【摘要】：背景和目的血管性癡呆（Vascular Dementia，VD）嚴重影響患者的身心健康和生存質(zhì)量。谷氨酸是哺乳動物中樞神經(jīng)系統(tǒng)內(nèi)的主要興奮性神經(jīng)遞質(zhì)，發(fā)揮重要的生理作用。但過量的谷氨酸對神經(jīng)系統(tǒng)具有興奮性毒性作用，可導(dǎo)致神經(jīng)元死亡，是引發(fā)VD的重要機制之一。膠質(zhì)細胞谷氨酸轉(zhuǎn)運體-1（glial glutamate transporter GLT-1）在終止谷氨酸能神經(jīng)傳遞、維持細胞外液谷氨酸濃度處于低水平、防止其興奮性毒性作用方面發(fā)揮重要的作用。通過調(diào)制GLT-1、增強其攝取細胞外液谷氨酸的能力有可能成為治療血管性癡呆的新思路。 β-內(nèi)酰胺類抗生素頭孢曲松（Ceftriaxone, Cef）可以特異性的上調(diào)GLT-1的表達及其對谷氨酸的攝取，降低細胞外液中谷氨酸濃度及其興奮性毒性作用，從而發(fā)揮抗局灶性及全腦缺血性腦損傷作用、以及抗神經(jīng)病理性痛和痛過敏作用。據(jù)此，我們推測Cef可能會具有抗VD的作用。為證實這一設(shè)想，本研究觀察Cef對VD小鼠學(xué)習記憶能力、海馬神經(jīng)元存活狀態(tài)、以及GLT-1表達的變化，探討Cef在預(yù)防和治療VD中的作用、和GLT-1在VD發(fā)生發(fā)展中的作用，為臨床上預(yù)防和治療VD提供新線索和依據(jù)。 方法健康雄性老齡（9-10月齡）C57BL/6小鼠70只，體重30±5克，隨機分為以下5組： 正常對照組(n=10)：正常動物，不作任何處理，直接測定其學(xué)習記憶能力、觀察海馬組織學(xué)改變和GLT-1的表達。 Sham組(n=10)：給動物進行VD的sham手術(shù)，于術(shù)后29天測定其學(xué)習記憶能力、觀察海馬組織學(xué)改變和GLT-1的表達。 VD模型組（n=10）：給動物進行VD造模手術(shù)，其它同sham組。 Cef預(yù)防（Cef prevention）組（n=20）：首先給動物腹腔注射Cef（200mg/kg），每日1次，連續(xù)7天。末次注射后1天給動物進行VD造模手術(shù)。其它同sham組。同時設(shè)NS對照組（n=10）。 Cef治療（Cef therapy）組（n=20）：首先給動物進行VD造模手術(shù)。于手術(shù)后第二天開始腹腔注射Cef（200mg/kg），每日1次，連續(xù)7天。其它同sham組。同時設(shè)NS對照組（n=10）。 以重復(fù)阻斷小鼠雙側(cè)頸總動脈血流法制作VD模型，每次阻斷20min間隔10min，重復(fù)3次。采用Morris水迷宮實驗測定動物的學(xué)習記憶能力應(yīng)用硫堇染色法觀察動物海馬組織學(xué)變化，依據(jù)Kitagawa和Kato的分級方法（Histological grade, HG）對海馬CA1區(qū)、CA3區(qū)和齒狀回（dentate gyrusDG）區(qū)進行組織病理學(xué)評價，標準如下：0級，無神經(jīng)元死亡；Ⅰ級，散在神經(jīng)元死亡；Ⅱ級，成片神經(jīng)元死亡；Ⅲ級幾乎全部神經(jīng)元死亡。應(yīng)用免疫組化技術(shù)觀察海馬GLT-1的表達情況。采用SPSS16.0統(tǒng)計軟件進行統(tǒng)計分析，應(yīng)用單因素方差分析(One-Way ANOVA)、重復(fù)測量分析（Repeated-Measures法）和秩和檢驗（Kruskal-Wallis法）進行數(shù)據(jù)分析以P0.05作為判斷差異顯著性的標準。結(jié)果1水迷宮實驗隨著訓(xùn)練次數(shù)的增加，正常對照組動物的尋臺潛伏期逐漸縮短，第十 次訓(xùn)練時縮短為25.7±13.1（sec）。