The Effect of Fluence on Macrophage Kinetics,Oxidative Stress,and Wound Closure Using Real-Time In Vivo Imaging

Abstract

Objective:

The aim of our study was to quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging.

Background:

Photobiomodulation has been reported to reduce tissue inflammation and accelerate wound closure; however, precise parameters of laser settings to optimize macrophage behavior have not been established. We hypothesized that quantitative and real-time in vivo imaging could identify optimal fluence for macrophage migration, reduction of reactive oxygen species, and acceleration of wound closure.

Methods:

Larval zebrafish Tg(mpeg-dendra2) were loaded with dihydroethidium for oxidative stress detection. Fish were caudal fin injured, treated with 635 nm continuous 5 mW He:Ne laser irradiation at 3, 9, or 18 J/cm² and time-lapsed imaged within the first 120 min postinjury. Images taken 1 and 24-h postinjury were compared for percentage wound closure.

Results:

A fluence of 3 J/cm² demonstrated significant increases in macrophage migration speed, fewer stops along the way, and greatest directed migration toward the wound. These findings were associated with a significant reduction in wound content reactive oxygen species when compared with control wounded fins. Both 3 and 9 J/cm² significantly accelerated wound closure when compared with nonirradiated control fish.

Conclusions:

Wound macrophage activity could be manipulated by applied fluence, leading to reduced levels of wound reactive oxygen species and accelerated wound closure. The zebrafish model provides a means to quantitatively compare wound macrophage behavior in response to a variety of laser treatment parameters in real time.

Introduction

Resolution of the inflammatory phase of wound repair is highly dependent on the recruitment and function of macrophages and removal of excessive oxidative stress.^1,2 Excessive recruitment or prolonged retention results in a persistent proinflammatory state leading to deficient wound healing.³ In diseased states, macrophage dysfunction can result in the failure to adequately remove neutrophils and large number of free radicals, such as superoxide anion (O₂ ^−.) corresponding to oxidative stress.⁴ Excess ROS may impair macrophages, delaying the initiation of tissue repair while low threshold levels may also impair a prohealing macrophage alternative activation.⁵ As a result, augmenting the ability of macrophages to mitigate oxidative stress and facilitate reparative tasks is a sought-after target for therapeutic intervention^1,6_ENREF_2.

Photobiomodulation (PBM), most commonly delivered by red laser light near the 635 nm wavelength, has been reported to enhance immune cell phagocytosis and secretion of chemical cues, crucial to oxidative stress clearance.^{1,6,7

–13} Studies have further suggested that red laser light treatment promotes inflammatory resolution and wound closure.^14

–18 Extension of in vitro studies to clinical application has been promising;^19

–22 however, guidelines defining laser treatment settings of wavelength and duration are not yet established.²³ The wide range of energy fluence studied both ex vivo (1–60 J/cm²) and in vivo to enhance macrophage function suggests a need for real-time quantification of the therapeutic effects of PBM. Without such granular and in vivo contextual analyses, a consensus in defining precise parameters for low-level red laser may lack precision and reproducibility.^24,25

The motivation for our work was to establish an in vivo model to interrogate the effects of energy fluence on real-time measures of macrophage kinetics and tissue oxidative stress in the context of tissue injury. The zebrafish model presents powerful transgenic phenotypes to facilitate in vivo real-time characterization of macrophage recruitment and oxidative stress after injury.^{26

–33} Insights into leukocyte migration behavior and reactive oxygen species generation at zebrafish wound sites have yielded high impact discoveries, providing novel insights translatable to mammalian systems.^{31,32,34
–36} To simultaneously track individual macrophage trajectories and tissue oxidative stress levels, zebrafish transgenic for a fluorescent macrophage marker, Tg(mpeg-dendra2), underwent dihydroethidium (DHE) loading. Fish were injured at the caudal fin and treated with red 635 nm, 5 mW output power, at treatment doses supported by the literature^37
–39 0, 3, 9, and 18 J/cm². This model enabled quantitative studies of the effects of energy fluence from a constant wave source on macrophage responses and tissue oxidative stress.

