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.