AMPA Receptor Antagonist NBQX Decreased Seizures By Normalization Of Perineuronal Nets

ObjectEpilepsy is a serious life-shortening neurological disorder,which affects approximately 1%of general population and leads to an increase of disability and mortality in the world.Epilepsy is a diverse phenotype including more than 15 different seizure types and more than 30 epilepsy syndromes.The core symptom of epilepsy is recurring,unprovoked seizures induced by the abnormal synchronous activity of cerebral neuronal networks.The abnormal hypersynchronous activity induced by impaired inhibition increased extracellular potassium and enhanced excitatory synaptic transmission that are involved in the pathological process in epilepsy.Glutamate is a predominant neurotransmitter released from excitatory neurons related to the fast synaptic excitation.The released glutamates diffuse across the synapticcleft and generate fast excitatory synaptic potentials(EPSPs)through binding to ionotropic glutamate receptors.The cumulative EPSPs of indivi dual neurons trigger action potentials,which are responsible for epileptic field potentials.Glutamate receptors of the AMPA(a-amino-3-hydroxy-5-methyl-4-iso xazolepropionic acid)subtype have been evidenced to play a key role in epilep togenesis.AMPA receptors regulate the fast synaptic excitation in brain regions that are related to epilepsy.Specifically,AMPA receptor antagonists markedly reduce epileptiform and inhibit spread of epileptic discharges in both animal seizure models and human epilepsy.In addition,growing evidence validate the critical role of AMPA receptors in epileptic seizures,and suggest that AMPA receptors may be a potential target for epilepsy therapy.Rodent models of epilepsy is used and it showed that perampanel,a potent,selective,orally active non-competitive AMPA receptor antagonist,exhibited a strong antiseizure activity in the maximal electroshock seizure test,and the 6 Hz seizure test.Moreover,clinical data showed that perampanel with daily dose is effective in patients with refractory partial-onset seizures.NBQX(2,3-dihydroxy-6-nitro-7-sulfamoyl-ben zoquinoxaline-2,3-dione)is a competitive AMPA receptor antagonist and has a property that suppressed focal electrographic seizures in epileptic mice.Recently,it was described that NBQX blocks the development of spontaneous recurrent seizures when treatment after neonatal seizures.Consistently,noncompetitive AMPA antagonist perampanel improved the performance of partial seizures,sug gesting a remarkable antiepileptogenic effect.Perineuronal nets(PNNs)are condensed extracellular matrix(ECM)structu res,which surround parvalbumin-positive inhibitory interneurons and play a critical role in neuronal cell development,activity and growth.Chondroitin sulfate proteoglycans(CSPGs)are crucial components of PNNs,which have been sug gested to be a critical factor regulating synaptic plasticity.Aberrant PNN signal ing was found to induce the dysfunctions of central nervous system such as epilepsy,stroke,Alzheimer's disease,schizophrenia and addiction.The plant lectins Wisteria floribunda agglutinin(WFA)can be used to visualize PNNs by binding to N-acetylgalactosamine residue.Therefore,we use WFA-labeled neurons to evaluate the numbers of PNNs in the current investigation.The three major ECM components of PNNs are tenascin-R,aggrecan and neurocan,which are important contributors to formation and stabilization of PNNs functions.Pentylenetetrazole(PTZ),ag-aminobutyric acid antagonist,is widely used to establish experimental models simulate human epilepsy.PTZ administration increased the glutamatergic transmitter and induced generalized tonic-clonic seizures in animals with higher doses.In the current study,we used PTZ at the dose of 50 mg/kg(i.p.)to induce chronic seizures to observe the anti-epileptic effect of NBQX and the possible involvement of PNNs.Materials and methods 1.AnimalsMale Wistar rats that weighed 220-240 gupon arrival were obtained fromthe Shandong University Experimental Animal Center and were used for all experiments.Rats were individually housed under constant temperature(23 ±2?)and humidity(50 ? 