Materials and Methods

Preparation of zebrafish

The Tg(mpeg:dendra2) zebrafish line, in which the promoter mpeg was used to drive dendra2 expression in macrophages,²⁷ was purchased through the Zebrafish International Resource Center (Cat.#ZL10389). Housing, water quality, and spawning were performed according to standard protocols.^40,41 E3 embryo medium was reconstituted in distilled water for a 1x solution (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) with 0.05% methylene blue for standard use with larval zebrafish for incubation at 28°C in 100 mm glass dishes. As per AVMA guidelines, larvae were anesthetized using buffered Tricaine, 3-aminobenzoic acid ethyl ester at certain time points as described hereunder (168 mg/L, adjusted to pH 7.0 using NaHCO₃; Sigma). Immediately following experimental procedures, larvae were humanely euthanized first with 400 mg/mL Tricaine solution and then 10% sodium hypochlorite solution as per 2013 AVMA guidelines. All animal procedures, conditions, and ethics described herein were approved by the Animal Care Committee at the University of Illinois at Chicago.

ROS probe loading

Dechorionated zebrafish larvae at 3 days postfertilization (dpf) were placed in 75 mm glass dishes with 5 mL of E3 medium. We used the following inclusion criterion for fish for study entry: demonstrated fluorescence, normal physiology, and no visible caudal fin nicks or wounds using a Zeiss inverted epifluorescent microscope. DHE (Sigma) dye was reconstituted in 1 mL of anhydrous dimethyl sulfoxide (Sigma-Aldrich) at 10 mM and stored in aliquots. Zebrafish, in groups of 8 to 15, were transferred to a 35 mm diameter glass bottom dish (MATEK) supplemented with 35 μM DHE solution in 1x Dulbecco's phosphate-buffered saline and incubated for 40 min in the absence of light, then transferred to a separate 75 mm glass dish, and washed with prewarmed E3 medium to remove excess DHE.

Wound placement

After ROS loading, zebrafish larvae were anesthetized (168 mg/L, Tricaine), immobilized (1% low melting point agarose solution), and underwent single caudal fin incisions (through 28 gauge sterile needle). Animals that did not meet uniformity in wound perimeter size (320 ± 50 μm), distance from notochord, or demonstrated small breaks or nicks in the embryonic outline were euthanized and not included in subsequent stages of the study.

Laser irradiation for time-lapse imaging

PBM laser treatment was administered through a research grade 5 mW (laser Power Source box: Zerona Medical Laser, Model #: EML-2245; Erchonia Corporation, McKinney, TX) emitting 635 nm continuous wave. Laser spot was elliptical at 0.25 cm² (4 × 2 mm) at the delivery site (Fig. 1). Laser dosing was quantified in energy fluency (unit Joules/cm²) equivalent, Table 1. Larvae were randomly assigned to five treatment groups: (1) no laser, no injury, (2) 0 J/cm² with injury, (3) 3 J/cm² laser dose with injury, (4) 9 J/cm² laser dose with injury, and (5) 18 J/cm² laser dose with injury. Laser irradiation was initiated 5–15 min postinjury.

FIG. 1.

Photobiomodulation therapy schematic shows laser treatment with a continuous laser of 635 nm wavelength at 5 mW output power. Laser is applied directly to the glass of 35 mm glass bottom dish to treat fish. The entirety of the fish with a spot size of 0.25 cm² is treated with either 150, 450, or 900 sec of irradiation for an energy fluence of 3, 9, and 18 J/cm², respectively.

Table 1.

Laser Treatment Parameters at Target

Treatment groups by energy fluence (J/cm²)	0 J/cm²	3 J/cm²	9 J/cm²	18 J/cm²
Beam spot size (cm²)	n/a	0.25	0.25	0.25
Irradiance (mW/cm²)	0	20	20	20
Radiant energy (J)	0	0.75	2.25	4.5
Number of points irradiated	n/a	1	1	1
Number of treatment sessions	0	1	1	1

n/a, not applicable.

Time-lapse imaging

All time-lapse images were acquired using ZEN^® software with a Zeiss LSM 510 confocal microscope, as described previously.³³ Fluorescent excitations 488 nm for dendra 2 and 561 nm for ethidium were targeted at 0.80 and 0.08 mW, respectively, through ZEN software. Time-lapse images were taken in 60–75 sec intervals from up to 25–150 min postinjury. Up to eight fish were imaged per session with at least one nonwound for group baseline comparative analysis.

