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Toll-like Receptor 4 deficiency ameliorates the Local Injury following Limb Ischemia and Reperfusion
Hassan Albadawi, Robert S Crawford, Marvin D Atkins, John E Jones, Michael T Watkins
Massachusetts General Hospital, Division of Vascular and Endovascular Surgery, Boston, MA
Introduction: Toll-like receptor 4 (TLR4) is a pattern recognition receptor. Studies in TLR4 deficient mice revealed protection against hepatic and cardiac ischemia-reperfusion injury (IR). These experiments were undertaken to evaluate the benefit of TLR4 deficiency in a murine model of hindlimb IR.
Methods: TLR4-mutant mice C3H/HeJ (TLR4m, n≥5) and wild type strain C3H/HeSnJ (WT, n≥5) were subjected to 1.5hrs of unilateral hindlimb ischemia followed by 48hrs of reperfusion. At the end of reperfusion, hindlimbs were harvested for histological evaluation or assessment of ATP using chemiluminescence and to assess the gene expression levels of inducible nitric oxide synthase (iNOS) using RT-PCR. Serum vascular endothelial growth factor-VEGF levels were quantified with ELISA.
Results: During reperfusion, TLR4m demonstrated markedly reduced percent-injured muscle fibers compared to WT (15.8±6% vs. 45.5±9%, p=0.031). This correlated with higher levels of ATP in the TLR4m (2.1±0.4 vs. 1.06±0.3 nmole/mg tissue, p=0.043). The difference in tissue injury was accompanied by significant 3.5 fold reduction in the steady state levels of iNOS mRNA as normalized to β-Actin mRNA in the TLR4m compared to WT (p=0.031). VEGF serum levels were significantly higher in the TLR4m (1272±377 vs. 314±260 pg/ml, p=0.031).
Conclusions: During reperfusion, TLR4 receptor pathway deficiency provides protection against skeletal muscle injury following IR. This was associated with preserved energy levels (ATP). TLR4 receptor pathway deficiency blunted the expression of proinflammatory gene iNOS and resulted in higher serum levels of the pro-angiogenic growth factor VEGF. These data suggest that targeting TLR4-pathways is important to ameliorate limb reperfusion injury.
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