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

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.

This method can help answer key questions in the tissue repair and regeneration field about the associations among the cytoprotective pathways, oxidative stress, and the wound healing response. The main advantage of this powerful technique is that you can measure reactive oxygen species in a wound, in real-time, to determine their impact on tissue regeneration. Demonstrating the procedure with me will be Jennifer Kwong, a research assistant in my laboratory.

Use a glucometer to measure the blood glucose of each anesthetized eight to 12 week old diabetic mouse. And use a hair trimmer and depilatory cream to remove the dorsal hair from the first animal. Use alcohol wipes two times to clean the exposed skin and drape the animal so that only the surgical area is exposed.

When the alcohol has dried, use sterile 10 millimeter biopsy punches to create two, 10 millimeter full thickness wounds extending through the panniculus carnosus according to a well established excisional wound healing technique. Use a 0.5 millimeter thick silicone sheet with 10 millimeter circular cutouts to splint the wounds open and secure the stints with interrupted 4/0 silk sutures. Then place the mice on a heat pad with monitoring until full recovery, before returning to its individual cage, providing paper towels as additional nesting material for two weeks.

The next day, apply either control nonsense small interfering RNA gel or experimental small interfering Keap1 gel to the top of the wound of each animal and wrap each torso with transparent film dressing to keep the gel in place while leaving the limbs free. On day three of the experiment, load a one millimeter syringe equipped with a 27-gauge needle with freshly prepared L-012 solution and wrap the syringe to protect the solution from light. To image the wounds, gently remove the transparent film dressing from each of the mice without disturbing the wounds and place the mice into the imaging chamber of a bioluminescence imaging system.

Set the imaging system inflow and the induction chamber oxygen levels to one liter per minute and image the mice by bioluminescence and bright field at baseline. Next, wipe the abdomens with alcohol wipes then inject five milligrams of L-012 solution per 200 grams body weight, IP, into each mouse once the alcohol has dried. Injecting the L-012 solution without disturbing the wounds on the dorsal skin is critical, because any distortion will affect the wound healing trajectory and data interpretation.

Ensure that the animal is securely in dorsal recumbency before the injection. Immediately following the injection, place the mice back into their respective locations in the imaging chamber and image the animals for one minute every four minutes over a 60 minute imaging period, defining the 10 millimeter wound as the region of interest for determining the level of reactive oxygen species. Then place the mice on a heat pad with monitoring until full recovery before returning the animals to their individual cages.

Three days after creating bilateral wounds according to the established excisional wound model, no bioluminescence is observed prior to L-012 injection. Following intraperintoneal L-012 solution injection, bioluminescence is observed in the areas of the wound where reactive oxygen species are detected. Recording of the bioluminescence for one minute every four to five minutes over the course of 60 minutes demonstrates the bioluminescent saturation of the region of interest over time, with the optimal imaging time to reach complete L-012 saturation observed at about 50 minutes.

The bioluminescence in each wound region of interest before and after L-012 injection into nonsense small interfering RNA treated or Keap1 small interfering RNA treated mice can then be calculated by dividing the total counts of light intensity by the area. Analysis of day 10 wound tissue sections to confirm the accuracy of reactive oxygen species level measurement by H&E staining reveals a reduced cellular infiltration into small interfering Keap1 treated wounds compared to small interfering nonsense treated tissues, indicating a reduced inflammatory morphology in the experimental gel-treated animals. Analysis of the immuno-reactivity of F4/80, a protein macrophage marker on wound tissue sections, indicates a reduced number of macrophages in small interfering Keap1 treated wounds compared to small interfering nonsense treated wounds, further underscoring the validity of this method.

When attempting this procedure, make sure to confirm the L-012 is not expired and to stir the solution in a place protected from light. Following this procedure, targeted probes and immuno-assay based methods can be used to study the sub-cellular localization of reactive oxygen species and their correlates, enabling a rapid assessment of interventions and mechanisms that affect the redux stats of wounds.