O metotrexato (MTX) é um antagonista do ácido fólico amplamente utilizado no tratamento de doenças neoplásicas e não neoplásicas. Entretanto, o uso terapêutico desse fármaco pode levar à neurotoxicidade que pode se manifestar na forma aguda, subaguda e crônica, abrangendo os seguintes sintomas: cefaléia, sonolência, confusão, edema cerebral, convulsões, encefalopatia, coma e prejuízo das funções cognitivas. Os achados neuropatológicos consistem de astrogliose reativa, desmielinização, dano axonal e necrose da substância branca. Em nível celular, o MTX parece afetar primeira e seletivamente os astrócitos, quando comparados com os neurônios.O exato mecanismo neurotóxico do MTX continua não esclarecido, e parece ser multifatorial. Visando ampliar os conhecimentos a respeito dos efeitos do MTX no SNC, foram desenvolvidos dois trabalhos com diferentes modelos em animais, nos quais foram estudados os possíveis mecanismos de ação tóxica desse fármaco, como também propomos a utilização do marcador bioquímico S100B na detecção de injúrias cerebrais associadas ao tratamento. A partir dos resultados obtidos nos experimentos de captação de glutamato in vitro, verificamos que o MTX interfere na remoção do glutamato da fenda sináptica, podendo levar à excitotoxicidade. Também, o aumento da proteína S100B auxilia no entendimento dos mecanismos de ação do MTX, pois sugere que os astrócitos estão respondendo a um insulto na tentativa de neuroproteção Além disso, a S100B, aliada a outros marcadores neuroquímicos e técnicas de diagnóstico por imagem, seria muito importante no monitoramento terapêutico, pois poderia detectar alterações celulares sutis e ajudaria a prevenir a neurotoxicidade pelo MTX. Os resultados que obtivemos nos experimentos de convulsões induzidas pelo MTX demonstraram a participação do sistema glutamatérgico na neurotoxicidade desse fármaco. Especificamente, evidenciamos o envolvimento dos receptores inotrópicos glutamatérgicos na patogênese das convulsões. Porém, neste modelo experimental, a captação de glutamato possivelmente diminuiu em decorrência das manifestações das convulsões e não por uma ação direta, ou indireta, do MTX. O entendimento dos mecanismos de ação é muito importante para a clínica médica, pois permite que novas ferramentas sejam criadas no intuito de prevenir os danos tóxicos induzidos por fármacos. Assim, mais estudos devem ser realizados para tentar desvendar os mecanismos de neurotoxicidade do MTX, como também para estudar potenciais marcadores bioquímicos de injúria cerebral que auxiliem no monitoramento terapêutico.

O metotrexato (MTX) é um antagonista do ácido fólico amplamente utilizado no tratamento de doenças neoplásicas e não neoplásicas. Entretanto, o uso terapêutico desse fármaco pode levar à neurotoxicidade que pode se manifestar na forma aguda, subaguda e crônica, abrangendo os seguintes sintomas: cefaléia, sonolência, confusão, edema cerebral, convulsões, encefalopatia, coma e prejuízo das funções cognitivas. Os achados neuropatológicos consistem de astrogliose reativa, desmielinização, dano axonal e necrose da substância branca. Em nível celular, o MTX parece afetar primeira e seletivamente os astrócitos, quando comparados com os neurônios.O exato mecanismo neurotóxico do MTX continua não esclarecido, e parece ser multifatorial. Visando ampliar os conhecimentos a respeito dos efeitos do MTX no SNC, foram desenvolvidos dois trabalhos com diferentes modelos em animais, nos quais foram estudados os possíveis mecanismos de ação tóxica desse fármaco, como também propomos a utilização do marcador bioquímico S100B na detecção de injúrias cerebrais associadas ao tratamento. A partir dos resultados obtidos nos experimentos de captação de glutamato in vitro, verificamos que o MTX interfere na remoção do glutamato da fenda sináptica, podendo levar à excitotoxicidade. Também, o aumento da proteína S100B auxilia no entendimento dos mecanismos de ação do MTX, pois sugere que os astrócitos estão respondendo a um insulto na tentativa de neuroproteção Além disso, a S100B, aliada a outros marcadores neuroquímicos e técnicas de diagnóstico por imagem, seria muito importante no monitoramento terapêutico, pois poderia detectar alterações celulares sutis e ajudaria a prevenir a neurotoxicidade pelo MTX. Os resultados que obtivemos nos experimentos de convulsões induzidas pelo MTX demonstraram a participação do sistema glutamatérgico na neurotoxicidade desse fármaco. Especificamente, evidenciamos o envolvimento dos receptores inotrópicos glutamatérgicos na patogênese das convulsões. Porém, neste modelo experimental, a captação de glutamato possivelmente diminuiu em decorrência das manifestações das convulsões e não por uma ação direta, ou indireta, do MTX. O entendimento dos mecanismos de ação é muito importante para a clínica médica, pois permite que novas ferramentas sejam criadas no intuito de prevenir os danos tóxicos induzidos por fármacos. Assim, mais estudos devem ser realizados para tentar desvendar os mecanismos de neurotoxicidade do MTX, como também para estudar potenciais marcadores bioquímicos de injúria cerebral que auxiliem no monitoramento terapêutico.

