笔者想承认DHMRI CLAS照顾动物的工作人员的大力支持下，基金经理丹尼尔·佩拉尔塔先生和主治兽医，Glicerio伊格纳西奥博士，DVM MRCVS。

所有动物程序由位于北卡罗莱纳州研究园（NCRC）戴维H Murdock的研究所IACUC批准，根据协议14-017，糖尿病和糖尿病前期/胰岛素抵抗和治疗的功效的非人灵长类动物模型的表征，以改善胰岛素敏感性和代谢功能。 1.动物的选择和学习做准备<ol><li>基于每月的食物摄入量和体重记录选择饮食和体重稳定的动物。 注:食欲都展出了近期的下跌动物不应该被定性，直到他们的食物摄入量已经稳定。 <ol><li>对于成熟的动物（&gt; 5 - 6年），不要选择动物的尸体连续两个月之间的权重超过10％不等，而不首先检查了猴子和排除不是改变为重戏剧性的变化代谢状态等原因。 </li></ol></li><li>对于血糖，胰岛素和C-pepti德分析，准备与蛋白酶抑制剂（抑肽酶和DPP4I）K 2 EDTA样品收集管。 注:DPP4I +抑肽酶鸡尾酒提供了抑制蛋白酶的广谱这应该是必需的额外的分析物（胰高血糖素，GLP-1）。在该附加的分析物没有收集的情况下，血管应该以同样的方式，以保持样品收集在一致性来制备。对于不验证该方法测定样品应根据制造商的建议分别收集。 <ol><li> 100毫克冻干抑肽酶用10ml DPP4I混合准备蛋白酶抑制剂。加10μl抑肽酶+ DPP4I混合物到每个管血的采集血液每毫升。 </li><li>额外的10微升蛋白酶抑制剂混合物添加到各管中可能集合超龄。在-20°C，直到使用存储处理的血管。保持湿冰管的procedu期间重新并在4℃下旋转。 </li><li>使用血清分离管中，在基线标准血清化学分析，收集血液样品。对于全血细胞计数（CBC），使用标准的K 2 EDTA无蛋白酶抑制剂。使用离心管到血样之后等分试样的血浆和血清已降速。 </li><li>与动物识别，日期，程序，时间点和样品体积正确标记血管和冷冻管。与在血浆中的分析物为合适的测定标记冷冻管。 </li></ol></li><li>通过注入0.15毫升每毫升肝素1000 USP单位到生理盐水250毫升袋子制备肝素化盐水冲洗。得到0.06毫克肝素/ ml的溶液。绘制40 - 60毫升该溶液放入盐水锁样品之间冲洗。绘制额外1毫升和5ml成单独的注射器的输注之前和之后冲洗葡萄糖输注端口，分别。 </li></ol> 2。动物镇静和准备<ol><li>从动物的笼子里取出的食物在手术前不低于14小时，不超过18小时。 注:重要的是，动物被禁食的程序，以避免在血糖值的任何餐后变化。这也是一个防范措施，避免反流胃内容物的吸入麻醉时。 </li><li>稳重动物的IVGTT过程用氯胺酮肌肉注射作为全身麻醉剂的持续时间，在10毫克/公斤。根据需要，在操作过程中30分钟，或 - 以20间隔 - （10毫克/千克5）管理附加氯胺酮。 <ol><li>称量镇静动物。将动物在加热过程表横向躺着的位置。 </li><li>监测临床参数，每15至20分钟，以确保动物在麻醉的稳定面。测量心脏速率（100 - 200 BPM）和SPO 2（&gt; 92％）与脉搏血氧仪。测量呼吸频率（20 - 50次/ min）W，<html>第i个秒表，计数呼吸视觉或用手超过十五秒钟，乘以4。测量温度（&gt; 97°F）直肠。显示器周围的牙龈和嘴唇（潮湿，粉红色）粘膜的颜色。 </li></ol></li><li>准备两个插管部位。用推剪修剪从其中导管将被插入的感兴趣的区域的毛发，并用氯己定的交替磨砂和70％的酒精消毒的整个区域。 <ol><li>将一个导管中的左侧或右侧头部或隐静脉的区域中，并将其与一个三路活塞附加到肝素盐水冲洗（0.06毫克肝素/毫升）。这是在采样抽血站点。 </li><li>放置第二个导管中另一个腿或臂在头侧或隐静脉的区域中，并附加一个端口。使用本网站的葡萄糖输液。使用小1毫升冲洗肝素生理盐水到插管专利保持葡萄糖输液前。 </li></ol></li></ol>e_title"&gt; 3。IVGTT过程注:IVGTT过程包括8抽血取样时间点（ 见表1）。 <ol><li>取基线样品，并使用手持血糖测计仪来测量空腹血糖水平。为了评估动物的总体健康状况获取标准化学分析为CBC血清样品，以及全血样品。收集血浆样品以测定使用验证与猕猴按照制造商的说明8,9使用的试剂盒从基线样品血糖和胰岛素水平。 注:以具有从套管采取任何样本的采血以除去残留的血液或肝素在插管的死空间之前采取为0.5ml预拉伸是很重要的。 </li><li>获得基线样品后，注入50％葡萄糖（250毫克/千克）的剂量在30秒到葡萄糖输注端口。 注意:高剂量模型（500毫克/千克），可以使用虽然剂量应以使纵向比较固定跨程序。 <ol><li>冲洗用5ml肝素生理盐水的输液口，以确保不存在葡萄糖留在端口。输液的到底是T0。有技术人员更换手套，如从输注残余葡萄糖可能污染后续血样。 </li></ol></li><li>第一次输注后的采样时间点是在T3分钟，从葡萄糖输注结束，接着T5分钟，T7分钟，T10分钟，T15分钟，T20分钟，和最后一个样本的时间点是在T30分钟。从每个时间点与基线样品测定血糖和胰岛素水平收集血浆（参见步骤3.1）。 <ol><li>在T3分钟的时间点，使用手持血糖测计仪再次检查血糖水平。 注意:在基线的血糖仪读数和T3只确认葡萄糖输液。在T3分钟的时间点的血糖值应为〜100毫克/分升高共mpared到空腹基线血糖水平。 </li></ol></li></ol> 4.动物恢复和来样加工<ol><li>取出插管和压力施加到T30分钟时间点后，插入导管位点的止血。监测动物，直到它已恢复了神志，坐起来。发售饲料一旦动物完全恢复。 </li><li>立即将每个全血样品放入冰K 2 EDTA管。离心机以3000rpm在4℃收集的10分钟内的温度。分装血浆样品到冷冻管，冷冻并储存在-80℃直至分析。 <ol><li>允许血液进入标准血清化学分析的血清管在室温下静置不少于20分钟，并在在室温下3,000rpm下离心前不超过30分钟。冻结血清样本采集之前的48小时内检测。 注:冷藏收集CBC分析直至全血样本收集24小时内赛义德。 </li></ol></li></ol> 5.数据处理<ol><li>建立血浆胰岛素和葡萄糖的曲线之后，确定从自然对数高于基线16，17的血糖值的斜率的葡萄糖清除率ķ。 注:健康NHP可以预期有一个葡萄糖清除率ķ远高于1，通常比2以上时，作为健康的动物将常在30分钟内恢复到其基线血糖值。由于胰岛素的产生脱落，葡萄糖清除率k，便会下降更为显着，跌破1。 </li><li>计算的AUC作为一个整体，作为代表的时间点之间的每个线段的曲线下面积的梯形的面积的总和的总和，通过T30的16，17。 注意:传统上，前十分钟的过程中的AUC被认为是acutÈ胰岛素响应于葡萄糖（ 空气 ），而从过程的最后20分钟的AUC被认为是晚期胰岛素反应（LIR）。当动物变得更加dysmetabolic，胰岛素曲线下面积将增加，这反映补偿增加胰岛素不敏感。作为动物转移到明显的糖尿病，然而，AUC将降低，经常最初在急性胰岛素相，由AIR捕获。 </li></ol>

