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This site has been established to host information about doping in the broadest sense of the word, and about doping prevention. This website was established because of the importance that the Doping Authority and the Ministry attach to the dissemination of information relevant to doping prevention. Disclosing and supplying relevant information is one of the cornerstones in the fight against doping in sport. However, in practice, a significant amount of information is still not available, or only available to a limited group of users. We therefore decided to bring together all the relevant information in a single site: Doping. The Doping Authority aims to supply as much information through this website as possible on an ongoing basis. As well as making documents available, the Doping Authority aims to supply searchable documents when possible, and to add relevant keywords to ensure easy access. In the future, Doping. This site has been designed for use by anti-doping professionals such as National Anti-Doping Organisations and International Federations but also for students, journalists and other people interested in the subject. More information explaining how to use this website can be found under ' help '. Executive Committee agrees to establish a Working Group that will focus on recommendations that can be reviewed and approved by the ExCo. In his opening remarks to the ExCo, Mr. The information in the file shows that WADA has done its work autonomously, independently and professionally, and that there is no evidence to the contrary. WADA has applied the rules to which its activity is subject. Cottier then noted that his final reasoned report went beyond the two questions that he was posed for the review, offering a number of findings, including that:. Clarification of certain anti-doping rules should be considered as part of the World Anti-Doping Code and International Standards Update Process that is currently underway, in particular with the regards to specific situations of group contamination; and. This is regrettable, but in the end, it does not change the outcome of the cases and the acceptance of the contamination hypothesis. Following notification a provisional suspension was imposed and the Athlete filed a statement in her defence. She explained with medical documents that she underwent medical treatment in a hospital. The AIU found that these medical documents were insufficient to explain the Athlete's positive test result. Because the Athlete did not respond within the deadline, the AIU determined in September that the Athlete was deemed to have waived her right for a hearing and accepted the consequences. Therefore the AIU decided, on 11 September , to impose a 4 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Following notification the Athlete timely admitted the violation, waived her right for a hearing, accepted a provisional suspension and the sanction proposed by the AIU. She could not explain how the substance had entered her system. The AIU deems that the Athlete failed to establish that the violation was not intentional. Therefore the AIU decides on 11 September to impose a 3 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. No administratieve review was requested and the Whereabouts Failures were recorded. Following notification the Athlete explained that her representative had not updated her Whereabouts on two occasions. She herself was unable to communicate in English, had not received anti-doping education, had limited access to internet and was completely dependent of her coach and her representative. The representative confirmed in his submission that the 2nd and 3rd Whereabouts Failure were entirely his fault. Thereupon the Athlete gave a timely admission, waived her right for a hearing, accepted a provisional suspension and the sanction proposed by the AIU. The AIU considers that the she has established that she was impaired by the circumstances in updating her Whereabouts information. Therefore the AIU decides on 11 September to impose a 20 month period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Thereupon the Athlete's request for lifting the provisional suspension was dismissed and analysis of his B sample confirmed the result of the A sample 1 sample. Hereafter the Athlete failed to respond to the AIU communications. The AIU determines that the Athlete's 1 and 2 positive test result shall be considered as one single anti-doping rule violation. Furthermore there are aggravating circumstances present in these case that justifies the imposition of a more severe sanction. Because the Athlete did not respond within the set deadline the AIU rules that the Athlete was deemed to have waived his right for a hearing and accepted the consequences. Therefore the AIU decides on 10 September to impose a 6 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Following notification the Athlete timely admitted the violation, waived his right for a hearing, accepted a provisional suspension and the sanction proposed by the AIU. Therefore the AIU decides on 10 September to impose a 3 