RESUMO
The inhibitor of tryptophan hydroxylase, para-chlorophenylalanine (PCPA), has been classically employed as a pharmacological tool to deplete serotonin (5-HT) in animal models and to evaluate whether this neurotransmitter is involved in the action of pharmacological compounds. PCPA is usually administrated by intraperitoneal (ip) injections, which are stressful and painful. To avoid ip injections, we designed and validated a protocol for PCPA oral administration. C57BL/6 elite male mice received PCPA during 7 days either ip or by giving the drug inside jelly cubes at an estimated dose of 500 mg/kg on days 1 and 2 and 250 mg/kg for the rest of the treatment. 5-HT levels decreased by 85% and 55% in the hippocampus of mice treated with oral or ip PCPA, respectively, whereas in the prefrontal cortex, 5-HT levels decreased by 65% (oral) and 50% (ip). Behavioral tests, like the forced swimming test (FST), the nestlet shredding test (NST), and the marble burying test (MBT), were performed. In the FST, mice received fluoxetine ip 30 min before the test. In mice with oral PCPA treatment, fluoxetine did not induce significant reductions of immobility, indicating that reduction of 5-HT levels was effective. No effect of ip or oral 5-HT depletion was observed in the NST nor in the MBT. In a second experiment, mice received oral PCPA for 8 weeks: again, serotonin levels were significantly decreased in both hippocampus and cortex, and effects on hippocampal neurogenesis replicated previous observations in hyposerotonergic mice. Therefore, neurochemical, behavioral, and neurogenic results allow us to validate this refined protocol for voluntary oral consumption of PCPA.
Assuntos
Fluoxetina , Serotonina , Animais , Fenclonina/farmacologia , Fluoxetina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NeurogêneseRESUMO
Most scientific journals ask authors to include a statement in their articles that animal studies have been carried out in agreement with international regulations on the use and care of laboratory animals. This statement implies that all the experiments conducted on animals have been evaluated and accepted by an Ethical Committee and, that animal welfare has been put as a priority throughout the experimental protocol. Nevertheless, discrepancies are commonly found between the described procedures and the guidelines that are claimed to have been followed; this reveals a double dilemma. First, animal welfare is not always considered, implicating discomfort or even worse, suffering to animals involved. Secondly, revisions of manuscripts are sometimes done without taking into account ethical and regulatory aspects concerning the use of animals. Underestimation of pain or suffering, disregard for physiological parameters, and other examples recently reported in scientific journals by neuroscientists from all over the world are discussed in this article. In a period of great debate about the ethical use of animals, with society being involved and engaged in the discussion, this Neuro-Opinion intends to call the attention of researchers, ethical committee members, and journal editors about the need of strictly endorsing international regulations and placing animal welfare as the top priority.
Assuntos
Experimentação Animal , Bem-Estar do Animal , Animais , Animais de LaboratórioRESUMO
We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.
Assuntos
Camundongos Nus , Fotografação/métodos , Doenças dos Roedores/diagnóstico por imagem , Neoplasias de Tecidos Moles/diagnóstico por imagem , Carga Tumoral , Animais , Feminino , Masculino , Camundongos , Organismos Livres de Patógenos EspecíficosRESUMO
It has been shown that the expression of the morphine (MOR) withdrawal syndrome precipitated by naloxone (NAL) is more intense in male mice than in females, but the reasons for this phenomenon remain uncertain. The purpose of the present study was to evaluate whether this sexual dimorphism might be due to differences in MOR and/or NAL plasma levels after a chronic treatment with MOR. Prepubertal Swiss male and female mice were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg), twice daily for 9 days. On day 10 dependent mice received NAL (6 mg/kg, i.p.) 60 min after MOR injection. Blood samples were taken at different times in order to determine MOR and NAL plasma levels by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC), respectively. Pharmacokinetic analysis showed no differences between male and female mice either for MOR or for NAL. In conclusion, although males and females respond differentially to NAL-precipitated withdrawal, this dimorphic behavior would not be influenced by a pharmacokinetic factor.