Analysis of time-lapse images

All time-lapse images were analyzed using open source Zirmi software in MATLAB R2015a, as described previously.³³ Quantification of cell kinetics was based on centroid positions, not whole cell morphology. Macrophage centroid positions were tracked using PhagoSight software's keyhole algorithm and visualization tools for proofing.⁴² Zirmi software was used to select macrophage tracks that matched a selection criterion or of distinguishable centroid positions in >70% of all frames and categorized as static when movement did not exceed 0.9 μm. Error-prone tracks of macrophages near wound region were eliminated from analysis if they aggregated together. Macrophage migration was analyzed 30–120 min postinjury. Time intervals were defined as T1 [30–60 min postinjury], T2 [30–90 min postinjury], and, an overview time interval, T3 [30–120 min postinjury]. To analyze the extracted data more efficiently, wound regions were normalized, orienting positions with respect to the epicenter or zero position (S_o) of the wound margin for discrete distance to wound determinations.³³ In unwounded fish and for baseline measurements, the most distal position of the caudal fin was selected as the zero position. Zirmi software was used to manually trace wound region to isolate background subtracted DHE mean fluorescent intensity values ∼65 μm radially extended from the wound margin.³³

Fin regeneration assay

Fish were imaged using a Zeiss inverted epifluorescent microscope in the first 60 min postinjury. Immediately after, fish were treated with laser 0 (n = 8), 3 (n = 12), 9 (n = 9), and 18 (n = 10) J/cm² doses. After treatment, fish were maintained at 28°C in E3 medium and, 24 h after injury, were anesthetized and imaged again. Total wound closure was reported as percentage of wound closure. The wound perimeter was measured using ImageJ (NIH) for 1- and 24-h fins per fish. Percent closure (R%) was an evaluation of 24-h wound closure defined as \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} R \% = 100 \; { \times } \; { \frac { 1 \;h \;wound \;perimeter - 24 \;h \;wound \;perimeter } { 1 \;h \;wound \;perimeter } } \tag { 1 } \end{align*} \end{document}

Statistical analysis

Statistical analyses were performed in GraphPad Prism and Microsoft Excel. Linear correlations were performed to derive Pearson r coefficients. p values were used for testing the hypothesis that there is no relationship between the observed phenomenon (null hypothesis). p values <0.05 were considered significant.

Results

Laser dose–response of oxidative stress after wounding

Control (Fig. 2A) untreated fish demonstrated higher pixel (red) intensity values around the margin of injury (Fig. 2B) than red laser-treated 3 J/cm² fish (Fig. 2C, D). DHE mean intensity values were used to calculate geometric means and 95% confidence intervals at 60 and 120 min postinjury of control (n = 21) and dose groups 3 J/cm² (n = 11), 9 J/cm² (n = 8), and 18 J/cm² (n = 8; Fig. 2E). At 60 min postinjury, a reverse dose trend was noted as 3 J/cm² exhibited an overall 29% ROS reduction in wound region (11,013.4 ± 2,000.59, p = 0.03) compared with control fish (13,599.55 ± 1,563.5). In contrast, 9 J/cm² exhibited values that were near control [14,108.78 ± 2,456.05, p = not significant (n.s.)] and 18 J/cm² exhibited an overall 19% increase in ROS values (16,337.55 ± 2,893.64, p = n.s.) at near wound region compared with control. At 120 min postinjury, the 3 J/cm² laser-treated group mean ROS remained significantly reduced when compared with the control group (p = 0.01), whereas the 9 J/cm² laser-treated group had no significant effect (p = n.s.), and the 18 J/cm²laser-treated group showed significantly increased ROS (p = 0.02). In addition, at 120 min postinjury, a more significant reverse dose–response was noted as mean oxidative stress in the 3 J/cm² dose group (10,190.45 ± 1,674.98) was significantly less than that in the 9 J/cm² dose group (26%, 13,753 ± 2,527.06, p = 0.02) and was significantly less than that in the 18 J/cm² dose group (37%, 16,161.02 ± 2,416.72, p < 0.001).

FIG. 2.

