Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy

Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 μm2) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.     

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar‐simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild‐type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.     

Effects of ultraviolet radiation on human cutaneous nerve fibres

Due to an increasing number of skin diseases as a result of exposure to ultraviolet (UV) radiation, it is necessary to evaluate the effectiveness of new skin care formulations with broad-spectrum sunscreens.
Objectives:  This study aims to assess the status of nerve fibres in healthy human skin, to quantify effects of UV radiation on nerve endings, and to evaluate neuroprotective effects of new skin care formulations against UV exposure damage.
Methods:  Samples were obtained from 34 female patients enrolled for plastic surgery and were immediately treated (10 min) with three emulsions: Cream 1, Cream 2 (placebo) and a sunscreen with sun protection factor 15 (SPF15). Control samples and those treated with the cream emulsions were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody (anti-human CD56/NCAM). Cell damage was assessed by image analysis.
Results:  Several cellular nervous structures were identified in the skin samples, including free nerve endings. UVA and UVB significantly decreased (40–60%) density of nerve endings in the control samples and those treated with placebo (Cream 2) or SPF15 (all P  < 0.001). Cream 1 completely blocked effects of UV radiation on nerve endings ( P  > 0.05 vs. control).
Conclusions:  Quantification of cell damage induced by UV radiation provides useful information for identification of new skin care compounds with neuroprotective properties.     

Photodamage to the cutaneous sensory nerves: role in photoaging and carcinogenesis of the skin?

Chronic exposure to ultraviolet radiation (UVR) plays a significant role in aging and carcinogenesis of the skin. Sensory nerve fibers densely innervate all layers of the skin and get in close anatomical as well as functional contact with cellular components of the epidermis and dermis. In this review, we address the impact of acute and chronic UVR exposure on the cutaneous sensory nervous system and its mediators. We suggest that skin cell-derived nerve growth factor (NGF) and skin nerve-derived neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) may play a central role in intrinsic aging as well as extrinsic (photo-) aging of the skin. In addition, we discuss the possible role of these mediators in photocarcinogenesis.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures. Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice. Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice. The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice. These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Effects of PGF2alpha on human melanocytes and regulation of the FP receptor by ultraviolet radiation

Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE2 receptors EP1 and EP3 and the PGF2alpha receptor FP, and that PGF2alpha stimulates melanocyte dendricity. We now show that PGF2alpha stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF2alpha by melanocytes. These results show that PGF2alpha binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF2alpha in melanocytes in response to ultraviolet radiation suggest that PGF2alpha could act as an autocrine factor for melanocyte differentiation.     

The role of melanocytes in protection against photooxidative stress Krystyna Stepień

Excessive exposure to solar ultraviolet radiation is an essential etiological factor for skin cancer. UV radiation, directly or indirectly through the generation of reactive oxygen species (ROS), causes damage to DNA, proteins and lipids, and induces inflammation and immunosuppression. Cutaneous pigmentation afforded by melanocytes is the main photoprotective mechanism in human skin. In response to UV, melanocytes produce melanin pigments and transfer them to adjacent keratinocytes. This review describes: (i) the photoprotective action of melanin; (ii) the regulation of UV-induced melanogenesis and the role of p53 in this process; (iii) the relation between melanogenic and antioxidant activities in melanocytes. The possible involvement of UV-induced ROS in the stimulation of melanin synthesis is also discussed.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

The role of afferent feedback in the control of hamstrings activity during human gait

In vertebrates, possibly also in man, the pattern of activation of muscles during locomotion can be generated by the spinal cord (locomotor CPG, central pattern generator). However, sensory feedback is crucial to adapt the functioning of the CPG to the external requirements during gait. It is postulated that afferent input from skin and muscles can contribute to the EMG activation patterns as observed in various limb muscles during gait. The activity of the hamstrings at end swing may be partially due to stretch reflexes of these muscles. At end stance the hamstring activity may be assisted by reflexes from natural skin activation from the dorsum of the foot. In addition, more specific actions are also incorporated. For example, sural nerve stimulation induces an activation of biceps femoris (BF) whereas a suppression is usually obtained for semitendinosus (ST), indicating that the induced activation is aimed at exorotation of the lower leg. Similarly, the preferential activation of medial versus lateral gastrocnemius (GM versus GL) in sural nerve induced reflexes could favor such exorotation. It is concluded that the present evidence points towards a possible contribution of various reflexes to the motor output seen during gait for movements both inside and outside the sagittal plane.     

