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 inactivation of proteins, lipids and sugars and the introduction of mutations in the DNA molecule^2,3,4,5. The gradual accumulation of oxidative damage in cells, tissues and whole organisms steadily increases with time and has been associated with the induction of cell death programs, several pathologies, and the ageing process^2,3,4,6.

Aerobic organisms have steadily evolved efficient molecular mechanisms to tackle excess ROS accumulation in cells and tissues. These mechanisms include members of the superoxide dismutase (SOD) protein family, which catalyze superoxide radical dismutation into molecular oxygen and hydrogen peroxide, as well as different catalases and peroxidases which use the antioxidant pool (glutathione, NADPH, peroxiredoxin, thioredoxin^7,8) to catalyze the subsequent conversion of hydrogen peroxide to water and molecular oxygen.

However, several reports support the role of ROS as key components of molecular circuits that regulate critical cell functions, including proliferation, differentiation and mobility^2,3,4. This concept is further supported by the initial identification and characterization of dedicated ROS-producing mechanisms in aerobic organisms, including lipoxygenases cyclooxygenases and NADPH oxidases^9,10. In this sense, ROS exhibit an active role during vertebrate embryo development^11,12,13 and key roles for these molecules in the regulation of specific in vivo physiological functions have been reported in different experimental systems, including the differentiation program of hematopoietic progenitors in Drosophila¹⁴, healing induction in zebrafish, or tail regeneration in Xenopus tadpoles¹⁵. In mammals, ROS have been involved in the self-renewal/differentiation potential of neural stem cells in a neurosphere model¹⁶ and in the deregulation of intestinal stem cell function during colorectal cancer initiation¹⁷. In the skin, ROS signalling has been associated with epidermal differentiation and the regulation of the skin stem cell niche and the hair follicle growth cycle^18,19.

In this perspective, a major experimental limitation to determine the physiological roles of ROS in biological systems, both in normal or pathological conditions, is the lack of adequate experimental tools to induce controlled production of these molecules in cells and tissues, accurately resembling their physiological production as second signalling messengers. At present, most experimental approaches involve the administration of exogenous ROS, mostly in the form of hydrogen peroxide. We have recently implemented an experimental approach to switch on a transient, nonlethal in vivo production of endogenous ROS in the mouse skin, based on the administration of precursors of the endogenous photosensitizer protoporphyrin IX (PpIX; e.g., aminolaevulinic acid or its methyl derivative methylaminolevulinate) and further irradiation of the sample with red light to induce the in situ formation of ROS from intracellular molecular oxygen (Figure 1). This photodynamic procedure may be efficiently used to stimulate resident stem cell niches, thus activating the regenerative programs of the tissue^19,20 and opening the way for new therapeutic modalities in skin regenerative medicine. Here, we present a detailed description of the protocol, showing representative examples of stimulation of stem cell niches, measured as an increase in the number of long-term 5-bromo-2’-deoxyuridine (BrdU) label retaining cells (LRCs) in the bulge region of the hair follicle^19,21, and subsequent activation of regeneration programs (acceleration of hair growth and burn healing processes) induced by transient, nonlethal ROS production in the skin of C57Bl6 mouse strain.

