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注: 所有用于动物实验的协议和方法已获台北医科大学动物保育及使用委员会 (IACUC) 批准 (协议编号: LAC-2014-0387)
1. 微渗透泵输液系统的研制
注: 准备泵, 人工脑脊液 (aCSF) 缓冲剂, 和药物 (Met-CCL5/RANTES 蛋白溶液 (10 ng/毫升在 aCSF)) 在无菌条件下使用的缓冲区过滤0.2 µm 过滤器, 并进行所有程序的文化遮光罩手套.手术程序如下:
<ol>
	<li>在手术前一天准备微渗透泵: 用人工脑脊液 (aCSF) 填充脑微渗透泵, 用1毫升注射器和钝头针连同试剂盒一起提供。将微渗透泵浸入 aCSF, 放在摇床上, 一夜之间轻轻摇动。 注意: 泵应充满 aCSF, 在泵内应避免气泡 (图 1A)。</li>
	<li>在开始手术前, 准备重组 Met-CCL5/RANTES 蛋白溶液 (10 ng/毫升, 稀释在 aCSF) 用于实验。从泵中取出 aCSF, 用药物溶液慢慢填充泵, 直到过量泄漏。 注:15 毫升 aCSF 或 Met-CCL5/RANTES 解决方案是足够的5-8 泵。 注意: 重复上述步骤, 确保泵内完全充满了没有气泡的药物。</li>
	<li>将导管管切割成所需长度, 并将其与钝端脑输液针连接在脑输液袋中。用药物填满输液盒和管子。</li>
	<li>最后, 在微渗透泵上组装和连接脑输液套件。 警告: 在管道或泵中不应形成气泡 (图 1A)。</li>
	<li>浸泡在 aCSF 的整个渗透泵-脑融合在一个消毒50毫升管, 以防止泵干燥。渗透泵-脑融合装置现在已经准备好用于外科手术。 注: 微渗透泵系统可用于长期药物输液。这确保了一个安全和方便的方式向老鼠大脑提供药物。</li>
</ol>
2. 脑室手术-微渗透泵的植入
注意: 用75% 乙醇消毒手术环境, 确保参与研究的人员佩戴无菌手套和干净的实验室大衣。手术工具/仪器必须经过蒸压和干燥后使用, 并随后消毒75% 乙醇 in-between 小鼠手术。
<ol>
	<li>用腹腔注射液 (IP) 和氯胺酮/嗪 (氯胺酮50毫克/千克, 嗪10毫克/千克) 称量鼠标和麻醉。 注意: 在渗透泵植入手术中, 不推荐使用小于24克的小鼠体重。</li>
	<li>安装并将鼠标头固定到立体定向设备 (图 1B)。</li>
	<li>使用一对手术剪刀和钳子, 以切断打开外皮肤覆盖的头骨。用碘清洗周围的头骨。</li>
	<li>用一对钝头钳在颈部附近的渗透泵-脑融合集植入 (图 1C) 的帮助下, 将皮下皮肤的最外层分开。</li>
	<li>使用立体定向仪将输液点标记为参考脑图 (图 1D)。在这个实验中, 针需要植入 3rd心室区 (Bregma: 0.0 毫米侧, 1.3 毫米后, 5.7 毫米腹)。</li>
	<li>在头骨上标记的区域 (图 1E) 上钻一个孔。 注意: 注意不要破坏老鼠的脑膜和血管, 从而避免脑部微血管的破坏。</li>
	<li>将含有 aCSF (as 控制) 或药物 (Met-CCL5/RANTES 蛋白溶液) 的微渗透泵-脑融合装置置于颈部后方的皮肤下, 并将脑输液针插入钻孔中, 将药物注入老鼠脑中 (图 1E)。针头会穿透脑膜进入心室使用表面敏凝胶 (图 1F) 将针固定在颅骨上, 等待1-2 分钟, 直到胶水干涸。接下来, 切断针顶部的凸出部分 (图 1G-H)。</li>
	<li>用纸巾粘胶来治疗头部的手术伤口。应用50µL 的胶水在伤口上, 把双方的皮肤拉在一起, 并举行三十年代, 让皮肤密封 (图 1I)。 注意: 手术后用100% 酒精垫清洗伤口, 100 ul 青霉素与链霉素预防感染。注意: 小鼠皮肤将形成疤痕组织和愈合在几天后的外科胶水的管理。胶水的主要优点是避免手术缝线, 这可能会引起皮肤发炎或炎症。</li>
	<li>把老鼠放在一个温暖的盘子里 (加热到37° c), 等到老鼠从麻醉效果中恢复过来。 注意: 保持小鼠的体温对提高手术后生存的几率至关重要。</li>
	<li>经过一周的恢复期, 小鼠将准备进一步的实验, 如口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT)。</li>
</ol>
3. 口服葡萄糖耐受试验 (OGTT)
注: 在注射 aCSF 和符合CCL5/RANTES (10 ng/毫升, 100 µL) 后7天进行口服葡萄糖耐受试验。在 OGTT 有足够的水供应前, 为小鼠保持6小时的快速。将动物放在相同的工作台上进行实验, 使它们能够适应到环境中, 减少在手术过程中的压力。
<ol>
	<li>葡萄糖溶液制备: 在进行实验之前, 在15毫升蒸馏的 H2O 中溶解3.75 克葡萄糖以制备葡萄糖溶液。</li>
	<li>在实验过程 (表 1) 中设置记录读数的时间表。 注意: 重要的是要建立一个时间表, 每个血液检查之间适当的时间间隔, 以便在实验中准确记录。</li>
	<li>空腹后称量每只老鼠, 并计算出适当的葡萄糖注射量。</li></ol>例如, 如果鼠标重30克, 那么要管理的葡萄糖溶液的量应该是300µL。
	<li>在工作台上准备下列仪器:<ol>
		<li>血糖 (按 "开始" 按钮检查电池状态, 确保它在测试之前正常工作。</li>
		<li>葡萄糖芯片</li>
		<li>胰岛素注射器 (0.3 毫升胰岛素注射器)</li>
		<li>刀片</li>
		<li>计时</li>
	</ol>
	</li>
	<li>一旦工作台设置, 测量和记录血糖水平如下: 把一个干净的和新的葡萄糖芯片到血糖, 并按开始按钮, 以零它。</li>
	<li>将鼠标靠在颈部后部, 并将尾巴划几次, 以确保足够的血液流向尾部区域。</li>
	<li>使用一个新的剃刀刀片切断一小块的尾巴, 挤出一小滴血 (约10-20 µL) 到葡萄糖芯片。血液应填补芯片, 以允许准确的测量。血糖将立即显示葡萄糖水平。如果机器显示 "错误", 重复的过程与一个新的葡萄糖芯片。 注: 葡萄糖芯片只需要一滴血。当血液样本需要收集不止一次时, 只需用手指沿着老鼠的尾巴跑几次, 而直接在芯片顶部收集血液就可以施加压力。在采集血样的时候, 不需要每次都切尾端。</li>
	<li>其次, 用胃胃技术对小鼠进行口服葡萄糖 (0.25 克/毫升) 的饲料。应使用该公式计算所需的葡萄糖量: 10X 体重 (BW) µL 葡萄糖溶液 (例如, 如果鼠标重30克, 将被管理的葡萄糖溶液的数量将是300µL)。在口服葡萄糖后立即启动计时器。</li>
	<li>重复葡萄糖测量过程在 15, 30, 60, 90 和 120 min。</li>
	<li>在所有的葡萄糖水平读数被记录下来后, 丢弃在生物危害容器中的刀片和葡萄糖芯片。把食物放回老鼠笼里, 然后把它们送到动物房间。</li>

