Modulation of anxiety-like behaviour by Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels located in the dorsolateral periaqueductal gray

The endocannabinoid anandamide is a possible agonist at the Transient Receptor Potential Vanilloid Type 1 (TRPV1) channel, in addition to its agonist activity at cannabinoid type 1 (CB1) receptor. In the midbrain dorsolateral periaqueductal gray (dlPAC) our previous data showed that CB1 activation induces anxiolytic-like effects. However, the rote of TRPV1 has remained unclear. Thus, in the present study we tested the hypothesis that this channel would contribute to the modulation of anxiety-like behaviour in the dlPAG. Mate Wistar rats received local injections of the TRPV1 antagonist capsazepine (10-60 nmol) and were submitted to the elevated plus-maze (EPM) and to the Vogel test. In addition, animals received local injections of capsaicin (0.01-1nmol), a TRPV1 agonist, and were tested in the same models. In accordance with our hypothesis, capsazepine produced anxiolytic-like effects both in the EPM and in the Vogel test. Capsaicin mimicked these results, which might be attributed to its ability to quickly desensitize the channel. Altogether, our data suggest that, while CB1 receptors seem to inhibit aversive responses in the dlPAG, TRPV1 could facilitate them. Thus, CB1 and TRPV1 may have opposite functions in modulating anxiety-like behaviour in this region. (C) 2008 Elsevier B.V. and ECNP. All rights reserved.

Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells

Yogi A, Callera GE, Tostes R, Touyz RM. Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells. Am J Physiol Regul Integr Comp Physiol 296: R201-R207, 2009. First published September 17, 2008; doi: 10.1152/ajpregu.90602.2008.-Transient receptor potential melastatin-7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in proinflammatory signaling by bradykinin. VSMCs from Wistar-Kyoto rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution, and that it colocalizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol/membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (phospholipase C inhibitor), but not by chelerythrine (PKC inhibitor). Expression of proinflammatory mediators VCAM-1 and cyclooxygenase-2 (COX-2) was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B(2) receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs: 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators VCAM-1 and COX2 are regulated, in part, via TRPM7- and phospholipase C-dependent pathways through B2 receptors. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B(2) receptor-mediated inflammatory responses in vascular cells.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia

Rationale Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. Objective To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. Methods and results Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 mu g/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-kappa B and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. Conclusion Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified. J Hypertens 29: 1400-1410 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats

The endocannabinoid anandamide, in addition to activating cannabinoid type 1 receptors (CB1), may act as an agonist at transient receptor potential vanilloid type 1 (TRPV1) channels. In the periaqueductal gray, CB1 activation inhibits, whereas TRPV1 increases, anxiety-like behavior. In the medial prefrontal cortex (mPFC), another brain region related to defensive responses, CB1 activation induces anxiolytic-like effects. However, a possible involvement of TRPV1 is still unclear. In the present study, we tested the hypothesis that TRPV1 channel contributes to the modulation of anxiety-like behavior in the mPFC. Male Wistar rats (n = 5-7 per group) received microinjections of the TRPV1 antagonist capsazepine (1-60 nmol) in the ventral portion of the mPFC and were exposed to the elevated plus maze (EPM) or to the Vogel conflict test. Capsazepine increased exploration of open arms in the EPM as well as the number of punished licks in the Vogel conflict test, suggesting anxiolytic-like effects. No changes in the number of entries into the enclosed arms were observed in the EPM, indicating that there were no changes in motor activity. Moreover, capsazepine did not interfere with water consumption or nociceptive threshold, discarding potential confounding factors for the Vogel conflict test. These data suggest that TRPV1 in the ventral mPFC tonically inhibits anxiety-like behavior. TRPV1 could facilitate defensive responses opposing, therefore, the anxiolytic-like effects reported after local activation of CB1 receptors.

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.