<ol>
	<li>Bergman, R., Phillips, L., Cobelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic+evaluation+of+factors+controlling+glucose+tolerance+in+man.">Physiologic evaluation of factors controlling glucose tolerance in man.</a> J. Clin. Invest. 68, 1456-1457 (1981).</li><li>Bergman, R., Prager, R., Volund, A., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equivalence+of+the+insulin+sensitivity+index+in+man+derived+by+the+minimal+model+and+the+euglycemic+glucose+clamp.">Equivalence of the insulin sensitivity index in man derived by the minimal model and the euglycemic glucose clamp.</a> J. Clin. Invest. 79, 790-800 (1987).</li><li>Hovorka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+glucose+distribution/transport,+disposal,+and+endogenous+production+during+IVGTT.">Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT.</a> Am. J. Physiol. Endocrinol. Metab. 282, E992-E1007 (2002).</li><li>Salinari, S., Guidone, C., Bertuzzi, A., Manco, M., Asnaghi, S., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-phase+insulin+secretion+restoration+and+differential+response+to+glucose+load+depending+on+the+route+of+administration+in+type+2+diabetic+subjects+after+beriatric+surgery.">First-phase insulin secretion restoration and differential response to glucose load depending on the route of administration in type 2 diabetic subjects after beriatric surgery.</a> Diabetes Care. 32 (3), 375-380 (2009).</li><li>Roden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Diabetes Research: Methods and Techniques. , (2007).</li><li>Cobelli, C., Pacini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+and+hepatic+extraction+in+humans+by+minimal+modeling+of+c-peptide+and+insulin+kinetics.">Insulin secretion and hepatic extraction in humans by minimal modeling of c-peptide and insulin kinetics.</a> Diabetes. 37, 223-231 (1988).</li><li>Lorenzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposition+index,+glucose+effectiveness,+and+conversion+to+type+2+diabetes:+the+insulin+resistance+atherosclerosis+study.">Disposition index, glucose effectiveness, and conversion to type 2 diabetes: the insulin resistance atherosclerosis study.</a> Diabetes Care. 33, 2098-2103 (2010).</li><li>Hansen, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+and+treatment+of+type+2+diabetes+in+nonhuman+primates.">Investigation and treatment of type 2 diabetes in nonhuman primates.</a> Methods Mol Biol. 933, 177-185 (2012).</li><li>Hansen, B. C., Bodkin, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+IVGTT.+Importance+of+method+used+to+calculate+glucose+disappearance.">Standardization of IVGTT. Importance of method used to calculate glucose disappearance.</a> Diabetes Care. 16 (5), 847 (1993).</li><li>Hardwood, J. H., Listrani, P., Wagner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhuman+primates+and+other+animal+models+in+diabetes+research.">Nonhuman primates and other animal models in diabetes research.</a> J Diabetes Sci Tech. 3, 503-514 (2012).</li><li>De Koning, E. J., Bodkin, N. L., Hansen, B. C., Clark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+in+Macaca+mulatta+monkeys+is+characterized+by+islet+amyloidosis+and+reduction+in+beta-cell+population.">Diabetes mellitus in Macaca mulatta monkeys is characterized by islet amyloidosis and reduction in beta-cell population.</a> Diabetologia. 36, 378-384 (1993).</li><li>Letiexhe, M. R., Scheen, A. J., Gerard, P. L., Desaive, C., Lefebvre, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion,+clearance+and+action+before+and+after+gastroplasty+in+severely+obese+subjects.">Insulin secretion, clearance and action before and after gastroplasty in severely obese subjects.</a> Int J Obes Relat Metab Disord. 18, 295-300 (1994).</li><li>Letiexhe, M. R., Scheen, A. J., Gerard, P. L., Desaive, C., Lefebvre, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postgastroplasty+recovery+of+ideal+body+weight+normalizes+glucose+and+insulin+metabolism+in+obese+women.">Postgastroplasty recovery of ideal body weight normalizes glucose and insulin metabolism in obese women.</a> J Clin Endocrinol Metab. 80, 364-369 (1995).