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. In May the United Kingdom Anti-Doping reported an anti-doping rule violation against the football player Jordan Cotterill after his sample tested positive for the prohibited substance Cocaine. Following notification the Athlete admitted the violation, waived his right for a hearing, accepted a provisional suspension and the sanction proposed by UKAD. The Athlete admitted that he had used Cocaine with alcohol recreational and out-of-competition. Therefore UKAD decides on 9 August to impose a 3 month period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Following notification a provisional suspension was ordered, yet ADAK was unable to locate the Athlete. The Panel finds that the presence of a prohibited substance has been established in the Athlete's sample and accordingly that he committed an anti-doping rule violation. The Panel determines that the Athlete was non responsive and didn't provide any statement in his defence. As a result the Panel deems that he failed to demonstrate that the violation was not intentional, nor how the substance had entered his system. Therefore the Tribunal decides on 25 July to impose a 4 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Following notification the Athlete timely admitted the violation, waived his right for a hearing, accepted a provisional suspension and the sanction proposed by ADAK. Therefore the ADAK decides on 4 July to impose a 3 year period of ineligibility on the Athlete, starting on the date of the provisional suspension, i. Following notification the Athlete admitted the violation, waived his right for a hearing and accepted the sanction proposed by ADAK. Goals This website was established because of the importance that the Doping Authority and the Ministry attach to the dissemination of information relevant to doping prevention. Activities The Doping Authority aims to supply as much information through this website as possible on an ongoing basis. Target readers This site has been designed for use by anti-doping professionals such as National Anti-Doping Organisations and International Federations but also for students, journalists and other people interested in the subject. Search Information.

A Novel Morphine Drinking Model of Opioid Dependence in Rats

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Official websites use. Share sensitive information only on official, secure websites. An animal model of voluntary oral morphine consumption would allow for a pre-clinical evaluation of new treatments aimed at reducing opioid intake in humans. However, the main limitation of oral morphine consumption in rodents is its bitter taste, which is strongly aversive. Taste aversion is often overcome by the use of adulterants, such as sweeteners, to conceal morphine taste or bitterants in the alternative bottle to equalize aversion. Thus, current animal models are not suitable to study treatments aimed at reducing consumption elicited by morphine itself. Since taste preference is a learned behavior, just-weaned rats were trained to accept a bitter taste, adding the bitterant quinine to their drinking water for one week. The latter was followed by allowing the choice of quinine or morphine 0. Then, quinine was removed, and the preference for morphine against water was evaluated. Morphine consumption led to significant analgesia and hyperlocomotion, and to a marked deprivation syndrome following the administration of the opioid antagonist naloxone. Voluntary morphine consumption was also shown to generate brain oxidative stress and neuroinflammation, signs associated with opioid dependence development. We present a robust two-bottle choice animal model of oral morphine self-administration for the evaluation of therapeutic interventions for the treatment of morphine dependence. Keywords: morphine, opioids, addiction, oral intake, animal model, dependence, quinine. Opioids are widely used to treat acute and chronic pain due to their potent analgesic and sedative properties. However, since opioids also generate the compulsion to take the drug, it has led to widespread non-medical opioid use \[ 1 \]. Opioid drugs are among the most addictive substances \[ 1 \]. Repeated daily use leads to physical dependence and rapid tolerance to their effects \[ 1 \]. It is consistently reported that the oral route is the preferred route of administration for prescription opioids. Thus, the generation of animal models of voluntary opioid oral intake has become a necessary pre-clinical tool to evaluate new pharmacological strategies for the treatment of opioid dependence. Studies evaluating the voluntary oral self-administration of drugs of abuse in animal models often utilize procedures in which the preference for drug-containing solutions is assessed against either water or palatable non-drug solutions. Among the different paradigms commonly used, the two-bottle free-choice exposure is widely used to evaluate drug avidity, being a non-invasive procedure in which animals have the option of voluntarily drinking the desired amount of drug. This paradigm has demonstrated relevant translational