A proliferação de neurônios granulares hipocampais aumenta e os dendritos dos novos neurônios são anormais no modelo experimental de ELT induzida por pilocarpina

Abstract

Hippocampal doublecortin-positive granular neurons have abnormal dendritic morphology in the temporal lobe epilepsy pilocarpine model To quantify the number and morphology of doublecortin-positive (DCX+) granular neurons in a temporal lobe epilepsy model, Wistar rats were treated with pilocarpine and, 90 min after status epilepticus (SE) onset, with diazepam. Controls received saline instead of pilocarpine. Rats were monitored 8 h per day to record spontaneous recurrent seizures (SRS) and perfused 30 days after SE. A series of sections containing the hippocampus was immunolabeled for DCX. The stereological optical fractionator method was used to count DCX+ neurons in granular cell layer (GCL) and subgranular zone of the dentate gyrus (DG). DCX+ neurons (n = 40) were digitally reconstructed and their dendritic distribution measured with Sholl’s concentric spheres analysis. The first sphere has the radius equal to the GCL thickness followed by spheres with increments of 50 μm in their radii. Neo-Timm staining was performed to evaluate the mossy fiber sprouting. Statistical difference in Student and Mann-Whitney tests at p < 0.05, values are mean ± SEM. Each animal had 8.4 ± 3.7 SRS recorded. The DCX+ counting revealed the presence of 8,987 ± 1,986 neurons in controls DG (n = 7) and 19,337 ± 4,715 neurons in epileptic DG (n = 7). Basal dendrites were observed in 85% of DCX+ neurons in epileptics and in 30% of DCX+ neurons in controls with a length of 63.9 ± 9.7 μm and 19.8 ± 5.3 μm, respectively. Apical dendrites of DCX+ neurons had ~500 μm in both groups, but the epileptics had more nodes (3.9 ± 0.3) and dendrites emerging from GCL (4.1 ± 0.4) than controls (2.47 ± 0.2 nodes and 2.95 ± 0.3 dendrites). The epileptic rats had more dendritic endings (4.55 ± 0.5) than controls (1.7 ± 0.4) in the first 50 μm of the molecular layer (GCL + 50). Epileptic rats had more DCX+ dendritic length in GCL (168 ± 14 μm against 126 ± 11 μm in controls) and GCL + 50 (231 ± 26 μm against 163 ± 17 μm in controls); the contrary occurred in GCL + 100 (94 ± 23 μm against 160 ± 23 μm in controls). Neo-Timm staining revealed a confluent band of silver granules in epileptic DG with a mean thickness of 48 μm (equivalent to GCL + 50) and no band in controls. Hilar basal dendrites were more frequent, long and ramified and the apical dendrites presented intense ramification in the inner molecular layer of epileptic rats. Dendrites were more abundant in regions with mossy fiber sprouting. Formation of synapses between these new dendrites and axons could contribute to the pathological process of hippocampal epileptogenesis.