Whale (Koiwashi kujira, Minke whale) Meat Extract Supplementation in Diet of Senescence-Accelerated Prone Mouse (SAMP8) Positively Affects Learning Memory Formation and Genome-Wide Changes in the Brain

The present study examines the effects of whale meat extract (WME) supplementation on the senescence-accelerated mouse prone 8 (SAMP8) model at the level of learning memory formation and gene expression profiles genome-wide. Our present study builds up on these previous studies by focusing on two sets of experiments examining WME-supplemented diet, on SAMP8 and SAMR1 (senescence-accelerated mouse resistant 1) learning and memory deficits (experiment 1) and whole-genome DNA microarray-based transcriptomics profiling in conjunction with Ingenuity Pathway Analysis (IPA) (experiment 2). We also examined the SAMP8 and SAMR1 mice fed with the regular (control; low-safflower oil, LSO) diets specifically to know the gene profiles in the brain of the SAMP8 mouse. Results revealed that WME supplementation on SAMP8 mouse resulted in an increase in the level of learning memory formation and positive changes in the transcriptome of the brain, suggested through the observation of recovery of gene expressions in the SAMP8 model over the not-supplemented mouse. 6-week-old mice (SAMP8 and SAMR1; CLEA, Tokyo, Japan) were housed at the Animal Institution Facility in Showa University, and maintained in individual cages in a ventilated animal room with controlled temperature and relative humidity under a 12-h light: 12-h dark regime (8:00 AM, lights turned on). Mice were fed chow (CE-2, CLEA Japan) and tap water ad libitum until 24-weeks-old. Then, 24-week-old SAMs mice were given experimental diet, LSO diet as a control diet and WME-supplemented diet, both in a powder form. The WME was made from red meat of Antarctic minke whale (Balaenoptera bonaerensis), taken from the Japanese Whale Research Program under Special Permit in the Antarctic-Phase II in 2009/2010 by heat, enzyme and drying treatments. The quality standard of WE were measured by Marugei Co. Ltd. (Hyogo, Japan). SAMP8 mice were randomly given LSO or WME diet, respectively. SAMR1 mice were given LSO diet only until 50-weeks-old. The behavioral tests were performed at the timing of 49-weeks-old for 8 days. At the end of the experiment (50 weeks of age) following the behavioral analysis (open field test, Y-maze test, new object recognition test (NOR), and water-filled multiple T-maze) and the last day of the feeding, the mice were removed from their cages, decapitated and their brains carefully removed on ice. The whole brains were quickly frozen in liquid nitrogen in a sterile freeze tube and stored at -80ºC till extraction of total RNA followed by DNA microarray analysis using a whole-genome mouse chip (Agilent-014868, 4 x 44K (G4122F)) with two-color dye-swap approach in conjunction with IPA bioinformatic analysis. All animal studies were conducted in accordance with the Standards Relating to the Care Management of Experimental Animals” (Notice No. 6 of the Office of Prime Minister dated March 27, 1980) and with approval from the Animal Use Committee of Showa University (Approval Number: #04093).

本研究旨在从学习记忆形成与全基因组基因表达谱层面，探究鲸肉提取物（whale meat extract, WME）补充剂对快速老化小鼠P8（senescence-accelerated mouse prone 8, SAMP8）模型的作用。本研究基于既往研究成果，聚焦两组实验：其一针对SAMP8与快速老化小鼠R1（senescence-accelerated mouse resistant 1, SAMR1）的学习记忆缺陷，探究WME补充日粮的干预效果（实验1）；其二结合Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）开展基于全基因组DNA微阵列的转录组谱分析（实验2）。此外，本研究还对饲喂普通（对照；低红花油（low-safflower oil, LSO））日粮的SAMP8与SAMR1小鼠进行研究，以明确SAMP8小鼠脑部的基因表达特征。

研究结果显示，对SAMP8小鼠补充WME可提升其学习记忆能力，并改善脑部转录组特征；通过观察到SAMP8模型的基因表达较未补充WME的小鼠得到恢复，佐证了上述结论。

本研究使用的实验动物为6周龄SAMP8与SAMR1小鼠（购自日本东京CLEA公司），饲养于昭和大学动物实验设施，于通风良好、温度与相对湿度可控的动物房内单笼饲养，采用12小时光照:12小时黑暗的光周期（每日早8:00开灯）。小鼠自由采食CE-2牌啮齿类饲料（CLEA日本公司）与自来水，饲养至24周龄。

24周龄时，对SAM系列小鼠分别饲喂实验日粮：对照日粮LSO粉末与WME补充日粮粉末。WME以2009/2010年日本南极特别许可捕鲸计划第二阶段获取的南极小须鲸（Balaenoptera bonaerensis）红肉为原料，经加热、酶解与干燥工艺制备而成。WME的质量标准由日本兵库县的Marugei有限公司测定。

将SAMP8小鼠随机分为两组，分别饲喂LSO日粮与WME日粮；SAMR1小鼠仅饲喂LSO日粮，直至50周龄。

于49周龄时开展为期8天的行为学测试，包括旷场实验、Y迷宫实验、新物体识别实验（new object recognition test, NOR）以及注水式多T迷宫实验。

在行为学分析结束后的实验终点（50周龄），即饲喂实验的最后一日，将小鼠脱颈椎处死，于冰上快速剥离脑部。将全脑置于无菌冻存管中，快速浸入液氮冷冻，随后保存于-80℃环境中，直至提取总RNA并开展全基因组小鼠芯片（Agilent-014868, 4 x 44K (G4122F)）的双色染料交换法DNA微阵列分析，并结合IPA生物信息学分析。

本研究所有动物实验均符合《实验动物饲养管理标准》（1980年3月27日日本首相办公室第6号通告），并获得昭和大学动物实验伦理委员会批准（批准号：#04093）。