中药Q0409筛选方改善SAM-P/8小鼠的学习记忆能力

目的:研究中药Q0409筛选方是否能够改善SAM-P/8小鼠的学习记忆能力及其初步机制。方法:4月龄SAM-P/8小鼠、SAM-R/1小鼠和昆明小鼠共91只,分别标记雌雄,根据动物在Morris水迷宫实验找到平台潜伏期的长短,按随机区组法分成5组,即正常对照组、SAM-R/1对照组、疾病模型组、中药Q0409筛选方治疗组和阳性药物(石杉碱甲)组,采用不同的药物或蒸馏水连续灌胃60 d(即从4月龄到6月龄)。第61~65天,先灌胃相应剂量药物或双蒸水,1 h后进行Morris水迷宫实验,观测行为学指标。在第65天,进行水迷宫测试结束后立即处死小鼠,取出脑海马组织,将其一半置玻璃匀浆器中加入预冷生理盐水制成10%(g/m L)匀浆,用于测定脑海马组织匀浆中乙酰胆碱酯酶(ACh E)的活性;将另一半海马组织用4%多聚甲醛固定,然后进行石蜡包埋及切片,进行免疫组化染色以观察β-淀粉样蛋白(Aβ)的表达情况。结果:与疾病模型组相比,中药Q0409治疗组小鼠Morris水迷宫实验中定位航行和空间探索实验的结果显著改善(P0.05),海马组织匀浆ACh E活性显著降低(P0.05);但上述指标与正常对照组和阳性药物组相比差异无统计学显著性。免疫组化观察结果显示,中药Q0409筛选方治疗组雄鼠未见典型的"老年斑",雌鼠可见"老年斑"颜色变浅,两者的海马CA1区Aβ蛋白沉积的阳性面积减少;这些结果与阳性药物组相似。结论:中药Q0409筛选方可有效改善SAM-P/8小鼠的学习和记忆能力,其机制可能与其抑制海马区ACh E活性并减少Aβ蛋白的沉积等有关,其效果与石杉碱甲相似。     

基于齿状回神经元树突棘形态探讨ROCK2敲减改善APP/PS1小鼠认知功能的作用机制

目的：观察海马区敲减Rho相关激酶2(ROCK2)对阿尔茨海默病模型APPswe/PS1dE9(APP/PS1转基因小鼠的干预效果，并探索其对海马齿状回区神经元树突棘形态的影响及潜在机制。方法：选用雄性8月龄的C57BL/6及APP/PS1小鼠，随机分为4组，分别为野生型（WT）组（8只）、APP/PS1小鼠生理盐水（NS）组（8只）、APP/PS1小鼠敲减对照（shRNA）组（12只）和APP/PS1小鼠ROCK2敲减（shROCK2）组（12只）。通过腺相关病毒共转染shRNA的方法，下调APP/PS1转基因小鼠双侧海马区神经元ROCK2蛋白表达，实现海马区局部神经元ROCK2敲减。利用水迷宫和Y迷宫实验检测小鼠的认知功能；免疫荧光染色和Western blot检测AD相关病理改变；激光共聚焦和Imairs软件观察并分析海马齿状回区神经元的树突棘形态变化；Western blot检测海马组织中突触相关蛋白的表达水平。结果：水迷宫实验中，与WT组的小鼠相比，NS和shRNA组小鼠到达平台的时间、到达平台的距离及第一次进入平台所在象限的时间均显著增加（P<0.05或P<0....     

APP/PS1双转基因AD小鼠早期内质网应激诱导的凋亡

目的观察APP/PS1双转基因AD小鼠神经细胞凋亡,及内质网分子伴侣葡萄糖调节蛋白（GRP78）和内质网促凋亡因子半胱氨酸蛋白酶-12（Caspase-12）表达的改变,探讨APP/PS1双转基因AD小鼠早期内质网应激诱导的凋亡。方法选取5、7月龄的APP/PS1双转基因小鼠和同月龄同背景的野生型小鼠（WT）,分为5月龄WT组、5月龄APP/PS1组、7月龄WT组和7月龄APP/PS1组,每组6只,应用原位细胞凋亡检测法（TUNEL）检测凋亡细胞,免疫组织化学方法检测其脑内GRP78和Caspase-12的表达水平。结果 TUNEL检测凋亡率分别为7月龄APP/PS1鼠（35.0±6.31）%、5月龄APP/PS1鼠（9.0±2.78）%、7月龄WT鼠（4.0±1.89）%、5月龄WT鼠（4.0±1.83）%,其中7月龄APP/PS1鼠凋亡率显著升高（P〈0.05）;免疫组织化学检测GRP78阳性率分别为7月龄APP/PS1鼠（30.0±5.43）%、5月龄APP/PS1鼠（10.0±2.12）%、7月龄WT鼠（2.0±1.71）%、5月龄WT鼠（3.0±1.41）%,7月龄APP/PS1鼠GRP78表达明显升高（P〈0.05）;免疫组织化学检测Caspase-12阳性率分别为7月龄APP/PS1鼠（33.0±5.98）%、5月龄APP/PS1鼠（12.0±2.60）%、7月龄WT鼠（4.0±2.56）%、5月龄WT鼠（2.0±1.79）%,7月龄APP/PS1鼠Caspase-12表达明显升高（P〈0.05）。结论 7月龄的APP/PS1双转基因小鼠出现了内质网应激诱导的凋亡。本实验结果为临床AD早期预防和治疗提供了重要依据。     