O metotrexato (MTX) é um antagonista do ácido fólico amplamente utilizado no tratamento de doenças neoplásicas e não neoplásicas. Entretanto, o uso terapêutico desse fármaco pode levar à neurotoxicidade que pode se manifestar na forma aguda, subaguda e crônica, abrangendo os seguintes sintomas: cefaléia, sonolência, confusão, edema cerebral, convulsões, encefalopatia, coma e prejuízo das funções cognitivas. Os achados neuropatológicos consistem de astrogliose reativa, desmielinização, dano axonal e necrose da substância branca. Em nível celular, o MTX parece afetar primeira e seletivamente os astrócitos, quando comparados com os neurônios.O exato mecanismo neurotóxico do MTX continua não esclarecido, e parece ser multifatorial. Visando ampliar os conhecimentos a respeito dos efeitos do MTX no SNC, foram desenvolvidos dois trabalhos com diferentes modelos em animais, nos quais foram estudados os possíveis mecanismos de ação tóxica desse fármaco, como também propomos a utilização do marcador bioquímico S100B na detecção de injúrias cerebrais associadas ao tratamento. A partir dos resultados obtidos nos experimentos de captação de glutamato in vitro, verificamos que o MTX interfere na remoção do glutamato da fenda sináptica, podendo levar à excitotoxicidade. Também, o aumento da proteína S100B auxilia no entendimento dos mecanismos de ação do MTX, pois sugere que os astrócitos estão respondendo a um insulto na tentativa de neuroproteção Além disso, a S100B, aliada a outros marcadores neuroquímicos e técnicas de diagnóstico por imagem, seria muito importante no monitoramento terapêutico, pois poderia detectar alterações celulares sutis e ajudaria a prevenir a neurotoxicidade pelo MTX. Os resultados que obtivemos nos experimentos de convulsões induzidas pelo MTX demonstraram a participação do sistema glutamatérgico na neurotoxicidade desse fármaco. Especificamente, evidenciamos o envolvimento dos receptores inotrópicos glutamatérgicos na patogênese das convulsões. Porém, neste modelo experimental, a captação de glutamato possivelmente diminuiu em decorrência das manifestações das convulsões e não por uma ação direta, ou indireta, do MTX. O entendimento dos mecanismos de ação é muito importante para a clínica médica, pois permite que novas ferramentas sejam criadas no intuito de prevenir os danos tóxicos induzidos por fármacos. Assim, mais estudos devem ser realizados para tentar desvendar os mecanismos de neurotoxicidade do MTX, como também para estudar potenciais marcadores bioquímicos de injúria cerebral que auxiliem no monitoramento terapêutico.

Orientador: Norma Sueli Pinheiro Módolo / Resumo: Introdução: A última atualização demográfica do Instituto Brasileiro de Geografia e Estatística realizada em 2017 mostrou que a população acima de 60 anos representava 14,6% dos 207,1 milhões de habitantes do Brasil, o que corresponde a aproximadamente 30 milhões de idosos, com expectativa de vida de 75,8 anos. Entre 2007 e 2011, o número acumulado de internações por fraturas de fêmur em pacientes acima de 60 anos, nos hospitais do Sistema Único de Saúde, foi de 175.781 O delirium pós-operatório é uma complicação comum nos pacientes idosos, e está associado a hospitalizações prolongadas, maiores taxas de institucionalização após a alta, deterioração cognitiva prolongada, diminuição da capacidade funcional, além de ser fator independente de mortalidade em 6-12 meses. Estudo disponível na literatura mostra que 14 a 24% dos pacientes idosos com fratura de quadril apresentam delirium já na admissão hospitalar, sendo que a prevalência durante a internação chega a 56%. Os mecanismos fisiopatológicos do desenvolvimento do delirium pós-operatório (DPO) e do déficit cognitivo pós-operatório têm sido estudados em nível molecular, porém ainda com pouco ganho. Maiores esforços de pesquisa são colocados na identificação de biomarcadores diagnósticos e prognósticos que estejam relacionados com mecanismos moleculares que levam ao DPO. Objetivos: Primariamente, relacionar os níveis séricos de dois biomarcadores de neuroinflamação (S100B e enolase neurônio-específica) com a ocorrência de deli... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Background: According to the latest census from IBGE (Instituto Brasileiro de Geografia e Estatística), population over 60 years-old accounted for 14,3% of a total 204,9 million habitants in Brazil. From 2007 to 2011, 175.781 elder patients were admitted after a hip fracture in brazilian public hospitals. Post-operative delirium (POD) is an usual complication in the elderly and is associated to longer hospital stay, cognitive impairment, functional decline and increased 6-12 months mortality rate. Delirium rates between the elder population can be as high as 14 to 24% already at admission, being the prevalence as high as 56% during hospital stay. Pathophysiologic mechanisms of delirium have been studied at molecular level, but research efforts are still needed in order to develop sensitive and specific early markers for this condition, so that diagnosis and effective treatment would be readily given. Objectives: This research project aims to correlate two neuroinflamation biomarkers serum levels (S100B protein and Neuron Specific Enolase - NSE) and the incidence of pre and postoperative delirium in elderly patients undergoing hip fracture repair. Also, we intent to evaluate how gender, ASA Physical Status classification and perioperative hypotension may contribute to changes in the levels of these biomarkers. Methods: An observational prospective study was proposed envolving patients aged 60 or more who were admitted at Clinics Hospital of Botucatu Medical School (Botucatu, S... (Complete abstract click electronic access below) / Doutor

