Vulnerability of postnatal hippocampal neurons to seizures varies regionally with their maturational stage

Abstract

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium-pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

Figure 1. SE induces necrosis in CA1 and apoptosis in DG

(A) Images of Fluoro-jade B staining in hippocampus from control animal and 24 h following SE. (B-C) Time course of neuronal injury in CA1 (B) and DG (C) following SE. Left panels: The graphs represent the number of eosinophilic cells detected with H&E staining from CA1 (B) and DG (C) 7–72 h after SE. Analyzed by Dunn’s test (***p<0.001, **p<0.01 vs control) or Mann Whitney test (^###p<0.001, ^##p<0.01 vs control). Middle panels: High magnification images of eosinophilic cells with pyknotic nuclei from CA1 (B) and fragmented nuclei from DG (C) obtained 24 h after SE. Arrows point to injured neurons. Right panels: Electron microscopy micrographs obtained 24 h after SE, showing a CA1 necrotic cell, characterized by swollen organelles, ruptured plasma membrane and tigroid fragmentation of the chromatin (B) and a DG cell with a late stage of apoptosis, characterized by large round chromatin clumps and mild organelle swelling (C). Scale bars in A = 100 μm; B-C = 25 μm (light microscopy) and 3 μm (electron microscopy).

Figure 2. SE activates caspase-3 in morphologically necrotic CA1 neurons

(A) Images of caspase-3a (green), NeuN (neuronal marker, red) IR and Hoechst staining (chromatin dye, blue). Arrows point to caspase-3a-IR neurons in CA1 24 h following SE. High magnification images show that caspase-3a IR is found in pyknotic nuclei. The graph represents the number of caspase-3a-IR neurons from CA1 7–72 h after SE (***p<0.001, **p<0.01, *p<0.05 vs control). (B) Images of caspase-3 activity (red) on unfixed frozen sections from control and experimental animal (24 h following SE). Arrowheads show that caspase-3 activity is mainly seen in pyknotic nuclei. (C) Example of a caspase-3a-IR neuron with necrotic morphology 24 h following SE. Gold particles, revealing caspase-3 localization (arrows), are mainly visible in the nucleus. Arrowheads show mitochondrial swelling. (D) The images show fluoro-Jade B staining in CA1 from a rat pretreated with vehicle and a rat pretreated with Q-VD-OPh 24 h following SE. The table shows that Q-VD-Oph did not modify the seizure severity (Chi-square= 0.619, df= 5, p = 0.987). The graph shows a reduction of the number of Fluoro-Jade B-positive cells (*p=0.003, Student’s t test vs vehicle). Scale bars in A-B = 100 μm (low magnification images) and 25 μm (high magnification images). Scale bars in D = 25 μm and C = 1 μm.

Figure 3. SE activates the extrinsic pathway in CA1 neurons

(A) Images of caspase-8 IR (red) show that many CA1 cells are IR 7 h following SE. High magnification images show that caspase-8 IR is mainly seen in the nuclei. The graph represents the number of caspase-8-IR cells from CA1 7–24 h after SE (***p<0.001 vs control). (B) Images showing the absence of caspase-9a IR in CA1. The graph represents the number of caspase-9a-IR cells from CA1 7–72 h after SE (*p<0.05 vs control). Scale bars in A-B = 100 μm (low magnification images) and 25 μm (high magnification images).

Figure 4. SE activates the intrinsic pathway in morphologically apoptotic DG neurons

(A-B) Images of caspase-3a or −9a IR (green) and Hoechst staining (chromatin dye, blue). Arrows point to IR cells in DG 7 h following SE. High magnification images show that IR is observed mainly in the cytosol of cells with fragmented nuclei. The graphs represent the number of IR cells in DG 7–72 h after SE (**p<0.001, *p<0.05 vs control). (C) Images showing the absence of caspase-8 IR in DG. The graph represents the number of caspase-8-IR cells in DG 7–24 h after SE. Scale bars = 100 μm (low magnification images) and 25 μm (high magnification images).

Figure 5. SE-injured DG cells have features of immature neurons

Caspase-3a-IR DG neurons (arrows) are calbindin-negative and doublecortin-positive. (A) Images of active caspase-3-IR (red), doublecortin-IR (immature neuron marker, green) and Hoechst staining (chromatin dye, white) in DG 24 h following SE. (B) Images of active caspase-3-IR (red), calbindin-IR (mature granule cell marker, green) and Hoechst staining (white) in DG 24 h following SE. Scale bars = 100 μm.