当归芍药散对SAM—P8小鼠海马CA1区、齿状回锥体细胞的影响

目的观察当归芍药散（DSS）对快速老化小鼠海马CA1区、齿状回锥体细胞数量的影响。方法选用雌性SAM-P8鼠36只，分成4组：预防组（3月龄）、预防对照组（3月龄）、治疗组（6月龄）、治疗对照组（6月龄），治疗组以DSS（浓度为0．5g／mL）灌胃1个月，预防组自由饮用DSS（0．02％，v／v）4个月，对照组给予冷开水，给药结束后取脑、冰冻切片、尼氏染色，Leica图像分析系统分析海马CA1区、齿状回锥体细胞层尼氏染色的平均灰度。结果海马CA1区锥体细胞层尼氏染色的平均灰度各组间差异不明显（P〉0．05）。预防组齿状回锥体细胞层尼氏染色的平均灰度明显多于其余各组（P〈0．05）。结论低剂量长期服用DSS可增加老年性痴呆模型SAM-P8小鼠的齿状回锥体细胞数量。     

APP转基因拟痴呆小鼠模型脑内α synuclein的增龄改变

目的： 观察不同时程APP转基因拟痴呆小鼠脑内α synuclein的改变，以探讨α synuclein在AD发病中的作用。方法: 拟AD动物模型为4 月龄、10月龄和16月龄APP695V717I转基因小鼠；同背景同月龄C57BL/6J小鼠设为正常对照组。表达谱基因芯片、RT PCR方法检测皮层、海马mRNA表达改变；Western blotting、免疫组化法检测蛋白表达的改变。结果: α-synuclein mRNA表达在不同时程APP转基因小鼠脑内均明显增多。α-synuclein蛋白表达在早期4月龄APP转基因小鼠即显著上调，10月龄继续增多，16月龄继续上调并形成蛋白的异常聚集。结论: APP转基因小鼠脑内AD老年斑非Aβ主要成分α-synuclein表达明显增多，并随增龄不断加重，可能是模型小鼠学习记忆障碍及AD发病的重要因素。     

阻滞和非阻滞品系小鼠次级卵母细胞基因表达水平的比较

目的:通过基因芯片检测阻滞品系昆明(KM)小鼠和非阻滞品系B6C3F1小鼠次级卵母细胞(MⅡ卵)基因表达差异,探讨KM小鼠植入前胚体外发育阻滞是否与母源基因表达差异相关。方法:分别收集KM小鼠和B6C3F1小鼠MⅡ卵,运用基因芯片分析和real-time PCR验证,母源基因的差异表达。结果:基因芯片检测共筛出差异表达基因995个,其中B6C3F1小鼠MⅡ卵上调基因有793个;下调基因有202个。B6C3F1小鼠MⅡ卵表达上调的基因主要与调节基因转录、蛋白质合成与转运、氧化还原、生长因子等相关。结论:KM与B6C3F1小鼠植入前胚体外发育潜能不同可能与母源基因表达差异相关。     

高脂高糖食诱导的糖尿病小鼠肝脏清道夫受体B1的表达

目的：探讨糖尿病小鼠肝脏SR-B1表达变化，及其与血脂的关系。 方法： 进正常食C57BL/6J小鼠10只、进高脂高糖食8周小鼠5只、进高脂高糖食16周小鼠10只，测定血脂、血清胰岛素（INS）、空腹血糖（FBG），及肝脏SR-B1蛋白表达。 结果： ① 高脂高糖食16周的小鼠血清甘油三酯（TC）、FBG明显高于正常小鼠（P＜0.05)，其胰岛素水平与正常小鼠无显著差异（P＞0.05）。② 高脂高糖食小鼠肝脏SB-B1表达高于正常动物，16周强于8周的小鼠。 结论： 糖尿病小鼠肝脏SR-B1表达高于正常小鼠，血清HDL-C降低可能与肝脏的SR-B1蛋白表达增高有关。     

