Matrix metalloproteinase-9 as a therapeutic target for the progression of fulminant liver failure with hepatic encephalopathy: A pilot study in mice.

AIM: If progressive liver injury and subsequent hepatic encephalopathy can be prohibited in fulminant liver failure (FLF), it would be ideal for intensive care of FLF and provide an expanded opportunity for liver transplantation (LT). We hypothesized that matrix metalloproteinase (MMP)-9 plays an important role in FLF progression, and investigated MMP-9 behaviors in a murine FLF model, especially at the coma stage. METHODS: The murine FLF model with azoxymethane recapitulates FLF in humans. The detailed coma status was evaluated, on the assumption that LT is indicated at early, but not late, stage 3. To investigate whether MMP-9 deletion or reduction has beneficial effects, an MMP-9 inhibitor (GM6001) and transfection of tissue inhibitor of metalloproteinases (TIMP)-1 cDNA were used. Mice were divided into five groups: control; FLF; FLF with GM6001 pretreatment; FLF with TIMP-1 plasmid transfection 24 h before disease onset; and FLF with TIMP-1 plasmid transfection 48 h before disease onset. Neurological findings, including survival, were followed. Samples were obtained at early and late stage 3. Biochemical examinations and histopathological assessments were performed. The expression and function of MMP-9 and TIMP-1 were evaluated by western blotting and zymography. A brain permeability study was also performed. RESULTS: MMP-9 was strongly increased in FLF. The MMP-9 inhibitions worked well, and prolonged the survival, interval to stage 3 and duration of early stage 3. MMP-9 inhibition improved the liver and subsequent brain injuries at early stage 3, with no remarkable improvements at late stage 3. CONCLUSION: MMP-9 has therapeutic potential for FLF progression.

Liver graft pretreated in vivo or ex vivo by γ-aminobutyric acid receptor regulation.

BACKGROUND: γ-Aminobutyric acid exists throughout the body, and the brain γ-aminobutyric acid receptor (GABAR) regulation reduces oxidative stress (OS). Effects of GABAR regulation in the liver are unknown. Ischemia or reperfusion injury after orthotopic liver transplantation (OLT) or shear stress after split OLT (SOLT) with a small-for-size graft causes OS-induced graft damage. Here, the strategic potential of graft pretreatment in vivo and ex vivo by GABAR regulation was investigated. MATERIALS AND METHODS: Recipient rats were divided into seven groups according to the graft pretreatments and graft types: (1) laparotomy, (2) OLT, (3) GABAR regulation in vivo and OLT, (4) GABAR regulation ex vivo and OLT, (5) SOLT, (6) GABAR regulation in vivo and SOLT, and (7) GABAR regulation ex vivo and SOLT. Survival study, biochemical assays, histopathologic or immunohistologic assessments, and Western blotting were performed at 6 h after OLT or SOLT. RESULTS: Graft pretreatment in vivo prolonged survival after SOLT. Histopathologic and biochemical profiles verified that graft pretreatment in vivo reduced graft damage after OLT or SOLT. Immunohistologically, graft pretreatment in vivo prevented apoptotic inductions after OLT or SOLT. The 4-hydroxynonenal confirmed the OS after OLT or SOLT, and graft pretreatment in vivo improved the OS. Graft pretreatment in vivo decreased ataxia-telangiectasia-mutated kinase and H2AX after OLT or SOLT. Graft pretreatment in vivo increased phosphatidylinositol 3 kinase and Akt after SOLT. In contrast, GABAR regulation ex vivo did not work. CONCLUSIONS: Graft pretreatment in vivo, not ex vivo, prevented the ischemia or reperfusion injury-mediated OS after OLT or SOLT via the ataxia-telangiectasia-mutated kinase/H2AX pathway and the shear stress-mediated OS after SOLT with small-for-size graft via the phosphatidylinositol 3 kinase/Akt pathway.

Hepatic arterial reconstruction for orthotopic liver transplantation in the rat.

BACKGROUND: Orthotopic liver transplantation (OLT) models in rats have been investigated in many studies. The reconstruction of hepatic artery is required for reliable OLT and also requires advanced skills. METHODS: The hepatic artery reconstructions by a hand-suture technique and a new method using a micro T-tube were investigated in rats with a whole-liver syngeneic graft. Operative time and postoperative patency were compared between the hand-suture and micro T-tube techniques. RESULTS: Our technique using the micro T-tube shortened the operative time of recipient surgery compared with the hand-suture technique and prolonged the operative time for the donor. The patency ratio was maintained at 24h after OLT with hand suturing but was significantly reduced with the micro T-tube, which had a patency ratio of 0.83 only up to 6h after OLT. CONCLUSION: The micro T-tube technique may have potential usefulness in the rat OLT model but requires further modification.

