鼻腔给予神经生长因子对铝毒性阿尔茨海默病大鼠模型神经保护作用的实验研究
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摘要
前言
阿尔茨海默病(Alzheimer's disease，AD)是最常见的神经变性疾病，是导致痴呆的主要病因，发病率随年龄而增加。AD患者典型的临床表现是进行性近期记忆力减退，疾病的晚期出现神经症状和行为异常。
AD的主要组织病理学特征是：患者脑内尤其是海马和新皮质出现大量的神经纤维缠结(Neurofibrillary tangles)、老年斑(Senile plagues)及胆碱能神经元缺失、颗粒空泡样变性。细胞内的神经纤维缠结由高度磷酸化的细胞骨架tau蛋白相互缠绕成神经丝构成，而细胞外的老年斑是β-淀粉样前体蛋白经蛋白水解产生的一系列长短不等的Aβ(39-43氨基酸)肽的沉积。
尽管AD病因未明，但大多数学者认为β-淀粉样蛋白(β-amyloid，Aβ)的神经毒作用是AD形成和发展的关键因素。
对AD患者的尸检发现，患者脑内胆硷能神经通路变性及胆碱乙酰基转移酶耗竭，这些改变被认为与AD患者的临床表现有关。所以，胆碱能替代疗法常被用于治疗AD引起的记忆及认知障碍。这类药包括：乙酰胆碱前体、促乙酰胆碱释放剂、胆碱酯酶抑制剂、毒蕈胆碱能受体激动剂。其他种类药物有：脑血管扩张剂、中枢神经系统兴奋剂、鸦片拮抗剂及神经肽。然而，上述药物都不能逆转AD患者的症状或阻止AD病情的进展。
近年来神经科学的研究发现，神经生长因子(nerve growth factor，NGF)对中枢神经系统多种类型神经元的生长、发育、分化、维持和损伤修复都具有重要作用。NGF也是胆碱能神经元重要的营养因子，它能增加乙酰胆碱转移酶的合成，还能阻止实验性损伤、生理性或病理性衰老以及AD等因素引起的胆碱能神经元变性。这些研究成果虽然为应用NGF治疗AD奠定了基础，但由于NGF分子量较大，难于透过血脑屏障进入脑内，使其临床应用受到限制。有人采用向侧脑室或脑实质内直接注射NGF、或向脑内植入表达NGF的基因片断等方法避开血脑屏障。由于这些方法不但手术风险大、费用昂贵而且会导致感染、副损伤等严重的并发症，因而不能在临床上广泛应用。直到1995年，Frey等人发现NGF及其它种类的营养因子(如IGF-Ⅰ)，可以通过鼻腔给药经嗅神经或三叉神经通路进入脑内，这一发现为NGF提供了一条到达脑内的非创伤性给药途径。此后，国外有学者相继采用经鼻腔给予转基因AD动物模型NGF的方法，预防转基因动物出现AD样病理改变，取得了比较理想的疗效。然而，AD病因错综复杂，基因变异导致的遗传性AD病例仅占AD患者的10％，绝大多数AD患者是由其它原因引起的散发病例。因此，研究经鼻腔给予NGF对非转基因AD动物模型是否具有神经保护作用是非常必要的，目前国内外尚无相关报道。
本研究利用向大鼠侧脑室注射三氯化铝的方法建立非转基因AD大鼠模型，通过观察经鼻腔给予NGF对铝中毒AD大鼠模型行为学、组织学及生物化学等方面的影响，综合评价经鼻腔给予NGF对铝毒性AD大鼠模型的神经保护作用，并初步探讨NGF可能的作用机理，为采用鼻腔给予NGF这一非创伤性途径治疗AD提供理论依据。
实验方法
利用向大鼠侧脑室注射三氯化铝(AlCl_3)的方法建立AD模型。应用Morris水迷宫、苏木素-伊红(Hematoxylin and Eosin，HE)染色、免疫印记(Western blot)、免疫组化ABC染色等技术，观察铝中毒大鼠空间学习记忆能力、大脑皮层和海马神经元数量形态、β-淀粉样蛋白(Aβ)沉积、β-淀粉样前体蛋白(β-amyloid precursor protein，β-APP)和ADAM10表达水平的变化，以及经鼻腔给予神经生长因子(NGF)后，对这些变化的影响。
实验结果
1、Morris水迷宫检测
实验组(侧脑室注射AlCl_3+鼻腔给予NGF)大鼠逃避潜伏期及穿越平台次数与对照组(侧脑室注射saline+鼻腔给予saline)比较，差异无统计学意义(P＞0.05)；而模型组(侧脑室注射AlCl_3+鼻腔给予saline)大鼠逃避潜伏期与对照组比较明显延长、穿越平台次数与对照组比较明显减少，差异均有统计学意义(P＜0.05)。
2、苏木素-伊红(HE)染色
对照组及实验组大鼠皮层、海马神经元排列密集、形态正常、胞膜完整；模型组大鼠神经元明显变性缺失、排列稀疏淡染、形态异常、胞膜破损、可见空泡样变。
3、免疫印记(Western blot)
各组大鼠β-APP表达水平：模型组(整合光密度值：皮层1.28±0.13、海马1.41±0.26)及实验组(整合光密度值：皮层1.16±0.15、海马1.32±0.23)大鼠皮层、海马β-APP含量均明显高于对照组(整合光密度值：皮层0.22±0.04、海马0.31±0.05)，差异有统计学意义(P＜0.05)。
各组大鼠ADAM10蛋白表达水平：ADAM10条带在实验组(整合光密度值：皮层0.93±0.16、海马0.98±0.20)颜色深，与对照组(整合光密度值：皮层0.19±0.07、海马0.21±0.06)比较，差异有统计学意义(P＜0.05)；ADAM10条带在模型组(整合光密度值：皮层0.16±0.04、海马0.19±0.05)及对照组颜色均较淡，两组差异无统计学意义(P＞0.05)。
4、免疫组化ABC染色
Aβ免疫组化ABC染色显示，对照组Aβ阳性反应物为阴性，说明Aβ在正常状态下几乎不表达或表达极微，实验组亦为阴性；而模型组大鼠皮层及海马均可见较多的Aβ阳性反应物。
结论
1、经鼻腔给予NGF能对抗铝的神经毒作用，有效地阻止铝中毒引起的学习记忆能力减退、神经元变性缺失等AD样病变的发生，对铝毒性AD大鼠模型具有神经保护作用。
2、上调ADAM10的蛋白表达水平，使过度表达的β-APP能通过α-分泌酶途径降解，增加可溶性的α-APPs，阻止不溶性Aβ的形成，可能是NGF对抗铝的神经毒性，发挥其神经保护作用的机理之一。
3、上调大鼠海马β-APP基因表达水平，可能是IBA损害海马神经元的分子毒理学机制之一。
Objective
Alzheimer's disease, the most common neurodegenerative disorder and the most frequent cause of dementia, becomes increasingly common with advancing age. Progressive impairment of recent memory is typically clinical findings of Alzheimer's disease. In the late stages psychiatric symptoms and behavioral disturbances may be prominent.
