Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons

Eugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na(+) channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications. eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is proinflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (dl-propargylglycine) or an H(2)S donor [Lawesson`s reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Morphine peripheral analgesia depends on activation of the PI3K gamma/AKT/nNOS/NO/K(ATP) signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3K gamma/AKT protein kinase B (AKT) and culminated in increasedactivation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ)

The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local (125)I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK(1) and NK(2)) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK(1) receptor antagonist, and in a lesser extent by SR48968, an NK(2) antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK(1) and NK(2) receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Participation of peripheral tachykinin NK(1) receptors in the carrageenan-induced inflammation of the rat temporomandibular joint

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK(1) receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNF alpha and IL-1 beta concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK(1) receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNF alpha and IL-1 beta into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK(1) receptors mediate the production of both TNF alpha and IL-1 beta in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component. 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity Of ot2/3-Na,K-ATPase varied with age. and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum. (C) 2007 Elsevier Inc. All rights reserved.

Differential effects of neuromuscular blockers on twitches and tetani in the isolated rat muscle: a multiple comparison study using simultaneous confidence intervals

In the present work a comparative quantitative evaluation of the differential effects of neuromuscular blockers on twitches and tetani was performed, encompassing: atracurium, cisatracurium, mivacurium, pancuronium, rocuronium and vecuronium. The sciatic nerve-extensor digitorum longus muscle of the rat was used, in vitro. Twitches were evoked at 0.1 Hz and tetani at 50 Hz. The differential effects of the studied compounds on twitches and tetani were statistically compared using simultaneous confidence intervals for the ratios between mean IC(50) for the block of twitches and mean IC(50) for the block of tetani. The results of ratios of mean IC(50) together with their corresponding 95% simultaneous confidence intervals were: vecuronium: 2.5 (1.8-3.5); mivacurium: 3.8 (3.0-4.9); pancuronium: 3.9 (2.0-7.6); rocuronium: 6.1 (3.8-9.9); atracurium: 9.0 (6.4-12.6); cisatracurium: 13.1 (6.0-28.4). Using the criteria that neuromuscular blockers displaying disjunct confidence intervals for the ratios of mean IC(50) differ statistically with regard to differential effects on twitches and tetani, significant differences in ratios of IC(50) were detected in the following cases: vecuronium vs. rocuronium, vs. atracurium and vs. cisatracurium and mivacurium vs: cisatracurium and vs. atracurium. The results show that the magnitude of the differential effects of neuromuscular blockers on twitches and tetani, as evaluated in the present work in the form of ratios of mean IC(50), does not depend on the chemical structure (comparing steroidal and isoquinolinic compounds), but seems to depend on differential pre- and post-synaptic effects of the compounds. It is also suggested that the greater the ability of a compound to block twitches and tetani in a differential manner, the safer is the compound from the clinical anesthesiology viewpoint.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(center dot-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection. (C) 2010 Elsevier B.V. All rights reserved.