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 the second telogen (resting) phase (about day 50 post-natal)
   1. Anesthetize mice with inhalation of 3% isoflurane. Confirm full deep anaesthesia by lack of a pedal reflex (firm toe pinch). Shave two independent side by side regions of the back skin in each single mouse using hair clippers and depilatory cream (Table of Materials). Use the left side for the control and the right side for treatment.
      NOTE: Check that, after shaving, the subjacent back skin is pinkish and not grey/black, an indicator of melanogenesis and entrance into the anagen (growing) phase in this mouse strain.
   2. Wash thoroughly with PBS to remove all cream remains and proceed to induction of transient in situ production of nonlethal ROS levels as described in section 2.1.
   3. Record hair follicle growth through daily acquisition of high-resolution images of the control and treated back skin areas in each animal (e.g., using an HD camera coupled to a 5−20x binocular lens).
2. Induction of 2^nd degree burn lesions in the back skin of mice in the second telogen (resting) phase (about day 50 post-natal)
   1. Anesthetize mice. Shave the whole back skin region in each single mouse using hair clippers and depilatory cream and wash thoroughly with PBS to remove all cream remains.
      NOTE: Administer in situ subcutaneous injections of lidocaine 0.5% (2 mg/ml) in sterile saline, not exceeding 7 mg/ml, just before the burning procedure.
   2. Preheat a brass bar (1 cm in cross-section) at 95 °C by immersing in boiling water and then apply on the central region of the dorsal back skin surface of each mouse for 5 s.
   3. Just after burn generation, intraperitoneally inject the animals with 1 mL of physiological solution (0.9% NaCl) on an electric blanket to prevent dehydration. Let the animals recover for 24 h and proceed to induction of transient in situ production of nonlethal ROS levels as described in section 2.3.
   4. Record burn wound progression through daily acquisition of high-resolution images of the control and treated back skin areas in each animal (e.g., using an HD camera coupled to a 5−20x binocular lens).
3. Generation and identification of long-term BrdU LRCs in the tail skin epithelium
   1. Inject 10/14-day old littermates intraperitoneally (no anaesthesia) once a day for 4 consecutive days with 50 mg/kg bodyweight BrdU dissolved in PBS. After the labelling phase, allow mice to grow for 50−60 days before any treatment.
      NOTE: Use a fresh needle for each injection. 
   2. Proceed as described in section 2.3 for the induction of transient in situ nonlethal ROS production in the tail skin at different times before the preparation of tissue wholemounts.
   3. To prepare wholemounts of tail epidermis, euthanize mice by cervical dislocation and clip the tails with surgical scissors.
      1. Use a scalpel to make a straight longitudinal incision all along the tail and peel the whole skin as a single piece from the backbone. Incubate the peeled skin in 5 mM EDTA in PBS in 5 mL tubes for 4 h at 37 °C and carefully separate intact sheets of epidermis from the dermis using forceps.
      2. Fix the tissue in 4% formaldehyde in PBS for at least 72 h at room temperature (RT) and proceed for BrdU detection using appropriate antibodies.
      3. Use fluorescence/confocal microscopy to identify and quantify LRCs in each experimental condition, including light controls and photodynamic treatments at different times before the preparation of tissue wholemounts, as previously described in detail^19,20.
         NOTE: Fixed epidermal sheets may be stored in PBS containing 0.02% sodium azide at 4 °C for up to three months. Fixed epidermal sheets may be used for immunolocalization of required proteins following standard histological sections procedures.

2. Induction of transient production of nonlethal ROS levels in mouse skin

NOTE: To induce transient production of nonlethal ROS levels in mouse skin, a photodynamic treatment using a precursor of the endogenous photosensitizer PpIX, in this case, methyl-aminolevulinate (mALA), and red light will be used.

1. To switch on transient ROS production for the induction of hair growth in the back skin, prepare the animals as indicated in section 1.1.
   1. Apply ~25 mg of mALA in the form of topical cream (Table of Materials) on the right region, keeping the left side as an internal control avoiding inter-individual differences. Incubate for 2.5 h in the dark, wash off the excess cream thoroughly with PBS. To confirm anesthesia depth, monitor the animals every 10 mins until fully recovered.
      NOTE: The production of PpIX in the back skin should be tested in situ by its red fluorescence under blue light (407 nm) excitation.
   2. Anesthetize the animals.
   3. Irradiate the whole back skin with an adequate red-light source (Table of Materials) for a total dose of 2.5−4 J/cm². Keep mice on an electric blanket until their complete recovery and proceed as described in step 1.1.3.
      NOTE: Irradiance should be adjusted by manipulating the distance between the light source and the tissue and measured using a power energy meter (Table of Materials). The experiment is considered finished when full hair growth is observed in any of the independent shaved regions of each animal. The whole procedure involves just a single photo treatment.
2. To switch on transient ROS production for healing improvement of 2^nd degree burn lesions, prepare the animals as indicated in section 1.2.
   1. Apply ~25 mg of mALA in the form of topical cream all along the burned surface, encompassing about 4 mm of adjacent tissue. Incubate for 2.5 h in the dark, wash off the excess cream thoroughly with PBS. To confirm anesthesia depth, monitor the animals every 10 mins until fully recovered.
   2. Anesthetize the animals.
   3. Irradiate the whole back skin with an adequate red-light source for a total dose of 2.5−4 J/cm². Keep the mice on an electric blanket until their complete recovery and proceed as described in step 1.2.4.
      NOTE: The experimental procedure for each animal is considered finished when full burn healing is observed. The whole procedure involves just a single photo treatment.
3. To switch on transient ROS production in tail skin, prepare mice as indicated in section 1.3, apply ~25 mg of mALA in the form of topical cream all along the dorsal tissue area and proceed as described in section 2.1 for back skin. Perform photo treatments and correspondent light controls 24 h, 48 h, or 72 h before animal euthanasia and further extraction of tail skin whole mounts.
   NOTE: In all experimental designs, assay the ROS-dependence of the process by using antioxidant ROS scavengers (e.g., daily inoculation of 100 mg/kg bodyweight N-acetyl-cysteine by intraperitoneal injection of a 20 mg/mL solution in PBS, pH 7.2, starting 5 days before mALA treatments or, alternatively, two doses of 100 mg/mL ascorbic acid in 50% ethanol, spaced 30 min, topically applied on the skin in the time interval between the mALA treatments and red-light irradiation).

3. ROS detection in the skin

1. Ex vivo evaluation of ROS production in tail skin after photodynamic treatment using hydroethidine
   NOTE: Hydroethidine is a non-fluorescent molecule that reacts specifically with ROS to produce fluorescent dye 2-hydroxyethidium (hET).
   1. Incubate whole tail skins, obtained as described in section 1.3.3, for 3 h at 37 °C in 5 mM EDTA in PBS solution (control samples) or additionally containing 2 mM mALA (photodynamic treatment samples).
   2. In all cases, add hydroethidine to a final concentration of 3.2 μM from a 25 mg/mL stock in dimethyl sulfoxide (DMSO) and incubate for 1 h in the dark at RT.
   3. Stretch the tail skin samples over a glass surface using fingertips and irradiate with 636 nm red light at a 10 J/cm² fluence.
   4. Proceed immediately to separate the epidermis from the dermis and to fix the epidermal sheets as described in section 1.3.3.
   5. Evaluate the hET red emission under a fluorescence/confocal microscope using green exciting light, capture high quality images and proceed with further analysis.
      NOTE: Use hET staining of tissue samples in the absence of photodynamic treatment as a negative control for hET autoxidation.
2. In vivo detection of ROS production in the back skin after induction of hair growth and burn healing followed by photodynamic treatment
   NOTE: This step is performed using 2′,7′-dichlorodihydrofluorescein diacetate (DHF-DA), a cell permeant non-fluorescent compound that, after cleavage by intracellular esterase enzymes, specifically reacts with ROS giving the 2′,7′-dichlorofluorescein (DCF) fluorescent dye.
   1. Use animals prepared for induction of hair growth (section 1.1) or burn healing (section 1.2). Just before topical mALA cream treatments (see sections 2.1 and 2.2), topically dispense 100 μL of 1 mg/mL in 50% ethanol of DHF-DA on all target control/treated skin areas, let the skin fully absorb the material and proceed forward with topical mALA cream application.
   2. Incubate treated animals for 4 h in the dark, wash the topical mALA cream thoroughly off the skin with PBS and dispense a second dose of 100 μL of DHF-DA solution on the skin. To confirm anesthesia depth, monitor the animals every 10 mins until fully recovered.
   3. Incubate treated animals for 50 min in the dark, anesthetize and irradiate the whole back skin with a fluence ranging from 2.5 to 4 J/cm² of 636 nm red light using a LED lamp.
   4. Immediately after irradiation, evaluate ROS levels generated in the skin using an in vivo imaging system (Table of Materials). Set the filter box for 445−490 nm excitation and 515−575 nm emission, capture high quality images and proceed with further analysis.
      NOTE: Use DHF-DA staining of tissue samples in the absence of photodynamic treatment as negative control for DHF-DA autoxidation.

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 ...

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

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