Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats.

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Neurochemical effects of photobiostimulation in the trigeminal ganglion after inferior alveolar nerve injury.

Orofacial pain is associated with peripheral and central sensitization of trigeminal nociceptive neurons. Nerve injury results in release of chemical mediators that contribute to persistent pain conditions. The activation of the transient receptor potential vanilloid 1 (TRPV1), promotes release of calcitonin gene-related peptide (CGRP) and substance P (SP) from trigeminal nerve terminals. CGRP and SP contribute to the development of peripheral hyperalgesia. The expression of SP and CGRP by primary afferent neurons is rapidly increased in response to peripheral inflammation. CGRP receptor activation promotes activation of AMPA receptors, leading to increased firing of neurons which is reflected as central sensitization. In this study we investigated whether inferior alveolar nerve (IAN) injury influences AMPA receptors, CGRP, SP and TRPV1 expression in the trigeminal ganglion (TG). The relative expression of the protein of interest from naive rats was compared to those from injured rats and animals that received low level laser therapy (LLLT). IAN-injury did not change expression of GluA1, GluA2 and CGRP, but increased the expression of TRPV1 and SP. LLLT increases GluA1 and GluA2 expression and decreases TVPV1, SP and CGRP. These results, together with previous behavioral data, suggest that IAN-injury induced changes in the proteins analyzed, which could impact on nociceptive threshold. These data may help to understand the molecular mechanisms of pain sensitization in the TG.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-κB (NF-κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-κB binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-κB, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-α (Tnf-α), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF-κB activation and increased NOS and α(2/3) -Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-κB activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-κB activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.

Genetic polymorphisms and protein levels in vocal fold leukoplakia: a systematic review.

Vocal fold leukoplakia (VFL) has a risk of malignant transformation. Therefore, patients can have symptoms such as dysphonia, vocal strain, difficulty breathing, and dysphagia. Additionally, there is a genetic predisposition that can be associated with genetic polymorphisms. We aimed to evaluate the influence of genetic polymorphisms and protein levels in the etiology of VFL. Our study followed the PRISMA checklist and was registered on PROSPERO database. The questions were: "Are genetic polymorphisms involved in the etiology of VFL? Are protein levels altered in patients with VFL?". Eligibility criteria were case control studies that compared the presence of polymorphisms or/and protein levels of subjects diagnosed with VFL and healthy controls. Of the 905 articles retrieved, five articles with a total of 1038 participants were included in this study. The C allele of the single nucleotide polymorphisms (SNP)-819 T/C IL-10, A allele of the SNP -592 A/C IL-10, CT genotype of the SNP rs11886868 C/T BCL11A, GG genotype of the SNP rs4671393 A/G BCL11A, LL genotype, and L allele of (GT)n repeat polymorphisms of the HO-1 were risk factors for VFL development. Nevertheless, there was a lack of association between VFL and the -1082 A/G IL-10, rs14024 CK-1, and -309 T/G Mdm2 SNPs. The concentrations of the MDM2, BCL11A, and HO-1 proteins were modified, while IL-10 levels were normally expressed in these subjects. In conclusion, most markers evaluated in this review could be potential indicators to develop effective therapies, avoiding a malignant transformation of the lesion.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety.

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 3'-untranslated region (3'-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3'-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety-like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety-related disorders in humans requires further investigation.

Antinociceptive effects of treadmill exercise in a rat model of Parkinson's disease: The role of cannabinoid and opioid receptors.

In addition to motor symptoms, Parkinson's disease (PD) presents high prevalence of painful symptoms responsible for worsening quality of life of PD patients. Physical exercise can improve such painful symptoms. This study evaluated the effects of exercise on nociceptive threshold using an unilateral rat model of PD, as well as the role played by cannabinoid and opioid receptors in areas responsible for pain pathways. For PD induction, Wistar rats were injected with 6-OHDA. 15 days after, rats either remained sedentary or were forced to exercise three times a week for 40 min. Motor and nociceptive behaviors were evaluated through cylinder and mechanical hyperalgesia tests, respectively. The animals were euthanized for analysis of cannabinoid receptor type 1 (CB1) and type 2 (CB2), and µ-opioid receptor (MOR) in the anterior cingulate cortex (ACC), periaqueductal gray matter (PAG), and thalamus areas by immunohistochemistry (IHC) and Western blotting. Our data revealed a decrease in the nociceptive threshold in both forepaws after surgery; in contrast, there was improvement in painful symptoms after the exercise protocol. For cannabinoid system there were an increase in CB2 expression in the ACC and PAG, and in CB1 levels in the PAG. And for opioid system there was an increase of MOR expression in the thalamus. Thus, modulation of those receptors by physical exercise can be an important non-pharmacological intervention to reduce painful symptoms in a rat model of PD, contributing to knowledge and promotion of better treatment aimed at improving the quality of life of PD patients.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes.

