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>

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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 inner ear function, especially in adult mice16,18,28,29,30, which is significant for the assessment of protective effects and for translational aspects. Several groups have used canalostomy 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. In the current study, we described step-by-step, detailed surgical techniques of canalostomy in adult and neonatal mice. Compared to previous studies, our study provides additional details on the positioning of the semicircular canals, which is a key procedure for successful surgery. As shown in Figure 2B and C, the PSC and LSC were generally located ~3 mm from the root of the pinna at 2-3 o&#39;clock. The LSC was at an approximately 30&#176; angle from the plane parallel to the calvarium, and the PSC was vertical to the LSC. Furthermore, we simplified the surgical procedures and shortened the surgical time to around 25 min with comparable results29.
During canalostomy, it is imperative to avoid leakage at the injection site and blockage of the cannula. Before switching on the pump, it is important to ensure that the tip of the cannula is inserted in the semicircular canal and that the cannula is not bent or blocked. Dry muscle is recommended to be used when sealing the hole in the semicircular canal of adult mice because, in the presence of semicircular canal fluid, it expands and plugs the hole (see steps 2.6 and 2.10). Because the wall of the semicircular canal of neonatal mice is soft and fragile, the opening should be sealed by covering with autogenous muscle or medical glue30. We also removed the cannula without covering the opening with muscle; the results revealed comparable AAV transduction efficiency in the inner ear (data not shown), indicating that the opening in the PSC of neonatal mice was closed satisfactorily.
Another advantage of canalostomy is the feasibility of multiple procedures, which enables repetitive applications of the same or different reagents (Figure 6). Because fibrotic and granulation tissues are frequently found at previous surgical sites, and the semicircular canal may be obstructed after surgery34, it is recommended to perform injections during repeated surgeries in a different area of the lateral and/or posterior semicircular canals18.
The main limitation of canalostomy is the difficulty in determining whether the injection cannula is inserted into the perilymphatic or endolymphatic space. After administration of adenoviral vectors into the inner ear16 via canalostomy, most transduced cells were located in the perilymphatic space, suggesting that the injection was likely performed in that area. Low molecular weight compounds (e.g., AAV, streptomycin, and siRNA) can pass through the barrier between the perilymph and endolymph and reach the sensory epithelium after injection into the perilymphatic or endolymphatic space18,19,34. However, after injection into the perilymph, reagents may remain in the perilymphatic space if they cannot penetrate the barrier between the perilymph and endolymph16,39,40. Therefore, the permeability of the injected reagents should be considered before using canalostomy.
In conclusion, canalostomy results in a broad distribution of reagents in the cochlea and vestibule with minimal disturbance of hearing and vestibular function. The surgery is easily implemented in adult and neonatal mice, and multiple procedures can be performed if required. Canalostomy is thus an effective and safe approach for drug delivery into the inner ear of rodents and may, in the future, be used clinically to treat human cochleovestibular diseases.

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&#956;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>
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			<td>Operating Miroscope</td>
			<td>Carl Zeiss Optical LLC.</td>
			<td>Pico</td>
			<td></td>
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			<td>Micro Forceps</td>
			<td>Dumont Dumostar</td>
			<td>10576</td>
			<td></td>
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		<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&#215;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>
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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 animals exhibited normal ABR thresholds and swimming test scores after AAV8-GFP injection18,30. Immunohistology revealed that, in the auditory sensory epithelium, inner hair cells (IHCs) and a few outer hair cells (OHCs) of the basal turn showed robust GFP expression30. In the vestibular sensory epithelium, GFP expression was detected in the utricle, saccule, and ampullae30. Both vestibular hair cells (Figure 5) and supporting cells were transduced with high efficiency18,30.
Infusion of streptomycin by canalostomy induces severe hair cell loss in the utricle18,34,38. To test the feasibility of multiple injections via canalostomy, mice were given streptomycin through the LSC followed 7 days later by AAV8-GFP suspensions through the PSC. This experiment was also designed to evaluate the transduction characteristics of AAV8-GFP in the lesioned utricle, which was useful for gene therapy studies in the damaged inner ear after HC loss18. GFP was distributed throughout the lesioned utricle (Figure 6). Numerous GFP-positive cells were negative for myosin VIIA, indicating that they were transduced SCs. A few HCs (myosin VIIa-positive cells) with immature hair bundles also expressed GFP.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57351/57351fig1.jpg" /> 
Figure 1: Device preparation. (A) The polyimide tubing (i) and the polyethylene tubing (ii) are sealed to make an injection cannula (iii). (B) The cannula is connected to a 10-&#181;L micro-syringe with a 30 G needle and then installed on a pump. Arrow indicates the cannula tip. (C) Injection reagent and normal saline are separated by an air gap. <a href="/files/ftp_upload/57351/57351fig1large.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/57351/57351fig2.jpg" /> 
Figure 2: Canalostomy in an adult mouse. (A) A post-auricular incision (arrow). (B) Muscles covering the temporal bone are exposed. If we define the root of the pinna (bold dot) as the origin and the plane parallel to the calvarium as 3/9 o&#39;clock, the posterior semicircular canal (PSC) and lateral semicircular canal (LSC) are generally located in the region between 2 and 3 o&#39;clock (pentagram), ~3 mm from the origin. Inset: lower-magnification image of the orientation. The dotted line indicates the calvarium plane. (C) The PSC and LSC are exposed (dotted lines). Inset: The LSC is at an approximately 30&#176; angle from the plane parallel to the calvarium, and the PSC is vertical to the LSC. (D) A small hole is made in the PSC. (E) The tip of the cannula is inserted into the PSC, and the reagent is injected. (F) The hole is sealed with a small piece of muscle. Scale bar in A is 5 mm, that in B is 1 mm (B for B-F), that in the inset to B is 1 cm, and that in the inset to C is 5 mm. <a href="/files/ftp_upload/57351/57351fig2large.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/57351/57351fig3.jpg" /> 
Figure 3: Canalostomy in a neonatal mouse. (A) A post-auricular incision (arrow). (B) Muscles covering the temporal bone are exposed, and the posterior semicircular canal (PSC) is ~2 mm from the root of the pinna (bold dot) at 2-3 o&#39;clock (pentagram). The orientation is identical to that in adult mice (Figure 2B). (C) The PSC and lateral semicircular canal (LSC) are exposed (dotted lines). (D) A small opening is made in the PSC. (E) The cannula is inserted into the PSC. (F) A small piece of muscle is used to cover the opening after injection. Scale bars in A and B inset are 5 mm, that in B is 1 mm, and that in C is 1 mm (C for C-F). <a href="/files/ftp_upload/57351/57351fig3large.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/57351/57351fig4.jpg" /> 
Figure 4: Stereo&#160;microscopic images of the inner ears of adult (A-A&#39;) and neonatal mice (B-B&#39;) administered fast-green dye via canalostomy. Samples were collected immediately after surgery. (A and B) The extracranial surface. (A&#39; and B&#39;) The intracranial surface. Fast-green dye distributes throughout the cochlea, vestibule, and semicircular canals. Scale bars are 1 mm (A&#39; for A-A&#39; and B&#39; for B-B&#39;). PSC, posterior semicircular canal. LSC, lateral semicircular canal. SSC, superior semicircular canal. <a href="/files/ftp_upload/57351/57351fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57351/57351fig5.jpg" /> 
Figure 5: Representative confocal images of whole mounts of the cochlea (A-A&#34;) and utricle (B-B&#34;) of adult mice, prepared 30 days after AAV8-GFP injection via canalostomy. Samples are stained with antibodies for GFP (green) and myosin VIIa (red). (A-A&#34;) GFP is expressed in most inner hair cells (IHC). (B-B&#34;) At the focal plane of the cuticular plate of the utricle, numerous hair cells express GFP (arrows). GFP, green fluorescent protein. OHC, outer hair cells. Scale bars are 25 &#181;m (A&#34; for A-A&#34;, B&#34; for B-B&#34;). <a href="/files/ftp_upload/57351/57351fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57351/57351fig6.jpg" /> 
Figure 6: Representative confocal images of a traumatized utricle obtained 30 days after AAV8-GFP injection via canalostomy. An adult mouse was injected with streptomycin via the lateral semicircular canal and 7 days later injected with AAV8-GFP via the posterior semicircular canal. GFP (green), myosin VIIa (red), and actin (blue). Arrowheads indicate representative supporting cells transduced with AAV8-GFP (GFP+/myosin VIIa- cells), and arrows indicate representative hair cells transduced with AAV8-GFP (GFP+/myosin VIIa+ cells) with immature hair bundles. Scale bar is 20 &#181;m (D for A-D). <a href="/files/ftp_upload/57351/57351fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

JoVE Journal

Genetics

10.6K Views.  Capital Medical University. Hier beschrijven we canalostomy procedure waarmee lokale drug levering tot de inner oren van volwassen en neonatale muizen door het semicircular kanaal met minimale schade aan gehoorbescherming en vestibulaire functie. Deze methode kan worden gebruikt om te enten virale vectoren, farmaceutische producten en kleine moleculen in het binnenoor muis.

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. ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

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Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

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

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