</li><li>Kim, S. H., Abbasi, F., Chu, J. W., McLaughlin, T. L., Lamendola, C., Polonsky, K. S., Reaven, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosiglitazone+reduces+glucose-stimulated+insulin+secretion+rate+and+increases+insulin+clearance+in+nondiabetic,+insulin-resistant+individuals.">Rosiglitazone reduces glucose-stimulated insulin secretion rate and increases insulin clearance in nondiabetic, insulin-resistant individuals.</a> Diabetes. 54, 2447-2452 (2005).</li><li>Toffolo, G., Breda, E., Cavaghan, M. K., Ehrmann, D. A., Polonsky, K. S., Cobelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+indexes+of+beta-cell+function+during+graded+up+and+down+glucose+infusion+from+C-peptide+minimal+models.">Quantitative indexes of beta-cell function during graded up and down glucose infusion from C-peptide minimal models.</a> Am J Physiol Endocrinol Metab. 280, E2-E10 (2001).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+beta-cell+insulin+secretory+function+using+a+graded+glucose-infusion+with+C-peptide+deconvolution+in+dysmetabolic,+and+diabetic+cynomolgus+monkeys.">Quantification of beta-cell insulin secretory function using a graded glucose-infusion with C-peptide deconvolution in dysmetabolic, and diabetic cynomolgus monkeys.</a> Diabetology and Metabolic Syn. 5, 40 (2013).</li><li>Xiao, Y. F., Wang, B., Wang, X., Du, F., Benzinou, M., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylazine-induced+reduction+of+tissue+sensitivity+to+insulin+leads+to+acute+hyperglycemia+in+diabetic+and+normoglycemic+monkeys.">Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys.</a> Anesthesiology. 13 (33), (2013).</li><li>Porte, D., Kahn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-cell+dysfunction+and+failure+in+type+2+diabetes+potential+mechanisms.">&#946;-cell dysfunction and failure in type 2 diabetes potential mechanisms.</a> Diabetes. 50, S160-S163 (2001).</li><li>DeFronzo, R. A., Tobin, J. D., Andres, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+clamp+technique:+a+method+for+quantifying+insulin+secretion+and+resistance.">Glucose clamp technique: a method for quantifying insulin secretion and resistance.</a> American Journal of Physiology. 237 (3), G214-G223 (1979).</li><li>Ferrannini, E., Gastaldelli, A., Miyazaki, Y., Matsuda, M., Mari, A., DeFronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-cell+function+in+subjects+spanning+the+range+from+normal+glucose+tolerance+to+overt+diabetes:+a+new+analysis.">&#946;-cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis.</a> J Clin Endocrinol Metab. 90 (1), 493-500 (2005).</li><li>Vaughan, K. L., Szarowicz, M. D., Herbert, R. L., Mattison, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anesthesia+protocols+for+intravenous+glucose+tolerance+testing+in+rhesus+monkeys.">Comparison of anesthesia protocols for intravenous glucose tolerance testing in rhesus monkeys.</a> J Med Primatol. 43, 162-168 (2014).</li><li>Kemnitz, J. W., Kraemer, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+glucoregulation+in+rhesus+monkeys+sedated+with+ketamine.">Assessment of glucoregulation in rhesus monkeys sedated with ketamine.</a> American Journal of Primatology. 3, 201-210 (1982).</li><li>Dutton, C. J., Parvin, C. A., Gronowski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+glycated+hemoglobin+percentages+for+use+in+the+diagnosis+and+monitoring+of+diabetes+mellitus+in+nonhuman+primates.">Measurement of glycated hemoglobin percentages for use in the diagnosis and monitoring of diabetes mellitus in nonhuman primates.</a> Am J Vet Res. 64, 562-568 (2003).</li><li>Rai, V., Iyer, U., Mani, I., Mani, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+biochemical+changes+in+insulin+dependent+and+non-insulin+dependent+diabetes+mellitus+and+their+role+in+the+development+of+secondary+complications.">Serum biochemical changes in insulin dependent and non-insulin dependent diabetes mellitus and their role in the development of secondary complications.</a> Int J Diab Dev Countries. 17, 33-37 (1997).</li><li>Shirasaki, Y., Yoshioka, N., Kanazawa, K., Maekawa, T., Horikawa, T., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+physical+restraint+on+glucose+tolerance+in+cynomolgus+monkeys.">Effect of physical restraint on glucose tolerance in cynomolgus monkeys.</a> J Med Primatol. 42, 165-168 (2013).</li></ol>

