胰岛素对大鼠脑缺血后记忆力损害的保护

Protective effects of insulin against memory deficits of rats with cerebral ischemic reperfusion

目的:观察胰岛素对大鼠脑缺血后酪氨酸羟化酶和多巴胺-β-羟化酶表达及学习记忆能力的影响。方法:实验于2001-01/2003-05在解放军第四军医大学完成。①选用纯种健康雄性SD大鼠54只,随机数字表法分为3组:假手术组6只、缺血对照组24只、胰岛素治疗组24只。Y型迷宫箱(张家港生物医学仪器厂制造,MGM-2型)。②缺血对照组、胰岛素治疗组采用改良四血管闭塞法建立大鼠全脑缺血模型。假手术组仅暴露双侧翼孔及颈动脉,各血管不行闭塞。③各组大鼠在麻醉状态下钻开颅骨,于前囟后1.2mm、左旁1.5mm处用微量加样器进针3.5mm穿刺脑室。成功后胰岛素治疗组于开放动脉夹行再灌注后立即注入1×105U/L的速效基因合成人胰岛素10μL,假手术组、缺血对照组注入等量生理盐水。④分别于全脑缺血再灌注1,3,5d,假手术组(1只/时相点)、缺血对照组(6只/时相点)、胰岛素治疗组(6只/时相点)大鼠断头取脑,行免疫组化染色,光镜下观察海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶表达的变化。⑤全脑缺血再灌注8周后,用Y型迷宫箱对假手术组(3只)、缺血对照组(6只)、胰岛素治疗组(6只)大鼠的学习记忆能力进行检测,以其测试时达到连续10次中9次正确反应所需的电击次数表示。并计数海马CA1区正常神经元。结果:实验选用纯种健康雄性SD大鼠54只,全部进入结果分析。①全脑缺血再灌注后不同时间点各组脑海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶的表达:假手术组海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶呈阴性表达。缺血对照组于缺血再灌注1d海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶表达阴性;3,5d时均呈阳性表达。而胰岛素治疗组于缺血再灌注1,3,5d海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶表达均呈阴性。②全脑缺血再灌注8周后各组大鼠学习记忆能力的变化:胰岛素治疗组记忆力损害较轻,达到9/10正确反应所需电击次数明显少于缺血对照组[(9.4±1.8),(18.6±2.7)次,P<0.01]。③全脑缺血再灌注8周后各组大鼠脑海马CA1区正常神经元计数结果:假手术组神经元形态正常,细胞核无固缩或溶解,正常神经元计数为(174.6±10.7)个/mm。缺血对照组神经元大部分死亡,正常神经元计数为(10.6±0.6)个/mm。胰岛素治疗组可见部分神经元死亡,死亡神经元胞体缩小或脱失,正常神经元计数为(113.4±9.1)个/mm,明显高于缺血对照组(P<0.01)。结论:胰岛素可通过调节儿茶酚胺合成与分解酶系的活性及含量来影响脑组织中单胺类神经递质的成分,纠正其代谢紊乱,减少神经元的凋亡,减轻脑缺血造成的学习记忆能力损害,从而发挥中枢的直接保护作用。

AIM： To study the effects of insulin on the expression of tyrosine hydroxylase （TH） and dopamine （DA） β as well as learning and memory abilities of rats with cerebral ischemia. METHODS： The experiment was conducted in the Fourth Military Medical University of Chinese PLA from January 2001 to May 2003.①54 healthy male SD rats were selected and randomly divided into 3 groups： sham-operation group （n=6）, ischemic control group （n=24, control group） and insulin treatment group （n=24, treatment group）. Ymaze box of MGM-2 type was adopted （manufactured by Zhangjiagang Biomedical Equipment Manufacturing Company）. ②Rats in the control group and treatment group were made into cerebral ischemic models by method modified four-vessel occlusion. Rats ,in the sham-operation group were exposed of bilateral foramen alares and carotid artery only without occluding the vessels. ③The skulls of all rats were drilled under anesthefization and the needle was injected for 3.5 nun to stab the brain ventricle at the 1.2 nun behind and 1.5 nun left to the anterior fontanel with a samplenjector of microamount. Rats in the treatment group were injected with 10 μL of human insulin of rapid gene synthesis （1 ×10%5 U/L）immediately after unclamped the bulldog clamp and reperfusion, while rats in the sham-operation group and control group were injected with normal saline at the same volume.④ At one, three and five days after cerebral ischemia-reperfusion, one rat in the sham-operation group, six rats in the control group and six rats in the treatment group were executed to obtain the brain. Changes of TH and DA-β in hippocampal CA1 region were detected by immunohistochemistry method under light microscope.⑤ 8 weeks after cerebral ischemia-reperfusion, the learning and memory abilities of 3 rats in the sham-operation group, 6 rats in the control group and 6 rats in the treatment group were tested by Y maze, and the shocking times of 9 correct responses out of 10 continuous times were tested. The number of normal neurons in hippocampal CA1 region were calculated. RESULTS： A total of 54 enrolled healthy male SD rats were involved in the analysis of results. ①Expressions of TH and DA-β after cerebral ischemia-reperfusion in hippocampal CA1 region at each time-peint： The expressions in the sham-operation group were negative, and the expressions of control group at one day after ischemia-reperfusion were negative, while those were positive at the 3^rd and 5^th days, while the expressions at 1, 3 and 5 days after ischemia-reperfusion were negative in the treatment group. ②The changes learning and memory abilities of rots at 8 weeks after cerebral ischemia-reperfusion： lesion of memory was slighter in the treatment group, and the shocking times of 9/10 correct response were significantly less than the control group [（9.4±1.8 ）, （18.6±2.7）,P 〈 0.01].③The number of normal neurons in hippocampal CA1 region at 8 weeks after reperfusion： the shape of neurons were normal in the sham-operation group without condensed cell nucleus or solution, and the number was （174.6±10.7）/mm, while most of the neurons in the control group were dead and the number was （ 10.6±0.6）/mm. Partial neurons died in the treatment group, and perikaryons contracted or withdrew with the normal neuron number of （113.4±9.1 ）/mm, which was obviously higher than the control group （P 〈 0.01）. CONCLUSION： Insulin can influence the ingredients Of monoamine neurotransmitter in brain tissues, correct the its metabolic disorder, reduce the apoptosis of neurons and the deficits of learning and memory abilities induced by cerebral ischema and directly protect the center by adjusting synthesis of catecholamine and the contents as well as the activities of catabolic enzyme.