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina.

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6brd1). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin I, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells.

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca(2+) transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X(4) pharmacology were responsible for ATP and ATP analogue-induced Ca(2+) transients. In neuronal-differentiated cells, P2Y(2,) P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca(2+)](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-betaS-induced proliferation in P19 cells was mediated by P2Y(1) and P2Y(2) receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y(1) and P2Y(2) receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca(2+) stores.

P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-D-aspartate-glutamate and acetylcholine receptors during neuronal differentiation.

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X2, P2X6, P2Y2, and P2Y6 receptors increased during the course of differentiation, whereas P2X3, P2X4, P2Y1 and P2Y4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X5 and P2X7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate-N-methyl-D-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y1 and possibly P2X2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X2 and P2Y2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.

The Impact of Short and Long-Term Exercise on the Expression of Arc and AMPARs During Evolution of the 6-Hydroxy-Dopamine Animal Model of Parkinson's Disease.

The loss of nigral dopaminergic neurons typical in Parkinson's disease (PD) is responsible for hyperexcitability of medium spiny neurons resulting in abnormal corticostriatal glutamatergic synaptic drive. Considering the neuroprotective effect of exercise, the changes promoted by exercise on AMPA-type glutamate receptors (AMPARs), and the role of activity-regulated cytoskeleton-associated protein (Arc) in the AMPARs trafficking, we studied the impact of short and long-term treadmill exercise during evolution of the unilateral 6-hydroxy-dopamine (6-OHDA) animal model of PD. Wistar rats were divided into sedentary and exercised groups, with and without lesion by 6-OHDA and followed up to 4 months. The exercised groups were subjected to a moderate treadmill exercise 3×/week. We measured the proteins tyrosine hydroxylase (TH), Arc, GluA1, and GluA2/3 in the striatum, substantia nigra, and motor cortex. Our results showed a higher reduction of TH expression in all sedentary groups when compared to all exercised groups in striatum and substantia nigra. In general, larger changes occurred in the striatum in the first and third months after training. After 1 month of exercise, there was significant increase of GluA2/3 with concomitant reduction of GluA1 and Arc. As a balanced system, these changes were reversed in the third month, showing an increase of Arc and GluA1 and decrease of GluA2/3. Similar results for GluAs and Arc were observed in the motor cortex of the exercised animals. These modifications may be relevant for corticostriatal circuits in PD, since the exercise-dependent plasticity can modulate GluAs expression and maybe neuronal excitability.

Carvacrol promotes neuroprotection in the mouse hemiparkinsonian model.

Carvacrol is a monoterpene that has been linked to neuroprotection in several animal models of neurodegeneration, including ischemia, epilepsy and traumatic neuronal injury. In this study, we investigated the effects of carvacrol (i.p.) upon the neurodegeneration induced by 6-hydroxy-dopamine unilateral intrastriatal injections in mice. We have also used the cylinder test to assess the behavioral effects of carvacrol in that model of Parkinson's disease, and immunoblots to evaluate the levels of caspase-3 and TRPM7, one of major targets of carvacrol. Behavioral testing revealed that carvacrol largely reduced the asymmetrical use of the forelimbs induced by unilateral 6-hydroxy-dopamine. Carvacrol dramatically reduced the loss of tyrosine hydroxylase immunostaining both in the substantia nigra and in the striatum that are typical of the model. Immunoblots for tyrosine hydroxylase confirmed this effect. Caspase-3 levels were very high after toxin injections, but carvacrol appeared to reduce them to control levels. Finally, TRPM7, observed by immunoblots, increased after 6-hydroxy-dopamine, suggesting the involvement of this cation channel in the ensuing neurodegenerative process. The present data suggest that carvacrol promotes a marked neuroprotection in the 6-hydroxy-dopamine model of Parkinson's disease, possibly by its non-specific blocking effect upon TRPM7 channels.

Peptidomic analysis of the anterior temporal lobe and corpus callosum from schizophrenia patients.

