Morphological study of the postnatal hippocampal development in the TRPV1 knockout mice.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with polymodal sensory function. TRPV1 links to fever, while, according to previous studies on TRPV1 knock-out (KO) mice, the role of the channel in the generation of febrile seizure is debated. In the hippocampal formation, functional TRPV1 channels are expressed by Cajal-Retzius cells, which have a role in guidance of migrating neurons during development. Despite the developmental aspects of febrile seizure as well as of Cajal-Retzius cells, no information is available about the hippocampal development in TRPV1 KO mouse. Therefore, in the present work postnatal development of the hippocampal formation was studied in TRPV1 KO mice. Several morphological characteristics including neuronal positioning and maturation, synaptogenesis and myelination were examined with light microscopy following immunohistochemical detection of protein markers of various neurons, synapses, and myelination. Regarding the cytoarchitectonics, neuronal migration, morphological, and neurochemical maturation, no substantial difference could be detected between TRPV1 KO and wild-type control mice. Our data indicate that synapse formation and myelination occur similarly in TRPV1 KO and in control animals. We have found slightly, but not significantly larger numbers of persisting Cajal-Retzius cells in the KO mice than in controls. Our result strengthens previous suggestion concerning the role of TRPV1 channel in the postnatal apoptotic cell death of Cajal-Retzius cells. However, the fact that the hippocampus of KO mice lacks major developmental abnormalities supports the use of TRPV1 KO in various animal models of diseases and pathological conditions.

Hydrogen peroxide production by epidermal dual oxidase 1 regulates nociceptive sensory signals.

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission. Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.

Noxious heat threshold temperature and pronociceptive effects of allyl isothiocyanate (mustard oil) in TRPV1 or TRPA1 gene-deleted mice.

AIMS: To investigate the roles of TRPV1 and TRPA1 channels in baseline and allyl isothiocyanate (AITC)-evoked nociceptive responses by comparing wild-type and gene-deficient mice. MAIN METHODS: In contrast to conventional methods of thermonociception measuring reflex latencies, we used our novel methods to determine the noxious heat threshold. KEY FINDINGS: It was revealed that the heat threshold of the tail measured by an increasing-temperature water bath is significantly higher in TRPV1(-/-), but not TRPA1(-/-), mice compared to respective wild-types. There was no difference between the noxious heat thresholds of the hind paw as measured by an increasing-temperature hot plate in TRPV1(-/-), TRPA1(-/-) and the corresponding wild-type mice. The withdrawal latency of the tail from 0°C water was prolonged in TRPA1(-/-), but not TRPV1(-/-), mice compared to respective wild-types. In wild-type animals, dipping the tail or paw into 1% AITC induced an 8-14°C drop of the noxious heat threshold (heat allodynia) of both the tail and paw, and 40-50% drop of the mechanonociceptive threshold (mechanical allodynia) of the paw measured by dynamic plantar esthesiometry. These AITC-evoked responses were diminished in TRPV1(-/-), but not TRPA1(-/-), mice. Tail withdrawal latency to 1% AITC was significantly prolonged in both gene-deleted strains. SIGNIFICANCE: Different heat sensors determine the noxious heat threshold in distinct areas: a pivotal role for TRPV1 on the tail is contrasted with no involvement of either TRPV1 or TRPA1 on the hind paw. Noxious heat threshold measurement appears appropriate for preclinical screening of TRP channel ligands as novel analgesics.

Transient receptor potential ankyrin 1 (TRPA1) receptor is involved in chronic arthritis: in vivo study using TRPA1-deficient mice.

BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable cation channel that is expressed on capsaicin-sensitive sensory neurons, endothelial and inflammatory cells. It is activated by a variety of inflammatory mediators, such as methylglyoxal, formaldehyde and hydrogen sulphide. Since only few data are available about the role of TRPA1 in arthritis and related pain, we investigated its involvement in inflammation models of different mechanisms. METHODS: Chronic arthritis was induced by complete Freund's adjuvant (CFA), knee osteoarthritis by monosodium iodoacetate (MIA) in TRPA1 knockout (KO) mice and C57Bl/6 wildtype mice. For comparison, carrageenan- and CFA-evoked acute paw and knee inflammatory changes were investigated. Thermonociception was determined on a hot plate, cold tolerance in icy water, mechanonociception by aesthesiometry, paw volume by plethysmometry, knee diameter by micrometry, weight distribution with incapacitance tester, neutrophil myeloperoxidase activity and vascular leakage by in vivo optical imaging, and histopathological alterations by semiquantitative scoring. RESULTS: CFA-induced chronic mechanical hypersensitivity, tibiotarsal joint swelling and histopathological alterations, as well as myeloperoxidase activity in the early phase (day 2), and vascular leakage in the later stage (day 7), were significantly reduced in TRPA1 KO mice. Heat and cold sensitivities did not change in this model. Although in TRPA1 KO animals MIA-evoked knee swelling and histopathological destruction were not altered, hypersensitivity and impaired weight bearing on the osteoarthritic limb were significantly decreased. In contrast, carrageenan- and CFA-induced acute inflammation and pain behaviours were not modified by TRPA1 deletion. CONCLUSIONS: TRPA1 has an important role in chronic arthritis/osteoarthritis and related pain behaviours in the mouse. Therefore, it might be a promising target for novel analgesic/anti-inflammatory drugs.

Analgesic topical capsaicinoid therapy increases somatostatin-like immunoreactivity in the human plasma.

The aim of the present study was to evaluate the therapeutic potential of local capsaicinoid (EMSPOMA(®) cream) treatment on chronic low back pain in patients with degenerative spine diseases and to investigate the possible mechanism of action of the therapy. The qualitative and quantitative analyses of capsaicinoids in EMSPOMA(®) cream were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the clinical study 20 patients with degenerative spine diseases were involved in a self-controlled examination. During the 21 day therapy they received 30 min daily treatment with capsaicinoid (EMSPOMA(®)) cream to the lumbar region of the back. The pain (VASs, Oswestry Disability Index) and the mobility of the lumbar region of the spine (Schober's, Domján's L and R test) were detected at baseline and at the end of the 1st, 2nd and 3rd weeks. The plasma level of somatostatin-like immunoreactivity (SST-LI) was measured by radioimmunoassay (RIA) before and after the treatment on the first and the last day of the therapy. Nonivamide (0.01%) was identified as the only capsaicinoid molecule in the cream. In the clinical study the 21 day local nonivamide treatment reduced the pain sensation. Oswestry Disability Index decreased from 39 ± 3.9% to 32.5 ± 4.4%. VASs showed 37.29%-59.51% improvement. In the plasma level of SST-LI threefold elevation was observed after the first nonivamide treatment. We conclude that nonivamide treatment exerts analgesic action in chronic low back pain and causes the release of the antinociceptive and anti-inflammatory neuropeptide somatostatin which may play pivotal role in the pain-relieving effect.

Barriers to the provision of smoking cessation assistance: a qualitative study among Romanian family physicians.

BACKGROUND: Smoking cessation is the most effective intervention to prevent and slow down the progression of several respiratory and other diseases and improve patient outcomes. Romania has legislation and a national tobacco control programme in line with the World Health Organization Framework for Tobacco Control. However, few smokers are advised to quit by their family physicians (FPs). AIM: To identify and explore the perceived barriers that prevent Romanian FPs from engaging in smoking cessation with patients. METHODS: A qualitative study was undertaken. A total of 41 FPs were recruited purposively from Bucharest and rural areas within 600 km of the city. Ten FPs took part in a focus group and 31 participated in semistructured interviews. Analysis was descriptive, inductive and themed, according to the barriers experienced. RESULTS: Five main barriers were identified: limited perceived role for FPs; lack of time during consultations; past experience and presence of disincentives; patients' inability to afford medication; and lack of training in smoking cessation skills. Overarching these specific barriers were key themes of a medical and societal hierarchy, which undermined the FP role, stretched resources and constrained care. CONCLUSIONS: Many of the barriers described by the Romanian FPs reflected universally recognised challenges to the provision of smoking cessation advice. The context of a relatively hierarchical health-care system and limitations of time and resources exacerbated many of the problems and created new barriers that will need to be addressed if Romania is to achieve the aims of its National Programme Against Tobacco Consumption.

