Copyright
©2013 Baishideng Publishing Group Co.
World J Gastroenterol. Mar 21, 2013; 19(11): 1683-1698
Published online Mar 21, 2013. doi: 10.3748/wjg.v19.i11.1683
Published online Mar 21, 2013. doi: 10.3748/wjg.v19.i11.1683
Table 1 Suzuki classification of liver ischaemia reperfusion injury
| Numerical assessment | Sinusoidal congestion | Vacuolisation/ballooning | Necrosis |
| 0 | None | None | None |
| 1 | Minimal | Minimal | Single cell |
| 2 | Mild | Mile | < 30% |
| 3 | Moderate | Moderate | 30%-60% |
| 4 | Severe | Severe | > 60% |
Table 2 Summary of knockout models of liver ischemia reperfusion injury pertaining to reactive oxygen species, cellular metabolism/adenosine and cells involved in the injurious mechanisms
| Ref. | Knockout model | IR protocol | Outcome measure | Agent | Adaptive responses | Injurious responses |
| Kuboki et al[29] | OTII; TCRd deficient | 70% I 90 min/R 4, 8 h | Histology; serum ALT; MPO | AntiCD1d Ab; anti NK1.1 Ab; anti CD25+ Ab | Antigen dependent CD4+ T cell activation via TCR and NKT cell activation increase IRI; GD T cell recruit PMN but not affect IRI | |
| Evans et al[2] | ob/ob or double knockout of leptin and UCP2 | Total hepatic ischaemia 15 min/R 1, 24 h | Histology (Neil and Hubscher scoring); serum ALT; WB; liver ATP assay; lipid peroxidation; 24 h survival | In steatotic livers of ob/ob mice only, UCP-2 depletes liver ATP which increases IRI 1 h onwards | ||
| Hanschen et al[12] | IL6 (-/-); CD4 (-/-); TNFR1 (-/-) | Left lobe I 90 min/R 30 min, 2, 3, 4 h | Kupffer cell activity (fluorescent latex beads and intravital microscopy, IVM); IH; serum AST and ALT | GdCl3 or glutathione to wild types (WT) only | Kupffer cells activation, ROS, IL6 and TNF-α increase SEC VAP-1 expression and CD4+ Tcell sinusoidal recruitment which increase IRI; CD4+ T cells inhibit Kupffer cell phagocytic activity | |
| Kim et al | Adenosine A1 receptor (A1AR) (-/-) | 70% I 1 h/R 24 h | Histology; serum ALT; IH; semiquantitative PCR; WB; TUNEL | CCPA (AIAR agonist); DPCPX (A1AR antagonist) | Endogenous adenosine via A1AR reduces IRI | Exogenous adenosine increase IRI most likely via a different adenosine receptor subtype to A1AR |
| Ben-Ari et al[18] | Bax (-/-); Bax (+/-) | Isolated liver perfused in environmental chamber: Global I 90 min/R 1 or 15 min | Histology (apoptosis features); serum ALT, AST, LDH; TUNEL and caspase-3 assay; WB | Bax activation after 15 min reperfusion activates caspase-3 which increases liver apoptosis | ||
| Lappas et al[30] | Rag1 (-/-), i.e., lack mature lymphocytes A2AR (-/-); IFNγ (-/-) | 70% I 72 min/R 2, 24 h | Histology; serum ALT; intracellular IFNγ | ip ATL146 (A2AR agonist); PK136 (NK1.1 depletion); CD1d Ab (inhibit NKT cell); NKT cell adoptive transfer from WT, A2AR and IFNγ KO to Rag1 KO | Exogenous and endogenous adenosine acts through A2AR to reduce NKT cell recruitment | NKT cell recruitment increases IRI through release of IFNγ from at least 2 h reperfusion onwards and increased neutrophil recruitment from at least 24 h after reperfusion |
| Shimamura et al[25] | Cd1d (-/-); nu/nu (no NKT cell, normal NK cells); perforin (-/-); gld/gld (Fas ligand deficient) | Total hepatic ischaemia 30 min/R 2 ,6, 12, 24, 48 h | Serum ALT; peroxide assay; cytotoxic assay; IH; ELISA | Anti-NK and anti-NKT Ab | NKT cell activation 1 to 24 h after reperfusion releases IFNγ and PMN activation 6 to 12 h after reperfusion with increased oxidative burst lead to increased apoptosis and necrosis in IRI | |