4. 胰岛素耐受试验 (ITT)
注: 胰岛素耐受试验和口服葡萄糖耐受试验应安排至少7天的间隔, 以减少对动物的禁食效果。对于胰岛素耐受性测试 (ITT), 人类胰岛素 (0.75 U/千克) 将通过 IP 注射进行管理。
<ol>
	<li>制备 0.25 U 型胰岛素溶液: 稀释100U 人胰岛素的比例为1:400 的盐水溶液。</li>
	<li>在禁食后称量每只老鼠, 并据此计算胰岛素注射量: 0.25 u 型胰岛素的体积 (µL) 被注入 IP = 3 X BW (0.75 u 胰岛素/千克体重)。例如: 对于重28.8 克的鼠标, 注射: 28.8 X 3 = 86.4 µL (0.25 U 稀释的胰岛素) (表 2)。 注意: 相同的动物在不同的日子禁食6小时后可能有不同的体重。因此, 有必要在禁食前后测量体重, 进行 OGTT 和 ITT 试验。根据物种、性别和禁食持续时间, 老鼠的体重会下降。高剂量的胰岛素会导致胰岛素休克, 并导致动物死亡。</li>
	<li>设置表 (表 2) 以在实验过程中记录读数。重复步骤3.4。到3.8。用于测量血糖水平。</li>
</ol>

