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 (380mg/kg i.p.) to induce
status epilepticus. The duration of
status epilepticus was controlled to last 1h by stopping it with
diazepam (4mg/kg i.p.). The progression of cellular damage was quantified at six specific time points following the initial
pilocarpine-induced insult: 3h, 6h, 12h, 24h, 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 3h 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 24h 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.