This work was supported by the National Natural Science Foundation of China (grant numbers 81570912, 81771016, 81100717).

All procedures and animal surgeries were conducted according to the guidelines of the Animal Care and Use Committee of the Capital Medical University of China.
1. Device Preparations
<ol>
	<li>To make the injection cannula (Figure 1A), connect polyimide tubing (inner diameter 114.3 &#181;m, outer diameter 139.7 &#181;m, length ~3 cm) to polyethylene tubing (inner diameter 280 &#181;m, outer diameter 640 &#181;m, length ~40 cm). Using superglue, seal the connection with at least three applications. Sterilize the injection cannula with ethylene oxide. 
	NOTE: When sealing the tubing, prevent the superglue from entering the polyimide tubing, which can result in a blockage of the cannula. Seal the tubing at least three times, because gas sterilization may cause leakage at the connection.</li>
	<li>Using a 30 G needle, connect the end of the polyethylene tubing to a 1 cc syringe containing normal saline. Fill the cannula with normal saline by injection with the 1-cc syringe to check for any leakage or blockage at the connection between the polyimide tubing and polyethylene tubing. 
	NOTE: If there is a leakage or a blockage of the connection, the cannula cannot be used in the following procedures.</li>
	<li>Evacuate a 10 &#181;L micro-syringe with normal saline and connect it to the aforementioned 30 G needle with the injection cannula. Install the micro-syringe on a microinjection pump (Figure 1B). Set the injection speed to 0.5 &#181;L/min and the volume to 1 &#181;L for fast-green dye and AAV8-GFP, or 2 &#181;L for streptomycin. 
	NOTE: The recommended range of injection speed is 0.1&#8722;0.5 &#181;L/min, and the recommended range of injection volume is 0.5&#8722;2 &#181;L.</li>
	<li>Pull back the micro-syringe to 1 &#181;L and extract the injection reagent. An air gap will form between the normal saline and the injected reagent (Figure 1C).</li>
	<li>Optionally label the position of the gas-fluid border on the polyethylene tubing using a marker pen. This can be used to monitor reagent flow during injection.</li>
</ol>
2. Canalostomy in Adult Mice
<ol>
	<li>Anesthetize an adult mouse (female, FVB/N, 5 to 6-week old) by intraperitoneal injection of ketamine HCl (120 mg/kg) and xylazine HCl (7 mg/kg). Wait for 5&#8722;10 min until the animal exhibits no response to painful stimuli (the toe-pinch reflex). Place the animal on a preheated electric pad after anesthesia.&#160;An analgesic, Meloxicam (1 mg/kg), was applied subcutaneously before surgery.</li>
	<li>Cover the animal&#39;s eyes with eye ointment. Shave the left post-auricular region with an electric animal clipper and disinfect the skin three times with 75% ethanol.</li>
	<li>Place the animal on a preheated electric pad. Set the temperature of the electric pad to ~37&#176;C. Place the animal in the right lateral position to facilitate surgery on the left ear.</li>
	<li>Make a 1-1.5-cm post-auricular incision ~3 mm from the left retroauricular groove (Figure 2A).</li>
	<li>Positioning the posterior semicircular canal (PSC) and lateral semicircular canal (LSC): When the root of the pinna (bold dot in Figure 2B) is defined as the origin and the plane parallel to the calvarium as 3 to 9 o&#39;clock, the PSC and the LSC are typically located ~3 mm from the root of the pinna between 2 and 3 o&#39;clock (Figure 2B).</li>
	<li>Bluntly dissect the muscle covering the temporal bone with micro-forceps to expose the PSC and LSC, whose margins are clearly visible as dark stripes in the temporal bone (Figure 2C). The LSC is at an approximately 30&#176; angle from the plane parallel to the calvarium, and the PSC is vertical to the LSC (Figure 2C). Collect a small piece of muscle with micro-forceps and let it dry. In the following step, this muscle will be used to seal the hole. 
	NOTE: Avoid excessive tearing of the tissues to minimize injury to muscles. Avoid damaging the adjacent vessel when exposing the PSC (Figure 2D).</li>
	<li>Make a small hole in the middle portion of the PSC using a 26 G needle (Figure 2D). Fluid leakage through the hole indicates successful penetration of the bony wall of the PSC. Enlarge the hole to an appropriate size, slightly larger than the diameter of the polyimide tubing. 
	NOTE: Drill and enlarge the hole gently and gradually to avoid fracture of the PSC.</li>
	<li>Clean the effusion surrounding the hole of the PSC using a cotton pellet.</li>