Progressive familial intrahepatic cholestasis: a single-center experience of living-donor liver transplantation during two decades in Japan.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) results in liver cirrhosis. Therefore, some PFIC patients require liver transplantation (LT). Although three types of PFIC have been identified, their etiologies include unknown mechanisms. PATIENTS: A total of 717 recipients who underwent living-donor LT (LDLT) at <20 yr old were enrolled in this study. Among these recipients, 14 PFIC recipients comprising 11 PFIC type 1 (PFIC1) and three PFIC type 2 (PFIC2) were evaluated. RESULTS: Three of 11 PFIC1 recipients died, while all three PFIC2 recipients survived. Eight of 11 PFIC1 recipients showed steatosis after LDLT. Among the eight steatosis-positive PFIC1 recipients, seven showed severe steatosis and seven were complicated with steatohepatitis. Nine of 11 PFIC1 recipients showed fibrosis after LDLT, and eight of the nine fibrosis-positive PFIC1 recipients showed severe fibrosis. In contrast to the PFIC1 recipients, the PFIC2 recipients did not show any steatosis or fibrosis after LDLT. CONCLUSIONS: The clinical courses and outcomes of PFIC1 recipients after LDLT are still not sufficient owing to steatosis/fibrosis, unlike the case for PFIC2 recipients. As PFIC1 patients will require LT during the long-term progression of the disease, further strategy improvements are required for PFIC1 patients.

Fulminant liver failure models with subsequent encephalopathy in the mouse.

BACKGROUND: A reliable model of fulminant liver failure (FLF) is urgently required in this research field. This study aimed to develop a murine FLF model. METHODS: We used three groups of male C57BL/6 mice: control, with azoxymethane treatment (AOM group), and with galactosamine and tumor necrosis factor-alpha treatment (Gal+TNF-alpha group). The effects of body temperature (BT) control on survival in all three groups were investigated. Using BT control, we compared the survival, histopathological findings and biochemical/coagulation profiles between the two experimental groups. The effects of hydration on international normalized ratios of prothrombin time (PT-INRs) were also checked. Dose-dependent survival curves were constructed for both experimental groups. Neurological behavior was assessed using a coma scale. RESULTS: No unexpected BT effects were seen in the control group. The AOM group, but not the Gal+TNF-alpha group, showed a significant difference in survival curves between those with and without BT care. Histopathological assessment showed consistent FLF findings in both experimental groups with BT care. There were significant differences between the experimental groups in aspartate aminotransferase levels and PT-INRs, and significant differences in PT-INRs between the sufficiently and insufficiently hydrated groups. There were significant differences between FLF models in the duration of each coma stage, with significant differences in stages 1 and 3 as percentages of the disease state (stages 1-4). The two FLF models with BT care showed different survival curves in the dose-dependent survival study. CONCLUSIONS: AOM provides a good FLF model, but requires a specialized environment and careful BT control. Other FLF models may also be useful, depending on the research purpose. Thoughtful attention to caregiving and close observation are indispensable for successful FLF models.

Liver transplantation for congenital biliary dilatation: a single-center experience.

BACKGROUND: Congenital biliary dilatation is a rare disease. Although the possibility of refractory cholangitis and/or the frequency of malignant tumors legitimize hepatobiliary surgery, repeated cholangitis and biliary obstruction result in secondary liver cirrhosis even after polysurgery. There are no definitive guidelines on liver transplantation for congenital biliary dilatation. PATIENTS: A total of 1,101 liver transplantation recipients were enrolled in this study. Eleven patients with congenital biliary dilatation including 5 patients with Caroli's disease were retrospectively analyzed in detail. RESULTS: Nine of 11 patients underwent initial operations before liver transplantation while 2 patients with Caroli's disease received liver transplantation as initial surgery, with good outcomes. All patients had intractable symptoms caused by liver cirrhosis, and growth delay was considerable in patients aged <20 years. Histopathological analysis of the native liver revealed hepatic fibrosis (≥F2). One patient with ABO incompatibility died. One patient with Caroli's disease accompanied with intrahepatic carcinoma survives 11.8 years after liver transplantation without any recurrences. CONCLUSIONS: Patients with congenital biliary dilatation with refractory symptoms and complications secondary to liver failure are appropriate candidates for liver transplantation. We suggest that liver transplantation is an effective therapeutic option for patients with congenital biliary dilatation with due consideration to many accompanying factors, such as clinical course, growth delay, image findings and histopathological analysis.