The number of people suffering from neurodegenerative diseases, such as Alzheimer`s disease, increased dramatically over the past centuries and is expected to increase even further within the next years. Based on predictions of the World Health Organization and Alzheimer`s Disease International, 115 million people will suffer from dementia by the year 2050. An additionally increase in other age related neurodegenerative diseases is also forecasted. Quite naturally, neurodegenerative diseases became a focus of attention of governments and health insurances, trying to control the uprising financial burden. Early detection and treatment of neurodegenerative diseases could be an important component in containing this problem. In particular, researchers focused on automatic methods to analyze patients’ imaging data. One way to detect structural changes in magnetic resonance images (MRI) is the voxel-based method approach. It was specifically implemented for various imaging modalities, e.g. T1-weighted images or diffusion tensor imaging (DTI). Voxel-based morphometry (VBM), a method specifically designed to analyze T1-weighted images, has become very popular over the last decade. Investigations using VBM revealed numerous structural brain changes related to, e.g. neurodegeneration, learning induced structural changes or aging. Although voxel-based methods are designed to be robust and reliable structural change detection methods, it is known that they can be influenced by physical and physiological factors. Dehydration, for example, can affect the volume of brain structures and possibly induce a confound in morphometric studies. Therefore, three-dimensional T1-weighted images were acquired of six young and healthy subjects during different states of hydration. Measurements during normal hydration, hyperhydration, and dehydration made it possible to assess consequential volume changes in gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). The datasets were analyzed using VBM, FreeSurfer and SIENA. A significant decrease of GM and WM volume, associated with dehydration, was found in various brain regions. The most prominent effects were found in temporal and parietal areas, in the left inferior orbito-frontal region, and in the extra-nuclear region. Moreover, we found consistent increases in CSF, an expansion around 6% of the ventricular system affecting both lateral ventricles, i.e. the third and fourth ventricle. Similar degrees of shrinkage in WM volume and increase of the ventricular system have been reported in studies of Alzheimer’s disease during disease progression and in its prestage mild cognitive impairment. Based on these findings, a potential confound in GM and WM or CSF studies due to the subjects’ hydration state cannot be excluded and should be appropriately addressed. These results underline the sensitivity of VBM and might also concern other voxel-based methods, such as Tract-Based Spatial Statistics (TBSS). TBSS was specifically designed for WM analyses and its sensitivity might be helpful for revealing the spatial relation of structural WM changes and related blood serum biomarkers. Two common brain related biomarkers are the glial protein S100B, a plasticity inducing neuro- and gliotrophin, and neuron-specific enolase (NSE), a marker for neuronal damage. However, the spatial specificity of these biomarkers for brain region has not been investigated in vivo until now. Therefore, we acquired two MRI parameters – T1- weighted and DTI - sensitive to changes in GM and WM, and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, the gene expression of S100B on the whole brain level in a male cohort of three subjects from the Allen Brain Database was analyzed. Furthermore, a female post mortal brain was investigated using double immunofluorescence labeling with oligodendrocyte markers. It could be shown that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity – the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes. The data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. This was the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. The results open a new perspective for future studies investigating major neuropsychiatric disorders. All above mentioned studies are mainly dependent on the sensitivity and accuracy of soft and hardware parameters. In particular, technical developments have improved acquisition accuracy in the field of MRI. Interestingly, very little is known about the confounding effects of variations due to hardware parameters and their possible impact on reliability and sensitivity of VBM. Recent studies have shown that different acquisition parameters may influence VBM results. Therefore age-related GM changes were investigated with VBM in 36 healthy volunteers grouped into 12 young, 12 middle-aged and 12 elderly subject. Six T1-weighted datasets were acquired per subject with a 12-channel matrix coil, as well as a 32-channel array, MP-RAGE and