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 |
KO: Transgenic knockout; I: Ischemia; R: Reperfusion; IR: Ischemia reperfusion; IRI: Ischemia reperfusion injury; ROS: Reactive oxygen species; ATP: Adenosine triphosphate; IH: Immunohistochemistry; HE: Hematoxylin and eosin; WB: Western blotting; MPO: Myeloperoxidase assay; PCR: Polymerase chain reaction; ELISA: Enzyme labelled immunosorbent assay; EMSA: Electrophoretic mobility shift assay; AST: Aspartate transaminase; ALT: Alanine transaminase; LDH; Lactate dehydrogenase; GOT: Glutamic oxaloacetic transaminase; NADPH; Nicotinamide adenine dinucleotide phosphate; IR: Ischemia reperfusion; IVM: Intravital microscopy; A2AR: Adenosine (subtype 2A) receptor; PMN: Polymorphonuclear cell; NKT: Natural killer T cell; NK: Natural killer cell; IFN: Interferon; Ab: Antibody; TNF: Tumour necrosis factor; TNFR1: Tumour necrosis factor receptor (subtype 1); TCR: T cell receptor; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling (assay for cell death); IL: Interleukin; ICAM: Intercellular adhesion molecule; VAP: Vascular adhesion protein.