Quantitative evaluation of skin disorders in type 1 diabetic mice by in vivo optical imaging

Abstract

Diabetes can affect the skin structure as well as the cutaneous vascular permeability. However, effective methods to quantitatively evaluate diabetes-induced skin disorders in vivo are still lacking. Here, we visualized the skin by using in vivo two-photon imaging and quantitatively evaluated the collagen morphology. The results indicated that diabetes could cause a significant reduction in the number of collagen fibers and lead to the disorder of skin collage fibers. And, the classic histological analysis also showed diabetes did lead to the change of skin filamentous structure. Additionally, the Evans Blue dye was used as an indicator to evaluate vascular permeability. We in vivo monitored cutaneous microvascular permeability by combining spectral imaging with the skin optical clearing method. This work is very useful for quantitative evaluation of skin disorders based on in vivo optical imaging, which has a great reference value in the clinical diagnosis.

Fig. 1

Two-photon imaging of skin in normal and T1D mice. (a) The image of each layer for the dorsal skin in normal and T1D mice from the skin surface to deep layer of the dermis. (b) and (c) were the fluorescence intensity profiles of normal and T1D mice, respectively (The green and blue curves are mean values, and the shadows represent standard error, n = 6). (d) The integrated fluorescence intensity profiles of normal and T1D mice (Error bars represent the standard error, n = 6). + p < 0.05 compared with the normal mice.

Fig. 2

The disorder degree of longitudinal orientated collagen of skin. (a) Typical maximum intensity projection maps (from 20 µm to 60 µm) of dermal SHG collagen in normal and T1D mice. (b) Quantitative evaluation of the skin collagen orientation from 0° to 180°corresponding to the blue and red regions of interest as indicated in (a) for normal mice and T1D mice. (The polar axis represents the proportion of collagen in each direction.)

Fig. 3

The texture analysis of skin SHG collagen images. The energy (a) and homogeneity (b) of SHG images at different dermal depths in normal and T1D mice. (The dotted lines represent the average values of gray-level co-occurrence matrix of SHG images in four directions. The red and blue shadows represent standard deviations.). The four-direction average values of energy (c) and homogeneity (d) of SHG maximum intensity projection maps from the normal and T1D mice. (Error bars represent the standard error, n = 6, + p < 0.05 compared with the normal mice.)

Fig. 4

The Masson staining of skin. (a) The images of Masson stained skin of normal and T1D mice. (b) The area ratio of collagen for normal and T1D mice. + p <0.01 compared with the normal mice (n = 9, mean ± standard deviations).

Fig. 5

The permeability of cutaneous microvessels. (a) The flow chart of monitoring the vascular EBd. (b) The skin microvascular EBd concentration maps obtained by spectral imaging with the help of in vivo skin optical clearing method (The colorbar represents the EBd concentration). The time-dependent changes of EBd concentration at vascular positions (c) and extravascular positions (d) (The vascular positions and extravascular positions were white and red rectangular areas as indicated in (b), respectively. Mean ± standard deviations). + + p <0.01 compared with EBd concentration at 5 min.