这项工作得到了中国国家自然科学基金 (批准号 81570912, 81771016, 81100717) 的支持。

所有的程序和动物手术都是按照中国首都医科大学动物保育和使用委员会的指导方针进行的。
1. 设备的准备工作
<ol><li>要制造注射套管 (图 1A), 请将聚酰亚胺油管 (内径114.3 µm, 外径139.7 µm, 长度 ~ 3 厘米) 与聚乙烯油管 (内径280µm, 外径640µm, 长度 ~ 40 厘米) 连接。使用超强力胶水, 密封连接至少三个应用程序。用环氧乙烷消毒注射套管。 注: 当密封油管, 防止超强力胶水进入聚酰亚胺油管, 这可能导致堵塞的套管。密封油管至少三次, 因为气体灭菌可能会导致泄漏的连接。</li>
	<li>使用30克针, 将聚乙烯油管的末端连接到含有生理盐水的1毫升注射器。用1毫升注射器用生理盐水填充套管, 检查聚酰亚胺油管和聚乙烯油管的连接是否有渗漏或堵塞。 注: 如果连接有泄漏或堵塞, 则不能在以下过程中使用套管。</li>
	<li>疏散10µL 微注射器与正常生理盐水, 并连接到上述30克针与注射套管。在微注射泵上安装微型注射器 (图 1B)。将注射速度设置为0.5 µL/分钟, 体积为1µL, 用于快速绿色染料和 AAV8-GFP, 或2µL 为链霉素。 注: 推荐的注塑速度范围为0.1−0.5 µL/分钟, 推荐的注射容积范围为0.5−2µL。</li>
	<li>将微注射器拉回1µL 并提取注射试剂。在生理盐水和注射试剂之间会形成空气间隙 (图 1C)。</li>
	<li>可以选择用标记笔标记聚乙烯油管上的气液边界位置。这可用于监测注射过程中的试剂流量。</li>
</ol>2. 成年小鼠的 Canalostomy
<ol><li>麻醉一只成年老鼠 (女性, FVB/N, 5 至6周) 通过腹腔注射氯胺酮盐酸盐 (120 毫克/千克) 和甲苯噻嗪盐酸 (7 毫克/千克)。等待5−10分钟, 直到动物对疼痛刺激 (脚趾捏反射) 没有反应。麻醉后将动物放在预热过的电垫上。镇痛, Meloxicam (1 毫克/千克), 在手术前皮下应用。</li>
	<li>用眼膏遮住动物的眼睛。用电动动物剪刀将左后耳区剃掉, 用75% 乙醇对皮肤进行三次消毒。</li>
	<li>把动物放在一个预热的电垫上。将电垫的温度设置为 ~ 37°c。将动物放置在右侧的侧位, 以促进左耳的手术。</li>
	<li>从左耳后凹槽 (图2A) 中发出1-1 英寸5厘米的耳后切口 ~ 3 毫米。</li>
	<li>定位后半圆管 (PSC) 和侧半圆管 (): 当耳廓的根 (图 2B中的粗点) 被定义为原点和平面平行于颅骨为3到9点时, PSC 和位于3毫米从耳廓的根在2和3点之间 (图 2B)。</li>
