The preceding data suggest immature retinal vessels may be particularly sensitive to LeTx. To test whether LeTx preferentially targets immature vessels, its effects on the retinas of P3 mice and P21 (fully mature) mice were compared. Examination of P3 retinas 4 days after LeTx treatment (P7) revealed a complete destruction of vessels (
Figs. 9,
9E). This effect persisted up to 8 days after LeTx administration (P11;
Supplementary Fig. S8). Histologic analysis revealed the vitreous cavity had filled with blood in LeTx-treated eyes compared with control-treated eyes, suggesting a loss of vascular integrity within the retina and continued destruction of developing vessels at this time (
Figs. 9B,
9F). Closer inspection of LeTx-treated retinas revealed a lack of organized superficial plexus formation compared with control-treated retinas (
Figs. 9C,
9G). Consistent with an earlier report,
40 these vessels contained both collagen IV and PDGFRβ staining in the superficial vessels (
Supplementary Figs. S7A, S7B). Interestingly, the extent of PDGFRβ staining in the superficial plexus at P3 looked remarkably like that of OIR P14 (compare
Supplementary Figs. S7B and S8B). Although the superficial staining of PDGFRβ at these immature and hypoxic time points was strong, covering the length of the superficial plexus, staining in the less hypoxic, more mature time points (OIR P17 [
Fig. 8D] and WT P14 [
Supplementary Fig. S7D]) was less intense and more sporadic over this region of the retina. This staining pattern suggests a shift in maturation level of the vessels, which correlates with LeTx sensitivity. Surprisingly, PDGFRβ was also found in regions of the retina that lacked collagen IV staining (open arrow in
Supplementary Fig. S7A), suggesting the recruitment of nonfunctional pericytes at this time. In contrast, LeTx had no effect on the organization of retinal vasculature when injected into the vitreous of adult (P21) mice (
Figs. 9D,
9H). Although it has been suggested that pericyte coverage (as demonstrated by PDGFRβ staining) alone is not sufficient to determine vessel maturation,
42 the correlation of PDGFRβ staining and LeTx sensitivity during OIR suggests that MKK activity is critical for activities associated with the formation of immature vessels in this model system.