Targeting Diagnosis And Treatment Value Of Functional Magnetic Nanoparticles In Temporal Lobe Epilepsy Model

Objective:Epilepsy is one of the most common diseases in neurology.Most patients with epilepsy are controlled by standardized antiepileptic drugs.However,20%-30%patients with epilepsy is difficult to alleviate and become refractory epilepsy and temporal lobe epilepsy is the most common type.The current treatment methods of epilepsy mainly include drug and surgical treatment.The key of surgical treatment is the accurate position of the epileptic focus and the key of drugs treatment is that antiepileptic drugs are better able to reach the epileptic tissues.MRI is one of the most common imaging methods in the diagnosis of epileptic foci due to high image definition,no ionizing radiation and no radiation damage to human body.In recent years,magnetic nanoparticles are widely used in various fields due to its unique advantages.The superparamagnetic iron oxide nanoparticles(SPIONs)can penetrate the physical barrier to the designated position.As a dye molecule in near infrared region,Cy5 can be used as a fluorescent probe molecule for optical imaging.Alpha-methyl-L-tryptophan(AMT)is an external synthetic tryptophan,which has high uptake and aggregation in epileptic foci.In this study,we chelate Cy5 and AMT on SPIONs to construct Cy5-AMT-SPIONs magnetic and optical dual-mode imaging and prove the active targeting positioning of AMT in temporal lobe epilepsy model.However,AMT can not own the function of targeting treatment of epilepsy lesions.Recent studies have shown that inflammation is closely related to the seizure and IL-1? is a common inflammatory factor.Using IL-1? monoclonal antibody to neutralize IL-1? concentration can reduce seizures.However,the presence of blood brain barrier prevents the IL-1? monoclonal antibody from entering the epileptic focus.We chelate IL-1 ? to superparamagnetic iron oxide nanoparticles as anti-IL-1?mAb-SPIONs.With the help of SPIONs easily via the blood brain barrier,we observe the targeting diagnosis and treatment value of anti-IL-1?mAb-SPIONs.Method:We used lithium-pilocarpine to induce temporal lobe epilepsy model and the experiment was divided into two parts and each experiment included three groups.In the first part of the experiment,we choose Cy5-AMT-SPIONs as the experimental group,Cy5-SPIONs and plain-SPIONs as control group.These particles wereinjected 48 hours after epileptic seizures.The MRI were performed before and four hours after the particles injection and the T2 values were measured and compared.Perl,s iron staining was observed the distribution of iron particles in the epileptic foci.Nissl's staining was observed the damage of hippocampal neurons.The immunofluorescence staining was observed the distribution of Cy5 in the epileptic foci and the situation of astrogliosis.In the second part of the experiment,we choose anti-IL-1? mAb-SPIONs as the experimental group,plain-SPIONs and normal saline(NS)as control group and injected these particles 3days and 14 days after epileptic seizures.The MRI were performed before and four hours after the particles injection and the T2 values were measured and compared and applied Perl's iron staining,Nissl's staining and the immunofluorescence staining.In addition,the Western blot and RT-PCR were to detecte the expression of IL-1? and NF-?B.Result:1.Most the SD rats appeared generalized tonic clonic seizure finally.The success rate of the model was 80%(40 rats in total,35 of them established a successful acute temporal lobe epilepsy model,and 3 died after,a total survival of 32finally)in the first part of the experiment.The success rate of the model was 76.7%(60 rats in total,49 of them established a successful acute temporal lobe epilepsy model,and 3 died after,a total survival of 46 finally)in the second part of the experiment.2.The T2 phase of MRI presented high signal lesions in the bilateral temporal lobe.In the first part of the experiment the T2 signal with the naked eyes and the T2 values were significantly decreased after injection of Cy5-AMT-SPIONs(P<0.05).In the second part of the experiment the T2 signals with the naked eyes and the T2 values were significantly decreased after injection of anti-IL-1?mAb-SPIONs(P<0.05).3.We can see blue brown particles in the cytoplasm by Perl,s staining.Injection of Cy5-AMT-SPIONs was the most dense distribution of iron particles,which was significant different in comparison with injection of plain-SPIONs and Cy5-SPIONs(P<0.05)in the first part of the experiment.In the second part of the experiment,there was no iron particle distribution after NS injection.We can observe blue-brown particles mainly distributed in the cytoplasm after injection of plain-SPIONs and anti-IL-1 ?mAb-SPIONs.Injection of anti-IL-1 ?mAb-SPIONs is the most dense distribution of iron particles,which was significant different in comparison withinjection of NS and plain-SPIONs(P<0.05).4.We can observe neuronal abnormalities,deletions,cell arrangement disorder,vacuolar degeneration in the CA1 and CA3 regions of the hippocampus by Nissl's staining.There were no obvious differences between each group of these phenomena in the first part of the experiment.These phenomena above were significantly improved after injection of anti-IL-1?mAb-SPIONs in comparison with injection of NS and plain-SPIONs in the second part of the experiment.5.In the first part of the experiment,we can observe no Cy5 distribution after injection of plain-SPIONs and the Cy5 distribution after injection of Cy5--SPIONs and Cy5-AMT-SPIONs.There was significant difference in Cy5 distribution between each two groups(P<0.05).We can see astrogliosis in each group and there was no significantly difference between each two groups in the first part of the experiment.These phenomena of astrogliosis and microglia activation were significantly improved after injection of anti-IL-1? mAb-SPIONs in comparison with injection of NS and plain-SPIONs in the second part of the experiment.6.We can observe that the expression of IL-1? and NF-?B p65 was significantly decreased after injection of anti-IL-1? mAb-SPIONs in comparison with injection of NS and plain-SPIONs by Western blot and RT-PCR in the second part of the experiment.Conclusion:1.Plain-SPIONs can penetrate the blood brain barrier and the concentration of iron particles may affect magnetic resonance imaging in the brain.2.Cy5 and plain-SPIONs were more likely to pass through the blood brain barrier under the active targeting of AMT.3.Nissl staining and immunofluorescence staining showed no obvious changes in neuron loss and astrogliosis after injection of plain-SPIONs,Cy5-plain-SPIONs and Cy5-AMT-SPIONs.It can be seen that the functional superparamagnetic nanoparticles have no obvious side effects on brain tissue.4.Imaging,histopathology and molecular biology results indicate that anti-IL-1?mAb-SPIONs can be used for targeting diagnosis and treatment of epileptic foci which provides a new way for the diagnosis and treatment of refractory epilepsy.