头孢曲松改善血管性痴呆小鼠学习记忆功能及其机制
发布时间：2018-08-25 09:56
【摘要】：背景和目的血管性痴呆（Vascular Dementia，VD）严重影响患者的身心健康和生存质量。谷氨酸是哺乳动物中枢神经系统内的主要兴奋性神经递质，发挥重要的生理作用。但过量的谷氨酸对神经系统具有兴奋性毒性作用，可导致神经元死亡，是引发VD的重要机制之一。胶质细胞谷氨酸转运体-1（glial glutamate transporter GLT-1）在终止谷氨酸能神经传递、维持细胞外液谷氨酸浓度处于低水平、防止其兴奋性毒性作用方面发挥重要的作用。通过调制GLT-1、增强其摄取细胞外液谷氨酸的能力有可能成为治疗血管性痴呆的新思路。 β-内酰胺类抗生素头孢曲松（Ceftriaxone, Cef）可以特异性的上调GLT-1的表达及其对谷氨酸的摄取，降低细胞外液中谷氨酸浓度及其兴奋性毒性作用，从而发挥抗局灶性及全脑缺血性脑损伤作用、以及抗神经病理性痛和痛过敏作用。据此，我们推测Cef可能会具有抗VD的作用。为证实这一设想，本研究观察Cef对VD小鼠学习记忆能力、海马神经元存活状态、以及GLT-1表达的变化，探讨Cef在预防和治疗VD中的作用、和GLT-1在VD发生发展中的作用，为临床上预防和治疗VD提供新线索和依据。 方法健康雄性老龄（9-10月龄）C57BL/6小鼠70只，体重30±5克，随机分为以下5组： 正常对照组(n=10)：正常动物，不作任何处理，直接测定其学习记忆能力、观察海马组织学改变和GLT-1的表达。 Sham组(n=10)：给动物进行VD的sham手术，于术后29天测定其学习记忆能力、观察海马组织学改变和GLT-1的表达。 VD模型组（n=10）：给动物进行VD造模手术，其它同sham组。 Cef预防（Cef prevention）组（n=20）：首先给动物腹腔注射Cef（200mg/kg），每日1次，连续7天。末次注射后1天给动物进行VD造模手术。其它同sham组。同时设NS对照组（n=10）。 Cef治疗（Cef therapy）组（n=20）：首先给动物进行VD造模手术。于手术后第二天开始腹腔注射Cef（200mg/kg），每日1次，连续7天。其它同sham组。同时设NS对照组（n=10）。 以重复阻断小鼠双侧颈总动脉血流法制作VD模型，每次阻断20min间隔10min，重复3次。采用Morris水迷宫实验测定动物的学习记忆能力应用硫堇染色法观察动物海马组织学变化，依据Kitagawa和Kato的分级方法（Histological grade, HG）对海马CA1区、CA3区和齿状回（dentate gyrusDG）区进行组织病理学评价，标准如下：0级，无神经元死亡；Ⅰ级，散在神经元死亡；Ⅱ级，成片神经元死亡；Ⅲ级几乎全部神经元死亡。应用免疫组化技术观察海马GLT-1的表达情况。采用SPSS16.0统计软件进行统计分析，应用单因素方差分析(One-Way ANOVA)、重复测量分析（Repeated-Measures法）和秩和检验（Kruskal-Wallis法）进行数据分析以P0.05作为判断差异显著性的标准。结果1水迷宫实验随着训练次数的增加，正常对照组动物的寻台潜伏期逐渐缩短，第十 次训练时缩短为25.7±13.1（sec）。Sham组动物的寻台潜伏期的变化趋势 与正常对照组相比无明显差别（P0.05）。VD模型组动物的寻台潜伏期 较Sham组和正常对照组显著延长（P0.05）。Cef预防组动物的寻台潜伏期较VD模型组明显缩短（P0.05），接近sham组。治疗组动物的寻台潜伏期与VD模型组相比无明显差异（P0.05）。空间记忆保持能力测定显示，正常对照组和Sham组动物在目标象限滞留时间比例无明显差别（P0.05）。VD模型组动物较正常对照组和Sham组动物显著缩短（P0.05）。Cef预防组动物在目标象限的滞留时间较VD 模型组动物显著升高（P0.05），接近sham组水平。治疗组动物在目标象限的滞留时间与VD模型组相比无明显差异（P0.05）。2海马组织病理学评价正常对照组动物海马CA1区轮廓清晰，锥体细胞为3-5层，细胞排列规则紧密，形状呈圆形或椭圆形，组织学分级（Histological grade, HG以0-Ⅰ级为主，0级动物占90%。Sham组动物海马组织病理学改变与正 常对照组动物类似。VD模型组动物海马锥体细胞层数减少，排列松散， 细胞数目减少，细胞形态不规则，有核固缩现象，部分区域出现神经元大面积缺失，仅有少量细胞存活；与正常对照和Sham组相比，其HG显著升高（P0.05），表现为0级动物比例降低至20%，，I级动物升至40%，并且出现2级和3级动物，比例均达到20%。Cef预防组小鼠海马锥体细胞排列较整齐，层次较清晰，细胞形态基本正常，无明显核固缩现象发生；与VD模型组相比，其HG显著下降（P0.05），表现为0级比例升高，达到70%，其余30%的动物为I级，无II和III级动物。与VD模型组相比，Cef治疗组海马CA1区组织学特征无明显变化。 海马CA3区和齿状回的组织病理学变化趋势与海马CA1区相类似。 3GLT-1表达 在正常对照组和Sham组海马CA1区锥体神经元层及其周边区域均可见棕黄色GLT-1的免疫反应颗粒，染色均匀。VD模型组GLT-1免疫反应性显著下降，染色较浅，积分光密度值明显降低，表明缺血再灌注损伤下调了GLT-1的表达。与VD模型组相比，Cef预防组GLT-1的免疫反应性显著增强，染色加深，积分光密度值较VD模型组有显著升高（P0.05），表明Cef可以上调GLT-1的表达，并抑制缺血-再灌注损伤引起的GLT-1表达的下降。Cef治疗组GLT-1的免疫反应性亦显著增强，染色加深，积分光密度值较VD模型组有显著升高（P0.05），但升高幅度低于Cef预防组。生理盐水对照组GLT-1的表达同VD模型组相比无明显变化。 海马CA3区和齿状回GLT-1表达的变化趋势与CA1区相类似。 结论： 1多次结扎松开双侧颈总动脉致大脑缺血-再灌注的方法可使老龄C57BL/6小鼠空间学习获得能力和空间记忆保持能力显著下降，此方法可以作为VD动物模型的制作方法。 2VD模型组小鼠海马各区出现明显的神经元死亡和GLT-1表达下调。这些变化可能是导致其学习记忆能力下降的原因。 3预防性应用Cef可以减轻缺血-再灌注引起的认知障碍、海马各区神经元死亡和GLT-1表达下降。 4上述结果提示，Cef可通过上调GLT-1的表达减轻缺血-再灌注引起的海马神经元损伤和认知功能障碍。
[Abstract]:BACKGROUND AND OBJECTIVE Vascular dementia (VD) seriously affects the physical and mental health and quality of life of patients. Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system and plays an important physiological role. However, excessive glutamate has an excitotoxic effect on the nervous system and can lead to neuronal death. Glutamate transporter-1 (GLT-1) plays an important role in terminating glutamatergic neurotransmission, maintaining a low concentration of extracellular glutamate and preventing its excitotoxicity. Force may become a new way of treating vascular dementia.
Ceftriaxone (Cef), a beta-lactam antibiotic, can specifically up-regulate the expression of GLT-1 and its uptake of glutamate, reduce the concentration of glutamate in extracellular fluid and its excitotoxicity, thereby exerting the effects of anti-focal and global cerebral ischemic brain injury, as well as anti-neuropathic pain and hyperalgesia. To confirm this hypothesis, we observed the effects of Cef on learning and memory ability, hippocampal neuronal survival, and GLT-1 expression in VD mice, explored the role of Cef in the prevention and treatment of VD, and the role of GLT-1 in the development of VD, providing new clues and evidence for clinical prevention and treatment of VD. According to it.
Methods 70 healthy male (9-10 month old) C57BL/6 mice weighing 30 + 5 grams were randomly divided into the following 5 groups:
Normal control group (n=10): Normal animals, without any treatment, directly measured their learning and memory ability, to observe the histological changes in the hippocampus and the expression of GLT-1.
Sham group (n=10): The animals were operated on with VD and their learning and memory abilities were measured at 29 days after operation. The histological changes of hippocampus and the expression of GLT-1 were observed.
VD model group (n=10): VD animal model surgery, and the other sham group.
Cef prevention group (n=20): Cef (200 mg/kg) was injected into the abdominal cavity of the animals once a day for 7 consecutive days.
Cef therapy group (n=20): VD model operation was performed on animals at first. Cef (200 mg/kg) was injected intraperitoneally the second day after operation, once a day for 7 consecutive days. The other groups were the same as sham group and NS control group (n=10).
VD model was established by repetitive blockade of bilateral common carotid artery blood flow in mice. The learning and memory abilities of mice were measured by Morris water maze test. Histological changes of hippocampus were observed by thionine staining. The hippocampal CA1 and CA3 regions were classified by Kitagawa and Kato grading (HG). Histopathological evaluation was carried out in the dentate gyrus and DG regions. The criteria were as follows: 0 grade, no neuronal death; 1 grade, scattered neuronal death; 2 grade, patchy neuronal death; and 3 grade almost all neuronal death. One-way ANOVA, Repeated-Measures and Kruskal-Wallis were used to analyze the data and P 0.05 was used as the criterion to judge the significance of the difference.
