阿尔茨海默病转基因小鼠早期记忆功能障碍与氧化应激损伤关系的实验研究

目的 观察转APP/PS1基因阿尔茨海默病(AD)小鼠(APP/PS1小鼠)早期空间学习记忆功能及相关氧化应激反应指标的变化,并探讨它们之间的相关性.方法 应用Morris水迷宫评定APP/PS1小鼠及相应野生型(WT)小鼠的空间学习记忆功能,采用ELISA方法检测脑组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性以及丙二醛(MDA)和蛋白质羰基的含量,并进行相关性分析.结果 2组小鼠的空间学习能力无明显差异(P＞0.05),而APP/PS1小鼠在目标象限(定位航行实验中平台所置第二象限)中航行时间占总时间的百分比(29.02±4.27)%较WT小鼠(47.39±6.01)%显著下降(t=0.000,P＜0.05),APP/PS1小鼠在目标象限中航行路程占总路程的百分比(28.85±3.77)%较WT小鼠(46.70±5.60)%也显著下降(t=0.000,P＜0.05),提示APP/PS1小鼠的空间记忆功能较WT小鼠显著下降.2组小鼠脑组织中的MDA含量、SOD和GSH-PX活性均无明显差异(P均＞0.05),而APP/PS1小鼠脑组织中的蛋白质羰基含量(2.67±0.19)较WT小鼠(2.38±0.15)显著增加(t=0.0088,P＜0.05).相关性分析表明:APP/PS1小鼠蛋白质羰基含量与目标象限航行时间百分比呈显著负性相关(r=-0.639,P＜0.05),APP/PS1小鼠蛋白质羰基含量与目标象限航行路程百分比呈显著负性相关(r=-0.636,P＜0.05).结论 APP/PS1小鼠早期空间记忆功能损害与脑组织中的蛋白质羰基含量升高呈负性相关,提示氧化应激导致的蛋白质羰基化在AD早期记忆损害发病过程中具有重要作用.

Abstract：

Objective To investigate the spatial learning and memory ability,the changes of indicators of oxidative stress,and their relationship in transgenic APP/PS1 mouse model of Alzheimer's disease(APP/PS1 mice). Methods The spatial learning and memory ability were assessed by Morris water maze test,and the activity or content of SOD, GSH-PX, MDA, and protein carbonyl in brain tissues were measured by ELISA in the APP/PS1 and wild type (WT) mice. Furthermore, the relationship between the learning and memory performances and the indicators of oxidative stress was examined. Results No significant difference in the spatial learning was observed between the APP/PS1 and WT mice (P ＜0. 05). The spatial memory which was measured as the percentage of time traveling in the targeted quadrant to the total traveling time was significantlydeclined in the APP/PS1 mice(29. 02 ± 4. 27) % as compared with the WT mice(47. 39 ± 6. 01) %(t =0. 000 ,P ＜0. 05). The percentage of length of traveling in the targeted quadrant to the total length traveled was significantly lower in the APP/PS1 mice(28. 85 ±3.77)% compared with the WT mice(46. 70 ±5.60)% (t =0. 000,P ＜0. 05). These findings indicated that the spatial learning and memory ability of APP/PS1 mice was significantly decreased compared to WT mice. There was no significant difference in activity or content of SOD,GSH-PX,and MDA in brain tissues between the APP/PS1 and WT mice (P ＜ 0. 05), while the content of protein carbonyl was significantly elevated in the APP/PS1 mice (2. 67 ±0. 19) than in the WT mice (2. 38 ±0. 15)(t = 0. 008, P ＜ 0. 05). Correlation analysis revealed that the elevated protein carbonyl was negatively correlated with the percentage of length traveled in the targeted quadrant(r = - 0. 639, P ＜ 0. 05) and the percentage of time traveled in the targeted quadrant(r = - 0. 636 ,P ＜ 0. 05). Conclusion The spatial memory impairment was negatively correlated with the elevated protein carbonyl in the APP/PS1 mice, suggesting that protein carbonylation caused by oxidative stress might play an important role in the development of memory impairment in the early stage of Alzheimer's disease.     