The characteristic histopathology is the presence of large numbers of neurofibrillary tangles, senile plaques, and cholinergic neurons granulovascuolar in the brain, especially in the neocortex and hippocampus of patients with Alzheimer's disease. Intra-neurofibrillary tangles consists of hyperphosphorylated, twisted filaments of the cytoskeletal protein tau, whereas extra-senile plaques are the depositions of amyloidp, a 39-43-amino-acid-long peptide derived from the proteolitic processing of amyloid precursor protein.
Although Alzheimer's disease is a disease of unknown etiology, but the neurotoxicity of amyloidp has been commonly accepted as the pivotal factor in the generation and progress of AD.
Degeneration of cholinergic neuronal pathways and depletion of the choline acetyltransferase have been found in the brains of patients dying of Alzheimer's disease, these changes may contribute to its clinical expression. Therefor, cholinergic replacement therapy has been used in an effort at symptomatic treatment of memonic and cognitive deficits. The drugs tried have included acetylcholine precursors, drugs that stimulate acetylcholine release, acetylcholinesterase inhibitors, muscarinic cholinergic receptor agonists. Several other pharmacologic treatments have been proposed for cognitive dysfunction in Alzheimer's disease, including cerebral vasodilators, central nervous system stimulants, opioid antagonists and neuropeptides. But of these, none has been proved unequivocally to reverse existing deficits or arrest the disease's progression.
Recent research advances in neuroscience show that nerve growth factor (NGF) plays an important role in the growth, development, differenciation, maintenance and regeneration of various type neurons in the CNS.It is also the important trophic factor for cholinergic neurons. NGF increases the synthesis of choline acetyltransferase and prevents cholinergic neurons degeneration caused by experimental injury or associated with physiological and pathological situation, such as aging and Alzheimer's disease. Although the results lay the groundwork to propose a therapeutic use of NGF in AD, the blood-brain barrier represents a major problem in developing a NGF-based treatment for Alzheimer's disease because it prevents this large molecule from reaching the brain. Intracere-broventricular administration NGF, directly infusing NGF into parenchyma, and delivering NGF ex vivo gene are used respectively in order to bypass the blood-brain barrier. However, these approaches not only require the use of risky surgical procedures, high cost but also may lead severe complications such as infections, extra damage and so on. Until 1995, Frey and coworkers showed that NGF and other trophic factors, such as IGF-Ⅰ, can be delivered to the brain via the olfactory and/or trigeminal pathways by intranasal administration, which provided a noninvasive delivery NGF to the brain. After that CapsoniS' work demonstrated that the intranasal NGF delivery was effective in rescuing AD-like neurodegeneration in transgenic mice. But the cause of AD is complicated. Most cases of AD are sporadic, only approximately 10% of AD cases are genetic basis. So it is very important to study the efficacy of intranasal NGF delivery in preventing AD-like changes in non transgenic model of AD. No currently available related data have been reported.
