High-Efficient FLPo Deleter Mice in C57BL/6J Background

Conditional gene manipulation in mice becomes a routine for genetic studies of mammalian gene functions. Additional site-specific recombinases such as FLP or φ31 provide one more level of gene manipulation flexibility. The recombination activity of the currently available FLP deleter mice remains low. We generated a new FLP deleter mouse line with the mouse codon-optimized FLPo gene in C57BJ/6 background, which showed superior recombination efficacy in comparison to FLPe deleter mice. 100% complete removal of FRT-flanked Neo cassette was observed in all F1 progeny mice carrying both FLPo and Neo cassette, which can be transmitted to F2 generation independent of FLPo activity. Our new FLPo transgenic mice (on pure C57BJ/6 background) will largely facilitate the gene targeting process and is valuable for conditional gene manipulation.     

不同方法建立C57BL/6J小鼠抑郁症模型的探讨

目的用不同方法建立C57BL/6J小鼠抑郁症模型,为探讨GalR蛋白对小鼠抑郁症的治疗作用打下基础。方法 C57BL/6J小鼠经体重、敞箱实验及反抗抓获实验初筛后,随机分为4组:Ⅰ.CUMS组,Ⅱ.CUMS+CORT组,Ⅲ.CORT组,Ⅳ.正常对照组。每天记录小鼠体重及摄食量。28d后进行液体消耗及强迫游泳实验测试。结果小鼠抑郁模型在第28d建立成功。Ⅱ组小鼠短期内体重迅速下降并死亡。与Ⅰ组小鼠相比,Ⅲ组小鼠液体消耗和强迫游泳实验指标改变更明显。结论成功建立C57BL/6J小鼠抑郁症模型。CUMS和CORT模型结合,小鼠不能耐受,短期内死亡。单独CORT模型造模效果要优于CUMS模型。在后续试验中,将用CORT法建立C57BL/6J小鼠抑郁症模型。     

Analysis of Bone Mineral Profile After Prolonged Every-Other-Day Feeding in C57BL/6J Male and Female Mice

Biological Trace Element Research - Intermitted fasting or every-other-day feeding (EOD) has many positive effects in rodents and humans. Our goal was to describe how EOD influences bone mineral...     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

高果糖高脂饮食对小鼠体内能量代谢的影响

目的:高热量物质的过度摄入是导致机体代谢紊乱,诱发2型糖尿病等代谢性疾病的主要原因,本文通过比较高果糖、高脂及高果糖高脂混合喂饲对小鼠体内能量代谢的影响,探索饮食诱发代谢紊乱性疾病的可能发病机制。方法:采用20%高果糖水,60%高脂饲料,及二者混合方式饲养C57BL/6小鼠3个月后,观察各组小鼠24小时内氧气消耗量,二氧化碳生成量,呼吸商及能量消耗的改变。结果:不同饮食喂饲3个月,与对照组小鼠相比,高果糖组、高脂组、及高果糖高脂组小鼠均表现出明显的肝内脂质蓄积,氧气消耗量增加,呼吸商下降,能量消耗增加。结论:过剩的高热量物质摄入导致机体内物质代谢、能量代谢发生改变,糖代谢受损,脂代谢增强,能量代谢方式从糖氧化为主转变为脂氧化供能。     