跑步对早期APP/PS1转基因阿尔茨海默病小鼠海马CA1区和齿状回神经元数量的影响

目的:应用体视学方法定量研究跑步对早期APP/PS1转基因阿尔茨海默病(AD)小鼠海马CA1和齿状回区域内神经元的影响。方法:将6月龄雄性APP/PS1转基因AD小鼠,随机分为跑步组和未跑步对照组。用Morris水迷宫检测2组小鼠的空间学习记忆能力,然后用体视学方法定量研究海马CA1和齿状回区域的体积和神经元的数量。结果:与对照组相比,跑步组AD小鼠Morris水迷宫的逃避潜伏期明显缩短,平台所在象限时间百分比和穿越平台次数均显著增加。体视学研究显示,跑步组AD小鼠海马CA1和齿状回的体积分别比对照组显著增大42.8%和27.3%,并且跑步组AD小鼠海马CA1和齿状回区域内的神经元数量分别比对照组显著增加61.8%和62.2%。结论:跑步能延缓早期AD小鼠空间学习记忆能力的下降;并且可降低AD小鼠海马CA1和齿状回区域内神经元的死亡。     

三七总皂苷对SAM-P/8小鼠海马中衰老相关基因表达的影响

目的探讨三七总皂苷对SAM-P/8小鼠海马中10个衰老相关基因表达的影响。方法采用RT-PCR技术检测10个衰老相关基因在各组小鼠海马中的表达及变化情况。结果各基因在海马中均有表达,成年三七组与成年对照组比较,SCN2B表达显著增加;老年三七组与老年对照组比较,MAP2、Sortilin-1、Rab6A、Calcineurin-B和MAPKK4的表达减少;MAP1B、Calmodulin-1、Regucalcin和RAP2A基因的表达在各组之间比较均没有变化。结论三七总皂苷可能通过调节部分衰老相关基因的表达来发挥其抗衰老的作用,但早期的预防比晚期治疗更加重要。     

天麻素对快速衰老小鼠大脑组织衰老相关基因表达的影响

目的研究天麻素对快速衰老小鼠(SAM)海马及其大脑额叶皮质中衰老相关基因表达的影响。方法采用RT-PCR方法对给予天麻素的SAM-P/8小鼠海马及额叶皮质中衰老相关基因的mRNA表达变化进行分析。结果所检测的衰老相关基因在SAM-P/8小鼠的海马和额叶皮质中均有表达。成年鼠应用天麻素组与成年组比较,在海马组织中SCN2BmRNA表达增加,Sortilin-1mRNA表达减少,MAP1BmRNA和RAP2AmRNA在额叶皮质中表达增加。老年鼠天麻素组MAPKK4、Sortilin-1和Rab6A在海马和额叶皮质中表达均比老年组减少。结论天麻素在分子水平上可能是通过调节部分衰老相关基因的表达水平来发挥其抗衰老作用,但对于衰老晚期,基因调控抗衰老的作用不明显。     

Senescence-accelerated mouse (SAM): age-related reduced anxiety-like behavior in the SAM-P/8 strain.

Age-related behavioral changes in the passive avoidance, food neophobia, elevated plus-maze, and water-lick conflict tests were studied using substrains of senescence-accelerated mouse (SAM-P/8 and SAM-R/1) at 2 to 20 months of age. SAM-P/8 mice exhibited a significant impairment of acquisition of passive avoidance compared with SAM-R/1 mice when they were trained repeatedly, and the acquired response in SAM-P/8 mice rapidly diminished in contrast to good retention in SAM-R/1 mice. SAM-P/8 mice showed an age-related decrease in the latency to eat novel food after a 24-h food deprivation as compared with SAM-R/1 mice at 2 to 12 months of age, despite no significant difference in latency to eat familiar food between the two strains. In the elevated plus-maze test, SAM-P/8 mice had apparent increases in the number of entries into open arms and time spent on open arms in comparison to SAM-R/1 mice at 4 through 12 months of age; this difference became obvious with aging, implying age-associated reduced anxiety in the SAM-P/8 strain. In addition, SAM-P/8 mice exhibited a significant increase in punished water drinking compared to SAM-R/1 mice in the water-lick conflict test, although unpunished water intake in SAM-P/8 mice did not differ from that in the SAM-R/1 control. Aged SAM-R/1 mice, 20 months old, exhibited low anxiety-like behavior in the food neophobia and elevated plus-maze tests such as was seen in SAM-P/8 mice, when compared with young (4-month-old) SAM-R/1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM)

For evaluation of the degree of senescence in SAM-P, accelerated senescence prone mouse, formerly called SAM or prone series or P-series, consisting of SAM-P/1, SAM-P/2, SAM-P/3 and SAM-P/4 corresponding to P-1, P-2, P-3 and P-4 series, respectively, in the previous reports, and in SAM-R, accelerated senescence resistant mouse, formerly called resistant series or R-series, consisting of SAM-R/1, SAM-R/2 and SAM-R/3 corresponding to R-1, R-2 and R-3 series, respectively, in the previous reports, the grading score system was adopted. The items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded from 0 to 4 according to the detailed criteria devised in our laboratory. After 8 months of age each mouse was examined every 4 months, and some of the mice were examined after 2 months of age.In almost all categories, the grading score and incidence began to increase from 4 or 6 months of age and continued to increase with advancing age in both SAM-P and SAM-R. The increase, however, was more marked in SAM-P than in SAM-R. The slow but steady increase in the SAM-R levelled out at 24 months of age and was comparable to that of 12 months of age in SAM-P. In both SAM-P/1 at 8 months of age and SAM-R/2 at 12 months of age, there was a significant reverse correlation between total score of this grading score system and length of residual life after examination.Systematic and extensive studies using the grading score system showed that if the validity of the system is, based on “irreversibility” and “universality” of the changes in     

Elevated concentrations of beta-nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8)

Levels of the beta-subunit of nerve growth factor (beta-NGF) were determined in various tissues from senescence-accelerated mice (SAM-P/8) and compared with those from senescence-resistant control mice (SAM-R/1) at 4 months of age. (1) In SAM-P/8, the testis was 30% larger in terms of wet weight than that from SAM-R/1, whereas the adrenal glands from males and females were smaller than those from the respective controls by 45% and 20%, respectively. (2) About 70% of SAM-P/8 individuals had high concentrations of testosterone in serum (greater than 5ng/ml). (3) In SAM-P/8, endogenous levels of beta-NGF were significantly higher in the adrenal gland (20 and 7 times higher on average in males and females, respectively), in the thymus (100 and 5 times higher in males and females, respectively) and in the testis (500 times higher) than those in the control tissues. In other tissues there were little or no differences in terms of levels of beta-NGF. (4) Morphological changes in the adrenal gland, thymus and testis of SAM-P/8 mice were not as marked as expected from the elevated levels of beta-NGF in these tissues. (5) These results show that, in SAM-P/8 mice at 4 months of age, an elevation in the endogenous level of beta-NGF has already occurred in some peripheral tissues before senescence becomes accelerated.     

Age-related changes in learning and memory in the senescence-accelerated mouse (SAM)

Age-related changes in learning ability were studied in senescence-accelerated mice (SAM) reared under specific pathogen-free (SPF) conditions. SAM-P/8/Ta (SAM-P/8, senescence-prone substrain) showed an age-associated increase in spontaneous motor activity (SMA) compared with SAM-R/1/Ta (SAM-R/1, senescence-resistant substrain) in a novel environment when the activity was measured in the light period, although there was no significant difference in the dark period. In observations of the circadian rhythm of SMA, SAM-P/8 showed a significant increase in diurnal SMA. In SAM-P/8 mice, the acquisition of passive avoidance response was slightly but significantly impaired even at 2 months of age, compared with SAM-R/1 control; the impairment became obvious with aging. In a one-way active avoidance task, SAM-P/8 did not show any impairment in the acquisition of avoidance response at 2 and 4 months of age. However, significant impairment was observed in SAM-P/8 at 12 months of age. The impairments of avoidance tasks were not due to a decrease in shock sensitivity, as indicated by no significant change in the flinch-jump threshold. In a water-filled multiple T-maze task, there was no difference in the number of errors between the two groups. With regard to the performance time to reach the goal, however, SAM-P/8 showed a mild prolongation at 2 months of age, and the prolongation became marked with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoimmune abnormalities in a murine model of accelerated senescence

Immunopathological abnormalities in senescence-accelerated mice (SAM) were studied by comparison of senescence-prone (SAM-P/1) and senescence-resistant (SAM-R/1) mice. Sera from SAM-P/1 mice contained a number of autoantibodies, including natural thymocytotoxic autoantibody (NTA), anti-nuclear antibodies (ANA) and IgG anti-single-stranded and anti-double-stranded (ss and ds) DNA antibodies. Furthermore, an earlier increase in serum IgG2 levels and an earlier appearance of IgG circulating immune-complexes (CIC) associated with low C3 levels, were observed in SAM-P/1 mice. These serological findings were distinctive features in SAM-P/1 mice, which could almost discriminate these mice from SAM-R/1 mice. In addition, age-associated glomerular mesangial and capillary lesions with granular IgG and C3 deposition were frequently observed in SAM-P/1 mice, whereas SAM-R/1 mice even at 10 months of age showed only mild mesangial lesions. These findings suggest that autoimmune abnormalities may contribute to the accelerated senescence in these mice.     