作者是附属于合同研究组织（冠科生物）活跃在代谢性疾病领域。

的IVGTT评估葡萄糖刺激的胰岛素释放的基于体重5，12，13的单一的葡萄糖输注的能力。从测定中，空腹血糖和胰岛素水平达到，并且它允许动物的能力来评估释放胰岛素和朝基线返回升高的血糖水平。这提供了信息来表征动物作为一个正常的血糖和胰岛素水平健康对照，具有正常血糖高胰岛素dysmetabolic动物，或高血糖的胰岛素抵抗性糖尿病动物的用户。 重要的是血液样品，特别是从早期的时间点，右旋糖的输注后，绘制并及时和一致的方式处理。这将减少可变性以内与主体之间，并减少在数据的时间顺序错误的机会。在T3的时机是至关重要的，因为游览Ø从葡萄糖输注后基线到三分钟˚F血浆葡萄糖是用于表征动物的代谢状态的端点中的一个。有头7内于葡萄糖清除的下降 - 10分钟葡萄糖输注后，如果这些抽血不采取准时可能被错过。它是葡萄糖和用来作为一个端点的胰岛素曲线两者的这一部分下的面积，强调这些早期时间点的重要性。如果T3后抽血取末，该过程可以继续，但拉伸的实际时间应以尽可能保持该曲线的实际形状进行报告。它是特别重要的是的一分多钟的偏差被捕获和报告。第10分钟后，葡萄糖清除趋于变得更平缓，和30分钟（ 图1）内可能不会返回到基线。该过程可通过T30到后取样每10分钟进行扩展捕捉需要多长时间动物回到基线。 IVGTTs通过它们直接测量胰岛素敏感性的能力受到限制。它是作为评估胰岛素产生和葡萄糖清除率的方法主要是有用的。然而，从门户供血肝胰岛素提取从全身血液供给损害"胰岛素分泌"的测量结果。本然而，可以部分地克服，通过测量C肽，它是在从胰腺等摩尔量分泌而由肝脏进入一般供血之前不会被删除。这使胰岛素生产的更清晰的视野，以更好地评估β细胞功能16。此外，IVGTTs不葡萄糖清除和非胰岛素依赖性机制1,6的胰岛素依赖型机制之间进行区分。虽然胰岛素敏感性可间接地通过从IVGTT数据的最小建模来评估，更可靠的措施，可通过第实现E使用的程序，如分级葡萄糖输注（GGI），它提供了一个β细胞的剂量-反应曲线14,15。 然而，对胰岛素的敏感性仍只评估了间接与GGI。一个标准的胰岛素剂量可以在IVGTT中施用给动物以改善胰岛素敏感性的估计，但是这将掩盖内源胰岛素生产，降低β细胞功能的评估。高血糖和高胰岛素钳技术通过测量控制胰岛素暴露直接葡萄糖调节。到GGI和钳技术两者的缺点是，它们是执行昂贵，并且需要更多的人员和时间（如长有时六到八个小时，这取决于本程序的设计）完成16。在IVGTT，在另一方面，可以一个小时内，并以最少的人力进行。 一般来说，一个健康的动物将部署葡萄糖非常迅速的LY，往往返回到其基线值在30分钟内，和它们的胰岛素曲线将显示出对于急性和晚期胰岛素反应两个截然不同的峰值。作为动物变得更加dysmetabolic，其禁食葡萄糖和在前三分钟他们的葡萄糖偏移输注后可能增加。但是，这些值可能会健康动物中广泛地变化，并与来自相同动物历史值时是最有信息的疾病进展。但总体而言，一个dysmetabolic动物的葡萄糖清除可能没有很大的时候，他们是健康的不同。疾病进展的第一个迹象将是最明显的胰岛素曲线。虽然空腹胰岛素水平可能只是轻度升高，胰岛素AUC通常会急剧dysmetabolic动物增加，反映了发展中胰岛素不敏感的补偿。作为β细胞功能下降，虽然，在急性期胰岛素反应的钝化可以看出。 ŧ他将反映在减小的空气 ，其中，归一化的时基线，可在糖尿病猴子接近0。这之后是在胰岛素生产整体下降并在葡萄糖间隙8，9，10，11的显着降低。 动物的代谢状态可能难以确定与单个IVGTT。试图向动物区分如任一初dysmetabolic或前糖尿病时，这可能是这种情况。当变得愈发dysmetabolic，胰岛素产量的增加来维持血糖正常。虽然空腹血糖值可能会变得稍隆起，直至β细胞功能已经受损和胰岛素生产开始下降18,20糖耐量处置不变得非常明显。有一段时间，在此之前显性糖尿病已设置，胰岛素值可能类似于在其中第一阶段的反应已经被入侵的早期dysmetabolic动物的曲线，但一些胰岛素STILL以上禁食，基线值而生成的。由于这个原因，一个早期dysmetabolic和糖尿病前期动物的胰岛素曲线的AUC可能是非常相似的。在这种情况下，升高的葡萄糖曲线，由这两种胰岛素依赖性和非胰岛素依赖型机制的影响，是不充分的动物区分的任一初dysmetabolic或前糖尿病。这种区别可以通过进行高胰岛素/血糖钳，这将直接表明胰岛素敏感性19制成。可替代地，这种区别可能在数个月后对动物进行另一IVGTT制成。在后一种情况下，如果胰岛素曲线的增加的AUC，动物可被认为已在第一个过程的时间是早dysmetabolic。如果AUC减少，但是，在动物的dysmetabolic状态可被认为已被提前/糖尿病前期在第一表征的时间。一个糖尿病前期也可以accompan通过减肥和灭蝇灯在时间介入两个IVGTT程序同期增加液体摄入量。这些情况说明为什么IVGTT是当在这样的步骤的记录的光被视为相对于被用作动物的健康的快照一个特别有用的工具。 使用椅子克制有意识动物的已被证实葡萄糖耐量测试25期间，以产生在血糖水平显著升高。出于这个原因，将动物服用镇静剂用于此过程。但是，必须注意麻醉的选择行使。据报道，使用ofα2肾上腺素受体激动剂，例如甲苯噻嗪和右旋美托咪镇静可以显著增加在非人灵长类17,21的血糖水平。这里报告的方法，仅使用氯胺酮镇静，不会引起血糖水平升高22。有时，动物可显示出这样的刚性和拉紧的技术人员可能会感到松弛是必要的。在这种情况下，地西泮和Telazol已证明对生产胰岛素和葡萄糖代谢然后α2肾上腺素受体激动剂21少得多深远的影响。因此，它需要考虑在代谢试验，如IVGTT麻醉方案是非常重要的。综上所述，IVGTT是常规的NHPS执行一个简单的代谢功能测试，但它可以提供有价值的信息，以集落内分类动物进入不同的代谢状态，因此，通知他们的动物保健和卫生管理，以及它们的潜在效用在代谢性疾病的模型。