P7C3 Attenuates the Scopolamine-Induced Memory Impairments in C57BL/6J Mice

Memory impairment is the most common symptom in patients with Alzheimer’s disease. The purpose of this study is to evaluate the memory enhancing effects of P7C3, a recently identified compound with robust proneurogenic and neuroprotective effects, on the cognitive impairment induced by scopolamine, a muscarinic acetylcholine receptor antagonist. Different behavior tests including the Y-maze, Morris water maze, and passive avoidance tests were performed to measure cognitive functions. Scopolamine significantly decreased the spontaneous alternation and step-through latency of C57BL/6J mice in Y-maze test and passive avoidance test, whereas increased the time of mice spent to find the hidden platform in Morris water maze test. Importantly, intraperitoneal administration of P7C3 effectively reversed those Scopolamine-induced cognitive impairments in C57BL/6J mice. Furthermore, P7C3 treatment significantly enhanced the level of brain-derived neurotrophic factor (BDNF) signaling pathway in the cortex and hippocampus, and the usage of selective BDNF signaling inhibitor fully blocked the anti-amnesic effects of P7C3. Therefore, these findings suggest that P7C3 could improve the scopolamine-induced learning and memory impairment possibly through activation of BDNF signaling pathway, thereby exhibiting a cognition-enhancing potential.     

Indomethacin Treatment Prevents High Fat Diet-induced Obesity and Insulin Resistance but Not Glucose Intolerance in C57BL/6J Mice

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.     

敲低肿瘤细胞来源的颗粒体蛋白前体抑制C57BL/6J小鼠黑色素移植瘤的生长

颗粒体蛋白前体 (progranulin, PGRN)在多种肿瘤中过表达。但PGRN在黑色素瘤发生发展中的作用尚无报道。为探究PGRN在黑色素肿瘤中的作用,本研究采用CRISPR-Cas9基因编辑技术建立了稳定敲低PGRN的小鼠黑色素瘤B16细胞株B16-PGRNlow。MTS法和BrdU掺入结合流式细胞（计量）术分析证明,敲低PGRN不影响B16细胞的细胞周期和增殖。将B16-ctrl（对照）和B16-PGRNlow细胞分别皮下接种野生型（WT）和PGRN敲除（KO）的C57BL/6J小鼠,比较观察黑色素移植瘤体积大小。移植瘤形成20 d后,与B16-ctrl细胞接种的移植瘤比较,无论在WT还是在KO荷瘤小鼠,B16-PGRNlow形成的移植瘤体积明显减小（WT鼠：P<0.05;KO鼠：P<0.01）。然而,比较B16-PGRNlow或B16-ctrl在WT鼠与KO鼠形成的移植瘤体积大小,并无显著差异,提示B16肿瘤细胞PGRN而非宿主PGRN影响移植瘤的生长。流式细胞术分析显示,在荷B16-PGRNlow移植瘤的WT型小鼠脾和淋巴结中,CD4+、CD8+T细胞数（百分比）比荷B16-ctrl移植瘤的WT鼠脾和淋巴结的CD4+、CD8+T细胞数明显增多（P<0.05,P<0.01）,而在KO鼠却未见明显差异。上述结果证明,敲低肿瘤细胞PGRN可抑制黑色素移植瘤的生长。上述结果还提示,抑制PGRN在黑色瘤的表达可引起脾和淋巴结CD4+和CD8+T细胞增加,提高宿主的细胞免疫能力。其机制尚待进一步研究。本文的发现为PGRN作为黑色素瘤治疗的潜在靶点提供了新证据。     

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models. Download video file.^{(81M, mov)}     

A High-Fat Diet Delays Age-Related Hearing Loss Progression in C57BL/6J Mice

Objective

Age-related hearing loss (AHL), or presbycusis, is the most common sensory disorder among the elderly. We used C57BL/6J mice as an AHL model to determine a possible association between AHL and a high-fat diet (HFD).

Methods

Forty C57BL/6J mice were randomly assigned to a control or HFD group. Each group was divided into the following subgroups: 1-, 3-, 5- and 12-month groups (HFD, n = 5/subgroup; control, n = 5/subgroup). Nine CBA/N-slc mice were also used as a 12-month control (n = 5) or 12-month HFD (n = 4) group. The mice were fed a HFD or normal (control) diet throughout this study. Hearing function was evaluated at 1, 3, 5 and 12 months using auditory evoked brainstem responses (ABRs). Spiral ganglion cells (SGCs) were also counted.

Results

The elevation of ABR thresholds (at 4 and 32 kHz) at 3 and 5 months was significantly suppressed in the HFD group compared with the control groups for C57BL/6J mice. After 12 months, the elevation of ABR thresholds was significantly suppressed in the HFD group at all frequencies for C57BL/6J mice. In contrast, CBA/N-slc mice displayed opposite outcomes, as ABR thresholds at all frequencies at 12 months were significantly elevated in the HFD group compared with the control group. For the C57BL/6J mice at 12 months, SGC numbers significantly decreased in all parts of the cochleae in the control group compared with the HFD groups. In contrast, for the CBA/N-slc mice, SGC numbers significantly decreased, particularly in the upper parts of the cochleae in the HFD group compared with the control groups.

Conclusions

The elevation in ABR thresholds and SGC loss associated with aging in the HFD-fed C57BL/6J mice were significantly suppressed compared with those in the normal diet-fed mice. These results suggest that HFD delays AHL progression in the C57B/6J mice.     

A Dual Drug Sensitive L. major Induces Protection without Lesion in C57BL/6 Mice

Leishmaniasis is a major health problem in some endemic areas and yet, no vaccine is available against any form of the disease. Historically, leishmanization (LZ) which is an inoculation of individual with live Leishmania, is the most effective control measure at least against cutaneous leishmaniasis (CL). Due to various reasons, LZ is not used today. Several live attenuated Leishmania have been developed but their use is limited. Previously, we developed a transgenic strain of L. major that harbors two suicide genes tk and cd genes (lmtkcd^+/+) for use as a challenge strain in vaccine studies. These genes render the parasite susceptible to Ganciclovir (GCV) and 5-flurocytosine (5-FC). The dual drug sensitive strain of L. major was developed using gene targeting technology using a modified Herpes Simplex Virus thymidine kinase gene (hsv-tk) sensitive to Ganciclovir antibiotic and Saccharomyces cerevisae cytosine deaminase gene (cd sensitive to 5-flurocytosine) that were stably introduced into L. major chromosome. BALB/c mice inoculated with lmtkcd^+/+ developed lesions which upon treatment with GCV and 5-FC completely healed. In the current study, the transgenic lmtkcd^+/+strain was assessed as a live vaccine model to determine the time necessary to develop a protective immune response. C57BL/6 mice were inoculated with the transgenic lmtkcd^+/+strain, and treated at the time of inoculation (day0) or at day 8 after inoculation. Immunized animals were challenged with wild-type L. major, and complete protection was induced in mice that were treated at day 8. The results show that in contrast to leishmanization, in group of mice inoculated with a dual sensitive L. major development and persistence of lesion is not necessary to induce Th1 response and protection.     

C57BL/6J小鼠超数排卵的研究

目的　确定C57BL 6J小鼠超排的最佳激素剂量和最合适的注射间隔时间 ,提高超排率。方法　 40只C57BL 6J雌鼠随机分为四组 ,分别用 5IU或 10IU的PMSG和HCG ,间隔 48h或 72h注射 ,比较排出卵母细胞的数量。结果　 5IU +5IU剂量的PMSG和HCG、间隔 48h注射组超排效果最好 ;8～ 10周龄雌鼠较 6～ 8周龄雌鼠超排效果好。结论　C57BL 6J小鼠超排的最佳激素剂量为 5IUPMSG +5IUHCG ,最合适的注射间隔时间为 48h ,处于繁殖期的雌鼠超排效果好。     

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

Aniracetam Does Not Alter Cognitive and Affective Behavior in Adult C57BL/6J Mice

