J. Xu1, X. Cui1, E. Tzeng1, U. Sachdev1 1University Of Pittsburgh,Vascular Surgery,Pittsburgh, PA, USA
Introduction: We have shown that mice with genetically absent MyD88, a critical mediator of TLR4 signaling, showed less ischemic damage and inflammation than controls after femoral artery ligation. Surprisingly, mice lacking TRIF, another important mediator of TLR4 signaling demonstrated necrosis rather than protection at the same time point. These earlier findings suggested that MyD88 and TRIF played complimentary roles in the recovery from limb ischemia. In order to further test this theory, we treated control C57B6 mice as well as TRIFKO with a peptide inhibitor of MyD88 to test the hypothesis that unopposed MyD88 inflammation in TRIFKO mice hindered muscle recovery after femoral ligation.
Method: C57B6 mice and TRIFKO mice were injected with 25 micrograms of peptide inhibitors to MyD88 or a nonspecific target one hour before the initiation of femoral artery ligation on the right hindlimb. On the left, the femoral vessels were exposed but not ligated. Laser Doppler Perfusion Imaging (LDPI) was performed after one week to assess perfusion recovery, expressed as a ratio of right to left flow. H and E was performed on ischemic muscle samples and the average area of each regenerating myocyte, identified by centrally located nuclei, was compared among groups.
Results: TRIFKO mice treated with a MyD88 inhibitor demonstrated an improvement in perfusion by LDPI 7 days after ligation (Figure 1). Neither control C57B6 mice nor mice treated with control peptide inhibitor demonstrated similar improvements in perfusion to the ischemic limb at the same time point. Similarly, the mean size of regenerating myocytes was significantly higher in TRIFKO mice treated with MyD88 inhibitor but not control inhibitor (295.12 ± 14.98 vs. 235.00 ± 14.72 micrometers; MyD88 inhibitor vs. control inhibitor, p=0.038, N=3-4/group). Similar improvements were not demonstrated in C57B6 mice treated with the MyD88 inhibitor.
Conclusion: Our data suggest that in the absence of TRIF, MyD88 activity may compromise muscle regeneration after ischemia. Blocking its activity with a peptide inhibitor improves perfusion recovery and increases regenerating myofiber size after one week. These results further support the hypothesis that TRIF attenuates MyD88 activity, acting to balance the deleterious inflammatory effects of intact MyD88 signaling in limb ischemia.
