Macrophage behavior is of great interest in response to tissue injury and promotion of regeneration. With increasing numbers of zebrafish reporter-based assays, new capabilities now exist to characterize macrophage migration, and their responses to biochemical cues, such as reactive oxygen species. Real time detection of macrophage behavior in response to oxidative stress using quantitative measures is currently beyond the scope of commercially available software solutions, presenting a gap in understanding macrophage behavior. To address this gap, we developed an image analysis pipeline solution to provide real time quantitative measures of cellular kinetics and reactive oxygen species content in vivo after tissue injury. This approach, termed Zirmi, differs from current software solutions that may only provide qualitative, single image analysis, or cell tracking solutions. Zirmi is equipped with user-defined algorithm parameters to customize quantitative data measures with visualization checks for an analysis pipeline of time-based changes. Moreover, this pipeline leverages open-source PhagoSight, as an automated keyhole cell tracking solution, to avoid parallel developments and build upon readily available tools. This approach demonstrated standardized space- and time-based quantitative measures of (1) fluorescent probe based oxidative stress and (2) macrophage recruitment kinetic based changes after tissue injury. Zirmi image analysis pipeline performed at execution speeds up to 10-times faster than manual image-based approaches. Automated segmentation methods were comparable to manual methods with a DICE Similarity coefficient > 0.70. Zirmi provides an open-source, quantitative, and non-generic image analysis pipeline. This strategy complements current wide-spread zebrafish strategies, for automated standardizations of analysis and data measures.

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/cm 2 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/cm 2 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/cm 2 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.

Background: Neutrophils participate in a cooperative defense strategy with macrophages following tissue injury. Application of low dose electromagnetic radiation via 635 nm wavelength can enhance macrophage recruitment to the wound, decrease tissue levels of ROS and speed healing. The impact of this wavelength on neutrophil activity in vivo is not well-described. Given that wound healing can be enhanced if collateral damage by neutrophils within the wound is decreased, we investigated how neutrophil kinetics changed during these accelerated reparative responses. Methods: Zebrafish, transgenic for the fluorescent neutrophil marker, mpx-dendra2, underwent defined fin tissue injury, and were randomized to one of two laser treatment groups, 3 J/cm2 (n=57), or 18 J/cm2 (n=69) or to the control group, 0 J/cm2 (n=164). Electromagnetic wave exposures were administered by a 635-nm continuous 5mW He:Ne laser with recipients randomized by dose delivered. Fish were three-dimensionally time-lapse imaged 30-120 minutes post injury (mpi) and wound healing documented at 24 hours post-injury. Individual neutrophil movement was tracked according to distance from wound center. Results: The lower treatment fluence, 3 J/cm2, significantly decreased neutrophil migration speed into the wound, increased reverse migration, and promoted stasis outside the adjacent wound area when compared to control and higher energy doses. The 3 J/cm2 treatment groups also exhibited more rapid wound closure when compared to control or higher fluence.  Conclusions: Unlike our previous work in macrophages in which low fluence treatment enhanced the speed of forward directed migration into the wound, the response of neutrophils was decreased speed into the wound, increased reverse migration, and stasis outside the areas of the wound edges. These findings advance the notion that low fluence treatments reduce neutrophil inflammatory responses within the wound by their reduced presence within the wound. Reduced neutrophil-mediated collateral damage may work in concert with enhanced macrophage wound activity to promote faster wound healing.

Purpose: Animal models that accurately reflect human responses to radiation injury are needed for advanced mechanistic investigation and development of effective therapeutics. The rabbit is an established animal model accepted by the FDA for studies of cardiovascular disease, lipid metabolism, the development of anticoagulants, testing of bone implants, and the development of treatments for infectious diseases such as HIV. The purpose of this study was to investigate the New Zealand White (NZW) Rabbit model as a model of acute radiation exposure because of its established similarity to human vascular, immune, and coagulation responses. Materials and methods: Two sequential studies were performed in a total of 81 male NZW rabbits, 16-20 weeks of age. All animals underwent clinical observations and peripheral blood analyses following a single dose of 0, 6, 7, 8, 8.5, 9, or 10 Gy of total body irradiation via a 6 MV Linear accelerator photon source on day 0. Animals were treated with timed release fentanyl patch (days 0-30), subcutaneous hydration (day 1, Study 2 only), and oral sulfamethoxazole/trimethoprim 30 mg/kg once daily (days 3-30) and were followed for 30 days or to time of mortality. Results: Study 1 revealed the estimated LD30, À50, À70, and À90 with 95% confidence intervals (CI) at 30 days to be 6.7 (

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