| PrefaceEpilepsy is a common type of nervous system disease.Among them about 25 percent patients couldn't control symptoms with drugs which are called intractable epilepsy.About 40 percent of human epilepsy is come form the temporal lobe,which is called the temporal lobe epilepsy(TLE).The most typical changes of the temporal lobe epilepsy are in the hippocampus,because of the unique structure of hippocampus. Patients of TLE accompanied with the loss of hippocampus neurons,and some researchers believed that the loss of hippocampus neurons is cell apoptosis.The curcumin is extracted from the ginger rhizome which has the function of antioxidant, anti-inflammatory,anti-coagulation,anti-tumor,anti-mutagenic,and many other pharmacological effects.Some studies showed that curcumin may play a role in the protection of neurons in the cerebral ischemia and traumatic brain injury.Recently, curcumin pretreatment has been shown to ameliorate damage at the chromatin level, attenuating histone modifications,expression of immediate early genes and the severity of KA-induced status epilepsy.The purpose of this study is to examine the effect of curcumin on hippocampus neuronal apoptosis in pilocarpine induced epileptic mice.Materials and Methods1.Experimental animalsThirty-two CD-1 mice,both female and male,weighing 18-22g,were randomly divided into four groups,and each group contains eight mice.(1)The first group was the control group(Ctl) (2)The second group was lithium-pilocapine induced epilepsy group(Pil)(3)The third group was injected curcumin advanced and then induced epilepsy group(Cur+Pil)(4)The fourth group was curcumin injection group(Cur)2.The behavior of epileptic miceThe mice by intraperitoneal injection of pilocarpine were observed their behavior changes,according to Simialowski 6(0-V)evaluation method.3.Preparation of tissueAnimals were anaesthetized with 4%sodium pentobarbital,72 hours after lithium-pilocarpine-induced epilepsy.They were then perfused with 4% paraformaldehyde from the left ventricles.The brains were removed and immersed in 30%sucrose solution at 4℃overnight.Frozen and paraffin sections were made for cresol violet staining,TUNEL staining,apoptosis factors Caspase-3 and Caspase-9 detection.4.Staining(1)Cresol violet stainingThe samples were cut into 10μm coronal sections in a cryostat and mounted on glass slides.After cresol violet staining,sections were observed under light microscope and the numbers of neurons in the CA1 and CA3 subareas of hippocampus were counted.(2)TUNEL assaySections were incubated in biotinylated nucleotide and TdT enzyme at 37℃for 30 min.Following washes and blocking endogenous peroxidase activity using H2O2,the biotin labels were detected with streptavidin-HRP(horseradish peroxidase)and a DAB (diaminobenzidine tetrahydrochloride dihydrate)color reaction.(3)In situ hybridization method The apoptosis factors of all animals are inspected by the in situ hybridization kit,according to the directions strictly.Sections are destroyed endogenous peroxydase,and then washed with PBS followed the proteinase K permeabilization. After then,the sections were hybridized followed the pre-hybridization,and then PBS washed,SABC 37℃20min incubated,horseradish peroxidase detected and DAB colored.Results1.The behavior changes of epileptic miceWe observed the behavior changes of 18 hours advanced injected curcumin and then induced epilepsy in experimental animals(Cur+Pil),and the epileptic animals (Pil).The two group animals mentioned above have the typical onset of epilepsy-Ⅳ-Ⅴgrades behaviors and some of them are in the status epilepsy or even spontaneous epilepsy.However,the animals in the control group(Ctl)and only received curcumin(Cur)group haven't the epilepsy occurrence.2.The protection effect of curcumin in epilepsy hippocampus neurons(1)The cresol violet stainingIn the sections,there is apparent vertebral neuronal death in the hippocampus CA1 and CA3 region of the animals which are induced epilepsy by pilocarpine.However,in the sections of the animals which were injected curcumin advanced,there was a clear role in the protection and effective inhibition of the neurons death in the hippocampus.(2)The results of TUNELThe results of TUNEL staining have the same conclusion with the cresol violet staining.A large number of TUNEL-positive cells in the sections of hippocampal CA1 and CA3 of pilocarpine induced epilepsy group(Pil)animals.But in sections of 18 hours injected curcumin advanced and then induced epilepsy in experimental animals (Cur+Pil),the TUNEL-positive cells decreased significantly,which means the curcumin can decreased the death rate of the hippocampal neurons in epilepsy mice and played a protective role in the number of neurons.(3)The results of apoptosis factors caspase-3 and caspase-9 in situ hybridization analysisIn the sections of pilocarpine epilepsy group(Pil)animals the apoptosis factors Caspase-3 and Caspase-9-positive cells are significantly increased in the hippocampus CA1 and animals CA3 area.But after the injection of curcumin advanced,the positive neurons in the hippocampus decreased.From the results mentioned above,we can believe that the curcumin can inhibit the apoptosis and decrease the apoptotic factors' production.DiscussionThis study showed that the frequent epileptic seizures can lead to neurons damage selectively,which resulting in the loss of neurons.In the epilepsy animal model of repeated seizures,this can lead to brain ischemia,hypoxia,activation the oxygen free radical,and large amounts of NO generation.All of them can induce the hippocampus neurons death.The morphological features of dead neurons are the same as the cell apoptosis,so the cell apoptosis is the main form of the neuronal death in patients with epilepsy maybe.The most common neuronal damage area is the CA1 and CA3 pyramidal cells in the hippocampus.It s known to all,the cell apoptosis is generated in two ways.One is endogenous apoptosis pathway,which the TNF receptor family members are involved and the apoptosis factor caspase-8 is activated.The other is exogenous apoptosis which the cytochrome C is participated and the apoptosis factor caspase-9 is activated.And both of them all activated the factors of caspase-3,7 to induce apoptosis.In the cresol violet staining,the results of the pilocarpine-induced epilepsy group sections displayed that the cells survived less in the CA1 and CA3 region of the hippocampus.In order to prove the cell death after the epilepsy 72 hours is closer to the cells apoptosis,we detect apoptotic factor caspase-3,9.In the results, the numbers of the positive apoptotic cells in the hippocampus CA1 and CA3 regions of the epilepsy group are larger than the other groups.This proves that the epilepsy may lead to the endogenous apoptosis way.Because of the curcumin's functions of antioxidant,anti-inflammatory,anti-coagulation,anti-tumor,anti-mutagenic,and other well-known pharmacological effects,we injected curcumin advanced in the epilepsy group animals,and then a comparative analysis have been done in the cell apoptosis detection,TUNEL assay and the endogenous apoptosis factors pathway.The results found that the numbers of the apoptosis factor positive cells in the animals injected curcumin advanced are lesser than the ones of the epilepsy group.This proved that the curcumin played an important role in the inhibition of apoptosis on the endogenous apoptosis pathway.The study provides a morphological proof of curcumin on the epilepsy's prevention and cure.ConclusionThis study used the methods of intraperitoneal injection curcumin advanced,the TUNEL assay and the in situ hybridization methods to the observed the hippocampus neurons changes in CD-1 mice brain.There are some conclusions about it such as:1.In mice by intraperitoneal injection of pilocarpine not only changes their behavior but also there are apparent vertebral neuronal death in the animal hippocampus CA1 and CA3 region in cresol violet and TUNEL staining.However the curcumin can decrease the hippocampus neuronal death rate and play a protective role in the hippocampus neurons.2.Curcumin can attenuate apoptosis factor-positive cells in the animals' hippocampus neurons by the in situ hybridization detection which showed that curcumin could effectively inhibit apoptosis and the apoptotic factors production.
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