In the present study, we injected AlCl_3 into rat lateral cerebral ventricle to establish non transgenic model of AD, and examined the spatial memorry and immunohistochemical and histological changes after injection, and the effects of intranasal administration of NGF on the changes. This study aims at elucidating the role of intranasal administration of NGF in preventing rats in Al exposure from Alzheimer-like changes and may provide new method for treatment of non genetic AD by noninvasive route of administration for delivery of NGF.
Methods
AlCl_3 was injected into rat lateral cerebral ventricle to establish non transgenic model of AD. Spatial learning and memory changes in aluminum (Al) exposure rats, and the effect on the changes induced by intranasal administration of NGF were measured by using Morris water maze. Immunohistochemical staining was used to detect the effect of intranasal administration of NGF onP-amyloid depositions in frontal cortex and hippocampus of Al exposure rats, and HE staining was used to detect neuronal changes, and the expression level of APP and ADAM 10 were determined by using Western blot.
Results
1. Morris water maze task
The escape latency in the experimental group rats did not differ significantly from those in the control group rats (P＞0.05), and there was no significant difference between the experimental group and the control group in terms of the number of annulus crossings (the number of times the previous platform location was crossed over) (P＞0.05). However, the escape latency in the model group rats prolonged significantly compared with those of control group rats, and there was a significant difference between the model group and the control group in terms of the number of annulus crossings, the difference was statistically significant (P＜0.05).
2. HE analysis
Nerve cells arrange intensive, eumorphism, cell membrane and nuclear membrane integrated in the control group experimental group rats of hippocampus. Nerve cells obvious depletion, arrange rarefaction and lightly dying, paramorphia, cell membrane and nuclear membrane pyknosis and rupture, vacuolus.
3. Western blot analysis
Compared with control group (integrated density value cortex: 0.22±0.04, hippocamp: 0.31±0.05), the expression of APP in the rat brain of model group (integrated density value cortex: 1.28±0.13, hippocamp: 1.41±0.26) and experimental group (cortex: 1.16±0.15, hippocamp: 1.32±0.23) increased remarkably, and the integrated density values of APP were statistically significant (P＜0.05).
A significant increase of integrated density value ratio of ADAM 10 in experimental group rat brain (integrated density value cortex: 0.93±0.16 hippocamp: 0.98±0.20) was noted, Comparing with control group (cortex: 0.19±0.07 hippocamp: 0.21±0.06) (P＜0.05). Compared with control group, the integrated density value ratio of AD AM10 in model group rat brain (cortex: 0.16±0.04, hippocamp: 0.19±0.05) was not increase (P＞0.05).
4. Immunohistochemical staining
The Aβ_(1-40) antibody revealedβ-amyloid depositions in the frontal cortex and hippocampus of model group animals. No labeling is seen in sections from control and experimental group rats.
Conclusion
1. Intranasal administration of nerve growth factor (NGF) can resist the nerve poisonous function of aluminum, can prevent availably from Alzheimer-like behavioral and histological deficits in aluminum-exposure rats, and have the nerve protection function.
2. Resisting the nerve toxicity of aluminum by up-regulation the ADAM 10 expression level, making over expressionβ-APP pass theα-Secretase degradation path, increasing theα-APPs of the dissolubility, and obstructing formation of Aβthat do not dissolve, may be one of the nerve protection mechanism of NGF.
3. Up-regulation the expression level of gene in the hippocampus of rats may be one of mechanism of molecular toxicology of IBA that damaged neurons in hippocampus.