There is a growing community of individuals who self-administer the nootropic aniracetam for its purported cognitive enhancing effects. Aniracetam is believed to be therapeutically useful for enhancing cognition, alleviating anxiety, and treating various neurodegenerative conditions. Physiologically, aniracetam enhances both glutamatergic neurotransmission and long-term potentiation. Previous studies of aniracetam have demonstrated the cognition-restoring effects of acute administration in different models of disease. No previous studies have explored the effects of aniracetam in healthy subjects. We investigated whether daily 50 mg/kg oral administration improves cognitive performance in naïve C57BL/6J mice in a variety of aspects of cognitive behavior. We measured spatial learning in the Morris water maze test; associative learning in the fear conditioning test; motor learning in the accelerating rotarod test; and odor discrimination. We also measured locomotion in the open field test, anxiety through the elevated plus maze test and by measuring time in the center of the open field test. We measured repetitive behavior through the marble burying test. We detected no significant differences between the naive, placebo, and experimental groups across all measures. Despite several studies demonstrating efficacy in impaired subjects, our findings suggest that aniracetam does not alter behavior in normal healthy mice. This study is timely in light of the growing community of healthy humans self-administering nootropic drugs.     

Long-Term Artificial Sweetener Acesulfame Potassium Treatment Alters Neurometabolic Functions in C57BL/6J Mice

With the prevalence of obesity, artificial, non-nutritive sweeteners have been widely used as dietary supplements that provide sweet taste without excessive caloric load. In order to better understand the overall actions of artificial sweeteners, especially when they are chronically used, we investigated the peripheral and central nervous system effects of protracted exposure to a widely used artificial sweetener, acesulfame K (ACK). We found that extended ACK exposure (40 weeks) in normal C57BL/6J mice demonstrated a moderate and limited influence on metabolic homeostasis, including altering fasting insulin and leptin levels, pancreatic islet size and lipid levels, without affecting insulin sensitivity and bodyweight. Interestingly, impaired cognitive memory functions (evaluated by Morris Water Maze and Novel Objective Preference tests) were found in ACK-treated C57BL/6J mice, while no differences in motor function and anxiety levels were detected. The generation of an ACK-induced neurological phenotype was associated with metabolic dysregulation (glycolysis inhibition and functional ATP depletion) and neurosynaptic abnormalities (dysregulation of TrkB-mediated BDNF and Akt/Erk-mediated cell growth/survival pathway) in hippocampal neurons. Our data suggest that chronic use of ACK could affect cognitive functions, potentially via altering neuro-metabolic functions in male C57BL/6J mice.     

链脲佐菌素诱导C57BL/6J小鼠2型糖尿病模型研究

目的建立与2型糖尿病(非胰岛素依赖型糠尿病、NIDDM)病人临床特征和发病过程相似的NIDDM动物模型.方法用高脂肪饲料喂养C57BL/6J雄性断乳小鼠3周,腹腔注射链脲佐菌素(STZ),继续喂养4周,测定给药前和给药后1、3、4周非空腹血糖、实验结束时非空腹胰岛素水平,观察胰腺形态学变化.结果喂养3周后(给药前)高脂饲料-STZ组及高脂饲料-柠檬酸组血糖浓度(7.0±0.39)mmol/L、( 6.8±0.45)mmol/L高于普通饲料-STZ组及普通饲料-柠檬酸组(5.3±0.40)mmol/L、(5. 4±0.39)mmol/L,P＜0.05;实验结束时,高脂饲料-STZ组血糖浓度(13 .1±2.01)mmo/L高于高脂饲料-柠檬酸(6.9±0.46)mmol/L、普通饲料-柠檬酸组(6.0± 0.46)mmol/L和普通饲料-STZ组(7.1±0.62)mmol/L(P＜0.05),各组间血浆胰岛素浓度、体重及饮水量差异无显著性,P＞0.05;实验过程中高脂饲料STZ组和柠檬酸组小鼠每天进食热量(64.49±9.2)kJ/只,(70.7±9.6)kJ/只, 显著高于普通饲料STZ组和柠檬酸组(52.7±7.9)kJ/只,(57.3±11.7)kJ/只;各组小鼠胰腺和胰岛细胞形态正常.结论高脂肪饲料和STZ是用C57BL/6J断乳幼鼠建立NIDDM模型所必须的,100mg/kg体重STZ对普通饲料小鼠血糖无影响;用高脂饲料和STZ 处理的小鼠血糖升高、胰岛素浓度正常,与NIDM病人临床特征和发病过程相似;C57BL/6J小鼠易得,建模方法简便,费用低,是在NIDDM实验研究中能广泛使用的较理想的非遗传性NID DM动物模型.     

