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^1,2,3,4. It has been successfully employed for in vivo imaging in murine models to study several inflammatory diseases, including arthritis and colitis^5,6. It has yet to be employed in an established cutaneous wound healing model. Measurement of ROS generated is equally relevant to assess the progression of wound healing under different conditions. The sensitivity and noninvasive nature of this method makes it a promising technique for studying wound healing across murine models.

Nrf2 is a major driver of the antioxidant response and a transcriptional factor with specificity for the antioxidant response element (ARE) common to the promoter regions of several antioxidant enzymes⁸. In the absence of oxidative stress, Nrf2 is sequestered in the cytoplasm by Keap1, which subsequently causes its ubiquitination and degradation. Imbalance of the Nrf2/Keap1 pathway has been implicated in inappropriate redox homeostasis and delayed wound healing in the setting of increased oxidative stress⁹. We have previously shown that suppression of Keap1 stimulates increased Nrf2 activity and promotes rescue of pathologic cutaneous wound healing in diabetic wounds⁹.

Here we describe a protocol that utilizes L-012-assisted bioluminescence imaging to measure ROS levels in an excisional cutaneous wound healing model, which is critical for highlighting the association between ROS and wound healing. This technique demonstrates real-time changes in oxidative burden within wounds and immediate periphery. Furthermore, this method allows for rapid assessment of interventions and mechanisms that affect redox handling. Here we use a model of Keap1 knockdown for the restoration of redox homeostasis to evaluate the applicability of our strategy. Because our technique is non-invasive and wounds are undisturbed, the same animal can be used for further confirmatory analyses on the basis of histology or cell lysates.

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 veterinary staff’s guidelines when anesthetizing animals using isoflurane.
2. Weigh the mice and record the body weight of each mouse. Confirm the diabetic status of animals by recording the blood glucose of each animal using a glucometer. 
3. Disinfect procedural workspace and anesthesia equipment. Remove dorsal hair of the mice using a hair trimmer, followed by application of hair removal lotion to wipe away excess hair. Use alcohol wipes to clean the exposed skin, twice, and allow to dry.
4. Create two 10 mm full-thickness wounds extending through the panniculus carnosus using sterile 10 mm biopsy punches according to a well-established excisional wound healing technique^7,8. Use sterile gloves for all survival surgery. Autoclave all surgical instruments in bags prior to surgery and open only in the surgical field.
5. Splint the wounds open using a 0.5 mm thick silicone sheet with 10 mm circular cutouts and secure the stents in place using interrupted 4-0 silk sutures.
6. Following surgery, remove animals from anesthesia and place on heating pad to facilitate proper recovery. Of note, if animal body temperature decreases during the procedure, the animal may be moved to the heating pad earlier to minimize body heat loss under anesthesia. Monitor the animals until they are awake and mobile.
7. Once fully recovered, return animals to individual cages, containing food and water (ensure some food is on the floor of the cage for post-operative enrichment). Provide shredded paper towels as additional nesting material for 2 weeks. Do not house animals that have undergone surgery with other animals, to prevent adverse interactions and changes to wound healing status.
8. For post-operative pain relief, inject mice subcutaneously with buprenorphine at 0.1 mg/kg of body weight twice a day, starting immediately following the procedure, for 3 days.

2. Day 1: Preparation of Keap1 siRNA

NOTE: Prepare all treatments inside a biosafety cabinet.

1. Prepare siRNA dilution by combining 37.5 µL of reduced serum medium with 12.5 µL of 20 µM Keap1 siRNA (siKeap1) (250 pmol) in a 1.5 mL microcentrifuge tube on ice.
2. Prepare liposome dilution by combining 25 µL of reduced serum medium with 25 µL of liposome mix in a 1.5 mL microcentrifuge tube.
3. Add 50 µL of siRNA dilution to 50 µL liposome dilution dropwise (1:1 volume), and gently mix.
4. Incubate for 20 minutes at room temperature.
5. Add 50 µL of 2% methylcellulose gel in water and mix gently by pipetting up and down.
6. Treat each animal with either a nonsense siNS (control) or siKeap1 (experimental). Apply the gel to the top of the wound. Wrap the animal’s torso with transparent film dressing to keep gel in place, keeping the limbs free to maintain mobility.

3. Day 3: Preparation of L-012 Solution

NOTE: Prepare all reagents in a biosafety cabinet.

1. In a 1.5 mL microcentrifuge tube, prepare L-012 in 1X PBS at a concentration of 0.5 mg/100 mL.
2. Manually vortex the microcentrifuge tube. The L-012 does not completely dissolve into the PBS, however it should be evenly suspended in the liquid. Of note, do not attempt to dissolve L-012 in water to avoid disturbing physiologic electrolyte balance following injection.
3. Transfer the solution into a 1 mL syringe using a 27 G needle.
   NOTE: Be sure to protect the L-012 solution from light.

4. Day 3: In Vivo Imaging of Diabetic Wounds

1. Anesthetize mice with inhalational 2% isoflurane. Researchers should follow their institutional veterinary staff’s guidelines when anesthetizing animals using 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.
2. Gently remove the transparent film dressing from the mice without disturbing the wounds.
3. Place the mice in the imaging chamber in their respective orders. To maintain proper O₂ levels in the chamber, set the imaging system inflow and the induction chamber O₂ levels to 1.0 L/min.
4. Image the mice for bioluminescence and photograph at baseline before injection of L-012 compound.
5. Wipe the abdomen with alcohol wipes and allow to dry. Perform an intraperitoneal injection of the L-012 solution at 5 mg per 200 g body weight using a 27-gauge needle. For example, a mouse weighing 20 g should receive 0.5 mg of L-012.
6. Immediately following the L-012 injection, place the mice back in their respective locations in the imaging chamber. Image the mice over the course of 60 minutes, for 1 minute at 4 minute intervals. Define the 10-mm wound as the region of interest for determining level of ROS.
7. Following surgery, remove animals from anesthesia and place on heating pad to facilitate proper recovery. Monitor the animals until they are awake and mobile.
8. Once fully recovered, return animals to individual cages, maintaining the same post-operative enrichment environment previously described in 1.6. Do not house animals that have undergone surgery with other animals, to prevent adverse interactions and changes to wound healing status.

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