阿尔茨海默病(Alzheimer's disease，AD)是最常见的神经变性疾病，是导致痴呆的主要病因，发病率随年龄而增加。AD患者典型的临床表现是进行性近期记忆力减退，疾病的晚期出现神经症状和行为异常。
AD的主要组织病理学特征是：患者脑内尤其是海马和新皮质出现大量的神经纤维缠结(Neurofibrillary tangles)、老年斑(Senile plagues)及胆碱能神经元缺失、颗粒空泡样变性。细胞内的神经纤维缠结由高度磷酸化的细胞骨架tau蛋白相互缠绕成神经丝构成，而细胞外的老年斑是β-淀粉样前体蛋白经蛋白水解产生的一系列长短不等的Aβ(39-43氨基酸)肽的沉积。
尽管AD病因未明，但大多数学者认为β-淀粉样蛋白(β-amyloid，Aβ)的神经毒作用是AD形成和发展的关键因素。
对AD患者的尸检发现，患者脑内胆硷能神经通路变性及胆碱乙酰基转移酶耗竭，这些改变被认为与AD患者的临床表现有关。所以，胆碱能替代疗法常被用于治疗AD引起的记忆及认知障碍。这类药包括：乙酰胆碱前体、促乙酰胆碱释放剂、胆碱酯酶抑制剂、毒蕈胆碱能受体激动剂。其他种类药物有：脑血管扩张剂、中枢神经系统兴奋剂、鸦片拮抗剂及神经肽。然而，上述药物都不能逆转AD患者的症状或阻止AD病情的进展。
近年来神经科学的研究发现，神经生长因子(nerve growth factor，NGF)对中枢神经系统多种类型神经元的生长、发育、分化、维持和损伤修复都具有重要作用。NGF也是胆碱能神经元重要的营养因子，它能增加乙酰胆碱转移酶的合成，还能阻止实验性损伤、生理性或病理性衰老以及AD等因素引起的胆碱能神经元变性。这些研究成果虽然为应用NGF治疗AD奠定了基础，但由于NGF分子量较大，难于透过血脑屏障进入脑内，使其临床应用受到限制。有人采用向侧脑室或脑实质内直接注射NGF、或向脑内植入表达NGF的基因片断等方法避开血脑屏障。由于这些方法不但手术风险大、费用昂贵而且会导致感染、副损伤等严重的并发症，因而不能在临床上广泛应用。直到1995年，Frey等人发现NGF及其它种类的营养因子(如IGF-Ⅰ)，可以通过鼻腔给药经嗅神经或三叉神经通路进入脑内，这一发现为NGF提供了一条到达脑内的非创伤性给药途径。此后，国外有学者相继采用经鼻腔给予转基因AD动物模型NGF的方法，预防转基因动物出现AD样病理改变，取得了比较理想的疗效。然而，AD病因错综复杂，基因变异导致的遗传性AD病例仅占AD患者的10％，绝大多数AD患者是由其它原因引起的散发病例。因此，研究经鼻腔给予NGF对非转基因AD动物模型是否具有神经保护作用是非常必要的，目前国内外尚无相关报道。
本研究利用向大鼠侧脑室注射三氯化铝的方法建立非转基因AD大鼠模型，通过观察经鼻腔给予NGF对铝中毒AD大鼠模型行为学、组织学及生物化学等方面的影响，综合评价经鼻腔给予NGF对铝毒性AD大鼠模型的神经保护作用，并初步探讨NGF可能的作用机理，为采用鼻腔给予NGF这一非创伤性途径治疗AD提供理论依据。
实验方法
利用向大鼠侧脑室注射三氯化铝(AlCl_3)的方法建立AD模型。应用Morris水迷宫、苏木素-伊红(Hematoxylin and Eosin，HE)染色、免疫印记(Western blot)、免疫组化ABC染色等技术，观察铝中毒大鼠空间学习记忆能力、大脑皮层和海马神经元数量形态、β-淀粉样蛋白(Aβ)沉积、β-淀粉样前体蛋白(β-amyloid precursor protein，β-APP)和ADAM10表达水平的变化，以及经鼻腔给予神经生长因子(NGF)后，对这些变化的影响。
实验结果
1、Morris水迷宫检测
实验组(侧脑室注射AlCl_3+鼻腔给予NGF)大鼠逃避潜伏期及穿越平台次数与对照组(侧脑室注射saline+鼻腔给予saline)比较，差异无统计学意义(P＞0.05)；而模型组(侧脑室注射AlCl_3+鼻腔给予saline)大鼠逃避潜伏期与对照组比较明显延长、穿越平台次数与对照组比较明显减少，差异均有统计学意义(P＜0.05)。
2、苏木素-伊红(HE)染色
对照组及实验组大鼠皮层、海马神经元排列密集、形态正常、胞膜完整；模型组大鼠神经元明显变性缺失、排列稀疏淡染、形态异常、胞膜破损、可见空泡样变。
3、免疫印记(Western blot)
各组大鼠β-APP表达水平：模型组(整合光密度值：皮层1.28±0.13、海马1.41±0.26)及实验组(整合光密度值：皮层1.16±0.15、海马1.32±0.23)大鼠皮层、海马β-APP含量均明显高于对照组(整合光密度值：皮层0.22±0.04、海马0.31±0.05)，差异有统计学意义(P＜0.05)。
各组大鼠ADAM10蛋白表达水平：ADAM10条带在实验组(整合光密度值：皮层0.93±0.16、海马0.98±0.20)颜色深，与对照组(整合光密度值：皮层0.19±0.07、海马0.21±0.06)比较，差异有统计学意义(P＜0.05)；ADAM10条带在模型组(整合光密度值：皮层0.16±0.04、海马0.19±0.05)及对照组颜色均较淡，两组差异无统计学意义(P＞0.05)。
4、免疫组化ABC染色
Aβ免疫组化ABC染色显示，对照组Aβ阳性反应物为阴性，说明Aβ在正常状态下几乎不表达或表达极微，实验组亦为阴性；而模型组大鼠皮层及海马均可见较多的Aβ阳性反应物。
结论
1、经鼻腔给予NGF能对抗铝的神经毒作用，有效地阻止铝中毒引起的学习记忆能力减退、神经元变性缺失等AD样病变的发生，对铝毒性AD大鼠模型具有神经保护作用。
2、上调ADAM10的蛋白表达水平，使过度表达的β-APP能通过α-分泌酶途径降解，增加可溶性的α-APPs，阻止不溶性Aβ的形成，可能是NGF对抗铝的神经毒性，发挥其神经保护作用的机理之一。
3、上调大鼠海马β-APP基因表达水平，可能是IBA损害海马神经元的分子毒理学机制之一。
Objective
Alzheimer's disease, the most common neurodegenerative disorder and the most frequent cause of dementia, becomes increasingly common with advancing age. Progressive impairment of recent memory is typically clinical findings of Alzheimer's disease. In the late stages psychiatric symptoms and behavioral disturbances may be prominent.
