Molecular mechanisms of liver ischemia reperfusion injury: Insights from transgenic knockout models

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.