Senescence grading system in mice

A grading system was devised to evaluate the degree of senescence in accelerated senescence prone (SAM-P) and resistant (SAM-R) mice, using 11 items considered to be associated with senescence. The items were graded from 0 to 4. For most items, the score and incidence began to increase from 4-6 month-old, continued to increase with age in both SAM-P and SAM-R and reached similar plateau levels at 12-16 months and 24 months, respectively. In both SAM-P/1 at 8 month-old and SAM-R/2 at 12 month-old, there was an inverse correlation between the total score and duration of survival after examination. Thus, the grading system is useful for evaluation of the degree of senescence of mice.     

Age-related changes of erythrocyte membrane in the senescence-accelerated mouse

Age-related changes in erythrocytes in senescence-accelerated mice (SAM-P) and control mice with normal aging characteristics (SAM-R) were examined. A significant decrease in the number of erythrocytes and significant increases in MCV and ATP levels were observed with aging in SAM-P, while no significant changes were seen in SAM-R. Erythrocytes in aged SAM-P were less fragile than those in aged SAM-R. The contents of cholesterol and phospholipids in erythrocyte membranes increased significantly in aged SAM-P, but the molar ratio of cholesterol/phospholipid decreased. The plasma cholesterol level of SAM-P decreased with aging. Changes such as those observed in SAM-P were not seen in SAM-R during the period of observation.     

Age-related changes of serum apoprotein SASSAM, apoprotein A-I and low-density lipoprotein levels in senescence accelerated mouse (SAM)

Age-related changes of serum concentration of apo SASSAM, an apoprotein of high density lipoprotein (HDL) which cross-reacts with antiserum against murine senile amyloid fibril protein (ASSAM) were estimated in senescence accelerated mouse (SAM-P) and in senescence resistant series (SAM-R), as a control, using a single radial immunodiffusion technique. Serum concentrations of apo A-I, a major apoprotein of HDL, and low density lipoprotein (LDL) were also measured. In SAM-P (SAM-P/1 and SAM-P/2) with a high incidence of senile systemic amyloidosis, we observed age-associated decreases in serum apo SASSAM levels. The concentrations of apo SASSAM at 16 months of age were below 40% of the concentration at 2 months of age, regardless of the sex. In contrast with SAM-P, we observed no age-associated decrease of serum apo SASSAM levels in SAM-R (SAM-R/1 and SAM-R/2) with a low incidence of amyloidosis. Serum apo SASSAM concentration was higher in SAM-R/1 than in any other strain of mice observed. Serum apo A-I concentration was highly and significantly correlated with the serum concentration of apo SASSAM and decreased with advancing age in SAM-P but not in SAM-R. Age-related changes of LDL were not observed in any strain, but the concentration was lower in the females. In old SAM-P (16 months' old), the concentration of apo SASSAM decreased to one-third of that in the young SAM-P (4 months' old) and the serum concentrations of albumin and total protein did not decrease, compared with those in the young mice. All these findings taken together suggest that abnormality of metabolism in apo SASSAM, putative precursor of ASSAM, might occur in SAM-P.     

Age-Related Changes in Bone Mass in the Senescence-Accelerated Mouse (SAM): SAM-R/3 and SAM-P/6 as New Murine Models for Senile Osteoporosis

Age-related changes of the femoral bone mass in several strains of the senescence-accelerated mouse (SAM) were investigated. Microdensitometrically, all strains exhibited essentially the same patterns of age changes, that is, bone mass corrected by the diameter of the shaft reached the peak value when the mice were 4 or 5 months of age and then fell linearly with age up to over 20 months of age. Two strains, SAM-R/3 and SAM-P/6, which originated from the same ancestry on pedigree, had a significantly lower peak bone mass than other strains (SAM-R/1, SAM-R/2, SAM-P/1, and SAM-P/2). On the other hand, the strains with a low peak bone mass had the same rate of decrease as other strains. Mineral and collagen contents per dry weight of bone showed little difference among the strains. Histologic studies of tibia, femur, and lumbar spine revealed that the osteopenia was not due to osteomalacia but, rather, to osteoporosis. The elderly mice in these two strains were prone to fracture, thus should be important models for study of senile osteoporosis seen clinically.