Oxidative stress and extracellular matrices after hepatectomy and liver transplantation in rats.

AIM: To investigate oxidative stress (OS)-mediated damage and the behavior of extracellular matrices in various rat models because shear stress with portal hypertension and cold ischemia/warm reperfusion injury trigger the liver regeneration cascade after surgery. These injuries also cause fatal liver damage. METHODS: Rats were divided into four groups according to the surgery performed: control; hepatectomy with 40% liver remnant (60% hepatectomy); orthotopic liver transplantation (OLT) with whole liver graft (100% OLT); and split OLT (SOLT) with 40% graft (40% SOLT). Survival was evaluated. Blood and liver samples were collected at 6 h after surgery. Biochemical and histopathological examinations were performed. OS-induced damage, 4-hydroxynonenal, ataxia-telangiectasia mutated kinase, histone H2AX, phosphatidylinositol 3-kinase (PI3K) and Akt were evaluated by western blotting. Behavior of extracellular matrices, matrix metalloproteinase (MMP)-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were also evaluated by western blotting and zymography. RESULTS: Although 100% OLT survived, 60% hepatectomy and 40% SOLT showed poor survival. Histopathological, immunohistological, biochemical and protein assays revealed that 60% hepatectomy, 100% OLT and 40% SOLT showed liver damage. PI3K and Akt were decreased in 60% hepatectomy and 40% SOLT. For protein expression, 40% SOLT showed differences in MMP-9, MMP-2 and TIMP-2. TIMP-1 showed differences in 60% hepatectomy and 40% SOLT. For protein activity, MMP-9 demonstrated significant differences in 60% hepatectomy, 100% OLT and 40% SOLT. CONCLUSION: Under conditions with an insufficient liver remnant, prevention of OS-induced damage via the Akt/PI3K pathway may be key to improve the postoperative course. MMP-9 may be also a therapeutic target after surgery.

Pretreatment of liver grafts in vivo by γ-aminobutyric acid receptor regulation reduces cold ischemia/warm reperfusion injury in rat.

BACKGROUND: Gamma-aminobutyric acid (GABA) is found throughout the body. The regulation of GABA receptor (GABAR) reduces oxidative stress (OS). Ischemia/reperfusion injury after orthotopic liver transplantation (OLT) causes OS-induced graft damage. The effects of GABAR regulation in donors in vivo were investigated. MATERIAL AND METHODS: Donor rats received saline, a GABAR agonist or GABAR antagonist 4 h before surgery. Recipient rats were divided into four groups according to the donor treatments: laparotomy, OLT with saline, OLT with GABAR agonist and OLT with GABAR antagonist. Histopathological, biochemical and immunohistological examinations were performed at 6, 12 and 24 h after OLT. Protein assays were performed at 6 h after OLT. The 4-hydroxynonenal (4-HNE), ataxia-telangiectasia mutated kinase (ATM), phosphorylated histone H2AX (gammaH2AX), phosphatidylinositol-3 kinase (PI3K), Akt and superoxide dismutase (SOD) were assessed by western blot analysis. RESULTS: In the univariate analysis, histopathological and biochemical profiles verified that the GABAR agonist reduced graft damage. Immunohistology revealed that the GABAR agonist prevented the induction of apoptosis. Measurement of 4-4-HNE levels confirmed OS-induced damage after OLT, and the GABAR agonist improved this damage. In the gammaH2AX, PI3K, Akt and antioxidant enzymes (SODs), ATM and H2AX were greatly increased after OLT, and were reduced by the GABAR agonist. In the multivariate analyses between multiple groups, histopathological assessment, aspartate aminotransferase level, immunohistological examinations for apoptotic induction and gammaH2AX showed statistical differences. CONCLUSIONS: A specific agonist demonstrated regulation of GABAR in vivo in the liver. This activation in vivo reduced OS after OLT via the ATM/H2AX pathway.

Pretreatment of Small-for-Size Grafts In Vivo by γ -Aminobutyric Acid Receptor Regulation against Oxidative Stress-Induced Injury in Rat Split Orthotopic Liver Transplantation.

