Name: stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ
Uploaded: 2020-05-08T12:30:00
Description: Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In

This work has been supported by grants from Ministerio de Econom&#237;a y Competitividad (RTC-2014-2626-1 to JE) and Instituto de Salud Carlos III (PI15/01458 to JE) of Spain. EC has been supported by the Atracci&#243;n de Talento Investigador grant 2017-T2/BMD-5766 (Comunidad de Madrid and UAM).

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 anestheti...

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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...

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 radical1,2,3. 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 molecule2,3,4,5. 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 process2,3,4,6.
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, thioredoxin7,8) 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 mobility2,3,4. This concept is further supported by the initial identification and characterization of dedicated ROS-producing mechanisms in aerobic organisms, including lipoxygenases cyclooxygenases and NADPH oxidases9,10. In this sense, ROS exhibit an active role during vertebrate embryo development11,12,13 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 cycle18,19.
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 tissue19,20 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&#8217;-deoxyuridine (BrdU) label retaining cells (LRCs) in the bulge region of the hair follicle19,21, 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:812px;" width="811">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">D6883-50MG</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5&#39;-bromo-2&#39;-deoxiuridine</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">B5002-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Anti-Bromodeoxyuridine-Fluorescein</td>
			<td style="width:104px;">Roche</td>
			<td>11202693001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream (e.g., Veet)</td>
			<td style="width:104px;">Veet</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Dihydroethidium</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">In Vivo imaging system, e.g., IVIS Lumina 2</td>
			<td style="width:104px;">Perkin Elmer</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td>
			<td style="width:104px;">Galderma</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Power energy meter (e.g., ThorLabs Model PM100D)</td>
			<td style="width:104px;">ThorLabs</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Red light source, e.g., 636 nm Aktilite LED lamp</td>
			<td style="width:104px;">Photocure ASA</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ

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.

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&#8722;4 J/cm2 promotes transient production of ROS in the tissue, particularly ...

Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In 

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

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.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

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