左旋谷氨酸单钠致大鼠认知障碍的实验观察

Experimental observation on cognitive handicap induced by monosodium glutamate in rats

目的:观察左旋谷氨酸单钠毁损下丘脑弓状核大鼠的认知功能,探讨其与下丘脑-垂体-肾上腺轴和中枢氨基酸能神经递质与受体的关系。方法:实验于2001-09/2002-03在河南中医学院动物实验中心和中药研究室进行。选取普通级SD大鼠12只,雌雄各半,作为种鼠供实验室繁殖。选取新生鼠23只,随机分为模型组(n=13)和对照组(n=10),分别于出生后第2,4,6,8,10天进行皮下注射左旋谷氨酸单钠4mg/g和生理盐水,每日1次。28d后离乳,再饲养8周后进行跳台试验和Morris水迷宫实验,分别记录各组大鼠的触电潜伏期,错误反应次数及寻台潜伏期,并计算跨台百分率,主要观察各组大鼠的学习记忆行为。实验结束后,全部大鼠用戊巴比妥钠腹腔注射麻醉,心脏取血,采用放免法测定血浆皮质醇、促肾上腺皮质激素。取模型组大鼠8只,对照组大鼠5只,不经灌注固定,冰台上迅速分离大脑,将大脑沿矢状缝分左右半球,左半球用于糖皮质激素受体mRNA原位杂交,右半球采用薄层扫描法测定谷氨酸和γ-氨基丁酸含量,同时取大鼠左侧肾上腺进行苏木精-伊红染色,观察肾上腺组织形态变化。取各组剩余大鼠,用含4%多聚甲醛和0.2%苦味酸的0.1mol/L磷酸盐缓冲液(PB,pH7.4)灌注固定,采用免疫组化法观察海马γ-氨基丁酸受体和N-甲基-D-天门冬氨酸受体1表达,并应用MotaMorph计算机图象分析系统计算免疫反应物质的光密度,取脑组织切片进行Nissl染色。组间比较采用t检验。结果:各组大鼠全部进入结果分析。①跳台实验观察结果:与对照组比较,模型组大鼠测试触电潜伏期显著缩短(t=4.940,P<0.001),训练期和测验期的错误反应次数显著增加(P<0.001～0.01)。②Morris水迷宫实验观察结果:模型组大鼠寻台潜伏期较对照组明显延长(P<0.01),跨台百分率较对照组明显降低(P<0.01)。③下丘脑-垂体-肾上腺-海马轴的观察:模型组大鼠血浆皮质醇及促肾上腺皮质激素水平均较对照组显著升高(t=2.943～4.013,P<0.001～0.01)。模型组海马糖皮质激素受体mRNA表达增强,阳性染色细胞较对照组明显增多。④中枢氨基酸能神经递质与受体的观察:模型组大鼠脑组织谷氨酸含量较对照组显著降低(t=3.063,P<0.05),γ-氨基丁酸含量及谷氨酸/γ-氨基丁酸值较对照组有降低趋势,与对照组比较,模型组海马N-甲基-D-天门冬氨酸受体1及γ-氨基丁酸受体免疫反应物质数密度明显降低(t=2.224～3.682,P<0.01～0.05)。⑤模型组大鼠海马Nissl染色显示模型组细胞稀疏、个数减少、有空泡出现,胞浆着色较浅,细胞轮廓不规整。图像分析模型组大鼠光密度较对照组有降低趋势。结论:模型组大鼠有明显学习记忆障碍,其机制与下丘脑-垂体-肾上腺轴功能亢进,氨基酸能神经递质与受体异常有关。

AIM：To observe the cognitive function in rats with arcuate nucleus of hypothalamus damaged by monosodium glutamate （MSG）,and to study the relation with hypothalamic-pituitary-adrenal axis （HPA） and central amino acid, neurotransmitter and recipient. METHODS： The experiment was done in the Animal Experimental Center and Traditional Chinese Medicine Laboratory of Henan College of Traditional Chinese Medicine from September 2001 to March 2002. Twelve common grade SD rats were selected, half male and half female as the breeders to reproduce news in the lab. Twenty-three SD neonatal rats were collected and randomly divided into model group with 13 rats and control group with 10 rats. and they were hypodermiely injected with 4 mg/g MSG and saline at the 2^th, 4^th, 6^th, 8^th and 10^th days after born, respectively, oncea day. After 28 days lactation, feed 8 weeks, then the jumping stand trial and Morris water maze trial were performed to record latency of electric shock, times of mistakes and latency of seeking the platform. The percentage of through the platform position was calculated. The learning and memory behavior was observed mainly in rats of every group. After the experiment, sodium pentobarbital was injected by intraperitoneal injection of all the rats for anesthesia, and the blood was gained from heart.The plasmcortisol and adrenocorticotrophic hormone were detected by the radioimmunoassay. Eight rats from model group and 5 rats in the control group were fixed without perfusion, and the brains that were separated directly on the ice stage were divided into left part and the right part along sagittal suture. The left part was used in the glucocorticoid receptor mRNA in situ hybridization. TLC measured the content of Glu andγ-GABA on the right part. Meanwhile, the left suprarenal gland was gained to perform the hematoxylineosin staining and observe the morphologic changes of tissue. The leftover rats in every group were collected and fixed by perfusion with 0.1 mol/L phosptat buffe （PB,pH7.4） contained of 4% citromint and 0.2% bitter acid. The expression ofγ-GABA receptor and N-methyl-D-aspartic acid receptor 1 in hippocampi was observed by immunohistochemical method. The light density of immunologic reaction materials was analyzed by the Mota Morph computer image analysis system. The brain tissue section was gained to perform Nissl staining, t-test was conducted to conduct the comparison among the groups. RESULTS： All the rats were involved in the result analysis.① Step down task results： Compared with the control group, the latency of electric shock was obviously shortened in the model group （t=4.940, P 〈 0.001 ）, and the times of mistakes obviously increased in the training period and testing period （P 〈 0.001-0.01 ）. ②Morris water maze task results： In the model group the latency of seeking the platform was obviously prolonged compared with that in the control group （P 〈 0.01）. The percentage of spaning the platform position was obviously decreased .compared with that in thecontrol group （P 〈 0.01 ）. ③ HPA-hippocampi observation： In the model group the levels of the plasm-cortisol and ACTH increased compared with that in the control group （t=2.943-4.013, P 〈 0.001-0.01 ）. The expression of GRmRNA increased in the model group, and the number of positive dyeing cell obviously increased compared with that in the control group.④ Observation on center aminophenol neurotransmitter and receptor： Compared with that in the control group, the content of Glu in brain tissues was obviously decreased in the model group （t=3.063, P 〈 0.05）, and the content ofγ-GABA and the value of Glu/γ-GABA had the trend of decrease compared with that in the control group. Compared with the control group, immunologic reaction materials density of the N-methyl-D-aspartic acid receptor 1 andγ-GABA receptor in hippocampi in the model group significantly decreased （t=2.224-3.682, P 〈 0.01-0.05）.⑤ The hippocampi Nissl staining in the model group indicated that the cells were sparse, and the number decreased with vacuole and irregular outline, without intimate pigmentation in the model group. The light density in the model group had the trend of decrease compared with that in the control group by the image analysis. CONCLUSION： The rats in the model group have significantly the handicap of learning and memory, and the mechanism is hyperfunetion with HPA. The aminophenol neurotransmitter has the relation with the abnormal receptor.