Name: guinea pig round window membrane explantation for ex vivo studies
Uploaded: 2024-02-23T13:20:00.000+00:00
Duration: 5 min 14 s
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

This work was supported in part by the NIDCD Grants No. 1K08DC020780 and 5T32DC000027-33, and the Rubenstein Hearing Research Fund.

All animal procedures were approved by the Institutional Animal Care and Use Committee (GP18M226). Hartley albino guinea pigs (both male and female, weighing 500-700 g) were used in the present study.
1. Procedure setup and preparation
<ol>
	<li>Sterilize all instruments with ethylene oxide before beginning the experiment.</li>
	<li>Euthanize the animals following the institutionally approved protocol. 
	NOTE: In the current study, a non-precharged chamber was employed to release 100% carbon dioxide (CO2) from a commercial cylinder. An inline restrictor was used to regulate the gas flow, maintaining it within the range of 30% to 70% of the chamber&#39;s volume per minute in accordance with the 2020 AVMA Guidelines11.</li>
	<li>Place the animal in the chamber and dispense carbon dioxide for 5 min. CO2 flow is maintained for 1 min following respiratory arrest.</li>
	<li>Perform decapitation following respiratory arrest to ensure euthanasia.</li>
</ol>
2. Surgical approach and explantation
<ol>
	<li>Extract the temporal bone from the guinea pig skull in the usual fashion12. Remove the excess soft tissue with a rongeur. Identify the external acoustic meatus, the temporal bulla, and the facial canal13 (Figure 1).</li>
	<li>Drill away the ventral aspects of the temporal bulla with a 6 mm diamond bit (see Table of Materials), exposing the middle ear space and the external auditory canal circumferentially.</li>
	<li>Using rongeurs, gently remove the external auditory canal and the tympanic ring, simultaneously separating the incudomalleolar joint. Identify the incus, incudostapedial joint, cochlea, horizontal semicircular canal, and facial canal13 (Figure 2A).</li>
	<li>Separate the incudostapedial joint and remove the incus using forceps. Identify the bony niche of the round window.</li>
	<li>Use a 6 mm diamond bit to drill away the bony lamina connecting the cochlea with the medial wall of the tympanic cavity toward the tensor tympani canal. Carefully decompress the bony channel of the tensor tympani and remove the tensor tympani muscle using a 28 G needle. 
	NOTE: The medial wall of the tensor tympani fossa connects directly with cochlear bone around the RWM, and care is taken not to cause fractures that can extend to the round window.</li>
	<li>Drill away the bony lamina connecting the cochlea to the inferior wall of the tympanic cavity until there is 1 mm of bony ledge abutting the cochlea remaining (Figure 2B).</li>
	<li>Using a 2 mm diamond bit (see Table of Materials), make a cochleostomy at the basal turn of the cochlea, leaving approximately 2 mm of bone to the round window. Continue the cochleostomy inferiorly in a plane parallel to the round window membrane to separate the base from the apex of the cochlea.</li>
	<li>Extend the cochleostomy cut through the skull base, which is much denser, resulting in a cross-sectional view of the basal turn of the cochlea. 
	NOTE: Aiming the drill toward the meatus of the internal auditory canal results in a trajectory that maximizes bone removal while avoiding traversing too close to the round window.</li>
	<li>Examine the specimen from the skull base side and, if not already done, identify the internal auditory canal and drill to the cochlear aperture. Remove the cochlear nerve with a 28 G needle.</li>
	<li>Examine the specimen from the intracochlear side. Identify and remove the osseous spiral lamina in the basal turn and the remaining modiolus with forceps or a 28 G needle.</li>
	<li>Irrigate the unified scala tympani-scala vestibuli cavity copiously to remove debris. The round window should be clearly visible from the cochectomy without any overlying debris (Figure 2C).</li>
	<li>Next, examine the specimen from the middle ear side. Drill the lateral semicircular canal and facial canal to the level of the oval window. Gently remove the stapes using forceps, exposing the oval window niche. Of note, there is a bony bridge between the crura of the stapes known as the crista stapedis.</li>
	<li>Using a 1 mm diamond drill (see Table of Materials), open the vestibule further by extending the oval window along the face of the round window, taking care to maintain 1-2 mm of cochlear bone abutting the round window niche (Figure 2D).</li>
	<li>Complete the temporal bone cuts by connecting the oval window cuts with the cochlectomy cuts on each side of the round window. 
	NOTE: Due to the fragility of the cochlear bone, preserving the tensor tympani fossa in the specimen and avoiding cuts through it will help prevent cochlear bone fractures that extend to the RWM and compromise its integrity.</li>
	<li>Make the final attachments to the dense bone of the skull base adjacent to the internal auditory canal and gently shave down to result in an excised RWM specimen (Figure 3A).</li>
</ol>

Guinea Pig Round Window Membrane Explantation

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	<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>

The authors have no disclosures to make.

