Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Deep brain stimulation (DBS) is currently being investigated as a therapy for treatment-resistant depression, with promising results. However, it is not clear whether or not DBS works via the same mechanisms as those induced by antidepressant medications. Processes currently implicated in antidepressant effects include neuroplastic changes and promotion of neurogenesis. We investigated the effects of chronic treatment with three different classes of antidepressants and DBS on markers of neuroplasticity (brain-derived neurotrophic factor, (BDNF), and phosphorylated cyclic-AMP regulatory element binding protein, (pCREB)) and neurogenesis (Ki-67, bromodeoxyuridine (BrdU) and doublecortin) in the rat hippocampus. No clear treatment effects were seen on BDNF, pCREB and Ki-67 levels. However all treatments caused increased levels of BrdU (range: 46%-96%) and doublecortin (8%-61%), although these effects were statistically significant only for DBS and amitriptyline, respectively. This overall pattern of results may suggest that diverse antidepressant treatments could possibly share common mechanisms involving cell survival and neuronal differentiation. Potentiated effects of DBS on cell survival may underlie its efficacy in treatment-resistant depression.

Naked mole-rats are eusocial rodents that live in subterranean colonies with a strict reproductive and social hierarchy. Breeders are socially dominant and other colony members are non-reproductive subordinates. The effects of manipulating the serotonergic system on aggression are well studied in many species, but not in eusocial rodents like the naked mole-rat. For the current study, the effects of fluoxetine hydrochloride (FLX) on status-specific behaviours of subordinates (Experiment 1) and queens (Experiment 2) were evaluated both in-colony and in a social-pairing paradigm to investigate how the serotonergic system influences aggression in this species. In accordance with our main hypothesis, chronic treatment of FLX attenuated the frequency and duration of aggression in queens, but not subordinates, when paired with an unfamiliar conspecific. Further exploration of pharmacological manipulation on status-specific behaviours of this eusocial species may elucidate the neurobiological mechanisms underlying their unique and rigid social hierarchy.

Deep brain stimulation (DBS) is currently being investigated as a therapy for treatment-resistant depression, with promising results. However, it is not clear whether or not DBS works via the same mechanisms as those induced by antidepressant medications. Processes currently implicated in antidepressant effects include neuroplastic changes and promotion of neurogenesis. We investigated the effects of chronic treatment with three different classes of antidepressants and DBS on markers of neuroplasticity (brain-derived neurotrophic factor, (BDNF), and phosphorylated cyclic-AMP regulatory element binding protein, (pCREB)) and neurogenesis (Ki-67, bromodeoxyuridine (BrdU) and doublecortin) in the rat hippocampus. No clear treatment effects were seen on BDNF, pCREB and Ki-67 levels. However all treatments caused increased levels of BrdU (range: 46%-96%) and doublecortin (8%-61%), although these effects were statistically significant only for DBS and amitriptyline, respectively. This overall pattern of results may suggest that diverse antidepressant treatments could possibly share common mechanisms involving cell survival and neuronal differentiation. Potentiated effects of DBS on cell survival may underlie its efficacy in treatment-resistant depression.

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo. In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls. In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups. In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo. In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC. In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Tyrimo objektas – kraujo plazma su vaistinių medžiagų mišiniu (buspirono hidrochloridu ir fluoksetino hidrochloridu). Šio tyrimo tikslas – nustatyti tinkamiausias ekstrakcijos sąlygas, reikalingas vaistų mišinio (buspirono ir fluoksetino) išskyrimui iš kraujo plazmos bei pritaikyti efektyviosios skysčių chromatografijos metodiką vaistų mišinio veikliųjų junginių identifikavimui ir kiekio nustatymui. Darbo uždaviniai buvo pritaikyti ir validuoti efektyviosios skysčių chromatografijos metodiką; išskirti buspirono ir fluoksetino vaistų mišinį iš kraujo plazmos skysčių – skysčių (SSE) ir kietafazės ekstrakcijos (KFE) metodais; optimizuoti geriausią ekstrakcijos metodą. Tyrimo metu buvo pritaikyta ESC metodika buspirono ir fluoksetino identifikavimui ir kiekio nustatymui. Atlikta ESC metodikos validacija: įrodytas specifiškumas, rezultatų glaudumas, remiantis koreliacijos koeficientu (R2) įvertintas metodikos teisiškumas, buspirono ir fluoksetino koreliacijos koeficientai atitinkamai lygūs 0,9997 ir 0,9998. Nustatytos aptikimo ribos (buspironui 0,4 µg/ml, fluoksetinui 0,75 µg/ml) ir nustatymo ribos (buspironui 0,75 µg/ml, fluoksetinui 1 µg/ml). Vaistinių medžiagų mišinys išskirtas iš kraujo plazmos SSE ir KFE metodais. Nustatyta, kad KFE yra greitesnis ir tikslesnis metodas lyginant su SSE, todėl KFE pasirinkta tolesniam metodo optimizavimui. Eksperimentai atlikti su 6 organiniais tirpikliais (metanoliu, etanoliu, propanoliu, trichlormetanu, dichlormetanu ir acetonitrilu)... [toliau žr. visą tekstą] / The object of study – human plasma with the mix of two drugs (buspirone hydrochloride and fluoxetine hydrochloride). The aim of this study was to determine extraction conditions for buspirone and fluoxetine isolation from human plasma and to apply high-performance liquid chromatography (HPLC) method for quantitative analysis of the drugs after extraction. The objective was to apply and validate HPLC procedure; to extract buspirone and fluoxetine from human plasma by liquid-liquid extraction (LLE) and solid phase extraction (SPE); to optimize efectiveness extraction method. The HPLC method for identification and quantitative analysis of drugs was optimized. The mobile phase was 0,1 per cent of trifluoroacetic acid and acetonitrile.Validation of the HPLC method was carried out. The specificity, precision, linearity, limits of detection (buspirone 0,4 µg/ml, fluoxetine 0,75 µg/ml) and quantification (buspirone 0,75 µg/ml, fluoxetine 1 µg/ml) were determined. Validate HPLC method was applied for analysis of buspirone and fluoxetine after extraction. Drugs were extracted from human plasma by LLE and SPE methods. The best recovery of analites gave SPE methods. The recoveries of the drugs using six different organic solvents (methanol, propanol, ethanol, trichloromethane, dichloromethane, acetonitrile) were examined. Selected the most appropriate environment (acidify) for methanol and the methanol percentage of the elution solvent (80 per cent). Selected two of the most appropriate... [to full text]

