The Protection Of Botulinum Neurotoxin A On Hippocampal Neurons In Lithium Chloride-Pilocarpine Rat Epileptic Model Via Intranasal Administration

BackgroundEpilepsy is a common neurological disease.Frequent epileptic seizures can cause severe brain damage,accidental injury and psychological trauma,which bring heavy burden to patients,families even society.The morbidity of epilepsy is about 7.2‰,involving more than 9,000,000 patients in China.Although the standard and routine anti-epileptic medicine were taken,seizure of nearly 1/3 patients could not be effectively controlled,even become refractory epilepsy,of which temporal lobe epilepsy(TLE)is the most common refractory epilepsy.The rat epileptic model of lithium chloride-pilocarpine was first reported by Honchar in 1983.The epileptic model is coincident with human temporal lobe epilepsy model in neuroelectrophysiology,neuronal injury and behavioral manifestations,so it is widely used in the study of status epilepticus(SE)and temporal lobe epilepsy.Botulinum neurotoxin A(BoNT/A)is a neurotoxic protein produced by the bacterium Clostridium botulinum,which can specifically cleave synaptic vesicle transport proteins to inhibit presynaptic neurotransmission.By inhibiting Na+ channels and the excitability of neurons,BoNT/A provides a theoretical basis on anti-epilepsy treatment.Recent studies have shown that injection of Botulinum neurotoxin(BoNT)in the intracranial hippocampus inhibited epileptic seizures in acute phase,alleviated hippocampal neuron impact,and reduced the frequency of epileptic seizures in the chronic phase,which further confirmed that BoNT was a potential antiepileptic drug.However,previous studies have used the method of intracranial injection,which is traumatic,complicated to operate,easily infected,and disadvantageous to long-term administration,and haven't further explored the potential mechanism of antiepileptic effect.Compared with intracranial injection,intranasal administration is a convenient and non-invasive method,which can bypass the blood-brain barrier,quickly enter the central nervous system and meet the needs of long-term administration for chronic diseases.While,it still required to further search the specific mechanism of BoNT/A on anti-epilepsy,and whether intranasal BoNT/A adiministration could reduce the neuronal damage induced by SE remains to be studied.ObjectiveIn this study,we observed the changes of neuronal morphology and the apoptotic autophagy-related proteins(Bax,Caspase-3,Bcl-2 and Beclin-1)in epileptic rats induced by lithium chloride-pilocarpine and investigated the mechanism of epilepsy-induced neuronal damage.We explored the feasibility of administration of BoNT/A by nasal instillation,observed the effect on behavioral and neuronal damage in epileptic rats,detected the level of apoptotic autophagy-related protein expression,and explored the underlying mechanism of BoNT/A antiepileptic effect.Materials and Methods120 healthy male SD rats(6-7 weeks,weight 220g-250g)were randomly divided into four groups: CON group(normal rats),PILO group(epileptic rats),PILO+NS group(epileptic rats + nasal instillation of normal saline)and PILO+BoNT/A group(epileptic rats + nasal instillation of BoNT/A).PILO group included 3h,8h,24 h,72h subgroup.Rats in the PILO+NS and PILO+BoNT/A groups were placed in supine position after intraperitoneal anesthesia and dripped the same amount of saline or BoNT/A into the nasal cavity.The CON and PILO groups were only given intraperitoneal anesthesia at the same time;body weight of all rats were recored.On the 50 th day after administration,four groups of rats were given intraperitoneal injection of lithium chloride(3mEq/kg).After 20 hours,the CON group was intraperitoneally injected with normal saline,and the others groups were given intraperitoneally pilocarpine(30mg/kg)to induce SE.After 60 minutes of SE,the rats were intraperitoneal injected of chloral hydrate(0.3 mg/kg)to terminate the SE.According to Racine criteria for epileptic seizures,IV or V grade were judged as model-making success,the success rate and latency of each group were recorded.The successful model was chose according to the behavioral performance.Rats of the PILO 3h,PILO 8h and PILO 24 h groups were anaesthetized and isolated bilateral hippocampus at the corresponding time.Western blot assay was used to detecte the expression levels of apoptotic autophagy-related proteins.Rats of the PILO 72 h group were anaesthetized and taken brain perfused at 72 h after SE.Nissl staining assay detected the injury of hippocampal neurons.The bilateral hippocampuses were removed from rats of PILO+NS and PILO+BoNT/A groups at 24 hours after SE,and detected the expression level of related proteins.The remaining rats were anesthetized and perfused at 72 h after SE.Paraffin and frozen sections were prepared to observe neuronal damage and detected cleaced-SNAP-25.Result: 1.Behavioral observationThe rats in CON group were normal.The rats in PILO,PILO+NS,and PILO+BoNT groups had seizures after injection of pilocarpine.There was no statistical significant difference on the success rate and incubation time between the three groups(p>0.05).2.Morphological changes in hippocampal neuronsFrom Nissl staining,compared to CON group,the neuron in PILO and PILO+NS group were apparently damaged,and had less quantity,with statistical significant difference(p<0.05);the neuron quantity in PILO+BoNT/A group was smaller than CON group without statistical significant difference(p>0.05);Compared to PILO group and PILO+NS group,the neuron quantity in PILO+BoNT/A group increased with statistical significant difference(p<0.05).3.Detection of cleaved-SNAP-25The cleaved-SNAP-25 protein was not detected in the olfactory bulb and hippocampal neurons in the PILO group,while the cleaved-SNAP-25 proteins was found in the PILO+BoNT/A group.4.Detection of apoptosis and autophagy related proteinsCompared to the CON group,status epilepticus could significantly inhibit the anti-apoptotic protein Bcl-2 expression and increased Bax,Beclin-1,and Caspase-3 levels within the first 24 hours after SE(p<0.05).Compared with PILO group,PILO+BoNT/A group could inhibit the changes of the above protein with statistically significant differences and had no significant difference(p> 0.05).Conclusions:1.Lithium chloride-pilocarpine epilepsy model is easy to operate and has high success rate,mainwhile the behavior of epilepsy in rats is obvious.2.Status epilepticus caused damage to hippocampal neurons,especially in CA1 and CA3 regions,involving neuronal autophagy and apoptotic pathways.3.BoNT/A can be administered intranasally into the central nervous system(olfactory bulb,hippocampus)of rats,confirming the feasibility of this administration routine.4.BoNT/A has a protective effect on neuronal damage in the lithium chloridepilocarpine rat model,and may participate in the neuronal autophagy and apoptosis pathway.5.Nasal administration can provide a new route of administration for BoNT/A in anti-epilepsy field.