Effects of acrous gramineus and its component, alpha-asarone, on apoptosis of hippocampal neurons after seizure in immature rats

BACKGROUND： α-asarone and acrous gramineus have been shown to play a necessary function in enhancing the reactivity and convulsant threshold to electric stimulation of immature rats. They have also been shown to effectively suppress epileptic seizures induced by pentylenetetrazol in young rats. However, the mechanisms for these roles have been still unclear. OBJECTIVE： To observe the effects in immature rats of acrous gramineus and α -asarone on apoptosis of hippocampal neurons after epileptic seizure at the protein level, and to analyze the mechanism for these effects. DESIGN： A randomized controlled animal experiment. SETTINGS： Department of Pediatrics, First Hospital of Jilin University; Department of Histology and Embryology, Norman Bethune Medical School of Jilin University; Department of Internal Medicine, Children＇s Hospital of Changchun City; Department of Neurology, First Clinical Hospital affiliated to Harbin Medical University. MATERIALS： Fifty 3-week old Wistar rats, 34-40 g, irrespective of gender, were provided by Gaoxin Research Center of Medical Animal Experiment, Changchun. The animals were treated according to the animal ethical standards. The following chemicals were used for this study： acrous gramineus powders or infusion （Batch No, 0307113, Tianjiang Medicine Company Limited, Jiangyin）, α-asarone tablets （Batch No. 030219, Tianwei Pharmaceutical Factory, Shenyang）, and phenobarbital sodium tablets （Batch No. 020608, Xinya Medicine Company Limited, Shanghai）. The animals were divided into five groups randomly. First, ten rats were chosen as the normal controls. The remaining rats were treated with i.p. injections of pentylenetetrazol to stimulate an epileptic model. METHODS： The experiments were performed at the Neurological Laboratory of the First Hospital of Jilin University between October and December 2004. The rats were treated with i.p. injections of pentylenetetrazol （60 mg/kg） to establish an epileptic model. According to Racine＇ s standard, animals that reached stage 4 and 5 were chosen and randomly divided into 4 groups： model group, phenobarbital sodium, acrous gramineus, and a-asarone group. The normal control group was treated with an i.p. injection of physiological saline （0.5 mL）. After modeling, the model groups were intragastrically administrated 0.5 mL saline. The phenobarbital sodium, acrous gramineus, and α-asarone groups were intragastrically administrated 18 mg/kg/d phenobarbital sodium, 2 350 mg/kg/d acrous gramineus and 29 mg/kg/d α-asarone, respectively. The course of treatment was twice a day for 7 days. The normal group received intragastric administration of 0.5 mL saline at the same time. The rats were sacrificed and brain sections were prepared for light microscopy and electron microscopy. MAIN OUTCOME MEASURES： （1） Pathological changes of CA1 and CA3 hippocampal region neurons were observed by light microscopy and electronic microscopy. （2） Neuronal apoptosis in the CA1 and CA3 region was measured by TUNEL staining. （3） Bcl-2 and Bax expression in CA1 and CA3 region neurons was detected by immunohistochemistry and a ratio of Bcl-2/Bax was calculated. RESULTS： All 50 immature rats were included in the final analysis. （1） Pathological changes of CA1 and CA3 region hippocampal neurons： there were different pathological changes in all groups other than the normal control group. The number of damaged neurons in the model group was highest. The phenobarbital sodium, acrous gramineus, and α -asarone group exhibited different degrees of improvement. （2） Neuronal apoptosis in the CA1 and CA3 regions： there were less TUNEL-positive cells in the CA1 and CA3 regions in the normal control group. One week after PTZ-induced seizure, numerous TUNEL-positive cells were detected in the CA1 and CA3 regions in the remaining four groups. There was a significant difference between the normal control group and the remaining four groups （t = 12.089-19.162, P 〈 0.0 1）. The number of TUNEL-positive cells was less in the phenobarbital sodium, acrous gramineus, and α-asarone groups compared to the model group （t = 4.707-5.268, P 〈 0.01）. （3）Bcl-2 and Bax expression of neurons in the CA1 and CA3 regions： The number of Bcl-2- and Bax-positive cells was less in the normal control group. The Bax-positive cells exhibited a normal shape and had large round nuclei that were predominant. One week after PTZ-induced epilepsy, the number of Bcl-2- and Bax-positive cells in the CA1 and CA2 regions was significantly increased in the remaining four groups compared to the normal control group （t = 11.606-27.042, P 〈 0.01）. The Bax-positive cells exhibited a reduced size and nuclear pyknosis was predominant. However, there was no significant difference among the four groups （P 〉 0.05）. The number of Bcl-2-positive cells in the phenobarbital sodium, acrous gramineus, and a-asarone groups were significantly increased compared to the model group （t = 4.051-6.404, P 〈 0.01）. However, the number of Bax-positive cells was not significantly different among the four groups. The ratio of Bcl-2 to Bax expression was approximately 6.0 in the normal controls, 0.7 in the model group, and 1.0 in the remaining three groups. CONCLUSION： Acrous gramineus and a-asarone increased Bcl-2 expression and decreased Bax expression, and also reduced the number of apoptotic hippocampal neurons during PTZ-induced epileptic seizures in immature rats.