	<li>Insert the tip of the polyimide tubing gently into the PSC toward the crus commune to a depth of 1&#8722;2 mm (Figure 2E). Start the injection by pressing the &#39;run&#39; button on the pump.</li>
	<li>After the injection, wait ~2 min to allow the reagent to spread. Trim the small piece of muscle collected in step 2.6 with micro-scissors. Next, remove the injection cannula and immediately place the muscle into the hole in the PSC. 
	NOTE: Check for any liquid leaking from the hole after plugging to ensure that the hole is completely sealed.</li>
	<li>Return the separated muscles and subcutaneous tissues. Suture the incision using a 5-0 suture. Disinfect the incision region with povidone iodine. 
	NOTE: All the above surgical procedures take about 25 min.</li>
	<li>Position the animal on an electric pad preheated to ~37 &#176;C. Place the animal in the right lateral position for recovery.</li>
	<li>Perform auditory brainstem response (ABR) measurements and swimming tests one week after the surgery18.</li>
	<li>If required, perform repeated injections in a different area of the PSC or LSC.</li>
</ol>
3. Canalostomy in Neonatal Mice
<ol>
	<li>Use hypothermia to induce and maintain sedation in neonatal mice (FVB/N) at postnatal days 1 to 2 (P1-2). Place the pup on a plastic-wrap-covered bed of crushed ice for ~4 min. Next, lay the pup on an ice-filled platform.&#160;Disinfect the surgical field three times with 75% ethanol. 
	NOTE: Ensure that the head of the pup is out of the ice. Perform the entire surgery with the pup on an ice-filled platform.</li>
	<li>The following steps differ in the neonate compared to the adult mice.
	<ol>
		<li>Make a ~3-mm postauricular incision from ~2 mm posterior to the auricular crease (Figure 3A - B).</li>
		<li>Gently make an opening in the soft PSC using a 26 G needle. Insert the cannula into the PSC without enlarging the opening (Figure 3C - E).</li>
		<li>After injection, use a piece of muscle to cover, rather than plug, the opening as the latter can lead to a fracture of the soft PSC (Figure 3F).</li>
		<li>Close the skin using a 6-0 suture.</li>
		<li>Perform ABR measurements and swimming tests at P3030.</li>
	</ol>
	</li>
	<li>Parental cannibalism is a common problem after neonatal surgery. The following steps reduce the likelihood of parental cannibalism.
	<ol>
		<li>Clean up the blood surrounding the incision with alcohol wipes after surgery.</li>
		<li>Ensure that the neonate can move freely before returning it to the dam.</li>
		<li>Smear neonates with soiled bedding from the mother&#39;s cage, and place the neonates back in the middle of the litter.</li>
		<li>Any that do not undergo surgery should receive a similar postauricular incision and suture.</li>
		<li>Separate the male from the breeding cage.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Stachler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice+guideline:+sudden+hearing+loss.">Clinical practice guideline: sudden hearing loss.</a> Otolaryngol Head Neck Surg. 146 (3 Suppl), S1-S35 (2012).</li><li>Lentz, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+hearing+and+vestibular+function+by+antisense+oligonucleotides+in+a+mouse+model+of+human+deafness.">Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.</a> Nat Med. 19 (3), 345-350 (2013).</li><li>Rivera, T., Sanz, L., Camarero, G., Varela-Nieto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+inner+ear:+strategies+and+their+therapeutic+implications+for+sensorineural+hearing+loss.">Drug delivery to the inner ear: strategies and their therapeutic implications for sensorineural hearing loss.</a> Curr Drug Deliv. 9 (3), 231-242 (2012).</li><li>El Kechai, 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=Recent+advances+in+local+drug+delivery+to+the+inner+ear.">Recent advances in local drug delivery to the inner ear.</a> Int J Pharm. 494 (1), 83-101 (2015).</li><li>Akil, O., Rouse, S. L., Chan, D. K., Lustig, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+method+for+virally+mediated+gene+delivery+to+the+mouse+inner+ear+through+the+round+window+membrane.">Surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane.</a> J Vis Exp. (97), e52187 (2015).</li><li>Ahmed, H., Shubina-Oleinik, O., Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Gene+Therapies+for+Genetic+Hearing+Loss.">Emerging Gene Therapies for Genetic Hearing Loss.</a> J Assoc Res Otolaryngol. 18 (5), 649-670 (2017).