Ultraviolet wavebands and melanoma initiation

In view of claims that ultraviolet radiation-emitting sunbeds are safe, or safe when they emit only longer wavelengths, research findings are reviewed here on the effects of ultraviolet wavebands A and B (UVA, 315-400 nm and UVB, 290-315 nm) on mutagenesis and carcinogenesis in skin, with particular reference to melanocytes and melanoma. Both UVA and UVB radiation have been shown to induce mutations, as well as mutagenic photoproducts such as cyclobutane pyrimidine dimers, in human skin. UVB can induce melanoma in susceptible mice and in xenografted human skin engineered to express melanocyte growth factors. There is evidence for photosensitization of melanocytes by melanin, especially pheomelanin. UVA can induce melanoma in pigmented fish, and melanocytic hyperplasia in pigmented opossums, but has not generally been tested for melanoma induction in pigmented mammals or in human skin. There is no experimental basis for a claim that UVA is safe, and recreational exposure to this known mutagen should be discouraged.     

Keratinocytes Control the Proliferation and Differentiation of Cultured Epidermal Melanocytes from Ultraviolet Radiation B‐Induced Pigmented Spots in the Dorsal Skin of Hairless Mice

Long‐term exposure of ultraviolet radiation B (UVB)‐induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR‐1 X HR//De) F₁. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts//melanocytes from UVB‐induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts//melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts//melanocytes and keratinocytes were prepared and co‐cultured in combination with control and irradiated mice in a serum‐free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non‐irradiated melanoblasts//melanocytes more greatly than those from non‐irradiated mice. In contrast, both non‐irradiated and irradiated adultmelanocytes proliferated and differentiated similarly when they were co‐cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB‐induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Pigmented spots in the wool-bearing skin on white merino sheep induced by ultraviolet light

Black-grey pigmented skin spots, some of which contained pigmented wool fibres, were observed in a flock of 8.5-year-old white Merino ewes. The spots were concentrated along the backline and increased in number following shearing, suggesting exposure to sunlight to be of importance in the development of these non-congenital pigmented skin spots in genetically white Merino sheep. To test the effect of ultraviolet light, white Merino sheep, ranging in age from 3 to 8 years, had a closely clipped midside area of wool-bearing skin irradiated on each of 28 consecutive days. Pigmented skin spots developed in 6 of the 16 white Merino sheep irradiated. Spots first appeared after 10 days of irradiation, the number subsequently increasing with time, and two skin spots were found to contain sparse numbers of black-grey pigmented wool fibres. Histological examination showed both the naturally occurring and irradiation-induced pigmented skin spots resulted from an increase in both number and activity of melanocytes localized along the epidermal-dermal border of the epidermis. With time, the melanocytes were observed to have entered, to varying depths, the outer-root sheath of follicles still producing white wool fibres. These ultraviolet-light-induced changes to epidermal melanocytes in white Merino sheep presumably occur due to alterations within the local tissue environment in which the melanocytes lie.     

Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Skin mild hypoxia enhances killing of UVB-damaged keratinocytes through reactive oxygen species-mediated apoptosis requiring Noxa and Bim

The naturally occurring skin hypoxia has emerged as a crucial host factor of the epidermal microenvironment. We wanted to systematically investigate how reduced oxygen availability of the epidermis modulates the response of keratinocytes and melanocytes to noxious ultraviolet B radiation (UVB). We report that the exposure of normal human keratinocytes (NHKs) or melanocytes (NHEMs) to mild hypoxia drastically impacts cell death responses following UVB irradiation. The hypoxic microenvironment favors survival and reduces apoptosis of UVB-irradiated NHEMs and their malignant counterparts (melanoma cells). In contrast, NHKs, but not the transformed keratinocytes, under hypoxic conditions display increased levels of reactive oxygen species (ROS) and are significantly sensitized to UVB-mediated apoptosis as compared to NHKs treated under normoxic conditions. Prolonged exposure of UVB-treated NHKs to hypoxia triggers a sustained and reactive oxygen species-dependent activation of the stress kinases p38(MAPK) and JNKs, which in turn, engage the activation of Noxa and Bim proapoptotic proteins. Combined silencing of Noxa and Bim significantly inhibits UVB-mediated apoptosis under hypoxic conditions, demonstrating that hypoxia results in an amplification of the intrinsic apoptotic pathway. Physiologically occurring skin hypoxia, by facilitating the specific removal of UVB-damaged keratinocytes, may represent a decisive host factor impeding important steps of the photocarcinogenesis process.     

