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...

The crypts of distal colon are submitted to frequent cell volume modifications resulting from fluctuating entry or exit of ion solutes and osmotically obliged water, and from variations in the osmotic pressure in the luminal compartment of the colon. The osmotically induced variations in crypt cell volume are rapidly compensated by uptake or efflux of osmotically active molecules. Thus, exposure of colon crypts to hypotonic media causes cell swelling followed by regulatory volume decrease (RVD) (Diener & Scharrer, 1995 1. A video-imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2. Transition from isotonic (310 mosmol l¢) to hypotonic (240 mosmol l¢) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3. Exposure of crypts to Cl¦-free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na¤-free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4. The chloride channel inhibitor NPPB (50 ìÒ) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5. The K¤ channel blockers barium (10 mÒ), charybdotoxin (10 nÒ) and TEA (5 mÒ) inhibited RVD by 51, 44 and 32%, respectively. 6. Intracellular [Ca¥] rose from a baseline of 174 ± 17 nÒ (n = 8) to 448 ± 45 nÒ (n = 8) during the initial swelling phase 7. The Ca¥ channel blockers verapamil (50 ìÒ) and nifedipine (10 ìÒ), the chelator of intracellular Ca¥ BAPTA AM (30 ìÒ), or the inhibitor of Ca¥ release TMB-8 (10 ìÒ), dramatically reduced volume recovery, leading to 51% (n = 9), 25% (n = 7), 37% (n = 6), 32% (n = 8) inhibition of RVD, respectively. TFP (50 ìÒ), an antagonist of the Ca¥-calmodulin complex, significantly slowed RVD. The Ca¥ ionophore A23187 (2 ìÒ) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca¥ from intracellular stores using bradykinin (1 ìÒ) or blockade of reabsorption with thapsigargin (1 ìÒ) decreased the duration of RVD. 8. Prostaglandin E2 (PGE2, 5 ìÒ) slightly delayed RVD, whereas leukotriene DÚ (LTD4, 100 nÒ) and arachidonic acid (10 ìÒ) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 ìÒ) inhibited RVD by 53%.

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...

Merkel cells (MCs) are rare multimodal epidermal sensory cells. Due to their interactions with slowly adapting type 1 (SA1) Aβ low-threshold mechanoreceptor (Aβ-LTMRs) afferents neurons to form Merkel complexes, they are considered to be part of the main tactile terminal organ involved in the light touch sensation. This function has been explored over time by ex vivo, in vivo, in vitro, and in silico approaches. Ex vivo studies have made it possible to characterize the topography, morphology, and cellular environment of these cells. The interactions of MCs with surrounding cells continue to be studied by ex vivo but also in vitro approaches. Indeed, in vitro models have improved the understanding of communication of MCs with other cells present in the skin at the cellular and molecular levels. As for in vivo methods, the sensory role of MC complexes can be demonstrated by observing physiological or pathological behavior after genetic modification in mouse models. In silico models are emerging and aim to elucidate the sensory coding mechanisms of these complexes. The different methods to study MC complexes presented in this review may allow the investigation of their involvement in other physiological and pathophysiological mechanisms, despite the difficulties in exploring these cells, in particular due to their rarity.

Adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum-free medium, stem cells from abdominal skin were cultured in serum-free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil-red-O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki-67 for proliferative cells, (v) FM1-43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1-43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown.