介导大鼠海马CA1区癫痫样活动的离子型谷氨酸受体的变化
发布时间：2018-07-28 11:35
【摘要】：目的 本研究采用正常大鼠海马脑片灌流离子型γ-氨基丁酸受体(GABA_A受体)拮抗剂荷包牡丹碱(bicuculline,Bic)引起癫痫样活动;并应用脑立体定位技术在大鼠海马CA3中心区通过微量注射海人草酸(Kainic acid,KA)来建立颞叶癫痫(temporal lobe epilepsy,TLE)大鼠模型。以离体脑片细胞外场电位记录方法观测离子型谷氨酸受体在大鼠海马CA1区癫痫样活动中的变化,为进一步探讨癫痫的发生机制提供依据。 方法 1模型建立 1.1颞叶癫痫模型雄性Wistar大鼠,体重200～240克,清洁级。大鼠经水合氯醛(350 mg/kg)腹腔麻醉后,固定于立体定位仪上,在右侧海马CA3中心区(AP: -4.0 mm, ML: 4.4 mm, DV: 3.8 mm)用微量注射针注入2.5～3μl KA (0.4μg/μl),注射15 min,留针10 min,术后缝合头皮。大鼠急性发作等级参照Racine标准分级,达到Ⅳ级及以上视为建模成功。正常对照组在海马CA3区注射等量生理盐水。 1.2急性癫痫样活动正常大鼠离体海马脑片灌流GABA_A受体拮抗剂Bic(30μmol/L)以引起癫痫样活动。 2离体脑片制备大鼠用2%戊巴比妥钠麻醉后迅速断头取脑,置于持续给予95% O2和5% CO_2混合气的4℃人工脑脊液(artificial cerebrospinal fluid, ACSF)中。待脑组织冷却后沿海马槽纤维走向切成400μm厚的脑片,并将其移至含(30±2)℃ACSF的孵育槽中,持续通入混合气,孵育1~2 h。 3场电位记录将孵育的脑片移至记录槽,持续灌流95% O2和5% CO_2混合气饱和的ACSF,灌流速度2 ml/min,温度32℃。刺激海马辐射层Schaffer侧支通路,在CA1区锥体细胞层记录群峰电位(population spike, PS)。 4统计学分析采用SPSS 13.0统计软件分析。实验结果以?x±s表示,组间均数用t检验进行比较,P 0.05具有统计学意义。 结果 1海马CA1区癫痫样活动PS的变化正常大鼠海马脑片CA1区锥体细胞层通常记录到单个PS;正常脑片灌流Bic(30μmol/L)后可引起多个PS的痫样放电,PS数为(5.07±0.30, n = 11),第1个PS的幅度明显增大,为灌流前的(150.86±22.56)%(n = 11, P㩳0.01);TLE模型大鼠脑片记录到痫样放电,PS数为(6.12±0.33, n = 9),与正常脑片相比,引起癫痫样活动的脑片记录到的PS数显著增加(P㩳0.01)。 2介导海马CA1区PS的离子型谷氨酸受体的变化正常脑片CA1区锥体细胞记录到的PS主要由非N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体介导。正常脑片灌流Bic后记录到的PS除了由非NMDA受体介导外,NMDA受体也参与痫样电位的介导。灌流NMDA受体拮抗剂DL-2-氨基-5-磷酸戊酸(DL-2-amino-5-phosphonovaleric acid,APV,50μmol/L),第1个PS幅度无明显变化(n = 11, P㧐0.05),而第4和第5个PS幅度明显降低(n = 11, P㩳0.05; n = 9, P㩳0.05)。TLE模型大鼠海马脑片灌流APV后,第1个PS幅度也无明显变化(n = 9, P㧐0.05),第4和第5个PS幅度明显降低(n = 8, P㩳0.05; n = 8, P㩳0.01),并可进一步抑制灌流非NMDA受体拮抗剂6-氰基-7-硝基喹VA啉-2,3-土卫四(6-cyano-7-nitroquinoxaline-2,3-dione,CNQX,10μmol/L)后的残留电位。 结论 1正常脑片灌流Bic和TLE模型大鼠脑片都能引起癫痫样活动,使CA1区PS数增加。 2大鼠海马CA1区癫痫样活动除了由非NMDA受体介导外,NMDA受体的激活也参与其发生。
[Abstract]:objective
In this study, epileptiform activity was induced by bicuculline (Bic), an antagonist of iontype gamma aminobutyric acid receptor (GABA_A receptor) receptor antagonist (bicuculline, Bic) in the hippocampus of normal rats, and the temporal lobe epilepsy (temporal lobe epilepsy) was established by microinjection of Kainic acid (KA) in the hippocampus CA3 center of rats. TLE) rat model. The changes in the epileptic activity of the ionotropic glutamate receptor in the CA1 region of the hippocampus of the rat were observed by the method of recording the field potential of the isolated brain slices, which provided the basis for further investigation of the mechanism of epilepsy.
