Erwin A. van Vliet

Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy.

To get insight into the mechanisms that may lead to progression of temporal lobe epilepsy, we investigated gene expression during epileptogenesis in the rat. RNA was obtained from three different brain regions [CA3, entorhinal cortex (EC), and cerebellum (CB)] at three different time points after electrically induced status epilepticus (SE): acute phase [group D (1 d)], latent period [group W (1 week)], and chronic epileptic period [group M (3–4 months)]. A group that was stimulated but that had not experienced SE and later epilepsy was also included (group nS). Gene expression analysis was performed using the Affymetrix Gene Chip System (RAE230A). We used GENMAPP and Gene Ontology to identify global biological trends in gene expression data. The immune response was the most prominent process changed during all three phases of epileptogenesis. Synaptic transmission was a downregulated process during the acute and latent phases. GABA receptor subunits involved in tonic inhibition were persistently downregulated. These changes were observed mostly in both CA3 and EC but not in CB. Rats that were stimulated but that did not develop spontaneous seizures later on had also some changes in gene expression, but this was not reflected in a significant change of a biological process. These data suggest that the targeting of specific genes that are involved in these biological processes may be a promising strategy to slow down or prevent the progression of epilepsy. Especially genes related to the immune response, such as complement factors, interleukins, and genes related to prostaglandin synthesis and coagulation pathway may be interesting targets.

Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy

Reactive gliosis is a prominent morphological feature of mesial temporal lobe epilepsy. Because astrocytes express glutamate receptors, we examined changes in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and transforming growth factor (TGF)‐β in glial cells of the hippocampal regions in an experimental rat model of spontaneous seizures. Rats that exhibited behavioural status epilepticus (SE) directly after 1 h of electrical angular bundle stimulation, displayed chronic spontaneous seizures after a latent period of 1–2 weeks as observed using continuous electrographic monitoring. SE resulted in hypertrophy of astrocytes and microglia activation throughout the hippocampus as revealed by immunolabelling studies. A dramatic, seizure intensity‐dependent increase in vimentin immunoreactivity (a marker for reactive astrocytes) was revealed in CA3 and hilar regions where prominent neuronal loss occurs. Increased vimentin labelling was first apparent 24 h after onset of SE and persisted up to 3 months. mGluR2/3 and mGluR5 protein expression increased markedly in glial cells of CA3 and hilus by 1 week after SE, and persisted up to 3 months after SE. Double immunolabelling of brain sections with vimentin confirmed co‐localization with glial fibrillary acidic protein (GFAP), mGluR2/3 and mGluR5 in reactive astrocytes. TGF‐β, a cytokine implicated in mGluR3‐mediated neuroprotection, was also upregulated during the first 3 weeks after SE throughout the hippocampus. This study demonstrates seizure‐induced upregulation of two mGluR subtypes in reactive astrocytes, which − together with the increased production of TGF‐β − may represent a novel mechanism for modulation of glial function and for changes in glial‐neuronal communication in the course of epileptogenesis.

Inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats

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.

Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain

Summary:  Purpose: Breast cancer resistance protein (BCRP) is a half adenosine triphosphate (ATP)‐binding cassette (ABC) transporter expressed on cellular membranes and included in the group of multidrug resistant (MDR)‐related proteins. Recently, upregulation of different MDR proteins has been shown in human epilepsy‐associated conditions. This study investigated the expression and cellular distribution of BCRP in human control and epileptic brain, including a large number of both neoplastic and nonneoplastic specimens from patients with chronic pharmacoresistant epilepsy.

Progression of temporal lobe epilepsy in the rat is associated with immunocytochemical changes in inhibitory interneurons in specific regions of the hippocampal formation

Immunocytochemical markers of specific rat hippocampal interneuron subpopulations, including the calcium binding proteins parvalbumin (PV), and calretinin (CR) were examined in relation to the evolution of spontaneous seizures after electrically induced status epilepticus (SE). PV/CR/NeuN immunoreactive neurons were counted in the hippocampal formation at different time intervals after SE and related to spontaneous hippocampal discharge activity. Decreased PV immunoreactivity was observed within 1 day after SE in the hilus, pre- and parasubiculum, and in the entorhinal cortex layers II and V/VI. In layer III, the density of detectable PV immunoreactive neurons did not decrease significantly, whereas the number of surrounding principal neurons was extensively decreased within a week in most post-SE rats, and after 3-4.5 months in all rats that had developed a progressive evolution of seizures. CR immunoreactive neuron number decreased in all hippocampal subregions except for the stratum lacunosum-moleculare and the EC layer II, in which the density did not decrease significantly. The apparent decrease in the number of PV and CR immunoreactive hilar neurons was correlated with the duration of the SE and was most extensive in rats with a progressive form of epilepsy. The loss of CR and PV expression or the loss of CR- and PV-containing neurons in specific regions of the hippocampal formation may play a role in the progressive nature of epilepsy possibly via increasing the entorhinal-hippocampal activity.

Inhibition of mammalian target of rapamycin reduces epileptogenesis and blood-brain barrier leakage but not microglia activation

Purpose:  Previous studies have shown that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin prevents epileptogenesis after pharmacologically induced status epilepticus (SE) in rat models of temporal lobe epilepsy. Because rapamycin is also known for its immunosuppressant properties we hypothesized that one of the mechanisms by which it exerts this effect could be via suppression of brain inflammation, a process that has been suggested to play a major role in the development and progression of epilepsy.

Hippocampal subregion-specific microRNA expression during epileptogenesis in experimental temporal lobe epilepsy

Since aberrant miRNA expression has been implicated in numerous brain diseases, we studied miRNA expression and miRNA regulation of important signaling pathways during temporal lobe epileptogenesis in order to identify possible targets for epilepsy therapy. The temporal profile of miRNA expression was analyzed in three brain regions (CA1; dentate gyrus, DG; parahippocampal cortex, PHC) associated with epileptogenesis in a rat model for temporal lobe epilepsy. Tissue was obtained after electrically-induced status epilepticus (SE) at 1day (n=5), 1week (n=5) and 3-4months (n=5), and compared with control tissue (n=10) using the Exiqon microRNA arrays which contain capture probes targeting all miRNAs for rat (p<0.01, and a 1.5 fold up- or downregulation). Expression of three blood plasma miRNAs from the same group of rats was also investigated in rats in order to determine whether plasma miRNAs could serve as potential biomarkers of the epileptogenic process. Molecular pathways potentially altered by the expression of multiple miRNAs were identified using a web-based algorithm, DIANA. In CA1 and DG, more upregulated than downregulated miRNAs were present during each stage after SE. The highest numbers of upregulated miRNAs were encountered during the chronic stage in the DG. In PHC, a high number of downregulated miRNAs were detected. Key pathways involved, based upon quantitatively altered miRNA expression were: axon guidance, MAPK signaling pathway, focal adhesion, TGFβ, ErbB-, Wnt- and mTOR signaling, and regulation of actin skeleton. Expression of plasma miRNAs was differentially regulated after induction of SE. This study identified several signaling pathways possibly involved in temporal lobe epileptogenesis, not previously indicated by RNA microarray studies. These include miRNAs that regulate the ErbB and Wnt pathways and focal adhesion, which may represent interesting new targets for therapeutic interventions.

Expression of Multidrug Transporters MRP1, MRP2, and BCRP Shortly after Status Epilepticus, during the Latent Period, and in Chronic Epileptic Rats

Summary:  Purpose: Overexpression of multidrug transporters may play a role in the development of pharmacoresistance by decreasing extracellular drug levels in the brain. However, it is not known whether overexpression is due to an initial insult or evolves more gradually because of recurrent spontaneous seizures. In the present study, we investigated the expression of different multidrug transporters during epileptogenesis in the rat. In addition, we determined whether these transporters affected phenytoin (PHT) distribution in the brain.

Altered hippocampal gene expression prior to the onset of spontaneous seizures in the rat post‐status epilepticus model

Neuronal loss, gliosis and axonal sprouting in the hippocampal formation are characteristics of the syndrome of mesial temporal sclerosis (MTS). In the post‐status epilepticus (SE) rat model of spontaneous seizures these features of the MTS syndrome can be reproduced. To get a global view of the changes in gene expression in the hippocampus we applied serial analysis of gene expression (SAGE) during the early phase of epileptogenesis (latent period), prior to the onset of the first spontaneous seizure. A total of 10 000 SAGE tags were analyzed per experimental group, resulting in 5053 (SE) and 5918 (control group) unique tags (genes), each representing a specific mRNA transcript. Of these, 92 genes were differentially expressed in the hippocampus of post‐SE rats in comparison to controls. These genes appeared to be mainly associated with ribosomal proteins, protein processing, axonal growth and glial proliferation proteins. Verification of two of the differentially expressed genes by in situ hybridization confirmed the changes found by SAGE. Histological analysis of hippocampal sections obtained 8 days after SE showed extensive cell loss, mossy fibre sprouting and gliosis in hippocampal sub regions. This study identifies new high‐abundant genes that may play an important role in post‐SE epileptogenesis.

COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats

Epileptic seizures drive expression of the blood-brain barrier efflux transporter P-glycoprotein via a glutamate/cyclooxygenase-2 mediated signalling pathway. Targeting this pathway may represent an innovative approach to control P-glycoprotein expression in the epileptic brain and to enhance brain delivery of antiepileptic drugs. Therefore, we tested the effect of specific cyclooxygenase-2 inhibition on P-glycoprotein expression in two different status epilepticus models. Moreover, the impact of a cyclooxygenase-2 inhibitor on expression of the efflux transporter and on brain delivery of an antiepileptic drug was evaluated in rats with recurrent spontaneous seizures. The highly selective cyclooxygenase-2 inhibitors SC-58236 and NS-398 both counteracted the status epilepticus-associated increase in P-glycoprotein expression in the parahippocampal cortex and the ventral hippocampus. In line with our working hypothesis, a sub-chronic 2-week treatment with SC-58236 in the chronic epileptic state kept P-glycoprotein expression at control levels. As described previously, enhanced P-glycoprotein expression in chronic epileptic rats was associated with a significant reduction in the brain penetration of the antiepileptic drug phenytoin. Importantly, the brain delivery of phenytoin was significantly enhanced by sub-chronic cyclooxygenase-2 inhibition in rats with recurrent seizures. In conclusion, the data substantiate targeting of cyclooxygenase-2 in the chronic epileptic brain as a promising strategy to control the expression levels of P-glycoprotein despite recurrent seizure activity. Cyclooxygenase-2 inhibition may therefore help to increase concentrations of antiepileptic drugs at the target sites in the epileptic brain. It needs to be further evaluated whether the approach also enhances efficacy.