Wrinkles are a major topic in dermocosmetology; the purpose of this work has been to go deeper into the knowledge of cutaneous damage underlying these modifications of skin surface. Up to now, the number of published works about the histological structure of wrinkles is not very large. Therefore to complete the findings, we studied 46 subjects of both sexes, between 57 and 98-year-old, enabling us to obtain 157 skin biopsies of wrinkles (face) and sun-protected areas (abdomen). We used different histological techniques involving histochemistry, immunohistochemistry, electron microscopy and quantification by image analysis in addition to classic standard techniques. This study has allowed us to confirm published structural modifications of wrinkles, but also to display many other original alterations. The increased thinning of the epidermis atrophied with age is confirmed by the study of desmoplakins outlining the cellular contours of keratinocytes. Such a thinning is accompanied by a decrease in several markers of epidermal differentiation at the bottom of the wrinkles: filaggrin, keratohyalin granules and transglutaminase I, disturbing desquamation and the capacity of the horny layer to retain water. The dermoepidermal junction is modified by a decrease of collagen IV and VII, which, combined with fewer and fewer oxytalan fibres under wrinkles, weakens this interface. The deposition of abnormal elastotic tissue in the dermis, with an interruption of these deposits under wrinkles and an atrophy of dermal collagen more pronounced under wrinkles, boosts the magnitude and depth of wrinkles. The composition of the other dermal constituents is also altered with, more particularly, a marked decrease of chondroitin sulphates in the papillary dermis under wrinkles, combined with an asymmetrical variation of glycosaminoglycans on both edges of wrinkles. The atrophy of the hypodermis, also more marked under wrinkles, with a thickening of fibrous lines, also makes the depth of wrinkles more pronounced. Wrinkle formation appears at the same time as numerous modifications in different cutaneous structures, which may be mutually amplified. Such a study by pointing out altered elements in skin physiology, makes the development of specific treatments possible in order to mitigate this unwelcome cutaneous deterioration.

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.

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 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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Using an ex vivo skin-nerve preparation, skin and nerve cells were reconstituted into a single unit and maintained in a nutrient medium bath until required experimentally. Our objective was to use the epidermis as a relay for the induction of an electric current to the neurons following the topical application of capsaicin on the skin epidermis of the skin explant, an agonist of the TRPV1 channel implicated in pruritus and pain. After 10-20 days of coculture to form the re-innervated skin model, we applied a solution of capsaicin directly on the epidermis of the skin explant (4 lM). The resulting current was recorded using a path-clamp technique on the neuronal fibres. Following the topical application of capsaicin, spontaneous activity was triggered, as characterised by repetitive spikes with periods of 125, 225 or 275 ms. This study demonstrates that the skin explant and nerve cells preparation may receive stimuli and be used to screen molecules or to study signal transmission.