Figure 1.
SCD mice show distinctly abnormal and disorganized BM vasculature. (A) Representative 3D reconstructed images of femoral diaphyses from healthy control (AA) and SCD (SS) mice. High-resolution whole-mount confocal images were acquired using a Leica SP8X confocal microscope after staining femurs with artery/arteriole marker Sca-1 and sinusoid marker endoglin. Arteries (Sca-1hi large vessels), arterioles (Sca-1hi small vessels), and sinusoids (endoglin+) are identified based on markers and vessel sizes. Scale bars, 100 μm. (B) Quantification of the percentage of BM volume of segmented Sca-1+ arterioles/arteries was performed using Volocity (PerkinElmer) from 3D reconstructed confocal images, as shown in (A). Percentage of BM volume of Sca-1+ = total volume of Sca-1+/total BM volume × 100; n = 4 mice per group (11-13 weeks old; 2 female mice and 2 male mice). *P < .05, 2-tailed unpaired Student t test. (C) Quantification of Sca-1+ BM area from 2D LaSC images was performed using iCys software (CompuCyte), as shown in supplemental Figure 1A. Sca-1+ BM area (μm2) per 100 000 BM cells = (Sca-1+ event number) × (mean area of Sca-1+ events)/(DAPI+ cell number) × 100 000; n = 4 mice per group (9-16 weeks old). A comparison between male and female SS mice does not show a significant difference. *P < .05, **P < .01, 2-way analysis of variance with Tukey’s multiple-comparisons test. (D) Frozen thin sections (5 μm thickness) of SCD mouse femurs were stained with antibodies against α smooth muscle actin (αSMA) and endoglin, followed by nuclear dye DAPI staining. Neovascularized arterioles were identified by αSMA+ arteriolar pericytes and quantitated by LaSC in the diaphysis of SCD mouse bones costained with endoglin. BM medullary area is divided by endosteal region (ER; BM zone within 100 μm from the endosteal surface) and non-ER. Percentage of BM area of αSMA+ = total area of αSMA+/total BM area × 100, n = 4 mice per group (11-15 weeks old; 2 female mice and 2 male mice). *P < .05, Two-tailed unpaired Student t test with Bonferroni correction for multiple comparisons. Scale bars, 100 μm. (E) The sizes of sinusoid lumens were quantitated by 2D LaSC (iCys by CompuCyte) analysis with laminin marker; n = 4 mice per group (13-15 weeks old; 2 female mice and 2 male mice). Scale bars, 50 μm. All bar graphs show mean + standard error of the mean. **P < .01, 2-tailed unpaired Student t test. NS, not significant.

SCD mice show distinctly abnormal and disorganized BM vasculature. (A) Representative 3D reconstructed images of femoral diaphyses from healthy control (AA) and SCD (SS) mice. High-resolution whole-mount confocal images were acquired using a Leica SP8X confocal microscope after staining femurs with artery/arteriole marker Sca-1 and sinusoid marker endoglin. Arteries (Sca-1hi large vessels), arterioles (Sca-1hi small vessels), and sinusoids (endoglin+) are identified based on markers and vessel sizes. Scale bars, 100 μm. (B) Quantification of the percentage of BM volume of segmented Sca-1+ arterioles/arteries was performed using Volocity (PerkinElmer) from 3D reconstructed confocal images, as shown in (A). Percentage of BM volume of Sca-1+ = total volume of Sca-1+/total BM volume × 100; n = 4 mice per group (11-13 weeks old; 2 female mice and 2 male mice). *P < .05, 2-tailed unpaired Student t test. (C) Quantification of Sca-1+ BM area from 2D LaSC images was performed using iCys software (CompuCyte), as shown in supplemental Figure 1A. Sca-1+ BM area (μm2) per 100 000 BM cells = (Sca-1+ event number) × (mean area of Sca-1+ events)/(DAPI+ cell number) × 100 000; n = 4 mice per group (9-16 weeks old). A comparison between male and female SS mice does not show a significant difference. *P < .05, **P < .01, 2-way analysis of variance with Tukey’s multiple-comparisons test. (D) Frozen thin sections (5 μm thickness) of SCD mouse femurs were stained with antibodies against α smooth muscle actin (αSMA) and endoglin, followed by nuclear dye DAPI staining. Neovascularized arterioles were identified by αSMA+ arteriolar pericytes and quantitated by LaSC in the diaphysis of SCD mouse bones costained with endoglin. BM medullary area is divided by endosteal region (ER; BM zone within 100 μm from the endosteal surface) and non-ER. Percentage of BM area of αSMA+ = total area of αSMA+/total BM area × 100, n = 4 mice per group (11-15 weeks old; 2 female mice and 2 male mice). *P < .05, Two-tailed unpaired Student t test with Bonferroni correction for multiple comparisons. Scale bars, 100 μm. (E) The sizes of sinusoid lumens were quantitated by 2D LaSC (iCys by CompuCyte) analysis with laminin marker; n = 4 mice per group (13-15 weeks old; 2 female mice and 2 male mice). Scale bars, 50 μm. All bar graphs show mean + standard error of the mean. **P < .01, 2-tailed unpaired Student t test. NS, not significant.

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