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

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

IH: Immunohistochemistry; WB: Western blotting; MPO: Myeloperoxidase assay; RT-PCR: Reverse transcriptase 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; NF: Nuclear factor; MMP: Matrix metalloproteinase; HSP: Heat shock protein; eNOS: Endothelial nitric oxide synthase; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase (subtype 1).