Fig 1.
Non-invasive detection of early stage of fetuses and enhanced visualization of fetal wound tissue.
(a) Ultrasound images of embryos at days E6.5, E13.5, and E15.5 (b) corresponding color-coded images for visual enhancement of tissue density in vivo, fetal positions shown by arrows. Wound-site marked with red arrow. Scale bar = 5 mm (c) Image panel showing fetal wound healing over time. Pre-wounding and post-wounding (3, 24, and 48h) (d) corresponding color-coded images are shown. Zoomed image of wounds at 3 and 48h post-wounding are marked by dotted circles as shown. Scale bar = 500 μm, n = 3. Wound-site marked with red arrow.
Fig 2.
Volumetric fetal wound measurement using three-dimensional (3D) reconstruction of ultrasound images.
(a) A 3D view of fetal wound (cyan) surface rendered 3D object constructed from 16 contours structure of fetal wound using the ultrasound B-mode images as shown by red arrow. (b) Semi-automated tracing of fetal wound border through different frames obtained from 3D visualization to measure wound volume (cyan). The tracing was performed manually in different frames of the 3D recorded video. The algorithm in ‘VevoStrain’ package then automatically traced the wound boarders on the frames that are in between the manually traced frames. (c) Line graph plot showing change in fetal wound volume over time at 3, 24, and 48h post-wounding. Data represented as the mean ± SE, n = 3 wounds. *p< 0.05.
Fig 3.
Histological characterization of fetal wound tissue depicted the re-epithelialization and vascularization of healing tissue.
(a) Microscopic image of hematoxylin and eosin (H&E) stained fetal wound tissue obtained at 48h post-wounding (at E17.5 post embryo formation), wound site is marked by dotted line and wound edge is marked by arrows (upper panel). Scale bar = 200 μm. Zoomed images from the wound edge and wound bed (lower panel). Scale bar = 20 μm. (b) Immunohistochemical staining of keratinocytes (Keratin 14, green) and nucleus of cell (DAPI, blue) of the fetal wound tissue obtained at 12 h and 48h post-wounding (upper panel), (Scale bar = 100 μm). Zoomed images (Scale bar = 20 μm). The break in the epithelium at 12 h panel and hyperproliferative epithelium at 48 h panel represented the site of wounding respectively.
Fig 4.
Noninvasive measurement of fetal cutaneous wound tissue stiffness.
(a) Region selected for fetal wound edge (red dot) and surrounding un-wounded skin (blue dot) to analyze biomechanical strain. A line was traced (green) around the wound site to help select the analysis sites using ‘Vevo Strain®’ software. (b) Strain curves of edge (red) and surrounding unwounded skin (blue) at different time intervals (3, 24 & 48h) post-wounding. (c) Fetal wound strain of wound edge, wound bed and normal skin was computed from the red, light green and blue curves respectively and the line graph was plotted over time (3h-48h for normal skin and wound edge and 24h-48h for wound bed). The standard error of mean of wound bed at time points 24h, and 48h are 0.001, and 0.001) respectively. Data represented as the mean ± SE, n = 3. *p< 0.05.
Fig 5.
Morphometry of adult diabetic cutaneous wound healing compared to the non-diabetic mice.
(a) Ultrasound B-mode image of full thickness stented wounds (d0,3,7,10,14) wound bed (red arrow, white dotted region), wound edge (yellow arrows) of db/db and control db/+ mice (b) Enhanced anatomical images using Matlab code to show the cellular density. Scale bar = 5 mm. Density index low (blue) to high (red) (c) Quantification of wound bed tissue cellularity over time in db/db and corresponding db/+ control. Data represented as the mean ± SE, n = 6. *p< 0.05. (d) Quantification of adult cutaneous wound volume over time. Data represented as the mean ± SE, n = 6. *p< 0.05.
Fig 6.
Non-invasive measurement of cutaneous wound tissue elastic-stiffness in adult mice.
Ultrasound imaging of adult murine normal skin and stented wounds tissue for the analysis of biomechanical property of db/+ mice (blue) and db/db mice (red). (a) Strain curves showing changes in wound edge tissue at 0, 3, 7, 10, and 14d post-wounding of db/+ mice and db/db mice. (b) Quantification of strains of normal skin (dotted lines, n = 3), wound edge of db/db and control db/+ mice post-wounding. Data represented as the mean ± SE, *p< 0.05, n = 6. (c) Strain curves showing changes in wound bed tissue at 0, 3, 7, 10, and 14d post-wounding of db/+ mice and db/db mice. (d) Quantification of normal skin (dotted lines, n = 3), wound bed strain of db/db and control db/+ mice post-wounding. Data represented as the mean ± SE, *p< 0.05, n = 6.
Fig 7.
Hemodynamics of fetal and adult cutaneous wound feeder vessels.
(a) Pulsed Doppler blood flow velocity profile of fetal cutaneous wounds at 3-, 24- and 48-hours post-wounding compared with pre-wounding condition. (b) Pulse pressure of feeder vessels unwounded and post-wounding plotted over time. Data presented as the mean ± SE. n = 3, *p< 0.05 compared to 3h. (c) Representative Doppler ultrasound images depicting blood flow in feeder vessel of adult cutaneous wound, db/+ (left panel) and db/db (right panel). Systolic peak shown with yellow arrow, diastolic peak shown in green. (d) Adult wound feeder artery pulse pressure db/+ (blue) and db/db (red) plotted as line graph over time. The comparison of pulse pressure was made between db/+ (blue line) and db/db (red line) at different time points. Data represented as the mean ± SE. n = 6. *, p< 0.05.