value for several drugs of abuse \[ 7 , 8 \]. However, the main limitation of oral morphine intake is its bitter taste. Thus, when the morphine bottle is paired with a bottle containing non-adulterated tap water, animals prefer the non-morphine solution \[ 9 \]. To overcome this limitation, researchers reported the addition of taste adulterants such as sweeteners, e. However, the neurobiology by which quinine and saccharin influence the ensuing preference for morphine in rodent models remains poorly understood. Ferraro et al. However, morphine and saccharin have known pharmacological interactions since saccharin potentiates morphine-induced antinociception in mice \[ 12 \]. Furthermore, saccharin consumption is also known to promote dopamine release in the nucleus accumbens, activating the mesolimbic reward system \[ 13 \]. Even after several days of high intake of morphine plus saccharin, the elimination of saccharin from the bottles markedly reduces morphine consumption \[ 11 \], thus limiting the translational value of this model. Similarly, rats given morphine adultered with sugar in their drinking water for 21 days do not develop a preference for the morphine-sugar bottle when evaluated against a bottle with only sugar dissolved in water \[ 14 \]. Thus, the taste-altering confounding factor must be eliminated before animals that consume morphine could be reliably used for pre-clinical testing of treatments aimed at reducing consumption elicited by morphine itself. On the other hand, when quinine is included in the alternative bottle, a preference for the morphine solution is observed, suggesting that quinine is effective in driving morphine consumption and generating dependence. However, several reports demonstrated that, in this paradigm, high morphine preference is not related to the rewarding properties of the drug, but its preference is influenced by the degree of quinine aversion \[ 15 , 16 \]. Thus, it was postulated that the aversion to the bitter taste of quinine is what leads to the maintenance of relatively high levels of fluid consumption from the morphine bottle \[ 15 \]. This interpretation is supported by the significant reduction in morphine intake observed when quinine is eliminated from the alternative bottle, despite weeks of morphine consumption \[ 11 , 15 \]. Thus, new paradigms to induce a preference for morphine-containing solutions without the influence of taste adulterant confounders are highly needed. In rodents, flavor preference learning usually occurs at the time of weaning and could influence food selection after weaning \[ 17 \]. Thus, repeated exposure to an aversive flavor early in life could improve flavor acceptance in adult life due to familiarity, reducing inherent neophobia \[ 18 \]. The bitter taste is one of these aversive flavors. Interestingly, exposure of young rats to the bitterant quinine as their only source of fluid immediately after weaning significantly increases quinine preference when evaluated in a two-bottle choice against water \[ 19 \]. Those results suggest that quinine consumption after weaning makes quinine, and possibly other bitterants, more palatable to weanling rats. In this study, we show a novel morphine drinking paradigm in which, immediately after weaning, rats are exposed for seven days to a quinine solution as their only fluid source, to make animals get used to a bitter taste. After that period, animals are exposed to a two-bottle choice of quinine or morphine solutions for two weeks. Finally, the quinine bottle is replaced by a water bottle, and animals now have the choice of drinking morphine or water. After four weeks of voluntary morphine intake, we evaluated classical markers of the pharmacological action of morphine, including thermal and mechanical analgesia and hyperlocomotion, as well as markers of morphine dependence including the appearance of somatic signs of morphine withdrawal triggered by the administration of naloxone, an opioid receptor antagonist. Upon weaning, three-week-old rats were exposed to a quinine solution 0. Young rats learn to drink a bitter-tasting quinine solution. Immediately after weaning, three-week-old female Wistar rats were exposed to water or 0. B Animal weights were determined daily. Following seven days of quinine exposure, a morphine-solution 0. While a modest downward intake of the morphine solution was seen thereafter, likely the result of the fast increases in body weight of recently weaned animals, the high preference for the morphine solution remained, which was consistent with a reduced total fluid consumption normalized by body weight Figure 2 C,D. Voluntary morphine intake by Wistar rats. One week after starting quinine intake Figure 1 , animals were exposed to 0. Thereafter, animals were exposed to 0. A Voluntary morphine intake is expressed as mg morphine consumed per kg of body weight per day. D Animal weights. After two weeks of free-choice between morphine and quinine solutions, the quinine bottle was replaced by one filled with only water to assess the morphine consumption without the confounding presence of quinine. It is noted that seven animals changed