介導大鼠海馬CA1區(qū)癲癇樣活動的離子型谷氨酸受體的變化
發(fā)布時間：2018-07-28 11:35
【摘要】：目的 本研究采用正常大鼠海馬腦片灌流離子型γ-氨基丁酸受體(GABA_A受體)拮抗劑荷包牡丹堿(bicuculline,Bic)引起癲癇樣活動;并應用腦立體定位技術在大鼠海馬CA3中心區(qū)通過微量注射海人草酸(Kainic acid,KA)來建立顳葉癲癇(temporal lobe epilepsy,TLE)大鼠模型。以離體腦片細胞外場電位記錄方法觀測離子型谷氨酸受體在大鼠海馬CA1區(qū)癲癇樣活動中的變化,為進一步探討癲癇的發(fā)生機制提供依據(jù)。 方法 1模型建立 1.1顳葉癲癇模型雄性Wistar大鼠,體重200～240克,清潔級。大鼠經(jīng)水合氯醛(350 mg/kg)腹腔麻醉后,固定于立體定位儀上,在右側海馬CA3中心區(qū)(AP: -4.0 mm, ML: 4.4 mm, DV: 3.8 mm)用微量注射針注入2.5～3μl KA (0.4μg/μl),注射15 min,留針10 min,術后縫合頭皮。大鼠急性發(fā)作等級參照Racine標準分級,達到Ⅳ級及以上視為建模成功。正常對照組在海馬CA3區(qū)注射等量生理鹽水。 1.2急性癲癇樣活動正常大鼠離體海馬腦片灌流GABA_A受體拮抗劑Bic(30μmol/L)以引起癲癇樣活動。 2離體腦片制備大鼠用2%戊巴比妥鈉麻醉后迅速斷頭取腦,置于持續(xù)給予95% O2和5% CO_2混合氣的4℃人工腦脊液(artificial cerebrospinal fluid, ACSF)中。待腦組織冷卻后沿海馬槽纖維走向切成400μm厚的腦片,并將其移至含(30±2)℃ACSF的孵育槽中,持續(xù)通入混合氣,孵育1~2 h。 3場電位記錄將孵育的腦片移至記錄槽,持續(xù)灌流95% O2和5% CO_2混合氣飽和的ACSF,灌流速度2 ml/min,溫度32℃。刺激海馬輻射層Schaffer側支通路,在CA1區(qū)錐體細胞層記錄群峰電位(population spike, PS)。 4統(tǒng)計學分析采用SPSS 13.0統(tǒng)計軟件分析。實驗結果以?x±s表示,組間均數(shù)用t檢驗進行比較,P 0.05具有統(tǒng)計學意義。 結果 1海馬CA1區(qū)癲癇樣活動PS的變化正常大鼠海馬腦片CA1區(qū)錐體細胞層通常記錄到單個PS;正常腦片灌流Bic(30μmol/L)后可引起多個PS的癇樣放電,PS數(shù)為(5.07±0.30, n = 11),第1個PS的幅度明顯增大,為灌流前的(150.86±22.56)%(n = 11, P㩳0.01);TLE模型大鼠腦片記錄到癇樣放電,PS數(shù)為(6.12±0.33, n = 9),與正常腦片相比,引起癲癇樣活動的腦片記錄到的PS數(shù)顯著增加(P㩳0.01)。 2介導海馬CA1區(qū)PS的離子型谷氨酸受體的變化正常腦片CA1區(qū)錐體細胞記錄到的PS主要由非N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受體介導。正常腦片灌流Bic后記錄到的PS除了由非NMDA受體介導外,NMDA受體也參與癇樣電位的介導。灌流NMDA受體拮抗劑DL-2-氨基-5-磷酸戊酸(DL-2-amino-5-phosphonovaleric acid,APV,50μmol/L),第1個PS幅度無明顯變化(n = 11, P㧐0.05),而第4和第5個PS幅度明顯降低(n = 11, P㩳0.05; n = 9, P㩳0.05)。TLE模型大鼠海馬腦片灌流APV后,第1個PS幅度也無明顯變化(n = 9, P㧐0.05),第4和第5個PS幅度明顯降低(n = 8, P㩳0.05; n = 8, P㩳0.01),并可進一步抑制灌流非NMDA受體拮抗劑6-氰基-7-硝基喹VA啉-2,3-土衛(wèi)四(6-cyano-7-nitroquinoxaline-2,3-dione,CNQX,10μmol/L)后的殘留電位。 結論 1正常腦片灌流Bic和TLE模型大鼠腦片都能引起癲癇樣活動,使CA1區(qū)PS數(shù)增加。 2大鼠海馬CA1區(qū)癲癇樣活動除了由非NMDA受體介導外,NMDA受體的激活也參與其發(fā)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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R338.8
本文編號：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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R338.8
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