Enhanced spinal neuronal responses as a mechanism for increased number and size of active acupoints in visceral hyperalgesia.

Acupuncture has been used to treat a variety of illness and involves the insertion and manipulation of needles into specific points on the body (termed "acupoints"). It has been suggested that acupoints are not merely discrete, static points, but can be dynamically changed according to the pathological state of internal organs. We investigated in a rat model of mustard oil (MO)-induced visceral hyperalgesia whether the number and size of acupoints were modified according to the severity of the colonic pain, and whether the changes were associated with enhanced activity of the spinal dorsal horn. In MO-treated rats, acupoints showing neurogenic inflammation (termed "neurogenic spots" or Neuro-Sps) were found both bilaterally and unilaterally on the leg. The number and size of these acupoints increased along with increasing doses of MO. Electroacupuncture of the acupoints generated analgesic effects on MO-induced visceral hypersensitivity. The MO-treated rats showed an increase in c-Fos expression in spinal dorsal horn neurons and displayed increased evoked activity and a prolonged after-discharge in spinal wide dynamic response (WDR) neurons in response to colorectal distension. Increased number and size of neurogenic inflammatory acupoints following MO treatment were reduced by inhibiting AMPA and NMDA receptors in the spinal cord. Our findings suggest that acupoints demonstrate increased number and size along with severity of visceral pain, which may be associated with enhanced neuronal responses in spinal dorsal horn neurons.

Ongoing pain in streptozotocin model of diabetes in the rat: correlation with cutaneous cheminociception.

Streptozotocin (STZ) is commonly used to induce diabetes mellitus in experimental animal studies on peripheral diabetic neuropathy (PDN). Animals with STZ model of diabetes commonly develop changes in test stimulus-evoked pain behavior. However, it is still unclear whether rats with STZ model of diabetes have ongoing pain. Here we assessed whether STZ-induced diabetes induces ongoing pain-like behavior in male rats using conditioned place-preference (CPP) paradigm. CPP was tested in the fourth week of diabetes by pairing one chamber of the CPP device with vehicle and another chamber with either pregabalin (an established analgesic; 30 mg/kg i.p.; n = 9) or Chembridge-5861528 (a TRPA1 channel antagonist; 30 mg/kg i.p.; n = 9). After drug-pairings, the animals were allowed to choose which chamber they preferred. Mechanical sensitivity was assessed with monofilaments and chemonociception in the skin by determining mustard oil-induced pain behavior. Diabetic animals developed in two weeks mechanical hypersensitivity that changed into hyposensitivity by the fourth week. Mustard oil-induced sustained pain was reduced by the 4th week. After 4 weeks of diabetes, neither pregabalin nor the TRPA1 antagonist induced a significant overall change in the median CPP, although both drugs significantly reduced median withdrawal responses evoked by noxious mechanical stimulation. Pregabalin-induced CPP, however, had a significant positive correlation with the sustained pain behaviour induced by topical mustard oil. In conclusion, the present results suggest that the response to topical mustard oil may predict ongoing pain-like behavior in the STZ model of diabetes.

Vascular Hyperpermeability Response in Animals Systemically Exposed to Arsenic.

The mechanisms underlying cardiovascular diseases induced by chronic exposure to arsenic remain unclarified. The objectives of this study were to investigate whether increased vascular leakage is induced by inflammatory mustard oil in mice systemically exposed to various doses of arsenic and whether an increased vascular leakage response is still present in arsenic-fed mice after arsenic discontinuation for 2 or 6 months. ICR mice were fed water or various doses of sodium arsenite (10, 15, or 20 mg/kg/day; 5 days/week) for 8 weeks. In separate experiments, the mice were treated with sodium arsenite (20 mg/kg) for 2 or 8 weeks, followed by arsenic discontinuation for 2 or 6 months. Vascular permeability to inflammatory mustard oil was quantified using Evans blue (EB) techniques. Both arsenic-exposed and water-fed (control) mice displayed similar basal levels of EB leakage in the ears brushed with mineral oil, a vehicle of mustard oil. The levels of EB leakage induced by mustard oil in the arsenic groups fed with sodium arsenite (10 or 15 mg/kg) were similar to those of water-fed mice. However, increased levels of EB leakage in response to mustard oil stimulation were significantly higher in mice treated with sodium arsenite (20 mg/kg; high dose) than in arsenic-fed (10 or 15 mg/kg; low and middle doses) or control mice. After arsenic discontinuation for 2 or 6 months, mustard oil-induced vascular EB leakage in arsenic-fed (20 mg/kg) mice was similar to that in control mice. Dramatic increases in mustard oil-induced vascular leakage were only present in mice systemically exposed to the high arsenic dose, indicating the synergistic effects of the high arsenic dose and mustard oil.