	<li>坦率地解剖用微钳覆盖颞骨的肌肉, 以暴露 PSC 和边缘, 在颞骨 (图 2C) 中, 其边距明显可见为深色条纹。在与颅骨平行的平面上, 所测得的距离约为30°角, PSC 垂直于该 (图 2C)。用微钳收集一小块肌肉, 让它干。在接下来的步骤中, 这种肌肉将被用来密封孔。 注意: 避免过度撕裂组织, 以尽量减少对肌肉的伤害。在暴露 PSC 时避免损坏相邻的容器 (图 2D)。</li>
	<li>使用 26 G 针 (图 2D) 在 PSC 的中间部分制作一个小孔。通过该孔的流体泄漏表明 PSC 的骨壁成功穿透。将孔放大到适当的尺寸, 稍大于聚酰亚胺管的直径。 注: 钻头轻轻地加大钻孔, 避免 PSC 断裂。</li>
	<li>使用棉花丸清洁 PSC 孔周围的积液。</li>
	<li>将聚酰亚胺油管的尖端轻轻插入 PSC, 向1−2毫米 (图 2E) 的深度。按泵上的 "运行" 按钮开始注射。</li>
	<li>注射后, 等待2分钟, 让试剂传播。用微剪刀修剪步骤2.6 中收集的小块肌肉。接下来, 取出注射套管, 立即将肌肉放到 PSC 的孔中。 注: 在堵塞后检查孔内是否有液体泄漏, 以确保孔完全密封。</li>
	<li>返回分离的肌肉和皮下组织。用5-0 缝线缝合切口。用聚维酮碘对切口区域进行消毒。 注: 以上手术步骤约25分钟。</li>
	<li>将动物放置在电垫上预热至摄氏37摄氏度。将动物置于正确的侧向位置以进行恢复。</li>
	<li>在手术后一周执行听觉脑干反应 (ABR) 测量和游泳测试18。</li>
	<li>如果需要, 在 PSC 的不同区域执行重复注射。</li>
</ol>3. 新生小鼠的 Canalostomy
<ol><li>使用低温诱导和维持新生鼠 (FVB/N) 产后1至 2 (P1-2) 的镇静。把小狗放在被压碎的冰层覆盖的塑料床上4分钟。接下来, 把小狗放在一个充满冰的平台上。用75% 乙醇消毒手术场三次。 注意: 确保幼崽的头部脱离冰层。完成整个手术与幼犬在一个充满冰的平台上。</li>
	<li>与成年小鼠相比, 新生儿的以下步骤有所不同。<ol>
<li>使3毫米耳后切口从2毫米后到耳折痕 (图3A -B)。</li>
		<li>使用26克针轻轻地在软 PSC 中打开一个开口。将套管插入 PSC 而不扩大打开 (图 3C -E)。</li>
		<li>注射后, 使用一块肌肉覆盖, 而不是插入, 因为后者可能导致软 PSC 的断裂 (图 3F)。</li>
		<li>用6-0 缝合线关闭皮肤。</li>
		<li>在 P3030上执行 ABR 测量和游泳测试。</li>
	</ol>
</li>
	<li>父母共食是新生儿手术后常见的问题。以下步骤减少了父母同类相食的可能性。<ol>
<li>手术后用酒精湿巾清理切口周围的血液。</li>
		<li>确保新生儿在返回大坝之前可以自由移动。</li>
		<li>把婴儿从母亲的笼子里弄脏的被褥涂上, 把新生儿放回垃圾的中间。</li>
		<li>任何不接受手术的人都应接受类似的耳后切口和缝合。</li>
		<li>把雄性从饲养笼中分离出来。</li>
	</ol>
</li>
</ol>