Sham組動物的尋臺潛伏期的變化趨勢 與正常對照組相比無明顯差別（P0.05）。VD模型組動物的尋臺潛伏期 較Sham組和正常對照組顯著延長（P0.05）。Cef預(yù)防組動物的尋臺潛伏期較VD模型組明顯縮短（P0.05），接近sham組。治療組動物的尋臺潛伏期與VD模型組相比無明顯差異（P0.05）�？臻g記憶保持能力測定顯示，正常對照組和Sham組動物在目標象限滯留時間比例無明顯差別（P0.05）。VD模型組動物較正常對照組和Sham組動物顯著縮短（P0.05）。Cef預(yù)防組動物在目標象限的滯留時間較VD 模型組動物顯著升高（P0.05），接近sham組水平。治療組動物在目標象限的滯留時間與VD模型組相比無明顯差異（P0.05）。2海馬組織病理學(xué)評價正常對照組動物海馬CA1區(qū)輪廓清晰，錐體細胞為3-5層，細胞排列規(guī)則緊密，形狀呈圓形或橢圓形，組織學(xué)分級（Histological grade, HG以0-Ⅰ級為主，0級動物占90%。Sham組動物海馬組織病理學(xué)改變與正 常對照組動物類似。VD模型組動物海馬錐體細胞層數(shù)減少，排列松散， 細胞數(shù)目減少，細胞形態(tài)不規(guī)則，有核固縮現(xiàn)象，部分區(qū)域出現(xiàn)神經(jīng)元大面積缺失，僅有少量細胞存活；與正常對照和Sham組相比，其HG顯著升高（P0.05），表現(xiàn)為0級動物比例降低至20%，，I級動物升至40%，并且出現(xiàn)2級和3級動物，比例均達到20%。Cef預(yù)防組小鼠海馬錐體細胞排列較整齊，層次較清晰，細胞形態(tài)基本正常，無明顯核固縮現(xiàn)象發(fā)生；與VD模型組相比，其HG顯著下降（P0.05），表現(xiàn)為0級比例升高，達到70%，其余30%的動物為I級，無II和III級動物。與VD模型組相比，Cef治療組海馬CA1區(qū)組織學(xué)特征無明顯變化。 海馬CA3區(qū)和齒狀回的組織病理學(xué)變化趨勢與海馬CA1區(qū)相類似。 3GLT-1表達 在正常對照組和Sham組海馬CA1區(qū)錐體神經(jīng)元層及其周邊區(qū)域均可見棕黃色GLT-1的免疫反應(yīng)顆粒，染色均勻。VD模型組GLT-1免疫反應(yīng)性顯著下降，染色較淺，積分光密度值明顯降低，表明缺血再灌注損傷下調(diào)了GLT-1的表達。與VD模型組相比，Cef預(yù)防組GLT-1的免疫反應(yīng)性顯著增強，染色加深，積分光密度值較VD模型組有顯著升高（P0.05），表明Cef可以上調(diào)GLT-1的表達，并抑制缺血-再灌注損傷引起的GLT-1表達的下降。Cef治療組GLT-1的免疫反應(yīng)性亦顯著增強，染色加深，積分光密度值較VD模型組有顯著升高（P0.05），但升高幅度低于Cef預(yù)防組。生理鹽水對照組GLT-1的表達同VD模型組相比無明顯變化。 海馬CA3區(qū)和齒狀回GLT-1表達的變化趨勢與CA1區(qū)相類似。 結(jié)論： 1多次結(jié)扎松開雙側(cè)頸總動脈致大腦缺血-再灌注的方法可使老齡C57BL/6小鼠空間學(xué)習獲得能力和空間記憶保持能力顯著下降，此方法可以作為VD動物模型的制作方法。 2VD模型組小鼠海馬各區(qū)出現(xiàn)明顯的神經(jīng)元死亡和GLT-1表達下調(diào)。這些變化可能是導(dǎo)致其學(xué)習記憶能力下降的原因。 3預(yù)防性應(yīng)用Cef可以減輕缺血-再灌注引起的認知障礙、海馬各區(qū)神經(jīng)元死亡和GLT-1表達下降。 4上述結(jié)果提示，Cef可通過上調(diào)GLT-1的表達減輕缺血-再灌注引起的海馬神經(jīng)元損傷和認知功能障礙。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.13;R965

【參考文獻】

相關(guān)期刊論文 前2條

1 魏居瑞;劉U

本文編號：2202531