Deep fried edible oils disturb hepatic redox equilibrium and heightens lipotoxicity and hepatosteatosis in male Wistar rats.

Hepatosteatosis is a complex disorder, in which insulin resistance and associated dyslipidemic and inflammatory conditions are fundamental. Dietary habit, especially regular consumption of fat and sugar-rich diet, is an important risk factor. Coconut and mustard oils (CO and MO) are medium-chain saturated and monounsaturated fats that are common dietary ingredients among the Indian populations. Present study analyzed the effect of prolonged consumption of the fresh and thermally oxidized forms of these oils on glucose tolerance and hepatosteatosis in male Wistar rats. Thermally oxidized CO (TCO) and MO (TMO) possessed higher amount of lipid peroxidation products and elevated p-anisidine values than their fresh forms. Dietary administration of TCO and TMO along with fructose altered glucose tolerance and increased hyperglycemia in rats. Dyslipidemia was evident by elevated levels of triglycerides and reduced high density lipoprotein cholesterol (HDLc) levels in fructose and edible oil-fed group ( p < 0.05). Additionally, hepatic antioxidant status was diminished and oxidative stress markers were elevated in TCO- and TMO-fed rats. Substantiating these, hike in liver function marker enzyme activities were also observed in these animals. Supporting this, histological analysis revealed higher incidence of microvesicles and hepatocellular ballooning. Results thus suggest that consumption of thermally oxidized fats may cause hepatic damage.

A novel zebrafish-based model of nociception.

Chronic pain affects the lives of millions yearly, but few new treatments are available. Due to decreasing budgets and increasing costs of preclinical research, alternatives are sought with high translatability and low cost. Here we demonstrate the utility of a zebrafish-based model of nociception to serve as a novel screening tool for analgesic drugs. Zebrafish swimming behavior was measured following administration of various algogens including histamine, cinnamaldehyde, mustard oil, acetic acid and complete Freund's adjuvant. All compounds reduce distance traveled, thought to be an expression of nociception. Additionally, the suppression of swimming was attenuated by administration of the common analgesic, morphine. Together these data provide support for the use of zebrafish as a cost-effective and translatable model of nociception.

Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways.

KEY POINTS: Voltage-gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage-gated sodium channel subtype NaV 1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV 1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality-specific manner and help to direct drug discovery efforts towards novel visceral analgesics. ABSTRACT: Voltage-gated sodium channel NaV 1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific NaV 1.7 knockout mouse (NaV 1.7Nav1.8 ) and selective small-molecule NaV 1.7 antagonist PF-5198007. NaV 1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV 1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV 1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of NaV 1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed NaV 1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV 1.7 (in NaV 1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV 1.7 antagonist PF-5198007. Our data demonstrate that NaV 1.7 (in NaV 1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV 1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

Protective effects of dietary glycine and glutamic acid toward the toxic effects of oxidized mustard oil in rabbits.

