Name: 作者聚焦：提取豚鼠圆窗膜促进内耳药物递送研究
Uploaded: 2024-02-23T13:20:00
Description: Watch this Scientific Journal Video about Author Spotlight: Extraction of Guinea Pig Round Window Membrane to Facilitate Inner Ear Drug Delivery Research at JoVE.com

这项工作得到了NIDCD拨款编号1K08DC020780和5T32DC000027-33以及鲁宾斯坦听力研究基金的部分支持。

所有动物程序均由机构动物护理和使用委员会 （GP18M226） 批准。本研究使用了哈特利白化豚鼠（雄性和雌性，体重 500-700 克）。
1. 程序设置和准备
<ol><li>在开始实验之前，用环氧乙烷对所有仪器进行灭菌。</li>
	<li>按照机构批准的方案对动物实施安乐死。 注意：在目前的研究中，采用非预充电室从商用气瓶中释放 100% 二氧化碳 （CO2）。根据 2020 年 AV...

豚鼠圆窗膜种植

<ol>
	<li>Duan, M. I., Zhi-qiang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+round+window+membrane+and+its+role+for+drug+delivery:+our+own+findings+and+literature+review.">Permeability of round window membrane and its role for drug delivery: our own findings and literature review.</a> J Otol. 4 (1), 34-43 (2009).</li><li>Kelso, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microperforations+significantly+enhance+diffusion+across+round+window+membrane.">Microperforations significantly enhance diffusion across round window membrane.</a> Otol Neurotol. 36 (4), 694-700 (2015).</li><li>Salt, A. N., Plontke, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+principles+in+the+inner+ear:+Influence+of+drug+properties+on+intratympanic+applications.">Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications.</a> Hear Res. 368, 28-40 (2018).</li><li>Szeto, B., 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=Inner+ear+delivery:+Challenges+and+opportunities.">Inner ear delivery: Challenges and opportunities.</a> Laryngoscope Investig Otolaryngol. 5 (1), 122-131 (2020).</li><li>Carpenter, A. M., Muchow, D., Goycoolea, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+the+human+round+window+membrane.">Ultrastructural studies of the human round window membrane.</a> Arch Otolaryngol Head Neck Surg. 115 (5), 585-590 (1989).</li><li>Forouzandeh, F., Borkholder, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtechnologies+for+inner+ear+drug+delivery.">Microtechnologies for inner ear drug delivery.</a> Curr Opin Otolaryngol Head Neck Surg. 28 (5), 323-328 (2020).</li><li>Leong, S., 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=Microneedles+facilitate+small-volume+intracochlear+delivery+without+physiologic+injury+in+guinea+pigs.">Microneedles facilitate small-volume intracochlear delivery without physiologic injury in guinea pigs.</a> Otol Neurotol. 44 (5), 513-519 (2023).</li><li>Singh, R., Birru, B., Veit, J. G. S., Arrigali, E. M., Serban, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+in+vitro+round+window+membrane+model+for+drug+permeability+evaluations.">Development and characterization of an in vitro round window membrane model for drug permeability evaluations.</a> Pharmaceuticals (Basel). 15 (9), 1105 (2022).</li><li>Du, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+targeted+delivery+of+dexamethasone+acetate+across+the+round+window+membrane+in+guinea+pigs.">Magnetic targeted delivery of dexamethasone acetate across the round window membrane in guinea pigs.</a> Otol Neurotol. 34 (1), 41-47 (2013).</li><li>Kopke, R. D., 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=Magnetic+nanoparticles:+inner+ear+targeted+molecule+delivery+and+middle+ear+implant.">Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant.</a> Audiol Neurootol. 11 (2), 123-133 (2006).</li><li>AVMA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AVMA+Guidelines+for+the+Euthanasia+of+Animals:+2020+Edition.">AVMA Guidelines for the Euthanasia of Animals: 2020 Edition.</a> AVMA. , (2020).</li><li>Goksu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+guinea+pig+temporal+bone.">Anatomy of the guinea pig temporal bone.</a> Ann Otolaryngol. 101 (8), 699-704 (1992).</li><li>Wysocki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+anatomy+of+the+guinea+pig+temporal+bone.">Topographical anatomy of the guinea pig temporal bone.</a> Hear Res. 199 (1), 103-110 (2005).</li><li>Veit, J. G. S., 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=An+evaluation+of+the+drug+permeability+properties+of+human+cadaveric+in+situ+tympanic+and+round+window+membranes.">An evaluation of the drug permeability properties of human cadaveric in situ tympanic and round window membranes.</a> Pharmaceuticals (Basel). 15 (9), 1037 (2022).</li><li>Kansara, V., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+ex+vivo+model+implication+for+carrier-mediated+retinal+drug+delivery).">Evaluation of an ex vivo model implication for carrier-mediated retinal drug delivery).</a> Curr Eye Res. 31 (5), 415-426 (2006).</li><li>Lundman, L., Bagger-Sj&#246;b&#228;ck, D., Holmquist, L., Juhn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+window+membrane+permeability.+An+in+vitro+model.">Round window membrane permeability. An in vitro model.</a> Acta Otolaryngol Suppl. 457, 73-77 (1989).</li><li>Moatti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+drug+permeability+through+an+ex+vivo+porcine+round+window+membrane+model.">Assessment of drug permeability through an ex vivo porcine round window membrane model.</a> iScience. 26 (6), 106789 (2023).</li><li>Lin, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+microbubble-facilitated+inner+ear+delivery+of+gold+nanoparticles+involves+transient+disruption+of+the+tight+junction+barrier+in+the+round+window+membrane.">Ultrasound microbubble-facilitated inner ear delivery of gold nanoparticles involves transient disruption of the tight junction barrier in the round window membrane.</a> Front Pharmacol. 12, 689032 (2021).</li><li>Jeong, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctional+modulation+of+round+window+membrane+enhances+dexamethasone+uptake+into+the+inner+ear+and+recovery+after+NIHL.">Junctional modulation of round window membrane enhances dexamethasone uptake into the inner ear and recovery after NIHL.</a> Int J Mol Sci. 22 (18), 10061 (2021).</li></ol>