Assuntos
Morfina/farmacocinética , Naloxona/farmacocinética , Síndrome de Abstinência a Substâncias/metabolismo , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/farmacocinética , Animais , Área Sob a Curva , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Meia-Vida , Injeções Intraperitoneais , Masculino , Camundongos , Morfina/administração & dosagem , Dependência de Morfina/sangue , Dependência de Morfina/complicações , Naloxona/administração & dosagem , Naloxona/sangue , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/farmacocinética , Fatores Sexuais , Maturidade Sexual , Síndrome de Abstinência a Substâncias/etiologia , Fatores de TempoRESUMO
Opioids make up a pharmacological group employed from antiquity for the treatment of various ailments. Nowadays they are far used like palliative of acute or chronic pains of high intensity considering their great analgesic effectiveness. During the last years, many articles have reported sex-related differences in the response of laboratory animals to several opioids. These data is according to the knowledge about sex determining both brain structure and functions. In addition, this sexual dimorphism has been recently reported for women and men treated with opioids. The relevance of this information could give rise to a revision of the dosage traditionally employed by physicians in either sex. The present article covers the main aspects of this subject considering its significance in the pharmacotherapy with opioids.
Assuntos
Tratamento Farmacológico/métodos , Tratamento Farmacológico/estatística & dados numéricos , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Transtornos Relacionados ao Uso de Opioides/reabilitação , Adulto , Feminino , Hormônios Esteroides Gonadais/sangue , Humanos , Masculino , Distribuição por SexoRESUMO
Although the expression of the morphine (MOR) withdrawal syndrome is more marked in male mice than in females, we have demonstrated that the GABAB agonist baclofen (BAC) is able to attenuate MOR withdrawal signs in either sex. In order to extend these previous observations, the aim of the present study was to evaluate the mu-opioid receptor labeling in various brain areas in mice of either sex, during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice were rendered dependent by intraperitonial (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the 10th day, dependent animals received naloxone (NAL; 6 mg/kg, i.p.) 60 min after MOR, and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL. Thirty minutes after NAL, mice were sacrificed and autoradiography with [3H]-[D-Ala2, N-Me-Phe4, -glycol5] enkephalin (DAMGO) was carried out on mice brains at five different anatomical levels. Autoradiographic mapping showed a significant increase of mu-opioid receptor labeling during MOR withdrawal in nucleus accumbens core (NAcC), caudate putamen (CPu), mediodorsal thalamic nucleus (MDTh), basolateral and basomedial amygdala, and ventral tegmental area vs. respective control groups in male mice. In contrast, opiate receptor labeling was not significantly modified in any of the brain areas studied in withdrawn females. BAC reestablished mu-opioid receptor binding sites during MOR withdrawal only in NAcC of males, and a similar tendency was observed in CPu and MDTh, even when it was not statistically significant. The sexual dimorphism observed in the present study confirms previous reports indicating a greater sensitivity of males in response to MOR pharmacological properties. The present results suggest that the effect of BAC in preventing the expression of MOR withdrawal signs could be related with the ability of BAC to reestablish the mu-opioid receptor labeling in certain brain areas.
Assuntos
Baclofeno/farmacologia , Dependência de Morfina/tratamento farmacológico , Morfina/efeitos adversos , Receptores Opioides mu/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Envelhecimento/metabolismo , Animais , Autorradiografia , Baclofeno/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Feminino , Agonistas GABAérgicos/farmacologia , Agonistas GABAérgicos/uso terapêutico , Masculino , Camundongos , Dependência de Morfina/metabolismo , Dependência de Morfina/fisiopatologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/efeitos adversos , Receptores Opioides mu/metabolismo , Caracteres Sexuais , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Morphine (MOR) withdrawal signs are more marked in males than in females. Considering that the influence of the dopaminergic system on these differences is unclear, we analyzed dopamine (DA) and dihydroxyphenylacetic-acid (DOPAC) brain levels during naloxone (NAL)-precipitated withdrawal as well as the involvement of D(1) and D(2) receptors in the expression of MOR withdrawal in either sex. Prepubertal Swiss-Webster mice received MOR (2 mg/kg, i.p.) twice daily for 9 days. On the tenth day, dependent animals received NAL (6 mg/kg, i.p.) after MOR and were sacrificed 30 min later. DA and DOPAC concentrations were determined in different brain areas using HPLC with electrochemical detection. Other pool of mice received either a D(1) (SCH 23390; 0.2 mg/kg, i.p.) or D(2) (raclopride; 0.3 mg/kg, i.p.) receptor antagonist before NAL and withdrawal signs were evaluated. DA and DOPAC levels only decreased in striatum and cortex of withdrawn males. Conversely, both DA receptor antagonists decreased the expression of MOR withdrawal signs in either sex. The neurochemical sex differences described here could partially explain the behavioral sex differences observed during MOR withdrawal. Additionally, SCH-23390 and raclopride effects suggest an important role of both DA receptors in the expression of MOR withdrawal in males and females.