转基因阿尔茨海默病小鼠脑内炎症反应的诱发因素研究

目的 观察炎症反应在转基因阿尔茨海默病(AD)小鼠脑组织中的变化,探讨AD脑内炎症反应的诱发因素.方法 选用3、12个月龄转人β-淀粉样前体蛋白/早老素-1(APP/PS1)基因AD小鼠及正常野生型(WT)小鼠,分别应用免疫组织化学法和ELISA法观察脑内淀粉样斑块、炎性因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)α、前列腺素(PGE)2]的变化.结果 3个月龄APP/PS1基因AD小鼠脑组织中无淀粉样斑块沉积,未发现激活的星型胶质细胞和小胶质细胞,炎性因子IL-1β、IL-6、TNFα、PGE2的含量与WT小鼠差异无统计学意义(P均＞0.05).12个月龄转APP/PS1基因AD小鼠脑组织中有大量淀粉样斑块沉积,并伴有大量激活的星型胶质细胞和小胶质细胞,炎性因子IL-1β、IL-6、TNFα、PGE2的含量[分别为(56.02±9.04)、(8.66±0.83)、(97.48±26.58)、(72.18±21.01)ng/g]较WT小鼠[分别为(29.81±6.03)、(7.73±0.74)、(61.98±11.11)、(37.23±10.96)ng/g]及3个月龄转APP/PS1基因AD小鼠[分别为(30.05±3.53)、(7.43±1.17)、(59.34±10.47)、(42.56±5.93)ng/g]显著增加(P＜0.05或P＜0.01).结论 在淀粉样斑块形成之前,转APP/PS1基因AD小鼠脑组织中无明确的炎症反应;而淀粉样斑块沉积之后,脑组织中有显著的炎症反应;AD脑内炎症反应与淀粉样斑块形成密切相关,淀粉样蛋白(Aβ)沉积是导致脑内炎症反应的直接诱发因素.

Abstract：

Objective To observe the changes of cerebral inflammation-related markers in brain of a transgenic mouse model of Alzheimer's disease (AD) ,and to determine the causative factor to the development of cerebral inflammation in AD. Methods 3- and 12-month-old β-amyloid protein precursor ( APP)/presenilin (PSI) transgenic mice and age-matched wild-type mice (WT) were used in the study. The changes of amyloid plaques, inflammatory factors ( interleukin 1β ( IL-1β ); interleukin 6( IL-6 ); tumor necrosis factor α (TNFα) ;prostaglandin E2 (PGE2)) in the brains among these mice were measured by immunohistochemistry and ELISA. Results Immunohistochemical analysis revealed that no amyloid plaques and activated astrocytes as well as microglia were observed in the 3-month-old APP/PS1 mice. There were no significant differences in the levels of inflammatory factors (IL-1β, IL-6 ,TNFα,and PGE2) between the 3-month-old APP/PS1 and WT mice ( Ps ＞ 0. 05 ). However, abundant amyloid plaques accompanied by a remarkable increase of activated astrocytes and microglia were found in the brain of the 12-month-old APP/PS1 mice. The levels of inflammatory factors (IL-1β,IL-6,TNFα, and PGE2 ) were significantly increased in the 12-month-old APP/PS1 mice ([56. 02 ±9. 04] ng/g, [8. 66 ±0.83] ng/g, [97.48 ±26.58] ng/g, [72. 18 ±21.01] ng/g) than in the WT mice ([29. 18 ± 6. 03] ng/g, [7. 73 ± 0. 74] ng/g, [61.98 ±11.11] ng/g, [37. 23 ± 10. 96] ng/g) and the 3-month-old APP/PS1 mice ( [30. 05 ± 3.53] ng/g, [7.43 ± 1.17] ng/g, [59.34 ± 10. 07] ng/g, [42. 56 ±5.93] ng/g) (P＜0.05,or P＜0.01,respectively). Conclusion This study demonstrates that the APP/PS1mice did not show cerebral inflammation before the appearance of amyloid plaques, and exhibited remarkable inflammation after amyloid plaque deposition. These findings suggest that the induction of cerebral inflammation is tightly associated with amyloid plaque formation, and deposition of amyloid-beta protein (Aβ) may be the direct causative factor to the development of cerebral inflammation in AD.     

