Imaging of P-glycoprotein-mediated pharmacoresistance in the hippocampus: proof-of-concept in a chronic rat model of temporal lobe epilepsy.

PURPOSE: Based on experimental findings, overexpression of P-glycoprotein at the blood-brain barrier has been suggested to be a contributor to pharmacoresistance of the epileptic brain. We test a technique for evaluation of interindividual differences of elevated transporter function, through microPET analysis of the impact of the P-glycoprotein modulator tariquidar. The preclinical study is intended for eventual translation to clinical research of patients with pharmacoresistant seizure disorders. METHODS: We made a microPET evaluation of the effects of tariquidar on the brain kinetics of the P-glycoprotein substrate [(18) F]MPPF in a rat model with spontaneous recurrent seizures, in which it has previously been demonstrated that phenobarbital nonresponders exhibit higher P-glycoprotein expression than do phenobarbital responders. RESULTS: Mean baseline parametric maps of the [(18) F]MPPF unidirectional blood-brain clearance (K(1) ; ml/g per min) and the efflux rate constant (k(2) ; per min) did not differ between the nonresponder and responder group. Tariquidar pretreatment increased the magnitude of [(18) F]MPPF K(1) in hippocampus by a mean of 142% in the nonresponders, which significantly exceeded the 92% increase observed in the responder group. The same treatment decreased the mean magnitude of [(18) F]MPPF k(2) in hippocampus by 27% in nonresponders, without comparable effects in the responder group. DISCUSSION: These results constitute a proof-of-concept for a novel imaging approach to evaluate blood-brain barrier P-glycoprotein function in animals. By extension, [(18) F]MPPF positron emission tomography (PET) with tariquidar pretreatment may be amenable for clinical applications exploring further the relevance of P-glycoprotein overexpression, and for enabling the rational design of pharmacotherapy according to individual differences in P-glycoprotein expression.

Cellular localization of Y-box binding protein 1 in brain tissue of rats, macaques, and humans.

BACKGROUND: The Y-box binding protein 1 (YB-1) is considered to be one of the key regulators of transcription and translation. However, so far only limited knowledge exists regarding its cellular distribution in the adult brain. RESULTS: Analysis of YB-1 immunolabelling as well as double-labelling with the neuronal marker NeuN in rat brain tissue revealed a predominant neuronal expression in the dentate gyrus, the cornu ammonis pyramidal cell layer, layer III of the piriform cortex as well as throughout all layers of the parahippocampal cortex. In the hilus of the hippocampus single neurons expressed YB-1. The neuronal expression pattern was comparable in the hippocampus and parahippocampal cortex of adult macaques and humans. Double-labelling of YB-1 with the endothelial cell marker Glut-1, the multidrug transporter P-glycoprotein, and the astrocytic marker GFAP did not indicate a co-localization. Following status epilepticus in rats, no induction of YB-1 occurred in brain capillary endothelial cells and neurons. CONCLUSION: In conclusion, our study demonstrates that YB-1 is predominantly expressed in neurons in the adult brain of rats, macaques and humans. Lack of a co-localization with Glut-1 and P-glycoprotein argues against a direct role of YB-1 in the regulation of blood-brain barrier P-glycoprotein.

Targeting epileptogenesis-associated induction of neurogenesis by enzymatic depolysialylation of NCAM counteracts spatial learning dysfunction but fails to impact epilepsy development.

Polysialylation is a post-translational modification of the neural cell adhesion molecule (NCAM), which in the adult brain promotes structural changes in regions of neurogenesis and neuroplasticity. Because a variety of plastic changes including neurogenesis have been suggested to be functionally involved in the pathophysiology of epilepsies, it is of specific interest to define the impact of the polysialic acid (PSA)-NCAM system on development of this disease and associated comorbidities. Therefore, we studied the impact of transient enzymatic depolysialylation of NCAM on the pathophysiology in an electrically induced rat post-status epilepticus (SE) model. Loss of PSA counteracted the SE-induced increase in neurogenesis in a significant manner. This effect of endoneuraminidase (endoN) treatment on hippocampal neurogenesis did not impact the subsequent development of spontaneous seizures. In contrast, transient lack of PSA during SE and in the early phase of epileptogenesis exhibited a cognition sparing effect as revealed in the Morris water maze paradigm. In conclusion, our data do not support a central role of neurogenesis in the development of a hyperexcitable epileptic network. However, in view of the cognition-sparing effect, the transient modulation of the PSA-NCAM system seems to allow beneficial long-term disease modification, which might be mediated by the partial normalization of neurogenesis.

In vivo down-regulation of mouse brain capillary P-glycoprotein: a preliminary investigation.

Over-expression of blood-brain barrier P-glycoprotein is considered as a major hurdle in the treatment of various CNS disorders. A down-regulation strategy is considered as one means to counteract disease- or therapy-associated induction of P-glycoprotein. Here, we evaluated whether a targeting of P-glycoprotein can be achieved in mouse brain capillary endothelial cells using siRNA. A 4-day treatment paradigm with once daily hydrodynamic intravenous injections of siRNA resulted in a significant reduction of the P-glycoprotein-labeled area in the hippocampal hilus and parietal cortex. P-glycoprotein expression proved to be down-regulated in these brain regions by 31 and 16%, respectively. An impact of siRNA administration on density of brain capillaries was excluded by quantification of the endothelial cell marker GLUT-1. In conclusion, the study provides first preliminary evidence that a down-regulation of P-glycoprotein can be achieved in brain capillary endothelial cells by administration of siRNA in vivo.

Pyrrolidine dithiocarbamate protects the piriform cortex in the pilocarpine status epilepticus model.

Pyrrolidine dithiocarbamate (PDTC) has a dual mechanism of action as an antioxidant and an inhibitor of the transcription factor kappa-beta. Both, production of reactive oxygen species as well as activation of NF-kappaB have been implicated in severe neuronal damage in different sub-regions of the hippocampus as well as in the surrounding cortices. The effect of PDTC on status epilepticus-associated cell loss in the hippocampus and piriform cortex was evaluated in the rat fractionated pilocarpine model. Treatment with 150 mg/kg PDTC before and following status epilepticus significantly increased the mortality rate to 100%. Administration of 50 mg/kg PDTC (low-dose) did not exert major effects on the development of a status epilepticus or the mortality rate. In vehicle-treated rats, status epilepticus caused pronounced neuronal damage in the piriform cortex comprising both pyramidal cells and interneurons. Low-dose PDTC treatment almost completely protected from lesions in the piriform cortex. A significant decrease in neuronal density of the hippocampal hilar formation was identified in vehicle- and PDTC-treated rats following status epilepticus. In conclusion, the NF-kappaB inhibitor and antioxidant PDTC protected the piriform cortex, whereas it did not affect hilar neuronal loss. These data might indicate that the generation of reactive oxygen species and activation of NF-kappaB plays a more central role in seizure-associated neuronal damage in the temporal cortex as compared to the hippocampal hilus. However, future investigations are necessary to exactly analyze the biochemical mechanisms by which PDTC exerted its beneficial effects in the piriform cortex.