Assessment of the formation of vascular structures following hind limb ischemia.

<p>(A) Vascular density was quantified by the number of CD31 structures per HPF in control, TLR2 KO and TLR4 KO mice two weeks following femoral artery ligation (black bars). Vessel area was also measured (gray bar) (N = 4 per group; *P<0.05 versus control and TLR4 KO). (B) Number of SMA staining vessels per HPF 2 weeks (N = 4 per group; *P = 0.003 vs. control and TLR4 KO).</p

Assessment of vascular structures in control, TRIF KO and MyD88 KO mice.

<p>(A) Representative photomicrographs demonstrating CD31 positive structures (arrowhead; scale bar = 25 µm). (B) Quantification of vascular density (black bars) and vessel area (gray bars) are shown (N = 4–8 per group; *P<0.001 vs. TRIF KO and control; ^¶P = 0.002 vs. control).</p

Vascular density in TLR2KO and TLR4KO mice.

<p>Representative photomicrographs of CD31 (A) and SMA (B) staining in tibialis anterior muscle from the ischemic hind limb of control, TLR2 KO and TLR4 KO mice two weeks after femoral artery ligation (Scale bar = 25 µm). (C) Quantification of vessels measuring greater than 30 µm² is shown (N = 4 per group; *P<0.05, TLR2 KO vs. TLR4 KO or control).</p

Interplay between TLR4, TLR2, MyD88 and TRIF in mediating inflammation, muscle regeneration and angiogenesis after muscle ischemia.

<p>Interplay between TLR4, TLR2, MyD88 and TRIF in mediating inflammation, muscle regeneration and angiogenesis after muscle ischemia.</p

Hind limb perfusion was determined by LDPI measurements and presented as ratios that compared the perfusion of the ischemic to nonischemic limb.

<p>(A) The perfusion for control, TLR2 KO, TLR4 KO, MyD88KO and TRIFKO mice at 1 and 13 days after femoral artery ligation. (N = 4–8 per group) is shown (N = 5–7 per group). (B) Representative 13 day LDPI in experimental groups demonstrating extent of perfusion recovery after ischemia.</p

Skeletal muscle responses to hind limb ischemia at 2 weeks following femoral artery ligation.

<p>(A) Representative sections of tibialis anterior muscle from control C57B6, TLR2 KO, TLR4 KO, MyD88 KO and TRIF KO mice were stained with H&E. Arrow represents regenerating muscle fiber with centrally located nucleus. Arrowhead depicts areas of fat replacement. Asterisk indicates necrotic muscle fiber, characterized by eosinophilia, loss of muscle architecture and inflammatory infiltrate. (B) Hemorrhage into ischemic tissue was seen in TLR2KO mice (double arrow). Scale bar = 50 µm. (C) Erythrocytes (orange) are seen outside of vasculature (green) in TLR2KO mice. Red box demonstrates area of magnification shown in images below originals. Scale bar = 25 µm.</p

Necrosis, fat replacement and muscle regeneration in control, TLR2 KO, and TLR4 KO mice two weeks following femoral artery ligation.

<p>Quantification of area was performed on 4–5 non-overlapping images per section with 3 sections per animal. Results are presented as % of total muscle area (mean ± SEM; *P<0.03 vs. control and TLR4 KO, **P<0.05 vs. control and TLR4 KO).</p

The role of TLR2 and TLR4 in angiogenesis.

<p>(A–B) In vitro endothelial tube formation was assessed in HDMVECS seeded on Matrigel treated with nonspecific IgG, anti-TLR2 antibody (T2.5), or anti-TLR4 antibody (HTA 125). Tube formation was quantified after 6 hours by measuring individual tube lengths (4–5 images per well, N = 3 per treatment; *P<0.03 vs. IgG and HTA 125 treated cells). Explants of anterior tibialis muscle from control (N = 5), TLR2 KO and TLR4 KO mice (N = 3) were embedded in Matrigel and cultured in a standard incubator. Endothelial tube sprouting from the muscle explants was imaged at 10x (C) after three weeks of culture (scale bar = 100 µm).</p