Our methodology is the first one to provide a reliable experimental tool to induce a transient activation of an endogenous reactive oxygen species or ROS production in a tissue in vivo. Our methodology provides a robust, user-friendly, and non-invasive procedure yielding a consistent production of physiological stimulating ROS levels in the skin basically using a red light source. As the physiological induction of ROS in the skin achieve the tissue stem needs, we have further applied our methodology for skin regeneration therapy including good healing and hair growth.
ROS biology and signaling is currently a research hotpot. A major limitation to investigate ROS function in vivo is the lack of adequate tools to induce a controlled endogenous ROS production. OUR methodology is user-friendly and easy, but all devices, lamp, IV, IS, et cetera, need to be adequately calibrated.
Critical chemical precursors must be fresh and all the steps or the protocol must be rigorously followed. To induce hair growth on the dorsal skin of mice in the second telogen phase, after confirming a lack of response to pedal reflex in about day 50 postnatal litter mate C57 black six mice, use a razor and depilatory cream to remove two independent side-by-side regions of skin from the back of each mouse. After a few minutes, remove the cream with PBS and record the hair follicle growth through the daily acquisition of high resolution images of the control and treated dorsal skin areas of each animal.
To induce the generation of second degree burn lesions on the dorsal skin of mice in the second telogen phase, remove the hair from the back of the mouse. Preheat a one centimeter cross-section brass bar to 95 degrees Celsius in boiling water and apply the bar to the central region of the mouse skin for five seconds. Immediately after burn generation, intraperitoneally inject the animals with one milliliter of physiological solution to prevent dehydration and allow the mice to recover for 24 hours before proceeding to the treatment.
For the generation of BrdU labeled retaining cells or LRCs, inject 10 or 14-day-old litter mates intraperitoneally once a day for four consecutive days with 50 milligrams per kilogram body weight of BrdU in PBS. After the labeling phase, allow mice to develop for 50 to 60 days before applying treatment. To induce the transient production of non-lethal reactive oxygen species or ROS levels in mouse skin for a hair growth analysis, after acquiring a baseline hair growth image, topically apply approximately 25 milligrams mALA cream on the right shaved skin region and place the animal in the dark for 2.5 hours.
At the end of the photosensitizer treatment period, thoroughly remove any excess cream with PBS and confirm PP9 production in situ by red fluorescence expression under blue light excitation, then irradiate the whole back skin with an adequate red light source for a total dose of 2.5 to 4 joules per square centimeter. Then allow the mice to recover on an electric blanket with monitoring until full recumbency before returning the animals to their cages. To switch on transient ROS production for second degree burn healing, apply approximately 25 milligrams of mALA cream along the right burn surface and about four millimeters of adjacent tissue and place the animals in the dark for 2.5 hours as demonstrated.
After removing the cream, irradiate the whole back skin and allow the mice to recover with monitoring as demonstrated. To induce transient ROS production in tail skin, apply approximately 25 milligrams of mALA cream all along the dorsal tissue area of the BrdU treated mice and irradiate the dorsal skin of the tail as demonstrated. After euthanizing the mice, harvest the tail skin for BrdU detection.
Use a scalpel to harvest the tail from each animal, then make straight longitudinal incisions and peel the whole skin as a single piece from the backbone. Incubate the peeled skin pieces in five millimolar EDTA and PBS and five milliliter tubes for four hours at 37 degrees Celsius to allow the careful separation of intact sheets of epidermis from the dermis with forceps. Fix the separated tissues in 4%formaldehyde and PBS for at least 72 hours at room temperature.
Then label the fixed tissues with the fluorescence conjugated antibodies of interest before visualizing the tissues by fluorescence confocal microscopy according to standard protocols to identify and quantify LRCs under each experimental condition. For the ex vivo evaluation of ROS production, after harvesting mouse tail skin pieces as demonstrated, incubate the control untreated tissues and five millimolar EDTA and PBS alone and the photodynamic treatment samples in EDTA supplemented with two millimolar mALA. Next, add hydroethidine to a 3.2 micromolar final concentration to each set of tissues and incubate all of the samples for one hour at room temperature in the dark.
At the end of the incubation, use fingertips to stretch the tail skin samples over a glass surface and irradiate the treated tissues with 636 nanometers of red light at a 10 joules per square centimeter fluence. At the end of the treatment, immediately separate the epidermis from the dermis and fix the epidermal sheets in 4%formaldehyde and PBS for at least 72 hours at room temperature. At the end of the fixation, evaluate the red 2-hydroxyethidium emission by fluorescence microscopy using green exciting light to allow the acquisition of high-quality images.
For in vivo detection of ROS production in the dorsal skin, just after red light irradiation, apply 100 microliters of one milligram per milliliter DH-FDA and 50%ethanol on all target control and treated skin areas. Topical administration of the mALA precursor onto the mouse back and tail skin results in a significant accumulation of protoporphyrin IX in the the whole tissue and noticeably in the hair follicle as demonstrated by the reddish pink fluorescence of this compound under blue light excitation. Subsequent irradiation of the treated tissue with red light promotes the transient production of ROS within the tissue particularly in the bulge region of the hair follicle.
Switching on non-lethal ROS production in mouse skin in vivo promotes a significant increase in the number of LRCs in the bulge region of the hair follicle two days after photodynamic treatment. Notably, the increase in LRC numbers is transient, restoring to normal levels six days after treatment. Transient ROS production accelerates skin healing after a second degree burn.
Quantification of the gradual reduction of the damaged skin demonstrates the robustness and statistical significance of the wound healing acceleration process tissue. Similarly, non-lethal ROS levels strongly promote hair growth after shaving during the second coordinated telogen, a phase during which the hair follicle is refractory to responding to growth stimuli. In addition, ROS production after photo treatments is also significantly reduced by the application of antioxidant compounds.
PP IX production in situ by red fluorescent expression under blue light excitation must be carefully confirmed. To induce ROS production, the irradiance of the red light source must be correctly established. versus tissue and total light dose.
After ROS induction, current biochemical and cell and molecular biology techniques may be used to analyze collected samples and evaluate the dynamic physiological response of the tissue. Our methodology provides a powerful tool to investigate the physiological effect of a transient production of endogenous ROS in a tissue in vivo. This is a new experimental approach that may be very useful for researchers in the field of ROS signaling.