5%)and maintained on a 12-hour light/dark cycle with free food and water available.Rats were monitored daily by experimenter at 8:00-12:00 am each day for eating,drinking,and activity,which were used for the physical health assessment.There are no animals died before the experimenta 1 endpoint.All of the animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The protocol of current animal experiments was approved by the Ethics of Animal Experiments of Jinan Central Hospital(Permit Number:SA-2014-008).All surgery was performed under sodium pentobarbital anesthesia,and all efforts were made to minimize suffering in animals.All of the behavioral tests and drug administrations were carried out in the dark phase.2.DrugsAMPAR antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline-2,3-dione(NBQX),pentylenetetrazole(PTZ)and chondroitinase ABC(ChABC)were supplied from Sigma-Aldrich Chemical Company(Sigma,St Louis.Missouri,USA).NBQX was freshly dissolved in saline as sodium salt.ChABC were dissolved in 0.1 M PBS(vehicle)for microinjection into the medial prefrontal cortex and prepared in stock solutions of 0.02 U/?l.3.Seizures induction and behavioral analysisRats were deprived of food but not water 12h before the experiments to prevent aspiration of food.Rats in PTZ group were treated with a single 50 mg/kg intraperitoneal(i.p.)administration of PTZ for 28 days.While rats were injected with saline(i.p.)in control group instead.Behavioral tests and neuroche mical analysis were performed on day 29 and 30,respectively.To observe anti-epileptic effects of NBQX in PTZ induced epilepsy,we divided rats into four groups:rats in saline + saline group were treated with saline only;rats in PTZ + saline group were treated with 50 mg/kg of PTZ(i.p.)and saline for 28 days;rats in saline + NBQX group were treated with saline for 28 days,and 20 mg/kg of NBQX(i.p.)for next 3 days;rats in PTZ +NBQX group were treated with 50 mg/kg of PTZ(i.p.)for 28 days and were treated with 20 mg/kg of NBQX(i.p.)for next 3 days.Behavioral tests and n eurochemical analysis were performed on the following 2 days.The doses for PDZ and NBQX were selected regarding to previous studies.To clarify the effects of degradation of PNNs caused by ChABC in mPFC on the seizures,we used four groups of rats:saline + penicillinase group was treated with saline plus penicillinase into mPFC on d24;saline + ChABC group was treated with saline and microinjection of ChABC(0.01 U/p,g/side/0.5?l)into mPFC on d24;PTZ + penicillinase group was treated with PTZ(50 mg/kg)for 28 days and microinjected with penicillinase on d24;PTZ + ChABC group was treated with PTZ for 28 days and microinjected with ChABC on d24.Behavioral tests and neurochemical analysis were performed on the folio wing 2 days.To determine whether PNNs degradation by ChABC can reverse the anti-e pileptic effect of NBQX,we injected rats with PTZ for 28 days and separated them into four groups:rats in vehicle group were treated with vehicle without NBQX,and were microinjected with penicillinase into mPFC on d 24;rats in vehicle + ChABC group were treated with vehicle plus microinjection of ChA BC into mPFC on d24;rats in NBQX + penicillinase group were treated with penicillinase on d24 and were treated with NBQX injection on d 29 to d31;rats in NBQX + ChABC group were treated with microinjection of ChABC into mPFC on d24 plus NBQX(20 mg/kg,i.p.)on d 29 to d31.Behavioral tests were performed on day 32.The epileptic seizure activity induced by PTZ was evaluated by latency to seizures(s),duration of the minor seizure onset(s),duration of the major sei zure onset(s),and scores for the severity of seizures in 1h after PTZ injection.The minor seizure onset was termed as isolated myoclonic jerks and clonic seizures accompanied by facial and front extremity muscle clonus.While the maj or seizure following the minimal seizure are characterized by head,neck,and tail extension with the loss of the tonic flexor reflex and tonic flexion-extensio n following the protracted clonus.The scores were used to measure the severit y of seizures after PTZ administration according to the following level:0:no changes in behavior;1:isolated myclonic jerks;2:only atypical minimal seizur es;3:minimal seizures;4:major seizures without atonic phase;and 5:complet ed tonic-clonic seizures.The performance of each rat was recorded by a video camera during the entire experimental procedures.The observers for the meas urement of seizure scores were blind to the treatment of each group.4.Immunofluorescence and image analysisAfter the behavioral measurement,rats were deeply anesthetized with sodium pentobarbital(100 mg/kg,i.p.)and were intracardially perfused with 200-250 ml of 0.1M phosphate-buffered saline,pH 7.4,followed by 200-250 ml of 4%paraformaldehyde phosphate buffer,pH 7.4.The brains were then postfixed at 4? for 24 hand dehydrated in 30%sucrose for at least 4 days.Serial coron al 30 ?m brain sections that contained the medial prefrontal cortex were cut on a Leica freezing