The protective role of glycine and glutamic acid against the toxic effects of oxidized oil was studied for the first time. Mustard seed oil was thermally oxidized and characterized for quality characteristics and polyphenolic composition using reversed phase HPLC-DAD. Significant changes in the quality characteristics occurred with thermal oxidation. Fourteen polyphenolic compounds were identified and quantified in oils. Quercetin-3-glucoside, quercetin-3-feruloylsophoroside, catechin, quercetin-3-rutinoside, quercetin-3,7-diglucoside, sinapic acid and vanillic acid hexoside were the major compounds in the fresh and oxidized oil. Oxidized, un-oxidized mustard oils, glycine and glutamic acid were given to rabbits alone or in combination. The biochemical responses were studied in terms of haematological and biochemical parameters and histopathology. It has been observed that biochemical and haematological parameters were adversely affected by the oxidized oil, while supplementation of both amino acids was beneficial in normalizing these parameters. Both amino acids alone have no significant effects, however, oxidized oil affected the liver by enhancing fat accumulation, causing hepatitis, reactive Kupffer cells and necrosis. The co-administration of oxidized oils with glycine or glutamic acid revealed significant recovery of the liver structure and function. In conclusion, glycine or glutamic acid is beneficial and protective against food toxicity and can be considered as an ameliorative food supplement.

Chalcone Derivatives Activate and Desensitize the Transient Receptor Potential Ankyrin 1 Cation Channel, Subfamily A, Member 1 TRPA1 Ion Channel: Structure-Activity Relationships in vitro and Anti-Nociceptive and Anti-inflammatory Activity in vivo.

Eleven compounds belonging to the chalcone family were tested for their ability to activate and subsequently desensitize the rat transient receptor potential ankyrin 1 cation channel, subfamily A, member 1 (TRPA1) in a heterologous expression system. Four of the tested compounds were more potent than the TRPA1 agonist mustard oil, and showed also a strong desensitizing effect. Some chalcone compounds were not pungent in the eye-wiping assay and quite remarkably inhibited in a long-lasting and dose-dependent manner the pain response in the formalin test. Chalcones can be considered as novel candidates for the development of antihyperalgesic preparations based on TRPA1 desensitization.

Neonatal vaginal irritation results in long-term visceral and somatic hypersensitivity and increased hypothalamic-pituitary-adrenal axis output in female mice.

Experiencing early life stress or injury increases a woman's likelihood of developing vulvodynia and concomitant dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. To investigate the outcome of neonatal vaginal irritation (NVI), female mouse pups were administered intravaginal zymosan on postnatal days 8 and 10 and were assessed as adults for vaginal hypersensitivity by measuring the visceromotor response to vaginal balloon distension (VBD). Western blotting and calcium imaging were performed to measure transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) in the vagina and innervating primary sensory neurons. Serum corticosterone (CORT), mast cell degranulation, and corticotropin-releasing factor receptor 1 (CRF1) expression were measured as indicators of peripheral HPA axis activation. Colorectal and hind paw sensitivity were measured to determine cross-sensitization resulting from NVI. Adult NVI mice had significantly larger visceromotor response during VBD than naive mice. TRPA1 protein expression was significantly elevated in the vagina, and calcium transients evoked by mustard oil (TRPA1 ligand) or capsaicin (TRPV1 ligand) were significantly decreased in dorsal root ganglion from NVI mice, despite displaying increased depolarization-evoked calcium transients. Serum CORT, vaginal mast cell degranulation, and CRF1 protein expression were all significantly increased in NVI mice, as were colorectal and hind paw mechanical and thermal sensitivity. Neonatal treatment with a CRF1 antagonist, NBI 35965, immediately before zymosan administration largely attenuated many of the effects of NVI. These results suggest that NVI produces chronic hypersensitivity of the vagina, as well as of adjacent visceral and distant somatic structures, driven in part by increased HPA axis activation.

Decreased face primary motor cortex (face-M1) excitability induced by noxious stimulation of the rat molar tooth pulp is dependent on the functional integrity of face-M1 astrocytes.