The trend of latency of the animals in the training group was shortened to 25.7 + 13.1 (SEC).Sham at the time of training.
There was no significant difference between the normal control group and the control group (P0.05) in the.VD model group.
Compared with the sham group and the normal control group, the stage-seeking latency of the Cef prevention group was significantly shorter than that of the VD model group (P 0.05), which was close to that of the sham group. There was no significant difference in the time ratio (P 0.05). The animals in VD model group were significantly shorter than those in normal control group and Sham group (P 0.05).
There was no significant difference in the retention time in the target quadrant between the treatment group and the VD model group (P 0.05). 2 Histopathological evaluation of the normal control group showed that the hippocampal CA1 area had clear contour, pyramidal cells were 3-5 layers, the cells were arranged regularly, the shape was round or oval, and the tissues were well-defined. Histological grade (HG) was mainly 0-I grade, and 90% of the animals were 0 grade. Histopathological changes and positive in the hippocampus of Sham group
In the control group, the number of pyramidal cells in the hippocampal pyramidal cells was reduced and arranged loosely in the.VD like model group.
Compared with the normal control and Sham group, the HG increased significantly (P 0.05), showing that the proportion of grade 0 animals decreased to 20%, grade I animals increased to 40%, and the proportion of grade 2 and grade 3 animals reached 20%. The pyramidal cells in the hippocampus of the preventive group were arranged regularly, with clear layers, normal cell morphology and no obvious nuclear pyknosis. Compared with the VD model group, the HG of the preventive group was significantly decreased (P There was no significant change in histological characteristics.
The histopathological changes in hippocampal CA3 and dentate gyrus are similar to those in hippocampal CA1 area.
3GLT-1 expression
The immunoreactive granules of brown GLT-1 were found in the pyramidal neuron layer and peripheral area of hippocampal CA1 area in normal control group and Sham group. The immunoreactivity of GLT-1 in VD model group was significantly decreased, the staining was lighter, and the integral optical density was significantly decreased, indicating that the expression of GLT-1 was down-regulated by ischemia-reperfusion injury. The immunoreactivity of GLT-1 in the prevention group was significantly enhanced and the staining was deepened. The integral optical density was significantly higher than that in the VD model group (P 0.05), indicating that Cef could up-regulate the expression of GLT-1 and inhibit the decrease of GLT-1 expression induced by ischemia-reperfusion injury. The expression of GLT-1 in normal saline control group was not significantly different from that in VD model group.
The expression of GLT-1 in hippocampal CA3 and dentate gyrus is similar to that in CA1.
Conclusion:
The method of multiple ligation and release of bilateral common carotid artery to induce cerebral ischemia-reperfusion can significantly reduce the spatial learning acquisition ability and spatial memory retention ability of aged C57BL/6 mice. This method can be used as a method of making VD animal model.
In the 2VD model group, neuronal death and GLT-1 expression were significantly down-regulated in the hippocampus. These changes may be responsible for the decline of learning and memory.
Preventive use of Cef can alleviate cognitive impairment induced by ischemia-reperfusion, neuronal death and decreased expression of GLT-1 in hippocampus.
These results suggest that Cef can alleviate hippocampal neuronal damage and cognitive impairment induced by ischemia-reperfusion by up-regulating the expression of GLT-1.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.13;R965
[Abstract]:BACKGROUND AND OBJECTIVE Vascular dementia (VD) seriously affects the physical and mental health and quality of life of patients. Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system and plays an important physiological role. However, excessive glutamate has an excitotoxic effect on the nervous system and can lead to neuronal death. Glutamate transporter-1 (GLT-1) plays an important role in terminating glutamatergic neurotransmission, maintaining a low concentration of extracellular glutamate and preventing its excitotoxicity. Force may become a new way of treating vascular dementia.
Ceftriaxone (Cef), a beta-lactam antibiotic, can specifically up-regulate the expression of GLT-1 and its uptake of glutamate, reduce the concentration of glutamate in extracellular fluid and its excitotoxicity, thereby exerting the effects of anti-focal and global cerebral ischemic brain injury, as well as anti-neuropathic pain and hyperalgesia. To confirm this hypothesis, we observed the effects of Cef on learning and memory ability, hippocampal neuronal survival, and GLT-1 expression in VD mice, explored the role of Cef in the prevention and treatment of VD, and the role of GLT-1 in the development of VD, providing new clues and evidence for clinical prevention and treatment of VD. According to it.