Effects of reference analgesics and psychoactive drugs on the noxious heat threshold of mice measured by an increasing-temperature water bath.

The study aimed at validating an increasing-temperature water bath suitable for determining the noxious heat threshold for use in mice. The noxious heat threshold was determined by immersing the tail of the gently held awake mouse into a water container whose temperature was near-linearly increased at a rate of 24°C/min. until the animal withdrew its tail, that is, heating attained the noxious threshold. The effects of standard analgesic, neuroleptic and anxiolytic drugs were investigated in a parallel way on both the noxious heat threshold and the psychomotor activity assessed by the open field test. Morphine, diclofenac and metamizol (dipyrone) elevated the heat threshold of mice with minimum effective doses of 6, 30 and 1000 mg/kg i.p., respectively. These doses of morphine and diclofenac failed to induce any remarkable effect on psychomotor activity in the open field test while that of metamizol exerted a profound inhibition. The anxiolytic diazepam and the neuroleptic droperidol at doses evoking a mild and moderate, respectively, psychomotor inhibition failed to alter the heat threshold. Combination of a subliminal dose of morphine (regarding both antinociceptive and psychomotor inhibitory action) with diclofenac, metamizol, diazepam or droperidol at doses also subliminal regarding the thermal antinociceptive effect elevated the noxious heat threshold without major additional effects in the open field test. It is concluded that the increasing-temperature water bath is suitable for studying the thermal antinociceptive effects of morphine and diclofenac as well as the morphine-sparing action of diclofenac, metamizol, droperidol and diazepam. Behavioural testing is recommended when testing analgesics.

The role of transient receptor potential ankyrin 1 (TRPA1) receptor activation in hydrogen-sulphide-induced CGRP-release and vasodilation.

Activation of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels on capsaicin-sensitive sensory neurons causes release of inflammatory neuropeptides, including calcitonin gene-related peptide (CGRP). We investigated whether the hydrogen sulphide (H(2)S)-evoked CGRP release from sensory neurons of isolated rat tracheae and H(2)S-induced increases in the microcirculation of the mouse ear were mediated by TRPA1 receptor activation. Allylisothiocyanate (AITC) or the H(2)S donor sodium hydrogen sulphide (NaHS) were used as stimuli and CGRP release of the rat tracheae was measured by radioimmunoassay. AITC or NaHS were applied to the ears of Balb/c, C57BL/6, TRPA1 and TRPV1 receptor gene knockout mice and blood flow was detected by laser Doppler imaging. Both AITC and NaHS increased CGRP release from isolated rat tracheae, and both responses were inhibited by the TRPA1 antagonist, HC-030031, but was not affected by the TRPV1 receptor blocker, BCTC. Application of AITC or NaHS increased the cutaneous blood flow in the mouse ears. Similarly to the effect of AITC, the vasodilatory response to NaHS was reduced by HC-030031 or in TRPA1 deleted mice. In contrast, genetic deletion of TRPV1 did not affect the increase in the ear blood flow evoked by AITC or NaHS. We conclude that H(2)S activates TRPA1 receptors causing CGRP release from sensory nerves of rat tracheae, as well as inducing cutaneous vasodilatation in the mouse ear. TRPV1 receptors were not involved in these processes. Our results highlight that TRPA1 receptor activation should be considered as a potential mechanism of vasoactive effects of H(2)S.