MP2RAGE, and with isotropic resolutions of 0.8 and 1 mm. DARTEL-VBM was applied on all images and GM, WM and CSF segments were statistically analyzed.. Paired t-tests and statistical interaction tests revealed significant effects of acquisition parameters on the estimated gray-matter-density (GMD) in various cortical and subcortical brain regions. MP2RAGE seemed slightly less prone to false positive results when comparing data acquired with different RF coils and yielded superior segmentation of deep GM structures. With the 12-channel coil, MP-RAGE was superior in detecting age-related changes, especially in cortical structures. Most differences between both sequences became insignificant with the 32-channel coil, indicating that the MP2RAGE images benefited more from the improved signal-to-noise ratio and improved parallel-imaging reconstruction). A possible explanation might be an overestimation of the GM compartment on the MP-RAGE images. In view of substantial effects obtained for all parameters, careful standardization of the acquisition protocol is advocated. While the current investigation focused on aging effects, similar results are expected for other VBM studies, like on plasticity or neurodegenerative diseases. This work has shown that voxel-based methods are sensitive to subtle structural brain changes, independent of imaging modality and scanning parameters. In particular, the studies investigated and discussed the analysis of T1- and diffusion weighted images with VBM and TBSS in the context of dehydration, blood serum sensitive biomarkers and aging were discussed. The major goal of these studies was the investigation of the sensitivity of voxel-based methods. In conclusion, sensitivity and accuracy of voxelbased methods is already high, but it can be increased significantly, using optimal hardand software parameters. It is of note, though, that these optimizations and the concomitant increase of detection sensitivity could also introduce additional confounding factors in the imaging data and interfere with the latter preprocessing and statistical computations. To avoid an interference e.g. originating from physiological parameters, a very careful selection and monitoring of biological parameters of each volunteer throughout the whole study is recommended. A potential impact of scanning parameters can be minimized by strict adherence to the imaging protocol for each study subjectwithin a study. A general increase in detection sensitivity due to optimized parameters selection in hard- and/or can not be concluded by the above mentioned studies. Although the present work addressed some of those issues, the topic of optimal selection of parameters for morphometric studies is still very complex and controversial and has to be individually decided. Further investigations are needed to define more general scanning and preprocessing standards to increase detection sensitivity without the concomitant amplification of confounding factors.

Fragestellung: Gegenstand dieser Untersuchung ist der diagnostische Nutzen der Neuro-Biomarker S100B-Protein und Neuronenspezifische Enolase (NSE) bei leichten Schädel-Hirn-Verletzungen im Kindesalter. Es wird untersucht, ob anhand der posttraumatischen S100B- und NSE-Serum-Konzentrationen Kinder mit einer Schädelprellung und einem leichten Schädel-Hirn-Trauma (SHT) differenziert werden können. Material und Methode: In einer prospektiven, klinischen Studie werden die posttraumatischen S100B- und NSE-Serum-Konzentrationen von Kindern im Alter zwischen 6 Monaten und 15 Jahren mit einer Schädelprellung oder einem leichten SHT untersucht. Kinder mit extrakraniellen Begleitverletzungen und Begleiterkrankungen sind ausgeschlossen. Die Blutentnahme erfolgt innerhalb von 6 Stunden nach dem Trauma. Es werden 2 diagnostische Gruppen definiert: Gruppe 1: asymptomatische Schädelprellungen (Glasgow-Coma-Scale [GCS] 15 Punkte), Gruppe 2: leichte SHT (GCS 13-15 Punkte) mit klinischen Zeichen einer Gehirnerschütterung (kurze Bewusstlosigkeit, Amnesie, Übelkeit, Erbrechen, Somnolenz, Kopfschmerzen, Schwindel, Sehstörungen, Kreislaufreaktion). Die S100B- und NSE- Konzentrationen werden zwischen beiden Diagnosegruppen verglichen. Die Korrelation zwischen S100B und NSE sowie zwischen den Markern und dem Alter der Kinder, dem Zeitraum zwischen Trauma und Blutentnahme, dem GCS-Wert und dem Vorhandensein von Kopfplatzwunden wird analysiert. Ergebnisse: 148 Kinder sind in die Studie eingeschlossen (53 Kinder mit einer Schädelprellung und 95 mit einem leichten SHT). Nach Adjustierung der gemessenen Marker-Konzentrationen auf Unterschiede im Alter und Zeitraum zwischen Trauma und Blutentnahme unterscheiden sich die S100B- und NSE-Konzentrationen nicht signifikant zwischen Kindern mit einer Schädelprellung und einem leichten SHT. Zwischen den S100B- und NSE-Konzentrationen besteht eine signifikant positive Korrelation. Beide Marker korrelieren signifikant negativ mit dem Alter und dem Entnahmezeitraum. Der GCS-Wert und das Vorhandensein von Kopfplatzwunden zeigen keinen Effekt auf die Marker-Konzentrationen. Schlussfolgerung: Die posttraumatischen S100B- und NSE-Serum-Konzentrationen zeigen keinen diagnostischen Nutzen bei der Differenzierung zwischen Kindern mit einer Schädelprellung und Kindern mit einem leichten SHT. S100B und NSE sind altersabhängige Marker.

