Marlen Petersen

Atopic Keratinocytes Induce Increased Neurite Outgrowth in a Coculture Model of Porcine Dorsal Root Ganglia Neurons and Human Skin Cells

Skin of patients suffering from atopic eczema displays a higher epidermal nerve fiber density, associated with neurogenic inflammation and pruritus. Using an in vitro coculture system, allowing a spatially compartmented culture of somata from porcine dorsal root ganglion neurons and human primary skin cells, we investigated the influence of dermal fibroblasts and keratinocytes on neurite outgrowth. In comparison with dermal fibroblasts, keratinocytes induced more branched and less calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. By adding neutralizing antibodies, we showed that nerve growth factor (NGF) and glial cell-line-derived neurotrophic factor (GDNF) are pivotal neurotrophic factors of skin cell-induced neurite outgrowth. Keratinocytes and dermal fibroblasts secreted different ratios of neurotrophic factors, influencing morphology and CGRP immunoreactivity of neurites. To investigate changes of the peripheral nervous system in the pathogenesis of atopic eczema in vitro, we analyzed neurite outgrowth mediated by atopic skin cells. Atopic keratinocytes produced elevated levels of NGF and mediated an increased outgrowth of CGRP-positive sensory fibers. Our results demonstrate the impact of dermal fibroblasts and keratinocytes on skin innervation and emphasize the role of keratinocytes as key players of hyperinnervation in atopic eczema.

Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human

&NA; Activity‐dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use‐dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C‐fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically‐induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano‐insensitive C‐fibers showed the largest conduction slowing (˜30%) upon electrical stimulation (2 Hz for 3 min). In addition, we found mechano‐insensitive cold nociceptors in pig that slowed only minimally (<10% at 2 Hz), and a similar slowing pattern was found in some human C‐fibers. Mechano‐sensitive afferents showed an intermediate conduction slowing upon 2 Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano‐insensitive afferents recovered the slowest, followed by mechano‐sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano‐insensitive C‐fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class‐specific changes of ADS.

Sensitization to bradykinin B1 and B2 receptor activation in UV-B irradiated human skin

&NA; Bradykinin B1 and B2 receptors contribute to nociceptor sensitization under inflammatory conditions. Here, we examined the vascular inflammatory responses and nociceptive effects resulting from activation of B1 and B2 receptors in healthy and UV‐B irradiated skin in human volunteers. The B1 receptor agonist des‐Arg10‐Kallidin (10−6–10−3 M) and the B2 receptor agonist bradykinin (10−9–10−4 M) were administered by dermal microdialysis to the ventral thigh. UV‐B irradiation was performed 24 h prior to the experiment with the threefold minimum erythemal dose. Pain sensation perceived during the stimulation with the bradykinin receptor agonists was estimated on a numeric scale. Local and axon reflex‐induced vasodilatations were recorded by laser Doppler imaging. For protein extravasation, total protein content in the dialysate was assessed as a measure of increased endothelial permeability. In normal skin, both B1 and B2 receptor activation dose‐dependently evoked pain, vasodilatation and protein extravasation. In UV‐B irradiated skin, pain sensation and axon reflex vasodilatation were enhanced by both B1 and B2 agonists, whereas local vasodilatation was increased only following B1 receptor activation. The UV‐B irradiation did not enhance B1 and B2 receptor‐induced protein extravasation indicating a differential sensitization of the neuronal, but not the vascular response.

Nerve growth factor selectively decreases activity-dependent conduction slowing in mechano-insensitive C-nociceptors

Summary Intracutaneous nerve growth factor reduces the activity‐dependent slowing of conduction of the mechano‐insensitive C‐fibers. This long‐term change of axonal characteristics might contribute to hyperalgesia. Parts of this study have been published in abstract form (Obreja et al., Society for Neuroscience Meeting, 2008, Washington, 668.5). ABSTRACT Nerve growth factor (NGF) induces acute sensitization of nociceptive sensory endings and long‐lasting hyperalgesia. NGF modulation of sodium channel expression might contribute to neurotrophin‐induced hyperalgesia. Here, we investigated NGF‐evoked changes of the activity‐dependent slowing of conduction in porcine C‐fibers. Animals received intradermal injections of NGF (2 μg or 8 μg) or saline in both hind limbs. Extracellular recordings from the saphenous nerves were performed 1 week later. Based on sensory thresholds and electrically induced activity‐dependent slowing (ADS) of axonal conduction, C‐fibers were classified as mechano‐sensitive afferents, mechano‐insensitive afferents, cold nociceptors, and sympathetic efferents. NGF (2 μg) increased conduction velocity in C‐fibers from 1.0 ± 0.05 m/s to 1.2 ± 0.07 m/s. In mechano‐insensitive afferents, NGF (8 μg) reduced activity‐dependent slowing of conduction, from 5.3 ± 0.2% to 3.2 ± 0.5% (0.125–0.5 Hz stimulation) and from 28.5 ± 1.3% to 20.9 ± 1.9% (2 Hz stimulation), such that ADS no longer differentiated between mechano‐sensitive and mechano‐insensitive fibers. Accordingly, the number of fibers with pronounced ADS decreased but more units with pronounced ADS were mechano‐sensitive. Spontaneously active C‐fibers were increased above the control level (1%) by NGF 8 μg (8%). The results demonstrate that NGF changes the functional axonal characteristics of mechano‐insensitive C‐fibers and enhances spontaneous activity thereby possibly contributing to hyperalgesia.

