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Bioanalytical Tools for Unraveling Part of the Puzzle of the Biochemical Phenomena in Epilepsy: Neuromediators and Antiepileptic Drugs

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Bioanalytical Tools for Unraveling Part of the Puzzle of the Biochemical Phenomena in Epilepsy: Neuromediators and Antiepileptic Drugs UNIVERSIDADE DA BEIRA INTERIOR Ciências Bioanalytical tools for unraveling part of the puzzle of the biochemical phenomena in epilepsy: neuromediators and antiepileptic drugs Beatriz Marques da Fonseca Tese para obtenção do Grau de Doutor em Bioquímica (3º ciclo de estudos) Orientador: Professor Doutor Gilberto Lourenço Alves Covilhã, fevereiro de 2019 i ii The experimental work presented in this thesis was carried out at the Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior (CICS-UBI), under the scientific supervision of Professor Gilberto Lourenço Alves. iii iv The work underlying the present thesis was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (SFRH/BD/86496/2012) through the POPH (Programa Operacional Potencial Humano) which is co-funded by FSE (Fundo Social Europeu), European Union. This work was also supported by FEDER funds through the POCI - COMPETE 2020 - Operational Programme Competitiveness and Internationalisation in Axis I - Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER-007491) and National Funds by FCT (Project UID/Multi/00709/2013). v vi A vocês, Família A ti, Ivo vii viii Agradecimentos Não há palavras suficientes para expressar gratidão a todas as pessoas que viajaram comigo nesta aventura. Agradeço ao meu orientador, o professor Doutor Gilberto Alves, pela possibilidade que me deu para prosseguir este desafio, apenas assim se garante a qualidade que um trabalho deste tipo requer. Também pela orientação científica, disponibilidade e por todo o conhecimento transmitido. A sua contribuição foi essencial para a realização e conclusão desta tese a todos os níveis, estando imensamente agradecida. Também quero agradecer a todos os meus coautores, em particular ao Márcio Rodrigues, pelas valiosas contribuições para o meu trabalho, e por toda a ajuda e conselhos. Agradeço a todos os meus amigos e colegas que acompanharam o meu conhecimento académico, pela força e amizade. Dirijo os meus mais profundos agradecimentos à minha família, os meus pais e irmão, pelo seu amor e apoio incondicionais para enfrentar este desafio emocionante. Um agradecimento muito especial a uma pessoa especial, meu pilar Ivo Moreno, pelo seu amor e por tudo. Além disso, agradeço a toda a família Moreno. Agradeço à Fundação para a Ciência e a Tecnologia pela bolsa de doutoramento e apoio financeiro. Finalmente, gostaria de agradecer ao Centro de Investigação em Ciências da Saúde da Universidade da Beira Interior (CICS-UBI). ix x TABLE OF CONTENTS RESUMO ALARGADO ........................................................................................ xv ABSTRACT ................................................................................................... xvii LIST OF FIGURES ............................................................................................ xix LIST OF TABLES ............................................................................................. xxi LIST OF ABBREVIATIONS................................................................................. xxiii LIST OF PUBLICATIONS ................................................................................. xxvii LIST OF COMMUNICATIONS ............................................................................. xxix CHAPTER I – General introduction ........................................................................ 1 I.1. Epilepsy .................................................................................................... 1 I.1.1. Historical perspective ............................................................................ 1 I.1.2. Epidemiology ....................................................................................... 1 I.1.3. Definitions and classification of epileptic seizures and epilepsy ....................... 2 I.1.4. Pathophysiology of epilepsy: A brief overview ............................................. 5 I.1.5. Signaling pathways: Focus on neurotransmitters ........................................ 11 I.1.5.1. Neuroactive amino acids ................................................................. 13 I.1.5.2. Catecholamines ............................................................................ 17 I.1.6. Epilepsy treatment .............................................................................. 19 I.1.6.1. An overview of the historical development of antiepileptic drugs ............ 20 I.1.6.2. Basic mechanisms of action of antiepileptic drugs................................. 22 I.1.6.3. Negative consequences of antiepileptic drug therapy ............................ 26 I.1.6.4. Drug resistance ............................................................................. 27 I.1.6.5. Antiepileptogenic therapies: Where are we now? ................................. 29 I.1.6.6. Non-pharmacologic treatment strategies ............................................ 30 I.1.7. Experimental models of epilepsy and seizures ........................................... 32 I.1.7.1. Acute seizure models ..................................................................... 33 I.1.7.2. Chronic epilepsy models ................................................................. 35 I.1.7.2.1. Kindling models ...................................................................... 35 I.1.7.2.2. Models based on status epilepticus .............................................. 36 I.1.7.2.2.1. Kainic acid models ............................................................ 36 I.1.7.2.2.2. Pilocarpine models ............................................................ 37 I.1.7.3. Genetic models of epilepsy .............................................................. 38 I.2. Bioanalysis .............................................................................................. 39 I.2.1. An overview....................................................................................... 39 I.2.2. Chromatographic analysis of neurotransmitters in brain ............................... 41 I.2.2.1. Sample preparation: Brain tissue ...................................................... 45 I.2.2.2. Chromatographic assays .................................................................. 48 I.2.2.3. Challenges in the quantification of endogenous compounds .................... 53 I.2.3. The role of bioanalysis in the context of epilepsy ....................................... 54 I.3. Global aims of this thesis ............................................................................ 55 xi CHAPTER II - Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography .............. 57 II.1. Introduction ............................................................................................ 57 II.2. Material and methods ................................................................................ 60 II.2.1. Chemicals and reagents ....................................................................... 60 II.2.2. Stock solutions, calibration standards and quality control samples ................. 60 II.2.3. Rat brain tissue .................................................................................. 61 II.2.4. Apparatus and chromatographic conditions .............................................. 61 II.2.5. Sample preparation ............................................................................ 62 II.2.6. Method validation ............................................................................... 62 II.2.7. Method application ............................................................................. 63 II.3. Results and discussion ............................................................................... 64 II.3.1. Method development .......................................................................... 64 II.3.2. Method validation ............................................................................... 65 II.3.3. Method application ............................................................................. 70 CHAPTER III - An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain .................................................................................................. 75 III.1. Introduction ........................................................................................... 75 III.2. Material and methods ............................................................................... 78 III.2.1. Standards and chemicals ..................................................................... 78 III.2.2. Rat brain tissue ................................................................................. 79 III.2.3. Stock solutions, calibration standards and quality control samples ................ 79 III.2.4. Sample preparation and derivatization ................................................... 80 III.2.5. Instrumentation and chromatographic conditions ...................................... 80 III.2.6. Method validation .............................................................................. 80 III.2.7. Method application ............................................................................ 81 III.3. Results and discussion .............................................................................. 82 III.3.1. Method development .........................................................................
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