Spontaneous Interblink Time Distributions in Patients with Graves` Orbitopathy and Normal Subjects

PURPOSE. To determine the shape of spontaneous interblink time interval distributions obtained in a long observation period in normal subjects and patients with Graves` orbitopathy. METHODS. The magnetic search coil technique was used to register the spontaneous blinking activity during 1 hour of video observation of two groups of 10 subjects each (normal controls aged 27-61 years, mean +/- SD = 46.0 +/- 13.6; patients with Graves` orbitopathy aged 33-61 years, mean +/- SD +/- 46.7 +/- 8.9). The spontaneous blink rate of each subject was calculated for the entire period of observation and for 56 five-minute bins. Histograms of the interblink time interval were plotted for each measurement of blink rate. RESULTS. Neither the overall mean blink rate (controls, 19.8 +/- 4.9; Graves`, 17.6 +/- 5.4) nor the interblink time (controls, 5.2 +/- 3.1, Graves`, 7.9 +/- 3.5) differed between the two groups. There was a large variation of both measurements when the 5-minute bins were considered. The interblink time distribution of all subjects was highly positively skewed when the 1-hour period was measured. A significant number of the 5-minute bin distributions deviated from the overall pattern and became symmetric. CONCLUSIONS. The normal blinking process is characterized by highly positively skewed interblink time distributions. This result means that most blinks have a short time interval, and occasionally a small number of blinks have long time intervals. The different patterns of distribution described in the early literature probably represent artifacts because of the small samples analyzed. (Invest Ophthalmol Vis Sci. 2011;52:3419-3424) DOI:10.1167/iovs.10-7060

Deuterium Equilibrium Time in Saliva of Newborn Infants

There is an increasing interest about the use of stable isotopes for body composition analysis in pediatrics. To ensure the success of total body water analysis by the deuterium dilution method, it is fundamental to determine the equilibrium tune (plateau) of deuterium in the body fluid studied. Objectives: We report here the equilibration time of deuterium oxide in the saliva of newborns after oral intake of the isotope. Methods: Twenty healthy term newborn infants, 10 males and 10 females, were analyzed. Saliva was collected from each newborn before the oral administration of a 100 mg/kg dose of deuterium oxide (baseline sample) and then at 1-hour intervals for 5 hours after administration. Deuterium enrichment of saliva was determined by isotope ratio mass spectrometry according to the recommendations of the International Atomic Energy Agency. Results: The plateau time of deuterium in saliva occurred 3 hours after oral administration of the stable isotope. Conclusion: These data are essential for further studies on the body composition of newborn infants. To the best of our knowledge, this is the first study regarding the equilibration time of deuterium in the saliva of term newborns. JPGN 48:471-474, 2009.

Rhipicephalus (Boophilus) microplus: Clotting time in tick-infested skin varies according to local inflammation and gene expression patterns in tick salivary glands

Ticks deposit saliva at the site of their attachment to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses. The anti-haemostatic properties of tick saliva have been described by many studies, but few show that tick infestations or its anti-haemostatic components exert systemic effects in vivo. In the present study, we extended these observations and show that, compared with normal skin, bovine hosts that are genetically susceptible to tick infestations present an increase in the clotting time of blood collected from the immediate vicinity of haemorrhagic feeding pools in skin infested with different developmental stages of Rhipicepahlus microplus; conversely, we determined that clotting time of tick-infested skin from genetically resistant bovines was shorter than that of normal skin. Coagulation and inflammation have many components in common and we determined that in resistant bovines, eosinophils and basophils, which are known to contain tissue factor, are recruited in greater numbers to the inflammatory site of tick bites than in susceptible hosts. Finally, we correlated the observed differences in clotting times with the expression profiles of transcripts for putative anti-haemostatic proteins in different developmental stages of R. microplus fed on genetically susceptible and resistant hosts: we determined that transcripts coding for proteins similar to these molecules are overrepresented in salivary glands from nymphs and males fed on susceptible bovines. Our data indicate that ticks are able to modulate their host`s local haemostatic reactions. In the resistant phenotype, larger amounts of inflammatory cells are recruited and expression of anti-coagulant molecules is decreased tick salivary glands, features that can hamper the tick`s blood meal. (C) 2010 Elsevier Inc. All rights reserved.