Aujourd’hui, malgré la description des mécanismes à l’origine du développement de la dépression et de la MA, aucun traitement curatif n’existe pour ces pathologies suggérant l’implication d’un autre phénomène. L’un des processus retrouvé communément dans ces pathologies est la neuroinflammation. Or pour le moment, les essais cliniques entrepris dans la MA afin de réduire la neuroinflammation n’ont pas permis d’aboutir à une amélioration significative des symptômes. L’une des raisons de cet échec serait une mauvaise fenêtre thérapeutique qui aurait pour conséquence d’exacerber les effets délétères de la neuroinflammation. Ceci met en lumière la méconnaissance de la cinétique de la neuroinflammation dans la MA. Ainsi notre travail de thèse avait pour but, d’une part, d’étudier l’impact d’anti-inflammatoires comparativement à celui d’antidépresseur dans la dépression chez la souris, et d’autre part, d’étudier l’impact de l’utilisation d’antidépresseur et d’anti-inflammatoires dans un modèle murin de MA. / Today, despite the description of the mechanisms underlying the development of depression and AD (Alzheimer’s disease), no cure exists for these diseases suggesting the involvement of another phenomenon. One of the processes commonly found in these pathologies is neuroinflammation. However, clinical trials undertaken in the AD to reduce neuroinflammation have not led to a significant improvement of symptoms. One reason for this failure could be a bad therapeutic window which would result in the increase of deleterious effects of neuroinflammation. This highlights the lack of understanding of the kinetics of neuroinflammation in AD.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The presence of pharmaceuticals in the aquatic environment has long been reported for some years in various studies. However, the impact of drugs on organisms present in aquatic ecosystems is still poorly known. Thus, we investigated the effects of acute exposure to diazepam or fluoxetine on the response to acute stress in zebrafish (Danio rerio). Fish were exposed to drugs for 15 minutes and, after this period, the group designated time 0 was collected; the other groups were subjected to a stimulus of stress, which consisted in persecution of fish with a net for two minutes, and after that, were collected at the times of 15, 60 and 240 minutes. Three concentrations of diazepam 0.88 μg/L (environmental concentration) were used; 16 μg/L; and 160 μg/L, concentrations that cause behavioral effects in zebrafish. Regarding fluoxetine at concentrations of 1 μg/L, 25 μg/L and 50 μg/L (25 to 50 times the concentration of the environment, respectively) were used. Our results show that diazepam and fluoxetine inhibit the axis of stress in the zebrafish. The intermediate concentration of diazepam is able to suppress the stress response, as measured by levels of cortisol whereas the fluoxetine is able to inhibit the stress response at concentrations similar to those found in the environment. Fish with an impaired stress response loses the ability to maintain homeostasis against stress factors, since it reduces the ability to promote ionic, metabolic and behavioral adjustments necessary for such response. Thus, these data suggest that the presence of psychoactive drugs in aquatic ecosystems may cause neuroendocrine dysfunction in fish. / A presença de produtos farmacêuticos no ambiente aquático tem sido relatada há alguns anos em vários estudos. No entanto, o impacto de medicamentos sobre os organismos presentes nesses ecossistemas ainda é pouco conhecido. Assim, investigamos os efeitos da exposição aguda ao diazepam e à fluoxetina sobre a resposta ao estresse agudo em zebrafish (Danio rerio). Os peixes foram expostos aos fármacos por 15 minutos e, após esse período, o grupo denominado tempo 0 foi coletado; os demais grupos foram submetidos a um estímulo de estresse, com perseguição dos peixes com uma rede durante dois minutos, e, após, foram coletados nos tempos de 15, 60 e 240 minutos. Foram utilizadas três concentrações de diazepam a 0,88 μg/L (concentração ambiental); 16 μg/L; e 160 μg/L, concentração com efeitos comportamentais relatados em zebrafish. Em relação à fluoxetina, foram utilizadas as concentrações de 1 μg/L, 25 μg/L e 50 μg/L (25 a 50 vezes a concentração do ambiente, respectivamente). Nossos resultados demonstram que o diazepam e a fluoxetina inibem o eixo de estresse no zebrafish. A concentração intermediária de diazepam é capaz de suprimir a resposta de estresse, conforme se mediu pelos níveis de cortisol, ao passo que a fluoxetina é capaz de inibir a resposta ao estresse em concentrações semelhantes às encontradas no ambiente. Peixes com resposta de estresse prejudicada perdem a capacidade de manter a homeostase contra fatores de estresse, uma vez que reduz a capacidade de promover ajustes iônicos, metabólicos e comportamentais necessários para tal resposta. Assim, esses dados sugerem que a presença de fármacos psicoativos em ecossistemas aquáticos pode causar disfunção neuroendócrina em peixes.