</li><li>Murillo-Cuesta, 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=A+Comparative+Study+of+Drug+Delivery+Methods+Targeted+to+the+Mouse+Inner+Ear:+Bullostomy+Versus+Transtympanic+Injection.">A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection.</a> J Vis Exp. (121), e54951 (2017).</li><li>Stevens, S. M., Brown, L. N., Ezell, P. C., Lang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mouse+Round-window+Approach+for+Ototoxic+Agent+Delivery:+A+Rapid+and+Reliable+Technique+for+Inducing+Cochlear+Cell+Degeneration.">The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration.</a> J Vis Exp. (105), e53131 (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=Principles+of+local+drug+delivery+to+the+inner+ear.">Principles of local drug delivery to the inner ear.</a> Audiol Neurootol. 14 (6), 350-360 (2009).</li><li>Akil, O., 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=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Pan, 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=Gene+therapy+restores+auditory+and+vestibular+function+in+a+mouse+model+of+Usher+syndrome+type+1c.">Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c.</a> Nat Biotechnol. 35 (3), 264-272 (2017).</li><li>Kilpatrick, L. 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=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Izumikawa, 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=Auditory+hair+cell+replacement+and+hearing+improvement+by+Atoh1+gene+therapy+in+deaf+mammals.">Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.</a> Nat Med. 11 (3), 271-276 (2005).</li><li>Chang, Q., 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=Virally+mediated+Kcnq1+gene+replacement+therapy+in+the+immature+scala+media+restores+hearing+in+a+mouse+model+of+human+Jervell+and+Lange-Nielsen+deafness+syndrome.">Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.</a> EMBO Mol Med. 7 (8), 1077-1086 (2015).</li><li>Chen, Z., Mikulec, A. A., McKenna, M. J., Sewell, W. F., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+intracochlear+drug+delivery+in+the+mouse.">A method for intracochlear drug delivery in the mouse.</a> J Neurosci Methods. 150 (1), 67-73 (2006).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+structural+outcome+of+inner+ear+gene+transfer+via+the+vestibular+and+cochlear+fluids+in+mice.">The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice.</a> Mol Ther. 4 (6), 575-585 (2001).</li><li>Bowers, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+transduction+attenuates+cisplatin+spiral+ganglion+neuron+ototoxicity+in+the+cochlea.">Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea.</a> Mol Ther. 6 (1), 12-18 (2002).</li><li>Wang, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+targeting+normal+and+traumatized+mouse+utricle.">Adeno-associated virus-mediated gene transfer targeting normal and traumatized mouse utricle.</a> Gene Ther. 21 (11), 958-966 (2014).</li><li>Isgrig, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+Restores+Balance+and+Auditory+Functions+in+a+Mouse+Model+of+Usher+Syndrome.">Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.</a> Mol Ther. 25 (3), 780-791 (2017).</li><li>Gassner, D., Durham, D., Pfannenstiel, S. C., Brough, D. E., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canalostomy+as+a+surgical+approach+for+cochlear+gene+therapy+in+the+rat.">Canalostomy as a surgical approach for cochlear gene therapy in the rat.</a> Anat Rec (Hoboken). 295 (11), 1830-1836 (2012).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+transgene+expression+after+adenoviral+vector+inoculation+in+the+endolymphatic+sac.">Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.</a> Hum Gene Ther. 10 (5), 769-774 (1999).</li><li>Xia, L., Yin, S., Wang, J. <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+gene+transfection+in+neonatal+mice+using+adeno-associated+viral+vector:+a+comparison+of+two+approaches.">Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches.</a> PLoS One. 7 (8), e43218 (2012).</li><li>Chien, W. W., McDougald, D. S., Roy, S., Fitzgerald, T. S., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+transfer+mediated+by+adeno-associated+virus:+Comparison+of+two+surgical+approaches.">Cochlear gene transfer mediated by adeno-associated virus: Comparison of two surgical approaches.</a> Laryngoscope. 125 (11), 2557-2564 (2015).</li><li>Zhu, B. Z., Saleh, J., Isgrig, K. T., Cunningham, L. L., Chien, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+Loss+after+Round+Window+Surgery+in+Mice+Is+due+to+Middle+Ear+Effusion.">Hearing Loss after Round Window Surgery in Mice Is due to Middle Ear Effusion.