Influence of pregnancy and smoking on brachial artery flow-mediated dilation values and time until maximum response

To evaluate the effect of pregnancy and smoking on endothelial function using brachial artery flow-mediated dilation (FMD) and to determine the time necessary until the occurrence of maximum brachial artery dilation after stimulus. This study was an observational study evaluating 133 women, who were grouped as follows: non-smoking pregnant women (N = 47), smoking pregnant women (N = 33), non-smoking women (N = 34), and smoking pregnant women (N = 19). The diameter of the brachial artery was measured at baseline and at 30, 60, 90 and 120 s after stimulus. The relative change of brachial artery was determined for each of these four moments. FMD measured at 60 s after stimulus was compared between the groups. The maximum FMD was observed at 60 s after cuff release in all groups. FMD was greater among non-smoking pregnant women compared to smoking pregnant women (11.50 +/- A 5.77 vs. 8.74 +/- A 4.83; p = 0.03) and also between non-smoking non-pregnant women compared to smoking non-pregnant women (10.52 +/- A 4.76 vs. 7.21 +/- A 5.57; p = 0.03). Maximum FMD was observed approximately 60 s after stimulus in all groups regardless of smoking and pregnancy status. The smoking habit seems to lead to endothelial dysfunction both in pregnant and non-pregnant women, as demonstrated by the lower FMD in smokers.

Genetic associations between carcass traits measured by real-time ultrasound and scrotal circumference and growth traits in Nelore cattle

The aim of the present study was to evaluate the genetic correlations among real-time ultrasound carcass, BW, and scrotal circumference (SC) traits in Nelore cattle. Carcass traits, measured by real-time ultrasound of the live animal, were recorded from 2002 to 2004 on 10 farms across 6 Brazilian states on 2,590 males and females ranging in age from 450 to 599 d. Ultrasound records of LM area (LMA) and backfat thickness (BF) were obtained from cross-sectional images between the 12th and 13th ribs, and rump fat thickness (RF) was measured between the hook and pin bones over the junction between gluteus medius and biceps femoris muscles. Also, BW (n = 22,778) and SC ( n = 5,695) were recorded on animals born between 1998 and 2003. The BW traits were 120, 210, 365, 450, and 550-d standardized BW (W120, W210, W365, W450, and W550), plus BW (WS) and hip height (HH) on the ultrasound scanning date. The SC traits were 365-, 450-, and 550-d standardized SC (SC365, SC450, and SC550). For the BW and SC traits, the database used was from the Nelore Breeding Program-Nelore Brazil. The genetic parameters were estimated with multivariate animal models and REML. Estimated genetic correlations between LMA and other traits were 0.06 (BF), -0.04 ( RF), 0.05 (HH), 0.58 (WS), 0.53 (W120), 0.62 (W210), 0.67 (W365), 0.64 ( W450 and W550), 0.28 (SC365), 0.24 (SC450), and 0.00 ( SC550). Estimated genetic correlations between BF and with other traits were 0.74 ( RF), -0.32 (HH), 0.19 (WS), -0.03 (W120), -0.10 (W210), 0.04 (W365), 0.01 (W450), 0.06 ( W550), 0.17 (SC365 and SC450), and -0.19 (SC550). Estimated genetic correlations between RF and other traits were -0.41 (HH), -0.09 (WS), -0.13 ( W120), -0.09 ( W210), -0.01 ( W365), 0.02 (W450), 0.03 (W550), 0.05 ( SC365), 0.11 ( SC450), and -0.18 (SC550). These estimates indicate that selection for carcass traits measured by real-time ultrasound should not cause antagonism in the genetic improvement of SC and BW traits. Also, selection to increase HH might decrease subcutaneous fat as correlated response. Therefore, to obtain animals suited to specific tropical production systems, carcass, BW, and SC traits should be considered in selection programs.

