The Study Of Effect On Apoptosis In The Hippocampal And Cerebellum Neurons And Cognitive Function Of The Rats By Carbamazepine And Dl-3-n-butylphthalide

Objective:Carbamazepine is similar to tricyclic antidepressants in structure also known as glutamine imipramine. By blocking the voltage-gated sodium channels,decrease the presynaptic glutamate and CAMP level and increasing the 5-HT leveal to reduce the neuronal excitability. Carbamazepine is widely used in the clinic for its low price and significant effect as one of the first-line anti-epileptic drugs. However,for its long course of treatment, carbamazepine may lead to adverse drug reactions which are quite different among individuals, and may affect multiple body systems and organs which take a variety of clinical manifestations. Recent studies have shown,dl-3-n-butylphthalide have significant effect on neural functional recovery after cerebral ischemia and improve cerebral blood supply not only by playing a protective effect on terms of morphology and the function of mitochondria but also reducing the apoptosis of neuron and increasing the express of BDNF in ischemic tissue. Dl-3-n-butylphthalide is very effective in treatment with a certain degree of neuroprotective effect. This experiment is based on the morphology and aimed to exclude the damage of cognition and apoptosis is the result of epileptic seizure.Through the observation of nerve cell apoptosis and cognitive impairment in the hippocampal CA1 area, CA3 area and cerebellar cortex of rats which were fed with regular doses of carbamazepine and dl-3-n-butylphthalide, study the side effects of carbamazepine on central nervous system and the possible mechanism of dl-3-n-butylphthalide for the apoptosis inhibition and cognitive improvement.So as to provide a theoretical basis for better clinical treatment and reduce the incidence of adverse drug reactions.Methods:1 Experimental animals and groups: 36 healthy male(7 weeks old) Sprague-Dawley rats, weighing(150±190) g. These rats were randomly divided into six groups, 6 rats in each group as follows:the physiological saline and peanut oil groups(YY);the physiological saline mixed with peanut oil group and dl-3-n-butylphthalide(NBP); the low-dose CBZ group(C1); the high-dose CBZ group(C2); the CBZ and NBP combined treatment groups: the low-dose CBZ and physiological saline mixed with peanut oil group and dl-3-n-butylphthalide groups(NC1);the high-dose CBZ and physiological saline mixed with peanut oil group and dl-3-n-butylphthalide groups(NC2).Drug interventions: according to the human and rat dose conversion formulas low and high dose CBZ were 125mg/kg/d, 500mg/kg/d respectively deliquescing in physiological saline(50mg/ml).The NBP groups were irrigated with dl-3-n-butylphthalide in each group was alawys 80mg/kg/d deliquescing in peanut oil(20mg/ml). The YY groups were treated with isodose saline and peanut oil. NBP groups were treated with dl-3-n-butylphthalide mixed with isodose saline and peanut oil. Above all were irrigated with drugs, once a day until 35 days. 2 Perfusion and preparation of samples: The rats were anesthetized by 10% chloral hydrate, supine fixed and exposed the heart. The perfusion needle was passed through the apex into the left ventricle-aortic valve-ascending aorta. First perfused with 0.9% normal saline simultaneously cutted open the right atrial appendage, then perfused with 4% paraformaldehyde. When perfusion was complete, the rats were decapitated immediately and placed in 4% paraformaldehyde for fixing. Specimens were dehydrated, transparent, embedded in paraffin and sliced for hematoxylin-eosin(HE) staining、Nissl staining and immunohistochemistry staining. Moreover selected two rats in every groups randomly used 1% paraformaldehyde-1.25% glutaric dialdehyde-0.1M phosphate buffer to perfuse and fix. Getted the brain tissue specimen of hippocampal region to observe the CA1 region and cerebellar purkinje cell by transmission electron microscope.3 Apoptosis and cognition detection: The paraffin sections of hippocampal CA1 region, CA3 region and cerebellar cortex purkinje cell were detected to observe the morphological changes under light microscopy by hematoxylin-eosin staining, to observe the change of nissl bodies by nissl staining, to evaluate cognitive behavioral changes by morris water maze,to observe the expression of apoptosis-related genes bax and bcl-2 and cognition-related genes bdnf by immumohistochemical staining. 4 Statics: The experimental data were statistically analyzed with SPSS 13.0 software, the One-Way ANOVA test was applied to compare the differences between groups. Assuming homogeneity of variance pairwise comparisons were applied and tested by the SNK or Tamhane’S T2 test,P <0.05 were considered statistically significant.Results:1 Results of hematoxylin-eosin staining: 1.1 Hippocampal CA1 region and CA3 region: Each group cells were clear and complete. Cells were occasionally dense and arranged in neat rows, reduced in size, separated from the surrounding cells, had dense cytoplasm and deeper eosinophilic staining, nucleus and chromatin gathered into lumps. 1.2 Cerebellar cortex purkinje cell: Each group cells were clear and complete.It could be seen rarely that shrinking cell and chromatin were gathered into lumpy.There were no obvious morphological differences compared with the YY groups. 2 Results of nissl staining 2.1 Hippocampal CA1 region and CA3 region: Hippocampal was stained purple-blue uniformly. The cells were arranged closely in neat rows and cytoplasm was stained clearly in which had some nissl bodys.There were no obvious morphological changes. 