Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli

Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmyelinated sensory neurons expressing the G protein-coupled receptor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1+ nociceptors, which constitute a nonoverlapping population, selectively abolishes noxious heat pain sensitivity. Combined elimination of both populations yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons

Epidemiology and risk factors of chronic kidney disease in India – results from the SEEK (Screening and Early Evaluation of Kidney Disease) study

Background: There is a rising incidence of chronic kidney disease that is likely to pose major problems for both healthcare and the economy in future years. In India, it has been recently estimated that the age-adjusted incidence rate of ESRD to be 229 per million population (pmp), and >100,000 new patients enter renal replacement programs annually. Methods: We cross-sectionally screened 6120 Indian subjects from 13 academic and private medical centers all over India. We obtained personal and medical history data through a specifically designed questionnaire. Blood and urine samples were collected. Results: The total cohort included in this analysis is 5588 subjects. The mean ± SD age of all participants was 45.22 ± 15.2 years (range 18–98 years) and 55.1% of them were males and 44.9% were females. The overall prevalence of CKD in the SEEK-India cohort was 17.2% with a mean eGFR of 84.27 ± 76.46 versus 116.94 ± 44.65 mL/min/1.73 m2 in non-CKD group while 79.5% in the CKD group had proteinuria. Prevalence of CKD stages 1, 2, 3, 4 and 5 was 7%, 4.3%, 4.3%, 0.8% and 0.8%, respectively. Conclusion: The prevalence of CKD was observed to be 17.2% with ~6% have CKD stage 3 or worse. CKD risk factors were similar to those reported in earlier studies. It should be stressed to all primary care physicians taking care of hypertensive and diabetic patients to screen for early kidney damage. Early intervention may retard the progression of kidney disease. Planning for the preventive health policies and allocation of more resources for the treatment of CKD/ESRD patients are imperative in India

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

Acute Lead Exposure Increases Arterial Pressure: Role of the Renin-Angiotensin System

Background: Chronic lead exposure causes hypertension and cardiovascular disease. Our purpose was to evaluate the effects of acute exposure to lead on arterial pressure and elucidate the early mechanisms involved in the development of lead-induced hypertension. Methodology/Principal Findings: Wistar rats were treated with lead acetate (i.v. bolus dose of 320 μg/Kg), and systolic arterial pressure, diastolic arterial pressure and heart rate were measured during 120 min. An increase in arterial pressure was found, and potential roles of the renin-angiotensin system, Na+,K+-ATPase and the autonomic reflexes in this change in the increase of arterial pressure found were evaluated. In anesthetized rats, lead exposure: 1) produced blood lead levels of 37±1.7 μg/dL, which is below the reference blood concentration (60 μg/dL); 2) increased systolic arterial pressure (Ct: 109±3 mmHg vs Pb: 120±4 mmHg); 3) increased ACE activity (27% compared to Ct) and Na+,K+-ATPase activity (125% compared to Ct); and 4) did not change the protein expression of the α1-subunit of Na+,K+-ATPase, AT1 and AT2. Pre-treatment with an AT1 receptor blocker (losartan, 10 mg/Kg) or an ACE inhibitor (enalapril, 5 mg/Kg) blocked the lead-induced increase of arterial pressure. However, a ganglionic blockade (hexamethonium, 20 mg/Kg) did not prevent lead's hypertensive effect. Conclusion: Acute exposure to lead below the reference blood concentration increases systolic arterial pressure by increasing angiotensin II levels due to ACE activation. These findings offer further evidence that acute exposure to lead can trigger early mechanisms of hypertension development and might be an environmental risk factor for cardiovascular diseaseThis study was supported by grants from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)/FAPES (Fundação de Amparo à Pesquisa do Espírito Santo)/FUNCITEC (Fundação de Ciência e Tecnologia)(39767531/07), Brazil and from MCINN (Ministerio de Ciencia e Innovación) (SAF 2009- 07201) and ISCIII (Instituto de Salud Carlos III) (Red RECAVA- Red Temática de Investigación en Enfermedades Cardiovasculares del Instituto de Salud Carlos III, RD06/0014/0011), Spai

Selective Targeting of TRPV1 Expressing Sensory Nerve Terminals in the Spinal Cord for Long Lasting Analgesia

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain

Lesion of the Cerebellar Noradrenergic Innervation Enhances the Harmaline-Induced Tremor in Rats

Abnormal synchronous activation of the glutamatergic olivo-cerebellar pathway has been suggested to be crucial for the harmaline-induced tremor. The cerebellum receives two catecholaminergic pathways: the dopaminergic pathway arising from the ventral tegmental area/substantia nigra pars compacta, and the noradrenergic one from the locus coeruleus. The aim of the present study was to examine a contribution of the cerebellar catecholaminergic innervations to the harmaline-induced tremor in rats. Rats were injected bilaterally into the cerebellar vermis with 6-hydroxydopamine (6-OHDA; 8 μg/0.5 μl) either alone or this treatment was preceded (30 min earlier) by desipramine (15 mg/kg ip). Harmaline was administered to animals in doses of 7.5 or 15 mg/kg ip. Tremor of forelimbs was measured as a number of episodes during a 90-min observation. Rats were killed by decapitation 30 or 120 min after harmaline treatment. The levels of dopamine, noradrenaline, serotonin, and their metabolites were measured by HPLC in the cerebellum, substantia nigra, caudate–putamen, and frontal cortex. 6-OHDA injected alone enhanced the harmaline-induced tremor. Furthermore, it decreased the noradrenaline level by ca. 40–80% in the cerebellum and increased the levels of serotonin and 5-HIAA in the caudate–putamen and frontal cortex in untreated and/or harmaline-treated animals. When 6-OHDA treatment was preceded by desipramine, it decreased dopaminergic transmission in some regions of the cerebellum while inducing its compensatory activation in others. The latter lesion did not markedly influence the tremor induced by harmaline. The present study indicates that noradrenergic innervation of the cerebellum interacts with cerebral serotonergic systems and plays an inhibitory role in the harmaline-induced tremor

Human keratinocytes are vanilloid resistant

BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials