Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures - PubMed (original) (raw)

Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures

J Bengzon et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase of in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by the N-methyl-D-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.

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Figures

Figure 1

Figure 1

Schematic drawing of the distribution of individual nuclei positive for fragmented DNA (dots) as assessed by TUNEL technique in the dentate gyrus and hilus from three control animals (A), three rats subjected to one hippocampal kindling stimulation and killed 5 h thereafter (B), three animals decapitated 2 h after 40 kindling stimulations (C), and three rats killed 4 h after systemic kainic acid (D). A micrograph of the boxed area is shown in Fig. 3_A_. (Bar = 800 μm.)

Figure 2

Figure 2

Number of nuclei positive for fragmented DNA as assessed by the TUNEL technique bilaterally in the dentate gyrus and hilus at various time points after a single hippocampal kindling stimulation (A), 40 kindling stimulations (B), and 4 h after systemic kainic acid (KA) injection (C). Mean ± SEM. ∗∗, P < 0.001, and ∗, P < 0.05 compared with control; one-way ANOVA followed by Bonferroni–Dunn post-hoc test.

Figure 3

Figure 3

(A) Nuclei labeled for fragmented DNA (arrows) along the hilar border of the granule cell layer 2 h after 40 hippocampal kindling stimulations. (B and C) High-power photomicrographs of labeled nuclei with morphological features characteristic of apoptosis, such as condensation and lobulation (arrow), following kindling stimulation. Note numerous apoptotic bodies (arrowheads). (D) Confocal scanning laser image showing nuclear TUNEL (green) and cytoplasmic NeuN immunolabeling (red) demonstrating the neuronal identity of a degenerating cell 2 h after 40 hippocampal kindling stimulations. (E) Light photomicrograph of an argyrophilic, degenerating neuron in the dentate gyrus granule cell layer 2 h following 40 rapid kindling stimulations. (F and G) Low-power confocal images of sections labeled by NeuN (green) and BrdU (red) immunofluorescence showing the medial dentate gyrus in a control rat (F) and in an animal 19 days after 40 kindled seizures (G). Arrowheads in G denote nuclei labeled by BrdU injections 5 days following stimulation. (H_–_K) Confocal images of a cell (arrow; same cell in all four views) double-labeled by NeuN (green) and BrdU (red) in the dentate gyrus after 40 kindled seizures. Individual signals for BrdU (J) and NeuN (K) are shown as well as composite images (H and I). I_–_K are 1 μm optical sections. (Bars: A = 80 μm; B = 15 μm; D, E, and K = 10 μm; G = 16 μm; magnification in C is the same as in B; magnification in H_–_J is the same as in K; magnification in F is the same as in G.)

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