Analysis of the chronic intake of and withdrawal from diazepam on emotional reactivity and sensory information processing in rats

It has been demonstrated that, on abrupt withdrawal, patients with chronic exposure can experience a number of symptoms indicative of a dependent state. In clinical patients, the earliest to arise and most persistent signal of withdrawal from chronic benzodiazepine (Bzp) treatment is anxiety. In laboratory animals, anxiety-like effects following abrupt interruption of chronic Bzp treatment can also be reproduced. In fact, signs that oscillate from irritability to extreme fear behaviours and seizures have been described already. As anxiety remains one of the most important symptoms of Bzp withdrawal, in this study we evaluated the anxiety levels of rats withdrawn from diazepam. Also studied were the effects on the motor performance and preattentive sensory gating process of rats under diazepam chronic treatment and upon 48-h withdrawal on three animal models of anxiety, the elevated plus-maze (EPM), ultrasonic vocalizations (USV) and startle + prepulse inhibition tests. Data obtained showed an anxiolytic- and anxiogenic-like profile of the chronic intake of and withdrawal from diazepam regimen in the EPM test, 22-KHz USV and startle reflex. Diazepam chronic effects or its withdrawal were ineffective in promoting any alteration in the prepulse inhibition (PPI). However, an increase of PPI was achieved in both sucrose and diazepam pretreated rats on 48-h withdrawal, suggesting a procedural rather than a specific effect of withdrawal on sensory gating processes. It is also possible that the prepulse can function as a conditioned stimulus to informing the delivery of an aversive event, as the auditory startling-eliciting stimulus. All these findings are indicative of a sensitization of the neural substrates of aversion in diazepam withdrawn animals without concomitant changes on the processing of sensory information

Influence of the starting time of preslaughter feed withdrawal on welfare of pigs and pork quality

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Depletion study and estimation of the withdrawal period for oxytetracycline in tilapia cultured in Brazil

Defining pharmacokinetic parameters and depletion intervals for antimicrobials used in fish represents important guidelines for future regulation by Brazilian agencies of the use of these substances in fish farming. This article presents a depletion study for oxytetracycline (OTC) in tilapias (Orechromis niloticus) farmed under tropical conditions during the winter season. High performance liquid chromatography, with fluorescence detection for the quantitation of OTC in tilapia fillets and medicated feed, was developed and validated. The depletion study with fish was carried out under monitored environmental conditions. OTC was administered in the feed for five consecutive days at daily dosages of 80 mg/kg body weight. Groups of ten fish were slaughtered at 1, 2, 3, 4, 5, 8, 10, 15, 20, and 25 days after medication. After the 8th day posttreatment, OTC concentrations in the tilapia fillets were below the limit of quantitation (13 ng/g) of the method. Linear regression of the mathematical model of data analysis presented a coefficient of 0.9962. The elimination half- life for OTC in tilapia fillet and the withdrawal period were 1.65 and 6 days, respectively, considering a percentile of 99% with 95% of confidence and a maximum residue limit of 100 ng/g. Even though the study was carried out in the winter under practical conditions where water temperature varied, the results obtained are similar to others from studies conducted under controlled temperature.

Fear state induced by diazepam withdrawal may be due to the sensitization of the neural substrates of aversion in the dPAG

This work was supported by FAPESP (P.N. 04/02859-0)

Similar ethanol drinking in adolescent and adult C57BL/6J mice after chronic ethanol exposure and withdrawal

BACKGROUND: Increasing evidence shows that excessive alcohol consumption during adolescence increases vulnerability to alcohol use disorders in adulthood. The aim of this study was to examine differences between adolescent and adult C57BL/6J mice in drinking behavior and blood ethanol (EtOH) concentrations (BECs) after chronic EtOH exposure and withdrawal. METHODS: Male adolescent (PND = 28 to 30) and adult (PND = 70) C57BL/6J mice were allowed to consume EtOH in a 2-bottle choice paradigm (15% EtOH vs. water) for 3 weeks (Baseline drinking, Test 1, and Test 2), which were interspersed with 2 cycles (Cycles I and II) of chronic EtOH vapor or air inhalation (16 hours) and withdrawal (8 hours). BECs were determined during both cycles. RESULTS: Chronic EtOH exposure led to increased EtOH intake during Test 1 and Test 2 in both adolescent and adult mice compared with air-exposed controls, and no differences between age groups were observed. During Cycle I adult mice showed higher BECs compared with adolescents. During Cycle II, BECs were lower in adult mice as compared to Cycle I, and BECs in adolescent mice did not change between the 2 cycles. CONCLUSIONS: Chronic EtOH exposure followed by withdrawal periods increases EtOH consumption similarly in both adolescent and adult mice, despite differences in BECs

