Leakage of the blood-brain barrier (BBB) is associated with various neurological disorders, including temporal lobe epilepsy (TLE). However, it is not known whether alterations of the BBB occur during epileptogenesis and whether this can affect progression of epilepsy. We used both human and rat epileptic brain tissue and determined BBB permeability using various tracers and albumin immunocytochemistry. In addition, we studied the possible consequences of BBB opening in the rat for the subsequent progression of TLE. Albumin extravasation in human was prominent after status epilepticus (SE) in astrocytes and neurons, and also in hippocampus of TLE patients. Similarly, albumin and tracers were found in microglia, astrocytes and neurons of the rat. The BBB was permeable in rat limbic brain regions shortly after SE, but also in the latent and chronic epileptic phase. BBB permeability was positively correlated to seizure frequency in chronic epileptic rats. Artificial opening of the BBB by mannitol in the chronic epileptic phase induced a persistent increase in the number of seizures in the majority of rats. These findings indicate that BBB leakage occurs during epileptogenesis and the chronic epileptic phase and suggest that this can contribute to the progression of epilepsy.

Summary:Purpose: Overexpression of multidrug transporters such as P-glycoprotein (P-gp) may play a significant role in pharmacoresistance, by preventing antiepileptic drugs (AEDs) from reaching their targets in the brain. Until now, many studies have described increased P-gp expression in epileptic tissue or have shown that several AEDs act as substrates for P-gp. However, definitive proof showing the functional involvement of P-gp in pharmacoresistance is still lacking. Here we tested whether P-gp contributes to pharmacoresistance to phenytoin (PHT) by using a specific P-gp inhibitor in a model of spontaneous seizures in rats.Methods: The effects of PHT on spontaneous seizure activity were investigated in the electrical post-status epilepticus rat model for temporal lobe epilepsy, before and after administration of tariquidar (TQD), a selective inhibitor of P-gp.Results: A 7-day treatment with therapeutic doses of PHT suppressed spontaneous seizure activity in rats, but only partially.However, an almost complete control of seizures by PHT (93 ± 7%) was obtained in all rats when PHT was coadministered with TQD. This specific P-gp inhibitor was effective in improving the anticonvulsive action of PHT during the first 3-4 days of the treatment. Western blot analysis confirmed P-gp upregulation in epileptic brains (140-200% of control levels), along with ∼20% reduced PHT brain levels. Inhibition of P-gp by TQD significantly increased PHT brain levels in chronic epileptic rats.Conclusions: These findings show that TQD significantly improves the anticonvulsive action of PHT, thus establishing a proof-of-concept that the administration of AEDs in combination with P-gp inhibitors may be a promising therapeutic strategy in pharmacoresistant patients.

Recent studies have suggested that overexpression of the multidrug transporter P-glycoprotein (P-gp) in the hippocampal region leads to decreased levels of antiepileptic drugs and contributes to pharmacoresistance that occurs in a subset of epileptic patients. Whether P-gp expression and function is affected in other brain regions and in organs that are involved in drug metabolism is less studied. Therefore, we investigated P-gp expression in different brain regions and liver of chronic epileptic rats, several months after electrically induced status epilepticus (SE), using Western blot analysis. P-gp function was determined by measuring phenytoin (PHT) levels in these brain regions using high-performance liquid chromatography, in the absence and presence of a P-gp-specific inhibitor, tariquidar (TQD). In addition, the pharmacokinetic profile of PHT was determined. PHT concentration was reduced by 20 to 30% in brain regions that had P-gp overexpression (temporal hippocampus and parahippocampal cortex) and not in brain regions in which P-gp expression was not changed after SE. Inhibition of P-gp by TQD significantly increased the PHT concentration, specifically in regions that showed P-gp overexpression. Despite increased P-gp expression in the liver of epileptic rats, pharmacokinetic analysis showed no significant change of PHT clearance in control versus epileptic rats. These findings show that overexpression of P-gp at the blood-brain barrier of specific limbic brain regions causes a decrease of local PHT levels in the rat brain.

Type II deiodinase (D2) plays a key role in regulating thyroid hormone-dependent processes in, among others, the central nervous system (CNS) by accelerating the intracellular conversion of T4 into active T3. Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid axis, D2 activity also appears to show daily variations. However, the mechanisms involved in generating these daily variations, especially in the CNS, are not known. Therefore, we decided to investigate the role the master biological clock, located in the hypothalamus, plays with respect to D2 activity in the rat CNS as well as the role of one of its main hormonal outputs, i.e. plasma corticosterone. D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Ablation of the biological clock abolished the daily variations of D2 activity in all four tissues studied. The main effect of the knockout of the suprachiasmatic nuclei (SCN) was a reduction of nocturnal peak levels in D2 activity. Moreover, contrary to previous observations in SCN-intact animals, in SCN-lesioned animals, the decreased levels of D2 activity are accompanied by decreased plasma levels of the thyroid hormones, suggesting that the SCN separately stimulates D2 activity as well as the hypothalamo-pituitary-thyroid axis.