高脂饮食加低剂量链脲霉素建立小鼠2型糖尿病模型

目的高脂饮食加低剂量链脲霉素（Streptozotocin,STZ）建立小鼠2型糖尿病模型。方法5周的雄性C57BL/6J小鼠,随机分为正常饲料组、正常饲料加STZ组、高脂饲料组和高脂饲料加STZ组。相应饲料喂养5周后,按照100 mg/Kg的剂量腹腔注射STZ,然后继续喂养4周。在第5周和第9周末测定小鼠的体重、收缩压、血糖、血胰岛素、血甘油三脂和胆固醇水平。结果STZ注射前各组小鼠的体重、血压、血糖、血胰岛素、血脂和血甘油三脂无明显差异（P〉0.05）。STZ注射后4周时,高脂饲料加STZ组小鼠的体重、血糖、血胰岛素、血压和血脂水平明显升高（P〈0.05）;而其他三组的这些指标无明显改变或仅部分升高。结论高脂饮食加低剂量链脲霉素可建立小鼠2型糖尿病模型,该模型具有人2型糖尿病的主要表型特征和相似的发病过程。     

高脂饮食诱导的C57BL／6J肥胖小鼠脂肪组织RIP140基因表达水平的研究

目的：探讨高脂饮食致肥胖小鼠脂肪组织RIP140mRNA表达水平的变化及其与胰岛素抵抗的关系。方法：将C57BL／6J雄性小鼠随机分为正常饮食（NFD）组、高脂饮食（HFD）纽分别喂养14周后,测量两组小鼠体重,以NFD组小鼠体重作为对照,选取HFD组中体重大于对照组小鼠平均体重20％的小鼠作为肥胖组小鼠。对照组和肥胖组小鼠取血测甘油三酯（TG）、总胆固醇（TC）、空腹血糖（FBG）、空腹胰岛素水平（FIns）,计算稳态模型胰岛素抵抗指数（HOMA-IR）;采用RT—PCR技术检测两组小鼠附睾脂肪组织RIP140 mRNA的表达水平,并进行统计学分析。结果：HDF组小鼠中有12只符合标准计入肥胖组。肥胖组小鼠TG、TC、FBG、Fins（P〈0．05）,HOMA-1R（P〈0．01）均明显高于对照组;肥胖组小鼠脂肪组织RIP140mRNA的表达高于对照组,差异具有统计学意义（P〈0．05）;相关分析显示小鼠脂肪组织R1P140 mRNA表达水平与TG水平呈正相关（r=0．536,P〈0．05）,与胰岛素抵抗指数呈正相关（r=0．465,P〈0．05）,而与TC、FBG、Fins水平相关分析无统计学意义（P〉0．05）。结论：高脂饮食诱导的肥胖小鼠脂肪组织RIP140 mRNA表达增加,并与胰岛素抵抗程度呈正相关。     

Age-Related Changes in EGF and Protease in Submandibular Glands of C57BL/6J Mice1,4

The submandibular glands of male C57BL/6J mice were studied cytologically and chemically at the following ages (months): 1–1.5, 6–8, 12–13, 28–32. The relative proportion of granular convoluted tubules (GCT) as well as the size and content of secretion granules of GCT cells, progressively increased throughout the first year of life. Correspondingly, the concentration within the glands of two GCT cell products, epidermal growth factor (EGF) and protease, also steadily increased. In senescent glands, GCTs formed relatively less of the gland parenchyma and were composed of shorter cells with reduced amounts of secretory granules. The concentration of EGF was reduced to 17% of its peak value at one year, while protease activity declined to 50% of its peak value. These morphologic and chemical findings imply a functional impairment in submandibular glands of the mouse with senescence.