的IVGTT是常规用于确定在人类中的β细胞功能在不同的代谢状态5,7的便利功能测定法。在T2D中的动物模型中，它被公认为表征的动物，显示从代谢疾病进展的工具一个健康的一个dysmetabolic高血糖状态8,9。T2D的最接近动物模型是表现在非人灵长类动物（NHPS），其中恒河猴和食蟹猴是显着的例子。这些动物自然发展随着年龄和肥胖促进其发生率相同的风险因素，如2型糖尿病的人类10。此外，还有一个类似的疾病进展和胰腺病理表示作为dysmetabolic疾病进展11淀粉样蛋白沉积物。 在这里，我们对我们在执行的NHPS作为IVGTT我们对这些动物代谢状态的殖民地特征的一部分的标准方法报告。此方法是容易进行相对于其它更耗时且昂贵的技术2。该IVGTT是一个用于快速和频繁的表征大群动物是有用的。当与糖化血红蛋白（HbA1c）的电平考虑在内的动物的饮食和食物摄取的历史，以及他们的百分之瘦体重和体脂肪，所述IVGTT通常足以用于向显性糖尿病6表征动物的代谢状态和进展8。 糖化血红蛋白代表了一个红血细胞的寿命的平均血糖水平，过去6周〜3个月，只要葡萄糖水平的可靠量度。当从IVGTT的禁食基线血样测定，该值在提供程序之间的几个月一个窗口，血糖控制。如果动物已经从dysmetabolic转变自上次IVGTT糖尿病，比前值高得多的糖化血红蛋白数值表明该过渡即将开始他们的最后IVGTT后，而，糖化血红蛋白值更接近其先前的数值表明，他们最近才转移。在一般情况下，在恒河猴，糖化血红蛋白值大于6％的被认为是不正常的，并指示血糖控制不佳10,23。 血糖水平应的动物作为一个整体的行为和一般健康状况的范围内进行解释。糖尿病猕猴 - 人一样 - 展览亢进，多饮，多尿。动物群的住房提供了这些指标和个人护理dysmetabolic和糖尿病的猴子所需的测量显著的挑战。我们建议单独容纳动物，以便可以提供更个性化的服务，并且更容易的猴的健康行为标记物来监测8。此外，糖尿病猕猴将表现出体重下降，以及升高的脂质分布（增加的胆固醇，高甘油三酯血症）和血清化学干扰矿物质代谢。它测量肝脏和血清化学肾功能的标志物是重要的，因为这些器官损伤往往前进代谢紊乱/糖尿病的并发症，并且可以是血糖，脂质和矿物质失衡9，11，18的共同决定因素，24 。 使用此方法时，来自多个，经常刻画产生的历史价值在猴子的生活中具有特殊的价值。如果其他程序，如葡萄糖钳夹或分级葡萄糖输注（GGI），需要充分评估动物的健康，这是常用于初步鉴定时，他们的历史是不可用。每三个月的频率。然而，一旦一个基线已经建立，重复IVGTTs通常足以跟踪动物的进展。当动物被注册到整个多项研究，这是特别重要历年的代谢状态为主。而他们的健康可能在一个时间数年保持相对稳定，当动物的代谢状况恶化，在胰岛素抵抗和葡萄糖不耐症的显着增加可以非常迅速地发生。糖化血红蛋白值允许的预定相隔3个月的程序之间的动物的健康状况的下降或改善一些插值。由于这个原因，这种方法是理想的特征在多个纵向研究对其自然寿命的过程中使用的动物。

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Allegra X-15R Centrifuge</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:188px;">plasma: 4C @3000 rpm for 10 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sorvall ST16R Centrifuge</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:188px;">serum: 22C @3000 rpm for 10 min</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:216px;">Thermo Scientific -86C Freezer, Forma 88000 Series</td>
			<td></td>
			<td>Model: 88500A</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose 50% (D50)</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8008986</td>
			<td style="width:188px;">I.V. glucose infusate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3mL Luer Lock Syringe</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;"></td>
			<td style="width:188px;">serial blood draws</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5ml Luer Lock Syringe</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;"></td>
			<td style="width:188px;">heparinized saline flush</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10mL Luer Lock Syringe</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;"></td>
			<td style="width:188px;">delivery of I.V. D50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gauze sponges 2x2</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">366.23000.4</td>
			<td style="width:188px;">Used Dry, w/ %70 Alcohol, and 2% Chlorohex Solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4 ml serum separator tubes&#160;</td>
			<td style="width:147px;">Midwest Veterinary Supply</td>
			<td style="width:119px;">366.45000.4</td>
			<td style="width:188px;">blood collection tube for superchem panel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2EDTA, 2mL</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">95057-239</td>
			<td style="width:188px;">blood collection tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aprotinin, 100mg</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">A1153-100MG</td>
			<td style="width:188px;">blood collection tube protease additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22g x 1&#34; Catheters</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">193.75250.2</td>
			<td style="width:188px;">I.V. catheter&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Injection Plug W/ Cap</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.11500.2</td>
			<td style="width:188px;">%50 dextrose infusion port</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Porus Tape, 1/2&#34; x 10yd&#160;</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.85000.2</td>
			<td style="width:188px;">maintain adherance of catheters and hep. Locks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chlorhexidine Solution 2%</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">193.08855.3</td>
			<td style="width:188px;">prep catheter site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">71001-654</td>
			<td style="width:188px;">prep catheter site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tourniquet</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8003432</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3 way stopcock</td>
			<td style="width:147px;">Midwest Veterinary Supply</td>
			<td style="width:119px;">366.28510.4</td>
			<td style="width:188px;">hep. lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37&#34; extension set</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8454200</td>
			<td style="width:188px;">hep. lock</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Exel 50-60cc LL Syringes</td>
			<td style="width:147px;">Midwest Veterinary Suppy</td>
			<td style="width:119px;">001.12250.2</td>
			<td style="width:188px;">Heparinized saline flush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 ml bag 0.9% saline</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-8365593</td>
			<td style="width:188px;">flush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,000 U Heparin, 10 ml</td>
			<td style="width:147px;">Webster</td>
			<td style="width:119px;">07-883-4916</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine, (Ketaset) 100mg/mL</td>
			<td style="width:147px;">Fort Dodge</td>
			<td style="width:119px;">(AV ordered)</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Precision Xtra glucose test strips 50/bx</td>
			<td style="width:147px;">Abbott (American Diabetes Wholesale)</td>
			<td style="width:119px;">9381599728K7</td>
			<td style="width:188px;">test baseline/ T3 blood glucose levels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Masimo Rad 57</td>
			<td style="width:147px;">DRE</td>
			<td style="width:119px;">6052057V</td>
			<td style="width:188px;">pulse-oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pavia rectal thermometer</td>
			<td style="width:147px;">Patterson</td>
			<td style="width:119px;">07-8391335</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision Xtra Glucometer</td>
			<td style="width:147px;">Abbott</td>
			<td style="width:119px;">9381599728K7</td>
			<td style="width:188px;">Handheld glucometer</td>
		</tr>
	</tbody>
</table>

characterization of metabolic status in nonhuman primates with the intravenous glucose tolerance test

静脉内葡萄糖耐量试验（IVGTT）起着葡萄糖稳态的表征了关键作用。当与血清生化型材，包括在这两个馈血糖水平和禁食状态，糖化血红蛋白，胰岛素水平，饮食的临床病史，身体组成和体重状态，正常和异常的血糖控制的评估一起可以制成。一个IVGTT的解释是通过在葡萄糖和胰岛素水平的变化的测量随时间的关系做了葡萄糖挑战。要考虑的关键组成部分是:在关系达到T0（葡萄糖输注结束），葡萄糖清除率ķ从快速葡萄糖清除的斜率在第20分钟，衍生（T1至T20），时间峰的葡萄糖和胰岛素水平返回到葡萄糖基线和曲线（AUC）下的面积。这些IVGTT措施将显示来自健康t特性变化的葡萄糖稳态举动OA患病代谢状态5。这里我们将描述非人灵长类动物（猕猴和食蟹猕猴），这是在人类中II型糖尿病（T2D）的最相关的动物模型和IVGTT和这些动物的临床概况从表征贫健康，肥胖dysmetabolic，和T2D状态8，10，11。

在图1中所示的结果是示范从成熟，健康和糖尿病猕猴在30分钟IVGTT的过程中典型的葡萄糖和胰岛素的曲线。从健康和晚期糖尿病猴子数据示为了对比，从代谢的表征的范围的两个极端处的动物之间的明显的差异。此IVGTT协议已经通过在具有类似的结果恒河猴作者成功使用。 一个健康的猕猴（恒河猴和食蟹猴）的禁食，基线葡萄糖值可以低至50至60毫克/分升8。如该图所示，最初的葡萄糖偏移 - 在T3测量 - 对于健康的NHP可能无法爬上高作为用于糖尿病动物，这常常是超过200毫克/分升的一个典型的基线葡萄糖值。在以下30个的过程中分钟，一个健康的动物的血糖水平经常回到自己的基线值，而dysmetabolic和糖尿病动物没有（ 图1;实线）。一个健康的动物伴随胰岛素曲线显示出两个峰（ 图 1;蓝色虚线），这对应于初始，快速的血糖由胰岛素对葡萄糖外周作用在比正常更大的部分介导的下降（相1）运输和摄取3,4。这些外周作用的大小，即使是在胰岛素反应的第一阶段，不等于肝脏的贡献为葡萄糖降低，这，较小但更持续第二胰岛素峰期间（第2阶段），具有通过内源性葡萄糖产生1的抑制对血糖水平的影响最大。 作为动物从健康到dysmetabolic的进展，有典型地第一个缩减Ë第一胰岛素响应于葡萄糖大丸剂，其可以在减小的空气被反射的相位。然而，AUC可以保持不变，因为会有更多的频繁在胰岛素产生的总体增加，在程序过程提供基本不变的血糖水平的增加胰岛素产生补偿了灵敏度的降低。一旦动物已成为公开的糖尿病，胰岛素产生响应于葡萄糖大丸剂急剧下降（ 图1;红色虚线）。血糖水平将保持在程序的过程中升高，如葡萄糖清除发生什么由非胰岛素依赖性机制（ 图1）现在介导的几乎完全。 <img alt="图1" src="/files/ftp_upload/52895/52895fig1.jpg" /> 图1:IVGTT葡萄糖清除和胰岛素的产生曲线糖尿病和Healthy控制动物。这里所示的是葡萄糖（实线）和胰岛素（虚线）的曲线为健康（蓝线）和糖尿病（红线）的动物。这些值是对猕猴进行IVGTTs期间所收集的实际数据的平均值（糖尿病血糖:N = 27;健康糖:N = 21;糖尿病胰岛素:N = 23;健康的胰岛素:N = 20;显示标准误差条）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52895/52895fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时钟时间 </td><td> 的时间点（分钟） </td><td> 即时的 </td><td> 血液样品量（ml） </td><td> 样本分析 </td><td colspan="2"> 葡萄糖抄表（毫克/分升） </td></tr><tr><td></tD&gt; <td>底线</td><td></td><td> 5毫升</td><td> 1.5毫升SST -化学 0.5毫升K 2 EDTA - CBC，糖化血红蛋白 3毫升K 2 EDTA + 50微升PROT -葡萄糖，胰岛素，C打气</td><td></td><td></td></tr><tr><td></td><td> 0 </td><td></td><td colspan="4">葡萄糖输注250毫克/千克IV，在30秒内给定的，接着用5 ml肝素盐水冲洗</td></tr><tr><td></td><td> 3 </td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td>五</td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td> 7 </td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td> 10 </td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td> 15 </td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td> 20 </td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr><tr><td></td><td>三十</td><td></td><td> 2.0毫升</td><td> K 2 EDTA + 30微升普罗特:葡萄糖，插件，C-佩普</td><td colspan="2"></td></tr></tbody></table> 表1:IVGTT过程表这里展示的是一个IVGTT的标准程序报告，详细介绍了手术过程中拍摄的所有相关信息。对于采样时钟时间得出以下的葡萄糖输液应从输注结束计算。实际时间应记录每个抽奖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52895/52895table1.docx" target="_blank">请点击此处下载此表为Microsoft Word文档。</a>

Watch this Scientific Journal Video about Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test at JoVE.com

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

该协议的目的是提出一种标准方法来执行静脉内葡萄糖耐量试验（IVGTTs）评估在非人灵长类动物的血糖控制和从健康评估dysmetabolic其代谢状态。

代谢状态的表征非人灵长类动物与静脉葡萄糖耐量试验

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

characterization-metabolic-status-nonhuman-primates-with-intravenous

Nanyang Technological University

Research

JoVE Journal

Medicine

10.8K 次观看.  Crown Bioscience. 该协议的目的是提出一种标准方法来执行静脉内葡萄糖耐量试验（IVGTTs）评估在非人灵长类动物的血糖控制和从健康评估dysmetabolic其代谢状态。 

视频: Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance

Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1.
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7.
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11 or sublingual nitrate12,13. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14, with a high positive response rate immediately after administration15. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1.
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1&plusmn;0.6 min16,17. This allows not only diagnosis and pathophysiological assessment19-22, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.

We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.

倾斜试验结合下体负压测量立位耐力的"金标准"

Orthostatic tolerance (OT)  refers to the ability to maintain cardiovascular stability when upright,  against the hydrostatic effects of gravity, and ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

通过长期进步力量训练提高力量，力量，肌肉有氧能力和葡萄糖耐量

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

球注射阿霉素对小鼠肾病综合征的诱导作用及缓释抑肽酶预防容积保留率的研究

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

在小鼠中纳入可存活的肝脏活检程序评估非酒精性脂肪性肝炎 (NASH) 的解决方法

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...