Schizophrenia is a complex disorder hypothesized to develop from a combination of genetic, neurodevelopmental, and environmental factors. Molecules that are directly involved in the pathogenesis of schizophrenia and may serve as biomarker candidates can be identified with "omics" approaches such as proteomics and peptidomics. In this context, we performed a peptidomic study in schizophrenia postmortem brains, to our knowledge the first such study in schizophrenia patients. We investigated the anterior temporal lobe (ATL) and corpus callosum (CC) by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and a label-free ion quantification technique based on data-dependent acquisition (DDA). Results indicated alterations in a specific intracellular neurogranin peptide in both the ATL and CC and a decrease of PepH, a fragment of histone H2B type 1-H intracellular peptide, in the ATL. PepH was tested in serum-deprived Neuro2A cells and showed a protective effect against cell death. Cells were also challenged with lipopolysaccharide (LPS), and PepH was able to prevent the endotoxic effects of LPS. Our data suggest that specific intracellular peptides are altered in schizophrenia patients. The potential biological activity of PepH supports intracellular peptides as novel targets in the study not only of schizophrenia but also of other neuropsychiatric diseases. BIOLOGICAL SIGNIFICANCE: Psychiatric disorders are considerably more difficult to diagnose in their early stages. Usually, by the time the diagnosis is clear and clinical treatment can be started, the disorder is already established and thus of greater severity. Consequently, the scientific community has been searching for biomarker candidates that can aid the early detection of such disorders and for novel therapeutics to improve treatment or at least delay disease progression. Moreover, key molecules involved in the establishment of psychiatric diseases may help the understanding of their pathogenesis and thus drive the development of more effective treatments. The present work screened peptides that might be possible novel targets to control cell machinery in schizophrenia and identified an intracellular peptide with potential cytoprotective activity. To our knowledge, this is the first peptidomic study in schizophrenia patients.

Genetic polymorphisms and protein levels in vocal fold leukoplakia: a systematic review

Vocal fold leukoplakia (VFL) has a risk of malignant transformation. Therefore, patients can have symptoms such as dysphonia, vocal strain, difficulty breathing, and dysphagia. Additionally, there is a genetic predisposition that can be associated with genetic polymorphisms. We aimed to evaluate the influence of genetic polymorphisms and protein levels in the etiology of VFL. Our study followed the PRISMA checklist and was registered on PROSPERO database. The questions were: "Are genetic polymorphisms involved in the etiology of VFL? Are protein levels altered in patients with VFL?". Eligibility criteria were case control studies that compared the presence of polymorphisms or/and protein levels of subjects diagnosed with VFL and healthy controls. Of the 905 articles retrieved, five articles with a total of 1038 participants were included in this study. The C allele of the single nucleotide polymorphisms (SNP)-819 T/C IL-10, A allele of the SNP -592 A/C IL-10, CT genotype of the SNP rs11886868 C/T BCL11A, GG genotype of the SNP rs4671393 A/G BCL11A, LL genotype, and L allele of (GT)n repeat polymorphisms of the HO-1 were risk factors for VFL development. Nevertheless, there was a lack of association between VFL and the -1082 A/G IL-10, rs14024 CK-1, and -309 T/G Mdm2 SNPs. The concentrations of the MDM2, BCL11A, and HO-1 proteins were modified, while IL-10 levels were normally expressed in these subjects. In conclusion, most markers evaluated in this review could be potential indicators to develop effective therapies, avoiding a malignant transformation of the lesion.

Monoaminergic markers in the optic tectum of the domestic chick.

The avian optic tectum has become a reliable model system to study the basic mechanisms that underlie the computation of visual stimuli. Many aspects of its cytoarchitecture, chemoarchitecture, connectivity and development are thoroughly characterized. However, knowledge about its monoaminergic innervation is still incomplete. As a prerequisite to understand a possible functional role of the monoaminergic neurotransmitters, the serotonergic, noradrenergic, and dopaminergic innervation of the optic tectum as well as the distribution of serotonin 2A receptors, the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and calbindin D-28K was studied in domestic chicks by immunohistochemical techniques. Serotonergic, noradrenergic, and tyrosine hydroxylase positive axons and axon terminals were present in all layers of the optic tectum. Generally, the highest densities of serotonergic, noradrenergic, and tyrosine hydroxylase positive fibers were found in the superficial tectal layers 1-8, whereas only moderate densities of serotonergic, noradrenergic, and tyrosine hydroxylase positive fibers became obvious in the deep tectal layers 9-15. Serotonergic fibers were particularly abundant in layers 4, 5a and 7 and serotonin 2A receptors in layer 13. Noradrenergic fibers were densest in layers 4 and 5a, whereas tyrosine hydroxylase positive fibers showed a slightly different distribution pattern with additional dense labeling in layer 7. As revealed by double-labeling immunohistochemistry, serotonergic fibers were closely related to the cell bodies of calbindin-positive horizontal cells in layer 5b and tyrosine hydroxylase positive fibers often contacted DARPP-32+ dendritic shafts in layers 9 and 10. These findings indicate that the catecholaminergic innervation of the optic tectum consists of a noradrenergic and a dopaminergic component and that the noradrenergic, serotonergic, and dopaminergic system may be potentially involved in the modulation of retinal input in the superficial layers of the optic tectum as well as in the modulation of tectal output via the deep tectal layers.

The C-terminus of murine S100A9 protein inhibits hyperalgesia induced by the agonist peptide of protease-activated receptor 2 (PAR2).

BACKGROUND AND PURPOSE: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2). EXPERIMENTAL APPROACH: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods. KEY RESULTS: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.

