Ex vivo characterization of neuroinflammatory and neuroreceptor changes during epileptogenesis using candidate positron emission tomography biomarkers

Objective: Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease. // Methods: As development of novel radiotracers and on‐site setup of existing radiotracers is highly time‐consuming and expensive, we used dual‐centre in vitro autoradiography as an approach to characterize the potential of innovative radiotracers in the context of epilepsy development. Using brain slices from the same group of rats, we aimed to characterise the evolution of neuroinflammation and expression of inhibitory and excitatory neuroreceptors during epileptogenesis using translational positron emission tomography (PET) tracers; 18F‐flumazenil (18F‐FMZ; GABAA receptor), 18F‐FPEB (metabotropic glutamate receptor 5; mGluR5), 18F‐flutriciclamide (translocator protein; TSPO, microglia activation) and 18F‐deprenyl (monoamine oxidase B, astroglia activation). Autoradiography images from selected time points after pilocarpine‐induced status epilepticus (SE; baseline, 24 and 48 hours, 5, 10 and 15 days and 6 and 12‐14 weeks after SE) were normalized to a calibration curve, co‐registered to an MRI‐based 2D region‐of‐interest atlas, and activity concentration (Bq/mm2) was calculated. // Results: In epileptogenesis‐associated brain regions, 18F‐FMZ and 18F‐FPEB showed an early decrease after SE. 18F‐FMZ decrease was maintained in the latent phase and further reduced in the chronic epileptic animals, while 18F‐FPEB signal recovered from day 10, reaching baseline levels in chronic epilepsy. 18F‐flutriciclamide showed an increase of activated microglia at 24 hours after SE, peaking at 5‐15 days and decreasing during the chronic phase. On the other hand, 18F‐deprenyl autoradiography showed late astrogliosis, peaking in the chronic phase. // Significance: Autoradiography revealed different evolution of the selected targets during epileptogenesis. Our results suggest an advantage of combined imaging of inter‐related targets like glutamate and GABAA receptors, or microglia and astrocyte activation, in order to identify important interactions, especially when using PET imaging for the evaluation of novel treatments

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy

Alteration in P-glycoprotein Functionality Affects Intrabrain Distribution of Quinidine More Than Brain Entry—A Study in Rats Subjected to Status Epilepticus by Kainate

This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20 mg/kg quinidine over 30 min or 4 h. Plasma, brain extracellular fluid (brain ECF), and end-of-experiment total brain concentrations of quinidine were determined during 7 h after the start of the infusion. Effect of pretreatment with tariquidar (15 mg/kg, administered 30 min before the start of the quinidine infusion) on the brain distribution of quinidine was assessed. This approach was repeated in kainate-treated rats. Quinidine kinetics were analyzed with population modeling (NONMEM). The quinidine microdialysis assay clearly revealed differences in brain distribution upon changes in P-glycoprotein functionality by pre-administration of tariquidar, which resulted in a 7.2-fold increase in brain ECF and a 40-fold increase in total brain quinidine concentration. After kainate treatment alone, however, no difference in quinidine transport across the blood–brain barrier was found, but kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This study did not provide evidence for the hypothesis that P-glycoprotein function at the blood–brain barrier is altered at 1 week after SE induction, but rather suggests that P-glycoprotein function might be altered at the brain parenchymal level

(R)-[11C]Verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus

<p>Abstract</p> <p>Background</p> <p>Increased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp) expression and functionality at an early stage after induction of status epilepticus by kainate.</p> <p>Methods</p> <p><it>(R)</it>-[¹¹C]verapamil, which is currently the most frequently used positron emission tomography (PET) ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control) treated rats, at 7 days after treatment. To investigate the effect of P-gp on <it>(R)</it>-[¹¹C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. <it>(R)</it>-[¹¹C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles) as well as by population mixed effects modelling (NONMEM).</p> <p>Results</p> <p>All data analysis approaches indicated only modest differences in brain distribution of <it>(R)</it>-[¹¹C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats.</p> <p>Conclusions</p> <p>P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.</p

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions

Ex vivo characterization of neuroinflammatory and neuroreceptor changes during epileptogenesis using candidate positron emission tomography biomarkers

Objective: Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease. // Methods: As development of novel radiotracers and on‐site setup of existing radiotracers is highly time‐consuming and expensive, we used dual‐centre in vitro autoradiography as an approach to characterize the potential of innovative radiotracers in the context of epilepsy development. Using brain slices from the same group of rats, we aimed to characterise the evolution of neuroinflammation and expression of inhibitory and excitatory neuroreceptors during epileptogenesis using translational positron emission tomography (PET) tracers; 18F‐flumazenil (18F‐FMZ; GABAA receptor), 18F‐FPEB (metabotropic glutamate receptor 5; mGluR5), 18F‐flutriciclamide (translocator protein; TSPO, microglia activation) and 18F‐deprenyl (monoamine oxidase B, astroglia activation). Autoradiography images from selected time points after pilocarpine‐induced status epilepticus (SE; baseline, 24 and 48 hours, 5, 10 and 15 days and 6 and 12‐14 weeks after SE) were normalized to a calibration curve, co‐registered to an MRI‐based 2D region‐of‐interest atlas, and activity concentration (Bq/mm2) was calculated. // Results: In epileptogenesis‐associated brain regions, 18F‐FMZ and 18F‐FPEB showed an early decrease after SE. 18F‐FMZ decrease was maintained in the latent phase and further reduced in the chronic epileptic animals, while 18F‐FPEB signal recovered from day 10, reaching baseline levels in chronic epilepsy. 18F‐flutriciclamide showed an increase of activated microglia at 24 hours after SE, peaking at 5‐15 days and decreasing during the chronic phase. On the other hand, 18F‐deprenyl autoradiography showed late astrogliosis, peaking in the chronic phase. // Significance: Autoradiography revealed different evolution of the selected targets during epileptogenesis. Our results suggest an advantage of combined imaging of inter‐related targets like glutamate and GABAA receptors, or microglia and astrocyte activation, in order to identify important interactions, especially when using PET imaging for the evaluation of novel treatments

99mTc-HMPAO SPECT imaging reveals brain hypoperfusion during status epilepticus

Status epilepticus (SE) is a clinical emergency with high mortality. SE can trigger neuronal death or injury and alteration of neuronal networks resulting in long-term cognitive decline or epilepsy. Among the multiple factors contributing to this damage, imbalance between oxygen and glucose requirements and brain perfusion during SE has been proposed. Herein, we aimed to quantify by neuroimaging the spatiotemporal course of brain perfusion during and after lithium-pilocarpine-induced SE in rats. To this purpose, animals underwen