2.2 Cerebellar cortex purkinje cell:Nissl staining only showed purkinje cell body and primary dendrite linked to the cell body.Nucleus and plasmosome were visible clearly. Cytoplasm had some nissl bodys gathered into lumps.There were no obvious morphological changes.3 Results of immunohistochemisty staining: 3.1 Expression of Bax: 3.1.1 Hippocampal CA1 region: CBZ groups can be seen a lot of Bax positive cells, which were stained brown in cytoplasm.YY and NBP groups had a little of Bax positive cells which were stained scattered and shallow.Statistically analyzed the OD value of Bax-positive cells in hippocampus in each group through Image Pro Plus 6.0 system showed that C1 and C2 groups were no statistical differences significantly(P>0.05) but had higher lever of Bax compaired with the other groups(P<0.01). The lever of Bax positive cells between NC1 and NC2 groups were no statistical differences significantly(P>0.05).The expression of Bax in this two groups was more than YY and NBP groups and less than C1 and C2 groups.The differences among them were statistically significant(P<0.05). 3.1.2 Hippocampal CA3 region: Each group had a few scattered Bax positive cells.Compared the OD value of the Bax-positive cells in different groups,C1 and C2 groups were no statistical differences significantly(P>0.05) but had higher lever of Bax compaired with the other groups(P<0.01). The lever of Bax positive cells between NC1 and NC2 groups were no statistical differences significantly(P>0.05).The expression of Bax in this two groups was more than YY and NBP group and less than C1 and C2 groups.The differences were statistically significant(P<0.01). 3.1.3 Cerebellar cortex purkinje cell: The Bax-positive cells in the NC1 and NC2 groups were no statistical differences significantly(P>0.05). The expression of Bax in this two groups were higher than C1 and C2 groups and lower than YY and NBP groups.The differences were statistically significant(P<0.01). Compared the OD value of the Bax-positive cells between YY and NBP groups were statistical differences significantly(P<0.01). 3.2 Bcl-2 expression results: 3.2.1 Hippocampal CA1 region: There was an evident expression of Bcl-2-positive cells in the C1 groups and the C2 groups with the cytoplasm stained brown, a medium expression in the CBZ and NBP combined groups,and a low expression in the NBP groups and YY groups in which the positive cells were scattered and stained shallow. Through Image Pro Plus 6.0 system to analyse the result that the differences of each group was not statistically significant(P>0.05). 3.2.2 Hippocampal CA3 region: Bcl-2 expression could be seen in all the groups with the cytoplasm stained brown. Compared the OD value of Bcl-2-positive cells in different groups were no statistical differences significantly(P>0.05). 3.2.3 Cerebellar cortex purkinje cell: Through Image Pro Plus 6.0 system to analyse the result that the differences of every groups were not statistically significant(P>0.05). 3.3 BDNF expression results: 3.3.1 Hippocampal CA1 region: There was an evident expression of BDNFpositive cells in different groups with the cytoplasm all stained brown mainly in the CA1 region and CA3 region observed under an optical microscope magnified 400 times. Compared the OD value of BDNF-positive cells in different groups, each groups was not statistically significant(P>0.05). 3.3.2 Hippocampal CA1 region: The expression of BDNF could be seen in every group, but the expression was low. Compared the OD value of the BDNF positive cells in different groups, the differences were not statistically significant(P>0.05). 3.3.3 Cerebellar cortex purkinje cell: BDNF-positive cells could be seen generally in the hippocampal region and were occasionally seen in the cerebellar cortex. Analyze the positive rate was not statistically significant in different groups(P>0.05). 4 The results of morris water maze: 4.1 Escape latency period: Escape latency period decreased significantly in different groups from the second day which showed train can improve memory ability(P<0.01),but there were no significant differences between each group(P>0.05). 4.2 The results of crossing platform frequency: The frequency of crossingplatform vary from different groups but there were no significant differences between each group(P>0.05). 5 The results of transmission electron microscope: 5.1 Hippocampal CA1 region: the subcellular structure of neurons has no abnormalities. 5.2 Cerebellar cortex purkinje cell:There were no abnormalities in the subcellular structure of neurons.Conclusions:1 Feeding healthy adult Sprague-Dawley rats with regular doses of carbamazepine affects the positive expression of Bax and Bcl-2 compared with control group but have no effect on cognitive behavioral and morphological changes of neurons.We can suggest that short-term use of carbamazepine on neuron damage may be reversible and the body’s own repairment increase the difficulty of apoptosis.These phenomenon shows the body has protective reaction to drug damages. 2 The carbamazepine has no effect on cognitive behavioral of healthy rats. 3 The dl-3-n-butylphthalide has little effect on the cognition and apoptosis of the normal rats.There was no impairment in hippocampal neurons. 4 The dl-3-n-butylphthalide can decrease the positive expression of Bax caused by carbamazepine in hippocampal CA1 region, CA3 region and cerebellar purkinje cell in healthy SD rats.