These results confirm a marked increase of AGEs during intrinsic ageing in normal human skin and also suggest that glycation is enhanced in photoaged skin.

The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.

Abstract:The nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re-innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co-culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co-culture. Epidermal thickness, cell density and quality of re-innervated skin explant were all higher when skin explants were re-innervated by sensory neurons at 10 days of culture.Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of cocultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system.Abbreviations: DRG, dorsal root ganglion; NF, neurofilaments; NI, without neurons condition; PGP9.5, protein gen product 9.5; PSN, primary sensory neurons condition; TEM, transmission electronic microscopy.Key words: homeostasis -human -innervation -organotypic skin model Accepted for publication 23 November 2011Skin organotypic in vitro systems are very interesting but are incomplete models because they lack innervation (1). Except for the models developed by Gingras to study innervation and myelinization (2,3), there is no available re-innervated skin organotypic model to study skin innervation and its effects.Skin is densely innervated, with the presence of both autonomic and sensory innervation. Furthermore, the nervous system plays an important role in skin homeostasis, health and disease (4). It acts directly on the epidermal organization and the renewal of keratinocytes (5-7). The epidermis is innervated by unmyelinated sensory fibres that ascend vertically between the keratinocytes to reach the stratum corneum (8). The peripheral nervous system and more specifically sensory neurons are part of the Neuro-ImmunoCutaneous System (9). Contact of sensory nerve fibres, component of the extracellular matrix, production of neurotransmitters and neurotrophins are able to modulate epidermal properties (9-13).We developed new model of skin explant co-cultured with primary sensory neuron for evaluating the possibility of neuron to re-innervate the skin explant and their potent effect on epidermis homeostasis.Primary sensory neurons (PSN) extracted from dorsal root ganglia (DRG) of rats were co-cultured with human skin explants from abdominoplasties since 10 days at air-liquid interface. Maintenance medium was constituted by a DMEM-F12 3:1 mixture (Lonza, BE12-719F and BE12-604 F ⁄ U1), with insulin at 5 lg ⁄ ml (Sigma-Aldrich, St Louis, MO, USA, I6634), hydrocortisone at 10 ng ⁄ ml (Sigma-Aldrich, H0135) and nerve growth factor 'NGF' at 25 ng ⁄ ml (Sigma-Aldrich...

Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied. In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [3H]-glucosamine and [35S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor's age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor's age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin. Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.

Skin aging is a complex process determined by genetic factors (intrinsic aging) and environmental factors (extrinsic aging). One of the most influential environmental factor is UV-B irradiation. Hyaluronic acid (HA) is an abundant component of skin extracellular matrix where it plays many roles such as hydration and architectural support. Downregulation of HA during photoaging was reported previously. Changes in expression and function of its degrading enzymes, the hyaluronidases (Hyals) might be involved in this decrease. In the present study, normal human keratinocytes were submitted to increasing doses of UV-B. The mRNA expression of HYAL1, HYAL2 and HYAL3 and the hyaluronidase enzymatic activity were quantified using real-time PCR and a microtiter-based assay, respectively. After UV-B irradiation, HYAL1 mRNA expression was upregulated whereas HYAL2 and HYAL3 mRNAs were downregulated and hyaluronidase enzymatic activity was increased in both cell layer and culture medium. In parallel, immunohistochemical studies performed on UV-B irradiated reconstructed epidermis confirmed that Hyal-1, Hyal-2 and Hyal-3 protein expression were differently regulated by UV-B. Taken together, our results demonstrate that UV-B irradiation induces differential regulations of hyaluronidase expression and enzymatic activity in human keratinocytes. These differential modulations of hyaluronidase expression and activity by UV-B could contribute to cutaneous photoaging.

The skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co-cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells cocultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells.Key words: neuron -neuronal growth -skin Accepted for publication 18 January 2013Nerve growth following a traumatic injury, such as an amputation, burns or skin graft, takes a long time and is often incomplete. Neurotrophins and semaphorins have been implicated in the guidance of neuronal growth (1,2) through interactions with each other to regulate the motility of the sensory neuronal growth cone. However, the mechanisms of the interactions between the peripheral neurons and the keratinocytes/epidermis/skin remain unclear. To study these interactions under normal physiological and injured conditions, the development of in vitro co-culture models is important. At present, models enabling the study of neuron-skin interactions are rare (3), with the majority involving either primary sensory neurons or a neuronal cell line co-cultured with keratinocytes (4-6), reconstructed skin (7), a skin explant (8) or lesional skin (9). Furthermore, the neurons used in these models are limited due to ethical problems involved in harvesting primary neurons and by the necessity to select an appropriate cell line. Today, adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering, but they are not co-cultured (10,11).PC12 is a versatile rat pheochromocytoma cell line that can be differentiated into neurons with sensory or autonomic characteristics using NGF (12,13). These differentiated PC12 cells have been used as a model for sensory or autonomic neurons (14-16). We developed an in vitro model of human skin explant, which can be considered as an equivalent of injured skin, co-cultured with PC12 cells differentiated into neurons.PC12 cells were co-cultured with human skin explants (E) until 10 days. Skin explants (three donors, one for each experiment) were cut using a 6-mm diameter biopsy punch and placed in 12-well culture plate with one skin explant per well. The skin explants were co-cultured with PC12 cells for more than 10 days at an airliquid interface, and the medium was changed every 2 days. The PC12 cells, which were obtained from the ATCC (ATCC, CRL-1721), were grown in DMEM-F12 media (Lonza, BE12-719F) supplemented with 10% ca...

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Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22–70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (H2O2). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.