A subpopulation of capsaicin‐sensitive porcine dorsal root ganglion neurons is lacking hyperpolarization‐activated cyclic nucleotide‐gated channels

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore, these channels could be a distinguishing marker.

No evidence for bradykinin B1 receptors in rat dorsal root ganglion neurons.

Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the B1 agonist was detected, whereas 12% of neurons responded to the B2 agonist. Northern blot analysis confirmed the lack of B1 receptor expression in dorsal root ganglia, as B1 mRNA was neither detected under normal conditions nor after nerve injury. In the calcium imaging experiments, agonists were applied with an elevated superfusion flow rate to avoid tachyphylaxis to the drug. Normal external solution applied at this flow rate constituted a mechanical stimulus causing a response in some neurons. Thus, in comparable set-ups mechanosensitivity has first to be tested to avoid masking effects.

Leukemia inhibitory factor differentially regulates capsaicin and heat sensitivity in cultured rat dorsal root ganglion neurons

Thermal hyperalgesia is one hallmark of neuropathic pain conditions. Although the exact pathophysiological mechanisms remain elusive, nerve growth factor (NGF) and leukemia inhibitory factor (LIF) are considered key mediators. Their local availability or synthesis are altered by nerve damage and, in turn, they entail changes in phenotype of affected neurons. We examined the effects of LIF on capsaicin sensitivity, heat responsiveness, and galanin immunoreactivity in rat dorsal root ganglion neurons cultured for up to 6 days without supplemented NGF. Using double labeling, the proportions of heat-sensitive/galanin-immunoreactive (GAL-IR) and capsaicin-sensitive/GAL-IR neurons were compared over time in culture with galanin immunoreactivity being a marker for nociceptive neurons. The time course of the proportions of neurons responding to heat (44 degrees C) or capsaicin (1 microM) which also were GAL-IR was differently affected by LIF. In the absence of LIF, within the population of heat-sensitive neurons, the proportion of neurons also GAL-IR increased from 17% to 32% between 6h and 1 day in culture to stay at this level. For the capsaicin-sensitive neurons, the proportion of neurons also GAL-IR increased from 10% after 6h to 18% at day 2 and then decreased to 4% at day 4. In contrast, LIF prevented the increase in the proportion of heat-sensitive/GAL-IR neurons and the decrease of capsaicin-sensitive/GAL-IR neurons. The results suggest that LIF partially prevents TRPV-1 downregulation in NGF-deprived nociceptive galaninergic DRG neurons. Furthermore, there is evidence that LIF regulates the expression of a heat receptor distinct from TRPV-1.

Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata

Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron’s functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.

Local NGF and GDNF levels modulate morphology and function of porcine DRG neurites, In Vitro

Nerve terminals of primary sensory neurons are influenced by their environment through target derived trophic factors, like nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF). In mice, subpopulations of DRG neurons express receptors either for NGF or GDNF and therefore differentially respond to these neurotrophic factors. We probed neurite endings from porcine DRG neurons cultured in either NGF or GDNF and examined their shape, elongation and stimulus-evoked CGRP release. A compartmentalized culture system was employed allowing spatial separation of outgrown neurites from their somata and use of different growth factors in the compartments. We show that neurites of GDNF cultured somata extend into lateral compartments without added growth factor, unlike neurites of NGF cultured ones. Neurites of NGF cultured somata extend not only into NGF- but also into GDNF-containing compartments. GDNF at the site of terminals of NGF responsive somata led to a strong neurite arborization and formation of large growth cones, compared to neurites in medium with NGF. Functionally, we could detect evoked CGRP release from as few as 7 outgrown neurites per compartment and calculated release per mm neurite length. CGRP release was detected both in neurites from NGF and GDNF cultured somata, suggesting that also the latter ones are peptidergic in pig. When neurites of NGF cultured somata were grown in GDNF, capsaicin evoked a lower CGRP release than high potassium, compared to those grown in NGF. Our experiments demonstrate that the compartmented culture chamber can be a suitable model to assess neurite properties from trophic factor specific primary sensory neurons. With this model, insights into mechanisms of gain or loss of function of specific nociceptive neurites may be achieved.

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of keratinocytes derived from new-born piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm2 for primary and 1000 viable cells/cm2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm2 had a PD of 4.36 ± 0.60 per passage and tg of 1.69 ± 0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation.