Differential progression of Dark Neuron and Fluoro-Jade labelling in the rat hippocampus following pilocarpine-induced status epilepticus

Abstract

To investigate the progression of cellular injury in a model of hippocampal epileptogenesis, we used two histochemical methods reported to specifically label injured neurons, the Dark Neuron stain and Fluoro-Jade. Pilocarpine was administered systemically (380 mg/kg i.p.) to induce status epilepticus. The duration of status epilepticus was controlled to last 1 h by stopping it with diazepam (4 mg/kg i.p.). The progression of cellular damage was quantified at six specific time points following the initial pilocarpine-induced insult: 3 h, 6 h, 12 h, 24 h, one week, and three weeks. To assess, in parallel, neuronal loss in specific hippocampal regions throughout epileptogenesis, the neuronal nuclear protein NeuN was used as a specific marker of neurons. Results revealed a different time-dependent progression of Dark Neuron and Fluoro-Jade labelling following status epilepticus. A significantly greater proportion of silver-impregnated cells labelled by the Dark Neuron stain was quantified in the stratum radiatum and stratum pyramidale of CA1 at the early time point of 3 h compared with the proportion of Fluoro-Jade labelling in adjacent sections. In contrast, the maximal staining with Fluoro-Jade appeared at a later stage during epileptogenesis (between 24 h and one week), with a significantly greater proportion of neurons labelled compared to the Dark Neuron stain in the stratum radiatum of CA1, stratum pyramidale of CA1, stratum radiatum of CA3 and the polymorphic layer of the dentate gyrus. Neurons from control animals were not significantly labelled by either of the two staining methods. Interestingly, the increase in Fluoro-Jade labelling corresponded in time to neuron loss. The two stains therefore appear to highlight separate processes of neuronal damage. This finding indicates that distinct cellular events take place at different stages of epileptogenesis, which may differ considerably from the permanent changes observed in chronically epileptic tissue.