Effects of Ipratropium on Exercise-Induced Bronchospasm

Exercise-induced bronchospasm (EIB) is the transient narrowing of the airways that follows vigorous exercise. Ipratropium bromide may be used to prevent EIB, but its effect varies among individuals. We hypothesized that time of administration of ipratropium interferes with its action. This was a prospective, double-blind, cross-over study carried out to evaluate the bronchoprotective and bronchodilatory effect of ipratropium at different times of day. The study consisted of 4 exercise challenge tests (2 at 7 am and 2 at 6 pm). In the morning, one of the tests was performed after placebo administration and the other one after ipratropium (80 mu g) and the two tests (placebo and ipratropium) were repeated in the evening. Twenty-one patients with severe or moderate asthma and previous confirmation of EIB were enrolled in this prospective trial. The bronchodilatory effect of ipratropium was 0.25 +/- 0.21 L or 13.11 +/- 10.99 % (p = 0.001 compared to baseline values) in the morning, and 0.14 +/- 0.25 L or 7.25 +/- 11.37 % (p > 0.05) in the evening. In the morning, EIB was 0.58 +/- 0.29 L on the placebo day and 0.38 +/- 0.22 L on the treatment day (p = 0.01). In the evening, EIB was 0.62 +/- 0.28 L on the placebo day and 0.51 +/- 0.35 L on the treatment day (p > 0.05). We suggest that the use of ipratropium for the treatment of asthma and EIB should take into consideration the time of administration.

Short communication: Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Subclinical mastitis is a common and easily disseminated disease in dairy herds. Its routine diagnosis via bacterial culture and biochemical identification is a difficult and time-consuming process. In this work, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows bacterial identification with high confidence and speed (1 d for bacterial growth and analysis). With the use of MALDI-TOF MS, 33 bacterial culture isolates from milk of different dairy cows from several farms were analyzed, and the results were compared with those obtained by classical biochemical methods. This proof-of-concept case demonstrates the reliability of MALDI-TOF MS bacterial identification, and its increased selectivity as illustrated by the additional identification of coagulase-negative Staphylococcus species and mixed bacterial cultures. Matrix-assisted laser desorption-ionization mass spectrometry considerably accelerates the diagnosis of mastitis pathogens, especially in cases of subclinical mastitis. More immediate and efficient animal management strategies for mastitis and milk quality control in the dairy industry can therefore be applied.

Use of the real time RT-PCR for immune related gene expression quantitation in experimentally infected Neospora caninum bovine calves

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-gamma and TNF-alpha genes showed increased expression in some samples. iNOS and TGF-beta 1 genes had some non-significant variations and IL-4 and IL-10 stayed pratically inaltered.

Time series evaluation of traumatic lesions and airsacculitis at one poultry abattoir in the state of Sao Paulo, Brazil (1996-2005)

Ten year official condemnation records of one officially inspected poultry abattoir in state of Sao Paulo. Brazil, were analyzed. Seasonal and cyclical trends were analyzed in relation to traumatic lesions and airsacculitis. which were the most relevant official condemnation causes Time series analysis of the records, seasonal indexes and moving averages was used to describe the adherence to the mathematical model and to offer preventive management strategies for the slaughter house industry Although cause-effect relationships were not defined, some insight was given into the causal mechanisms that generated the series (C) 2010 Elsevier B V All rights reserved

Influence of etching time on bond strength in dentin irradiated with erbium lasers

The purpose of this in vitro study was to evaluate the effect of etching time on the tensile bond strength (TBS) of a conventional adhesive bonded to dentin previously irradiated with erbium:yttrium-aluminum-garnet (Er:YAG) and erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) lasers. Buccal and lingual surfaces of 45 third molars were flattened until the dentin was exposed and randomly assigned to three groups (n = 30) according to the dentin treatment: control (not irradiated), irradiated with Er:YAG (1 W; 250 mJ; 4 Hz; 80.6 J/cm(2)) laser or Er,Cr:YSGG (4 W; 200 mJ; 20 Hz; 71.4 J/cm(2)) laser, and into three subgroups (n = 10) according to acid etching time (15 s, 30 s or 60 s) for each experimental group. After acid etching, the adhesive was applied, followed by the construction of an inverted cone of composite resin. The samples were immersed in distilled water (37A degrees C for 24 h) and subjected to TBS test [50 kilogram-force (kgf), 0.5 mm/min]. Data were analyzed by analysis of variance (ANOVA) and Tukey statistical tests (P a parts per thousand currency signaEuro parts per thousand 0.05). Control group samples presented significant higher TBS values than those of all lased groups. Both irradiated groups exhibited similar TBS values. Samples subjected to the different etching times in each experimental group presented similar TBS. Based on the conditions of this in vitro study we concluded that Er:YAG and Er,Cr:YSGG laser irradiation of the dentin weakens the bond strength of the adhesive. Moreover, increased etching time is not able to modify the bonding strength of the adhesive to irradiated dentin.