The characteristic histopathology is the presence of large numbers of neurofibrillary tangles, senile plaques, and cholinergic neurons granulovascuolar in the brain, especially in the neocortex and hippocampus of patients with Alzheimer's disease. Intra-neurofibrillary tangles consists of hyperphosphorylated, twisted filaments of the cytoskeletal protein tau, whereas extra-senile plaques are the depositions of amyloidp, a 39-43-amino-acid-long peptide derived from the proteolitic processing of amyloid precursor protein.
Although Alzheimer's disease is a disease of unknown etiology, but the neurotoxicity of amyloidp has been commonly accepted as the pivotal factor in the generation and progress of AD.
Degeneration of cholinergic neuronal pathways and depletion of the choline acetyltransferase have been found in the brains of patients dying of Alzheimer's disease, these changes may contribute to its clinical expression. Therefor, cholinergic replacement therapy has been used in an effort at symptomatic treatment of memonic and cognitive deficits. The drugs tried have included acetylcholine precursors, drugs that stimulate acetylcholine release, acetylcholinesterase inhibitors, muscarinic cholinergic receptor agonists. Several other pharmacologic treatments have been proposed for cognitive dysfunction in Alzheimer's disease, including cerebral vasodilators, central nervous system stimulants, opioid antagonists and neuropeptides. But of these, none has been proved unequivocally to reverse existing deficits or arrest the disease's progression.
Recent research advances in neuroscience show that nerve growth factor (NGF) plays an important role in the growth, development, differenciation, maintenance and regeneration of various type neurons in the CNS.It is also the important trophic factor for cholinergic neurons. NGF increases the synthesis of choline acetyltransferase and prevents cholinergic neurons degeneration caused by experimental injury or associated with physiological and pathological situation, such as aging and Alzheimer's disease. Although the results lay the groundwork to propose a therapeutic use of NGF in AD, the blood-brain barrier represents a major problem in developing a NGF-based treatment for Alzheimer's disease because it prevents this large molecule from reaching the brain. Intracere-broventricular administration NGF, directly infusing NGF into parenchyma, and delivering NGF ex vivo gene are used respectively in order to bypass the blood-brain barrier. However, these approaches not only require the use of risky surgical procedures, high cost but also may lead severe complications such as infections, extra damage and so on. Until 1995, Frey and coworkers showed that NGF and other trophic factors, such as IGF-Ⅰ, can be delivered to the brain via the olfactory and/or trigeminal pathways by intranasal administration, which provided a noninvasive delivery NGF to the brain. After that CapsoniS' work demonstrated that the intranasal NGF delivery was effective in rescuing AD-like neurodegeneration in transgenic mice. But the cause of AD is complicated. Most cases of AD are sporadic, only approximately 10% of AD cases are genetic basis. So it is very important to study the efficacy of intranasal NGF delivery in preventing AD-like changes in non transgenic model of AD. No currently available related data have been reported.
In the present study, we injected AlCl_3 into rat lateral cerebral ventricle to establish non transgenic model of AD, and examined the spatial memorry and immunohistochemical and histological changes after injection, and the effects of intranasal administration of NGF on the changes. This study aims at elucidating the role of intranasal administration of NGF in preventing rats in Al exposure from Alzheimer-like changes and may provide new method for treatment of non genetic AD by noninvasive route of administration for delivery of NGF.
Methods
AlCl_3 was injected into rat lateral cerebral ventricle to establish non transgenic model of AD. Spatial learning and memory changes in aluminum (Al) exposure rats, and the effect on the changes induced by intranasal administration of NGF were measured by using Morris water maze. Immunohistochemical staining was used to detect the effect of intranasal administration of NGF onP-amyloid depositions in frontal cortex and hippocampus of Al exposure rats, and HE staining was used to detect neuronal changes, and the expression level of APP and ADAM 10 were determined by using Western blot.
Results
1. Morris water maze task
The escape latency in the experimental group rats did not differ significantly from those in the control group rats (P＞0.05), and there was no significant difference between the experimental group and the control group in terms of the number of annulus crossings (the number of times the previous platform location was crossed over) (P＞0.05). However, the escape latency in the model group rats prolonged significantly compared with those of control group rats, and there was a significant difference between the model group and the control group in terms of the number of annulus crossings, the difference was statistically significant (P＜0.05).