Heat Pain Increases the Perceived Magnitude of an Innocuous Electrical Stimulus: Evidence for a Peripheral Mechanism

Painful heat produced an increase in the perceived magnitude of an innocuous electrical stimulus applied either to the sural nerve or to the skin of the dorsum of the foot. The increased sensitivity was observed when the painful heat was spatially coincident with the electrical stimulus, and when it was not coincident but adjacent within the same dermatome. Painful heat had no effect when it was applied to the contralateral foot, which makes it unlikely that attention or arousal played any role in the increased electrical sensitivity produced by ipsilateral heat. The painful heat also produced an increase in the amplitude of the sural nerve compound action potential (CAP). The heat-pain-related changes in the CAP and subjective magnitude ratings were in the same direction, which suggests that the latter were due at least in part to a temperature-dependent change in the electrical sensitivity of the peripheral afferents     

Pigmentation effects of solar-simulated radiation as compared with UVA and UVB radiation

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre-existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2-week repetitive exposure of human skin to solar-simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin-related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5-S-cysteinyldopa (5SCD) were elevated about 4-fold in SSR- or UVB-exposed skin compared with UVA-exposed or control skin. Levels of protein-bound form of 5SCD tended to be higher in SSR- or UVB-exposed skin than in UVA-exposed or control skin. Total levels of 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR- than in UVB-exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.     

Characteristics of dorsal horn neurons expressing subliminal responses to sural nerve stimulation

The present study was designed (1) to characterize the subliminal responses of dorsal horn neurons to stimulation of the sural nerve, and (2) to correlate the type of response to this stimulus with the responses to natural mechanical stimulation of the skin. To accomplish this, intracellular and extracellular recordings were carried out in L6 and L7 dorsal horn neurons in the cat. The excitatory responses of each cell to electrical stimulation of the sural nerve and to mechanical stimulation of the skin were noted. Of 35 dorsal horn cells recorded intracellularly, 11 responded with impulses to sural nerve stimulation, 9 responded with excitatory postsynaptic potentials (EPSPs) but not impulses, and 15 had no excitatory responses to this stimulus. The type of response to sural nerve stimulation was strongly correlated with receptive field modality. Most cells receiving an input from high-threshold cutaneous mechanoreceptors responded with impulses or gave no excitatory response to sural nerve stimulation, whereas most cells that had only low-threshold mechanoreceptor input responded with EPSPs only or gave no response. In cells with only low-threshold (LT) mechanoreceptive input, response to sural nerve stimulation was highly correlated with receptive field locus. Those LT cells with no excitatory responses to sural nerve stimulation had receptive fields confined to the foot and/or toes, whereas those that gave EPSPs had more proximal receptive fields. The possible significance of these data with reference to changes observed after lesions, such as increased response to sural nerve stimulation, increased receptive field size, and somatotopic reorganization, is discussed.     

15th Annual spring brain conference,march 10–13, 2004,summary report

The present study was designed (1) to characterize the subliminal responses of dorsal horn neurons to stimulation of the sural nerve, and (2) to correlate the type of response to this stimulus with the responses to natural mechanical stimulation of the skin. To accomplish this, intracellular and extracellular recordings were carried out in L⁶ and L⁷ dorsal horn neurons in the cat. The excitatory responses of each cell to electrical stimulation of the sural nerve and to mechanical stimulation of the skin were noted.

Of 35 dorsal horn cells recorded intracellularly, 11 responded with impulses to sural nerve stimulation, 9 responded with excitatory postsynaptic potentials (EPSPs) but not impulses, and 15 had no excitatory responses to this stimulus. The type of response to sural nerve stimulation was strongly correlated with receptive field modality. Most cells receiving an input from high-threshold cutaneous mechanoreceptors responded with impulses or gave no excitatory response to sural nerve stimulation, whereas most cells that had only low-threshold mechanoreceptor input responded with EPSPs only or gave no response. In cells with only low-threshold (LT) mechanoreceptive input, response to sural nerve stimulation was highly correlated with receptive field locus. Those LT cells with no excitatory responses to sural nerve stimulation had receptive fields confined to the foot and/or toes, whereas those that gave EPSPs had more proximal receptive fields. The possible significance of these data with reference to changes observed after lesions, such as increased response to sural nerve stimulation, increased receptive field size, and somatotopic reorganization, is discussed.