their preference ratio from the morphine solution to water during the two weeks of observation and were removed from the study. As a measure of the analgesic effect of morphine, rats that had voluntarily consumed morphine for four weeks were tested for their withdrawal latency following a thermal stimulus and for the withdrawal threshold to a mechanical stimulus. Voluntary morphine intake induces analgesia in Wistar rats. Hargreaves plantar test A and Von Frey test B showing the withdrawal latency delayed response to thermal stimulus and withdrawal threshold to mechanical stimulus, respectively, of rats that had voluntarily ingested a morphine solution for four weeks. Animals drinking only water were used as controls. Withdrawal latency and withdrawal threshold were measured during the first hour of light. Voluntary morphine intake induces hyperlocomotor activity in Wistar rats. C Trajectory plot of rats drinking morphine and water showing entries to the center of the open field. The open field test was conducted during the first hour of light. Animals that had voluntarily consumed morphine also developed physical dependence. This was shown by the eliciting of withdrawal syndrome signs following the administration of the opioid antagonist naloxone. A clear opioid deprivation syndrome is induced by naloxone administration in Wistar rats that had voluntarily consumed morphine. To study whether rats that had voluntarily consumed morphine displayed brain neurochemical alterations, we determined the presence of neuroinflammation, oxidative stress levels, and morphine-related protein expression. Hippocampal neuroinflammation: Neuroinflammation was evaluated by determining glial reactivity in the hippocampus, assessed as morphological changes in astrocytes and microglial density. Voluntary morphine intake induces hippocampal neuroinflammation in Wistar rats. Nuclei were counterstained with DAPI blue, nuclear marker , scale bar 25 mm. B left Quantification of total length and B right thickness of primary astrocytic process and C Quantification of microglial density evaluated by confocal microscopy and FIJI image analysis software. Hippocampal oxidative stress levels. Voluntary morphine intake induces hippocampal oxidative stress in Wistar rats. Morphine-related signaling pathway. Voluntary morphine intake induces an increase in opioid receptor mRNA levels in pre-frontal cortex but not in nucleus accumbens of Wistar rats. While several models of intravenous opioid self-administration are reported \[ 27 , 28 \], these do not fully reflect the changes that result from the pharmacokinetic of oral opioid administration. Thus, the generation of a novel and reliable animal model of voluntary oral opioid intake resulting in marked dependence, as shown by a clear naloxone-induced precipitated withdrawal, would constitute a valuable pre-clinical tool to evaluate new pharmacological strategies for the treatment of oral opioid dependence. The main limitation in the development of an animal model of oral morphine intake is its bitter taste. Thus, the aim of the present study was to train rats to accept the taste of morphine. In rodents, learning taste preference occurs primarily at weaning time and can influence food selection later in life \[ 17 \]. It was postulated that repeated exposure to a known aversive flavor at a young age could improve animal flavor acceptance in adult life \[ 18 \]. Indeed, exposure of rats immediately after weaning to the bitter taste of a quinine solution as the only source of fluid for seven days significantly increases subsequent quinine preference \[ 19 \]. This was confirmed in the present study since, after only two days of access to a quinine solution as the only source of fluid, rats ingested the quinine solution at the same rate as rats that were allowed water access only. These findings suggest that exposure to quinine after weaning renders morphine more palatable. Although we did not explore the pharmacological effect of quinine consumption during the seven-days period of exclusive consumption of just-weaned rats, we do not expect persisting alterations that may confound the findings associated with the morphine consumption. In patients following that regime, mild and reversible adverse reactions are common. In our study, morphine consumption against water was analyzed 15 to 28 days after rats were drinking high doses of quinine, ensuring the disappearance of the possible adverse effects of quinine exposure. A limitation of our model is the number of subjects required to be removed from the study. Because our goal was to develop a robust model that could be used to study therapeutics to reduce morphine consumption of dependent subjects, we rejected animals that did not show a preference for morphine against the alternative bottle. This suggests that the morphine and quinine solutions were similarly aversive, representing a preference before the animals became dependent. Seven additional rats were removed from the study when the quinine bottle was replaced by one containing only water, since those animals changed their preference from morphine to water. These