The number of people suffering from neurodegenerative diseases, such as Alzheimer`s disease, increased dramatically over the past centuries and is expected to increase even further within the next years. Based on predictions of the World Health Organization and Alzheimer`s Disease International, 115 million people will suffer from dementia by the year 2050. An additionally increase in other age related neurodegenerative diseases is also forecasted. Quite naturally, neurodegenerative diseases became a focus of attention of governments and health insurances, trying to control the uprising financial burden. Early detection and treatment of neurodegenerative diseases could be an important component in containing this problem. In particular, researchers focused on automatic methods to analyze patients’ imaging data. One way to detect structural changes in magnetic resonance images (MRI) is the voxel-based method approach. It was specifically implemented for various imaging modalities, e.g. T1-weighted images or diffusion tensor imaging (DTI). Voxel-based morphometry (VBM), a method specifically designed to analyze T1-weighted images, has become very popular over the last decade. Investigations using VBM revealed numerous structural brain changes related to, e.g. neurodegeneration, learning induced structural changes or aging. Although voxel-based methods are designed to be robust and reliable structural change detection methods, it is known that they can be influenced by physical and physiological factors. Dehydration, for example, can affect the volume of brain structures and possibly induce a confound in morphometric studies. Therefore, three-dimensional T1-weighted images were acquired of six young and healthy subjects during different states of hydration. Measurements during normal hydration, hyperhydration, and dehydration made it possible to assess consequential volume changes in gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). The datasets were analyzed using VBM, FreeSurfer and SIENA. A significant decrease of GM and WM volume, associated with dehydration, was found in various brain regions. The most prominent effects were found in temporal and parietal areas, in the left inferior orbito-frontal region, and in the extra-nuclear region. Moreover, we found consistent increases in CSF, an expansion around 6% of the ventricular system affecting both lateral ventricles, i.e. the third and fourth ventricle. Similar degrees of shrinkage in WM volume and increase of the ventricular system have been reported in studies of Alzheimer’s disease during disease progression and in its prestage mild cognitive impairment. Based on these findings, a potential confound in GM and WM or CSF studies due to the subjects’ hydration state cannot be excluded and should be appropriately addressed. These results underline the sensitivity of VBM and might also concern other voxel-based methods, such as Tract-Based Spatial Statistics (TBSS). TBSS was specifically designed for WM analyses and its sensitivity might be helpful for revealing the spatial relation of structural WM changes and related blood serum biomarkers. Two common brain related biomarkers are the glial protein S100B, a plasticity inducing neuro- and gliotrophin, and neuron-specific enolase (NSE), a marker for neuronal damage. However, the spatial specificity of these biomarkers for brain region has not been investigated in vivo until now. Therefore, we acquired two MRI parameters – T1- weighted and DTI - sensitive to changes in GM and WM, and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, the gene expression of S100B on the whole brain level in a male cohort of three subjects from the Allen Brain Database was analyzed. Furthermore, a female post mortal brain was investigated using double immunofluorescence labeling with oligodendrocyte markers. It could be shown that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity – the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes. The data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. This was the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. The results open a new perspective for future studies investigating major neuropsychiatric disorders. All above mentioned studies are mainly dependent on the sensitivity and accuracy of soft and hardware parameters. In particular, technical developments have improved acquisition accuracy in the field of MRI. Interestingly, very little is known about the confounding effects of variations due to hardware parameters and their possible impact on reliability and sensitivity of VBM. Recent studies have shown that different acquisition parameters may influence VBM results. Therefore age-related GM changes were investigated with VBM in 36 healthy volunteers grouped into 12 young, 12 middle-aged and 12 elderly subject. Six T1-weighted datasets were acquired per subject with a 12-channel matrix coil, as well as a 32-channel array, MP-RAGE and MP2RAGE, and with isotropic resolutions of 0.8 and 1 mm. DARTEL-VBM was applied on all images and GM, WM and CSF segments were statistically analyzed.. Paired t-tests and statistical interaction