在局部药物递送至耳部时，RWM 是治疗药物到达内耳的主要途径。需要一个准确可靠的台式模型来更好地了解新型递送载体和药物开发的运输机制和渗透性。在这项研究中，我们证明了豚鼠 RWM 移植是一种可行且可靠的程序，可以对药物-膜相互作用进行系统研究。Lundman 等人和 Kelso 等人先前使用类似的 RWM 渗透率模型 2,16 进行了描述;然而，到目前为止，?...

已经出现了用于治疗感音神经性听力损失的新型疗法。这些疗法在临床人群的转化受到安全有效的内耳运输途径的限制。目前在动物研究中 的体内 递送方法依赖于开窗进入内耳或通过圆窗膜 （RWM） 扩散，圆窗膜是一种将中耳间隙与耳蜗分开的非骨屏障1。
手术开窗和显微注射到内耳都是侵入性的，可能会对残余内耳功能构成风险2.因此，RWM 是局部药物递送的重要途径，豚鼠是主要的临床前动物模型，用于研究 RWM 和内耳的局部药物药代动力学，用于药物开发 3,4。虽然比人类RWM更薄，但豚鼠RWM具有相同的三层结构。它的直径约为 1 mm，厚度为 15-25 μm，由夹在结缔组织层5 中的两个上皮细胞层组成。面向中耳的上皮层密集堆积，并通过紧密的连接连接，而面向内耳和鳞状鼓膜的层结构较松，没有明显的细胞间粘连。
目前调查豚鼠 RWM 药物渗透性的临床前研究依赖于体内中耳注射，然后对内耳内的外淋巴液进行采样，这不允许对 RWM 转运进行特异性研究 6,7。RWM 外植体的片段已用于临床前研究，但由于其脆弱性和小尺寸，它们不适合对需要跨 RWM2 的防水密封的药物和载体运输进行系统的微流体研究。其他研究小组使用培养的人上皮细胞的体外模型来近似RWM 8,9,10。然而，这些结构中的大多数只关注外上皮层，并没有捕捉到天然组织结构的复杂性。为了更详细地了解RWM的转运机制，需要有针对性的离体研究。
在这项研究中，我们展示了豚鼠 RWM 的移植与周围的骨支撑以保持膜完整性，并说明了它们在专为特定研究药物递送载体的 RWM 运输而设计的实验范式中的使用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mm Diamond Ball Drill Bit</td>
			<td>Anspach</td>
			<td>1SD-G1</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mm Diamond Ball Drill Bit</td>
			<td>Anspach</td>
			<td>2SD-G1</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm Diamond Ball Drill Bit</td>
			<td>Anspach</td>
			<td>6D-G1</td>
			<td></td>
		</tr>
		<tr>
			<td>ANSPACH EMAX 2 Plus System</td>
			<td>Anspach</td>
			<td>EMAX2PLUS</td>
			<td>Any bone cutting drilling system will work</td>
		</tr>
		<tr>
			<td>BD Eclipse Needle 27 G x 1/2 in. with detachable 1 mL BD Luer-Lok Syringe</td>
			<td>Becton, Dickinson, and Co.&#160;</td>
			<td>382903057894</td>
			<td>Any 27-28 G needle</td>
		</tr>
		<tr>
			<td>Gorilla Epoxy</td>
			<td>Gorilla</td>
			<td>4200101</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik-CAST</td>
			<td>World Precision Instruments</td>
			<td>KWIK-CAST</td>
			<td></td>
		</tr>
	</tbody>
</table>