| Caldwell et al[28] | CD4 (-/-); B cell (-/-) | 70% I 90 min/R 1, 2, 4, 8 h | Histology; serum ALT; MPO | Adoptive transfer CD4+T cell to CD4(-/-); anti-IL17 Ab | CD4+ T cell only 1-4 h after reperfusion secrete IL17 releasing MIP-2 increasing neutrophil infiltration, but inhibiting their oxidative burst, and reducing necrosis 8 h reperfusion onwards | |
| Baskin-Bey et al | Cathepsin B (-/-) | Two weeks fed methionine choline deficient (MCD) diet to induce steatosis; liver stored 24 h 4 °C UWS then perfused in isolated apparatus at 37 °C for 1 h | Histology; electron microscopy (EM); TUNEL; IH; liver tissue ALT and LDH | R-3032 ip 2 h preop (cathepsin B inhibitor) | Reduced lysosomal integrity more pronounced in steatotic livers with increased cathepsin B release into cytosol associated with increased apoptosis and necrosis | |
| Khandoga et al[22] | ICAM (-/-) | Left lobe I 90 min/R 20 min | Serum AST and ALT; IH; caspase-3 assay; lipid peroxidation assay; IVM | Anti-fibronectin Ab | Platelets bind fibronectin deposited on ICAM-1 expressed on SECs, associated with reduced sinusoidal perfusion, increased lipid peroxidation and apoptosis | |
| Shen et al[33] | nu/nu; CD154 (-/-) | 70% I 90 min/R 4 h | Serum ALT; histology; MPO; WB | Anti-CD154 Ab to WT; adoptive transfer spleen lymphocytes into KO or Ab treated group | IRI induces HO-1 protein | CD4-CD154 T cell costimulation is associated with increased IRI |
| Wyllie et al[69] | Natural resistance associated macrophage protein 1 (Nramp) (-/-) | 70%I 45 min/R 30, 60 min | Plasma GOT and TNF-α; histology; WB; Northern Blot; IH; EMSA (NFκβ) | HO-1 expressed in this model is protective in IRI | Macrophage activation after reperfusion increases TNF-α release and NFκβ activity which increases IRI | |
| Young et al[21] | P-selectin/ ICAM-1 double KO | 70% I 90 min/R 1.5, 3, 6 h | Serum ALT; histology | P-selectin and ICAM-1 do affect the severity of IRI up to 6 h reperfusion in this model, although PMN infiltration is slightly increased in midzonal area | ||
| Ozaki et al[13] | gp91 phox component of phagocyte NADPH oxidase (-/-) | 70% I 60 min/R 5, 8, 24 h +/- iv injection 3 d preop of adenovirus | Serum GOT; histology (HE; ELISA for DNA histone fragments); TUNEL; IH; WB; assays for lipid peroxidation, hydrogen peroxide and superoxide; EMSA (NFκβ) | Replication deficient adenovirus encoding Rac1 (control: Adβgal) | Rac1 is activated in IRI and is protective | Liver tissue releases ROS within 5 min of reperfusion and PMN from 8 h onwards, associated with increased lipid peroxidation, apoptosis and necrosis. NFκβ DNA binding is associated with increased IRI; NADPH oxidase regulated by Rac1 small GTP binding protein is a source of ROS in IRI |
| Sawaya et al[19] | P-selectin (-/-) | Left lobe I 30 min/R 15, 30, 60, 120 min | Serum AST, ALT, LDH; histology; IVM in terminal hepatic venule (THV) | Radiolabelled anti P-selectin Ab | P selectin expression on SECs increases rolling, saltating and adherent leucocytes in THV peaking at 30 min reperfusion | |
| Singh et al[20] | P-selectin (-/-) | Left lobe I 30 min/R 20 min, 2, 5, 12, 24 h | Serum AST, ALT, LDH; histology; WB | Radiolabelled anti P-selectin Ab | P-selectin expression peaks at 20 min and 5 h after reperfusion and is associated with worse IRI |
Table 3 Cytokine, chemokine, tolllike receptor, complement knockout models of liver ischemia reperfusion injury
| Ref. | Knockout model | IR protocol | Outcome measure | Agent | Adaptive responses | Injurious responses |
| Kuboki et al[26] | CXCR2 | 70% I 90 min/R 12, 24, 48, 96 h | Histology; MPO; serum ALT, TNF-α, IL6; WB and NFκβ activity | CXCR2 activates STAT3 hepatocyte proproliferative pathway | MIP2 activates CXCR2 which increases neutrophil recruitment and IRI. Nuclear factor (NF) κβ activity reduced in IRI | |
