Lindsay B. Gardner

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.

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.

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.

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.

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.

Left-sided grafts for living-donor liver transplantation and split grafts for deceased-donor liver transplantation: Their impact on long-term survival

BACKGROUND A small-for-size graft is important in living-donor liver transplantation (LDLT) and deceased-donor liver transplantation (DDLT). SUBJECTS AND METHODS First, we confirmed the effect of initial graft volume on survival using a rat model of liver transplantation (LT). We then evaluated the actual long-term survival based on graft type in 1421 LTs (including 1364 LDLTs) at Kyoto University and 2000 DDLTs at the Mayo Clinic, to evaluate donor safety in LDLT and the possibility of shifting to split orthotopic liver transplantation (SOLT) in DDLT. RESULTS In the rat model, SOLTs with 40%- and 20%-grafts had a poor survival. A total of 697 pediatric LTs showed good long-term outcomes (survival rate was 0.764 at 21.2 years). The survival rate of 724 adult LTs was 0.664 at 17.8 years. The survival rates of auxiliary partial orthotopic liver transplantation with a left-sided graft (0.421 at 15.0 years) and SOLT with a left-sided graft (0.000 at 0.8 years) need to be improved. Although the survival rate of 1965 adult DDLTs with a whole-liver graft in the Mayo Clinic was 0.727 at 12.8 years, that of adult SOLT was 0.595 at 11.0 years. CONCLUSION From the viewpoint of greater donor safety and expanded donor candidates in LDLT, the choice of a left-sided graft still remains controversial. A shift to SOLT to achieve excellent results should be established to resolve a donor shortage in DDLT.

Impact of Hepatic Arterial Reconstruction on Orthotopic Liver Transplantation in the Rat

ABSTRACT 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.

Matrix metalloproteinase-9 after the cold ischemia/reperfusion injury and/or shear stress with portal hypertension: an overview

Cold ischemia/warm reperfusion injury (CIWRI) during liver transplantation (LT) and shear stress with portal hypertension (SSPH) associated with hepatectomy are critical issues [1, 2], and has been confirmed from the early postoperative period [3, 4]. They trigger a liver regeneration cascade, but can also cause fatal damage in liver remnants/ grafts. Matrix metalloproteinases (MMPs) are a family of enzymes capable of degrading the constituents of the extracellular matrix and the basement membrane. A total of 28 MMPs have been identified so far. MMP-9 is especially important for liver regeneration, and many studies have focused on the role of MMP-9 after liver surgery, including LT with small-for-size grafts (SFSGs) [3–5]. Although inhibition of MMP-9 attenuated acute graft injury in the drastic SFSG model (a rat LT model with a 25 % graft) [5], the behavior of MMP-9 in CIWRI and/or SSPH is still unclear. We herein investigated the behaviors of MMPs during the early postoperative period in various clinically relevant conditions which clearly showed liver damage in the conventional liver test. For these studies, Lewis rats were divided into five groups according to the surgical treatments: (1) laparotomy, (2) temporary clamping for 15 min (Pringle maneuver) as temporary inflow occlusion, (3) orthotopic liver transplantation (OLT) with a wholeliver graft (100 % OLT), (4) partial hepatectomy without temporal inflow occlusion (60 % hepatectomy) and (5) split orthotopic liver transplantation (SOLT) with SFSG (40 % SOLT). In this study, we employed a cold ischemia time of 3 h at 4 C in 100 % OLT (3) and 40 % SOLT (5), although this term of cold preservation is considered to be minimal in the rat liver transplantation model. Liver samples were collected 6 h after surgery (n = 4 in each group). Rat liver transplantation requires skillful techniques, and surgical issues cannot be ignored because they will affect the results. The anhepatic phase is a key for a successful surgery, and this phase in all recipients was \20 min in this study. We also omitted samples if any complications are observed at autopsy. Western blotting for MMP-9 expression and zymography for MMP-9 activity were performed. The expression of MMP-9 was not significantly different following temporary clamping, 100 % OLT and 60 % hepatectomy compared with the levels following laparotomy, T. Hori (&) S. Uemoto T. Hata L. B. Gardner F. Chen A.-M. T. Baine Division of Hepato-Biliary-Pancreatic and Transplant Surgery, Department of Surgery, Kyoto University Graduate School of Medicine, 54 Shogoinkawara-cho, Sakyo-ku, Kyoto 606-8507, Japan e-mail: horit@kuhp.kyoto-u.ac.jp

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.