(A) and (B) display bright field image of fish after 60 min postinjury of a wounded untreated control and of a wounded and treated fish with 3 J/cm², respectively. (C) and (D) display a color map of the ROS probe (DHE) intensity; red hue indicating saturated DHE and blue hue indicated absence of DHE. Fish of untreated wounds 0 J/cm² (n = 21) were compared with laser-treated wounds: 3 J/cm² (n = 11), 9 J/cm² (n = 8), and 18 J/cm² (n = 8). (E) displays group overall geometric mean ± 95% confidence intervals of DHE mean intensity values at 60 and 120 min postinjury. Student's t-tests (*p < 0.05), (**p < 0.01), (***p < 0.001) were performed in Microsoft Excel. DHE, dihydroethidium; ROS.

Laser dosing exhibits different levels of macrophage kinetics in response to wounding

We investigated the following number of available individual macrophages and their trajectories through three time periods [T₁, T₂, and T₃]: 0 J/cm² (n = 19; 227 macrophages tracked), 3 J/cm² (n = 12; 196 macrophages tracked), 9 J/cm² (n = 10; 96 macrophages tracked), and 18 J/cm² (n = 8; 118 macrophages tracked). When compared with control, 3 J/cm² macrophages demonstrated significantly more wound-directed migration (Fig. 3A, B). Speed (Fig. 3C) and meandering index (Fig. 3D) were used for determining persistent travel. Cumulatively, 3 J/cm² macrophages exhibited significantly faster speeds (T₃: 4.5 ± 0.7 vs. 3.9 ± 0.5 μm/min, p = 0.01) and more “straightness” or higher meandering index (T₃: 0.44 ± 0.09 vs. 0.37 ± 0.09, p = 0.04) than nontreated control fish. Higher static ratio (Fig. 3E) was used to provide more insight into the pattern of travel, to provide insights into “pausing” and henceforth efficiency in travel. Compared with controls (T₃: 0.177 ± 0.05, p = 0.03), 3 J/cm² exhibited significantly less “pausing” overall (T₃: 0.135 ± 0.04, p = 0.03), whereas 9 J/cm² (T₃: 0.139 ± 0.04, p = 0.1) and 18 J/cm² (T₃: 0.154 ± 0.02, p = 0.25) were only slightly less than control in a dose-dependent manner. Because persistence through speed, meandering index, and static ratio does not demonstrate a migration bias but instead reflects a consistent direction orientation, we used vector distances from macrophage centroid positions to the centroid position of the wound gap to identify and compare movement orientation to the site of injury. Higher net distances traveled toward the wound were used to indicate a preferential wound travel behavior or migration bias (Fig. 3F). Laser demonstrated significantly higher net distances toward wound in 3 J/cm² overall (T₃: 125 ± 50 μm) when compared with 9 J/cm² (T₃: 92 ± 52 μm, p = 0.8) and when compared with nontreated fish (T₃: 89 ± 38 μm, p = 0.02). The 18 J/cm² laser-treated group demonstrated an overall decrease in macrophage wound-oriented net distances (T₃: 75 ± 38, p = 0.2).

FIG. 3.

Wound recruited macrophages of 0, 3, 9, 18 J/cm² treatment groups of fish (n = 20, 12, 10, and 8, respectively) were tracked using software analysis. Representative control fish (A) and 3 J/cm² (B) are shown with trajectories with the highest total distances hued as dark red and lowest values hued as dark blue. Quantitative measures were algorithmically derived at T1 (30–60 min postinjury), T2 (30–90 min postinjury), and T2 (30–120 min postinjury) temporal domains postinjury with means and 95% CIs for each independent group measure. Macrophage measures of absolute velocity (C) meandering index (D) were determined to evaluate persistence. Wound-oriented net distance traveled (E) and static ratio (F) were determined to evaluate wound bias efficiency. Statistical differences were computed with Student's t-tests (*p < 0.05), (**p < 0.01) in Graphpad^® Prism.