2. HE analysis
Nerve cells arrange intensive, eumorphism, cell membrane and nuclear membrane integrated in the control group experimental group rats of hippocampus. Nerve cells obvious depletion, arrange rarefaction and lightly dying, paramorphia, cell membrane and nuclear membrane pyknosis and rupture, vacuolus.
3. Western blot analysis
Compared with control group (integrated density value cortex: 0.22±0.04, hippocamp: 0.31±0.05), the expression of APP in the rat brain of model group (integrated density value cortex: 1.28±0.13, hippocamp: 1.41±0.26) and experimental group (cortex: 1.16±0.15, hippocamp: 1.32±0.23) increased remarkably, and the integrated density values of APP were statistically significant (P＜0.05).
A significant increase of integrated density value ratio of ADAM 10 in experimental group rat brain (integrated density value cortex: 0.93±0.16 hippocamp: 0.98±0.20) was noted, Comparing with control group (cortex: 0.19±0.07 hippocamp: 0.21±0.06) (P＜0.05). Compared with control group, the integrated density value ratio of AD AM10 in model group rat brain (cortex: 0.16±0.04, hippocamp: 0.19±0.05) was not increase (P＞0.05).
4. Immunohistochemical staining
The Aβ_(1-40) antibody revealedβ-amyloid depositions in the frontal cortex and hippocampus of model group animals. No labeling is seen in sections from control and experimental group rats.
Conclusion
1. Intranasal administration of nerve growth factor (NGF) can resist the nerve poisonous function of aluminum, can prevent availably from Alzheimer-like behavioral and histological deficits in aluminum-exposure rats, and have the nerve protection function.
2. Resisting the nerve toxicity of aluminum by up-regulation the ADAM 10 expression level, making over expressionβ-APP pass theα-Secretase degradation path, increasing theα-APPs of the dissolubility, and obstructing formation of Aβthat do not dissolve, may be one of the nerve protection mechanism of NGF.
3. Up-regulation the expression level of gene in the hippocampus of rats may be one of mechanism of molecular toxicology of IBA that damaged neurons in hippocampus.
引文
1 Smith,D.E.Roberts,J.Gage,F.H.etal.Aged-associated neuronal atrophy occurs in the primate brain and reversible by nerve growth factor gene therapy[J].Neurobiology,1999,96(19): 10893-1089.
2 Chaturvedi RK,Shukla S,Seth K,et al.Nerve growth factor increases survival of dopaminergic graft,rescue negral dopaminergic neurons and restores functional deficits in rat model of Parkinsons disease.NeurosciLett.2006,398(1-2):44-49
3 Frey WH, Liu J, Chen X,et al.Delivery of 125I-NGF to the brain via the olfactory route[J].Drug Deliv, 1997,4(1):87-92.
4 DeRosaR, Garcia AA, BraschiC, et al.Intranasal administration of nerve growth factory (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice.Proc Natl Acad Sci U S A,2005,102(10):3811-3816.
5 Capsoni S, Giannotta S, Cattanaeo A. Nerve growth factor and galantamine ameliorate early signs of neurodegeneration in anti-nerve growth factor mince. Proc Natl Acad Sci U S A, 2002, 99(19):12432-12437.
6 BAO XM，SHU SY．The Stereotaxic Atlas of The Rat Brain．[M]北京：人民卫生出版社．1991：44-45
7 赵宪林，方秀斌，李东培．大鼠血管性痴呆模型制作 中国医科大学学报 2002；31(3)：166—177．
8 秦俊法．微量元素与脑功能．第一版，北京．原子能出版社．1994，52．
9 Klatzo I, Wishie wisk H, Streicher E. experimental production of neurofibrillary degeneration. J Neuropath Exp Neural, 1965, 24:187.
10 Crapper DG. Krishnan SS, Dakton AJ. Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science 1973, 180(4085): 511.
11 PerL DP. Brady AR. Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle bearing neurons. Science 1980, 208(18): 297.
12 Morris R G M. Garrud P, Raulins J NP, et al. Place navigation impaired in rats with hippocampal lesions [J] Nature, 1982, 297: 681.
13 Squire L R. Zola-Morgan S. Memory brain system and behavior [J] Trends in Neuro Science, 1988, 11: 170.
14 Andrei C. MIU et, al. Abehavioral and histological study of the effects of exposure of adult rats to aluminum J. Neuroscience, 2003, 113:1197-1211.
15 Boegman, R. J., & Bates, L. A. (1984). Neurotoxicity of aluminum. Canadian Journal of Physiology and Pharmacology, 62, 1010-1014.
16 郑观成Alzheimer's病的脑形态学．脑老化与老年性痴呆．上海：上海科学技术文献出版社．1995．55-69．
17 Pepeu G; Giovannelli L, Casamenti F et, al. Amyloidβ-peptides injection into the cholinergic nuclei: morphological, neurochomical and behavioral effects [J]. Prog Brain Res, 2006, 109: 273-82.
