RESUMEN
Blood-brain barrier dysfunction in epilepsy contributes to seizures and resistance to antiseizure drugs. Reports show that seizures increase brain glutamate levels, leading to barrier dysfunction. One component of barrier dysfunction is overexpression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Based on our previous studies, we hypothesized that glutamate released during seizures activates cytosolic phospholipase A2 (cPLA2), resulting in P-gp and BCRP overexpression. We exposed isolated rat brain capillaries to glutamate ex vivo and used an in vivo-ex vivo approach of isolating brain capillaries from rats after status epilepticus (SE) and in chronic epileptic (CE) rats. Glutamate increased cPLA2, P-gp, and BCRP protein and activity levels in isolated brain capillaries. We confirmed the role of cPLA2 in the signaling pathway in brain capillaries from male and female mice lacking cPLA2. We also demonstrated, in vivo, that cPLA2 inhibition prevents overexpression of P-gp and BCRP at the blood-brain barrier in rats after status epilepticus and in CE rats. Our data support the hypothesis that glutamate signals cPLA2 activation, resulting in overexpression of blood-brain barrier P-gp and BCRP.-Hartz, A. M. S., Rempe, R. G., Soldner, E. L. B., Pekcec, A., Schlichtiger, J., Kryscio, R., Bauer, B. Cytosolic phospholipase A2 is a key regulator of blood-brain barrier function in epilepsy.
Asunto(s)
Barrera Hematoencefálica/enzimología , Epilepsia/enzimología , Fosfolipasas A2 Grupo IV/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/enzimología , Capilares/enzimología , Epilepsia/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Genotipo , Ácido Glutámico/farmacología , Fosfolipasas A2 Grupo IV/genética , Masculino , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo/ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage.SIGNIFICANCE STATEMENT The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A2, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.
Asunto(s)
Barrera Hematoencefálica/patología , Epilepsia/patología , Metaloproteinasas de la Matriz/metabolismo , Animales , Capilares/efectos de los fármacos , Femenino , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Convulsiones/patología , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Proteínas de Uniones Estrechas/metabolismoRESUMEN
A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Epilepsia/metabolismo , Convulsiones/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Capilares/metabolismo , Modelos Animales de Enfermedad , Femenino , Ácido Glutámico/metabolismo , Humanos , Ratas , Ratas Wistar , Regulación hacia Arriba/fisiologíaRESUMEN
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.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Epilepsia del Lóbulo Temporal/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Animales , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Radioisótopos de Carbono , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Hipocampo/diagnóstico por imagen , Hipocampo/metabolismo , Humanos , Fenobarbital/metabolismo , Fenobarbital/farmacología , Fenobarbital/uso terapéutico , Tomografía de Emisión de Positrones , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Convulsiones/diagnóstico por imagen , Convulsiones/metabolismoRESUMEN
Up-regulation of the blood-brain barrier efflux transporter P-glycoprotein in central nervous system disorders results in restricted brain access and limited efficacy of therapeutic drugs. In epilepsies, seizure activity strongly triggers expression of P-glycoprotein. Here, we identified the prostaglandin E2 receptor, EP1, as a key factor in the signaling pathway that mediates seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier. In the rat pilocarpine model, status epilepticus significantly increased P-glycoprotein expression by 92 to 197% in the hippocampal hilus and granule cell layer as well as the piriform cortex. The EP1 receptor antagonist 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-[1-oxo-3-(4-pyridinyl)propyl]hydrazide hydrochloride (SC-51089) abolished seizure-induced P-glycoprotein up-regulation and retained its expression at the control level. The control of P-glycoprotein expression despite prolonged seizure activity suggests that EP1 receptor antagonism will also improve antiepileptic drug efficacy. Preliminary evidence for this concept has been obtained using a massive kindling paradigm during which animals received a subchronic SC-51089 treatment. After withdrawal of the EP1 receptor antagonist, a low dose of the P-glycoprotein substrate phenobarbital resulted in an anticonvulsant effect in this pretreated group, whereas the same dosage of phenobarbital did not exert a significant effect in the respective control group. In conclusion, our data demonstrate that EP1 is a key signaling factor in the regulatory pathway that drives P-glycoprotein up-regulation during seizures. These findings suggest new intriguing possibilities to prevent and interrupt P-glycoprotein overexpression in