Author(s):
Alves, Raquel Maria Azeitão, 1986- 
Date: 2011
Persistent ID: http://hdl.handle.net/10451/6395
Origin: Repositório da Universidade de Lisboa
Subject(s): Monóxido de carbono; Apoptose; Stress oxidativo; Cérebro; Teses de mestrado - 2011
Description
Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011 A hipoxia isquemia e reperfusao leva a lesoes cerebrais graves que rapidamente culminam na morte das celulas neuronais. O monoxido de carbono tem vindo a ser estudado como molecula neuroprotectora e, em pequenas concentracoes, confere proteccao contra a apoptose em diversos tipos de celulas e tecidos. Neste trabalho, propusemo-nos evidenciar a capacidade modeladora do CO, explorando a comunicacao celular e o trafego metabolico entre neuronios e astrocitos apos o pre-condicionamento de astrocitos com CO. O estudo baseou-se num sistema de coculturas (astrocitos-neuronios) no qual foi inicialmente observado que o precondicionamento de astrocitos e posterior co-cultura leva a um aumento significativo na viabilidade neuronal apos um insulto por stress oxidativo (uma consequencia da HIR), pela adicao de tert-butylhydroperoxide (t-BHP) 0 a 60 μM. Assim, foram investigadas duas moleculas libertadas pelos astrocitos apos o insulto: o ATP e o seu produto de degradacao, a adenosina, que sao conhecidas como neuroprotectoras e que estao envolvidas na comunicacao celula-celula. Em monoculturas de neuronios a adicao de adenosina e/ou ATP previne a morte celular induzida por stress oxidativo, indicando que ambas as moleculas estao envolvidas no mecanismo de proteccao neuronal. Nos astrocitos, quando se bloqueia a saida de ATP, pela inibicao da Cx43 atraves do antagonista AGA, houve um aumento na morte neuronal. Tambem a inibicao do enzima ectonucleotidase (que permite a degradacao do ATP em adenosina) pelo antagonista ARL67156 apresentou tal efeito. Nos neuronios o uso de antagonistas de receptores purinergicos P1 como e o exemplo do SCH59261 e de receptores P2, PPADS e Suramina, revelaram um defice na proteccao neuronal por reversao do efeito protector dos astrocitos pre-tratados com CO. Juntas, ambas as moleculas parecem estar envolvidas no mecanismo de comunicacao (neuronios-astrocitos) induzido pelo CO e que leva a proteccao neuronal. Contudo, o efeito observado pela inibicao dos receptores de adenosina e maior em relacao aos receptores de ATP. A adenosina parece assim ter maior importancia na comunicacao neuronio-astrocito e na proteccao neuronal pois: (i) a inibicao quimica do seu receptor A2A, (ii) a inibicao da degradacao do ATP pela ectonucleotidase nos astrocitos, (iii) a diminuicao de adenosina no meio extracelular (como produto da degradacao do ATP pela inibicao da Cx43 nos astrocitos), comprometem a sobrevivencia neuronal. Assim, a adenosina libertada pela adicao do CO, e considerada um elemento chave no mecanismo de proteccao, sendo uma grande aposta no aperfeicoamento e desenvolvimento de novas estrategias no estudo do metabolismo e comunicacao celular. Hypoxia ischemia and reperfusion leads to severe brain injuries and can rapidly culminate in neuronal cells death. Carbon monoxide has been studied as a neuroprotective molecule and, when applied at low concentrations, protects against apoptosis in several cell types and tissues. Herein we propose to demonstrate that CO modulates neuronal cells death, by exploring cell to cell communication and metabolic traffic occurring between neurons and astrocytes after astrocytic CO-preconditioning. The study was based on a co-culture system (astrocytes - neurons). Astrocytes were pre-treated with CO, followed by co-cultures establishment and challenging neurons to death by oxidative stress (a HIR consequence) by addition of tert-butylhydroperoxide (t-BHP) at 0 to 60 μM. We targeted two molecules which are released by astrocytes after CO treatment: ATP and its degradation product , adenosine, which are known to be neuroprotective, and to be involved in cell to cell communication. ATP and adenosine prevented oxidative stress induced-apoptosis in monoculture of neurons, indicating that both molecules are involved in neuronal protection. In astrocytes, inhibition of ATP release by addition of Cx43´s antagonist AGA, led to an increase in neuronal death. Still, ectonucleotidase inhibition (which is involved in ATP degradation to adenosine) by addition of its antagonist ARL67156 presented the same effect. In neurons, the use of P1 purinergic receptor antagonist such as SCH59261, and P2 receptor antagonist namely PPADS and Suramin, reversed the neuroprotective role of CO-treated astrocytes. Taken all together, ATP and adenosine seem to be involved in CO-induced astrocyte-neuron communication, improving neuronal survival. However, the effect observed by inhibition of adenosine receptors is higher than ATP receptors. Adenosine appears to present a more effective role in neuronal protection by CO-pretreated astrocytes because: (i) chemical inhibition of A2A receptor, (ii) inhibition of ectonucleotidase in astrocytes, (iii) decrease in extracellular adenosine by inhibition of Cx43 in astrocytes markedly decreased neuronal survival in the co-culture system. Based in our study, adenosine combined with CO can be considered a key element in HIR damage prevention. All these results prove to be important on the improvement and development of new strategies in neuronal metabolism and cell to cell communication.