Method
1 model establishment
1.1 temporal lobe epilepsy model male Wistar rats, body weight 200~240 grams, clean grade. Rats were fixed on the stereotaxic after intraperitoneal anesthesia with chloral chloral (350 mg/kg). In the right hippocampal CA3 Center (AP: -4.0 mm, ML: 4.4 mm, DV: 3.8 mm), 2.5 to 3 mu L KA (0.4 mu) was injected with microinjection needle, 10 injection of 0.4 mu, and 10 needle retention, suture after operation. Scalp. The rat acute attack grade was classified according to the Racine standard, and the model was considered to be a successful model. The normal control group was injected with the same amount of saline in the CA3 area of the hippocampus.
1.2 acute epileptic activity in normal rat hippocampal slices was perfused with GABA_A receptor antagonist Bic (30 mol/L) to induce epileptogenic activity.
2 the rat brain slices prepared with 2% pentobarbital sodium anaesthesia and quickly cut the head after the anesthesia, and placed the 4 centigrade artificial cerebrospinal fluid (artificial cerebrospinal fluid, ACSF), which continued to give 95% O2 and 5% CO_2 mixture. After the brain tissue was cooled, the coastal manger fibers were cut into 400 m thick slices of brain and moved to the incubating slot containing (30 + 2) C ACSF. Continue to enter mixed gas and incubate 1~2 H.
The incubated brain slices were moved to the recording slot by 3 field potential records, and the ACSF of mixed gas saturated with 95% O2 and 5% CO_2 was perfused. The perfusion velocity was 2 ml/min and the temperature was 32. The Schaffer lateral branch of the hippocampal radiation layer was stimulated, and the peak potential (population spike, PS) was recorded in the pyramidal layer of the CA1 region.
4 statistical analysis was conducted by SPSS 13 statistical software. The results of the experiment were expressed as x + s, and the mean values between groups were compared by t test. P 0.05 had statistical significance.
Result
1 the epileptic activity PS in the hippocampal CA1 region was changed in normal rats. The pyramidal layer of the hippocampal CA1 region of the hippocampus was usually recorded on a single PS; after the normal brain slices were perfused with Bic (30 u mol/L), multiple PS epileptiform discharges were induced, PS number was (5.07 + 0.30, n = 11), and the amplitude of first PS increased significantly, which was (150.86 + 22.56)% (n = 11, P? 0.01) before perfusion. The number of epileptiform discharges recorded in the brain slices was PS (6.12 + 0.33, n = 9). Compared with normal brain slices, the number of PS recorded in brain slices that caused epileptic activity increased significantly (P? 0.01).
2 mediated changes in the ionotropic glutamate receptor of PS in the CA1 region of the hippocampus. The PS of normal brain slices CA1 pyramidal cells is mainly mediated by the non N- -D- aspartic acid (N-methyl-D-aspartate, NMDA) receptor. The PS of the normal brain slices after perfusion of Bic is mediated by the non NMDA receptor, and the NMDA receptor is also involved in the mediation of the epileptic potential. The receptor antagonist DL-2- amino -5- phosphate valerate (DL-2-amino-5-phosphonovaleric acid, APV, 50 u mol/L), first PS amplitudes did not change significantly (n = 11, P? 0.05), while fourth and fifth PS significantly decreased (n = 11, P? 0.05, 9, 0.05) after perfusion of hippocampal slices in rat model rats, there were no significant changes in first amplitude (9, 0.05), The amplitude of fourth and fifth PS decreased significantly (n = 8, P? 0.05; n = 8, P? 0.01), and could further inhibit the residual potential of the perfusion non NMDA receptor antagonist, 6- cyanobased -7- NITROQUINE VA -2,3- (6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, 10 micron).
conclusion
1 normal brain slice perfusion of Bic and TLE brain slices could induce epileptic activity and increase the number of PS in CA1 region.
2 the epileptic activity in hippocampal CA1 area of rats is mediated by non NMDA receptors, and the activation of NMDA receptors is also involved.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R338.8
本文编号：2150005
[Abstract]:objective
In this study, epileptiform activity was induced by bicuculline (Bic), an antagonist of iontype gamma aminobutyric acid receptor (GABA_A receptor) receptor antagonist (bicuculline, Bic) in the hippocampus of normal rats, and the temporal lobe epilepsy (temporal lobe epilepsy) was established by microinjection of Kainic acid (KA) in the hippocampus CA3 center of rats. TLE) rat model. The changes in the epileptic activity of the ionotropic glutamate receptor in the CA1 region of the hippocampus of the rat were observed by the method of recording the field potential of the isolated brain slices, which provided the basis for further investigation of the mechanism of epilepsy.
Method
1 model establishment
1.1 temporal lobe epilepsy model male Wistar rats, body weight 200~240 grams, clean grade. Rats were fixed on the stereotaxic after intraperitoneal anesthesia with chloral chloral (350 mg/kg). In the right hippocampal CA3 Center (AP: -4.0 mm, ML: 4.4 mm, DV: 3.8 mm), 2.5 to 3 mu L KA (0.4 mu) was injected with microinjection needle, 10 injection of 0.4 mu, and 10 needle retention, suture after operation. Scalp. The rat acute attack grade was classified according to the Racine standard, and the model was considered to be a successful model. The normal control group was injected with the same amount of saline in the CA3 area of the hippocampus.
1.2 acute epileptic activity in normal rat hippocampal slices was perfused with GABA_A receptor antagonist Bic (30 mol/L) to induce epileptogenic activity.
2 the rat brain slices prepared with 2% pentobarbital sodium anaesthesia and quickly cut the head after the anesthesia, and placed the 4 centigrade artificial cerebrospinal fluid (artificial cerebrospinal fluid, ACSF), which continued to give 95% O2 and 5% CO_2 mixture. After the brain tissue was cooled, the coastal manger fibers were cut into 400 m thick slices of brain and moved to the incubating slot containing (30 + 2) C ACSF. Continue to enter mixed gas and incubate 1~2 H.
The incubated brain slices were moved to the recording slot by 3 field potential records, and the ACSF of mixed gas saturated with 95% O2 and 5% CO_2 was perfused. The perfusion velocity was 2 ml/min and the temperature was 32. The Schaffer lateral branch of the hippocampal radiation layer was stimulated, and the peak potential (population spike, PS) was recorded in the pyramidal layer of the CA1 region.
4 statistical analysis was conducted by SPSS 13 statistical software. The results of the experiment were expressed as x + s, and the mean values between groups were compared by t test. P 0.05 had statistical significance.
Result
1 the epileptic activity PS in the hippocampal CA1 region was changed in normal rats. The pyramidal layer of the hippocampal CA1 region of the hippocampus was usually recorded on a single PS; after the normal brain slices were perfused with Bic (30 u mol/L), multiple PS epileptiform discharges were induced, PS number was (5.07 + 0.30, n = 11), and the amplitude of first PS increased significantly, which was (150.86 + 22.56)% (n = 11, P? 0.01) before perfusion. The number of epileptiform discharges recorded in the brain slices was PS (6.12 + 0.33, n = 9). Compared with normal brain slices, the number of PS recorded in brain slices that caused epileptic activity increased significantly (P? 0.01).
2 mediated changes in the ionotropic glutamate receptor of PS in the CA1 region of the hippocampus. The PS of normal brain slices CA1 pyramidal cells is mainly mediated by the non N- -D- aspartic acid (N-methyl-D-aspartate, NMDA) receptor. The PS of the normal brain slices after perfusion of Bic is mediated by the non NMDA receptor, and the NMDA receptor is also involved in the mediation of the epileptic potential. The receptor antagonist DL-2- amino -5- phosphate valerate (DL-2-amino-5-phosphonovaleric acid, APV, 50 u mol/L), first PS amplitudes did not change significantly (n = 11, P? 0.05), while fourth and fifth PS significantly decreased (n = 11, P? 0.05, 9, 0.05) after perfusion of hippocampal slices in rat model rats, there were no significant changes in first amplitude (9, 0.05), The amplitude of fourth and fifth PS decreased significantly (n = 8, P? 0.05; n = 8, P? 0.01), and could further inhibit the residual potential of the perfusion non NMDA receptor antagonist, 6- cyanobased -7- NITROQUINE VA -2,3- (6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, 10 micron).
conclusion
1 normal brain slice perfusion of Bic and TLE brain slices could induce epileptic activity and increase the number of PS in CA1 region.
2 the epileptic activity in hippocampal CA1 area of rats is mediated by non NMDA receptors, and the activation of NMDA receptors is also involved.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R338.8
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