tests revealed significant effects of acquisition parameters on the estimated gray-matter-density (GMD) in various cortical and subcortical brain regions. MP2RAGE seemed slightly less prone to false positive results when comparing data acquired with different RF coils and yielded superior segmentation of deep GM structures. With the 12-channel coil, MP-RAGE was superior in detecting age-related changes, especially in cortical structures. Most differences between both sequences became insignificant with the 32-channel coil, indicating that the MP2RAGE images benefited more from the improved signal-to-noise ratio and improved parallel-imaging reconstruction). A possible explanation might be an overestimation of the GM compartment on the MP-RAGE images. In view of substantial effects obtained for all parameters, careful standardization of the acquisition protocol is advocated. While the current investigation focused on aging effects, similar results are expected for other VBM studies, like on plasticity or neurodegenerative diseases. This work has shown that voxel-based methods are sensitive to subtle structural brain changes, independent of imaging modality and scanning parameters. In particular, the studies investigated and discussed the analysis of T1- and diffusion weighted images with VBM and TBSS in the context of dehydration, blood serum sensitive biomarkers and aging were discussed. The major goal of these studies was the investigation of the sensitivity of voxel-based methods. In conclusion, sensitivity and accuracy of voxelbased methods is already high, but it can be increased significantly, using optimal hardand software parameters. It is of note, though, that these optimizations and the concomitant increase of detection sensitivity could also introduce additional confounding factors in the imaging data and interfere with the latter preprocessing and statistical computations. To avoid an interference e.g. originating from physiological parameters, a very careful selection and monitoring of biological parameters of each volunteer throughout the whole study is recommended. A potential impact of scanning parameters can be minimized by strict adherence to the imaging protocol for each study subjectwithin a study. A general increase in detection sensitivity due to optimized parameters selection in hard- and/or can not be concluded by the above mentioned studies. Although the present work addressed some of those issues, the topic of optimal selection of parameters for morphometric studies is still very complex and controversial and has to be individually decided. Further investigations are needed to define more general scanning and preprocessing standards to increase detection sensitivity without the concomitant amplification of confounding factors.

Este trabalho apresenta a continuidade da avaliação farmacológica do derivado Nfenilpiperazínico LASSBio-579 em busca de um novo candidato a antipsicótico de segunda geração. Em estudos anteriores, demonstramos que LASSBio-579 base livre é um potencial candidato a antipsicótico atípico capaz de modular três diferentes sistemas neurotransmissores envolvidos na patofisiologia da esquizofrenia: a neurotransmissão dopaminérgica, serotonérgica e glutamatérgica; entretanto, LASSBio-579 na forma de cloridrato apresenta baixa biodisponibilidade. Neste trabalho avaliamos inicialmente a ação de LASSBio-579.HCl. -ciclodextrina, proposto como alternativa para melhorar a biodisponibilidade. Porém, quando avaliado no modelo de escalada induzida por apomorfina, preditivo de atividade antipsicótica, essa preparação não foi efetiva. Assim, seguimos a avaliação farmacodinâmica com LASSBio-579 base livre, utilizando modelos preditivos de atividade antipsicótica, em camundongos. Neste trabalho foram realizados ainda ensaios in vitro, onde se avaliou a ação de LASSBio-579 sobre a viabilidade celular, captação de glutamato e secreção de proteína S100B, utilizando-se para isso fatias hipocampais de ratos tratadas de forma aguda com LASSBio-579 nas concentrações de 0,1; 1,0; 10 e 20μM. / This study presents the continuity of the pharmacological evaluation of the Nphenilpiperazine derivative LASSBio-579, searching a new second generation antipsychotic compound. In previous studies we have demonstrated that LASSBio- 579 in form of base is a potential atypical antipsychotic able to modulate three different neurotransmitter systems involved in the pathophysiology of schizophrenia: dopaminergic, glutamatergic and serotonergic. However, LASSBio-579 hydrochloride has low bioavailability. In this study we evaluated LASSBio-579.HCl. -cyclodextrin, prepared with the aim of increasing oral bioavailability, in the apomorphine induced climbing in mice, which is a model predictive of antipsychotic activity; and it was not effective. Thus, we continue the study with LASSBio-579 in form of base by testing it in others mice models predictive of antipsychotic activity. In this study, also were made in vitro studies performed in hippocampal acute slices which demonstrated that LASSBio-579 induced a glutamate uptake inhibition and also inhibited the S100B protein secretion.