运动对快速老化小鼠海马β-淀粉样蛋白及淀粉样蛋白前体蛋白的影响

目的 研究运动对快速老化小鼠(SAMP)8海马β-淀粉样蛋白(Aβ)及淀粉样蛋白前体蛋白(APP)的影响,以探讨运动改善阿尔茨海默病(AD)学习记忆功能的机制.方法 将40只3月龄SAMP8小鼠采用单纯随机抽样法分为运动组和对照组,运动组进行跑笼运动训练,第1个月每周训练5 d,每天10 min,第2个月每周训练5 d,每天20 min.2个月后采用H-E染色观察2组小鼠海马神经元形态改变;免疫组织化学技术检测海马Aβ免疫阳性细胞的表达;逆转录-聚合酶链反应(RT-PCR)技术检测海马APP mRNA的表达水平.结果 对照组5月龄SAMP8小鼠海马部分神经元细胞变性、死亡,核浓缩,空泡变性;运动组偶有神经元细胞变性、死亡,大部分细胞形态正常.运动组海马Aβ免疫阳性细胞表达水平为(0.192±0.018),明显低于对照组的(0.274±0.017)(P＜0.05);运动组海马APP mRNA表达水平为(0.168±0.059),明显低于对照组的(0.574±0.115)(P＜0.01).结论 运动可以延缓SAMP8小鼠海马神经元变性,降低海马Aβ及APP的表达,这可能是运动改善AD学习记忆功能的重要机制之一.

Abstract：

Objective To explore the effects of movement on hippocampal β-amyloid protein ( Aβ ) and amyloid precursor protein (APP) in senescence-accelerated and senescence-prone (SAMP8) mice, and the mechanism by which movement improves learning and memory in mice with a model of Alzheimer's disease. Methods Forty 3-month-old SAMP8 mice were divided randomly into a movement group and a control group. The movement group was trained with a running wheel 10 min daily, 5 days a week in the first month, and 20 min daily in the second month. Morphological changes in the hippocampus were observed under the microscope after HE staining. The expression of Aβ in the hippocampus was detected by immumohistochemical methods and APP mRNA expression was detected by RT-PCR two months later. Results HE staining showed neuron degeneration and death, chromatin condensation and vacuolar degeneration in the hippocampus of the 5-mouth-old SAMP8 mice of the control group. The movement group showed less neuron degeneration and death, and the morphology of most cells was normal The expression of Aβ in the hippocampus of the 5-month-old SAMP8 mice in the movement group was significantly lower than that in the control group. APP mRNA expression levels in the movement group were also significantly lower.Conclusions Movement can delay neuron degeneration and down-regulate Aβ and APP mRNA expression levels in the hippocampus of SAMP8 mice. It may be an important mechanism by which movement improves learning and memory in mice with a model of Alzheimer's disease.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.     

电针配合神经生长因子对脑缺血大鼠学习记忆能力的影响

Objective To observe the effect of nerve growth factor(NGF)and electro-acupuncture(EA) on learning and memory in rats with cerebral ischemia.Methods An experimental model of cerebral ischemiareperfusion was established in 40 rats,who were then randomly divided into a model group,a sham operated group,a NGF group,an acupuncture group and a NGF plus acupuncture group.Three weeks were allowed for the recovery of the blood-brain barrier lesion caused by ischemia-reperfusion before intravenous injection of NGF(10μg/kg,once daily for 15 days)via the vena caudalis.In the two acupuncture groups,this was administered in combination with EA at the Baihui(DU20)and Yamen(DU15)acupoints(frequency 100 Hz,2 mA,20 min/time,once daily for 15 days).Morris' water maze test was used to assess any changes in spatial learning and memory abilities.Results The ischemic rats showed significant learning and memory disorders compared with the sham-operated group.In the NGF group,the rats' learning and memory abilities did not impmve significantly compared with the model group and the EA group.Significantly better learning was observed in the NGF+EA group compared with the model group,the EA group and the NGF group.Conclusion NGF combined with EA can improve the learning and memory abilities of rats with learning and memory dysfunction caused by cerebral ischemia-repedusion.