Background. Graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation (LT). The strategic potential of graft pretreatment in vivo by a specific agonist for γ -aminobutyric acid receptor (GABAR) was investigated in the rat LT model with a small-for-size graft (SFSG). Methods. Recipient rats were divided into three groups according to donor treatments and recipient surgeries: (i) saline and laparotomy, (ii) saline and split orthotopic liver transplantation (SOLT) with 40%-SFSG, and (iii) GABAR agonist and SOLT with 40%-SFSG. Survival was evaluated. Blood and liver samples were collected 6 h after surgery. Immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal, ataxia-telangiectasia mutated kinase (ATM), histone H2AX, phosphatidylinositol-3 kinase (PI3K), Akt, and free radical scavenging enzymes were performed. Results. Pretreatment by GABAR showed improvement in survival, histopathological assessment, and biochemical tests. Apoptotic induction and oxidative stress were observed after SOLT with an SFSG, and this damage was limited by GABAR regulation. GABAR regulation appeared to reduce DNA damage via the ATM/H2AX pathway and to promote cell survival via the PI3K/Akt pathway. Conclusions. Pretreatment in vivo by GABAR regulation improves graft damage after SOLT with an SFSG. This strategy may be advantageous in LT.

TIMP-1 attenuates blood-brain barrier permeability in mice with acute liver failure.

Blood-brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and D-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF.

Effect of specific activation of γ-aminobutyric acid receptor in vivo on oxidative stress-induced damage after extended hepatectomy.

AIM: γ-Aminobutyric acid (GABA) is a multifunctional molecule with various physiological effects throughout the body. The regulation of GABA receptor (GABAR) plays a key role in reducing the damage mediated by oxidative stress (OS). Extended hepatectomy causes fatal OS-induced injury in the liver remnant. We aimed to investigate the effect of a GABAR agonist in extended hepatectomy. METHODS: Saline or a GABAR agonist (43.56 nmol/g bodyweight of muscimol) was administrated intravenously at 4 h preoperatively. C57BL/6 mice were divided into three groups: laparotomy only, 90% hepatectomy with saline and 90% hepatectomy with a GABAR agonist. Liver samples were obtained at 6 h after surgery. RESULTS: Survival curves were prolonged by the GABAR agonist. Histopathological findings and biochemical profiles showed that the GABAR agonist reduced liver damage. Immunohistological assessment demonstrated that the GABAR agonist prevented apoptotic induction. As shown by 4-hydroxynonenal, which reflects OS-induced damage, 90% hepatectomy caused OS and the GABAR agonist reduced OS. We measured ataxia-telangiectasia mutated kinase (ATM), H2AX, Akt and free radical scavenging enzymes because they may be affected by GABAR regulation, and found that Akt was greatly decreased after 90% hepatectomy, but it recovered with the GABAR agonist. CONCLUSION: GABAR is activated by a specific agonist in the liver in vivo. This activation reduces OS-mediated damage after extended hepatectomy in vivo, and the mechanism via an Akt-dependent pathway may be a key.

Impact of hepatic arterial reconstruction on orthotopic liver transplantation in the rat.

Orthotopic liver transplantation (OLT) models in rats have been investigated in many studies, but detailed information on the impact of hepatic artery (HA) reconstruction on postoperative factors remains to be investigated. HA reconstruction also requires advanced skills. The effect of the reconstruction of the HA by a hand-suture technique in rats with a whole-liver syngeneic graft was investigated. Long-term survival, histopathological assessment, immunohistological evaluation, and blood biochemistry were investigated until postoperative day (POD) 28. From the early postoperative period, significant differences between OLTs with or without HA reconstruction were found in graft parenchymal damage, induction of apoptosis, and transaminase levels, though survival curves and the coagulation profile showed no differences. In OLT without HA reconstruction, biliary proliferation was decreased at POD 5-14, and total bilirubin level was increased at PODs 10 and 14. The study indicates that HA reconstruction is required for reliable OLT in rats.

Simple and reproducible hepatectomy in the mouse using the clip technique.

AIM: To investigate the reliability of massive hepatectomy models by using clip techniques. METHODS: We analyzed anatomical findings in 100 mice following massive hepatectomy induced by liver reduction > 70%. The impact of various factors in the different models was also analyzed, including learning curves, operative time, survival curves, and histopathological findings. RESULTS: According to anatomical results, models with 75%, 80%, and 90% hepatectomy produced massive hepatectomy. Learning curves and operative times were most optimal with the clip technique. Each hepatectomy performed using the clip technique produced a reasonable survival curve, and there were no differences in histopathological findings between the suture and clip techniques. CONCLUSION: Massive hepatectomy by the clip technique is simple and can provide reliable and relevant data.

Simple and sure methodology for massive hepatectomy in the mouse.