In local drug delivery to the ear, the RWM is the primary route of passage for therapeutics to reach the inner ear. An accurate and reliable benchtop model is needed to better understand transport mechanisms and permeability across novel delivery vehicles and for drug development. In this study, we demonstrate that guinea pig RWM explantation is a feasible and dependable procedure to allow for systematic investigations of drug-membrane interactions. Lundman et al. and Kelso et al. previously described utilizing a similar RWM permeability model2,16; however, the specific steps of the surgical extraction have not been detailed until now, and the fragility of the cochlear bone and complex anatomy have posed a challenge for the consistent, en bloc harvest of intact RWMs.
The guinea pig RWM is located at the end of the scala tympani of the cochlea and is surrounded by a thin layer of cochlear bone. Fracture of this osseous structure during the explantation process can result in an unusable specimen, as the RWM tends to tear away if the surrounding cochlear bone does not remain intact. Our group noted that fractures most often occurred when drilling near the junction between the highly-dense skull base bone and the brittle cochlea, as the transition in bone density increased the likelihood of fracture propagation through the round window. For this reason, it is suggested that maintaining the denser bone of the tensor tympani fossa, which is attached to the cochlea in close proximity to the RW, will increase the yield of specimen harvest, particularly in older animals. Disruption of the bone can also occur during the removal of the cochlear contents; care must be taken to gently remove the osseous spiral lamina with minimal manipulation of the surrounding cochlear bone.
An important detail to consider with this model is the viability of the RWM after explantation. Prior groups have suggested that mammalian RWM remains viable for 24-48 h after extraction17. The present study has reflected these findings; consistent transport studies and histological analyses demonstrating intact cell structures (Figure 2A) have both supported the viability of the guinea pig RWM at the time of experiments. To maintain the overall health of the extracted specimen, the RWM is extracted and embedded within 3 h of euthanasia.
In the study of drug pharmacokinetics across the RWM in vivo, there remains considerable technical difficulty in making measurements of perilymphatic distribution and concentration1. Changes in intra-tympanic delivery methods and applied amount have resulted in varied therapeutic results. These difficulties are compounded by the complex fluid dynamics within the labyrinth, as well as irregular egress of injected material through the eustachian tube. The described method of explantation allows for the isolated study of the RWM and factors that affect its permeability as an ex vivo model. Furthermore, the explant also allows for the direct interrogation and visualization of various methods currently employed to increase RWM permeability, such as ultrasound microbubbles18 and chemically induced junctional modulation19. Future studies on specific endocytosis mechanisms implicated in drug delivery would also benefit from this benchtop model.

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.

<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

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.

