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 AVMA指南 11，使用在线限流器调节气体流量，将其保持在每分钟腔室体积的 30% 至 70% 范围内。</li>
	<li>将动物置于腔室中并分配二氧化碳5分钟，呼吸停止后保持CO2 流动1分钟。</li>
	<li>呼吸骤停后进行斩首，以确保安乐死。</li>
</ol>2. 手术入路和移植
<ol><li>以通常的方式从豚鼠头骨中提取颞骨12.用 rongeur 去除多余的软组织。识别外耳道、颞大疱和面管13 （图 1）。</li>
	<li>用 6 mm 的金刚石钻头钻掉颞大疱的腹侧（见 材料表），圆周暴露中耳间隙和外耳道。</li>
	<li>使用rongeurs，轻轻去除外耳道和鼓室环，同时分离incudomalleolar关节。识别砧骨、耳蜗关节、耳蜗、水平半规管和面管13 （图 2A）。</li>
	<li>分离镊子关节并用镊子取出砧骨。识别圆窗的骨质壁龛。</li>
	<li>使用 6 mm 金刚石钻头钻掉连接耳蜗和鼓室内侧壁朝向鼓膜张管的骨层。小心地减压鼓膜张肌的骨通道，并使用 28 G 针去除鼓膜张肌。 注意：鼓膜张肌窝的内侧壁直接与RWM周围的耳蜗骨相连，注意不要引起可能延伸到圆窗的骨折。</li>
	<li>钻掉连接耳蜗和鼓室下壁的骨层，直到剩下 1 毫米的骨架与耳蜗相邻（图 2B）。</li>
	<li>使用 2 毫米的金刚石钻头（见 材料表），在耳蜗基底处进行人工耳蜗造口术，将大约 2 毫米的骨头留给圆窗。在与圆窗膜平行的平面上继续下方的耳蜗造口术，以将耳蜗的底部与耳蜗的顶点分开。</li>
	<li>将耳蜗造口切口延伸到颅底，颅底密度要大得多，从而获得耳蜗基底转弯的横截面视图。 注意：将钻头对准内耳道的鼻道会产生一个轨迹，最大限度地去除骨头，同时避免过近圆窗。</li>
	<li>从颅底侧检查标本，如果尚未完成，则识别内耳道并钻入耳蜗孔。用 28 G 针取出耳蜗神经。</li>
	<li>从耳蜗侧检查标本。用镊子或 28 G 针识别并去除基底转弯处的骨螺旋层和剩余的 modiolus。</li>
	<li>大量冲洗统一的鳞状鼓膜-鳞状前庭腔以清除碎屑。圆窗应从结肠切除术中清晰可见，没有任何覆盖碎屑（图2C）。</li>
	<li>接下来，从中耳侧检查标本。将外侧半规管和面管钻至椭圆形窗口的水平。用镊子轻轻取下镫骨，露出椭圆形窗龛。值得注意的是，镫骨的嵴之间有一座骨桥，称为镫骨。</li>
	<li>使用 1 毫米的金刚石钻头（见 材料表），通过沿圆窗的表面延伸椭圆形窗口来进一步打开前庭，注意保持 1-2 毫米的耳蜗骨与圆窗壁龛相邻（图 2D）。</li>
	<li>通过将椭圆形窗口切口与圆窗两侧的耳蜗切除切口连接起来完成颞骨切口。 注意：由于耳蜗骨的脆弱性，将鼓膜张肌窝保留在标本中并避免切开它将有助于防止延伸到 RWM 并损害其完整性的耳蜗骨折。</li>
	<li>将最后的附着物连接到靠近内耳道的颅底致密骨上，然后轻轻剃掉以产生切除的 RWM 标本（图 3A）。</li>
</ol>

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>

作者没有披露。

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><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.&amp;nbsp;</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。

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

作者聚焦：提取豚鼠圆窗膜促进内耳药物递送研究

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。

用于体外研究的豚鼠圆窗膜移植

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

610 次观看.  Johns Hopkins School of Medicine. 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?...

视频: 作者聚焦：提取豚鼠圆窗膜促进内耳药物递送研究

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

内淋巴管闭塞手术诱导的内淋巴积水

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

科研

生物学

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

豚鼠耳蜗植入术

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

医学

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

小型猪载体上 RPE 的视网膜下植入:术前准备、手术技术和术后护理指南

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

生物工程

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

几内亚猪诱导内部水解的修正手术方法

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

神经科学

使用胶原支架进行猪单阶段尿流改道手术

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

作者聚焦:改善先天性畸形和后天性疾病重建手术结果的创新方法

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

小型猪单期组织工程尿路上皮管手术模型

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans. 
To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements. 
The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

微型猪:人工耳蜗研究的大型动物模型

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

用于体外研究的豚鼠圆窗膜移植

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内淋巴管闭塞手术诱导的内淋巴积水

豚鼠耳蜗植入术

小型猪载体上 RPE 的视网膜下植入:术前准备、手术技术和术后护理指南

几内亚猪诱导内部水解的修正手术方法

小型猪单期组织工程尿路上皮管手术模型

小型猪单期组织工程尿路上皮管手术模型

小型猪单期组织工程尿路上皮管手术模型

微型猪:人工耳蜗研究的大型动物模型

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

猪视网膜色素上皮细胞原代培养