microtome and stored in a cryoprotectant solution at-20?.The sections were incubated overnight at 4? in a solution of biotin-conjugated lectin wisteria floribunda.All of the sections were then washed 3 times in PBS and then incubated in FITC-conjugated streptavidin in 25 ? for 3 h.Four or five sections from each brain region of each rat were selected.Fluorescence microscope with an image-analysis program was used for measuring the nu mber of WFA-positive PNNs.The average number of PNNs on either side of target brain region was taken as the positive immunoreactive cell number for each rat as previously reported.5.Tissue sample preparationRats were killed 30 min after the last administration of PTZ.Their brains were extracted and removed.Subsequently,bilateral tissue punches of the mPFC were obtained from approximately 1 mm thick coronal sections cut in a Reichert-Jung 2800 Frigocut E cryostat at-20?.The rostral faces of the coronal sections were approximately 3.8 mm from bregma.Tissue punches were homogenized with an electrical disperser after being lysed with RIPA lysis buffer with protease-inhibitor for 30 min.Afterward,the homogenate was subjected to 10,000 xg centrifugation at 4?for 20 min.All of the above procedures were performed under low temperature(0-4?).The protein concentrations of all samples were determined using the BCA assay kit.The protein concentration was normalized by diluting the samples with RIPA lysis buffer.6.Western blot assaysSamples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for approximately 30 min at 80 V in stacking gel and approximately 1 h at 120 V in resolving gel.Proteins were transferred electrophoretically to Immobilon-P transfer membranesat 0.25 A for 3 h.Membranes were washed with TBST before dipping in blocking buffer overnight at 4?.Membranes were then incubated for 1 h at room temperature with anti-tenascin-R,anti-aggrecan,anti-neurocan,and anti-?-actin antibody in TBST plus 5%bovine serum albumin.After the membrane was shaken in 4×6 min washes in TBST buffer,the blots were incubated for 45 min at room temperature with horseradish peroxidase-conjugated secondary antibody diluted 1:5000 in blocking buffer.The blots were then shaken in 4 x 6 min washes in TBST.Afterward,the blots were incubated with a layer of Super Signal enhanced chemiluminescence substrate mixture for 1 min at room temperature.Finally,the blots were exposed against X-ray film.Band intensities were quantified using Quantity One software from Bio-Rad Corporation.7.Intracerebral cannula implantation and intracranial injectionsRats were anesthetized with sodium pentobarbital(60 mg/kg,i.p.),and guide cannulae were implanted bilaterally 1 mm above the medial prefrontal cortex with the following stereotaxic coordinates:anterior/posterior(A/P),-3.2 mm;medial/lateral(M/L),±2.5 mm;dorsal/ventral(D/V),-3.3 mm.The rats were allowed to recover for at least 7 d before intracranial injections.Vehicle or ChABCwere intracranial microinjected using 10?l Hamilton syringes connected via polyethylene-50 tubing to 30-gauge injectors into the mPFC.The dose of ChABC was used as previously evidenced.A total volume of 0.5 ?l was infused into each side over 1 min,and the injection syringe was left in place for an additional 1 min to allow for diffusion.At the end of the behavioral tests,the rats were anesthetized with sodium pentobarbital(100 mg/kg,i.p.)and transcardially perfused.Cannula placements were assessed using Nissl staining with a thickness of 30 ?m under a light microscopeto determine the infusion site.Subjects with misplaced cannulae were excluded from the statistical analysis.Results1.The behavioral patterns of epileptic seizures induced by PTZ2.Chronic PTZ treatment reduced WFA,tenascin-R,aggrecan and neurocan in mPFC3.AMPA receptor antagonist NBQX decreased seizures induced by PTZ 4.NBQX normalized WFA,tenascin-R,aggrecan and neurocan in mPFC5.The degradation of PNNs caused by ChABC in mPFC exacerbated seizures.6.ChABC reversed the anti-epileptic effects of NBQX in PTZ-induced seizuresConclusionIn summary,the present data showed that AMPA receptor antagonist NBQ X decreased the onset of epileptic seizures induced by PTZ through regulation of PNNs in the medial prefrontal cortex.Degradation of PNNs caused by ChABC in mPFC not only exacerbated seizures but also reversed the anti-epileptic effect of NBQX in PTZ-treated rats.These findings therefore revealed that PNNs in the medial prefrontal cortex is related to the anti-epileptic effect of NBQX and enhancement of PNNs may be effective for the treatment of epilepsy.