guinea pig round window membrane explantation for ex vivo studies

高效和微创的药物输送到内耳是一项重大挑战。圆窗膜（RWM）是内耳为数不多的入口点之一，已成为研究的重要焦点。然而，由于分离RWM的复杂性，我们对其药代动力学的理解仍然有限。RWM 由三个不同的层组成：外上皮层、中间结缔组织层和内上皮层，每个层都可能具有独特的递送特性。
目前用于研究跨 RWM 转运的模型利用依赖于细胞培养物或膜片段的体内动物模型或离体 RWM 模型。豚鼠是研究内耳内药物药代动力学的经过验证的临床前模型，也是耳蜗递送载体转化开发的重要动物模型。在这项研究中，我们描述了一种移植具有周围耳蜗骨的豚鼠 RWM 用于台式药物递送实验的方法。这种方法允许保留原生 RWM 架构，并且可以提供比当前台式模型更真实的运输障碍表示。

Novel classes of therapeutics have emerged for the treatment of sensorineural hearing loss. The translation of these therapeutics to clinical populations is limited by safe and efficacious routes of transport into the inner ear. Current methods of in vivo delivery in animal studies rely on either fenestration into the inner ear or diffusion through the round window membrane (RWM), a non-osseous barrier that separates the middle ear space from the cochlea1.
Surgical fenestration and microinjection into the inner ear are both invasive and can pose risks to residual inner ear function2. Therefore, the RWM is an important route for local drug delivery, and guinea pigs are the primary preclinical animal model used to study local drug pharmacokinetics across the RWM and in the inner ear for pharmaceutical development3,4. Although thinner than the human RWM, the guinea pig RWM shares an identical three-layered structure. It is approximately 1 mm in diameter, 15-25 &#181;m thick, and comprised of two epithelial cell layers sandwiching a connective tissue layer5. The epithelial layer facing the middle ear is densely packed and connected via tight junctions, while the layer facing the inner ear and scala tympani has looser architecture and does not have significant intercellular adhesions.
Current pre-clinical studies investigating drug permeability in the guinea pig RWM rely on in vivo middle ear injections followed by the sampling of the perilymph fluid within the inner ear, which does not allow for the specific study of RWM transport6,7. Fragments of RWM explants have been used in preclinical studies, but due to their fragility and small size, they are not suitable for systematic, microfluidic investigations of drug and vehicle transport requiring a watertight seal across the RWM2. Other groups have employed in vitro models with cultured human epithelial cells to approximate the RWM8,9,10. However, the majority of these constructs focus solely on the outer epithelial layer and do not capture the complexity of native tissue architecture. For a more detailed understanding of transport mechanisms across the RWM, targeted, ex vivo studies are required.
In this study, we demonstrate the explantation of a guinea pig RWM with surrounding bony support to preserve membrane integrity and illustrate their use in an experimental paradigm designed for the specific study of the RWM transport of drug delivery vehicles.