18 Bondy, S. C., & Truong, A. (1999). Potentiation of beta-folding of β-amyloid peptide 25-35 by aluminum salts. Neuroscience Letters, 1999, 267, 25-28.
19 Glenner GG, Wong CW. Alzheimer's disease and Down's syndrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res, 1983;122:1131-1135.
20 Colciaghi F, Marcello E, Borroni B, et al. Platelet APP, ADAM10 and BACE alterations in the early stages of Alzheimer disease. Neurology, 2004, 62; 498-501.
21 Di Luca M, Grossi E, Borroni B, et al. Artificial neural networks allow the use of simultaneous measurements of Alzheimer disease markers for early detection of the disease. J Transl Med, 2005, 3: 30.
22 Lammich S, Kojiro E,Postinqa R, et al.Constitutive and regulated secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. PROC Natl Acad USA, 1999, 96: 3922-3927.
23 刘春风，戴永萍，包仕尧．β淀粉样蛋白对大鼠额叶和海马细胞外液单胺类递质的影响．[J]．临床神经病学杂志．2002，15：260．
24 丁新生，程虹，张雪玲，等．脑脊液中β淀粉样蛋白检测对老年期痴呆的诊断意义[J]．临床神经病学杂志．2001，14：3．
25 Hardy J, Selkoe DJ. The amyloid hypohesis of Alzheimers diease: progress and problems on the road to theropeutics [J] science, 2002, 297: 353.
26 Flood JF, Roberts E.An amyloid β-protein fragment, Aβ(12-28), equiptently impairs postrtaining memory pressing when injection into different limbic system atructure. Brain Res, 2004, 663: 271.
27 吴正治等．清肝泻火对老年痴呆鼠模型学习记忆及中枢单胺神经递质的影响．中国中医基础医学杂志．1998．12．4(12)：136—137．
28 贾怡昌等．铝中毒痴呆鼠海马脂质过氧化和谷氨酰胺合成酶活性变化．中华老年医学杂志．2002，21(2)：125—127．
29 Ahmed MM, Hoshino H, Chikun T, Kato T. Effect of memantine on the levels of glial cells, neuropeptides, and peptide-degrading enzymes in rat brain lesions of ibotenic acid treated Alzheimer's disease model [J]. Neuroscience, 2004, 126(3): 639-649.
30 Spowart-Manning L, van der Staay FJ. Spatial discrimination deficits by excitotoxic lesions in the Morris water escapetask [J]Behav Brain Res, 2005,156 (2): 269-276.
31 Csernansk JG, Bardgett ME, Sheline YI, Morris JC, Olney JW. CSF excitatory amino acids and severity of illness in Alzheimer's disease [J].Neurology, 1996, 46(6): 1715-1720.
32 Henneberry RC, Novelli A, Vigano MA, Reilly JA, Cox JA, Lysko PG. Energy-related neurotoxicity at the NMD receptor: a possible role in Alzheimers disease and related disorders.Prog Clin BioRes. 1989, 317:143-56.Res. 1989; 317:143-56.
33 Paul T Francis, Alan p Palmer, Iichael Snape, Gordon K Wilcock. The cholinergic hypothesis of Alzheimers disease: a review of progress J Neurol Neurosurg Psychiatry 1999:66:137-147 (February)
34 姚志斌．海马——研究神经科学的理想模型．广东解剖学通报．1989，11：17-21．
35 郭长杰．三七总皂苷对痴呆大鼠模型学习记忆行为的影响及机理探讨 中国药房 2004，10．
36 知母活性成分对脑内定位注射鹅膏蕈酸痴呆大鼠的影响 核技术 2004，4：20—22．
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2 Chaturvedi RK,Shukla S,Seth K,et al.Nerve growth factor increases survival of dopaminergic graft,rescue negral dopaminergic neurons and restores functional deficits in rat model of Parkinsons disease.NeurosciLett.2006,398(1-2):44-49
3 Frey WH, Liu J, Chen X,et al.Delivery of 125I-NGF to the brain via the olfactory route[J].Drug Deliv, 1997,4(1):87-92.
4 DeRosaR, Garcia AA, BraschiC, et al.Intranasal administration of nerve growth factory (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice.Proc Natl Acad Sci U S A,2005,102(10):3811-3816.
5 Capsoni S, Giannotta S, Cattanaeo A. Nerve growth factor and galantamine ameliorate early signs of neurodegeneration in anti-nerve growth factor mince. Proc Natl Acad Sci U S A, 2002, 99(19):12432-12437.
6 BAO XM，SHU SY．The Stereotaxic Atlas of The Rat Brain．[M]北京：人民卫生出版社．1991：44-45
7 赵宪林，方秀斌，李东培．大鼠血管性痴呆模型制作 中国医科大学学报 2002；31(3)：166—177．
8 秦俊法．微量元素与脑功能．第一版，北京．原子能出版社．1994，52．
9 Klatzo I, Wishie wisk H, Streicher E. experimental production of neurofibrillary degeneration. J Neuropath Exp Neural, 1965, 24:187.
