Name: canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice
Uploaded: 2018-05-25T13:00:00.000+00:00
Duration: 9 min 34 s
Description: 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

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. P., 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=Notch+signaling+and+Atoh1+expression+during+hair+cell+regeneration+in+the+mouse+utricle.">Notch signaling and Atoh1 expression during hair cell regeneration in the mouse utricle.</a> Hear Res. 267 (1-2), 61-70 (2010).</li><li>Pietola, L., 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=HOX-GFP+and+WOX-GFP+lentivirus+vectors+for+inner+ear+gene+transfer.">HOX-GFP and WOX-GFP lentivirus vectors for inner ear gene transfer.</a> Acta Otolaryngol. 128 (6), 613-620 (2008).</li><li>Han, J. J., 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=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li></ol>

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<su...

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&amp;mu;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><tr><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&amp;times;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<sup class=...

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

Research

JoVE Journal

Genetics

10.7K 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 ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

异位黏膜移植程序直接给药到小鼠的大脑

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

医学

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

神经科学

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

后半圆管法在新生小鼠内耳基因传递中的研究

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

针对鼠标内耳给药方法的比较研究:Bullostomy<em&gt;对战</em&gt;鼓室注射

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

生物学

Canalostomy for Local Drug Delivery into the Inner Ear of an Adult Mouse

Source: Guo, J. et al., <a href="https://app.jove.com/v/57351">Canalostomy as a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice.</a>&#160;J. Vis. Exp. (2018)
This video showcases canalostomy, a procedure that allows local drug delivery into the inner ear of a mouse through the semicircular canal with minimal damage to hearing and vestibular function. This method enables the inoculation of viral vectors, pharmaceuticals, and small molecules into the mouse&#39;s inner ear.

耳道造口术用于将局部药物输送到成年小鼠的内耳

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Device ...

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

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