Figure 4
Figure 4. Inhibition of CXCR4 results in morphologic defects in the developing retinal vasculature. Treatment with AMD3100 results in spiky appearance to the distal vasculature and a reduction in the lateral intercellular connections normally seen between tip cells. Newborn mice were treated with 40 mg/kg AMD3100 or PBS control (A-B) twice daily for 2 days. Retinas were harvested 48 hours after first injections and stained with fluorescently labeled isolectin and imaged using 5× (A; scale bar represents 500 μm) or 40× (B; scale bar represents 100 μm) objectives. For each of the measurements in panels C to F, a litter was divided into treatment groups, and 1 eye from each neonate was quantified. Typical results for 1 litter are shown. Average vascularized retinal area is moderately but not significantly reduced after AMD3100 treatment. One-way analysis of variance indicates P = .18 for this dataset (C). Filopodia numbers are significantly reduced in AMD3100-treated retinas; 40× images of the vascular periphery were acquired in z-stacks encompassing the full vascular layer. A maximum projection image of each stack was generated, and filopodia were counted manually using ImageJ software and normalized to the length of the distal vascular front (D). Numbers of unbranched vascular protrusions between 50 and 100 mm and greater than 100 mm in length are increased in AMD3100-treated retinas. Total counts of protrusions were identified for each retina in each of 3 length bins (30-50 μm, 50-100 μm, and > 100 μm). The Student t test was used to test whether the means of the distributions for the vehicle or AMD-treated counts were different for a particular length bin. The resulting P values were corrected for multiple testing using the Bonferroni method (E). Sholl analysis indicates significantly reduced vascular densities at 100 and 250 μm from the vascular front; these regions correlate with areas of CXCR4 expression in the retinal vasculature (F). Error bars represent SEM. Statistical analysis was performed using the unpaired Student t test assuming equal variance for pairwise comparison of treatment groups unless otherwise indicated: *P < .05, **P < .005.

Inhibition of CXCR4 results in morphologic defects in the developing retinal vasculature. Treatment with AMD3100 results in spiky appearance to the distal vasculature and a reduction in the lateral intercellular connections normally seen between tip cells. Newborn mice were treated with 40 mg/kg AMD3100 or PBS control (A-B) twice daily for 2 days. Retinas were harvested 48 hours after first injections and stained with fluorescently labeled isolectin and imaged using 5× (A; scale bar represents 500 μm) or 40× (B; scale bar represents 100 μm) objectives. For each of the measurements in panels C to F, a litter was divided into treatment groups, and 1 eye from each neonate was quantified. Typical results for 1 litter are shown. Average vascularized retinal area is moderately but not significantly reduced after AMD3100 treatment. One-way analysis of variance indicates P = .18 for this dataset (C). Filopodia numbers are significantly reduced in AMD3100-treated retinas; 40× images of the vascular periphery were acquired in z-stacks encompassing the full vascular layer. A maximum projection image of each stack was generated, and filopodia were counted manually using ImageJ software and normalized to the length of the distal vascular front (D). Numbers of unbranched vascular protrusions between 50 and 100 mm and greater than 100 mm in length are increased in AMD3100-treated retinas. Total counts of protrusions were identified for each retina in each of 3 length bins (30-50 μm, 50-100 μm, and > 100 μm). The Student t test was used to test whether the means of the distributions for the vehicle or AMD-treated counts were different for a particular length bin. The resulting P values were corrected for multiple testing using the Bonferroni method (E). Sholl analysis indicates significantly reduced vascular densities at 100 and 250 μm from the vascular front; these regions correlate with areas of CXCR4 expression in the retinal vasculature (F). Error bars represent SEM. Statistical analysis was performed using the unpaired Student t test assuming equal variance for pairwise comparison of treatment groups unless otherwise indicated: *P < .05, **P < .005.

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