-size grafts but increased 28% in half-size grafts. Ad-Smad7 restored BrdU labeling to 32%, and the graft weight increased to 43% in quarter-size grafts. Serum total bilirubin increased approximately 30-fold after the implantation of quarter-size grafts. Ad-Smad7 blunted hyperbilirubinemia by 80%. The basal hepatic TGF-beta(1) level was 7 ng/g of liver wet weight, and this increased to 30 ng/g at 1.5 hours after the transplantation of full-size grafts but decreased rapidly afterwards. After the transplantation of quarter-size grafts, however, TGF-beta(1) progressively increased to 159 ng/g in 38 hours. Nuclear phosphorylated Smad2/3 was barely detectable, and p21Cip1 expression was negligible in full-size grafts but increased markedly in quarter-size grafts. Ad-Smad7 blocked Smad2/3 activation and expression of p21Cip1. Together, these data show that TGF-beta is responsible, at least in part, for the defective liver regeneration in small-for-size grafts by activating the Smad signaling pathway.

Mitochondrial calcium and the permeability transition in cell death.

Dysregulation of Ca(2+) has long been implicated to be important in cell injury. A Ca(2+)-linked process important in necrosis and apoptosis (or necrapoptosis) is the mitochondrial permeability transition (MPT). In the MPT, large conductance permeability transition (PT) pores open that make the mitochondrial inner membrane abruptly permeable to solutes up to 1500 Da. The importance of Ca(2+) in MPT induction varies with circumstance. Ca(2+) overload is sufficient to induce the MPT. By contrast after ischemia-reperfusion to cardiac myocytes, Ca(2+) overload is the consequence of bioenergetic failure after the MPT rather than its cause. In other models, such as cytotoxicity from Reye-related agents and storage-reperfusion injury to liver grafts, Ca(2+) appears to be permissive to MPT onset. Lastly in oxidative stress, increased mitochondrial Ca(2+) and ROS generation act synergistically to produce the MPT and cell death. Thus, the exact role of Ca(2+) for inducing the MPT and cell death depends on the particular biologic setting.

NIM811 (N-methyl-4-isoleucine cyclosporine), a mitochondrial permeability transition inhibitor, attenuates cholestatic liver injury but not fibrosis in mice.

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were approximately 1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor alpha, transforming growth factor-beta1, procollagen alpha1(I) mRNA, alpha-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion.

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.

BACKGROUND: Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. METHODS: IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. RESULTS: After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2'-deoxyuridine labeling and increases of graft weight were only approximately 3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. CONCLUSION: Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.