Acute inflammatory dental pain is a prevalent condition often associated with limited jaw movements. Mustard oil (MO, a small-fiber excitant/inflammatory irritant) application to the rat molar tooth pulp induces increased excitability (i.e., central sensitization) of trigeminal medullary dorsal horn (MDH) nociceptive neurons that can be modulated by MDH application of the astrocytic inhibitor methionine sulfoximine (MSO). The objectives of the study were to determine whether MO application to the rat right maxillary first molar tooth pulp affects left face-M1 excitability manifested as altered intracortical microstimulation thresholds for evoking electromyographic activity in the right anterior digastric (RAD, jaw-opening muscle), and whether MSO application to face-M1 can modulate this MO effect. Under Ketamine general anesthesia, Sprague-Dawley male rats had a microelectrode positioned at a low-threshold (≤30 µA) face-M1 site. Then MO (n = 16) or control solution (n = 16) was applied to the previously exposed tooth pulp, and RAD threshold was monitored for 15 min. MSO (0.1 mM, n = 8) or saline (n = 8) was then applied to the face-M1, and RAD thresholds were monitored every 15 min for 120 min. ANOVA followed by post hoc Bonferroni was used to analyze data (p < 0.05). Within 15 min of MO (but not control) pulp application, RAD thresholds increased significantly (p < 0.001) as compared to baseline. One hour following MSO (but not saline) application to the face-M1, RAD thresholds decreased significantly (p = 0.005) toward baseline. These novel findings suggest that acute inflammatory dental pain is associated with decreased face-M1 excitability that may be dependent on the functional integrity of face-M1 astrocytes and related to mechanisms underlying limited jaw movements in acute orofacial pain conditions.

Epidemic dropsy 2013: case series.

Epidemic dropsy (ED) is caused due to intoxication with Argemone mexicana. Here we report a case series of three families, all of whom were residents of Uttar Pradesh, India, who presented in August 2013 with all the classical features of ED. We aim to highlight the importance of this malady even though the sale of unbottled mustard oil is illegal in India.

pERK1/2 immunofluorescence in rat dorsal horn and paraventricular nucleus neurons as a marker for sensitization and inhibition in the pain pathway.

The aim of the present study was to investigate whether the phosphorylation of ERK1/2 in the rat lumbar dorsal horn and in the parvocellularis part of the paraventricular nucleus can be used to visualize neuronal activity. pERK1/2 fluorescence-immunohistochemistry is specifically suited to mirror neuronal activity in the pain pathway following an acute noxious stimulation. The rat hind paw was either stimulated by noxious heat or by a sequence of mustard oil and noxious heat. Two and 10 min after the thermal stimulation a 3-4-fold increase in cells with pERK1/2 immunoreactivity was observed in lamina I/II of the L3-L5 dorsal horn. The combination of mustard oil with heat led to a 5-6-fold increase in the pERK1/2 signal. The pERK1/2 immunoreactivity in the parvocellularis part of the paraventricular nucleus increased by 2-fold following the heat stimulus, with no further increase following the sequential mustard oil and heat stimulus. A pretreatment with the opioid analgesic morphine or the NMDA antagonist MK-801 markedly attenuated ERK1/2 phosphorylation in both areas of the pain pathway. The present findings support the concept that the pERK1/2 immunofluorescence signal can be used as a quantitative marker for sensitization or inhibition in the pain pathway at spinal and hypothalamic level.

Start-up of a combined anaerobic/partial nitritation/anammox process for high-salt mustard wastewater treatment.

To treat high salinity wastewater from the mustard pickling industry, a combined anaerobic, partial nitritation (PN), and anammox process was employed using three connected reactors: an anaerobic sequencing batch biofilm reactor (ASBBR) for anaerobic treatment, a sequencing batch reactor (SBR) for PN, and an upflow anaerobic sludge blanket (UASB) for anammox. The start-up of the three individual reactors was investigated. Results showed that each reactor started up successfully, notwithstanding the stepwise increase of influent salinity to about 16.1 g NaCl/L. In the ASBBR, 89.7 % of chemical oxygen demand in the influent was removed and organic nitrogen was converted to ammonium (NH4 (+)-N). The SBR performed well with NO3 (-)-N concentration of 4.9 mg/L and ratio of NO2 (-)-N to NH4 (+)-N at the range of 1.0 to 1.3 in the effluent, which favored the anammox process. After the start-up of the UASB, the anammox process also showed stability and efficiency with a high total nitrogen removal efficiency of 86.2 % under high salinity of 12.0 g NaCl/L and nitrogen loading rate of 258 mg/(L · day).