作者聚焦：提取豚鼠圆窗膜促进内耳药物递送研究

用于体外研究的豚鼠圆窗膜移植

Our group is investigating drugs and drug delivery vehicles for the treatment of inner ear diseases such as hearing loss. The round window membrane is the critical barrier that prevents locally applied drugs from getting into the cochlea and we're currently working to understand how nanoparticle delivery vehicles can be engineered to pass through this barrier. Understanding how the round window membrane works in terms of drug transport is crucial in developing effective drug delivery strategies to the ear.Guinea pig is an important preclinical animal model for studying drug delivery to the ear. However, isolating this fragile membrane, which is only 17 microns thick, for drug transport studies remains a significant challenge. Here, we describe a method for extracting the guinea pig round window membrane that allows for efficient bench top drug transport studies.Current investigations of drug delivery to the inner year rely primarily on in vivo experimental models followed by perilymph sampling which do not allow for a specific and targeted study of round window membrane transport mechanisms. In this study, we demonstrate the extraction of the guinea pig round window membrane with surrounding bony support. This method preserves native tissue architecture integrity and allows for precise and targeted studies of drug delivery across the round window membrane.

如 图3A所示，该方法允许移植完整的豚鼠圆窗膜，并带有周围的刚性骨环。RWM 应在圆周上与骨环完全连接。不应意识到耳蜗骨折。与人类圆窗标本相比，豚鼠RWM没有覆盖的假膜。此外，与人类不同的是，豚鼠镫骨的骨桥之间有一座骨桥，需要在提取镫骨上部结构之前将其压裂和移除。代表性RWM的组织学分析（图3B）显示清晰的三层上皮结构，具有相邻?...

Watch this Scientific Journal Video about Author Spotlight: Extraction of Guinea Pig Round Window Membrane to Facilitate Inner Ear Drug Delivery Research at JoVE.com

该协议概述了一种从豚鼠颞骨中移植圆窗膜的方法，为 离体 研究提供了宝贵的资源。

Efficient and minimally invasive drug delivery to the inner ear is a significant challenge. The round window membrane (RWM), being one of the few entry ...

我们小组正在研究用于治疗听力损失等内耳疾病的药物和药物输送载体。圆窗膜是防止局部应用的药物进入耳蜗的关键屏障，我们目前正在努力了解如何设计纳米颗粒递送载体以通过这一屏障。了解圆窗膜在药物转运方面的工作原理对于制定有效的药物递送策略至关重要。豚鼠是研究药物递送至耳部的重要临床前动物模型。然而，分离这种只有17微米厚的脆弱膜用于药物转运研究仍然是一个重大挑战。在这里，我们描述了一种提取豚鼠圆窗膜的方法，该方法允许有效的台式药物转运研究。目前对药物递送至内年的研究主要依赖于体内实验模型，然后是外淋巴取样，这不允许对圆窗膜转运机制进行特异性和有针对性的研究。在这项研究中，我们演示了具有周围骨支撑的豚鼠圆窗膜的提取。该方法保留了天然组织结构的完整性，并允许对跨圆窗膜的药物递送进行精确和有针对性的研究。

guinea-pig-round-window-membrane-explantation-for-ex-vivo-studies

Research

JoVE Journal

Biology

Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies

Efficient and minimally invasive drug delivery to the inner ear is a significant challenge. The round window membrane (RWM), being one of the few entry points to the inner ear, has become a vital focus of investigation. However, due to the complexities of isolating the RWM, our understanding of its pharmacokinetics remains limited. The RWM comprises three distinct layers: the outer epithelium, the middle connective tissue layer, and the inner epithelial layer, each potentially possessing unique delivery properties.
Current models for investigating transport across the RWM utilize in vivo animal models or ex vivo RWM models which rely on cell cultures or membrane fragments. Guinea pigs serve as a validated preclinical model for the investigation of drug pharmacokinetics within the inner ear and are an important animal model for the translational development of delivery vehicles to the cochlea. In this study, we describe an approach for explantation of a guinea pig RWM with surrounding cochlear bone for benchtop drug delivery experiments. This method allows for preservation of native RWM architecture and may provide a more realistic representation of barriers to transport than current benchtop models.

This protocol outlines a method for the explantation of the round window membrane from guinea pig temporal bones, providing a valuable resource for ex vivo studies.