Differential regulation of TRP channels in a rat model of neuropathic pain

Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory Stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these Stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real-time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception OF neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. fit situ hybridisation showed a widespread increase of expression ill neurons of small, medium and large cell sizes, indicating expression ill multiple subtypes. Co-localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad Subtype distribution. Expression studies during development showed that TRPML3 is all embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus. the current results link for the first time a re-expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5. (C) 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O(2) for 40 s, every 9 min, 8 h day(-1)) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n = 14) exhibited higher levels of mean arterial pressure than controls (101 +/- 3 versus 89 +/- 3 mmHg, n = 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart-brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 +/- 5 versus 40 +/- 3%; n = 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post-inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic-respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Increased Capsaicin Receptor TRPV1 in the Peritoneum of Women With Chronic Pelvic Pain

Objective: To investigate the expression of capsaicin receptor [transient receptor potential vanilloid type-1 (TRPV1)] in the peritoneum of women with chronic pelvic pain (CPP). Methods: A case-control study was conducted on 25 women with CPP and 10 controls. Samples of the rectouterine excavation (2 cm 2) were obtained by laparoscopy, fixed in 4% formaldehyde, and underwent immunohistochemistry analysis using rabbit anti-TRPV1 (1:400) polyclonal antibodies and anti-protein gene product 9.5 (PGP 9.5) (1:2000) as a neuronal marker. Ten sequential images of high magnification fields ( x 40) were captured from each slide and the area identified with the antibody was calculated with Kontron V2.0 software. Results: Immunoreactivity to TRPV1 was sparsely detected in the nervous tissue and epithelium of endometriotic lesions. The percent area of immunoreactivity for TRPV1 [expressed as median (range)] was greater in specimens from women with CPP, 1.02% (0.54 to 2.93), than from women without the disease, 0.14% (0.07 to 1.12) (P<0.0001). This greater expression was not secondary to an increase in neuronal fibers because there was also a significant difference in the percent area TRPV1:PGP 9.5 ratio between women with CPP, 1.18 (0.26 to 4.63), and controls, 0.15 (0.06 to 0.95) (P = 0.0003). Discussion: TRPV1 may play an important role in the maintenance and perpetuation of symptoms in women with CPP. In view of the immunoreactivity detected for TRPV1, the endometriotic lesion may have the ability to interfere with nociception or with the inflammatory peritoneal environment in women with CPP. Further Studies are needed to elucidate the participation of TRPV1 in CPP and its association with endometriosis.

POSTOPERATIVE ANALGESIA INDUCED BY INTRATHECAL NEOSTIGMINE OR BETHANECHOL IN RATS

P>Cholinergic agonists and acetylcholinesterase inhibitors, such as neostigmine, produce a muscarinic receptor-mediated antinociception in several animal species that depends on activation of spinal cholinergic neurons. However, neostigmine causes antinociception in sheep only in the early, and not late, postoperative period. In the present study, a model of postoperative pain was used to determine the antinociceptive effects of bethanechol (a muscarinic agonist) and neostigmine administered intrathecally 2, 24 or 48 h after a plantar incision in a rat hind paw. Changes in the threshold to punctate mechanical stimuli were evaluated using an automated electronic von Frey apparatus. Mechanical hyperalgesia was obtained following plantar incision, the effect being stronger during the immediate (2 h) than the late post-surgical period. Bethanechol (15-90 mu g/5 mu L) or neostigmine (1-3 mu g/5 mu L) reduced incision-induced mechanical hyperalgesia, the effects of both drugs being more intense during the immediate (2 h) than the late post-surgical period. The ED(50) for bethanechol injected at 2, 24 and 48 h was 5.6, 51.9 and 82.5 mu g/5 mu L, respectively. The corresponding ED(50) for neostigmine was 1.62, 3.02 and 3.8 mu g/5 mu L, respectively. The decline in the antinociceptive potency of neostigmine with postoperative time is interpreted as resulting from a reduction in pain-induced activation of acetylcholine-releasing descending pathways. However, the similar behaviour of bethanechol in the same model points to an additional mechanism involving intrinsic changes in spinal muscarinic receptors.

Inflammatory mediators involved in the nociceptive and oedematogenic responses induced by Tityus serrulatus scorpion venom injected into rat paws.