Assuntos
Dopamina/fisiologia , Morfina/efeitos adversos , Síndrome de Abstinência a Substâncias/fisiopatologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Feminino , Masculino , Camundongos , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/fisiologia , Maturidade Sexual/fisiologia , Síndrome de Abstinência a Substâncias/metabolismoRESUMO
We have previously shown that the GABA(B) agonist baclofen (BAC) prevents the expression of morphine (MOR) withdrawal syndrome in male as well as female mice. In addition, we have demonstrated that BAC reestablishes the dopamine levels modified by MOR withdrawal syndrome in male mice. The aim of the present study was to evaluate the micro-opioid receptor binding parameters in striatum and frontal cortex of male and female mice during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice of either sex were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the tenth day, dependent animals received naloxone (NAL) (6 mg/kg, i.p.) 60 min after the last dose of MOR and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL injection. Thirty min after NAL or saline injection mice were sacrificed, brains were collected, and the striatum and frontal cortex were dissected in order to perform binding studies with [(3)H][DAMGO]. The density of micro-opioid receptor increased significantly during MOR withdrawal in male and female striatum as well as in male cortex. In addition, in both brain areas the B(max) was higher in male than in female mice during MOR withdrawal. Finally, BAC pretreatment of MOR withdrawn mice reestablished the levels of micro-opioid receptor by significantly decreasing the B(max) in either sex. In conclusion, although there were sex differences in the micro-opioid receptor density during MOR withdrawal syndrome, BAC was able to reestablish the changes in binding parameters induced by the NAL-precipitated withdrawal in female and male mice.
Assuntos
Baclofeno/farmacologia , Agonistas GABAérgicos/farmacologia , Entorpecentes/farmacologia , Receptores Opioides mu/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Feminino , Masculino , Camundongos , Morfina/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Fatores Sexuais , Síndrome de Abstinência a Substâncias/tratamento farmacológicoRESUMO
The present study analyzes the effects of baclofen (BAC) on mice brain neurochemical alterations during the morphine (MOR) withdrawal syndrome. Male Swiss-Webster albino mice (27-33 g) were rendered dependent by intraperitoneal (i.p.) injection of MOR (2mg/kg), twice daily for 9 days. On day 10, the dependent animals were divided into two groups: one receiving naloxone (NAL; 6 mg/kg i.p.) to precipitate the withdrawal syndrome 60 min after the last dose of MOR and the other received BAC (2mg/kg, i.p.) followed by NAL (6 mg/kg, i.p.), injected 30 and 60 min after the last dose of MOR, respectively. Ten minutes after these treatments, mice were killed by decapitation and the striatum, cortex and hippocampus were dissected to determine endogenous concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites using HPLC with electrochemical detection. Striatal DA, dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) concentrations as well as cortical DA concentrations of the withdrawal groups decreased significantly with respect to the control groups. BAC attenuated the decrease in DA and DOPAC concentrations observed during the withdrawal, without modifying per se the control DA concentrations. No changes on 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) concentrations were observed during the MOR abstinence syndrome. The prevention caused by BAC on the decreased concentrations of DA induced by MOR withdrawal could have a therapeutic interest for the management of withdrawal syndrome.