<ol>
	<li>Plata-Salaman, C. R., Borkoski, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines/intercrines+and+central+regulation+of+feeding.">Chemokines/intercrines and central regulation of feeding.</a> Am J Physiol. 266, R1711-R1715 (1994).</li><li>Milanski, M., 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=Inhibition+of+hypothalamic+inflammation+reverses+diet-induced+insulin+resistance+in+the+liver.">Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver.</a> Diabetes. 61, 1455-1462 (2012).</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=Increased+hypothalamic+inflammation+associated+with+the+susceptibility+to+obesity+in+rats+exposed+to+high-fat+diet.">Increased hypothalamic inflammation associated with the susceptibility to obesity in rats exposed to high-fat diet.</a> Experimental diabetes research. 2012, 847246 (2012).</li><li>Benomar, Y., 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=Insulin+and+leptin+induce+Glut4+plasma+membrane+translocation+and+glucose+uptake+in+a+human+neuronal+cell+line+by+a+phosphatidylinositol+3-kinase-+dependent+mechanism.">Insulin and leptin induce Glut4 plasma membrane translocation and glucose uptake in a human neuronal cell line by a phosphatidylinositol 3-kinase- dependent mechanism.</a> Endocrinology. 147, 2550-2556 (2006).</li><li>Gual, P., Le Marchand-Brustel, Y., Tanti, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+regulation+of+insulin+signaling+through+IRS-1+phosphorylation.">Positive and negative regulation of insulin signaling through IRS-1 phosphorylation.</a> Biochimie. 87, 99-109 (2005).</li><li>Werner, E. D., Lee, J., Hansen, L., Yuan, M., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+due+to+phosphorylation+of+insulin+receptor+substrate-1+at+serine+302.">Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302.</a> The Journal of biological chemistry. 279, 35298-35305 (2004).</li><li>Boura-Halfon, S., Zick, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+IRS+proteins,+insulin+action,+and+insulin+resistance.">Phosphorylation of IRS proteins, insulin action, and insulin resistance.</a> American journal of physiology. Endocrinology and metabolism. 296, E581-E591 (2009).</li><li>Chou, S. Y., 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=CCL5/RANTES+contributes+to+hypothalamic+insulin+signaling+for+systemic+insulin+responsiveness+through+CCR5.">CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5.</a> Sci Rep. 6, 37659 (2016).</li><li>Calegari, V. 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=Inflammation+of+the+hypothalamus+leads+to+defective+pancreatic+islet+function.">Inflammation of the hypothalamus leads to defective pancreatic islet function.</a> J Biol Chem. 286, 12870-12880 (2011).</li><li>Mighiu, P. I., Filippi, B. M., Lam, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+inflammation+to+the+brain-liver+axis.">Linking inflammation to the brain-liver axis.</a> Diabetes. 61, 1350-1352 (2012).</li><li>Tavares, E., Minano, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANTES:+a+new+prostaglandin+dependent+endogenous+pyrogen+in+the+rat.">RANTES: a new prostaglandin dependent endogenous pyrogen in the rat.</a> Neuropharmacology. 39, 2505-2513 (2000).</li><li>Appay, V., Rowland-Jones, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANTES:+a+versatile+and+controversial+chemokine.">RANTES: a versatile and controversial chemokine.</a> Trends in immunology. 22, 83-87 (2001).</li><li>DeVos, S. L., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intraventricular+delivery+of+drugs+to+the+rodent+central+nervous+system.">Direct intraventricular delivery of drugs to the rodent central nervous system.</a> J Vis Exp. , e50326 (2013).</li><li>Wang, N., 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=Admission+blood+glucose+and+in-hospital+clinical+outcome+among+patients+with+acute+stroke+in+Inner+Mongolia,+China.">Admission blood glucose and in-hospital clinical outcome among patients with acute stroke in Inner Mongolia, China.</a> Clin Invest Med. 32, E151-E157 (2009).</li><li>Olsen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+glucose+in+acute+stroke.">Blood glucose in acute stroke.</a> Expert Rev Neurother. 9, 409-419 (2009).</li><li>De Jong, W. H., Borm, 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=Drug+delivery+and+nanoparticles:applications+and+hazards.">Drug delivery and nanoparticles:applications and hazards.</a> Int J Nanomedicine. 3, 133-149 (2008).</li><li>Kitade, H., 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=CCR5+plays+a+critical+role+in+obesity-induced+adipose+tissue+inflammation+and+insulin+resistance+by+regulating+both+macrophage+recruitment+and+M1/M2+status.">CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status.</a> Diabetes. 61, 1680-1690 (2012).</li><li>Kennedy, A., 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=Loss+of+CCR5+results+in+glucose+intolerance+in+diet-induced+obese+mice.">Loss of CCR5 results in glucose intolerance in diet-induced obese mice.</a> Am J Physiol Endocrinol Metab. 305, E897-E906 (2013).</li></ol>

作者没有什么可透露的。

慢性炎症的机制和相关的趋化因子, 如 CCL5 及其受体-CCR5 在 type-2 糖尿病的发展仍然不清楚。慢性炎症导致巨噬细胞渗入脂肪组织, 影响脂肪的调节;同时, 它也吸引β细胞, 并损害胰岛素分泌从胰岛的反应血糖。大脑下丘脑在调节食欲、外周血糖代谢和胰岛素反应等系统外周组织胰岛素和脂肪信号方面起着重要作用。许多研究还表明, 下丘脑炎症导致能量稳态的缺陷调节以及胰岛和肝功能的缺陷2,3,9,10。CCL5 在大脑中有助于食物摄入和体温调节在下丘脑的11,12;然而, CCL5 与下丘脑和全身胰岛素信号的相关性还不清楚。一个 CCL5 的整体体击倒鼠标 (CCL5/-) 已经生成, 以解决这个问题, 这表明胰岛素抵抗表型与较高的胰岛素水平和高血糖水平的血液中8。然而, 由于可能的长期代偿效应, T2DM 表型的发育需要较长的时间, 很难研究 CCL5 在下丘脑胰岛素信号中的作用和机制。因此, 直接操纵 CCL5 信号在下丘脑神经元是最好的方法。然而, 在下丘脑区域有多种类型的神经元, 这是相当昂贵和耗时的, 以产生细胞特异性敲除小鼠。利用 ICV 的输液系统, 可以节省时间, 并提供一个更具体的方法来操纵 CCL5 功能直接在大脑中, 绕过可能的周围炎症反应。
利用渗透泵的研究已经发表过, 提供了很好的例子和示范的技术参与了在啮齿动物的渗透泵植入13。然而, 在我们的研究中, 我们在遵循这些协议时面临着一些挑战。首先, 协议中使用的一些设备相当昂贵, 包括 1) 电气系统到达的位置, 绘制和插入到小鼠脑针, 2) 的热系统保持鼠标的体温和 3) 氧异氟醚对小鼠进行麻醉的供应系统。其次, 在其他文章中描述的技术很难复制, 因为我们只能够在一个小范围的体重和在一定年龄的研究中使用动物。我们知道更大的老鼠更适合做手术和植入。然而, 在我们的研究中, 我们不得不使用小的和年轻的老鼠来避免超重和衰老对胰岛素和血糖调节的影响: 研究中选择了体重25±2克的雄性小鼠和2月龄左右的年龄。因此, 很难进行手术和缝合伤口的鼠标头部。第三, 手术后的炎症反应必须减少, 因为炎症细胞因子是本研究的目标。在手术后, 小鼠和大鼠可以很容易地切除缝线和开放性伤口, 从而引起炎症, 增加趋化因子反应。因此, 一项策略, 以达到的位置, 并绘制和插入到小鼠脑针, 避免继发感染是必要的。因此, 我们修改了以前描述的协议, 使这项技术的成本效益, 更容易, 并减少对动物的伤害, 如下段所述。
首先, 我们使用了一个指甲钻, 以手动钻一个洞周围的目标区域标记的头骨, 如步骤2.6 所述。这种方法是成本效益, 使我们能够监测整个过程, 以避免损害小鼠脑膜和血管。急性中风后血糖调节受损, 如脑出血。急性高血糖和糖尿病样综合征也观察中风后临床设置14,15。同样, 我们也发现在脑出血和脓液的小鼠中, 葡萄糖水平和胰岛素反应受损。我们知道, 更好地控制手工手术是必要的, 以确保结果的一致性。其次, 我们利用了一种新开发的医用生物材料, 通常用于临床、组织胶黏剂 (步骤 2.8), 在手术后用鼠标封住皮肤, 从而避免缝合, 加快愈合速度。这使得手术过程更容易执行, 并减少了继发性炎症的几率。第三, 完成整个手术过程所需时间较短, 增加了小鼠生存的机会, 降低了注射腹腔的麻醉药用量。我们观察到了高生存率 (95%), 并获得了相对准确的结果, 遵循这一修改的协议。
这一技术的局限性是药物传递的相对较短的时间框架。虽然渗透泵可以被放置到鼠标的身体或者不重开大脑, 我们的研究只关注炎症趋化因子对大脑的影响, 以调节周围系统胰岛素信号。外周组织的额外手术可能引起周围组织的炎症反应, 从而增加炎症趋化因子的表达, 并影响结果。其次, 药物的半衰期也限制了研究的持续时间。重组蛋白, 如趋化因子, 通常有一个较短的半衰期, 它失去了它的活动随着时间的推移, 虽然它也允许我们研究的影响, 阻断 CCL5 信号在大脑在短期内。我们以前的研究还描述了一种用于生成 CCL5 击倒鼠标的遗传修改方法, 它提供了一个具有长期影响的模型8。
有一些新的技术和替代方法, 以提供药物进入大脑。纳米技术是一种强有力的方法, 可用于将药物输送到中枢神经系统。然而, 许多药物是热敏的, 可以销毁时, 试图包装成纳米颗粒16。此外, 纳米粒子可以通过血脑屏障和吸细胞, 适合 siRNA 或最常见的药物, 但它不是一个理想的方法, 配体受体结合。CCL5 需要结合其受体, CCR5, 在下丘脑弧神经元生效8, 并交付 CCL5 拮抗剂MetCCL5 进入神经元通过纳米粒子可能会导致失去的能力绑定和阻止 CCR5 在细胞表面.
在口服葡萄糖耐受试验中, 与对照组 (aCSF 管小鼠) 相比, CCL5-antagonist MetCCL5 的小鼠血糖水平明显增高。另外的胰岛素管理 (胰岛素耐受性测试) 也不能降低血糖水平在MetCCL5 接收小鼠 (图 4B), 这表明内源性和外部胰岛素不能降低血糖水平当阻断下丘脑 CCL5 信号。小鼠在下丘脑没有 CCL5 活动就成了胰岛素抵抗。serine302 磷酸化的 IRS-1 被发现在接受 Met-CCL5 的小鼠相比, 控制小鼠接受 aCSF (图 5A-b)。丝氨酸302磷酸化 IRS-1 已被证明是诱导 IRS-1 从胰岛素受体的物理离解, 这是一个主要原因胰岛素抵抗6;胰岛素无法激活下游信号, 如 PI3K-Akt 通路。一个前体内胰岛素刺激研究证实胰岛素下游信号分子 Akt (p-AktS473) 没有激活胰岛素在小鼠下丘脑组织注入 Met-CCL5, 而相反, 丝氨酸302磷酸化增加。总之, 生理数据 (OGTT 和 ITT) 和分子研究表明, 下丘脑 CCL5 信号介导的下丘脑胰岛素信号调节, 这有助于系统性胰岛素抵抗和葡萄糖代谢。
CCL5 和 CCR5 在肥胖相关糖尿病中的作用和机制尚不清楚。出口et al.报告, CCR5 缺乏保护小鼠的肥胖引起的炎症, 巨噬细胞招募和胰岛素抵抗17。然而, 其他的研究由肯尼迪et al.发现相反的结果表明, CCR5 缺乏损害全身葡萄糖耐受以及脂肪细胞和肌肉胰岛素信号传递18。两项研究都采用高脂饮食诱导肥胖, 导致全身慢性炎症和代偿性反应。这些研究没有提供清洁和明确的机制, CCL5 和 CCR5 在胰岛素信号调节。另一方面, 渗透泵技术允许大脑特定的输液和避免补偿反应, 其有时限的分娩。
总之, 虽然渗透泵与脑输液系统似乎是一个 "old-fashioned" 技术, 它提供了一个更便宜, 更容易, 更少的有害的药物传递的方法, 并帮助调查配体受体信号的功能在大脑.

胰岛素会影响各种各样的组织, 包括大脑。胰岛素通过血脑屏障, 进入中枢神经系统 (CNS), 并结合胰岛素受体 (IR) 在下丘脑, 以调节食物摄入量, 交感神经活动, 和外周胰岛素反应。外周脂肪组织的慢性炎症已被建议用于2型糖尿病 (T2DM), 但这些炎症反应如何影响下丘脑中的胰岛素信号以调节全身胰岛素反应和葡萄糖不耐受仍不清楚。一些趋化因子参与食欲调节和体温调节在下丘脑1 , 如肿瘤坏死因子α (TNFα), 白细胞介素 (IL)-6, IL-1β, 单核分子趋化 protein-1 (MCP-1), 和 CCL5 (c-c 主题配体5).此外, 下丘脑炎症导致胰岛素抵抗 T2DM2,3。
在这些趋化因子中, CCL5 及其受体 CCR5 在脂肪组织中的表达水平的改变, 在人类和动物的 T2DM 中都与动脉硬化和葡萄糖不耐受有关。CCL5 也有神经内分泌功能, 包括调节食物摄入量和体温, 在下丘脑。因此, 重要的是要研究 CCL5 参与胰岛素信号激活在下丘脑或周围组织。
胰岛素信号在细胞内受到严格的调控。胰岛素受体结合 (IR) 激活胰岛素受体基质 (IRS) 蛋白, 其次是醇3激酶 (PI3K) 和蛋白激酶 B (PKB/AKT) 活化和葡萄糖 transporter-4 (GLUT4) 膜易位4.IRS 蛋白是这一信号传导通路的关键调节器: 它们有多个酪氨酸和丝氨酸残留物, 可以磷酸化反应阳性或负胰岛素信号5。例如, IRS-1 丝氨酸302磷酸化可导致 IRS-1 从 IR 和阻断胰岛素信号转导的物理离解, 导致胰岛素抵抗6。在下丘脑中, IRS 蛋白的活性损伤已被证明能诱发小鼠的胰岛素抵抗和葡萄糖不耐受7。
一个共同的方法来研究一个特定的基因的功能是操纵的表达目标基因分布在整个身体的有机体。然而, 这可能有几个缺点: 1) 它可能产生不同的反馈调节或补偿作用, 随着时间的推移和 2) 这种方法并没有帮助我们说明目标蛋白在特定脑区的作用。此外, 组织和细胞特异的基因剔除动物需要很长的时间来繁殖和昂贵。因此, 我们使用短期的脑输液渗透泵系统-一个相对快速和方便的方式来干扰的信号的目标蛋白在大脑中使用拮抗剂药物克服上述问题。立体定向注射用于要求复杂的手术技巧和广泛的投资在仪器和时间。在本协议中, 我们提供了一个简单和安全的方法来执行立体定向注射和快速, 不那么有害, 瞬时的方法来检测血糖浓度和研究的作用, CCL5 在下丘脑胰岛素信号调节。

<table><tbody><tr><td>Vetbond Tissue Adhesive</td><td>3M</td><td>#1049SB</td><td>The glue used to seal the lesion site on the mouse head</td></tr><tr><td>LOCTITE 454 instant adhesive</td><td>Durect Corporation</td><td>#8670</td><td>The glue used to fix the needle on the mouse skull</td></tr><tr><td>Alzet Micro- Osmotic Pump</td><td>Durect Corporation</td><td>#9922</td><td>0.11 &amp;mu;l per hour, 28 days</td></tr><tr><td>Brain infusion system</td><td>Durect Corporation</td><td>#8851</td><td>1-3 mm, used to perfuse the drug in to the mice brain</td></tr><tr><td>Glucometer</td><td>Roche</td><td>#06870244001</td><td>Used to measure the blood glucose level</td></tr><tr><td>Glucose chip</td><td>Roche</td><td>#06454011020</td><td>Used to load the blood sample</td></tr><tr><td>Evan&#039;s blue</td><td>Sigma</td><td>#MKBK0523V</td><td>To demonstrate the drug infusion area</td></tr><tr><td>Insulin syringe</td><td>Becton, Dickinson and Company</td><td>#3232145 C</td><td>Used to administer insulin intraperitoneally</td></tr><tr><td>MIO NE116 CONTROL UNIT&lt;br/&gt; (nail drill)</td><td>Mio System</td><td>#E235-015</td><td>To drill a hole in the skull of the mouse</td></tr><tr><td>CCL5/Met-RANTES Protein</td><td>R&amp;amp;D</td><td>#ADB0111081</td><td>Recombinant Human CCL5, E-coli derived</td></tr><tr><td>aCSF formula</td><td>119 mM NaCl&lt;br/&gt; 26.2 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;br/&gt; 2.5 mM KCl&lt;br/&gt; 1 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 1.3 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 10 mM glucose</td><td></td><td>Filter sterilize with a 0.22 &amp;mu;m filter apparatus, and store at 4&amp;deg;C.&lt;br/&gt; aCSF is stable for 3-4 weeks</td></tr><tr><td>Phospho-IRS-1 Serine302 antibody</td><td>Cell Signaling</td><td>#12879</td><td>1:1000 dilution</td></tr><tr><td>IRS-1 (D23G12) antibody</td><td>Cell Signaling</td><td>#12879</td><td>1:1000 dilution</td></tr><tr><td>Phospho-Akt Serine 473 antibody</td><td>Cell Signaling</td><td>#9916</td><td>1:2000 dilution</td></tr><tr><td>Akt (pan) (C67E7) antibody</td><td>Cell Signaling</td><td>#9916</td><td>1:1000 dilution</td></tr><tr><td>Animals: C57BL/6</td><td>NAR Labs</td><td></td><td>Wild type mice strain used in the study</td></tr></tbody></table>

studying the hypothalamic insulin signal to peripheral glucose intolerance with a continuous drug infusion system into the mouse brain

胰岛素调节下丘脑和外周胰岛素反应的系统性代谢。外周脂肪组织的炎症反应有助于2型糖尿病 (T2DM) 的发育和下丘脑的食欲调节。在2型糖尿病 (T2DM) 中, 已经提出了趋化因子 CCL5 和 c-c 因子受体型 5 (CCR5) 水平来调和动脉硬化和葡萄糖不耐受。此外, CCL5 在下丘脑通过调节食物摄入量和体温来发挥神经内分泌作用, 从而促使我们研究其在下丘脑胰岛素信号传导和周围葡萄糖代谢调节中的作用。
微渗透泵脑输液系统是一种快速、精确的控制 CCL5 功能的方法, 研究其在脑内的作用。它还提供了一种方便的替代方法, 以产生一个转基因的淘汰赛动物。在这个系统中, CCL5 信号被脑室 (ICV) 的拮抗剂--使用微渗透泵的 CCL5--的输注阻断. 口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 检测了周围葡萄糖代谢和胰岛素应答。然后通过从动物身上提取的组织样本中的蛋白质印迹来分析胰岛素信号的活性。
在7-14 天的MetCCL5 输液后, 小鼠的葡萄糖代谢和胰岛素反应能力受损, 如 OGTT 和 ITT 的结果所示。CCL5 抑制后, IRS-1 serine302 磷酸化增加, 小鼠下丘脑神经元 Akt 活性降低。总之, 我们的数据表明, 阻断 CCL5 在老鼠的大脑增加磷酸化的 IRS-1 S302 和中断下丘脑胰岛素信号, 导致胰岛素功能下降的外周组织以及损害的葡萄糖代谢.

手术植入的渗透输液泵含有 aCSF 作为控制或 CCL5 拮抗剂满足CCL5 (阻止 CCL5 的影响在大脑) 进行了对小鼠。在手术后7和14天, 使用 OGTT (7 天后) 和 ITT (14 天后) 对小鼠的周围葡萄糖耐受和胰岛素反应进行了分析, 如协议所述。在禁食6小时后进行小鼠口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT)。小鼠用葡萄糖口服, 其数量根据其各自的体重。记录血糖水平的变化, 如图 3所示。采用腹腔 (IP) 胰岛素注射对小鼠进行胰岛素敏感性试验, 并立即测定血糖水平的变化。记录了不同输液药物对小鼠胰岛素刺激时血糖水平的变化, 如图 4所示。血糖管理 (图 3B) 和胰岛素注射 (图 4B) 在小鼠 CCL5 拮抗剂 (MetCCL5) 输液后, 血糖水平仅略有下降, 与 aCSF 输注的小鼠相比。这些结果提示胰岛素功能损害的小鼠的外周糖代谢的MetCCL5 管理的大脑。
其次, 通过对下丘脑组织中 IRS-1 磷酸化和 Akt 活化水平的评价, 分析了胰岛素信号的活化作用。302 IRS-1 的丝氨酸磷酸化上调在老鼠被喂食时用拮抗剂 (MetCCL5) (图 5B-c) 处理。在对照组, aCSF 被管理的小鼠下丘脑和胰岛素的挑战激活下游信号分子 Akt (磷酸化 Akt 丝氨酸 473) (图 5D, F) 不增加 IRS-1 serine302 激活 (图 5D-e)和 Akt serine473 磷酸化。相比之下, Akt 信号在注入MetCCL5 的小鼠中没有增加, 但 IRS-1 丝氨酸302的磷酸化量反而增加了。同时, 阻断 CCL5 信号在小鼠脑内阻断胰岛素活动的下丘脑和受损的外周胰岛素功能。从我们的整体发现, 如来自 ITT, OGTT, 和前体胰岛素挑战的结果, 我们得出结论, CCL5 在下丘脑有助于胰岛素信号激活和周围葡萄糖代谢后, 胰岛素刺激。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56410/56410fig1.jpg"> 
图1。渗透泵的制备及在小鼠中的植入手术方法.(A) 用药物溶液灌注脑输液试剂盒和泵制剂。红色箭头表示充满液体的导管管。(B) 固定并将鼠标头安装到立体定向设备上。(C) 将皮肤的最外层与皮下皮肤分开, 用于微渗透泵-脑输液装置的植入;虚线表明渗透泵植入物的位置。(D) 箭头指示输液侧。(E) 在头骨上的标记区域周围钻一个洞。(F) 将渗透泵-脑输液置于鼠标后部, 并将脑输液针插入钻洞 (虚线圆圈)。(g) 将针固定在头骨上, 使用组织粘胶, 并将针 (剪刀尖在 G) 上的顶部分离出来, 如下所示 (H)。(I) 用组织胶黏剂封住伤口。<a href="//ecsource.jove.com/files/ftp_upload/56410/56410fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56410/56410fig2.jpg"> 
图 2.药物扩散区的代表性图像, 当药物在心室区使用渗透泵管理。埃文的蓝色是代表药物用于渗透泵药物输液例证入心室区域 (A) 和扩散入侧向和第三脑室 (B)。缩放条 = 0.5 厘米.<a href="//ecsource.jove.com/files/ftp_upload/56410/56410fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56410/56410fig3.jpg"> 
图 3.通过口服葡萄糖耐受试验 (OGTT) 测定小鼠术后的葡萄糖代谢。在注射 aCSF (A) 和拮抗剂的小鼠口服葡萄糖后, 血糖水平的分布发生变化, MCCL5 (B)。数据显示为平均± SE. (图从8中修改)。* p 和 #60; 0.05, 由 two-way 方差分析。<a href="//ecsource.jove.com/files/ftp_upload/56410/56410fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56410/56410fig4.jpg"> 
图 4.胰岛素在小鼠血糖中的作用-胰岛素耐受试验 (ITT)。注入 aCSF (A) 的小鼠胰岛素注射后血糖水平的分布变化, 并注入拮抗剂, MCCL5 (B)。数据显示为平均± SE (图修改自8)。p & #60; 0.001, 由 two-way 方差分析。<a href="//ecsource.jove.com/files/ftp_upload/56410/56410fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56410/56410fig5.jpg"> 
图5。小鼠术后胰岛素信号的活性.(A) 抑制丝氨酸302磷酸化形式的 IRS-1 (胰岛素反应 substrate-1, pIRS1S302) 在小鼠下丘脑组织治疗 aCSF 或 CCL5 拮抗剂, MetCCL5 (MCCL5)输液泵。(B) 与正常喂养小鼠下丘脑输注后的 phosphor-IRS-1S302相关的水平。(C) S302 磷酸化 IRS-1 和 Akt 活化 (磷-Akt S473, pAktS473) 的西印迹, 在下丘脑组织 aCSF 或 CCL5 输液后有或没有胰岛素刺激. (D-E)pIRS-1S302、pS6KT421和 pAktS473的相对级别。("2" 在每个酒吧图代表: n=2 为所有 quantifications)。(空白的酒吧在 5 d-e, 左: 没有胰岛素; 带酒吧在 5 d e, 权利: 与胰岛素)。<a href="//ecsource.jove.com/files/ftp_upload/56410/56410fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#</td>
			<td>鼠标 ID</td>
			<td>身体</td>
			<td>葡萄糖</td>
			<td>开始</td>
			<td>0</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>重量</td>
			<td>μ l = 10 xbw</td>
			<td>时间</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
		</tr>
		<tr>
			<td>1</td>
			<td>501</td>
			<td>25.8g</td>
			<td>258</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:15</td>
			<td>九点半</td>
			<td>10:00</td>
		</tr>
		<tr>
			<td>2</td>
			<td>502</td>
			<td>25.3g</td>
			<td>253</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:22</td>
			<td>9:37</td>
			<td>10:07</td>
		</tr>
	</tbody>
</table>
表 1.口服葡萄糖耐受试验 (OGTT) 记录时间表
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#</td>
			<td>鼠标 ID</td>
			<td>身体</td>
			<td>胰岛素0.25IU</td>
			<td>开始</td>
			<td>0</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>重量</td>
			<td>μ l = 3 xbw</td>
			<td>时间</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
			<td>分钟</td>
		</tr>
		<tr>
			<td>1</td>
			<td>501</td>
			<td>28.8g</td>
			<td>86。4</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:15</td>
			<td>九点半</td>
			<td>10:00</td>
			<td>十点半</td>
		</tr>
		<tr>
			<td>2</td>
			<td>502</td>
			<td>25.3g</td>
			<td>75。9</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:22</td>
			<td>9:37</td>
			<td>10:07</td>
			<td>10:37</td>
		</tr>
	</tbody>
</table>
表 2.胰岛素耐受测试时间表 (ITT) 记录

Watch this Scientific Journal Video about Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain at JoVE.com

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

该协议研究的作用趋化因子 (c-c 主题) 配体 5 (CCL5) 在下丘脑通过提供拮抗剂,满足CCL5, 进入鼠标的大脑使用微渗透泵脑输液系统。这种瞬态抑制 CCL5 活动阻断了下丘脑胰岛素信号, 导致葡萄糖不耐受和外周系统胰岛素敏感性。

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 ...

studying-hypothalamic-insulin-signal-to-peripheral-glucose

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Insulin and Hypoglycemic Drugs

SCIENTISTS IN ACTION

10.2K 次观看.  Taipei Medical University and National Health Research. 该协议研究的作用趋化因子 (c-c 主题) 配体 5 (CCL5) 在下丘脑通过提供拮抗剂,满足CCL5, 进入鼠标的大脑使用微渗透泵脑输液系统。这种瞬态抑制 CCL5 活动阻断了下丘脑胰岛素信号, 导致葡萄糖不耐受和外周系统胰岛素敏感性。

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

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

在自觉大鼠高胰岛素 - 正常血糖钳

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

科研

医学

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

神经科学

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 ...

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

<meta charset="utf8"></head><body>腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

小鼠高血糖和低血糖夹的手术安装

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively.
Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

作者聚焦:使用经济高效的高血糖和低血糖钳夹技术研究小鼠大脑中的血糖稳态

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

清醒小鼠的高血糖钳夹和低血糖钳夹

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, ...

Simple Continuous Glucose Monitoring in Freely Moving Mice

Mice are a common model organism used to study metabolic diseases such as diabetes mellitus. Glucose levels are typically measured by tail-bleeding, which requires handling the mice, causes stress, and does not provide data on freely behaving mice during the dark cycle. State-of-the-art continuous glucose measurement in mice requires inserting a probe into the aortic arch of the mouse, as well as a specialized telemetry system. This challenging and expensive method has not been adopted by most labs. Here, we present a simple protocol involving the utilization of commercially available continuous glucose monitors used by millions of patients to measure glucose continuously in mice as a part of basic research. The glucose-sensing probe is inserted into the subcutaneous space in the back of the mouse through a small incision to the skin and is held in place tightly using a couple of sutures. The device is sutured to the mouse skin to ensure it remains in place. The device can measure glucose levels for up to 2 weeks and sends the data to a nearby receiver without any need to handle the mice. Scripts for the basic data analysis of glucose levels recorded are provided. This method, from surgery to computational analysis, is cost-effective and potentially very useful in metabolic research.

Here, we describe a simple method to implant a commercial continuous glucose monitor designed for patients onto mice and provide the scripts to analyze the results.

自由移动小鼠的简单连续血糖监测

Mice are a common model organism used to study metabolic diseases such as diabetes mellitus. Glucose levels are typically measured by tail-bleeding, which ...

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

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在自觉大鼠高胰岛素 - 正常血糖钳

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

海马胰岛素显微注射和

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)

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

清醒小鼠的高血糖钳夹和低血糖钳夹

清醒小鼠的高血糖钳夹和低血糖钳夹

清醒小鼠的高血糖钳夹和低血糖钳夹

自由移动小鼠的简单连续血糖监测