Distinct Spatial and Temporal Activation of Caspase Pathways in Neurons and Glial Cells After Excitotoxic Damage to the Immature Rat Brain Sonia Villapol, * Laia Acarin, * Maryam Faiz, Bernardo Castellano, and Berta Gonzalez Medical Histology, Faculty of Medicine, Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Autonomous University of Barcelona, Bellaterra, Spain Although cleaved caspase-3 is known to be involved in apoptotic cell death mechanisms in neurons, it can also be involved in a nonapoptotic role in astrocytes af- ter postnatal excitotoxic injury. Here we evaluate partic- ipation of upstream pathways activating caspase-3 in neurons and glial cells, by studying the intrinsic path- way via caspase-9, the extrinsic pathway via caspase- 8, and activation of the p53-dependent pathway. N- methyl-D-aspartate (NMDA) was injected intracortically in 9-day-old postnatal rats, which were sacrificed at several survival times between 4 hr postlesion (pl) and 7 days pl. We analyzed temporal and spatial expression of caspase-8, caspase-9, and p53 and correlation with neuronal and glial markers and caspase-3 activa- tion. Caspase-9 was significantly activated at 10 hpl, strongly correlating with caspase-3. It was present mainly in damaged cortical and hippocampal neurons but was also seen in astrocytes and oligodendrocytes in layer VI and corpus callosum (cc). Caspase-8 showed a diminished correlation with caspase-3. It was present in cortical neurons at 10–72 hpl, showing layer specificity, and also in astroglial and microglial nuclei, mainly in layer VI and cc. p53 Expression increased at 10–72 hpl but did not correlate with caspase-3. p53 Was seen in neurons of the degenerating cortex and in some astrocytes and microglial cells of layer VI and cc. In conclusion, after neonatal excitotoxicity, mainly the mito- chondrial intrinsic pathway mediates neuronal caspase- 3 and cell death. In astrocytes, caspase-3 is not widely correlated with caspase-8, caspase-9, or p53, except in layer VI-cc astrocytes, where activation of upstream cas- cades occurs. V V C 2007 Wiley-Liss, Inc. Key words: neonatal; microglia; astrocyte; p53; oligo- dendrocyte Caspases are cystein proteases expressed as inactive precursors that undergo proteolytic maturation into a large subunit (p20) and a small subunit (p10), which combine to form an active tetramer. Upon activation, initiator caspases such as caspase-8, -9, -10, and -2 cleave executioner caspases-3, -6, and -7, which are effector proteases degrading structural proteins, signaling mole- cules, and DNA repair enzymes (Marks and Berg, 1999; Salvesen and Dixit, 1999; Hengartner, 2000; Fischer et al., 2003; Kumar, 2007). Accordingly, activation of executioner caspases, mainly caspase-3, has been viewed as the terminal event in apoptosis. However, emerging evidence suggests a broader role for caspases in cell proliferation, cell cycle regulation, differentiation, and gene expression (Schwerk and Schulze-Osthoff, 2003; McLaughlin, 2004) and implies that the cell type and environment play an important role in defining the con- sequences of caspase activation. The most common events ensuing from caspase-3 activation are the intrinsic and extrinsic pathways of apo- ptotic cell death through caspase-9 and caspase-8 activa- tion, respectively. The mitochondrial intrinsic pathway is triggered by cellular stress signals such as DNA damage, ionic dysfunction, and subcellular relocalization of proa- poptotic proteins. The key event in this pathway is mi- tochondrial cytochrome c release and its binding with APAF, which facilitates apoptosome formation that acti- vates caspase-9 by proteolysis and dimerization, activat- ing downstream effector caspase-3 (Costantini et al., 2002; Kumar, 2007). The extrinsic pathway can be induced by several stimuli that activate death receptors Contract grant sponsor: Ministry of Sciences; Contract grant number: BFU2005-02783; Contract grant sponsor: Marato TV3 Foundation; Contract grant number: TV3-061710; Contract grant number: 2005SGR-00956. *Correspondence to: Sonia Villapol or Laia Acarin, Medical Histology, Torre M5, Department Cell Biology, Physiology and Immunology and Institute of Neurosciences, Autonomous University of Barcelona, Bellaterra 08193, Barcelona, Spain. E-mail: sonia.villapol@uab.es or laia.acarin@ uab.cat Received 30 March 2007; Revised 29 May 2007; Accepted 30 May 2007 Published online 1 August 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21450 Journal of Neuroscience Research 85:3545–3556 (2007) ' 2007 Wiley-Liss, Inc.