Central α-adrenoceptors contribute to mustard oil-induced central sensitization in the rat medullary dorsal horn.

Our previous studies have demonstrated that application of the inflammatory irritant mustard oil (MO) to the tooth pulp produces trigeminal central sensitization that includes increases in mechanoreceptive field size and responses to noxious stimuli and decrease in activation threshold in brainstem nociceptive neurons of trigeminal subnucleus caudalis (the medullary dorsal horn, MDH). The aim of the present study was to test if central noradrenergic processes are involved in the central sensitization of MDH neurons and if α1-adrenoceptors or α2-adrenoceptors or both are involved. In urethane/α-chloralose-anesthetized rats, the activity of extracellularly recorded and functionally identified single nociceptive neurons in the MDH was studied. Continuous intrathecal (i.t.) superfusion of the adrenergic modulator guanethidine and α-adrenoceptor blocker phentolamine or selective α1-adrenoceptor antagonist prazosin over the medulla strongly attenuated all three MO-induced parameters of central sensitization in the MDH nociceptive neurons, compared to phosphate-buffered saline (as vehicle control). In contrast, i.t. superfusion of the selective α2-adrenoceptor antagonist yohimbine had little effect on the mechanoreceptive field expansion and the decreased mechanical activation threshold, and indeed facilitated responses to noxious stimuli of sensitized nociceptive neurons. Superfusion of each of the four chemicals alone did not affect baseline nociceptive neuronal properties. These findings provide the first documentation of the involvement of central noradrenergic processes in MDH in the development of the central sensitization, and that α1- and α2-adrenoceptors may be differentially involved.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: a microdialysis study.

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.

Inhibition of sensory afferents activation and visceral pain by a brominated algal diterpene.

BACKGROUND: In the search of new therapeutic options for the treatment of pain, isolation, and testing of secondary metabolites from plant extracts has raised significant attention. We have investigated the effects of the brominated diterpene O(11) 15- cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (that we have named VLC5), extracted from the Mediterranean red algae Laurencia glandulifera. METHODS: The pure extract was tested on primary afferent calcium signals induced by high concentration of KCl, transcient receptor potential vanilloid (TRPV)1 (capsaicin) or TRPV4 agonists, histamine, or protease-activated receptor-2 (PAR(2) ) agonist. It was also tested in mice in a model of mustard oil-induced colonic hypersensitivity. KEY RESULTS: VLC5 was inhibited PAR(2) agonist or histamine-induced calcium mobilization in mouse primary afferents, but did not modify calcium signals induced by high concentrations of KCl, TRPV1 or TRPV4 agonists. The effect of VLC5 on histamine-induced calcium signal in primary afferent was inhibited by pertussis toxin pretreatment and was dependent on the activation of mu- or kappa-opioid receptor agonists, as it was inhibited by selective antagonists of those two receptors, but not by selective antagonist of the delta-opioid receptor. Intraperitoneal treatment of mice with VLC5 (10 mg kg(-1)) significantly reduced visceral pain behaviors induced by the intracolonic administration of mustard oil, in an opioid receptor-dependent manner. CONCLUSIONS & INFERENCES: We have demonstrated significant analgesic properties for the algal metabolite VLC5, which is able to signal directly to primary afferents, through a mechanism dependent on the activation of opioid receptors. This identifies a new natural compound capable of activating peripheral opioidergic systems, exerting analgesic properties.

Systemic pregabalin attenuates sensorimotor responses and medullary glutamate release in inflammatory tooth pain model.

