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. 2017 Dec;14(6):1225-1236.
doi: 10.1111/iwj.12789. Epub 2017 Jul 20.

Changes in the extracellular matrix surrounding human chronic wounds revealed by 2-photon imaging

Jessica E S Sutcliffe  1 , Christopher Thrasivoulou  1 , Thomas E Serena  2 , Leigh Madden  3 , Toby Richards  4 , Anthony R J Phillips  5 , David L Becker  3   6
Affiliations

Changes in the extracellular matrix surrounding human chronic wounds revealed by 2-photon imaging

Jessica E S Sutcliffe et al. Int Wound J. 2017 Dec.

Abstract

Chronic wounds are a growing problem worldwide with no effective therapeutic treatments available. Our objective was to understand the composition of the dermal tissue surrounding venous leg ulcers and diabetic foot ulcers (DFU). We used novel 2-photon imaging techniques alongside classical histology to examine biopsies from the edges of two common types of chronic wound, venous leg ulcers and DFU. Compared to normal intact skin, we found that collagen levels are significantly reduced throughout the dermis of venous leg ulcer biopsies and DFU, with a reduction in both fibril thickness and abundance. Both wound types showed a significant reduction in elastin in the upper dermis, but in DFU, the loss was throughout the dermis. Loss of extracellular matrix correlated with high levels of CD68- and CD18-positive leukocytes. 2-photon imaging of the extracellular matrix in the intact tissue surrounding a chronic wound with a hand-held device may provide a useful clinical indicator on the healing progression or deterioration of these wounds.

Keywords: Chronic wounds; Extracellular matrix; Second harmonic imaging.

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Figures

Figure 1
Figure 1
2‐photon imaging of the extracellular matrix of normal arm and leg skin. (A) Second harmonic generation imaging of collagen (green, backscatter; red, forward detection) and autofluorescence imaging and elastin fibres and nuclei (blue) in normal arm and leg skin. (A) Montage of a 4‐mm biopsy of arm skin. (B) Lambda scans of the SHG of collagen signal and autofluorescence of elastin. (C) High‐power images of upper and lower dermis from leg skin. (D) Normal arm skin with Verhoeff van Gieson (VvG) staining collagen red and elastin black. Montage followed by high‐power images of the upper, lower and deep dermis. (E) Normal arm skin with Herovici staining differentiating between collagen type I, stained purple, and type III, blue. Scale bars A, 1 mm; C, D and E, 1 mm and 200 µm.
Figure 2
Figure 2
Imaging of the extracellular matrix of venous leg ulcer biopsies. (A) Second harmonic generation and autofluorescence imaging of collagen (green, backscatter; red, forward detection) and elastin fibres and nuclei (blue) in a venous leg ulcer biopsy. Montage of high‐power SHG and autofluorescence images of a 4‐mm skin biopsy from the edge of a venous leg ulcer. High‐power images of the upper, middle and lower dermis. (B) Verhoeff van Gieson (VvG) staining collagen red and elastin black in a sister section. Montage followed by high‐power images of the upper, middle and lower dermis. (C) Herovici staining differentiates between collagen type I, stained purple, and type III, blue. (D) Graphs quantifying SHG images and the % reduction in signal from collagen and elastin. Arrow and WE marks wound edge on the left side of the images. Error bars: mean ± SEM. Scale bars, montage 1 mm and high power 200 µm.
Figure 3
Figure 3
Imaging of the extracellular matrix of diabetic foot ulcer biopsies. (A) Second harmonic generation and autofluorescence imaging of collagen (green, backscatter; red, forward detection) and elastin fibres and nuclei (blue) in a diabetic foot ulcer. Montage of high‐power SHG and autofluorescence images of a 4‐mm skin biopsy from the edge of a diabetic foot ulcer. High‐power images of the upper and lower dermis. (B) Verhoeff van Gieson (VvG) staining collagen red and elastin black in a sister section. Montage followed by high‐power images of the upper and lower dermis. (C) Herovici staining differentiates between collagen type I, stained purple, and type III, blue. (D) Graphs quantifying SHG images and the % reduction in signal from collagen and elastin. Arrow and WE marks wound edge to the left of the image. Error bars: mean ± SEM. Scale bars, montage 1 mm and high power 200 µm.
Figure 4
Figure 4
Staining of CD68 and CD18 inflammatory cells in normal skin and venous leg ulcer biopsies. (A) Montage of a section from a 4‐mm biopsy from normal arm skin and (B) the edge of a venous leg ulcer stained for CD68 (purple). (C) Higher‐power images taken in the upper dermis at the wound edge (WE) 1 mm in and at the far edge (FE). Graphs showing counts of CD68‐positive cells demonstrated a significant (P < 0·001 WE & 1 mm and FE P < 0·01) elevation compared to normal skin biopsies. (D) Montage of a section from a 4‐mm biopsy from normal arm skin and (E) the edge of a venous leg ulcer stained for CD18 (purple). (F) Higher‐power images taken in the upper dermis at the wound edge (WE) 1 mm in and at the far edge (FE). (H) Graphs showing counts of CD18‐positive cells demonstrated a significant (P < 0·01 WE, P < 0·05 FE) elevation compared to normal skin biopsies. Error bars: mean ± SEM. Arrow and WE mark the wound edge. Scale bars, montage 500 µm and high power 100 µm.
Figure 5
Figure 5
Staining of CD68 and CD18 inflammatory cells in normal skin and diabetic foot ulcer biopsies. (A) Montage of a section from a 4‐mm biopsy from normal arm skin and (B) the edge of a diabetic foot ulcer stained for CD68 (purple). (C) Higher‐power images taken in the upper dermis at the wound edge (WE) 1 mm in and at the far edge (FE). Graphs of counts of CD68‐positive cells showed a significant (P < 0·05 WE) elevation compared to normal skin biopsies. (D) Montage of a section from a 4‐mm biopsy from normal arm skin and (E) the edge of a diabetic foot ulcer stained for CD18 (purple). (F) Higher‐power images taken in the upper dermis at the wound edge (WE) 1 mm in and at the far edge (FE). Graphs of counts of CD18‐positive cells showed a significant (P < 0·05 1 mm) elevation compared to normal skin biopsies. Error bars: mean ± SEM. Arrow and WE mark the wound edge. Scale bars, montage 500 µm and high power 100 µm.
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