Classical tone-shock conditioning induces Zenk expression in the pigeon (Columba livia) hippocampus.

The hippocampus is involved in fear conditioning, although the molecular events underlying this function are still under investigation. The authors analyzed the expression of the Zenk proto-oncogene product within the pigeon (Columba livia) hippocampus after training with a classical aversive conditioning protocol using tone-shock associations. Control groups were trained with shock or tone alone or were only exposed to the experimental chamber and manipulated. Experimental pigeons showed significant increases of Zenk expression in the ventromedial region of the hippocampus, whereas both the experimental and shock groups had increased Zenk expression in the dorsal region. The expression of Zenk in specific neuronal populations within the pigeon hippocampus may be indicative of plasticity-associated aversive classical conditioning.

The effect of exposure to irrigant solutions on apical dentin biofilms in vitro.

This study assessed the effectiveness of different concentrations of sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) (Vista Dental Products, Racine, WI), and BioPure MTAD (Dentsply Endodontics-Tulsa Dental, Tulsa, OK). Intracanal contents were collected from 10 patients diagnosed with chronic apical periodontitis. The samples were cultured on hemisections of root apices to generate a polymicrobial biofilm. Each biofilm was separately immersed in 6% NaOCl, 3% NaOCl, 1% NaOCl, 2% CHX, 1% NaOCl followed by BioPure MTAD, and sterile phosphate buffered solution (PBS). SEM analysis showed 6% NaOCl and 3% NaOCl were capable of disrupting and removing the biofilm; 1% NaOCl and 1% NaOCl followed by MTAD were capable of disrupting the biofilm, but not eliminating bacteria; 2% CHX was not capable of disrupting the biofilm. Viable bacteria could not be cultured from specimens exposed to 6% NaOCl, 2 % CHX, or 1% NaOCl followed by BioPure MTAD. These results indicate that 6% NaOCl was the only irrigant capable of both rendering bacteria nonviable and physically removing the biofilm.

Social stress in adolescents induces depression and brain-region-specific modulation of the transcription factor MAX.

MAX is a conserved constitutive small phosphoprotein from a network of transcription factors that are extensively studied in tumorigenesis and whose functions affect cell proliferation, differentiation and death. Inspired by its higher expression during development and in regions involved in emotional behaviors, we hypothesized its involvement in cerebral changes caused by early-life stress. We studied the effects of repeated social stress during adolescence on behaviors and on MAX and its putative partner MYC. Thirty-day-old C57BL/6 male mice underwent brief daily social defeat stress from an adult aggressor for 21 days. Following social stress episodes and housing in social groups after each defeat, adolescent mice exhibit depressive-like, but not anxiety-like behaviors and show higher MAX nuclear immunoreactivity in hippocampal (HC) but not prefrontal cortical (PFC) neurons. Conversely, MAX immunoreactivity is lower in the striatum (ST) of defeated adolescents. The positive correlation between MAX and MYC levels in the PFC revealed disruptions in both the HC and ST. The changes in MAX protein levels are not due to differential gene expression or protein degradation in those regions, suggesting that posttranscriptional modifications occurred. These findings indicate that repeated, brief social defeat in adolescent male mice, combined with group housing, is a useful protocol to study a subtype of depression that is dissociated from generalized (non-social) anxiety. To our knowledge, this is the first report of an association between dysregulation of the MAX-MYC network in the brain and a behavior, suggesting a novel approach for exploiting the neuroplasticity associated with depression.

Respiratory deficits in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of the dopaminergic nigrostriatal pathway. In addition to deficits in voluntary movement, PD involves a disturbance of breathing regulation. However, the cause and nature of this disturbance are not well understood. Here, we investigated breathing at rest and in response to hypercapnia (7% CO2) or hypoxia (8% O2), as well as neuroanatomical changes in brainstem regions essential for breathing, in a 6-hydroxydopamine (6-OHDA) rat model of PD. Bilateral injections of 6-OHDA (24µg/µl) into the striatum decreased tyrosine hydroxylase (TH(+))-neurons in the substantia nigra pars compacta (SNpc), transcription factor phox2b-expressing neurons in the retrotrapezoid nucleus and neurokinin-1 receptors in the ventral respiratory column. In 6-OHDA-lesioned rats, respiratory rate was reduced at rest, leading to a reduction in minute ventilation. These animals also showed a reduction in the tachypneic response to hypercapnia, but not to hypoxia challenge. These results suggest that the degeneration of TH(+) neurons in the SNpc leads to impairment of breathing at rest and in hypercapnic conditions. Our data indicate that respiratory deficits in a 6-OHDA rat model of PD are related to downregulation of neural systems involved in respiratory rhythm generation. The present study suggests a new avenue to better understand the respiratory deficits observed in chronic stages of PD.