Fig 1.
Definiens® analysis of NRP-1 expression in epidermis.
(A) Selection of epidermal area using Hoechst nuclear counterstain. (B) Selected epidermal area shown in NRP-1 layer. (C) Total epidermal area in orange. (D) NRP-1 positive marker area as recognized by software in red. Scale bar: 50μm.
Fig 2.
Definiens® analysis of NRP-1 expression in dermis.
(A) NRP-1 (red)/CD31 (green) double labeling immunofluorescence in the dermis of human skin biopsy. Dashed line represents the delineation of the ROI in dermis. (B) NRP-1 (green) immunoreactivity subtracted by Definiens® technology in delineated ROI (grey). (C) CD31 (orange) immunoreactivity subtracted by Definiens® technology in delineated ROI (grey). (D) NRP-1/CD31 double staining (white) subtracted by Definiens® technology in delineated ROI (grey). Scalebar: 50μm.
Table 1.
Baseline demographics.
Fig 3.
Accuracy of NRP-1 receptor antibody.
(A) Western blot analysis of NRP-1 transfected (+) HEK293 cells and wild-type (-) HEK293 cells. (B) NRP-1 immunohistochemistry performed on wild type (B1), NRP-1- (B2) and NRP-2- (B3) transfected HEK293 cells. Scale bar: 50μm.
Fig 4.
NRP-1 receptor expression related to epidermis and dermal vasculature in skin of healthy volunteers.
(A) NRP-1 receptor expression in human skin biopsy. NRP-1 was present in dermal vasculature (arrows) and basal keratinocytes of epidermis (asterix). (B and C) NRP-1 receptor (red) and CD31 (green) expression patterns in dermis of human skin biopsy. NRP-1 was present in dermal vasculature (yellow, co-localization of NRP-1 and CD31), adjacent to vessels (arrow; CD31, green) and in cells with a fibroblastic phenotype (asterix). Hoechst counterstain (blue). Scalebar: 20μm.
Fig 5.
NRP-1 Receptor expression in the epidermis of control, D1 capsaicin treated and DM Type 2 NP+ subjects.
NRP-1 receptor (red) and CD31 (green) expression pattern in epidermis as obtained by NRP-1/CD31 immunofluorescence in human skin biopsy of (A) healthy subject,(B) capsaicin treated healthy subject on day 1 (D1), (C) DM Type 2 NP+ diabetic subject. The lower panel (A2, B2, and C2) shows a grey scale image of NRP-1. Hoechst nuclear counterstain (blue). Scalebar: 20μm. (D) Multiple comparison graph of NRP-1 Marker area (%) in epidermis of control, D1 and DM Type 2 NP+ subjects. * p < 0.05.
Fig 6.
Definiens analysis of NRP-1 receptor expression in dermis.
(A) Number of NRP-1 positive structures (#/mm2) and (B) NRP-1 positive area/ROI (%) in dermis of controls, healthy volunteers who received topical capsaicin at day 1 (D1), variable time point (Dvar) and day 54 (D54) after capsaicin was applied. (C) Number of NRP-1 positive structures (#/mm2) and (D) NRP-1 positive area/ROI (%) in dermis of controls, diabetic subjects Type 1 (DM Type 1), Type 2 (DM Type 2) and diabetic subjects suffering from polyneuropathy (DM Type 2 NP+).
Table 2.
Definiens analysis results for dermal NRP-1 Receptor expression.
Fig 7.
Definiens analysis of vascular NRP-1 receptor expression in dermis.
(A) Number of NRP-1/CD31 positive structures (#/mm2) and (B) NRP-1/CD31 positive area/ROI (%) in dermis of controls, healthy volunteers who received topical capsaicin at day 1 (D1), a variable time point (Dvar) and day 54 (D54) after capsaicin was applied. (C) Number of NRP-1/CD31 positive structures (#/mm2) and (D) NRP-1/CD31 positive area/ROI (%) in dermis of controls, diabetic subjects Type 1 (DM Type 1), Type 2 (DM Type 2) and diabetic subjects suffering from polyneuropathy (DM Type 2 NP+).
Fig 8.
Definiens analysis of dermal vasculature.
(A) Number of CD31 positive structures (#/mm2); (B) CD31 positive area/ROI (%); (C) Sum of CD31 positive border in dermis of controls, healthy volunteers who received topical capsaicin at day 1 (D1), a variable time point (Dvar) and day 54 (D54) after capsaicin was applied. (D) Number of CD31 positive structures (#/mm2); (E) CD31 positive area/ROI (%); (F) Sum of CD31 positive border in dermis of controls, diabetic subjects Type 1 (DM Type 1), Type 2 (DM Type 2) and diabetic subjects suffering from polyneuropathy (DM Type 2 NP+).