[en] Although it is well established that apoptotic mechanisms play a central role in neuronal death after different types of CNS damage, little is known about the activation of apoptotic pathways in glial cells which have been postulated to be important for the termination of the glial response. It is remarkable that the classically defined main executioner of programmed cell death, caspase-3, has recently been attributed a non-apoptotic role, participating in cellular events including cell proliferation, cell cycle regulation, or cellular differentiation. Accordingly, the general aim of this thesis was to analyze the putative role of the classic apoptotic and anti-apoptotic pathways after acute excitotoxic damage to the immature rat brain. For this purpose, a well-characterized in vivo model of excitotoxicity was used, where damage was induced by the intracortical injection of N-methyl-D-asparate (NMDA) in postnatal day 9 rats. Cell death, apoptotic and anti-apoptotic proteins were analyzed at different survival times after the lesion by enzymatic assays and double immunofluorescence for confocal microscope analysis. The first set of results demonstrated that although cleaved caspase-3 was found in some damaged neurons showing TUNEL+ nuclei, cleaved caspase-3 was mainly observed in the nucleus of activated astrocytes in the lesioned hemisphere as early as 4 h post-lesion, persisted until the glial scar was formed at 7-14 days, and was marginally associated with TUNEL labeling. Caspase-3 enzymatic activity was significant during the first hours post-lesion and co-localized with the presence of caspase-cleaved fragments of glial fibrillary acidic protein (CCP-GFAP) in astrocytes. However, at longer survival times, when astroglial hypertrophy was observed, astroglial caspase-3 did not generally correlate with GFAP cleavage but instead was associated with de novo expression of vimentin. Moreover, astroglial caspase-3 cleavage was not associated with cell proliferation. These first results provided evidence for a non-traditional role of caspases in astroglial function, suggesting that caspase activation may be important for astroglial cytoskeleton remodeling following injury. Secondly, in order to evaluate upstream pathways activating caspase-3 in neurons and glial cells, the spatio-temporal activation of the intrinsic and extrinsic apoptosis pathways were analyzed. In damaged neuronal cells of the ipsilateral cortex and hippocampus an important contribution of the intrinsic mitochondrial pathway through caspase-9 activation was shown to account for most of neuronal caspase-3 activation and apoptotic nuclei. However, neuronal caspase-8 showed a diminished correlation with caspase-3 and characteristically showed cortical layer specificity. In glial cells, activation of caspase-9, caspase-8, and also p53, another putative caspase-3 activating molecule, was only significant in cortical layer VI and the corpus callosum, suggesting that astroglial caspase-3 does not generally correlate with classical upstream activating pathways. In the third part of this thesis, inhibitor of apoptosis proteins (IAPs) and heat shock proteins (HSPs), known inhibitors of cleaved caspase-3 in other cell types, were analyzed. Whereas neuronal cells showed noticeable expression of HSC70/HSP70 and cIAP-2, these poorly correlated with caspase-3 in this cell type. In astrocytes, survivin and the small HSP25/27 showed a strong correlation with caspase-3 in several regions and throughout different survival times, pointing to these proteins as relevant candidates for the blockade of caspase-3 enzymatic activity, which could account for the absence of astroglial cell death. In conclusion, this thesis has discerned new non-apoptotic roles of caspase-3 in astroglial cells and suggests mechanisms for caspase-3 blockade in these glial cells, improving the understanding of the mechanisms employed by astrocytes to cope with damage in the immature brain.

[gl] Os mecanismos apoptóticos estudiáronse en profundidade, constituíndo un papel substancial na morte neuronal despois de diferentes tipos de dano no SNC. Nembergantes, moi pouco se sabe acerca da activación dos mecanismos apoptóticos nas células gliais e a sua relevancia na terminación da resposta glial. A caspasa-3 definiuse clásicamente coma principal executura da morte celular programada, fóronlle recentemente atribuídos outros papeis non-apoptóticos, participando en acontecementos celulais, incluíndo proliferación, regulación do ciclo celular, e vías apoptóticas e antiapoptóticas despois dun dano excitotóxico no cerebro inmaduro de rata. Para este propósito, utilizáronse un modelo de excitotoxicidade ben caracterizado, que consiste na inducción dun dano cerebral mediante unha inxección intracortical de N-methyl-D-asparate (NMDA) en ratas postnatais no día 9 de vida (P9). A morte celular, as proteínas apoptóticas e antiapoptóticas foron analizadas a distintos tempos de supervivencia despois da lesión por ensaios enzimáticos e análise de doble fluorescencia con microscopía confocal. Os primeiros resultados coa caspasa-3 clivada presentábanse en neuronas dañadas cos núcleos TUNEL+, aunque foi observada maioritariamente no núcleo de astrocitos activados no hemisferio lesionado sen asociación con fragmentación do ADN, dende tempos máis cedos, ás 4 horas post-lesión (hpl) ata a formación da cicatriz glial ós 7-14 días, sen encontrarse asociación co marcaxe con TUNEL. A actividade enzimática da caspasa-3 foi detectada as primeiras horas post-lesión e coincidindo cos fragmentos clivados por caspasas da proteína acídica fibrilar (CCP-GFAP) en astrocitos. Sen embargo, a tempos longos de supervivencia, cando surxe a hipertrofia astroglial, a caspasa-3 astroglial xeralmente non correlacionou co clivaxe de CCP-GFAP, pero si se relacionou coa expresión de novo de vimentina. Por outra banda, a caspasa-3 clivada non se vinculou á proliferación celular. Estes resultados iniciais mostraron a evidencia dun non-tradicional papel das caspasas na función astroglial, suxerindo que a sua activación pode ser importante para a reestructuración do citoesqueleto despois dun dano. Secundariamente, ó evaluar as vías de activación da caspasa-3 en neuronas e nas células gliais, a activación espacio-temporal das vías apoptóticas intrínseca e extrínseca foron analizadas. Expresouse principalmente nas neuronas dañadas corticais e hipocampais, unha importante contribución da vía intrínseca através da gran proporción de caspasa-9 activa coa activación neuronal da caspase-3 e morte celular. Por outra banda, a caspasa-8 presentou unha reducida correlación coa caspasa-3, e característicamente presentou especificidade nas capas corticais. Nas células gliais, a activación da caspasa-9, caspasa-8, e tamén p53, outra molécula que activa a caspasa-3 indirectamente, foi soamente significante na capa cortical VI e no corpus callosum, suxerindo que a caspasa-3 astroglial non se correlaciona xeralmente coas clásicas vías de activación. A terceira parte desta tesis focalizouse no estudio das moléculas antiapoptóticas, coma varios inhibitores de apoptosis (IAPs) e as heat shock proteins (HSPs), coñecidas coma inhibidoras da caspasa-3 clivada noutros tipos celulares. As neuronas presentaron unha importante expresión de HSC70/HSP70 e cIAP-2, anque estas pobremente correlacionábanse coa caspasa-3 neste tipo celular. En astrocitos , a survivina e a chaperona HSP25/27 mostraron unha forte correlación con caspasa-3 en varias rexións cerebrais e a varios tempos de supervivencia, sendo estas proteínas destacadas candidatas para o bloqueo da actividade enzimática da caspasa-3, a cal podría acumularse en ausencia de morte astroglial. En conclusión esta tesis destapou novos roles non apoptóticos da caspasa-3 en células astrogliais, suxerindo mecanismos de bloqueo da caspasa-3 e axudou á mellor comprensión dos mecanismos empregados polos astrocitos para enfrentarse ó dano no cerebro inmaduro.