Genomisches Imprinting Beckwith-Wiedemann-Syndrom assoziierter Gene

In der vorliegenden Arbeit wurde das Imprinting von Genen der Chromosomenregion 11p15.5 des Menschen und des orthologen murinen Abschnitts 7F5 untersucht. Bei der Analyse der humanen Gene H19, IGF2 und KCNQ1OT1 stand deren Regulation durch differentiell methylierte Regionen (DMR) und die Identifizierung von Methylierungsfehlern bei Patienten mit Verdacht auf Beckwith-Wiedemann Syndrom (BWS) im Vordergrund. Hierzu wurden unmethylierte Cytosinnukleotide durch Bisulfitbehandlung in Uracilnukleotide umgewandelt und PCR-amplifizierte DNA-Fragmente sequenziert. Die elterliche Herkunft der Allele wurde mit Hilfe von Einzelnukleotidpolymorphismen (SNP) bestimmt. Während in der H19-Promotorregion in Lymphozyten eine nur tendenziell allelspezifische Methylierung festgestellt werden konnte, wurde im B1-Repeat der H19/IGF2-Region in allen Kontroll- und 20 Patienten-DNAs eine spezifische Methylierung des väterlichen Allels nachgewiesen. Vier BWS-DNAs zeigten hingegen eine nahezu vollständige Hypomethylierung. In der zwe

Die regulatorische Funktion des Usher-Syndrom-Proteins SANS in Proteinnetzwerken

Die vorliegende kumulative Arbeit umfasst Analysen zur Aufklärung der molekularen Grundlagen des humanen Usher-Syndroms (USH), der häufigsten Ursache kombinierter vererblicher Taub-Blindheit. Ziel dieser Arbeit war es, neue Erkenntnisse zur Funktion der USH-Proteine und den von ihnen organisierten Protein-Netzwerken in der Photorezeptorzelle zu erhalten. Dadurch sollten weitere Einsichten in die molekularen Ursachen des retinalen Phänotyps von USH gewonnen werden. Die Ergebnisse dieser Analysen wurden in einem Übersichtsartikel (I) und zwei Originalarbeiten (II, III) zusammengestellt.rn Im Übersichtsartikel (I) wurden die vorliegenden Hinweise zusammengefasst, die USH auf Grundlage der molekularen Verbindungen ebenfalls als Ciliopathien definiert. Zudem wird die Bedeutung des periciliären USH-Proteinnetzwerkes für das sensorische Cilium (Außensegment) der Photorezeptorzelle herausgestellt. rn In Publikation II wurde der Aufbau des USH1-USH2-Proteinnetzwerkes als Teil des periciliären Komplexes analysiert, der beim cargo handover von vesikulärer Fracht vom Innensegment- auf den ciliären Transport für die Photorezeptorzelle essentiell ist. Experimentell wurde Ush2a als neuer SANS-Interaktionspartner validiert. Des Weiteren wurde ein ternärer Komplex aus den USH-Proteinen SANS, Ush2a und Whirlin identifiziert, dessen Zusammensetzung durch die phosphorylierungsabhängige Interaktion zwischen SANS und Ush2a reguliert werden könnte. Dieser ternäre Komplex kann sowohl der Integrität der Zielmembran dienen als auch am Transfer von Molekülen ins Außensegment beteiligt sein.rn In Publikation III wurde das MAGUK-Protein Magi2 als neuer Interaktionspartner von SANS identifiziert und die Interaktion durch komplementäre Interaktionsassays validiert. Dabei wurde ein internes PDZ-Binde-Motiv in der SAM-Domäne von SANS identifiziert, das die Interaktion zur PDZ5-Domäne von Magi2 phosphorylierungsabhängig vermittelt. Dadurch wurde bestätigt, dass SANS durch post-translationale Modifizierung reguliert wird. Weiterführende Experimente zur Funktion des Magi2-SANS-Komplexes zeigen, dass Magi2 an Prozess der Rezeptor-vermittelten Endocytose beteiligt ist. Die Phosphorylierung von SANS durch die Kinase CK2 spielt bei der Endocytose ebenfalls eine wichtige Rolle. Der Phosphorylierungsstatus von SANS moduliert die Interaktion zu Magi2 und reguliert dadurch negativ den Prozess der Endocytose. In RNAi-Studien wurde die durch Magi2-vermittelte Endocytose darüber hinaus mit dem Prozess der Ciliogenese verknüpft. Die Analyse der subzellulären Verteilung der Interaktionspartner lokalisieren Magi2 im periciliären Komplex und assoziieren das periciliäre USH-Proteinnetzwerk dadurch mit dem Prozess der Endocytose in der ciliary pocket. Der SANS-Magi2-Komplex sollte demnach für Aufbau und Funktion des sensorischen Ciliums der Photorezeptorzelle eine wichtige Rolle spielen.rn Die Gesamtheit an Informationen, die aus den Publikationen dieser Dissertation und aus den Kooperationsprojekten (*) resultieren, haben die Kenntnisse zur zellulären Funktion der USH-Proteine und ihrer Interaktionspartner und damit über die pathogenen Mechanismen von USH erweitert. Dies bildet die Basis, um fundierte Therapiestrategien zu entwickeln.