Laser dose–response of 24-h wound closure

To examine the 24-h regenerative capacity of the caudal fin, wounds were measured 1-h (Fig. 4A, B) and 24-h postinjury (Fig. 4C, D). Single wounds were induced and randomly assigned to laser treatment. The following mean baseline wound perimeters were measured at 1-h postinjury: control 0 J/cm² wound margin 343 ± 102 μm (n = 8), 3 J/cm² wound margin 309 ± 64 μm (n = 12), 9 J/cm² wound margin 318 ± 81 μm (n = 9), and 18 J/cm² wound margin 318 ± 88 μm (n = 10). To ensure 1 h wound margin size did not influence 24 h wound size outcomes, a linear regression was performed indicating no correlation with 1 h wound size and 24 wound size per group: control (r = 0.22, p = n.s.), 3 J/cm² (r = 0.14, p = n.s.), 9 J/cm² (r = 0.37, p = n.s.), and 18 J/cm² (r = 0.09, p = n.s.). All laser-treated fish exhibited accelerated wound closure outcomes 24 h postinjury when compared with nonirradiated controls. Percentage wound closure (geometric mean ± 95% confidence interval) of unirradiated controls (38% ± 8%) was compared with mean closure for each treatment group: 3 J/cm² (51% ± 12%, p = 0.02), 9 J/cm² (48% ± 11%, p = 0.04), and 18 J/cm² (45% ± 12%, p = n.s.; Fig. 4E). These results indicate that a fluence of <9 J/cm² may be an ideal therapeutic threshold for zebrafish caudal fin-accelerated wound closure.

FIG. 4.

(A) and (B) show caudal fin wound region 1 h post wounding. (C) and (D) display the wound region 24 h post wounding, untreated and treated with (3 J/cm²) laser, respectively. (E) displays the geometric mean ± 95% confidence interval of percentage wound closure from 1 to 24 h postwound of untreated control group (n = 8) and 3, 9, and 18 J/cm² laser-treated groups (n = 12, 9, and 10, respectively). Student's t-tests (*p < 0.05) were performed in Microsoft Excel and wound measurements were performed in ImageJ.

Discussion

The zebrafish model facilitated the real-time detection of a dose–response in wound-oriented macrophage trajectories and oxidative stress. A dose of 3 J/cm² decreased wound oxidative stress and elevated macrophage speeds and meandering index values for a resultant contribution for more “straightness” and less “pausing” in migration when compared with either nontreated or higher dose-treated groups. Significantly, a dose of 18 J/cm² resulted in slower macrophage responses and highest oxidative stress values at the wound region. The 9 J/cm² cell kinetic measures consistently fell between both high and low energy fluence. Both 3 and 9 J/cm² demonstrated significantly greater wound closure than nontreated controls. Given the deleterious effects of excessive reactive oxygen species on wound closure rate observed in clinical practice,⁴³ our results support the need for a selection of laser dose to quantitatively optimize macrophage activity for the reduction of ROS and facilitation of wound closure.

Based on the large data sets involved in time-lapse imaging, this study focused on macrophage initial recruitment; selection of the quantitative measures of migration was important for quantifying an elicited effect on cellular kinetics. Our study outlines macrophage kinetics speeds, 3–6 μm/min on average, and meandering index, 0.4 to 0.6, that fall within the range in zebrafish literature.^27,44 However, the time postinjury is important in considering the average macrophage migration behavior. Initial recruitment 30–60 min postinjury demonstrated elevated speeds and less meandering for all zebrafish groups. These values lessened as time progressed. Laser-treated zebrafish did not appear to experience supernatural kinetic behavior; speeds, for instance, did not exceed the range of macrophage ranges previously reported. Macrophage static ratios were significantly correlated to respective speeds (for 3 J/cm² group, r = −0.56, p < 0.01). Laser-treated macrophages with 3 J/cm² demonstrated more movement, shown by decreased static ratio, implicating macrophages were simply more active rather than traveling at abnormally faster speeds frame-by-frame.

It is well accepted that speed and meandering index can be used for determining persistence, also defined as the period by which a cell moves in the same direction.⁴⁵ The 3 J/cm² laser-treated group demonstrated considerably higher net distances toward wound and higher meandering index value. These macrophages demonstrated a preferential wound migration bias with less “stopping” and lower static ratios than the untreated controls, indicating more persistent or efficient wound-oriented tracks. Recently, reports have highlighted the role of enhanced levels of ATP, as a means of stimulating changes in speed and migration behaviors of immune cells.^46
–48 Since laser treatment has been linked to enhanced ATP production,^49

–52 it is possible that increased available ATP spurred increased macrophage persistence.⁵³ Together our findings coupled with previous reports support the notion that application of 635 nm energy at a fluence of 3 J/cm² provides a threshold of utilizable energy that promotes greater macrophage activity, increased efficiencies of action, and a reduction in the time required for wound closure. The dose-dependent relationship may suggest that higher dose ranges are not as optimal as lower dose ranges for facilitating the resolution of early inflammatory events.