Our previous studies have demonstrated that application of inflammatory irritant mustard oil (MO) to the tooth pulp induces medullary glutamate release and central sensitization in the rat medullary dorsal horn (MDH), as well as nociceptive sensorimotor responses in craniofacial muscles in rats. There is recent evidence that anticonvulsant drugs such as pregabalin that influence glutamatergic neurotransmission are effective in several pain states. The aim of this study was to examine whether systemic administration of pregabalin attenuated glutamate release in the medulla as well as these nociceptive effects reflected in increased electromyographic (EMG) activity induced by MO application to the tooth pulp. Male adult rats were anesthetized with isofluorane (1.0-1.2%), and jaw and tongue muscle EMG activities were recorded by needle electrodes inserted bilaterally into masseter and anterior digastric muscles and into the genioglossus muscle, and also the medullary release of glutamate was assessed by in vivo microdialysis. Pregabalin or vehicle control (isotonic saline) was administered 30 min before the pulpal application of MO or vehicle control (mineral oil). Application of mineral oil to the maxillary first molar tooth pulp produced no change in baseline EMG activity and glutamate release. However, application of MO to the pulp significantly increased both the medullary release of glutamate and EMG activity in the jaw and tongue muscles for several minutes. In contrast, pre-medication with pregabalin, but not vehicle control, significantly and dose-dependently attenuated the medullary glutamate release and EMG activity in these muscles after MO application to the tooth pulp (analysis of variance (ANOVA), p<0.05). These results suggest that pregabalin may attenuate the medullary release of glutamate and associated nociceptive sensorimotor responses in this acute inflammatory pulpal pain model, and that it may prove useful for the treatment of orofacial inflammatory pain states.

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch.

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.

Salicylic acid minimizes nickel and/or salinity-induced toxicity in Indian mustard (Brassica juncea) through an improved antioxidant system.

INTRODUCTION: This study was hypothesized that salicylic acid elevates the level of antioxidant system that will protect plants from the stress generated by nickel and/or salinity. MATERIALS AND METHODS: Seeds of Brassica juncea were sown in sand amended with NiCl(2) (100 mg kg(-1)) and/or 15-day-old seedlings supplied for 3 days with NaCl (150 mM) and were then, at 20-day stage, sprayed with salicylic acid (10(-5) M) to assess selected morphological, physiological, and biochemical parameters at 30-day stage. RESULTS: The combination of Ni and NaCl proved most deleterious and exhibited significant decline in growth, leaf water potential, the level of pigments, and photosynthetic attributes. However, the follow-up treatment with salicylic acid detoxified the stress-generated damages caused by the combination (NiCl(2) and NaCl) and also significantly improved values for the above parameters. The NiCl(2) and/or NaCl increased electrolyte leakage, lipid peroxidation, and H(2)O(2) content but decreased the membrane stability index and activity of nitrate reductase and carbonic anhydrase. However, the salicylic acid treatment in the presence or absence of the stress improved the activity of nitrate reductase and carbonic anhydrase. The activity of antioxidative enzymes and the level of proline exhibited a significant increase in response to NiCl(2) and/or NaCl stress and which enhanced further with the spray of salicylic acid. CONCLUSIONS: It is concluded that the elevated level of antioxidative enzymes and level of proline might be responsible for minimizing the Ni and/or salinity-induced toxicity in Indian mustard which is manifested in terms of improved growth and photosynthesis.

Isothiocyanate-containing mustard protects human cells against genotoxins in vitro and in vivo.

BACKGROUND: Human intervention trials in which cytogenetic biomarkers are used as intermediate endpoints in carcinogenesis are implicitly required to support the assumption of chemo-preventive efficacy. METHODS: To evaluate the genotoxic and anti-genotoxic properties of defined isothiocyanate-containing mustard, we first used a human liver cell-line and then conducted a controlled pilot human intervention trial. Blood from volunteers served as surrogate tissue for time-kinetic analysis of the chemo-preventive effect of mustard consumption. RESULTS: Mustard extracts displayed significant anti-genotoxicity against benzo(a)pyrene in human HepG2 hepatoma cells. At high concentrations, the extracts induced genotoxicity by themselves without compromising cell viability. The protective effect of mustard supplementation against DNA damage induced ex vivo was detected in blood of volunteers within 12h after the start of the intervention, and increased over time. No genotoxicity was induced in human peripheral mononuclear blood cells by mustard intake over the whole period of the study. Also, liver parameters remained within the normal range at all times. Although no change in total plasma GST activity was detected, plasma alpha-GST levels increased over time, peaking at 48 h. CONCLUSIONS: The results suggest the capacity of small amounts of isothiocyanate-containing food to protect cells from DNA damage, even with short-term application.