Methods 70 healthy male (9-10 month old) C57BL/6 mice weighing 30 + 5 grams were randomly divided into the following 5 groups:
Normal control group (n=10): Normal animals, without any treatment, directly measured their learning and memory ability, to observe the histological changes in the hippocampus and the expression of GLT-1.
Sham group (n=10): The animals were operated on with VD and their learning and memory abilities were measured at 29 days after operation. The histological changes of hippocampus and the expression of GLT-1 were observed.
VD model group (n=10): VD animal model surgery, and the other sham group.
Cef prevention group (n=20): Cef (200 mg/kg) was injected into the abdominal cavity of the animals once a day for 7 consecutive days.
Cef therapy group (n=20): VD model operation was performed on animals at first. Cef (200 mg/kg) was injected intraperitoneally the second day after operation, once a day for 7 consecutive days. The other groups were the same as sham group and NS control group (n=10).
VD model was established by repetitive blockade of bilateral common carotid artery blood flow in mice. The learning and memory abilities of mice were measured by Morris water maze test. Histological changes of hippocampus were observed by thionine staining. The hippocampal CA1 and CA3 regions were classified by Kitagawa and Kato grading (HG). Histopathological evaluation was carried out in the dentate gyrus and DG regions. The criteria were as follows: 0 grade, no neuronal death; 1 grade, scattered neuronal death; 2 grade, patchy neuronal death; and 3 grade almost all neuronal death. One-way ANOVA, Repeated-Measures and Kruskal-Wallis were used to analyze the data and P 0.05 was used as the criterion to judge the significance of the difference.
The trend of latency of the animals in the training group was shortened to 25.7 + 13.1 (SEC).Sham at the time of training.
There was no significant difference between the normal control group and the control group (P0.05) in the.VD model group.
Compared with the sham group and the normal control group, the stage-seeking latency of the Cef prevention group was significantly shorter than that of the VD model group (P 0.05), which was close to that of the sham group. There was no significant difference in the time ratio (P 0.05). The animals in VD model group were significantly shorter than those in normal control group and Sham group (P 0.05).
There was no significant difference in the retention time in the target quadrant between the treatment group and the VD model group (P 0.05). 2 Histopathological evaluation of the normal control group showed that the hippocampal CA1 area had clear contour, pyramidal cells were 3-5 layers, the cells were arranged regularly, the shape was round or oval, and the tissues were well-defined. Histological grade (HG) was mainly 0-I grade, and 90% of the animals were 0 grade. Histopathological changes and positive in the hippocampus of Sham group
In the control group, the number of pyramidal cells in the hippocampal pyramidal cells was reduced and arranged loosely in the.VD like model group.
Compared with the normal control and Sham group, the HG increased significantly (P 0.05), showing that the proportion of grade 0 animals decreased to 20%, grade I animals increased to 40%, and the proportion of grade 2 and grade 3 animals reached 20%. The pyramidal cells in the hippocampus of the preventive group were arranged regularly, with clear layers, normal cell morphology and no obvious nuclear pyknosis. Compared with the VD model group, the HG of the preventive group was significantly decreased (P There was no significant change in histological characteristics.
The histopathological changes in hippocampal CA3 and dentate gyrus are similar to those in hippocampal CA1 area.
3GLT-1 expression
The immunoreactive granules of brown GLT-1 were found in the pyramidal neuron layer and peripheral area of hippocampal CA1 area in normal control group and Sham group. The immunoreactivity of GLT-1 in VD model group was significantly decreased, the staining was lighter, and the integral optical density was significantly decreased, indicating that the expression of GLT-1 was down-regulated by ischemia-reperfusion injury. The immunoreactivity of GLT-1 in the prevention group was significantly enhanced and the staining was deepened. The integral optical density was significantly higher than that in the VD model group (P 0.05), indicating that Cef could up-regulate the expression of GLT-1 and inhibit the decrease of GLT-1 expression induced by ischemia-reperfusion injury. The expression of GLT-1 in normal saline control group was not significantly different from that in VD model group.
The expression of GLT-1 in hippocampal CA3 and dentate gyrus is similar to that in CA1.
Conclusion:
The method of multiple ligation and release of bilateral common carotid artery to induce cerebral ischemia-reperfusion can significantly reduce the spatial learning acquisition ability and spatial memory retention ability of aged C57BL/6 mice. This method can be used as a method of making VD animal model.
In the 2VD model group, neuronal death and GLT-1 expression were significantly down-regulated in the hippocampus. These changes may be responsible for the decline of learning and memory.
Preventive use of Cef can alleviate cognitive impairment induced by ischemia-reperfusion, neuronal death and decreased expression of GLT-1 in hippocampus.
These results suggest that Cef can alleviate hippocampal neuronal damage and cognitive impairment induced by ischemia-reperfusion by up-regulating the expression of GLT-1.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.13;R965
【参考文献】
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