Dor neuropática (DN) é definida como “dor iniciada ou causada por lesão primária ou disfunção em sistema nervoso”, porém sua prevalência depende do tipo de trauma e da disfunção relacionada. Apesar desta condição dolorosa ser considerada altamente prevalente e debilitante, os tratamentos disponíveis são relacionados a efeitos adversos dificultando a adesão. Devido a isso, buscam-se alternativas não farmacológicas para o tratamento deste tipo de dor, entre elas, as técnicas de neuromodulação periférica, como acupuntura (AC) e eletroacupuntura (EA). Estas técnicas podem ser combinadas com intervenções farmacológicas e não farmacológicas e têm apresentado resultados promissores no tratamento da dor neuropática. No entanto, seus mecanismos de ação não estão totalmente elucidados, desta forma a utilização de modelos animais é de grande valia para o estudo destes mecanismos no tratamento da dor neuropática e da patofisiologia deste tipo de dor crônica. É importante salientar que a aplicação de AC e EA em animais acordados é complexa, visto que gera desconforto e pode alterar a analgesia induzida pelo tratamento. Em muitos estudos a anestesia com isoflurano é utilizada durante a aplicação dos tratamentos, porém sua utilização pode gerar um viés no estudo, considerando a possível interferência do fármaco nos resultados comportamentais e neuroquímicos. Outro importante foco de estudo consiste em comparar as duas técnicas, AC e EA, buscando determinar qual destas é a mais eficaz no tratamento da dor neuropática. Considerando o exposto acima, os objetivos desta tese foram: 1) avaliar os parâmetros comportamentais e neuroquímicos dos efeitos da utilização de anestesia na aplicação de AC e EA em ratos submetidos ao modelo de DN; 2) comparar os efeitos da AC e EA em modelo animal de DN por meio de parâmentros comportamentais, neuroquímicos e histológicos. Considerando os resultados obtidos nesta tese, concluímos que o isoflurano aumenta a analgesia promovida por AC e EA, provavelmente diminuindo o efeito do estresse gerado pela aplicação dos tratamentos em animais acordados, resultado que é corroborado pela diminuição do nível de S100β periférico (marcador de morte neuronal central); Por outro lado, o isoflurano diminuiu os níveis de fator de crescimento neuronal (NGF) no nervo periférico lesado, indicando diminuição do processo de regeneração neural, enquanto a EA aumentou. Ao mesmo tempo, o isoflurano alterou os efeitos dos tratamentos nos comportamentos exploratórios e nos níveis de N-metil D-aspartato em tronco encefáfio e medula espinhal. A AC apresentou-se mais eficaz no tratamento da DN em comparação à EA, porém nenhum dos tratamentos foi capaz de alterar os danos causados pela indução da DN no músculo gastrocnemio esquerdo dos animais demonstrado na histologia. Todavia, este resultado não alterou a analgesia gerada pelos tratamentos. / Neuropathic pain (NP) is defined as "pain initiated or caused by primary injury or dysfunction in the nervous system," but its prevalence depends on the type of trauma and related dysfunction. Although this painful condition is considered to be highly prevalent and debilitating, the available treatments are related to adverse effects, making adherence difficult. Because of this, non-pharmacological alternatives for the treatment of this type of pain are sought, among them, the techniques of peripheral neuromodulation, such as acupuncture (AC) and electroacupuncture (EA). These techniques can be combined with pharmacological and non-pharmacological interventions and have shown promising results in the treatment of neuropathic pain. However, its mechanisms of action are not fully elucidated, so the use of animal models is of great value for the study of these mechanisms in the treatment of neuropathic pain and the pathophysiology of this type of chronic pain. It is important to emphasize that the application of AC and EA in awake animals is complex, since it generates discomfort and can alter the analgesia induced by the treatment. In many studies, anesthesia with isoflurane is used during the application of the treatments, but its use may generate a bias in the study, considering the possible interference of the drug in the behavioral and neurochemical results. Another important focus of the study is to compare the two techniques, AC and EA, seeking to determine which is the most effective in the treatment of neuropathic pain. Considering the above, the objectives of this thesis were: 1) to evaluate the behavioral and neurochemical parameters of the effects of the use of anesthesia in the application of AC and EA in rats submitted to the DN model; 2) to compare the effects of AC and EA on animal model of DN by means of behavioral, neurochemical and morphological parameters. Considering the results obtained in this thesis, we conclude that isoflurane increases the analgesia promoted by AC and EA, probably decreasing the effect of the stress generated by the application of the treatments in agreed animals, a result that is corroborated by the decrease in the level of peripheral S100β (biomarker of central neuronal injury); On the other hand, isoflurane decreased the levels of neural grown factor (NGF) in the injured peripheral nerve, indicating a decrease in the neural regeneration process, while the EA increased. At the same time, isoflurane altered the effects of treatments on exploratory behaviors and N-metil-D-aspartato (NMDA) levels in the brainstem and spinal cord. AC was more effective in the treatment of DN compared to EA, but none of the treatments was able to alter the damage caused by DN induction in the left gastrocnemius muscle of the animals showed in histology. However, this result did not alter the analgesia generated by the treatments.