10 Crapper DG. Krishnan SS, Dakton AJ. Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science 1973, 180(4085): 511.
11 PerL DP. Brady AR. Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle bearing neurons. Science 1980, 208(18): 297.
12 Morris R G M. Garrud P, Raulins J NP, et al. Place navigation impaired in rats with hippocampal lesions [J] Nature, 1982, 297: 681.
13 Squire L R. Zola-Morgan S. Memory brain system and behavior [J] Trends in Neuro Science, 1988, 11: 170.
14 Andrei C. MIU et, al. Abehavioral and histological study of the effects of exposure of adult rats to aluminum J. Neuroscience, 2003, 113:1197-1211.
15 Boegman, R. J., & Bates, L. A. (1984). Neurotoxicity of aluminum. Canadian Journal of Physiology and Pharmacology, 62, 1010-1014.
16 郑观成Alzheimer's病的脑形态学．脑老化与老年性痴呆．上海：上海科学技术文献出版社．1995．55-69．
17 Pepeu G; Giovannelli L, Casamenti F et, al. Amyloidβ-peptides injection into the cholinergic nuclei: morphological, neurochomical and behavioral effects [J]. Prog Brain Res, 2006, 109: 273-82.
18 Bondy, S. C., & Truong, A. (1999). Potentiation of beta-folding of β-amyloid peptide 25-35 by aluminum salts. Neuroscience Letters, 1999, 267, 25-28.
19 Glenner GG, Wong CW. Alzheimer's disease and Down's syndrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res, 1983;122:1131-1135.
20 Colciaghi F, Marcello E, Borroni B, et al. Platelet APP, ADAM10 and BACE alterations in the early stages of Alzheimer disease. Neurology, 2004, 62; 498-501.
21 Di Luca M, Grossi E, Borroni B, et al. Artificial neural networks allow the use of simultaneous measurements of Alzheimer disease markers for early detection of the disease. J Transl Med, 2005, 3: 30.
22 Lammich S, Kojiro E,Postinqa R, et al.Constitutive and regulated secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. PROC Natl Acad USA, 1999, 96: 3922-3927.
23 刘春风，戴永萍，包仕尧．β淀粉样蛋白对大鼠额叶和海马细胞外液单胺类递质的影响．[J]．临床神经病学杂志．2002，15：260．
24 丁新生，程虹，张雪玲，等．脑脊液中β淀粉样蛋白检测对老年期痴呆的诊断意义[J]．临床神经病学杂志．2001，14：3．
25 Hardy J, Selkoe DJ. The amyloid hypohesis of Alzheimers diease: progress and problems on the road to theropeutics [J] science, 2002, 297: 353.
26 Flood JF, Roberts E.An amyloid β-protein fragment, Aβ(12-28), equiptently impairs postrtaining memory pressing when injection into different limbic system atructure. Brain Res, 2004, 663: 271.
27 吴正治等．清肝泻火对老年痴呆鼠模型学习记忆及中枢单胺神经递质的影响．中国中医基础医学杂志．1998．12．4(12)：136—137．
28 贾怡昌等．铝中毒痴呆鼠海马脂质过氧化和谷氨酰胺合成酶活性变化．中华老年医学杂志．2002，21(2)：125—127．
29 Ahmed MM, Hoshino H, Chikun T, Kato T. Effect of memantine on the levels of glial cells, neuropeptides, and peptide-degrading enzymes in rat brain lesions of ibotenic acid treated Alzheimer's disease model [J]. Neuroscience, 2004, 126(3): 639-649.
30 Spowart-Manning L, van der Staay FJ. Spatial discrimination deficits by excitotoxic lesions in the Morris water escapetask [J]Behav Brain Res, 2005,156 (2): 269-276.
31 Csernansk JG, Bardgett ME, Sheline YI, Morris JC, Olney JW. CSF excitatory amino acids and severity of illness in Alzheimer's disease [J].Neurology, 1996, 46(6): 1715-1720.
32 Henneberry RC, Novelli A, Vigano MA, Reilly JA, Cox JA, Lysko PG. Energy-related neurotoxicity at the NMD receptor: a possible role in Alzheimers disease and related disorders.Prog Clin BioRes. 1989, 317:143-56.Res. 1989; 317:143-56.
33 Paul T Francis, Alan p Palmer, Iichael Snape, Gordon K Wilcock. The cholinergic hypothesis of Alzheimers disease: a review of progress J Neurol Neurosurg Psychiatry 1999:66:137-147 (February)
34 姚志斌．海马——研究神经科学的理想模型．广东解剖学通报．1989，11：17-21．
35 郭长杰．三七总皂苷对痴呆大鼠模型学习记忆行为的影响及机理探讨 中国药房 2004，10．
36 知母活性成分对脑内定位注射鹅膏蕈酸痴呆大鼠的影响 核技术 2004，4：20—22．
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