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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=siRNA+targeting+Hes5+augments+hair+cell+regeneration+in+aminoglycoside-damaged+mouse+utricle.">siRNA targeting Hes5 augments hair cell regeneration in aminoglycoside-damaged mouse utricle.</a> Mol Ther. 21 (4), 834-841 (2013).</li><li>Kamiya, K., 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=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> Am J Pathol. 171 (1), 214-226 (2007).</li><li>Pfannenstiel, S. C., Praetorius, M., Plinkert, P. K., Brough, D. E., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+gene+therapy+prevents+aminoglycoside-induced+degeneration+of+auditory+and+vestibular+hair+cells.">Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.</a> Audiol Neurootol. 14 (4), 254-266 (2009).</li><li>Kawamoto, K., Izumikawa, M., Beyer, L. A., Atkin, G. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+hair+cell+regeneration+in+the+mouse+utricle+following+gentamicin+ototoxicity.">Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.</a> Hear Res. 247 (1), 17-26 (2009).</li><li>Wang, G. 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不声明利益冲突。

在本研究中, 我们表明 canalostomy 的药物传递导致了整个耳蜗和前庭端器官的试剂的广泛分布。作为内耳基因传递方法, canalostomy 导致在成年和新生小鼠内耳内的 GFP 表达, 对听力和前庭功能的损伤最小。此外, 在同一动物中可以很容易地进行多次注射。
canalostomy 最大的优点之一是它会对内耳功能造成最小的损害, 尤其是成年小鼠16,18,28,29,30, 这对于评估保护效果和翻译方面。几个小组使用 canalostomy 提供各种试剂, 如病毒载体, siRNA, 干细胞和氨基糖苷, 进入小鼠内耳18,19,28,29, 34,35,36,37。在目前的研究中, 我们描述了一步一步, 详细的手术技术 canalostomy 在成年和新生小鼠。与以前的研究相比, 我们的研究提供了关于半圆形运河定位的更多细节, 这是成功手术的关键步骤。如图 2B和 C 中所示,在2-3 点, PSC 和耳廓通常位于3毫米的根上。在与颅骨平行的平面上, 该离轴的距离约为30°角, PSC 垂直于该线。此外, 我们简化了手术程序, 将手术时间缩短到25分钟左右, 结果可比29。
在 canalostomy 期间, 必须避免注射部位渗漏和套管堵塞。在切换泵之前, 重要的是要确保套管的尖端插入到半圆管中, 并且套管没有弯曲或堵塞。建议在封闭成年小鼠半圆管孔时使用干肌, 因为在半圆管液的存在下, 它会膨胀并堵塞孔 (见步骤2.6 和 2.10)。由于新生小鼠半圆管壁柔软易碎, 开口应用自体肌肉或医用胶水覆盖30进行密封。我们也去掉了套管, 不覆盖开口与肌肉;结果显示, 内耳的 AAV 转导效率可比性高 (数据未显示), 表明新生小鼠 PSC 的开放度已令人满意。
canalostomy 的另一个优点是多个过程的可行性, 它允许重复应用相同或不同的试剂 (图 6)。由于在以前的外科手术部位经常发现纤维化和肉芽组织, 而半圆形运河在手术后可能会受阻34, 建议在另一侧的不同区域进行反复手术时进行注射。和/或后半圆运河18。
canalostomy 的主要局限性是在确定注射套管是否插入淋巴或淋巴空间中的难点。在通过 canalostomy 将腺病毒载体管理到内耳16后, 大多数转基因细胞位于淋巴空间, 这表明注射可能在该区域进行。低分子量化合物 (例如, AAV、链霉素和 siRNA) 可以穿过外淋巴液和 endolymph 之间的屏障, 并在注入淋巴或淋巴空间18, 19 中到达感觉上皮. ,34。但是, 在注入外淋巴液后, 如果无法穿透外淋巴液和 endolymph 16、39、40之间的屏障, 则试剂可能会留在淋巴空间中.因此, 在使用 canalostomy 前应考虑注入试剂的渗透性。
总之, canalostomy 结果在耳蜗和前庭的试剂广泛分布, 听力和前庭功能的干扰最小。这项手术很容易在成年和新生小鼠中实施, 如果需要, 可以执行多个程序。Canalostomy 是一种有效和安全的方法, 以药物传递到啮齿目动物的内耳, 并可能在将来用于临床治疗人类 cochleovestibular 疾病。

