In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Summary

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

Abstract

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C₁₃H₈ClN₄NaO₂) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

Introduction

Oxygen metabolites generated through inflammatory processes contribute to various signaling cascades as well as destructive alteration of cellular components¹. Utilizing sensitive and specific techniques to measure ROS is critical for studying inflammatory processes and characterizing the effects of oxidative stress. In vivo imaging is valuable because of its ability to provide dynamic spatial and temporal data in living tissue. L-012 is a synthetic chemiluminescent probe that is highly sensitive for superoxide anions and produces a higher light intensity than other fluorescent probes in cells, tissues, and whole blood

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. All mice are housed behind a barrier and all personnel wear appropriate personal protective equipment.

1. Day 0: Preparation of Murine Model of Excisional Wound Healing

1. Anesthetize diabetic (Lepr^db/db) mice, aged 8–12 weeks, with inhalational 2% isoflurane. Confirm that each mouse has been properly anesthetized using the foot pad pinch test. Apply sterile ocular lubricant to each eye to prevent irritation from dryness. Researchers should follow their institutional veter....

Results

Three days after creating bilateral wounds according to an established excisional wound model (Figure 1A), diabetic mice are positioned in the imaging chamber. An initial photograph and a measure of bioluminescence are taken before injection of L-012 to account for background signal (Figure 1B). Following intraperitoneal injection with the L-012 solution, the mice are repositioned in the chamber and bioluminescence is visualized .......

Discussion

Common techniques for measuring ROS have been limited by complex protocols requiring tissue extraction or similarly invasive techniques. In recent years, measurements of oxidative stress have been reported on the basis of innovative imaging modalities, thereby allowing for spatiotemporal assessments^9,10,11. L-012 has several advantages as a chemiluminescent probe relative to luminol, lucigenin, and MCLA¹<.......

Materials

Name	Company	Catalog Number	Comments
BKS.Cg-Dock7m+/+ Leprdb/J mice	Jackson Laboratories	000642
13 cm x 18 cm Silicone sheet (0.6 mm)	Sigma Aldrich	665581
3M Tegaderm Transparent Film Dressings	3M	88-1626W
Lipofectamine 2000 Transfection Reagent	Life Technologies	11668027
Keap1 Stealth siRNA	Thermofisher Scientific	1299001
Silencer negative control	Thermofisher Scientific	AM4635
Opti-MEM Reduced Serum	ThermoFisher Scientific	11058021
DPBS	ThermoFisher Scientific	14040133
Methyl-cellulose	Sigma Aldrich	9004-67-5
L-012	Wako Chemicals	120-04891
IVIS Lumina III XR In Vivo Imaging System	PerkinElmer