Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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<su…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Polymide Tubing | A-M Systems | 823400 | |
| Polyethylene Tubing | Scientific Commodities Inc. | BB31695-PE/1 | |
| 10μl Microsyringe | Hamilton Company | 80001 | |
| Xylazine HCL | Sigma-Aldrich Co. Llc. | X-1251 | |
| Operating Miroscope | Carl Zeiss Optical LLC. | Pico | |
| Micro Forceps | Dumont Dumostar | 10576 | |
| Fast-green Dye | Sigma-Aldrich Co. Llc. | F7252 | |
| AAV8-GFP | BioMiao Biological Technology Co. Ltd (Beijing, China) | 20161101 | Titer: 2×10e12 vg/mL |
| Streptomycin Sulfate | Sigma-Aldrich Co. Llc. | S9137 | |
| Microinjection Pump | Stoelting Co. | 789100S | |
| Electric Pad | Pet Fun | 11072931136 | |
| 1 cc Syringe | Mishawa Medical Industries Ltd. (Shanghai, China) | 2011-3151258 | |
| Ketamine HCL | Gutian Pharmaceutical Co., Ltd. (Fujian, China) | H35020148 | |
| Electric Animal Clipper | Codos Electrical Appliances Co., Ltd. (Guangdong, China) | CP-8000 | |
| Cotton Pellet | Yatai Healthcare Ltd. (Henan, China) | Yu-2008-1640081 | |
| Suture | Shanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China) | Hu-2013-2650207 | |
| Eye Ointment | Beijing Shuangji Pharmaceutical Ltd. (Beijng China) | H11021270 |
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