It is possible that increased macrophage cellular kinetics may play a direct role in the reduction of oxidative stress by hastening the pace of neutrophil clearance. Karu et al. reported that red laser (638.2 nm) at low energy fluence (0.01 to 0.03 J/cm²) could induce respiratory burst, important for clearing debris, in murine phagocytes in vitro measured by chemiluminescence.¹⁰ Lu et al. demonstrated that red laser (632.8 nm) at lower energy fluence (1 and 2 J/cm²) increased mouse peritoneal macrophage phagocytosis of microspheres in vitro when compared with nonirradiated controls.¹¹ Additional findings demonstrate a reduction in proinflammatory macrophage behavior after red laser (660 nm at 4.5 J/cm²),¹² commonly tied to reactive oxygen species generation. It is possible that in vivo reduction of oxidative stress is a consequence of decreased neutrophil persistence and increased clearance.

Our findings agree with previous reports demonstrating enhanced wound closure after red laser treatment; however, wavelength and fluence varied. Rats treated with 660 nm at 4 J/cm² demonstrated accelerated dermal healing at 7 and 14 days postinjury.¹⁴ Alternatively, a review of laser treatments in rats demonstrated reductions in wound size and inflammatory infiltrates⁵⁴; however, these findings varied in dosing parameters (600–1000 nm; 1–60 J/cm²). Such widened parameters may be optimized for improved and perhaps more consistent therapeutic results.

We conclude that the zebrafish model provides a means to quantitatively test real-time macrophage responses to a variety of laser settings, using ROS and wound closure as clinically relevant outcome measures. Investigations examining each parameter of PBMT on macrophage and ROS behavior individually and in concert with each other in zebrafish provide a new means of defining this therapy. Previously, laser parameters tested comparatively in vivo using real-time quantitative methods have been difficult to accomplish. Future zebrafish investigations could provide direct comparisons of effects per laser-specific attribute such as constant versus intermittent energy delivery and differences in wavelength. The model can be interrogated to characterize macrophage polarization, neutrophil migration, and neutrophil and ROS clearance for optimization of the therapeutic in inflammatory conditions. Reduction of oxidative stress and facilitated tissue repair with PBMT would be clinically relevant for the reduction of ischemia–reperfusion injury after organ preservation or reperfusion injury after myocardial infarction, as well as a variety of additional macrophage-driven inflammatory conditions.

Footnotes

Acknowledgments

A.D.P. was funded by Diversifying Higher Education Faculty in Illinois Program Fellowship. This study was funded by a grant from the UIC Department of Surgery. We would like to thank Steve Shanks, Erchonia, for the use of the laser.

Author Disclosure Statement

No competing financial interests exist.

References

Koh

, DiPietro

. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med, 2011; 13:e23.

Ross

, Odland

. HUMAN WOUND REPAIR: II. Inflammatory cells, epithelial-mesenchymal interrelations, and fibrogenesis. J Cell Biol, 1968; 39:152–168.

Mirza

, Koh

. Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice. Cytokine, 2011; 56:256–264.

Ushio-Fukai

. Compartmentalization of redox signaling through NADPH oxidase–derived ROS. Antioxid Redox Signal, 2009; 11:1289–1299.

Zhang

, Choksi

, Chen

, Pobezinskaya

, Linnoila

, Liu

Z-G

. ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages. Cell Res, 2013; 23:898–914.

Silva

. Macrophage phagocytosis of neutrophils at inflammatory/infectious foci: a cooperative mechanism in the control of infection and infectious inflammation. J Leukoc Biol, 2011; 89:675–683.

Wynn

, Chawla

, Pollard

. Macrophage biology in development, homeostasis and disease. Nature, 2013; 496:445–455.

Chawla

, Nguyen

, Goh

. Macrophage-mediated inflammation in metabolic disease. Nat Rev Immunol, 2011; 11:738–749.

Young

, Bolton

, Dyson

, Harvey

, Diamantopoulos

. Macrophage responsiveness to light therapy. Lasers Surg Med, 1989; 9:497–505.

10.

Karu

, Ryabykh

, Fedoseyeva

, Puchkova

. Helium-neon laser-induced respiratory burst of phagocytic cells. Lasers Surg Med, 1989; 9:585–588.

