Fig 1.
Micro-plasma diagnosis and illustrations of animal setting and treatment area.
(a) Plasma plume temperature versus supply power for 0.1% N2/Ar micro-plasma. Error bars indicated the standard deviation of the mean for n = 6 independent experiments. (b) Relative intensities of plasma species versus percentage (0, 0.1 and 0.2%) of N2 addition in Ar plasma, as determined from optical emission spectrum (OES). (c) Illustrations of micro-plasma system, target mouse, and OES system (1. hollow stainless steel inner electrode, 2. dielectric quartz tube, 3. outer copper electrode, 4. fiber optic thermometer, 5. OES device, 6. radio frequency power supply, and 7. mass flow controller). (d) Dorsal region treated with laser fluences can generate wound with ablative 3 mm × 20 mm column, penetrating the mid dermis. 5 mm × 2.5 mm treatment area was exposed to 0.1% N2/Ar micro-plasma.
Fig 2.
NO production in micro-plasma treated tissue lysate.
NO concentrations under micro-plasma and gas flow exposure for 30, 60, or 90 secs, respectively. The NT group was as an experimental control, because pure gas flow with varied exposure time did not have significant change with NT. NO concentrations are expressed as the means ± standard deviation of the mean (SD) (*p < 0.05 compared with all other groups).
Fig 3.
Laser-induced wounds were created on day 0 and measured over 21-day observation period. Representative photographs for (a) NT, (b) PT1, and (c) PT3. (d) Measurements of open surface in mice conducted on days 0, 3, 7, 14, and 21. Percentages of open surface are expressed as the means ± standard deviation of the mean (SD) (*p < 0.05 compared with all other groups) as a function of post wounding day (n = 6).
Fig 4.
The OCT images of wound tissue after micro-plasma treatment.
(a) Representative OCT images were acquired on days 3, 7, and 14 for each group. Specific characteristics of OCT images were identified as crust (white arrow), edema (green arrow), granulation (red arrow), and new epithelium (yellow arrow). (b) The backscattered light of OCT images was measured in ROI. Arbitrary units of integrated density are displayed as the means ± standard deviation of the mean (SD) (*p < 0.05 compared with all other groups) as a function of post wounding days (n = 6). Scale bar = 200 μm.
Fig 5.
Histological observations of all groups on days 3 and 7 after wound generation.
Wound tissue sections were stained with H&E. Red dotted lines demarcate central zone of laser ablation. On day 3, crusts (black arrows) could be seen in superficial layer in all groups. Inflammatory cells (green arrows) between crusted superficial layer and damaged dermal papillae were observed in all groups. Faster wound healing was found for PT3 group (indicated by earlier expression of new epithelium (yellow arrows)) compared to those for NT and PT1 groups. On day 7, crusts and inflammatory cells were still presented in the same area continuously in all groups. PT1 showed increased granulation tissue (red arrows) beneath damaged dermal papillae compared to that for NT. PT3 exhibited more visible new epithelium (yellow arrows) and granulation tissue (red arrows) in reticular dermis compared to those for NT and PT1. Scale bar = 200 μm.
Fig 6.
Immunohistochemical stain and semi-quantification of MMP-3 and laminin on days 3 and 7 after wound generation.
(a) MMP-3 was localized in all layers of wounded skin. Red dotted lines demarcate areas of reticular dermis on side of wound margin toward central zone of laser ablation. Inside red dotted areas, weaker staining of MMP-3 appeared for PT3 compared to that for NT and PT1 on days 3 and 7. (b) Laminin was localized between basal layer of epithelium and dermal papillae, displayed with a fibrous connective tissue as dermo-epidermal junction. Red dotted lines demarcate areas between superficial layer and reticular dermis on side of wound margin toward central zone of laser ablation. Laminin immunoreactivity was more intense in PT3 than in NT and PT1 on day 7. No difference was found between PT1 and PT3 on day 3. (c) Semi-quantification analysis of MMP-3 showed significant MMP-3 protein expression localized in demarcated (a) area on days 3 and 7. (d) Semi-quantification analysis of laminin showed significant laminin protein expression localized in demarcated (b) area on days 3 and 7. MMP-3 or laminin positive stained cells of total nuclei in (c-d) are shown as the means ± standard deviation of the mean (SD) (*p < 0.05 compared with all other groups) as a function of post wounding days 3 and 7 (n = 6). Scale bar = 100 μm.
Fig 7.
Assessment of blood flow of wound was detected by laser Doppler scanning.
(a) Representative blood flow cytometry images were obtained on days 7, 14, and 21 for each group. Red areas represent increased (normal) blood flow, and blue areas represent reduced (or non-existent) blood flow. (b) Quantitative data for (a) show blood flow in ROI through flux intensity. Arbitrary units of flux intensity are expressed as the means ± standard deviation of the mean (SD) (*p < 0.05 compared with all other groups) as a function of post wounding days (n = 6).