Fig. 4

a CD31 Immunofluorescence and DAPI Staining in T1DM and Control Rabbits. Scale bars in top-left corners indicate magnification. Dashed lines: Growth plate at metaphysis; White boxes: Neovascular sprouts on growth plate; Blue: DAPI; Red: CD31. a, e: DAPI staining in control and T1DM rabbits; b, f: CD31 staining in control and T1DM rabbits; c, g: Magnified CD31 staining in metaphysis of control and T1DM rabbits; d, h: Magnified CD31 staining in diaphysis of control and T1DM rabbits. b EMCN Immunofluorescence and DAPI Staining in T1DM and Control Rabbits. Scale bars in top-left corners indicate magnification. Dashed lines: Growth plate at metaphysis; Blue: DAPI; Cyan: EMCN. a, e: DAPI staining in control and T1DM rabbits; b, f: EMCN staining in control and T1DM rabbits; c, g: Magnified EMCN staining in metaphysis of control and T1DM rabbits; d, h: Magnified EMCN staining in diaphysis of control and T1DM rabbits. c CD31 + EMCN Dual Immunofluorescence in Metaphysis of T1DM and Control Rabbits. Scale bars in top-left corners indicate magnification. Dashed lines: Growth plate at metaphysis; Red: DAPI; Cyan: EMCN; Merge: CD31 + EMCN co-staining. d Estimated marginal means of CD31 and EMCN immunofluorescence intensity in T1DM and control rabbit bone marrow. Error bars represent 95% confidence intervals. T1DM rabbits showed significantly reduced intensity compared to controls for both markers (both p < 0.001). In both groups, metaphyseal values were significantly higher than diaphyseal values (both p < 0.001). e HIF-1α Immunofluorescence and DAPI Staining in Tibial Bone Marrow. Dashed lines: Growth plate at metaphysis; Red circles: Metaphysis; Yellow circles: Diaphysis; Blue: DAPI; Green: HIF-1α. a, e: Tibial DAPI staining in control and T1DM rabbits; b, f: Tibial HIF-1α staining in control and T1DM rabbits; c, g: Magnified HIF-1α staining in metaphysis of control and T1DM rabbits; d, h: Magnified HIF-1α staining in diaphysis of control and T1DM rabbits. f VEGF Immunofluorescence and DAPI Staining in Tibial Bone Marrow. Dashed lines: Growth plate at metaphysis; Red circles: Metaphysis; Yellow circles: Diaphysis; Blue: DAPI; Purple: VEGF. a, e: Tibial DAPI staining in control and T1DM rabbits; b, f: Tibial VEGF staining in control and T1DM rabbits; c, g: Magnified VEGF staining in metaphysis of control and T1DM rabbits; d, h: Magnified VEGF staining in diaphysis of control and T1DM rabbits. g Estimated marginal means of HIF-1α and VEGF immunofluorescence intensity and neovascular sprout quantity in T1DM and control rabbits. (A-B) Error bars represent 95% confidence intervals. T1DM rabbits showed significantly higher HIF-1α intensity (p < 0.001) and lower VEGF intensity (p < 0.001) compared to controls. (C) Neovascular sprout quantity (mean ± SEM) was significantly reduced in T1DM rabbits compared to controls (p < 0.001). Within the T1DM group, metaphyseal HIF-1α intensity was significantly higher than diaphyseal intensity (p < 0.001). In the control group, metaphyseal VEGF intensity was significantly higher than diaphyseal intensity (p < 0.001).