| Zhai et al[44] | IFNAR type1 (-/-); IFNAR type 2 (-/-) | 70% I 90 min/R 6 h | Histology; quantitative PCR | IFNβ (not IFNγ) mediates IRI by binding to IFNAR type 1 | ||
| Zhao et al | CXCL10 (-/-) | 70% I 90 min/R 1, 2, 4, 8 h | Histology; serum ALT; IH; quantitative PCR; WB | CXCL10 activation increases TNF-α, IL6, IL1b, iNOS, MIP-2 mRNA and PMN and Kupffer cell activation contributes to IRI | ||
| Fondevilla et al | C6 deficient rats | Donor/recipient: WT/WT, KO/WT, WT/KO, KO/KO; | Serum GOT; histology; MPO; IH; TUNEL; WB; PCR; ELISA | Membrane attack complex (C5b-C9) activation in this OLT model of cold/warm IRI increases apoptosis, necrosis, PMN and macrophage infiltration and TNF-α, IFNγ and IFNβ expression | ||
| OLT and organ storage 24 h 4 °C UWS | ||||||
| Shen et al[32] | Toll like receptor 4 (TLR4) (-/-) | Donor/recipient: WT/WT, KO/WTWT/KO, KO/KO; OLT with dearterialisation, organ stored 24 h 4 °C UWS | Histology; IH; MPO; quantitative PCR; capsase-3 activity; WB | TLR4 activation increases IL4 and IL10, but inhibits HO-1 | TLR4 activation increases TNF-α, IL1b, IL2, IFNγ, ICAM1, CXCL10, PMN and CD4+ T cell recruitment leading to increased liver necrosis and apoptosis | |
| Conzelmann et al | TNFR (-/-) | Donor/recipient: WT/WT, KO/WTWT/KO, KO/KO; organ storage 12 h 4 °C UWS; 8 h graft harvest | Histology; serum ALT; MPO; TUNEL and caspase-3 assay; IH | TNFR within liver mediates reduced IRI | TNFR outside liver increases IRI in terms of necrosis, apoptosis and neutrophil infiltration | |
| Tsung et al[37] | Interferon regulatory factor-1 (IRF-1) (-/-) | 70% I 60 min/R 1, 3, 6, 12 h | Histology; serum ALT; WB; PCR | Adenovirus IRF-1 vector | IFNγ, IFNβ, TNF-α, IL1β all activate IRF-1 which increase JNK (not p38 MAPK) and TNF-α and iNOS expression in IRI | |
| Tian et al[40] | TNFR1 (-/-); IL6 (-/-) | Donor/recipient: WT/WT, KO/WT, WT/KO, KO/KO; OLT: 50% or small for size 30% arterialised graft | Histology; serum AST; portal flow measurement; IVM; IH; PCR; 30 d mortality | GdCl3 (ip to donor); pentoxifylline (to donor and recipient sc); recombinant IL6 to KO only | Increased IL6 | Increased activation of Kupffer cells and TNF-α mediated activation of IFNR1 from 3 h reperfusion onwards increases liver necrosis, nonperfused sinusoids, adherent leucocytes and reduces hepatocyte regeneration |
| Shen et al[38] | TLR4 (-/-); TLR2 (-/-) | 70% I 90 min/R 6 h | Histology; serum ALT; MPO; WB; PCR | Snpp (inhibit HO-1); CoPP | HO-1 is expressed which inhibits TLR4 | TLR4 activation increases TNF-α expression associated with increased IRI |
| Lagoa et al[81] | PAI-1 (-/-) | MAP 25-30 mmHg for 2.5 h (2.25 mL/100 g blood withdrawn)/Resuscitation MAP > 80 mmHg for 4 h (30 min with shed blood and crystalloid) | Serum ALT, IL6, IL10; histology; Electron microscopy; IH; zymography for plasminogen activators; DNA microarray; PCR; WB | PAI-1 to PAI-1 (-/-) mice | PAI-1 expression in SEC contributes to IRI with periportal/pericentral injury, loss of sinusoidal fenestra and prominent SEC injury; PAI-1 inhibits u-PA which reduces formation of active HGF and increases active TGF-β1, but no effect on IL6 or IL10; this is associated with reduced activation of ERK-1/-2 pathway. | |
| Teoh et al[36] | TNF-α (-/-) | 70%I 90 min/R 2, 4, 24 h | Serum ALT; IH; serum TNF-α; EMSA (NFκβ); WB | Low dose or high dose TNF-αip | TNF-α from at least 2 h reperfusion onwards is injurious to ischaemic but not normal liver, increasing NFκβ DNA binding | |