The present study investigated the role of kinins, prostaglandins (PGs) and nitric oxide (NO) in mechanical hypernociception, Spontaneous nociception and paw oedema after intraplantar (ipl) injection of Tityus serrulatus venom (Tsv) in male Wistar rats. Tsv was ipl-injected in doses of 0.01-10 mu g/paw. Pre-treatment (30 min prior) with DALBK (100 nmol/paw) and icatibant (10 nmol/paw), B1 and B2 selective kinin receptor antagonists, L-NAME (50 mg/kg, i.p., a non-selective nitric oxide synthase inhibitor) or celecoxib, selective COX-2 inhibitor, was given 1 h prior per os (5 mg/kg, p.o.), significantly reduced the hypernociceptive response (Von Frey method), the spontaneous nociception (determined by counting the number of flinches) and paw oedema (plethysmometer method) induced by Tsv at doses of 1.0 and 10 mu g/paw for both nociceptive and oedematogenic responses, respectively. Nevertheless, indomethacin (5 mg/kg, i.p.. 30 min prior) was ineffective in altering all of these events. The results of the present study show that Tsv, injected ipl into the rat paw, causes a dose-dependent paw oedema, mechanical hypernociception and flinches (a characteristic biphasic response) in which kinins and NO are substantially involved. Although celecoxib was effective against the oedema and pain caused by Tsv, COX-2 does not seem to be involved in the inflammatory response caused by Tsv. (C) 2008 Elsevier Ltd. All rights reserved.

Sildenafil improves the beneficial haemodynamic effects of intravenous nitrite infusion during acute pulmonary embolism

Acute pulmonary embolism produces acute pulmonary hypertension, which can be counteracted by activating the nitric oxide-cyclic guanosine 3`,5`-monophosphate (cGMP) pathway. While previous studies have shown that sildenafil (an inhibitor of cGMP-specific phosphodiesterase type 5) or nitrite (a storage molecule for nitric oxide) produces beneficial effects during acute pulmonary embolism, no previous study has examined whether the combination of these drugs can produce additive effects. Here, we expand previous findings and examine whether sildenafil enhances the beneficial haemodynamic effects produced by a low-dose infusion of nitrite in a dog model of acute pulmonary embolism. Haemodynamic and arterial blood gas evaluations were performed in non-embolized dogs treated with saline (n = 4), and in embolized dogs (intravenous injections of microspheres) that received nitrite (6.75 mu mol/kg intravenously over 15 min. followed by 0.28 mu mol/kg/min.) and sildenafil (0.25 mg/kg over 30 min.; n = 8), or nitrite followed by saline (n = 8), or saline followed by sildenafil (n = 7), or only saline (n = 8). Plasma thiobarbituric acid-reactive substances (TBARS) concentrations were determined using a fluorometric method. Acute pulmonary embolism increased pulmonary artery pressure by similar to 24 mmHg. While the infusion of nitrite or sildenafil infusions reversed this increase by similar to 42% (both P < 0.05), the combined infusion of both drugs reversed this increase by similar to 58% (P < 0.05). Similar effects were seen on the pulmonary vascular resistance index. Nitrite or sildenafil alone produced no significant hypotension. However, the combined infusion of both drugs caused transient hypotension (P < 0.05). Both dugs, either alone or combined, blunted the increase in TBARS concentrations caused by acute pulmonary embolism (all P < 0.05). These results suggest that sildenafil improves the beneficial haemodynamic effects of nitrite during acute pulmonary embolism.

REDUCED ERYTHROCITARY GLUTATHIONE AND HEINZ BODIES IN CATS EXPERIMENTALLY INFECTED WITH THE FELINE IMMUNODEFICIENCY VIRUS

Santos, K.B.; Daniel, A.G.T. & Hagiwara, M.K. [Reduced erythrocitary Glutathione and Heinz bodies in cats experimentally infected with the Feline Immunodeficiency Virus.] Glutationa Reduzida Eritrocitaria e Corpusculos de Heinz em gatos infectados experimentalmente pelo Virus da Imunodeficieneia Felina. Revista Brasileira de Medicina Veterinaria, 30(1):26-30, 2008. Centro Universitario Serra dos Orgaos, Rua Comendador Queiroz 52, Apto. 1403, Niteroi, RJ 24230-220, Brasil. E-mail: keilabsantos@)gmail.com The Feline Immunodeficiency Virus (FIV) produces a chronic infection with immunologic system disfunctions in domestic cats developing non-responsive anaemia. The feline immunologic status depression produces free radicals, whose imbalance between production and removal in the organism favour the oxidative injury occurrence. Heinz bodies in large amounts also can evident in vivo oxidative damage. Glutatione, a tripeptide found in the animal cells, is an important protection mechanism against oxidative damages. In this work erythrocyte reduced glutathione (GSH) concentrations for healthy and cats experimentally infected by FIV were determinated in relation to the hemogram and the Heinz bodies presence. All of the animals presents normal values for hemogram and Heinz bodies, however the GSH erythrocyte concentrations in the FIV positive cats were below the normality probably due to an passive depletion to GSH.

