The Role Of Thrombospondin-1 In Angiogenesis In Kainic Acid-induced Acute Generalized Seizures And Synaptogenesis In Amygdala-kindling Epilepsy

Background and Purpose:Angiogenesis and synaptogenesis is the common pathological change in the development of epilepsy and epileptic seizure.The relevant study referred to thrombospondin-1(TSP-1)play a vital role in synaptogenesis and angiogenesis.We have reason to suspect that TSP-1 may be the key factor in the development of epileptic seizures.But the role of TSP-1 in the development epilepsy was not clear.The study aim to investigate the change of TSP-1 in kainic acid induced acute generalized seizure and the progression of amygdala kindling induced epielpsy and analysis the role and mechanism of TSP-1 in synaptogenesis and angiogenesis.Methods:The study consists of two parts.Firstly,kainic acid(KA)was injected in the left lateral ventricle to induce acute generalized seizure model.Western blotting,immunohistochemical and flow cytometry were used to detect the marker of angiogenesis(VEGF/CD34).We assessed the amount of Evans Blue in the brain as a measure of BBB breakdown.By genetic and pharmacological interventions(inhibiting TSP-1 expression with small interfering RNA(siRNA)or TGF-?1 activation by Leu-Ser-Lys-Leu peptide(LSKL),or blocking purinergic receptor(P2R)or P2Y4 receptors(PPADS or Reactive Blue 2)),the expression of relevant protein in the TSP-1/TGF-?1/Smad2/3 pathway and the amount of Evans Blue were detected to evaluate the impact of those interventions in BBB and angiogenesis.Animals behaviors and electroencephalography(EEG)were recorded to evaluate the severity of epileptic seizure.Secondly,in amygdala kindling-induced epilepsy,western blotting,immunohistochemical and flow cytometry were used to detect the marker of synapse/excitatory synapse(Synapsin-I/PSD95/GluT)and the expression of relevant protein in the TSP-1/TGF-?1 pathway expression.The effects of siRNA interference and pharmacological inhibition(LSKL,PPADS and Reactive Blue 2)were used to interfere the different target in the P2Y4/TSP-1/TGF-?1 pathway.Then we observed the expression of downstreams molecular protein in the pathway and evaluated the epileptic progression and synaptogenesis.Key Results:1)The increased expression of VEGF/CD34(the marker of angiogenesis)synchronized to those of TSP-1 immunoreactivity in KA-induced acute generalized seizures.Rats treated with PPADS,Reactive Blue 2,LSKL or siRNA showed that significantly reduced level of Smad2/3 phosphorylation and attenuated angiogenesis,BBB damage,and seizure severity.2)In amygdala kindling-inducing seizure model,the marker of synapses/excitatory synapse(Synapsin-I/PSD95/GluT)regularly increased in several brain regions(such as hippocampus)which were associated with progressing of epileptic discharges after kindling,accompanied with the increased TSP-1/TGF-?1 expression.Genetic or pharmacologic methods were used to inhibit TSP-1 expression or the activation of TGF-?1.The results indicate that genetic or pharmacologic methods partly reversed the increased synapses/excitatory synapses and inhibited the generalization of focal epilepsy.Conclusions:The P2R/TSP-1/TGF-?1 pathway may participate in KA-induced acute generalized seizures and angiogenesis,as well as amygdala kindling-induced epileptic progression and synaptogenesis.Appropriately inhibiting this pathway prevented synaptogenesis and angiogenesis,inhibited development of epilepsy and reduced seizure severities.