Switching on a transient endogenous ROS production in mammalian cells and tissues.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

DNA labeling in vivo: quantification of epidermal stem cell chromatin content in whole mouse hair follicles using Fiji image processing software.

DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cells (LRC) in long-term experiments. A major hindrance of this methodology relies on the selection of adequate procedures to quantify the nucleoside analogue content from image data files. Here we propose a simple procedure using Fiji image processing software to accurately calculate nucleoside analogue retaining chromatin/total chromatin (LRC/DAPI) signal ratios in the well-known mouse hair follicle stem cell niche.

Standard DNA methylation analysis in mouse epidermis: bisulfite sequencing, methylation-specific PCR, and 5-methyl-cytosine (5mC) immunological detection.

In mammals, methylation of cytosine C-5 position is a major heritable epigenetic mark on the DNA molecule. Maintenance of proper DNA methylation patterns is a key process during embryo development and in the maintenance of adult tissue homeostasis. The use of experimental procedures based on the chemical modification of cytosine by sodium bisulfite and the development of antibodies recognizing 5mC have essentially contributed to our knowledge on DNA methylation dynamics in normal and disease states. Here we describe standard procedures for bisulfite sequencing, methylation-specific PCR, and 5mC immunodetection using mouse skin and the hair follicle stem cell niche as model tissues.

Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation.

Photodynamic therapy using methyl 5-aminolevulinate (MAL) as a precursor of the photosensitizing agent protoporphyrin IX is widely used in clinical practice for the treatment of different pathologies, including cancer. In this therapeutic modality, MAL treatment promotes the forced accumulation of the endogenous photoactive compound protoporphyrin IX in target malignant cells. Subsequent irradiation of treated tissues with an appropriate visible light source induces the production of reactive oxygen species (ROS) that, once accumulated above a critical level, promote cell death. Here we demonstrate that a photodynamic treatment with low MAL concentrations can be used to promote a moderate production of endogenous ROS, which efficiently stimulates cell growth in human immortalized keratinocytes (HaCaT). We also show that this proliferative response requires Src kinase activity and is associated to a transient induction of cyclin D1 expression. Taken together, these results demonstrate for the first time that a combination of light and a photoactive compound can be used to modulate cell cycle progression through Src kinase activation and that a moderate intracellular increase of photogenerated ROS efficiently stimulates cell proliferation.

A preventative foot care programme for people with diabetes with different stages of neuropathy.

The aim of this study was to assess the efficacy of a preventative foot care programme, applied in a normal outpatient setting to decrease the incidence of foot ulcers in people with diabetes diagnosed as having neuropathy by neuropathy disability score (NDS), in relation to the severity of neuropathy based on the vibration perception threshold (VPT). A structured continuous preventative foot care programme was designed to ensure proper footwear, walking foot hygiene, callus care, nailcutting, water temperature checks, use of warming devices, bathroom surgery, foot care products and self-inspection. Continual foot-care education and treatment, including podiatry, were available. Evaluation was at least every 6 months. Diabetic patients (n=308) with neuropathy (NDS > or =6), 72.3+/-10.7 years old, 45% men, 10.9+/-8.8 years duration of diabetes, and HbA(1c) 6.5+/-1.3%, without a history of foot lesions were recruited over 3 years and followed-up for 4.6 (3-6) years. A low risk group (n=124) had a VPT<25 V while 184 had a VPT > or =25 V (high risk). In all 220 patients (71%) complied with the programme, compliance being 76 and 68% in low and high risk groups. The low risk group developed nine ulcers in nine patients, and the high risk group 24 ulcers in 19 patients. Of these eight and 19 ulcers, respectively, were in the non-compliant patient group, giving relative risk of 22 and eight compared with people attending the programme. Thus compliance with a preventative foot programme reduces the incidence of foot ulceration in people with diabetes with neuropathy. This decrease is relatively greater in patients with less severity of neuropathy. The simple design should be widely generalisable.