Renormalizing the kinetic energy operator in elementary quantum mechanics

In this paper, we consider solutions to the three-dimensional Schrodinger equation of the form psi(r) = u(r)/r, where u(0) not equal 0. The expectation value of the kinetic energy operator for such wavefunctions diverges. We show that it is possible to introduce a potential energy with an expectation value that also diverges, exactly cancelling the kinetic energy divergence. This renormalization procedure produces a self-adjoint Hamiltonian. We solve some problems with this new Hamiltonian to illustrate its usefulness.

Long-Term Maternal Separation Differentially Alters Serum Corticosterone Levels and Blood Neutrophil Activity in A/J and C57BL/6 Mouse Offspring

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used. Copyright (C) 2011 S. Karger AG, Basel

Behavioral effects of acute stimulation of kappa-opioid receptors during lactation

The behavioral effects of the K-opioid receptor agonist U69593 were examined in lactating rats. On day 5 of lactation, animals were treated with 0.1 mg/kg of U69593 to determine whether it influences general activity and maternal latencies toward pups. Because little attention has been given to the possibility that pre-mating treatment with morphine may modulate the response to K-opioid receptor stimulation, another group of animals was submitted to the same acute challenge after abrupt withdrawal from repeated treatment with morphine sulfate during the pre-mating period (5 mg/kg on alternate days for a total of five doses). Acute F;opioid stimulation reduced total locomotion, rearing frequency, and time spent self-grooming and increased immobility duration. These K agonist effects were not observed in animals pretreated with morphine. Similarly, latencies to retrieve pups were longer only in animals pretreated with saline and challenged acutely with U69593. None of these effects were observed in morphine sulfate-pretreated animals. The present results suggest that pre-mating repeated exposure to morphine produces a tolerance-like effect on behavioral responses to low-dose K-opioid receptor stimulation in active reproductive females. (c) 2008 Elsevier Inc. All rights reserved.

Esthetic restorative materials and opposing enamel wear

This in vitro study compared the effects of a gold alloy (Degulor M), four dental ceramics (IPS Empress, IPS Empress 2, Duceram Plus, Duceram. LFC) and a laboratory-processed composite (Targis) on the wear of human enamel. The amount of wear of the enamel (dental cusps) and restorative materials (disks) were tested in water at 37 degrees C under standard load (20 N), with a chewing rate of 1.3 Hz and was determined after 150,000 and 300,000 cycles. Before the test, the average surface roughness of the restorative materials was analyzed using the Ra parameter. The results of this study indicate that Targis caused enamel wear similar to Degulor M and resulted in significantly less wear than all the ceramics tested. IPS Empress provoked the greatest amount of enamel wear and Degulor M caused less vertical dimension loss. Targis could be an appropriate alternative material to ceramic, because it is esthetic and produces opposing enamel wear comparable to gold alloy.

Use of laser phototherapy on a delayed wound healing of oral mucosa previously submitted to radiotherapy: case report

Radiotherapy produces both acute and delayed effects on mucosal tissues, disturbing their healing. This report shows a successful treatment with laser phototherapy (LPT) on a delayed wound healing in oral mucosa previously submitted to radiotherapy with a follow up of 3 years. A 47-year-old patient treated 6 months earlier for tongue squamous cell carcinoma by surgery and radiotherapy presented with a mass in the operated area. Biopsy showed chronic inflammatory infiltrate around a residual polyglactin suture. After 2 months there was a painful mucosal dehiscence on the biopsy site. LPT was performed using a semiconductor laser with 660-nm wavelength (InGaAlP) and spot size of 0.04 cm(2). The parameters applied were 40 mW, 4 Jcm(2)/point, 0.16 J/point, 2.4 J/session. The irradiation was performed punctually, through contact mode in 15 points (4 seconds/point), on top of and around the lesion, during ten sessions. The wound healed completely after ten sessions. This treatment proved to be conservative and effective, inducing healing of a chronic wound in a tissue previously submitted to radiotherapy.

Involvement of the heme oxygenase-carbon monoxide-cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase-carbon monoxide-cGMP (HO-CO-cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO-CO-cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5-7 days) were subjected to the measures of baseline tail flick test Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na(2)CO(3)), heme-lysinate (substrate overload) or vehicle (L-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO-CO-cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain. (C) 2011 Elsevier B.V. All rights reserved.

Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 mu mol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10 mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 mu mol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26 +/- 4 vs. 37 +/- 4 mmHg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine. (C) 2009 Elsevier B.V. All rights reserved.