The nociceptive withdrawal reflex: Normative values of thresholds and reflex receptive fields

Assessments of spinal nociceptive withdrawal reflexes can be used in human research both to evaluate the effect of analgesics and explore pain mechanisms related to sensitization. Before the reflex can be used as a clinical tool, normative values need to be determined in large scale studies. The aim of this study was to determine the reference values of spinal nociceptive reflexes and subjective pain thresholds (to single and repeated stimulation), and of the area of the reflex receptive fields (RRF) in 300 pain-free volunteers. The influences of gender, age, height, weight, body-mass index (BMI), body side of testing, depression, anxiety, catastrophizing and parameters of Short-Form 36 (SF-36) were analyzed by multiple regressions. The 95% confidence intervals were determined for all the tests as normative values. Age had a statistically and quantitatively significant impact on the subjective pain threshold to single stimuli. The reflex threshold to single stimulus was lower on the dominant compared to the non-dominant side. Depression had a negative impact on the subjective pain threshold to single stimuli. All the other analyses either did not reveal statistical significance or displayed quantitatively insignificant correlations. In conclusion, normative values of parameters related to the spinal nociceptive reflex were determined. This allows their clinical application for assessing central hyperexcitability in individual patients. The parameters investigated explore different aspects of sensitization processes that are largely independent of demographic characteristics, cognitive and affective factors.

Variability of lung function predicts loss of asthma control following withdrawal of inhaled corticosteroid treatment

BACKGROUND One aspect of a multidimensional approach to understanding asthma as a complex dynamic disease is to study how lung function varies with time. Variability measures of lung function have been shown to predict response to beta(2)-agonist treatment. An investigation was conducted to determine whether mean, coefficient of variation (CV) or autocorrelation, a measure of short-term memory, of peak expiratory flow (PEF) could predict loss of asthma control following withdrawal of regular inhaled corticosteroid (ICS) treatment, using data from a previous study. METHODS 87 adult patients with mild to moderate asthma who had been taking ICS at a constant dose for at least 6 months were monitored for 2-4 weeks. ICS was then withdrawn and monitoring continued until loss of control occurred as per predefined criteria. Twice-daily PEF was recorded during monitoring. Associations between loss of control and mean, CV and autocorrelation of morning PEF within 2 weeks pre- and post-ICS withdrawal were assessed using Cox regression analysis. Predictive utility was assessed using receiver operator characteristics. RESULTS 53 out of 87 patients had sufficient PEF data over the required analysis period. The mean (389 vs 370 l/min, p<0.0001) and CV (4.5% vs 5.6%, p=0.007) but not autocorrelation of PEF changed significantly from prewithdrawal to postwithdrawal in subjects who subsequently lost control, and were unaltered in those who did not. These changes were related to time to loss of control. CV was the most consistent predictor, with similar sensitivity and sensitivity to exhaled nitric oxide. CONCLUSION A simple, easy to obtain variability measure of daily lung function such as the CV may predict loss of asthma control within the first 2 weeks of ICS withdrawal.

Test-retest reliability of the nociceptive withdrawal reflex and electrical pain thresholds after single and repeated stimulation in patients with chronic low back pain

Recent studies have shown that the nociceptive withdrawal reflex threshold (NWR-T) and the electrical pain threshold (EP-T) are reliable measures in pain-free populations. However, it is necessary to investigate the reliability of these measures in patients with chronic pain in order to translate these techniques from laboratory to clinic. The aims of this study were to determine the test-retest reliability of the NWR-T and EP-T after single and repeated (temporal summation) electrical stimulation in a group of patients with chronic low back pain, and to investigate the association between the NWR-T and the EP-T. To this end, 25 patients with chronic pain participated in three identical sessions, separated by 1 week in average, in which the NWR-T and the EP-T to single and repeated stimulation were measured. Test-retest reliability was assessed using intra-class correlation coefficient (ICC), coefficient of variation (CV), and Bland-Altman analysis. The association between the thresholds was assessed using the coefficient of determination (r (2)). The results showed good-to-excellent reliability for both NWR-T and EP-T in all cases, with average ICC values ranging 0.76-0.90 and average CV values ranging 12.0-17.7%. The association between thresholds was better after repeated stimulation than after single stimulation, with average r (2) values of 0.83 and 0.56, respectively. In conclusion, the NWR-T and the EP-T are reliable assessment tools for assessing the sensitivity of spinal nociceptive pathways in patients with chronic pain.

Effect of ketamine on the limb withdrawal reflex evoked by transcutaneous electrical stimulation in ponies anaesthetised with isoflurane

The purpose of this study was to evaluate the anti-nociceptive activity of ketamine and isoflurane in horses using a limb withdrawal reflex (WR) model. Single and repeated stimulations were applied to the digital nerve of the left forelimb in ponies anaesthetised with isoflurane before, during and after intravenous administration of racemic ketamine. Surface electromyographic activity was recorded from the deltoid muscle. Higher stimulation intensity was required to evoke a reflex during ketamine administration. Furthermore, the amplitudes of response to stimulations were significantly and dose-dependently depressed and a flattening of the stimulus-response curves was observed. The reflex activity recovered partially once the ketamine infusion finished. The results demonstrated that the limb WR can be used to quantify the temporal effect of ketamine on the sensory-motor processing in ponies anaesthetised with isoflurane.