| Objective: Epilepsy, an ancient and common disease, is a chronic braindisease due to the highly synchronized paradoxical discharge in the brainneurons. In the clinical treatment, antiepileptic drugs (AEDs) are mainly usedto treat the patients whose etiology is not clear, or who can’t be cured despite aclear etiology. Among the numerous AEDs, Phenytoin Sodium (PHT) remainsthe drug of first choice as the treatment of different types of epilepsy, due toits long half-life, non-sedative side effects, low prices, etc. Yet because of itsnarrow treatment spectrum (about10-20ug/ml) and great individualdifferences among patients, the toxic side effects in different bodily systemsare easily seen during PHT clinical treatment. The common adverse reactionsin the nervous system mainly include the cerebellar vestibular dysfunction,damage in peripheral nervous system, extrapyramidal dysfunction, PHTencephalopathy, high intracranial pressure, impaired consciousness, vegetativenerve functional disturbance and other aspects. In clinical, tibetan medicineRannasangpei (RNSP) is commonly used in the co-treatment of epilepsytogether with AEDs, because this medicine has a sedative and anticonvulsanteffect, which promotes neuronal repair and protection, and reduces suchneurotransmitters as NA, β-EP,5-HT after a brain injury. This experiment isaimed at a study on the relativity between PHT, RNSP and apoptosis of theparietal lobe and hippocampus neurons of normal adult rats to learn moreabout the neurotoxic effects of PHT and the effect of RNSP in neurons whichare damaged by PHT. To be specific, a labeling technique which is calledterminal deoxynucleotidyl transferse-mediated biotinylated deoxyuridinetriphosphate nickel end labeling (TUNEL) is used to detect the apoptosis indifferent groups of parietal lobes and hippocampal neurons. Besides, immunohistochemical method is used to detect protein expression levels ineach group of Bcl-2and Bax.Methods:1Groupings and administration:40healthy and clean male SD(SpragueDawley) rats aged ten weeks, weighing (200±20)g, were randomly dividedinto NS (saline) group, RNSP group, PHT group and PHT+RNSP mixedgroup,10rats in each group. Then each group divided into two subgroupsaccording to the time factor of10days or20days,5in a subgroup. Theadministered dosage given to a rat for each day was calculated25times asmuch as the conventional treatment dose on an adult per weight. So thespecific dose were NS1ml/100g/d, PHT150mg/kg/d, RNSP250mg/kg/d. Therats were gavaged twice a day by gavage needle, which lasted for10days and20days respectively in different groups.2Sampling and detection: Fetch some rats in each group, which weredeeply anesthetized by intraperitoneal injection of10%chloral-hydrate, cutopen the chest and exposed the hearts, then punctured from the apex throughthe left ventricle to the aorta by gavage needle, and flushed rats with saline,fixed by4%paraformaldehyde. Next soak specimens in paraformaldehyde for24hours and then dehydrate, embed and section them. At last, detect theapoptotic neurons in the specimens with TUNEL and detect the apoptosisrelated protein expression Bcl-2and Bax by Immunohistochemical method.3Statistical analysis: This experiment was completely randomized designdata. ANOVA of SPSS13.0statistical software was used to compare thedifferences among different groups，and the Dunnett-t test or SNK-q test wasused to compare the differences between any two groups. At last, linear relatedanalysis was used to detect the relativity between Bcl-2and Bax proteinexpression.Results:1The comparison of parietal lobe neurons apoptosis between differentexperimental groups:1.1Results of HE dyeing: The parietal cortical neurons of the rats in each drug group were aligned,a small number of apoptotic cells could be found with membrane shrinkage,nucleus pyknosis, and vacuolization around chromatin. Compared with NSgroup, there was no significant difference in morphology.1.2Results of TUNEL:Ns group had a small number of TUNEL positive apoptotic cells, whichlooked like brown granular or lumpy. RNSP group, PHT group, PHT+RNSPmixed group could be seen scattered TUNEL positive cells. The group meanwas used for pairwise comparison and got the result of P values>0.01,indicating the difference between the groups was not statistically significant.1.3Results of the Bcl-2and Bax protein positive expression:The Bcl-2protein was the anti-apoptotic protein, its positive cells werebrown particles redyed in the cytoplasm and karyotheca. The Bax protein wasthe pro-apoptotic protein, and its positive cells were tawny cytoplasm. Thegroup AO values(Average optical density) were used for pairwise comparisonand got the result of P values>0.01, indicating the difference between thegroups was not statistically significant.2The result of hippocampus neurons apoptosis in every experimental group:2.1Results of HE dyeing:The hippocampal neurons of the rats in NS group were aligned, with aclear cell profile, pink-staining cytoplasm and blue-staining nuclei, a smallnumber of apoptotic cells could be seen with membrane shrinkage, nucleuspyknosis, vacuolization around chromatin. Only a few scattered apoptotic cellscould be seen in RNSP group. PHT group had more apoptotic cells, with amessy arrangement, increasing cell spacing, unclear cell profile and nucleuspyknosis with vacuolization around; the apoptosis was more obviously seen inthe subgroup of long-course treatment. The apoptosis of PHT+RNSP mixedgroup ranged between PHT group and RNSP group.2.2Results of TUNEL:Ns group had a small number of TUNEL positive cells, RNSP groupcould be seen scattered TUNEL positive cells. The group mean was used for pairwise comparison and got the result of P values>0.01, indicating thedifference between two groups was no statistically significant. Apoptotic cellswere increased sharply in PHT group, especially in the PHT group oflong-course treatment; The apoptosis of PHT+RNSP mixed group was onmedium; two group mean and NS group mean were used for pairwisecomparison and got the result of P values<0.01, indicating the differencebetween the groups had statistically significant.2.3Results of the Bcl-2and Bax protein positive expression:A small number of Bax positive cells could been seen in NS group andRNSP group; PHT+RNSP mixed group had more positive expression cells ofBax; PHT group had the most Bax positive cells and the deepest coloring,especially in the long-course treatment subgroup. The aspect of Bcl-2proteinexpression was opposite to Bax protein expression in each group. The groupAO values were used for pairwise comparison, got the result of P values>0.01between NS group and RNSP group, indicating the difference between the twogroups was not statistically significant. The other groups AO values and NSgroup AO values were used for pairwise comparison and got the result of Pvalues<0.01, indicating the difference between the groups had statisticallysignificant.3The relationship between Bcl-2protein and Bax protein:In hippocampal neurons of experimental group, there was a negativecorrelation between Bcl-2and Bax protein expression.Conclusions:1PHT group, RNSP group, PHT+RNSP group, whether the drug andthe period are different, have not significantly affection on the parietal cortexneurons apoptosis and the expression of apoptosis-related protein Bcl-2, Baxin normal rats.2RNSP group has no significant effect on the hippocampal neuronsapoptosis and the expression of apoptosis-related protein Bcl-2, Bax in normalrats whether the period are different; the hippocampal neurons apoptosis inPHT group is obviously seen, the expression of Bcl-2protein is decreased and the expression of Bax protein is increased, the expression of positive cells aremore obvious in the long-course treatment subgroup; the apoptosis in PHT+RNSP mixed group and the expression of apoptosis-related protein Bcl-2, Baxis between that in RNSP group and PRT group, indicating that RNSP mayinhibit hippocampus neurons apoptosis which is caused by Phenytoin Sodium.3In the experimental groups, there may be a negative correlationbetween Bcl-2and Bax protein expression, and Bcl-2, Bax protein may beinvolved in the regulation of neuronal apoptosis path. |
PDF Full Text Request