As demonstrated in Figure 3A, this method allows for the explantation of the intact guinea pig round window membrane with a surrounding ring of rigid bone. The RWM should be fully connected to the bony annulus circumferentially. No fractures of the cochlear bone should be appreciated. In comparison with human round window specimens, guinea pig RWM does not have an overlying pseudomembrane. Additionally, unlike humans, there is a bony bridge between the crura of the guinea pig stapes, which requires fracturing and removal prior to the extraction of the stapes superstructure. Histological analysis (Figure 3B) of the representative RWM shows a clear three-layered epithelial structure with an adjacent, intact round window niche.
From a technique perspective, there are two critical steps. Firstly, when making the cochectomy in step 2.7, it is important to use the equator of the drill bit, rather than the tip, to make the cut, as jitters at the burr tip can lead to traumatic fractures in the cochlear bone that may extend to the round window membrane. Secondly, it is important to fully drill out the internal auditory canal, as this allows for the complete removal of the cochlear nerve and easier dissection of the osseous spiral lamina, resulting in a unified cavity on the intracochlear side for experimental sampling (Figure 2C).
After the RWM has been successfully extracted, our group utilized a modified Ussing chamber to evaluate pharmacokinetic properties of the membrane. The modified Ussing chamber has been validated by other groups in tympanic and round window membranes as well as retinal tissue to evaluate transport properties of epithelial membranes14,15. This 3D printed device was constructed using Poly-Jet Vero and consists of two triangular base pieces, each with a 400 &#181;L fluid chamber (Figure 3). The bony rim of the RWM is affixed to the base using 2-part epoxy (Gorilla, see Table of Materials), followed by silicon sealant (see Table of Materials) to ensure a water-tight seal (Figure 4). Meticulous care is taken to avoid any epoxy or sealant contacting the membrane. With the RWM sandwiched in place, the two bases are then glued together with epoxy. During transport experiments, the loading (middle-ear facing) is filled with phosphate-buffered saline (PBS) containing the delivery vehicle (or any molecule of interest), and the sampling chamber (scala tympani facing) is filled with PBS only. At regular time intervals, fluid from the sampling chamber is completely aspirated and replaced with fresh PBS.
Quality control measures are taken during each experiment to ensure that there is both a water-tight seal around the specimen within the apparatus, as well as a fully intact membrane without microperforations. Representative results with a brown, iron-core nanoparticle solution are shown below. Quantitative verification of a water-tight seal is accomplished by aspirating the full volume of the sampling chamber during each sampling interval; fluid leaking out of the sampling chamber would result in less than the full aspiration volume. At the conclusion of each experiment, the fluid volume in the loading chamber should also remain the same. Additionally, as our target fluid is brown in color, it is also readily visible in the event of a leak.
To ensure no leak within the membrane, several approaches were taken. First, RWM specimens used in this study are all immediately extracted, and light, confocal, and electron microscopy images confirm intact cellular and membrane structures without microperforations (Figure 3A,B). Second, microperforations were deliberately created in a subset of RWM specimens to compare the effect on nanoparticle delivery. Visual inspection of the sampling and loading chamber aspirate serves as further confirmation of specimen integrity and watertight attachment. In RWM with perforations, there is rapid equilibration of the loading and sampling chamber, which can be observed via a distinct color change (Supplementary Figure 1). Additionally, it was found that transport in microperforated RWMs was higher than the maximal variance observed in intact membranes (Supplementary Figure 2). Together, these serve as quality control mechanisms for the integrity of the water-tight seal surrounding the specimen, as well as the RWM.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65816/65816fig01.jpg" /> 
Figure 1: Preoperative image of the guinea pig temporal bone. The tympanic bulla, external auditory canal, and facial canal (*) are shown in this preoperative image of the guinea pig temporal bone. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/65816fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65816/65816fig02.jpg" /> 
Figure 2: Intraoperative images. Intraoperative images demonstrating the relationship between the round window membrane and the incus (*), cochlea (**), stapes (&#8224;), and decompressed internal auditory canal (&#8225;). Shaded areas indicate portions of the specimen that will be removed. (A) Guinea pig middle ear cavity after removal of tympanic membrane and facial nerve (step 2.3). (B) Middle ear cavity after decompression of the tensor tympanic canal (step 2.6). (C) Basal view of RWM after cochleostomy and removal of inner ear contents (step 2.11). (D) View of RWM after removal of stapes and identification of the vestibule (step 2.13). Scale bar = A,B (1 cm); C,D (5 mm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/65816fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65816/65816fig03.jpg" /> 
Figure 3: Final RWM specimen and histology. (A) Guinea pig RWM gross specimen with a bony annulus. Scale bar = 1 cm. (B) Histology of the explanted guinea pig round window membrane demonstrates an intact three-layered structure. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/65816fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65816/65816fig04.jpg" /> 
Figure 4: Microfluidic device for transmembrane transport experiments. (A) Diagram of the microfluidic device used for transmembrane transport experiments. (B,C) Printed microfluidic device for the experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/65816fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Loading and sampling chamber contents over a 5 h iron-core nanoparticle delivery experiment. (A) Loading chamber of intact RWM shows a gradual decrease in color intensity as the concentration of nanoparticles decreases. (B) Sampling chamber of intact RWM shows stable coloration over time, consistent with low levels of nanoparticle delivery. (C) Loading chamber of perforated RWM demonstrates a rapid decrease in color intensity as the contents equilibrate with the sampling chamber. (D) Sampling chamber of perforated RWM shows a gradual increase in color over time, consistent with high levels of nanoparticle delivery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/Supplemental Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Representative transport results for delivery of nanoparticles. Representative transport results for the delivery of nanoparticles (Fe3O4 core with polyethylene glycol coating, mean radius of 77 nm) across guinea pig RWM, both intact and with perforations (mean &#177; SD, n &#8805; 3 RWM experiments). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65816/Supplemental Figure 2.zip" target="_blank">Please click here to download this File.</a>

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

Author Spotlight: Extraction of Guinea Pig Round Window Membrane to Facilitate Inner Ear Drug Delivery Research

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.

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 ...

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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.

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

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.

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

Medicine

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.

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

Bioengineering

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.

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

Neuroscience

Accessing Collagen Scaffold for Single-Stage Urinary Diversion Surgery in Pigs

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.

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.

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.

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.

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.

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

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine Eye

Source: Bascuas T. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
In this video we describe a technique to isolate iris and retinal pigment epithelial cells from porcine eyes to obtain their primary cultures for in vivo and ex vivo studies. These pigment cells could be genetically modified to study regenerative approaches to treat ocular disorders.

Source: Bascuas T. et al., Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies

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