11.

, Song

, Tang

, Zhou

. Effect of low power laser irradiation on macrophage phagocytic capacity. Proc. SPIE 7900, Biophotonics and Immune Responses VI, 79000H (10 February 2011); doi: 10.1117/12.874325; https://doi.org/10.1117/12.874325.

12.

Souza

NHC

, Marcondes

, Albertini

, Mesquita-Ferrari

, Fernandes

KPS

, Aimbire

. Low-level laser therapy suppresses the oxidative stress-induced glucocorticoids resistance in U937 cells: relevance to cytokine secretion and histone deacetylase in alveolar macrophages. J Photochem Photobiol B, 2014; 130:327–336.

13.

Fernandes

, Souza

, Mesquita-Ferrari

, et al. Photobiomodulation with 660-nm and 780-nm laser on activated J774 macrophage-like cells: effect on M1 inflammatory markers. J Photochem Photobiol B, 2015; 153:344–351.

14.

de Medeiros

, Araújo-Filho

, da Silva

, et al. Effect of low-level laser therapy on angiogenesis and matrix metalloproteinase-2 immunoexpression in wound repair. Lasers Med Sci, 2017; 32:35–43.

15.

Suzuki

, Takakuda

. Wound healing efficacy of a 660-nm diode laser in a rat incisional wound model. Lasers Med Sci, 2016; 31:1683–1689.

16.

Figurová

, Ledecký

, Karasová

, et al. Histological assessment of a combined low-level laser/light-emitting diode therapy (685 nm/470 nm) for sutured skin incisions in a porcine model: a short report. Photomed Laser Surg, 2016; 34:53–55.

17.

Houreld

. Irradiation at 660 nm modulates different genes central to wound healing in wounded and diabetic wounded cell models. Proc. SPIE, 8932, Mechanisms for Low-Light Therapy IX, 89320A (18 February 2014); doi: 10.1117/12.2041430; https://doi.org/10.1117/12.2041430.

18.

Dawood

, Salman

. Low level diode laser accelerates wound healing. Lasers Med Sci, 2013; 28:941–945.

19.

Kazemi-Khoo

. Successful treatment of diabetic foot ulcers with low-level laser therapy. Foot, 2006; 16:184–187.

20.

Houreld

, Abrahamse

. Low-level laser therapy for diabetic foot wound healing. The Diabetic Foot. 2005.

21.

Heidari

, Paknejad

, Jamali

, Nokhbatolfoghahaei

, Fekrazad

, Moslemi

. Effect of laser photobiomodulation on wound healing and postoperative pain following free gingival graft: a split-mouth triple-blind randomized controlled clinical trial. J Photochem Photobiol B, 2017; 172:109–114.

22.

Vaghardoost

, Momeni

, Kazemikhoo

, et al. Effect of low-level laser therapy on the healing process of donor site in patients with grade 3 burn ulcer after skin graft surgery (a randomized clinical trial). Lasers Med Sci, 2018; 33:603–607.

23.

Tchanque-Fossuo

, Ho

, Dahle

, et al. A systematic review of low-level light therapy for treatment of diabetic foot ulcer. Wound Repair Regen, 2016; 24:418–426.

24.

Wang

, Yuan

, Zhang

, Dong

, Mao

, Hu

. Phototherapy for treating foot ulcers in people with diabetes. Cochrane Database Syst Rev, 2017 Jun 28; 6:CD011979. doi: 10.1002/14651858. CD011979. pub2.

25.

Tunér

. The laser wound healing contradiction. Photomed Laser Surg, 2015; 33:343–344.

26.

Renshaw

, Trede

. A model 450 million years in the making: zebrafish and vertebrate immunity. Dis Model Mech, 2012; 5:38–47.

27.

Ellett

, Pase

, Hayman

, Andrianopoulos

, Lieschke

. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood, 2011; 117:e49–e56.

28.

Hall

, Flores

, Crosier

. Live cell imaging of zebrafish leukocytes. Methods Mol Biol, 2009; 546:255–271.

29.

Cvejic

, Hall

, Bak-Maier

, et al. Analysis of WASp function during the wound inflammatory response—live-imaging studies in zebrafish larvae. J Cell Sci, 2008; 121:3196–3206.

30.

