Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat

Abstract

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Fig. 1.

The time course of apoptosis within the core infarct as indicated by TUNEL. At 6 hr after injury there is no evidence of DNA damage within the core infarct (A,B). At 24 hr there is strong staining in a moderate number of cells distributed throughout lamina II/III (arrows, C) and lamina V (arrows, D). By 48 hr most of the cells within lamina II/III show robust staining (E), as do the majority of cells within lamina V (F). Scale bar: A–F, 250 μm.

Fig. 2.

Gel analysis of DNA. Brain homogenates from 24 hr after injury were selected based on the TUNEL findings and basic histological analysis, which indicated the presence of apoptotic bodies at this time (A). A laddered pattern of DNA was observed in samples taken from the ipsilateral cortex (Ip; side of injury) but not from the uninjured contralateral cortex (Co) (B).St, Molecular weight standards.

Fig. 3.

Western blots demonstrating antibody specificity. SK441 (left) detects full-length endogenous caspase-8, a 55 kDa band, in all lanes of Jurkat extract and brain. In addition, SK441 recognizes the processed prodomain (there is autoprocessing of the full-length recombinant caspase-8, resulting in a mixture of active an inactive enzyme) as bands at 25 and 14 kDa in the lane spiked with recombinant full-length caspase-8. SK440 detects the cleaved p20 subunit of caspase-8 only (middle). SK398 (right) reacts with the p20 subunit of human caspase-8 (18 kDa) and the prodomain plus p20 of human caspase-3 (21.5 kDa) and the p20 subunit of human caspase-3 (17 kDa). A single band corresponding to active caspase-3 is detected in extracts of mouse liver after intravenous administration of anti-Fas. Mw, Molecular weight standards;8A, Jurkat extract spiked with recombinant active human caspase-8 (p20/p10 fusion); 8F, Jurkat extract spiked with full-length recombinant human caspase-8; Jk, Jurkat extract; Br, Rat brain; 3A, Jurkat extract spiked with recombinant human caspase-3 that has been cleaved to its active form; 3F, Jurkat extract spiked with full-length recombinant human caspase-3; Liver, mouse liver extract from animals that were not (−) injected with anti-Fas and animals that were injected with anti-Fas (+).

Fig. 4.

Expression of caspase-8 in cortical neurons of normal and ischemic rat brain. Punctate cytoplasmic staining for procaspase-8 is evident in two large pyramidal neurons of lamina V (A). The active caspase-8 antibody fails to demonstrate positive immunostaining in normal rat brain (B). At 6 hr after injury, positive immunoreactivity is readily detectable in the large pyramidal neurons of lamina V (C). Both positive and negative (arrow) immunostaining is still apparent in the large pyramidal neurons of lamina V at 48 hr (D). Colocalization of TUNEL reaction product (E) and positive immunofluorescence for active caspase-8 (F) could be found. Scale bars: (inA) A, B, 60 μm;C, D, 60 μm; (in E)E, F, 60 μm.

Fig. 5.

Detection of active caspase-3 in neurons and microglia located within the core infarct. All images are from 24 hr after injury. Positive immunostaining localized to the nucleus is seen in the small- and medium-sized pyramidal neurons of lamina II/III (A); several immunopositive neurons are indicated (arrows). No staining is evident in the deeper cortical lamina (B). Doubling labeling of active caspase-3-positive cells with NeuN confirms their neuronal phenotype (C). A mixed nuclear–cytoplasmic compartmentalization of immunostaining is only rarely observed (D). As early as 6 hr, small fragmenting cells with robust cytoplasmic immunostaining (arrows) could be seen (E). Immunopositive cells often had either a classical microglia morphology (F) or appeared atrophic (G) as evidenced by an abnormal cell shape and an eccentrically located pyknotic nuclei bounded by a thin rim of cytoplasm (arrow, G,H). Double labeling with the microglia-specific marker OX42, which is localized to the plasma membrane, confirms the microglial phenotype (H). Scale bars: (inA) A, B, 100 μm;C, E, 40 μm; D, 20 μm; (in F) F–H, 15 μm.

Fig. 6.

The specificity of SB440 and SB398 was further evaluated by adsorption with caspase peptides. Adsorption of SB440 with recombinant human caspase-3 (irrelevant peptide control) did not have an affect on immunostaining (A), whereas adsorption with recombinant human caspase-8 completely abolished immunostaining (B). Adsorption of SB398 with recombinant human caspase-8 (irrelevant peptide control) did not have an affect on immunostaining (C), whereas adsorption with recombinant caspase-3 abolished all immunostaining (D).

Fig. 7.

Quantitative analysis of cell death and active caspase expression. The quantitation of NeuN-positive cells (A, gray bars) indicated that there was a significant loss of neurons at 24 hr (*p < 0.05) compared with naïve, which was followed by a plateauing of neuronal cell loss (24–48 hr) and a subsequent significant loss of neurons detectable at 5 d (**p < 0.05) compared with 48 hr. The analysis of TUNEL (A,black bars) indicated a significant number of cells with DNA damage at 48 hr after injury (φp < 0.05) compared with naïve. The absence of TUNEL before 24 hr after injury indicates that the initial wave of neuronal loss was nonapoptotic. Temporal expression of active caspase-8 (B, ♦) preceded expression of caspase-3 (B, ▪). Peak expression of both caspases was observed at 24 hr, before the peak in TUNEL-positive cells (A). Analysis focusing on lamina V (C) indicated that the number of active caspase-8 (▪)-immunopositive neurons at 24 hr accounted for 19% of the total neuronal population of lamina V and that this peak of active caspase-8-expressing neurons preceded the rise of TUNEL (gray bars) within this lamina.