DNA damage response in epidermis sections of 140 µm unit length after in vivo exposure to 50 Gy γ-irradiation studied by radiation-induced focus analysis.

<p>The mean and SD of 3 pigs are presented, except for the 20 h and 3 day values (2 pigs). (A) 4 h and 20 h post IR all cells contained RIF. This percentage dropped after 3days post IR. (B) Average number of γ-H2AX and 53BP1 RIF per cell in skin biopsies at the consecutive time points investigated. At 4 h post IR the focus number was derived from cells with distinct γ-H2AX labeling (see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039521#pone-0039521-g001" target="_blank">Fig.1A</a>). “Overlap” addresses foci with γ-H2AX and 53BP1 signal colocalization, which is complete at late time points. (C) Average foci numbers in keratinocytes that carry ≥1 RIF are significantly increased relative to control, 4 h and 20 h time points (**p<0.001). (D) Percentage of cells carrying ≥1 53BP1 RIF at 70 days post IR. There is a significant difference (**, p<0.001) between keratinocytes of the hair sheath (70d dermis) and all other conditions. (E) At ≥49 days post IR the mean 53BP1 foci diameter (maximum contour radius ± SD) is significantly increased (**, p<0.002). Error bars reflect differences of 3 different animals. Significant differences were noted ^a relative to control, ^b relative to 4 h, ^c to 3 days, ^e to 28 days, ^f to 49 days and, ^g relative to 70 days.</p

IR-induced changes of proliferation index (Ki67) and in epidermal cellularity as displayed by nuclear counterstaining (DAPI, blue).

<p>(A) Control minipig skin samples showing Ki67-positive cells (pink) at the basal layer of the densely populated epidermis. (B) Minipig skin sample 3 days post IR displaying a dearth of replicating cells, while at 28 (C), 49 (D) and 70 (E) days proliferating cells are abundant. (F) At 98 days epidermal hyperplasia was associated with a drop of replicating cell number. Magnification bar for all details: 20 µm. (G) Frequency of Ki67 positive (+) cells per unit area revealing a significant reduction (**p<0.001) of Ki-67-positive cells 3 days after IR relative to control and 4 h. A raise of replicating cells was noted ≥ 49 days post IR being significant at day 70 relative to control and the earlier time points. The fraction of replicating cells was significantly reduced at 96 days post IR, being associated with epithelia hyperplasia. (H) Epidermal cellularity during the time window investigated as determined by enumeration of the total number of DAPI-positive nuclei (blue, A-F) of keratinocytes per 169 µm unit contour length (average of 3 pigs). There is a significant decrease in the cellularity of the epidermis from 3 days up to 28 days post IR, as compared to control. A constant raise of the cellularity of the epidermis was observed from 28 days post irradiation onwards. The mean and SD of 3 pigs is given, except for 3 days (2 pigs); *, p<0.05; **, p<0.001. (G,H) Significant difference: ^a relative to control, ^b relative to 98 d, ^c relative to all other time points, ^d relative to 4 h, 3d, 48d and 72 days.</p

Average number of DSB-related foci/cell in pig epidermis.

<p>−, sample not available. Con, non-irradiated control.</p

Acute IR exposure of the skin leads to caspase-dependent apoptosis.

<p>(A) TUNEL staining renders the epidermis largely positive for fragmented DNA in control (con) and (B) 50Gy-exposed skin sections (4 h post IR). (C-F) Immunofluorescence for activated-caspase 3 (a.casp.3; red) and γ-H2AX (green). Nuclei are counterstained with DAPI (blue). (C) Unirradiated control is largely lacking activated caspase 3-positive cells. (D) Three days post IR numerous activated-caspase 3-positive cells are present in the basal layer of the epidermis. (E) At 28 days post IR only few cells are positive for activated caspase 3. (F) 98 days post IR activated-caspase 3-positive cells often occur in the spinous layer of the epidermis (nuclei, blue) relative to the earlier time points. Activated-caspase 3-positive apoptotic cells at late time points often are also strongly positive for γ-H2AX nuclear staining (green; yellow due to signal overlap). Magnification bar: 20 µm.</p

Epidermal and dermal radio-induced injuries in the skin of PBS-treated minipigs.

<p>Kinetic study from four iterative biopsies performed few days after each PBS-treatment in representative controls (magnification ×100). Progressive disorganisation of the epidermal layers, keratinocytes degeneration, vacuolation and barrier disruption. Numerous dermal cavities two weeks after the 4^th PBS-treatment.</p

Study design.

<p>Study design.</p

Engraftment of ASCs into the irradiated skin.

<p>Location of transplanted ASCs (in red) in the epidermis (A), in the dermis (B), and in the subcutis (C) of skin biopsies centred on injection plots, performed three days after the 3rd graft, on day 70. Brightly red fluorescing quantum dots marked ASCs (arrows) around blue nuclei (DAPI) near the cutis/subcutis border and among adipose tissue. The immunostaining of CD3 and lambda light chains (in pink) in the subcutis of healthy (D), PBS-treated (F), and ASC-grafted (F) skin underlined the infiltration by T and B lymphocytes three days after the ASC graft. Magnification: ×100.”</p

Decline of mean fpc values in PBL of ∼49 Gy PBI γ-exposed minipigs.

<p>Mean fpc values ± SD are shown. While the study comprises only 3 time points the shape of the obtained function is consistent with that known for DNA repair after <i>in vitro</i> irradiation of minipig cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087458#pone.0087458-Moroni2" target="_blank">[9]</a>.</p

ASC grafting stimulated cutaneous vascularization.

<p>The red immunofluorescent staining of Von Willebrand factor revealed the increase of the vasculature in the dermis of the ASC-grafted minipig (A) in comparison with PBS-treated control (B), two weeks after the fourth treatment (day119). Magnification: ×100.</p

After local irradiation ASCs favoured reepithelialisation and enhanced lymphocyte infiltration and angiogenesis in dermis.

<p>Kinetic study from four iterative biopsies performed few days after each ASC-graft in representative animals (magnification in the original ×100). Early cleaning of damaged epidermis was followed by complete epidermis recovery/hyperproliferative keratinocyte activity. Two weeks after the 4^th ASC-graft, early T and B lymphocyte infiltration in dermis near the subcutis, with a strong increase in vascularisation.</p