Walters

, Green

, Surfus

, Yoo

, Huttenlocher

. Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome. Blood, 2010; 116:2803–2811.

31.

Niethammer

, Grabher

, Look

, Mitchison

. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature, 2009; 459:996–999.

32.

Yoo

, Starnes

, Deng

, Huttenlocher

. Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature, 2011; 480:109–112.

33.

Paredes

, Benavidez

, Cheng

, Mangos

, Donoghue

, Bartholomew

. An automated quantitative image analysis pipeline of in vivo oxidative stress and macrophage kinetics. J Biol Methods, 2018; 5:e101. DOI: 10.1444/0/jbm.2018.259.

34.

Lisse

, King

, Rieger

. Comparative transcriptomic profiling of hydrogen peroxide signaling networks in zebrafish and human keratinocytes: implications toward conservation, migration and wound healing. Sci Rep, 2016; 6:20328.

35.

Oliveira

, Boudinot

, Calado Â, Mulero

. Duox1-derived H2O2 modulates Cxcl8 expression and neutrophil recruitment via JNK/c-JUN/AP-1 signaling and chromatin modifications. J Immunol, 2015; 194:1523–1533.

36.

Robertson

, Holmes

. A zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism. Sci Transl Med, 2014; 6:225ra29.

37.

Huang

Y-YY

, Sharma

, Carroll

, Hamblin

. Biphasic dose response in low level light therapy—an update. Dose Response, 2011; 9:602–618.

38.

Hawkins

, Houreld

, Abrahamse

. Low level laser therapy (LLLT) as an effective therapeutic modality for delayed wound healing. Ann N Y Acad Sci, 2005; 1056:486–493.

39.

Hamblin

. Mechanisms and applications of the anti-inflammatory effects of photobiomodulation. AIMS Biophys, 2017; 4:337–361.

40.

Mugoni

, Camporeale

, Santoro

. Analysis of oxidative stress in zebrafish embryos. J Vis Exp, 2014; e51328.

41.

Renaud

, Herbomel

, Kissa

. Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells. Nat Protoc, 2011; 6:1897–1904.

42.

Henry

, Pase

, Ramos-Lopez

, Lieschke

, Renshaw

, Reyes-Aldasoro

. PhagoSight: an open-source MATLAB^® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model. PLoS One, 2013; 8:e72636.

43.

Frykberg

, Banks

. Challenges in the treatment of chronic wounds. Adv Wound Care, 2015; 4:560–582.

44.

, Yan

, Shi

, Zhang

, Wen

. Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem, 2012; 287:25353–25360.

45.

Svensson

, Medyukhina

, Belyaev

, Al-Zaben

, Figge

. Untangling cell tracks: quantifying cell migration by time lapse image data analysis. Cytometry A, 2018; 93:357–370.

46.

Kotwal

, Chien

. Macrophage differentiation in normal and accelerated wound healing. Results Probl Cell Differ, 2017; 62:353–364.

47.

Kelly

, O'Neill

LAJ

. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res, 2015; 25:771–784.

48.

Sáez

, Vargas

, Shoji

, Harcha

, Lennon-Duménil

A-M

, Sáez

. ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X7 receptors. Sci Signal, 2017; 10:eaah7107.

49.

de Oliveira

, López-Muñoz

, Candel

, Pelegrín

, Calado Â, Mulero

. ATP modulates acute inflammation in vivo through dual oxidase 1-derived H2O2 production and NF-κB activation. J Immunol, 2014; 192:5710–5719.

50.

Anders

, Ketz

, Wu

. Basic principles of photobiomodulation and its effects at the cellular, tissue, and system levels. Laser Therapy in Veterinary Medicine: Photobiomodulation, 2017.

51.

Karu

. Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP. Photomed Laser Surg, 2010; 28:159–160.

52.

Hawkins

, Abrahamse

. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg Med, 2006; 38:74–83.

53.

Kotwal

, Sarojini

, Chien

. Pivotal role of ATP in macrophages fast tracking wound repair and regeneration. Wound Repair Regen, 2015; 23:724–727.

54.

de Lima

, de Neto

, Barbosa

, et al. Is there a protocol in experimental skin wounds in rats using low-level diode laser therapy (LLDLT) combining or not red and infrared wavelengths? Systematic review. Lasers Med Sci, 2016; 31:779–787.