Effects of acrous gramimeus and its main component alpha-asarone on the reactivity and convulsive threshold of immature rats to electric stimulation

BACKGROUND： The traditional Chinese medicine acrous gramimeus is the dry rhizome of Acrous gramimeus Soland, a kind of Araceae familial perennial herb, which has a sedation action, anticonvulsant and antiepileptic effect. Its effective component has not been known yet, and α-asarone, the major component of the volatile oil extracted from acrous gramineus, has been supposed to play a necessary role in it. OBJECTIVE： To explore the effects of acrous gramimeu and α-asarone on the reactivity and convulsive threshold to electric stimulation in immature rats, furthermore, attempt to definitize the anticonvulsant effect of α-asarone. DESIGN： A randomized controlled study.SETTINGS： Department of Pediatrics, First Hospital of Jilin University; Department of Histology and Embryology, School of Basic Medical Sciences of Jilin University; Department of Neurology, First Clinical Hospital affiliated to Harbin Medical University; Department of Internal Medicine, Children＇s Hospital of Changchun City. MATERIALS ： Seventy 3-week immature Wistar rats （either males or females） of 34-40 g were used. Acrous gramimeu （1 g/bag, the content of α-oasarone was 0.046 26%-0.070 16%） with the batch number of 0307113 was provided by Tianjiang Medicine Company Limited, Jiangyin City. α-asarone tablet （60 mg per tablet） with the batch number of 030219 was provided by Tianwei Pharmaceutical Factory, Shenyang City. α-asarone injectable preparation （2 mL per piece） with the batch number of 030105 was provided by Shuanghe Medicine Limited Company, Beijing City. METHODS ： The experiments were carried out in the Neurological Laboratory of the First Hospital of Jilin University between August and October in 2004.① The 70 rats were randomly divided into intragastric subset and intraperitoneal subset. The intragastric subset included four groups of control, phenobarbital sodium, acrous gramimeu and α-asarone; the intraperitoneal subset included three groups of control, phenobarbital sodium and α-asarone. There were 10 rats per group. ② In the intragastric subset, different group was treated with saline （1 mL for each time, phenobarbital sodium （18 mg/kg per day）, acrous gramineu （2 350 mg/kg per day） and α-asarone （29 mg/kg per day） respectively twice every day for 5 days. In the intraperitoneal subset, different group was treated with saline （0.5 mL）, phenobarbital sodium （29 mg/kg） and α-asarone （2.9 mg/kg） respectively. ③ Before and after administration for 5 days in the intragastric subset as well as before and after administration for about 1 hour in the intraperitoneal subset respectively, the rats were given electric stimulation with the NIHOM KOMDEM multifunctional electrophysiological recorder, and the reactivity and convulsive threshold to electric stimulation of the rats were recorded. MAIN OUTCOME MEASURES： The reactivity and convulsive threshold to electric stimulation in immature rats were compared. RESULTS： All the rats were involved in the analysis of results. ① Results for intragastric administration： Before intragastric administration, there were no obvious differences in the reactivity and convulsive threshold to electric stimulation among the groups （P 〉 0.05）. After intragastric administration for 5 days, the reactivity and convulsive threshold to the electric stimulation had no obvious changes in the control group, but those were significantly higher than before administration in the drug administration groups （t=-3.317-7.401, P 〈 0.01）, which were also obviously higher than those in the control group （t=3.027-8.941, P 〈 0.01）, and those in the acrous gramimeu group and α-asarone group were not markedly different from those in the phenobarbital sodium group. ② Results for intraperitoneal injection： Before intraperitoneal injection, the reactivity and convulsive threshold to the electric stimulation had no obvious differences among the groups. After the intraperitoneal injection for 1 hour, the reactivity and convulsive threshold to the electric stimulation had no obvious change in the control group, but those were significantly higher than before administration in the drug administration groups （P 〈 0.01）, which were also obviously higher than those in the control group （t=6.211-7.237, P 〈 0.01; t=4.085-5.633, P 〈 0.05）, and there was no marked difference between α-asarone group and phenobarbital sodium group （P 〉 0.05）.CONCLUSION ： ① As effective anticonvulsants, both acrous gramineu and α-asarone can enhance the reactivity and convulsive threshold of immature rats to electric stimulation. ② As one of the major effective components against convulsion of acrous gramineu, α-asarone is equivalent to phenobarbital sodium.

MORPHOLOGICAL STUDY OF FETAL NEOCORTICAL TRANSPLANT GRAFTED TO THE CEREBRAL CORRESPONDING AREA IN YOUNG RATS DIFFERENTIATION OF IMMATURE NEURONS AND RECIPROCAL CONNECTIONS OF FIBERS BETWEEN GRAFT-HOST BRAIN

The fetal neocortical transplant (E15-17 days gestation) of Wistar rat was grafted to the corresponding neocortical region (frontal-parietal lobe) of the same strain in young rats (4-5 weeks old). On the 7th, 15th, 30th, 60th, 150th day after transplantation, the sections cut through the middle area of graft-ost brain were examined by HE, Nissl, Glees stain, immunohistochemical technique for GFAP and NF, Nissl, Glees stain, immunohistochemical technique for GFAP and NF, acetylcholinesterase (AChE) histochemistry as well as horseradish peroxidase (HRP) retrograde tracing with light microscope. Some of the sections were also examined with TEM. The result showed that most immature neurons within the graft can survive, grow, differentiate and mature, and are similar to the structure of the neocortical neurons of host brain. This study also provides patterns of integration of the interface between graft-host brain varying with the proliferation of reactive astrocyte as well as graft-host reciprocal connection of fibers.