Resumo

Objective: The Pilocarpine Model of Epilepsy (PME) reproduces the main features of human temporal lobe epilepsy in rats. Evidences suggest that blood-brain barrier (BBB) failure may be an etiological factor contributing to the development of seizures. The aim of this work was to investigate the relationship between the seizures and the BBB integrity during acute phase of the PME in Wistar male adult rats. Methods: The animals were killed at 30 min after pilocarpine injection (320-350 mg/kg i.p.), 1h, 5h and 24h after status epilepticus (SE) onset. The rats of groups 5h and 24h SE received an injection of diazepam (7-10 mg/kg i.p.) 3-5 hours after the onset of seizures. The BBB integrity was assessed using the Evans blue (EB; 80 mg/kg) or sodium fluorescein (NaFl; 100 mg/kg) dyes. EB dye was injected intravenously immediately before PILO injection (group 30 min) or at the beginning of the SE (1h, 5h and 24h groups). It binds to serum albumin forming a complex of 68,500 Da, which has been used as a marker of macromolecules extravasation. NaF was injected intravenously thirty minutes before animal sacrifice. It is a vital dye of 376 Da used as marker of micromolecules extravasation. Rats were perfused transcardially until the fluid from the right atrium became colourless. The brain was dissected into 7 regions: left/right hippocampus (LH/RH), left/right entorhinal cortex (LE/RE), left/right cerebral neocortex (LN/RN) and cerebellum. Samples were homogenized, proteins were precipitated and the supernatants were measured for EB absorbance or NaFl fluorescence. Results were expressed in ug of total EB or NaF(mean +/- SEM). Results: Animals developed a self-sustained SE approximately 30 min after PILO injection. Macroscopic EB dye extravasation was found after 5h and 24h of SE in hippocampus, entorhinal cortex and cerebral neocortex. These results were corroborated by quantitative measures which showed significant extravasation of EB 5h after SE in hippocampus (LH: 0.57+0.155* and RH: 0.52+0.006***), entorhinal cortex (LE: 0.51+0.071*** and RE: 0.44+0.08**) and neocortex (LN: 1.62+0.314*); 24 hours after SE EB dye was detected in hippocampus (RH: 0.45+0.037* and LE: 0.52+0.043***), entorhinal cortex (RE: 0.48+0.028*** and LN: 2.21+0.232***) and neocortex (RN: 2.11+0.173***), when compared to respective controls LH (0.297+0.034), RH (0.26+0.03), LE (0.24+0.02), RE (0.26+0.02), LN (0.89+0.09) and RN(0.94+0.09). Quantitative measures of the NaFl showed significant extravasation of dye from blood to brain 30 minutes after PILO injection in hippocampus (LH: 0.029+0.003* and RH: 0.029+0.004*); 1h after SE in hippocampus (LH: 0.033+0.001** and RH: 0.029+0.001*); 5h after SE development in hippocampus (LH: 0.03+0.004* and RH: 0.031+0.004*), entorhinal cortex (LE: 0.038+0.007* and RE: 0.037+0.007*) and neocortex (LN: 0.154+0.035** and RN: 0.151+0.035**); 24h after SE in hippocampus (LH: 0,032+0,003** and RH: 0,031+0,003**) and entorhinal cortex (LE: 0.041+0.007** and RE: 0.040+0.006**), when compared to controls LH (0.017+0.002), RH (0.015+0.002), LE (0.015+0.002), RE (0.015+0.003), LN (0.052+0.013) and RN (0.053+0.012). ).* p<0,05; ** p<0,01 and ***p<0,001. Conclusion: BBB permeability to micromolecules is increased during acute phase of epilepsy model since the pilocarpine injection until 24 hours after SE onset. BBB breakdown to macromolecules occurs between 5 and 24h after SE development.