Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Summary

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Abstract

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

Introduction

Reactive oxygen species (ROS) are the result of the chemical reduction of molecular oxygen to form water, and include singlet oxygen, superoxide anion, hydrogen peroxide and the hydroxyl radical^1,2,3. ROS have a very short lifespan due to their extremely chemical reactive nature. In aerobic organisms, ROS are incidentally formed inside the cells as a major leaky by-product of aerobic respiration (electron transport chain) in the mitochondria. Transient accumulation of high levels of ROS in the cell results in an oxidative stress condition that may provoke the irreversible ina....

Protocol

All mouse husbandry and experimental procedures must be conducted in compliance with local, national, international legislation and guidelines on animal experimentation.

1. Induction of hair growth, burn induction and identification of long-term BrdU LRCs in the tail skin epithelium wholemounts

NOTE: Use 10-day or 7-week old C57BL/6 mice, preferably littermates, for the experimental designs described below. In all the experimental procedures, animals will be anesthetized by 3% isoflurane inhalation or euthanized by cervical dislocation as indicated.

1. Induction of hair growth in the back skin of mice in....

Results

The topical administration of the mALA precursor in the mouse back and tail skin results in a significant accumulation of PpIX in the whole tissue and, noticeably, in the hair follicle, as demonstrated by the reddish-pink fluorescence of this compound under blue light (407 nm) excitation (Figure 2A,C). Subsequent irradiation of treated tissue with red light (636 nm) at a fluence of 2.5−4 J/cm² promotes transient production of ROS in the tissue, particularly in the.......

Discussion

Here, we present a methodology that allows a transient activation of endogenous ROS production in vivo in mouse skin with physiological effects. The methodology is based on a photodynamic procedure to induce a controlled and local stimulation of the endogenous photosensitizer PpIX (Figure 1B). This experimental approach is an interesting tool to study ROS biology in in vivo experimental systems constituting a significant advance over methodologies using external ROS sources (usually hydrogen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2′,7′-Dichlorofluorescin diacetate	Sigma Aldrich	D6883-50MG
5'-bromo-2'-deoxiuridine	Sigma Aldrich	B5002-500MG
Anti-Bromodeoxyuridine-Fluorescein	Roche	11202693001
Depilatory cream (e.g., Veet)	Veet
Dihydroethidium	Sigma Aldrich	37291-25MG
In Vivo imaging system, e.g., IVIS Lumina 2	Perkin Elmer
mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g	Galderma
Power energy meter (e.g., ThorLabs Model PM100D)	ThorLabs
Red light source, e.g., 636 nm Aktilite LED lamp	Photocure ASA