| Objective: Phentoin is diphenylhydantoin sodium,which remains one of the most commonly prescribed and effective antiepileptic drugs.The rang of its safe plasma level is narrow, the thearaputic level is approximate to its toxic level and the individual difference of drug level is great under the commonly-used dosage which usually result in the toxicity involved in many systems such as nervous system, hematopoietic system, endocrine system, lymphatic system.Within the central neuron system, the cerebellar and vestibular systems, extracorticospinal tract and cognition function are the most often affected by PHT-mediated toxicity.The syndrom of PTH toxicity is different because of the difference of the route and period of and the drug consumption.The cases of PHT toxicity are reported frequently in the clinic.The side effect of PHT may be corrected with the apoptosis,but epilepsy can also lead to apoptosis.This experiment is to investigate the effect of PHT on the normal rats,which is grouping according to the dosage and the period of drug administration.It detects apoptotic neurons in the cortex of the frontal lobe and the cerebellar by TUNEL(terminal deoxynucleotidyl transferse-medi- ated biotinylated deoxyuridine triphosphate nickel end labeling)and the expressed Bax or Bcl-2 protein by immunohistochemistry.It is to to observe the relation of the side effect of PTH with neuron apoptosis and the the expressed Bax or Bcl-2 protein.Methods:1 Divided into six groups randomly:30 adolescent male healthy Sprague Dawley rats aged ten weeks, weighing(200+20)g, were randomly divided into three groups. The first group was treated with normal saline by stomach lavaging, the second group was treated with low-dosage PHT adminstration and the third group was treated with high-dosage PHT adminstration. Fifteen days later, five rats were sacrificed randomly from each group and the rest were sacrificed another fifteen days later with phenytoin administration.2 Drug administration: the four experimental groups: The first group respectively administrated equal dose of normal saline for 15 days and 30 days. the second group were respectively treated with PHT 100 mg/kg/d for 15 days and 30 days with intragastric administration. the third group were respectively treated with PHT 200 mg/kg/d for 15 days and 30 days with the same way.3 Preparation of samples: After one and half an hour infusion with 4% glutaraldehyde from the left ventricle, SD rat brain was obtained and the anterior and posterior segment were cutted respectively as the frontal lobe and cerebellum by 4mm thick and quickly add to 4% of the fixative of polyformaldehyde, conventional biopsy done by TUNEL and immunohistochemical technique after 24 hours paraffinembedd.4 Detecting the apoptotic neurons in the frontal lobe and cerebellum: after the brains were made to paraffin block, the apoptotic neurons in the frontal lobe and cerebellum were detected by TUNEL.5 Detecting the positive expression of protein Bcl-2 and Bax: After the paraffin sections, the antigen retrieval, goat serum closed, Bcl-2 and Bax polyclonalantibodies used, the second-antibody, third-antibody incubated, DAB color and observed under the microscope.Result:1 The results of neuron apoptosis in the cortical neuron of the frontal lobe and cerebellar granular neuron: In the cortical neuron of the frontal lobe, every group has few TUNEL positive cells and there are no significant differences compared each other. In the cerebellar neuron,the groups observed greater TUNEL positive cells except the normal control groups;there are significant differences in the different dosage groups and different period groups.2 The results of The expression of Bcl-2 in the cortical neuron of the frontal lobe and cerebellar neuron: There are some positive expression cells of Bcl-2 in short-term and long-term saline groups in the two locations, there is no significant difference between two groups. In frontal lobes ,the low and high dosage PHT groups have some positive expression cells of Bcl-2, but they have no significance compared with the normal control groups respectively. In the cerebellar neuron, the low dosage PHT groups have fewer positive expression cells of Bcl-2 than the control groups and have significance difference in contrast with the control groups, and have significance differences between the two groups; the hight dosage PHT groups have fewer positive expression cells of Bcl-2 than the low dosage groups and have significance difference in contrast with the low dosage groups,and have significance difference between the two groups.3 The results of The expression of Bax in the cortical neuron of the frontal lobe and cerebellar granular neuron: There are few positive expression cells of Bax in short-term and long-term saline groups in the two locations, there is no significant difference between two groups. In frontal lobes ,the experimental groups have few positive expression cells of Bax and have no significance difference between each two groups and in contrast with the control groups. In the cerebellar granular neuron, the low dosage PHT groups have more positive expression cells of Bax than the control groups and have significance difference in contrast with the control groups,and have significance difference between the two groups; the hight dosage PHT groups have more positive expression cells of Bax than the low dosage groups and have significance difference in contrast with the low dosage groups,and have significance difference between the two groups.Conclusions:1 PHT, whether the dosage and the period are different, have little effect on the apoptosis of the neurons and make few positive expression cells of Bcl-2 decrease and positive expression cells of Bax increase in the frontal lobe of the adult rats.2 PHT, whether the dosage and the period are different, cause the apoptosis of the neurons and make positive expression cells of Bcl-2 decrease and positive expression cells of Bax increase in the cerebellar neuron of the adult rats.The different results may be dependent on the different dosage and period.3 The reason why PHT cause the apoptosis of the cerebellar neuron may be concerned with positive expression cells of Bcl-2 decreaseing and positive expression cells of Bax increasing.
|
PDF Full Text Request