A epilepsia do lobo temporal (ELT) é a um dos casos mais frequente epilepsia em humanos e de maior refratariedade nos pacientes. A maioria dos fármacos antiepilépticos são moduladores da atividade neuronal e atuam sobre canais iônicos do receptor GABAA. Estudos vêm demonstrando o papel das células gliais e da neuroinflamação na epileptogênese e a modulação desta resposta pode ser um alvo potencial para drogas adjuvantes aos fármacos anti-epilépticos. Astrócitos são células gliais participantes da sinapse tripartite, moduladores da atividade neuronal. Os astrócitos são capazes de promover a homeostase de íons e de neurotransmissores, são responsáveis pelo metabolismo energético e da produção de fatores neurotróficos, glutationa, glutamina, S100B e citocinas. Neste trabalho, induzimos status epilepticus (SE) em ratos jovens (PN28) através do modelo lítio-pilocarpina que mimetiza alterações neuronais, bioquímicas e morfológicas similares à ELT em humanos. Os animais foram divididos nos tempos 1, 14 e 56 dias após a indução de status epilepticus (SE). Estes períodos são caracterizados respectivamente como a fase aguda, latente e crônica da epilepsia. Inicialmente, analisamos as mudanças neuroquímicas e astrocitárias ao longo do tempo. Foi observada neuroinflamação inicial e transitória que promove morte neuronal e mudanças ao longo do tempo de astrogliose e disfunção astrocitária. Também foi observado que a proteína S100B, proteína ligante de cálcio, predominantemente astrocitária, pode ser considerado um marcador da disfunção neuronal e astrocitária promovida neste modelo de epilepsia. Em seguida, demonstramos que a modulação da secreção de S100B pelo anti-inflamatório dexametasona um dia após indução de SE reverte a neuroinflamação, astrogliose e disfunção astrocitária à curto e à longo prazo. Por conseguinte, observamos que a modulação do receptor GABAA através de agonistas e antagonistas GABAérgicos altera a secreção de S100B em fatias hipocampais agudas e em cultura de astrócitos. Portanto, pode-se sugerir que as alterações astrogliais e a neuroinflamação dependentes do tempo podem estar ligadas à excitabilidade neuronal e/ou à morte neuronal em ratos jovens em modelo de epilepsia; que a proteína S100B pode ser considerada um marcador deste modelo de epilepsia e que a modulação da sua secreção pode ser um possível alvo farmacológico no tratamento da epilepsia. / Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy in humans and is more associated to refractory to anti-epileptic drugs (AED) in patients. The most AEDs are modulators of neuronal activity and act on ion channels, such as GABAA receptor. Studies have been demonstrating the role of glial cells and neuroinflammation in epileptogenesis. The modulation of this response may be a potential target for adjunctive drugs to anti-epileptic drugs. Astrocytes are glial cells that participated in the tripartite synapse and modulated neuronal activity. Astrocytes are able to promote homeostasis of ions and neurotransmitters, are responsible for energy metabolism and the production of neurotrophic factors, glutathione, glutamine, S100B and cytokines. In this work, we induced status epilepticus (SE) in young rats (PN28) through the lithiumpilocarpine model that mimics neuronal, biochemical and morphological alterations similar to ELT in humans. The animals were divided at times 1, 14 and 56 days after the induction of SE. These periods are characterized respectively as the acute, latent and chronic phase of epilepsy. Initially, we analyzed neurochemical and astrocytic changes over time. Initial and transient neuroinflammation was observed and promoted over time neuronal death, astrogliosis and astrocytic dysfunction. It has also been observed that the protein S100B, a calcium-binding protein, predominantly astrocytic, can be considered a marker of neuronal and astrocytic dysfunction promoted by this model of epilepsy. Next, we demonstrate that the modulation of S100B secretion by the antiinflammatory dexamethasone one day after SE induction reverses neuroinflammation, astrogliosis and astrocytic dysfunction in the acute and chronic time. Therefore, we analyzed that modulation of the GABAA receptor through GABAergics agonists and antagonists alters the secretion of S100B in acute hippocampal slices and in astrocyte culture. Therefore, it may be suggested that astroglial changes and time dependent neuroinflammation may be related to neuronal excitability and/or neuronal death in young rats in this epilepsy model; that S100B protein can be considered a marker of this epilepsy model and that the modulation of its secretion may be a possible pharmacological target in the treatment of epilepsy.