感音性听力丧失和前庭功能障碍影响大量患者, 并与内耳紊乱密切相关。将治疗药物送进内耳, 对内耳疾病的治疗有一定的保证。系统的或局部的方法可以用来向内耳输送药物。一些内耳疾病成功地治疗与系统性药物管理, 如特发性突然听力损失, 这是通常治疗全身类固醇1。此外, 蓝兹et 等表明, 系统管理反义寡核苷酸能够改善听觉和平衡功能的 Ush1c 突变鼠模型2。然而, 由于血液迷宫障碍, 导致内耳疾病的很大一部分无法有效地治疗, 因为它限制了对内耳的药物访问3,4。相反, 局部药物传递策略可以更有效地治疗内耳疾病。事实上, 内耳可能是当地药物传递的理想目标;它充满了液体, 这有助于药物的传播后, 一个站点的扩散或注射, 它是相对孤立的邻近器官, 这限制副作用5,6。
当地的药物传递战略包括鼓室和 intralabyrinthine 方法。鼓室路线的有效性主要依靠药物渗透性通过圆的窗口膜 (RWM) 和药物的停留时间在 RWM3,4,7,8。因此, 它不适合提供不能穿透 RWM 的药物或试剂。Intralabyrinthine 方法包括直接将药物接种到内耳内, 导致高剂量和广泛分布。然而, intralabyrinthine 方法需要精细的手术和侵入, 导致内耳功能受损。目前, intralabyrinthine 注射药物仅用于动物研究, 因为它没有被证明是足够安全的用于人类的9。因此, 必须简化手术程序, 并减少受伤的风险, 将 intralabyrinthine 方法转化为临床。
通过 RWM5、10、11并进入 scala 媒体12、13、14、scala 索, 在动物中进行了几种 intralabyrinthine 方法的评估15,16、scala 前庭、17、半圆运河16、18、19、20和淋巴 sac21。每种方法都有优缺点6。通过 RWM 传递的是非创伤性的新生老鼠5, 22.然而, 在成年小鼠 RWM 注射后出现轻度听力减退23, 可能是由于手术后的中耳积液24。Scala 介质注射, 包括直接注射试剂进入含有感官上皮的淋巴空间, 达到目标端器官的高试剂浓度12,14,25,26. 但是, 这种方法需要一个复杂的过程, 并在比产后 5 (P5)25、27(限制其应用程序) 的后期执行时, 导致听力阈值显著升高。
与上述 intralabyrinthine 方法相比, canalostomy 会对内耳造成最小的损伤, 尤其是成年小鼠16,18,28,29,30, 这是对评估保护效果和平移方面很重要。此外, 在啮齿类动物中, 半圆形的运河位于泡之外, 这有利于外科手术, 避免了中耳在手术过程中的干扰。在临床上, 半圆形运河手术用于顽固性良性阵发性位置眩晕31,32,33, 提示 canalostomy 的临床可行性。因为它最初是由 Kawamoto et描述的。16 2001年, canalostomy 已被用来提供各种试剂, 如病毒载体, siRNA, 干细胞和氨基糖苷, 进入小鼠内耳18,19,28,29 ,34,35,36,37。canalostomy 腺相关病毒 (AAV) 载体的接种使耳蜗和前庭末端器官的感觉上皮和原发神经元的外源基因表达18,28, 29,30。Whirlin 基因治疗的 canalostomy 恢复平衡功能和改善听力的小鼠模型的人引种综合征19, 表明 canalostomy 是有用的基因治疗的遗传 cochleovestibular 疾病的研究。canalostomy 骨髓间充质干细胞移植治疗急性感音性聋大鼠耳蜗纤维细胞和听力恢复的重组研究 35.此外, canalostomy 可用于将氨基糖苷类引入内耳, 以建立前庭病变18, 34, 38, 如果需要, 可以执行多个注射18,34。
在本篇文章中, 我们详细描述了 canalostomy 技术在成年和新生小鼠。我们接种了各种试剂, 包括快速绿色染料和 AAV 血清 8 (AAV8), 连同绿色荧光蛋白 (GFP) 基因 (AAV8-GFP) 和链霉素, 进入小鼠内耳, 以评估直接和长期的结果后 canalostomy。

