Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging  Min-Ho Kim, Wei Liu, Dori L. Borjesson, Fitz-Roy E. Curry, Lloyd S. Miller, Ambrose L. Cheung, Fu-Tong Liu, R. Rivkah Isseroff, Scott I. Simon  Journal of Investigative Dermatology  Volume 128, Issue 7, Pages 1812-1820 (July 2008) DOI: 10.1038/sj.jid.5701223 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Tissue fluorescence intensity correlates with EGFP-PMN recruitment into the wound area. (a) Flow cytometric detection of bone marrow-isolated neutrophils (Gr-1-positive cells) expressing EGFP fluorescence. Left panel is an EGFP/anti-Gr-1-PE plot and right panel is a cell count histogram showing EGFP+ cells, in which Gr-1+ cells were gated to determine the percentage of EGFP+ cells from total Gr-1+ cells. Representatives of two separate experiments. (b) In vivo titration of bone marrow-isolated neutrophils. GFP fluorescent intensity correlates linearly with number of EGFP neutrophils placed in back skin wound site of wild-type mice (n=2, fluorescence intensity=241*PMN+7.57 × 107). Data are expressed as mean±SEM. (c) GFP fluorescence intensity correlates linearly with number of GFP+ cells in histological skin sections of wounded site viewed by fluorescence microscopy, in which counted GFP+ cells in histological sections of skin at days 0, 2, 5, 6, and 7 after wounding were correlated with GFP fluorescence intensity measured using Xenogen imaging system just before the preparation of histological skin sections. (d) Immunofluorescent detection of macrophages (F4/80+ cells, red) expressing EGFP fluorescence in histological sections of skin at days 0, 2, 5, 6, and 7 after wounding. White arrow indicates EGFP+ cells coexpressing F4/80 (shown as yellow). Bar=100μm. (e) Percentage of GFP+ monocyte/macrophage coexpressing F4/80 from total GFP+ cells. Either GFP+ or F4/80+ cells was counted from 5 to 6 different regions of each samples and average was taken for mean value. Journal of Investigative Dermatology 2008 128, 1812-1820DOI: (10.1038/sj.jid.5701223) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dynamics of neutrophil infiltration over time course of wound healing. (a) Time course of wound EGFP fluorescence during initial 24hours after wounding (n=4). (b) Time course of wound EGFP fluorescence during initial 10 days after wounding (n=5). (c) Representative fluorescent images of EGFP neutrophil infiltration during entire wound healing process. Data were expressed as means±SEM. Journal of Investigative Dermatology 2008 128, 1812-1820DOI: (10.1038/sj.jid.5701223) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Spatial mapping and lifetime of EGFP-PMN in the wound. (a) Representative fluorescent image of GFP intensity (photon per second per cm2 per sr) emitted from infiltrated EGFP-PMN in circular (3mm in radius) full thickness wound at 0, 24, and 48hours after wounding. Where r and dotted line indicate radius and boundary of wound edge, respectively. (b) Dynamic changes in number of EGFP-PMN per area at regions from edge (r=3mm) to center (r=0) within wound area at 0, 24, and 48hours after wounding (n=3). *Significant difference between r=3 vs r=2, r=1, and r=0mm (P<0.05). (c) Ex vivo time-dependent decay of GFP fluorescence emitted from bone marrow-isolated EGFP-PMN (1 × 106 cells) on back skin wound of WT mice. Fluorescence intensity in the presence of EGFP-PMN was normalized to the value before application (normalized fluorescence intensity=1.136 exp(-0.17t)-0.1892). Data are expressed as mean±SEM. Journal of Investigative Dermatology 2008 128, 1812-1820DOI: (10.1038/sj.jid.5701223) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 GM-CSF and S. aureus inoculation increase systemic neutrophil count and wound recruitment, but not wound healing time. (a) Dynamics of neutrophil infiltration over time course of wound healing for saline-injected control (n=5), GM-CSF (n=5), and S. aureus (n=4) groups. (b) Percentage increase of circulating neutrophil numbers from basal value at day 0 (baseline value: 0.28±0.08 × 106PMNml−1 for saline control, 0.21±0.01 × 106PMNml−1 for GM-CSF, and 0.15±0.15 × 106PMNml−1 for S. aureus group). (c) Dynamics of in vivo bioluminescence of actively metabolizing bacteria in wounded skin of EGFP mice inoculated with bioluminescent and non-bioluminescent strain of S. aureus. (d) Wound area over time course of wound healing for saline-injected control (n=5), GM-CSF (n=4), and S. aureus (n=4) groups. (e) Representative images of in vivo S. aureus bioluminescence and EGFP neutrophil fluorescence. Data are expressed as mean±SEM. Journal of Investigative Dermatology 2008 128, 1812-1820DOI: (10.1038/sj.jid.5701223) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions