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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>

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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 &#956;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&#39;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 
			(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&#38;D</td>
			<td>#ADB0111081</td>
			<td>Recombinant Human CCL5, E-coli derived</td>
		</tr>
		<tr>
			<td>aCSF formula</td>
			<td>119 mM NaCl 
			26.2 mM NaHCO3 
			2.5 mM KCl 
			1 mM NaH2PO4 
			1.3 mM MgCl2 
			10 mM glucose</td>
			<td></td>
			<td>Filter sterilize with a 0.22 &#956;m filter apparatus, and store at 4&#176;C. 
			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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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Insulin and Hypoglycemic Drugs

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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

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

科研

医学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

药品再利用产生假设使用"RE:精药品"系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

胰腺管输液:药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...