Dynamics of neutrophil infiltration during cutaneous wound healing and infection using fluorescence imaging - PubMed (original) (raw)

Dynamics of neutrophil infiltration during cutaneous wound healing and infection using fluorescence imaging

Min-Ho Kim et al. J Invest Dermatol. 2008 Jul.

Abstract

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations.

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Figures

Figure 1

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=100 μm. (e) Percentage of GFP+ monocyte/macrophage coexpressing F4/80 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.

Figure 2

Figure 2. Dynamics of neutrophil infiltration over time course of wound healing

(a) Time course of wound EGFP fluorescence during initial 24 hours 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.

Figure 3

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 (3 mm in radius) full thickness wound at 0, 24, and 48 hours 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_=3 mm) to center (_r_=0) within wound area at 0, 24, and 48 hours 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.

Figure 4

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±0.08×106PMN ml-1 for saline control, 0.21±0.01×106PMN ml-1 for GM-CSF, and 0.15±0.15×=106PMN ml-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.

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