<table>
	<tbody>
		<tr>
			<td>Polymide Tubing</td>
			<td>A-M Systems</td>
			<td>823400</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing</td>
			<td>Scientific Commodities Inc.</td>
			<td>BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#956;l Microsyringe</td>
			<td>Hamilton Company</td>
			<td>80001</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine HCL</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>X-1251</td>
			<td></td>
		</tr>
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			<td>Operating Miroscope</td>
			<td>Carl Zeiss Optical LLC.</td>
			<td>Pico</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Forceps</td>
			<td>Dumont Dumostar</td>
			<td>10576</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-green Dye</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV8-GFP</td>
			<td>BioMiao Biological Technology Co. Ltd (Beijing, China)</td>
			<td>20161101</td>
			<td>Titer: 2&#215;10e12 vg/mL</td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate</td>
			<td>Sigma-Aldrich Co. Llc.</td>
			<td>S9137</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection Pump</td>
			<td>Stoelting Co.</td>
			<td>789100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric Pad</td>
			<td>Pet Fun</td>
			<td>11072931136</td>
			<td></td>
		</tr>
		<tr>
			<td>1 cc Syringe</td>
			<td>Mishawa Medical Industries Ltd. (Shanghai, China)</td>
			<td>2011-3151258</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCL</td>
			<td>Gutian Pharmaceutical Co., Ltd. (Fujian, China)</td>
			<td>H35020148</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric Animal Clipper</td>
			<td>Codos Electrical Appliances Co., Ltd. (Guangdong, China)</td>
			<td>CP-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pellet</td>
			<td>Yatai Healthcare Ltd. (Henan, China)</td>
			<td>Yu-2008-1640081</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China)</td>
			<td>Hu-2013-2650207</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Beijing Shuangji Pharmaceutical Ltd. (Beijng China)</td>
			<td>H11021270</td>
			<td></td>
		</tr>
	</tbody>
</table>

canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice

局部向内耳提供治疗药物是治疗内耳疾病的一种有前景的疗法。通过半圆渠道注射 (canalostomy) 已被证明是一种有用的方法, 以当地药物传递到内耳。本文的目的是详细描述 canalostomy 在成年和新生小鼠中所涉及的外科手术技术。正如绿色荧光蛋白基因的快速绿色染料和腺相关病毒8型所示, canalostomy 促进了在耳蜗和前庭端器官中广泛分布的注射试剂, 对听力的损伤最小,前庭功能。成功地实现了成人和新生小鼠的手术;事实上, 如果需要, 可以执行多项手术。总之, canalostomy 是一种有效、安全的方法, 将药物传递到成年和新生小鼠的内耳, 并可用于治疗人内耳疾病的未来。

