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Live Imaging of Cutaneous Wound Healing after Rotary Tool Injury in Zebrafish

2023; Elsevier BV; Volume: 144; Issue: 4 Linguagem: Inglês

10.1016/j.jid.2023.10.015

1523-1747

Leah J. Greenspan, Keith Ameyaw, Daniel Castranova, Caleb A. Mertus, Brant M. Weinstein,

Angiogenesis and VEGF in Cancer

Cutaneous wounds are common afflictions that follow a stereotypical healing process involving hemostasis, inflammation, proliferation, and remodeling phases. In the elderly and those suffering from vascular or metabolic diseases, poor healing after cutaneous injuries can lead to open chronic wounds susceptible to infection. The discovery of new therapeutic strategies to improve this defective wound healing requires a better understanding of the cellular behaviors and molecular mechanisms that drive the different phases of wound healing and how these are altered with age or disease. The zebrafish provides an ideal model for visualization and experimental manipulation of the cellular and molecular events during wound healing in the context of an intact, living vertebrate. To facilitate studies of cutaneous wound healing in zebrafish, we have developed an inexpensive, simple, and effective method for generating reproducible cutaneous injuries in adult zebrafish using a rotary tool. We demonstrate that our injury system can be used in combination with high-resolution live imaging to monitor skin re-epithelialization, immune cell recruitment and activation, and vessel regrowth in the same animal over time. This injury system provides a valuable experimental platform to study key cellular and molecular events during wound healing in vivo with unprecedented resolution. Cutaneous wounds are common afflictions that follow a stereotypical healing process involving hemostasis, inflammation, proliferation, and remodeling phases. In the elderly and those suffering from vascular or metabolic diseases, poor healing after cutaneous injuries can lead to open chronic wounds susceptible to infection. The discovery of new therapeutic strategies to improve this defective wound healing requires a better understanding of the cellular behaviors and molecular mechanisms that drive the different phases of wound healing and how these are altered with age or disease. The zebrafish provides an ideal model for visualization and experimental manipulation of the cellular and molecular events during wound healing in the context of an intact, living vertebrate. To facilitate studies of cutaneous wound healing in zebrafish, we have developed an inexpensive, simple, and effective method for generating reproducible cutaneous injuries in adult zebrafish using a rotary tool. We demonstrate that our injury system can be used in combination with high-resolution live imaging to monitor skin re-epithelialization, immune cell recruitment and activation, and vessel regrowth in the same animal over time. This injury system provides a valuable experimental platform to study key cellular and molecular events during wound healing in vivo with unprecedented resolution. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwNTViY2NmZDU3YWI0NTdjMzZhNmI0N2E2NTA5MGYwNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNzE2NzE4NDQ3fQ.QTf9g-5n3RXKNKk6mypTuWTJmncLtMjPnOqFzX_cSfX9eOGLcrK59S5_FIL46oTpogOi69iYo79mmn-Vw-2V_B9YH9Ikj9rMS3V0AUbNSlZQGHHvFXSEjYsxgQFrAo2QUl3d9ky4VXnzJWkLeyLf1M-KwG3XQu5NaS3jIU5QDtzjllmvg7z5d-aSlaUdrS2XTCHdzVdPM3kshvOZJuraCQPWx9QnBKqkK25amU1c2v4dilu9PcNphDUnT5kbObfS6iDw86KunCuAG7Mn4mWr8cF-JPxO3hJs80Dvx1eTOLKblbTe9lgFyXP6T57NOyfRIOg7nxhoq2aOJTgrfLyMRQ (mp4, (11.42 MB) Download video Cutaneous wounding in zebrafish. Movie showing preparation and generation of a cutaneous wound in zebrafish using a rotary tool. The following steps are shown: (i) fish swimming before injury, (ii) addition of filter paper with Tricaine water to the holding platform, (iii) loading of the fish onto the holding platform, (iv) securing fish onto the holding platform using anchoring rods, (v) rotating insert to angle fish, (vi) lining up the rotary tool to the site of injury, (vii) creating the injury with the rotary tool, (viii) final wounding outcome, and (ix) fish swimming normally after injury. Time-lapse imaging of skin re-epithelialization after wounding. Still and timelapse images of a Tg(actb2:GFP) (β-actin) fish before and after cutaneous wounding. 0–5": still images of fish prior to injury, including a zoomed-out image and close-up image of the wound site. 6–16": time-lapse of skin re-epithelization 0.5–17.6 hours after injury. 17–19": high magnification timelapse 18 hours after injury showing the infiltration of many cell types into the wound area. 20–24": still image of wound area 2 days after injury showing that the wound has been closed. Neutrophil recruitment after cutaneous wounding. Still and timelapse images of a Tg(lyz:DsRed2)nz50 fish stained with BODIPY before and after cutaneous wounding. Schematic of fish shows approximate site of wounding. 0–4": still image of the wound area before injury shows scales intact and neutrophils evenly scattered throughout. 5–16": timelapse of neutrophil recruitment 0.5–17.25 hours after injury. A mass influx of neutrophils is seen during the indicated time, suggesting that neutrophils respond quickly to cutaneous injury. BODIPY labeling shows skin re-epithelialization occurring simultaneously with neutrophil recruitment. Neutrophil behavior after cutaneous wounding. Still and timelapse images of a Tg(lyz:DsRed2)nz50 fish before and after cutaneous wounding showing neutrophils in magenta and scales in blue (autofluorescence). 0–2": still image of the wound area before injury shows intact tissue with neutrophils scattered throughout. 3–6": still image of the wound soon after injury, including a zoomed-out image and close-up image of the wound site. 7–36": zoomed-in timelapse of neutrophil behavior 0.5–17.5 hours after injury showing individual cells (left movie) and tracked cells containing spots (magenta) with dragon tails (cyan) depicting the last 15 minutes of movement (right movie). Movies are paused at the following times to indicate specific neutrophil behavior: 13" initial migration, 20" amplified recruitment, 28" neutrophil clustering, and 35" wound infiltration. Neutrophil behavior in uninjured fish. Still and timelapse images of a Tg(lyz:DsRed2)nz50 uninjured fish showing neutrophils in magenta and scales in blue (autofluorescence). 0–2": still image of fish flank shows neutrophils scattered throughout the skin. 3–8": timelapse of neutrophil behavior in uninjured skin showing individual cells (left movie) and tracked cells containing spots (magenta) with dragon tails (cyan) depicting the last 15 minutes of movement (right movie). Macrophage movement after cutaneous wounding. Still and timelapse images of a Tg(mpeg:eGFP)gl22 fish before and after cutaneous wounding showing macrophages in green. The 0–3": still image of the wound area before injury shows macrophages completely covering the skin. 4–23": timelapse of macrophage behavior 0.5–16.5 hours after injury shows macrophages streaming into the wound site after injury. 24–26": high magnification timelapse 16.5 hours after injury.