</a> Audiol Neurootol. 21 (6), 356-364 (2017).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+postnatal+virus+inoculation+into+the+scala+media+achieved+extensive+expression+of+exogenous+green+fluorescent+protein+in+the+inner+ear+and+preserved+auditory+brainstem+response+thresholds.">Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.</a> J Gene Med. 15 (3-4), 123-133 (2013).</li><li>Lee, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+human+embryonic+stem+cells+implanted+in+the+guinea+pig+auditory+epithelium.">Survival of human embryonic stem cells implanted in the guinea pig auditory epithelium.</a> Sci Rep. 7, 46058 (2017).</li><li>Ishimoto, S., Kawamoto, K., Kanzaki, S., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+supporting+cells+of+the+organ+of+Corti.">Gene transfer into supporting cells of the organ of Corti.</a> Hear Res. 173 (1-2), 187-197 (2002).</li><li>Okada, 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=Gene+transfer+targeting+mouse+vestibule+using+adenovirus+and+adeno-associated+virus+vectors.">Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.</a> Otol Neurotol. 33 (4), 655-659 (2012).</li><li>Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L. H., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy+with+ancestral+AAV+in+adult+mice:+complete+transduction+of+inner+hair+cells+without+cochlear+dysfunction.">Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction.</a> Sci Rep. 7, 45524 (2017).</li><li>Guo, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochleovestibular+gene+transfer+in+neonatal+mice+by+canalostomy.">Cochleovestibular gene transfer in neonatal mice by canalostomy.</a> Neuroreport. 28 (11), 682-688 (2017).</li><li>Beyea, J. A., Agrawal, S. K., Parnes, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmastoid+semicircular+canal+occlusion:+a+safe+and+highly+effective+treatment+for+benign+paroxysmal+positional+vertigo+and+superior+canal+dehiscence.">Transmastoid semicircular canal occlusion: a safe and highly effective treatment for benign paroxysmal positional vertigo and superior canal dehiscence.</a> Laryngoscope. 122 (8), 1862-1866 (2012).</li><li>Naples, J. G., Eisen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+and+Evolution+of+Surgery+on+the+Vestibular+Labyrinth.">The History and Evolution of Surgery on the Vestibular Labyrinth.</a> Otolaryngol Head Neck Surg. 155 (5), 816-819 (2016).</li><li>Hamilton, L., Keh, S., Spielmann, P. M., Hussain, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+we+do+it:+locating+the+posterior+semicircular+canal+in+occlusion+surgery+for+refractory+benign+paroxysmal+positional+vertigo:+a+cadaveric+temporal+bone+study.">How we do it: locating the posterior semicircular canal in occlusion surgery for refractory benign paroxysmal positional vertigo: a cadaveric temporal bone study.</a> Clinical Otolaryngology. 41 (2), 190-193 (2016).</li><li>Jung, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+targeting+Hes5+augments+hair+cell+regeneration+in+aminoglycoside-damaged+mouse+utricle.">siRNA targeting Hes5 augments hair cell regeneration in aminoglycoside-damaged mouse utricle.</a> Mol Ther. 21 (4), 834-841 (2013).</li><li>Kamiya, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> Am J Pathol. 171 (1), 214-226 (2007).</li><li>Pfannenstiel, S. C., Praetorius, M., Plinkert, P. K., Brough, D. E., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+gene+therapy+prevents+aminoglycoside-induced+degeneration+of+auditory+and+vestibular+hair+cells.">Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.</a> Audiol Neurootol. 14 (4), 254-266 (2009).</li><li>Kawamoto, K., Izumikawa, M., Beyer, L. A., Atkin, G. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+hair+cell+regeneration+in+the+mouse+utricle+following+gentamicin+ototoxicity.">Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.</a> Hear Res. 247 (1), 17-26 (2009).</li><li>Wang, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+and+Atoh1+expression+during+hair+cell+regeneration+in+the+mouse+utricle.">Notch signaling and Atoh1 expression during hair cell regeneration in the mouse utricle.</a> Hear Res. 267 (1-2), 61-70 (2010).</li><li>Pietola, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HOX-GFP+and+WOX-GFP+lentivirus+vectors+for+inner+ear+gene+transfer.">HOX-GFP and WOX-GFP lentivirus vectors for inner ear gene transfer.</a> Acta Otolaryngol. 128 (6), 613-620 (2008).</li><li>Han, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li></ol>