BACKGROUND: Reliable models for massive hepatectomy in the mouse are required for experimental liver research. METHODS: We analyzed anatomical findings in 100 mice following massive hepatectomy induced by liver reduction >70%. The impact of various factors in the different models was also analyzed, including learning curves, operative time, survival curves and histopathological findings. RESULTS: According to anatomical results, murine models with 75%, 80% and 90% of liver resection produced massive hepatectomy. Learning curves and operative times were most optimal with the clip technique. Each hepatectomy performed using the clip technique produced a reasonable survival curve, and there were no differences in histopathological findings between the suture and clip techniques. CONCLUSION: Massive hepatectomy by the clip technique is simple and can provide reliable and relevant data.

Fulminant liver failure model with hepatic encephalopathy in the mouse.

AIM: To develop a reliable murine model for fulminant liver failure (FLF). MATERIAL AND METHODS: We treated three groups of male C57BL/6 mice:as controls, with azoxymethane (AOM), and with galactosamine (Gal) and tumor necrosis factor-alpha (TNFα). Effects of body temperature (BT) control on survival, in all three groups were investigated. Using BT control, survival, histopathological findings and biochemical/coagulation profiles were compared between the experimental groups. Effects of hydration on international normalized ratios of prothrombin time (PT-INR) were also checked. Dose-dependent survival curves were made for both experimental groups. Neurological behaviors were assessed using a coma scale. RESULTS: No unexpected BT effects were seen in the control group. The AOM group, but not the Gal+TNFα group, showed significant differences in survival curves between those with and without BT care. Histopathological assessment showed consistent FLF findings in both experimental groups with BT care. Between the experimental groups, there were significant differences in aspartate aminotransferase levels and PT-INR; and significant differences in PT-INRs between sufficiently- and insufficiently-hydrated groups. There were significant differences between FLF models, in the duration of each coma stage, with significant differences in stages 1 and 3 as percentages of the diseased state (stages 1-4). The two FLF models with BT care showed different survival curves in the dose-dependent survival study. CONCLUSION: Azoxymethane can provide a good FLF model, but requires a specialized environment and careful BT control. Other FLF models may also be useful, depending on research purpose. Thoughtful attention to caregiving and close observation are indispensable for successful FLF models.

Thrombotic microangiopathy-like disorder after living-donor liver transplantation: a single-center experience in Japan.

AIM: To investigate thrombotic microangiopathy (TMA) in liver transplantion, because TMA is an infrequent but life-threatening complication in the transplantation field. METHODS: A total of 206 patients who underwent living-donor liver transplantation (LDLT) were evaluated, and the TMA-like disorder (TMALD) occurred in seven recipients. RESULTS: These TMALD recipients showed poor outcomes in comparison with other 199 recipients. Although two TMALD recipients successfully recovered, the other five recipients finally died despite intensive treatments including repeated plasma exchange (PE) and re-transplantation. Histopathological analysis of liver biopsies after LDLT revealed obvious differences according to the outcomes. Qualitative analysis of antibodies against a disintegrin-like domain and metalloproteinase with thrombospondin type 1 motifs (ADAMTS-13) were negative in all patients. The fragmentation of red cells, the microhemorrhagic macules and the platelet counts were early markers for the suspicion of TMALD after LDLT. Although the absolute values of von Willebrand factor (vWF) and ADAMTS-13 did not necessarily reflect TMALD, the vWF/ADAMTS-13 ratio had a clear diagnostic value in all cases. The establishment of adequate treatments for TMALD, such as PE for ADAMTS-13 replenishment or treatments against inhibitory antibodies, must be decided according to each case. CONCLUSION: The optimal induction of adequate therapies based on early recognition of TMALD by the reliable markers may confer a large advantage for TMALD after LDLT.

Comprehensive and innovative techniques for liver transplantation in rats: a surgical guide.

AIM: To investigate our learning curves of orthotopic liver transplantation (OLT) in rats and the most important factor for successful surgery. METHODS: We describe the surgical procedures for our rat OLT model, and determined the operator learning curves. The various factors that contributed to successful surgery were determined. The most important surgical factors were evaluated between successful and unsuccessful surgeries. RESULTS: Learning curve data indicated that 50 cases were required for operator training to start a study. Operative time, blood loss, warm ischemic time, anhepatic phase, unstable systemic hemodynamic state, and body temperature after surgery significantly affected surgery success by univariate analysis, while the anhepatic phase was the most critical factor for success by multivariate analysis. CONCLUSION: OLT in rats is the only liver transplantation model that provides clinically relevant and reliable results. Shortened anhepatic phase is key to success in this model.