epilepsy. Future studies are necessary to further evaluate the appropriateness of the strategy to enhance the efficacy of antiepileptic drugs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Receptores de Prostaglandina E/efectos de los fármacos , Convulsiones/prevención & control , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Anticonvulsivantes/farmacología , Western Blotting , Capilares/efectos de los fármacos , Electrodos Implantados , Femenino , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Excitación Neurológica/efectos de los fármacos , Agonistas Muscarínicos , Fenobarbital/farmacología , Pilocarpina , Ratas , Ratas Wistar , Subtipo EP1 de Receptores de Prostaglandina E , Convulsiones/genética , Estado Epiléptico/inducido químicamente , Estado Epiléptico/prevención & control , Regulación hacia Arriba/fisiologíaRESUMEN
PURPOSE: The aim of this study is to non-invasively assess early, irradiation-induced normal tissue alterations via metabolic imaging with 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT). PROCEDURES: Twenty-nine male C57BL/6 mice were investigated by [(18) F]FLT positron emission tomography for 7 days after total body irradiation (1, 4, and 8 Gy) versus 'sham' irradiation (0 Gy). Target/background ratios were determined. The imaging results were validated by histology and immunohistochemistry (Thymidine kinase 1, Ki-67). RESULTS: [(18) F]FLT demonstrated a dose-dependent intestinal accumulation post irradiation. Mean target/background ratio (±standard error) 0 Gy: 1.4 (0.2), 1 Gy: 1.7 (0.1), 4 Gy: 3.1 (0.3), 8 Gy: 4.2 (0.6). Receiver operating characteristic analysis (area under the curve, p value): 0 vs. 1 Gy: 0.81, 0.049; 0 vs. 4 Gy: 1.0, 0.0016; and 0 vs. 8 Gy: 1.0, 0.0020. Immunohistochemistry confirmed the results. CONCLUSIONS: [(18) F]FLT seems to provide dose-dependent information on radiation-induced proliferation in the bowel. This opens the perspective for monitoring therapy-related side-effects as well as assessing, e.g., radiation accident victims.
Asunto(s)
Didesoxinucleósidos/farmacocinética , Intestino Grueso/metabolismo , Intestino Grueso/efectos de la radiación , Radiofármacos/farmacocinética , Irradiación Corporal Total/métodos , Animales , Didesoxinucleósidos/química , Relación Dosis-Respuesta en la Radiación , Inmunohistoquímica , Intestino Grueso/química , Masculino , Ratones , Ratones Endogámicos C57BL , Radiofármacos/químicaRESUMEN
UNLABELLED: The progression of ß-amyloid deposition in the brains of mice overexpressing Swedish mutant ß-amyloid precursor protein (APP-Swe), a model of Alzheimer disease (AD), was investigated in a longitudinal PET study using the novel ß-amyloid tracer (18)F-florbetaben. METHODS: Groups of APP-Swe and age-matched wild-type (WT) mice (age range, 10-20 mo) were investigated. Dynamic emission recordings were acquired with a small-animal PET scanner during 90 min after the administration of (18)F-florbetaben (9 MBq, intravenously). After spatial normalization of individual PET recordings to common coordinates for mouse brain, binding potentials (BPND) and standardized uptake value ratios (SUVRs) were calculated relative to the cerebellum. Voxelwise analyses were performed using statistical parametric mapping (SPM). Histochemical analyses and ex vivo autoradiography were ultimately performed in a subset of animals as a gold standard assessment of ß-amyloid plaque load. RESULTS: SUVRs calculated from static recordings during the interval of 30-60 min after tracer injection correlated highly with estimates of BPND based on the entire dynamic emission recordings. (18)F-florbetaben binding did not significantly differ in APP-Swe mice and WT animals at 10 and 13 mo of age. At 16 mo of age, the APP-Swe mice had a significant 7.9% increase (P < 0.01) in cortical (18)F-florbetaben uptake above baseline and at 20 mo there was a 16.6% increase (P < 0.001), whereas WT mice did not show any temporal changes in tracer uptake during the interval of follow-up. Voxelwise SPM analyses revealed the first signs of increased cortical binding at 13 mo and confirmed progressive binding increases in both the frontal and the temporal cortices (P < 0.001 uncorrected) to 20 mo. The SUVR strongly correlated with percentage plaque load (R = 0.95, P < 0.001). CONCLUSION: In the first longitudinal PET study in an AD mouse model using the novel ß-amyloid tracer (18)F-florbetaben, the temporal and spatial progression of amyloidogenesis in the brain of APP-Swe mice were sensitively monitored. This method should afford the means for preclinical testing of novel therapeutic approaches to the treatment of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Compuestos de Anilina/farmacocinética , Encéfalo/metabolismo , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Estilbenos/farmacocinética , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/diagnóstico por imagen , Estudios Longitudinales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución TisularRESUMEN
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.