| Inderbitzin et al[57] | CI inhibitor overexpressed | Total hepatic ischaemia 30 min/R 2 h | Endothelial permeability index (measured using radiolabelled albumin iv into inferior vena cava) of liver, lung and gut | C1 inhibitor overexpression is protective in IRI | Classical complement pathway is activated in IRI; liver ischaemia and reperfusion causes liver and gut, but not lung, IRI in this model | |
| Zhai et al | TLR4 (-/-); TLR2 (-/-) | 70%I 90 min/R 6 h | Serum ALT; histology; PCR | TLR4 activation increases expression of IRF3 which upregulates IFNβ associated with increased IRI | ||
| Rudiger et al[39] | TNFR (-/-); Fas (-/-); FasL (-/-) | 70% I 75 min/R 3 h | Serum AST; TUNEL; caspase-3 assay; ELISA; WB | Pentoxifylline | TNF-α binds to TNFR1 which increases apoptosis in IRI; fas and FasL not involved in this model | |
| Kato et al[82] | IL1R (-/-) | 70%I 90 min/R 1, 2, 4, 8, 16, 24 h | Serum ALT, IL1β, TNF-α and MIP-2; histology (PMN score); MPO; EMSA (NFκβ); PCR | IL1R not involved in IRI | ||
| Calmargo et al | IL6 (-/-) | Median lobe (45%) I 90 min/R 30, 60, 90, 120 min | Serum AST and ALT; histology; PCR | Recombinant IL6 | IL6 released in IRI is protective | TNF-α expression during reperfusion is associated with worse IRI |
Table 4 Nitric oxide synthase, HSP/heme oxygenase-1, matrix metalloproteinase knockout models of liver ischemia reperfusion injury
| Ref. | Knockout model | IR protocol | Outcome measure | Agent | Adaptive responses | Injurious responses |
| Hamada et al[26] | iNOS (-/-); MMP-9 (-/-) | 70% I 90 min/R 3, 6, 24 h | Histology; serum ALT, NO2-/NO3-; myeloperoxidase activity (MPO); immunohistochemistry; PCR, Western blotting; MMP-9 activity assay; MMP-9 protein levels; neutrophil (PMN) migration assay; TUNEL and caspase-3 activity | ONO-1714 (iNOS inhibitor); NO donor (DETA NONOate) | Increased macrophage iNOS producing NO increases PMN MMP-9 and PMN transmigration over fibronectin | |
| Hamada et al[42] | MMP-9 (-/-) | 70% I 90 min/R 6, 24 h | Histology; serum GPT and GOT; MPO; IH; PCR | Anti MMP-9 iv; MMP-2/9 inhibitor; anti MMP-2 (all to WT only) | MMP-9 (not MMP-2) increase TNF-α, IFNg, IL2, IL6 and increase PMN and CD4+ T cell recruitment leading to increased liver necrosis | |
| Kuboki et al[73] | HSP70 (-/-) | 70% I 90 min/R 1, 8 h | Histology; serum AST; TNF-α; IL6; MIP-2; MPO; WB; EMSA (NFκβ) | Sodium arsenite iv to induce HSP70; recombinant HSP70 | No involvement of HSP70 in IRI; NFκβ activity associated with IRI | |
| Theruvath et al[59] | eNOS (-/-) | Donor (WT/KO) to WT recipient; organ stored 18 h, 4 °C, UWS | Histology; serum ALT; IVM; TUNEL; IH (macrophage infiltration) | eNOS activation reduces necrosis and apoptosis, with associated inhibition of macrophage infiltration, increased sinusoidal diameter and blood flow | ||
| Tsuchiashi et al | HO-1 (+/-); HO-1 (-/-) | 70% I 90 min/R 6 h | Histology; serum GOT; MPO; quantitative real time RT-PCR; WB; TUNEL | CoPP (induces HO-1) 24 h preop | HO-1 upregulated which inhibits expression of cytokines TNF-α and IFNγ | TNF-α and IFNγ expression increased overall in IRI associated with increased apoptosis and necrosis |
| Hines et al[23] | eNOS (-/-); iNOS (-/-) | 70% I 45 min/R 1, 3 h | Serum ALT; histology; PCR | Increased eNOS expression in IRI inhibits TNF-α and IL12 expression; iNOS activates eNOS in this model | No PMN infiltration at 3 h reperfusion | |
| Lee et al[58] | eNOS (-/-); iNOS (-/-) | 70% I 1 h/R 1, 3, 6 h | Serum ALT and AST; perfusion studies; PCR | eNOS activated during IRI is protective | Increased iNOS mRNA expression from 3 h reperfusion onwards regulates reperfusion and is associated with worse IRI | |
