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

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

KO: Transgenic knockout; WT: Wild type (normal animals); IH: Immunohistochemistry; 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; GOT: Glutamic oxaloacetic transaminase; I: Ischemia; R: Reperfusion; IR: Ischemia reperfusion; IRI: Ischemia reperfusion injury; IVM: Intravital microscopy; IFN: Interferon; Ab: Antibody; TNF: Tumour necrosis factor; TNFR1: Tumour necrosis factor receptor (subtype 1); TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling (assay for cell death); IL: Interleukin; CXCR: Chemokine receptor; PAI: Plasminogen activator inhibitor; NF: Nuclear factor; C1-9: Complement protein 1 to 9; MAP: Mean arterial pressure; MIP: Major intrinsic protein.