将快速绿色染料注入成人和新生小鼠的 PSC 中, 以评估其在内耳中的即刻分布。在手术后立即检测到整个耳蜗、前庭和半圆形运河的染料 (图 4)。
为评价 canalostomy 对内耳基因传递的安全性和有效性, AAV8-GFP 在成年和新生小鼠内耳内注射。所有动物在 AAV8-GFP 注射后显示正常的 ABR 阈值和游泳测试分数18,30。Immunohistology 透露, 在听觉感觉上皮, 内毛细胞 (IHCs) 和一些外毛细胞 (OHCs) 的基底轮显示健壮的 GFP 表达式30。在前庭感觉上皮中, 在囊、囊和 ampullae30中检测到 GFP 表达式。前庭毛细胞 (图 5) 和支持单元格均转基因高效1830。
在囊18,34,38中, 通过 canalostomy 注入链霉素会导致严重的毛细胞丢失。为了检验通过 canalostomy 进行多次注射的可行性, 在7天后通过 PSC 通过 AAV8-GFP 悬浮剂, 小鼠被给予链霉素。本实验还旨在评价损伤囊中 AAV8-GFP 的转导特性, 对于 HC 损失后内耳损伤的基因治疗研究具有重要意义18。GFP 分布在整个损伤囊 (图 6) 中。许多 GFP 阳性细胞为肌球蛋白 VIIA 阴性, 表明它们是转基因。一些 HCs (肌球蛋白 VIIa 阳性细胞) 与未成熟的发束也表达了 GFP。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57351/57351fig1.jpg"> 图 1: 设备准备。(A)聚酰亚胺油管 (i) 和聚乙烯管材 (ii) 密封以制造注射套管 (iii)。(B)套管连接到10µL 微型注射器, 用30克针, 然后安装在泵上。箭头指示套管尖端。(C)注射试剂和生理盐水由空气间隙隔开。<a href="/files/ftp_upload/57351/57351fig1large.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/57351/57351fig2.jpg"> 图 2: 成人鼠标中的 Canalostomy。(a)耳后切口 (箭头)。(B)覆盖颞骨的肌肉暴露。如果我们将耳廓的根 (粗点) 定义为起源和平行于颅骨的平面为3/9 点, 则后半圆管 (PSC) 和外侧半圆管 () 通常位于2至3点之间的区域 (五角星)。, 距原点3毫米。插入: 方向的低放大图像。虚线表示颅骨平面。(C)将公开 PSC 和分隔线 (虚线)。嵌入: 在与颅骨平行的平面上, 所测得的距离约为30°角, 而 PSC 垂直于该边距。(D)在 PSC 中进行了一个小孔。(E) 套管尖端插入 PSC, 并注射试剂。(F)孔用一小块肌肉密封。刻度条在 A 是5毫米, 那在 b 是1毫米 (b 为 b F), 那在插入到 b 是 1 cm, 并且那在插入到 C 是5毫米.<a href="/files/ftp_upload/57351/57351fig2large.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/57351/57351fig3.jpg"> 图 3: 新生儿鼠标中的 Canalostomy.(A)耳后切口 (箭头)。(B)覆盖颞骨的肌肉暴露, 后半圆管 (PSC) 从耳廓 (黑体点) 的根部到2-3 点 (五角星) 的2毫米左右。方向与成年小鼠的定位相同 (图 2B)。(C)公开了 PSC 和侧半圆管 (点划线)。(D)在 PSC 中进行小的打开。(E) 套管插入 PSC。(F)一小块肌肉用于覆盖注射后的开口。A 和 B 中的刻度条为5毫米, 即 b 为1毫米, c 为1毫米 (c F)。<a href="/files/ftp_upload/57351/57351fig3large.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/57351/57351fig4.jpg"> 图 4: 成人 (a) 和新生小鼠内耳的立体显微图像 (b-b) 通过 canalostomy 管理快速绿色染料.手术后立即采集标本。(A 和 B)颅外表面。(A ' 和 B ')颅内表面。快速绿色染料分布在耳蜗、前庭和半圆形运河中。刻度条是1毫米 (a ' 为 a ' 和 b ' 为 b ')。PSC, 后半圆管。外侧半圆管。SSC, 优越的半圆形运河。<a href="/files/ftp_upload/57351/57351fig4large.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/57351/57351fig5.jpg"> 图 5: 成年小鼠耳蜗 (a a) 和囊 (b b) 的整体装入的代表共焦图像, 在 AAV8-GFP 注射液通过 canalostomy 后30天内制备.样品染色荧光蛋白 (绿色) 和肌球蛋白 VIIa (红色) 的抗体。(a a)GFP 在大多数内毛细胞 (IHC) 中表达。(b b)在囊表皮板的焦平面上, 许多毛细胞表达 GFP (箭头)。绿色荧光蛋白。OHC, 外毛细胞。刻度条为25µm (a a a, b "为 b b")。<a href="/files/ftp_upload/57351/57351fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57351/57351fig6.jpg"> 图 6: 在 AAV8-GFP 注射液通过 canalostomy 后30天获得外伤性囊的代表性共焦图像.一只成年小鼠通过外侧半规管注射链霉素, 7 天后通过半圆形导管注射 AAV8-GFP。GFP (绿色), 肌球蛋白 VIIa (红色), 和肌动蛋白 (蓝色)。箭头表示有代表性的支持细胞转基因与 AAV8-GFP (gfp +/肌球蛋白 VIIa 细胞), 和箭头表示有代表性的毛细胞转基因与 AAV8-GFP (gfp +/肌球蛋白 VIIa + 细胞) 与未成熟的发束。刻度条为20µm (d)。<a href="/files/ftp_upload/57351/57351fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice at JoVE.com

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

在这里, 我们描述了 canalostomy 程序, 允许当地药物传递到成年和新生小鼠的内耳通过半圆形运河, 对听力和前庭功能的最小损害。该方法可用于将病毒载体、药物和小分子接种到小鼠内耳内。

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

canalostomy-as-surgical-approach-to-local-drug-delivery-into-inner

Nanyang Technological University

Research

JoVE Journal

Genetics

10.6K 次观看.  Capital Medical University. 在这里, 我们描述了 canalostomy 程序, 允许当地药物传递到成年和新生小鼠的内耳通过半圆形运河, 对听力和前庭功能的最小损害。该方法可用于将病毒载体、药物和小分子接种到小鼠内耳内。

视频: Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...