| Hines et al[60] | iNOS (-/-) | 70% I 45 min/R 1, 3, 6 h | Serum ALT; histology; MPO | L-NIL (iNOS inhibitor) | Reduced IRI in iNOS (-/-), but no iNOS mRNA or L-NIL effect in WT; may be genetic compensation effect in KO | |
| Kawachi et al[24] | eNOS (-/-); iNOS (-/-) | 70% I 45 min/R 5 h | Serum ALT; histology; MPO | eNOS is activated in IRI and is protective | There is no PMN infiltration up to 5 h reperfusion and iNOS is not activated in IRI in this model |
Table 5 Knockouts of downstream mediators and models of liver ischemia reperfusion injury
| Ref. | Knockout model | IR protocol | Outcome measure | Agent | Adaptive responses | Injurious responses |
| Theruvath et al[14,15] | JNK (-/-) | 70% I 1 h/R 4, 8 h | Histology; serum ALT; IVM (dyes to probe mitochondrial function and cell death); survival 14 d | JNK2 activation leads to mitochondrial depolarization and increased necrosis only | ||
| Theruvath et al[14,15] | JNK (-/-) | WT or KO donor; 30 h, 4 °C, UWS preservation; WT recipient | Histology 8 h posttransplant; serum ALT, TUNEl and caspase-3 assay; IVM; IH; lipid peroxidation | JNK2 activation leads to caspase-3 activation, mitochondrial depolarisation and release of cytochrome c, lipid peroxidation which all translate into reduced survival posttransplant | ||
| Beraza et al[72] | Conditional hepatocyte specific NEMO knockout | 70% I 1 h (caudate lobe resected)/R 3, 6 h | TUNEL and caspase-3 assay; WB; IH; Southern blotting; EMSA (NFκβ) | NFκβ activity reduces necrosis and apoptosis, inhibits TNF-α, JNK and iNOS | ||
| Okaya et al[35] | PPARα (-/-) | 70%I 90 min/R 4, 8 h | Serum ALT; TNF-α; MIP-2; MPO; liver NO2/NO3; WB; EMSA (AP-1, NFκβ) | WY14643 iv (PPARα agonist) | PPARα protective in IRI | PPARα independent release of TNF-α and MIP-2 and increased NO2/NO3 associated with IRI |
| Shen et al[78] | STAT4 (-/-); STAT6 (-/-); nu/nu | 70%I 90 min/R 6 h | Serum ALT; histology; MPO; WB; PCR | Adoptive transfer of CD4+ T cells from WT or other KO to nu/nu; SnPP ip | HO-1 expressed at very low levels after 6 h in this model, but protective in IRI | CD4+T cell activation involving T cell STAT4 activation, but not STAT6 associated with increased IRI |
| Khandoga et al[61] | PARP (-/-) | Left lobe I 90 min/R 30 min | Serum ALT; IVM; IH; PCR | PARP activation in IRI upregulates E-selectin, ICAM1 and VCAM1, associated with increased platelet and leucocyte endothelial interaction and reduced sinusoidal perfusion | ||
| Kato et al[82] | P50 NFκβ (-/-) | 70% I 90 min/R 1, 8 h | Serum ALT; histology; MPO WB; EMSA (p50 and p65 subunits of NFκβ) | No effect of p50 subunit deletion, but increased p50/p65 heterodimer in WT and some p65 in KO, so there may be some functional redundancy of NFκβ subunits | ||
| Kato et al[83] | STAT4 (-/-) | 70% I 90 min/R 30 min, 1, 2, 4, 8 h | Serum ALT; histology; MPO; WB | Anti IL12 Ab | IL12 expression associated with IRI. STAT4 not activated in IRI in this model | |
| Kato et al[70] | STAT6 (-/-) | 70% I 90 min/R 1, 4, 8 h | Serum ALT, TNF-α; MPO; PCR; EMSA (NFκβ) | IL4 or IL13 iv | STAT6 is not activated in IRI in this model, although STAT6 activation by iv IL4 or IL13 is protective. IRI is associated with increased NFκβ DNA binding |
- Citation: Datta G, Fuller BJ, Davidson BR. Molecular mechanisms of liver ischemia reperfusion injury: Insights from transgenic knockout models. World J Gastroenterol 2013; 19(11): 1683-1698
- URL: https://www.wjgnet.com/1007-9327/full/v19/i11/1683.htm
- DOI: https://dx.doi.org/10.3748/wjg.v19.i11.1683