Orientadora: Profa Dra Elizabete Campos de Lima / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental, 2017. / Nos efluentes, estão presentes as mais variadas misturas de fármacos, produtos de limpeza, de higiene, derivados de processos industriais, entre outros. Mesmo após o tratamento, estudos indicam que esses compostos podem continuar presentes, sendo despejados nos corpos hídricos, representando riscos para o ecossistema aquático, os quais só se tornam conhecidos após estudos científicos. Dentre essas substâncias, os fármacos são grandes representantes e a fluoxetina é um antidepressivo massivamente utilizado, capaz de promover alterações comportamentais em espécies aquáticas expostas a mesma concentração em que essa substância é encontrada em matrizes ambientais. Para controle de compostos considerados potencialmente nocivos ao ecossistema e à saúde humana, é indispensável conhecer suas concentrações em efluentes e corpos d'água. Contudo, esse tipo de matriz contém impurezas e compostos que não são de interesse, necessitando preparo de amostra para limpeza e pré-concentração do analito alvo. Nesse contexto, o objetivo deste trabalho foi desenvolver um polímero de impressão molecular para extração em fase sólida como preparo de amostra para determinação de fluoxetina em efluentes por cromatografia líquida acoplada a detector de arranjo de diodos, HPLC-DAD. Foram sintetizados polímeros de impressão molecular com ácido acrílico, ácido metacrílico e estireno como monômeros funcionais. Os rendimentos de síntese foram de 67,2% para estireno, 64,0% para ácido acrílico e 62,2% para ácido metacrílico. Foi avaliada a seletividade entre cafeína e fluoxetina, resultando em melhor afinidade do polímero com o antidepressivo. A adsorção ocorre como reação de pseudo segunda ordem, com melhor adsorção do polímero impresso do que o polímero não impresso. Ao aplicar a extração em fase sólida em efluente e analisá-lo pelo método HPLC-DAD desenvolvido e validado, o valor de recuperação do polímero molecularmente impresso (30,6%), apesar de estar abaixo do desejado, se mostrou bastante superior ao valor de recuperação do cartucho comercial C18 (2,1%). / In the sewage, there are the most varied mixtures of medicines, cleaning products, hygiene products, industrial processes derivatives, among others. Even after treatment, studies indicate that these compounds may remain in wastewater, being discharged into the water bodies, representing risks to the aquatic ecosystem, risks which only become known after scientific studies. Among these substances, the pharmaceuticals are great representatives and fluoxetine is a massively used antidepressant, capable of promoting behavioral changes in aquatic species exposed to the same concentration in which this substance is found in environmental matrices. For control of compounds considered potentially harmful to the ecosystem and to human health, it is indispensable to know their concentrations in effluents and water bodies. However, such matrix contains impurities and compounds that are not of interest, thus it is necessary some sample preparation for cleaning and preconcentration of the target analyte. In this context, the objective of this work was to develop a molecularly imprinted polymer for solid phase extraction as sample preparation for the determination of fluoxetine in effluents by liquid chromatography coupled to a diode arrangement detector, HPLC-DAD. Molecularly imprinted polymers were synthesized with acrylic acid, methacrylic acid and styrene as functional monomers. The yields of synthesis were 67.2% for styrene, 64.0% for acrylic acid and 62.2% for methacrylic acid. The selectivity between caffeine and fluoxetine was evaluated, resulting in better affinity of the polymer with the antidepressant. The adsorption occurs as a pseudo second order reaction, with better adsorption of the imprinted polymer than the non-imprinted polymer. By applying the solid phase extraction in effluent and analyzing it by the developed and validated HPLC-DAD method, the recovery value for the molecularly imprinted polymer (30.6%), although below the desired one, was shown to be much higher than the recovey value for commercial C18 cartridge (2.1%).