Figure 8. Systemic delivery using retro-orbital injection of recombinant annexin A6 protected against muscle damage in vivo.

(A) Wild-type mice were injected retro-orbitally (RO) with recombinant human annexin A6 (rANXA6) or control solution. Following this, muscles were damaged with cardiotoxin (CTX). (B and C) Immunofluorescence imaging revealed approximately 38% less dye uptake (red) in muscle pretreated with rANXA6. Dotted lines outline the tibialis anterior muscle sections (top panel). DAPI (blue) marks nuclei. Surface plots of dye uptake depict reduced fluorescence in muscle pretreated with rANXA6. (D) Whole-tissue spectroscopic analysis of injured gastrocnemius/soleus muscles revealed a 58% reduction in dye uptake with rANXA6 pretreatment compared with control muscle. Abs, absorbance at 620 nm. (E) Wild-type mice were injected intravenously with rANXA6 or control solution. Two hours later, tibialis anterior muscles were damaged with cardiotoxin. Muscles were harvested 7 days after injury. (F and G) Hematoxylin and eosin images were quantified and show a reduction in percentage myofiber damage (dotted lines), in rANXA6-treated mice compared with controls. Scale bars: 1 mm (B) and 500 μm (F). Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test. *P < 0.05. n = 3 mice, n = 6 legs per condition (B–D); n = 6 mice; n = 11 muscles per condition (F and G). EBD, Evans blue dye; TA, tibialis anterior.