rejection rates are in concordance to what was previously reported for other drugs since a diversity in the willingness to consume a drug is observed when outbred animals, i. Another limitation of our model is the specific age requirement for starting experiments, that being at weaning age, which results in the experiments being performed using young rats. Animals were eight weeks old when euthanasia was performed. On the other hand, our model could be useful for the study of morphine consumption in young individuals, an age segment with high prevalence. In humans, the average age of opioid initiation is between 22 and 23 years old, and teenagers report high availability of illicit opioids \[ 32 \]. Other research groups also show that morphine is voluntarily consumed by animals when quinine is given in the alternative bottle. In the present study, we show that after four weeks of voluntary morphine intake versus water in controls , the classical acute effects and chronic dependence on morphine effects are observed, including: i thermal and mechanical analgesia; ii morphine-induced hyperlocomotion, and importantly iii naloxone precipitated withdrawal. The later included a significant diarrhea, b marked weight loss, c wet-dog shakes, d forepaw tremors, e chewing, and f reduced rearing, as previously reported \[ 20 , 21 \]. Other studies also aimed at developing animal models for oral opioid self-administration. It is shown that mice \[ 33 , 34 \] and rats \[ 35 \] voluntarily consume oxycodone. In studies in mice, a period of forced oxycodone intake was initially imposed under post-prandial conditions in the early dark cycle which was followed by operant self-administration oxycodone intake conditioned by light and tones. In the studies using rats, the animals were initially water deprived for 22 h a day and exposed to oxycodone solutions as their only fluid, also followed by operant self-administration. While, in all these studies, the animals learned to self-administer oxycodone orally, a naloxone precipitated withdrawal was not reported under these oral self-administration conditions. Indeed, in studies using mice, authors induced the naloxone precipitated withdrawal in animals that were administered oxycodone systemically, rather than orally. In contrast, in the rat study of oral operant self-administration of oxycodone, a high dose of naltrexone was not reported to generate the signs of opioid withdrawal. Data suggests that means to induce adult animals to consume oxycodone orally do not readily result in consumption levels that lead to the development of clear physical dependence. These alterations were not observed in the nucleus accumbens, suggesting that they are region-specific. An animal model for voluntary morphine consumption should also reproduce relevant molecular and histological brain alterations that are commonly observed in human patients after morphine exposure. These phenomena are shown to characterize opioid tolerance and may represent the engines of relapse \[ 38 , 40 \]. Indeed, the administrations of antioxidant and anti-inflammatory agents are shown markedly to reduce morphine withdrawal \[ 41 \] and conditioned place preference \[ 42 \]. In general, antioxidant and anti-inflammatory agents are shown to reduce the self-administration and relapse of many drugs of abuse, supporting the hypothesis that brain oxidative stress and neuroinflammation are required for an addiction to develop \[ 43 \]. In the present oral morphine intake model, molecular markers of morphine-induced dependence were clearly observed, including a significant increase in morphine-induced neuroinflammation evidenced by an increase in the length and thickness of primary astrocytic processes and an increase in microglial density in the hippocampus. Overall, studies conducted show a novel two-bottle choice paradigm to induce a preference for morphine-containing solutions, that starts with young weaning rats, with no influence of taste adulterant confounders nor the need for the animals to consume it to avoid an externally imposed punitive condition. These animals voluntarily drink morphine-containing solutions in amounts that lead to clear morphine dependence, as seen by the classical naloxone-induced signs, and display the molecular and histological brain alterations commonly observed in opioid addicted patients. Thus, this animal model could be valuable for the testing of new therapeutic interventions for the treatment of this devastating and extending condition. Three-week-old female Wistar rats were used in the experiments. Female rats were chosen since it is reported that female rats consume or self-administer higher levels of morphine than male rats \[ 44 , 45 , 46 \]. Immediately after weaning, three-week-old rats were randomly assigned to one of two groups. One group was exposed to 0. Quinine intake, water intake, and body weight were recorded daily. Seven days after starting quinine exposure, animals were changed for two weeks to a two-bottle choice paradigm in which one bottle contained 0. Thereafter, the quinine bottle was removed, and animals were exposed to 0. Animals drinking only water were maintained as controls. Morphine intake, morphine preference, total fluid consumption, and animal weights were measured daily Figure S1. For two-bottle choice tests, the position of each bottle was alternated daily to prevent the accustoming of the animal to drinking from the bottle of a specific position. There were two instances when subjects were removed: i when a morphine bottle was presented to rats drinking only quinine and ii when the quinine bottle was replaced with a water bottle. Methanol extracted samples were vacuum dried, resuspended in the provided buffer, and fold diluted before the analysis. Following four weeks of voluntary morphine intake, analgesia was assessed by the determination of thermal and mechanical sensitivity. For thermal sensitivity determinations, the rats were placed for 20 min inside an acrylic box provided with a mobile on-off infrared light Ugo Basile Plantar Test below the surface of the box. Thereafter, the infrared light was placed beneath the mid-plantar surface of a hind paw, and the paw withdrawal response was automatically recorded. The maximum exposure was set at 15 s to avoid hind paw damage. The infrared light exposure was performed to either paw until completing three measurements, with an interval of five minutes between stimuli, and measurements were repeated the following day. Data in seconds were expressed as the mean withdrawal latency registered each day as we previously reported \[ 47 \]. For mechanical sensitivity determination, animals were placed inside an acrylic box with a mesh floor that allows free access to the plantar surface of the paw. Then, the mid-plantar surface of one of the hind paws was stimulated with an electronic Von Frey filament Electronic Von Frey; Ugo Basile, Comerio, Varese, Italy with increasing strength, and the paw withdrawal response was automatically recorded. The stimulation was performed to either paw until completing three measurements, with an interval of five minutes between stimuli, and measurements were repeated the following day. Following four weeks of voluntary morphine intake, locomotor activity was assessed by the open field test. Rats were placed in the center, and their locomotor activity was recorded for five minutes with a video camera placed in a zenithal position. The room was lit by a soft white light 50—60 lux. The experiments were performed during the first hour of light to expect the highest plasmatic morphine levels. Total distance traveled and thigmotaxis time tendency to remain close to the walls were evaluated using the ANY-maze video tracking system ver. Immediately after naloxone administration, animals were placed in a glass beaker mm height and mm diameter and monitored for weight loss, diarrhea, wet-dog shakes, forepaw tremors, chewing, and rearing events over 30 min. Neuroinflammation was evaluated, determining astrocyte activation and microglial density in the hippocampus of rats after four weeks of voluntary morphine intake. Double-labeling immunofluorescence against the astrocyte marker glial fibrillary acidic protein GFAP and the microglial marker ionized-calcium-binding adaptor molecule 1 Iba-1 were evaluated in coronal 30 mm thick cryo-sections of hippocampus as we previously reported \[ 48 \]. Briefly, coronal sections were washed with 0. Thereafter, sections were rinsed in PBS containing 0. Then, sections were rinsed, incubated in blocking solution 0. Samples were rinsed with PBS, incubated with goat anti-mouse secondary antibody Alexa Fluor , Thermo Fisher Scientific, dilution in blocking solution for 1 h in the dark at room temperature. Microphotographs were taken from the stratum radiatum of the hippocampus using a confocal microscope Olympus FV10i. The area analyzed for each stack was 0. The total length and thickness of the GFAP-positive astrocyte primary process and density of Ibapositive microglial cells were assessed using FIJI image analysis software as previously reported \[ 24 \]. To this end, the hippocampi were extracted and mixed with three volumes of ice-cold potassium buffer 0. The excess of 2-vinyl pyridine was neutralized with triethanolamine. To ensure that amplicons were generated from mRNA and not from genomic DNA, controls without RT during the reverse transcription reactions were included. Statistical analyses were performed using GraphPad Prism software. GraphPad Prism version 9. To facilitate text reading, the full statistical analyses are presented in the figure legends. All authors have read and agreed to the published version of the manuscript. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Int J Mol Sci. Find articles by Mauricio Quezada. Find articles by Daniela Santapau. Find articles by Paola Morales. Find articles by Carolina Ponce. Find articles by Cristian De Gregorio. Find articles by Marcelo Ezquer. Find articles by Mario Herrera-Marschitz. Find articles by Yedy Israel. Find articles by Fernando Ezquer. Zachary A Rodd : Academic Editor. Open in a new tab. Click here for additional data file. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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