No conflicts of interest are declared.

In this study, we showed that drug delivery by canalostomy resulted in widespread distribution of the reagent throughout the cochlea and vestibular end-organs. As an inner ear gene delivery method, canalostomy resulted in GFP expression in the inner ears of adult and neonatal mice with minimum damage to hearing and vestibular function. Furthermore, multiple injections can be easily performed in the same animal.
One of the greatest strengths of canalostomy is that it causes minimal damage to in...

Sensorineural hearing loss and vestibular dysfunction affect a substantial number of patients and are closely associated with inner ear disorders. Delivery of therapeutic drugs into the inner ear shows promise for the treatment of inner ear disorders. A systemic or local approach can be used to deliver drugs into the inner ear. Some inner ear diseases are successfully treated with systemic drug administration, such as idiopathic sudden hearing loss, which is commonly treated with systemic steroid1. In addition, Lentz et al. showed that systemic administration of antisense oligonucleotide was able to improve hearing and balance functions in the Ush1c mutant mouse model2. However, a large portion of inner ear diseases are not effectively treated by systemic drug administration because of the blood-labyrinth barrier, which limits drug access to the inner ear3,4. In contrast, local drug delivery strategies can treat inner ear disorders more efficiently. Indeed, the inner ear is potentially an ideal target for local drug delivery; it is filled with fluid, which facilitates dissemination of the drug after one-site diffusion or injection, and it is relatively isolated from neighboring organs, which limits side-effects5,6.
Local drug delivery strategies include intratympanic and intralabyrinthine methods. The effectiveness of the intratympanic route largely relies on drug permeability through the round window membrane (RWM) and the residence time of the drug on the RWM3,4,7,8. Thus, it is not suitable for the delivery of drugs or reagents that cannot penetrate the RWM. Intralabyrinthine methods involve inoculation of drugs directly into the inner ear, resulting in a high dose and widespread distribution. However, intralabyrinthine methods require delicate surgeries and are invasive, leading to damage to inner ear function. Currently, the intralabyrinthine injection of drugs is used only in animal studies as it has not been demonstrated to be sufficiently safe for use in humans9. Therefore, surgical procedures must be simplified, and the risk of injury reduced to translate intralabyrinthine approaches into the clinic.
Several intralabyrinthine approaches have been assessed in animals by injection through the RWM5,10,11 and into the scala media12,13,14, the scala tympani15,16, the scala vestibule17, the semicircular canals16,18,19,20, and the endolymphatic sac21. Each of these approaches has advantages and disadvantages6. Delivery through the RWM is atraumatic in neonatal mice5,22. However, a mild hearing loss is observed in adult mice after RWM injection23, possibly due to middle ear effusion after the surgery24. Scala media injection, which involves the injection of the reagent directly into the endolymphatic space containing the sensory epithelium, achieves a high reagent concentration in target end-organs12,14,25,26. However, this approach requires a complex procedure and results in significant elevation of the hearing threshold if performed later than postnatal day 5 (P5)25,27, which limits its application.
Compared to the above-mentioned intralabyrinthine approaches, canalostomy causes minimal damage to the inner ear, especially in adult mice16,18,28,29,30, which is important for the assessment of protective effects and translational aspects. Furthermore, in rodents, the semicircular canals are located beyond the bulla, which facilitates surgical procedures and avoids disturbance of the middle ear during surgery. In the clinic, semicircular canal surgeries are used for intractable benign paroxysmal positional vertigo31,32,33, suggesting the clinical feasibility of canalostomy. Since it was first described by Kawamoto et al.16 in 2001, canalostomy has been used to deliver various reagents, such as viral vectors, siRNA, stem cells, and aminoglycoside, into the murine inner ear18,19,28,29,34,35,36,37. Inoculation of adeno-associated virus (AAV) vectors by canalostomy enable overexpression of exogenous genes in the sensory epithelium and primary neurons of the cochlea and vestibular end-organs18,28,29,30. Whirlin gene therapy by canalostomy restores balance function and improves hearing in a mouse model of human Usher syndrome19, suggesting that canalostomy is useful for studies of gene therapy for genetic cochleovestibular diseases. Transplantation of mesenchymal stem cells by canalostomy results in reorganization of cochlear fibrocytes and hearing recovery in a rat model of acute sensorineural hearing loss35. Additionally, canalostomy can be used to introduce aminoglycosides into the inner ear to establish vestibular lesions18,34,38, and multiple injections can be performed if required18,34.
In the present article, we describe, in detail, canalostomy techniques in adult and neonatal mice. We inoculated various reagents, including fast-green dye and AAV serotype 8 (AAV8), together with the green fluorescent protein (GFP) gene (AAV8-GFP) and streptomycin, into the mouse inner ear to evaluate the immediate and long-term outcomes after canalostomy.

<table><tbody><tr><td>Polymide Tubing</td><td>A-M Systems</td><td>823400</td><td></td></tr><tr><td>Polyethylene Tubing</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/1</td><td></td></tr><tr><td>10&amp;mu;l Microsyringe</td><td>Hamilton Company</td><td>80001</td><td></td></tr><tr><td>Xylazine HCL</td><td>Sigma-Aldrich Co. Llc.</td><td>X-1251</td><td></td></tr><tr><td>Operating Miroscope</td><td>Carl Zeiss Optical LLC.</td><td>Pico</td><td></td></tr><tr><td>Micro Forceps</td><td>Dumont Dumostar</td><td>10576</td><td></td></tr><tr><td>Fast-green Dye</td><td>Sigma-Aldrich Co. Llc.</td><td>F7252</td><td></td></tr><tr><td>AAV8-GFP</td><td>BioMiao Biological Technology Co. Ltd (Beijing, China)</td><td>20161101</td><td>Titer: 2&amp;times;10e12 vg/mL</td></tr><tr><td>Streptomycin Sulfate</td><td>Sigma-Aldrich Co. Llc.</td><td>S9137</td><td></td></tr><tr><td>Microinjection Pump</td><td>Stoelting Co.</td><td>789100S</td><td></td></tr><tr><td>Electric Pad</td><td>Pet Fun</td><td>11072931136</td><td></td></tr><tr><td>1 cc Syringe</td><td>Mishawa Medical Industries Ltd. (Shanghai, China)</td><td>2011-3151258</td><td></td></tr><tr><td>Ketamine HCL</td><td>Gutian Pharmaceutical Co., Ltd. (Fujian, China)</td><td>H35020148</td><td></td></tr><tr><td>Electric Animal Clipper</td><td>Codos Electrical Appliances Co., Ltd. (Guangdong, China)</td><td>CP-8000</td><td></td></tr><tr><td>Cotton Pellet</td><td>Yatai Healthcare Ltd. (Henan, China)</td><td>Yu-2008-1640081</td><td></td></tr><tr><td>Suture</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China)</td><td>Hu-2013-2650207</td><td></td></tr><tr><td>Eye Ointment</td><td>Beijing Shuangji Pharmaceutical Ltd. (Beijng China)</td><td>H11021270</td><td></td></tr></tbody></table>

canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Fast-green dye was injected into the PSC of adult and neonatal mice to evaluate its immediate distribution in the inner ear. The dye was detected throughout the cochlea, vestibule, and semicircular canals immediately after surgery (Figure 4).
To evaluate the safety and efficiency of canalostomy for inner ear gene delivery, AAV8-GFP was injected into the inner ear of adult and neonatal mice. All anim...

Watch this Scientific Journal Video about Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice at JoVE.com

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

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Research

JoVE Journal

Genetics

10.6K Views.  Capital Medical University. Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Video: Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

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

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

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

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

Neuroscience

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

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

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

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

Medicine

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Biology

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

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

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Summary

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Chapters in this video

In Vitro Transcription Assays and Their Application in Drug Discovery

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Controllable Ion Channel Expression through Inducible Transient Transfection

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

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

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

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

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

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

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