eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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 adeno-associated virus tagged with green fluorescent protein (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 hydrochloride (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&#160;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.&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
		<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&#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>
</table>

canalostomy for local drug delivery into the inner ear of an adult mouse

Source: Guo, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

<img alt="Figure 1" src="/files/ftp_upload/22275/22275_Figure1.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.
<img alt="Figure 2" src="/files/ftp_upload/22275/22275_Figure2.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.

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

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

Begin with an anesthetized mouse placed in a right lateral position.
Make an incision behind the ear and dissect the underlying muscles to expose the semicircular canals.
Excise a small muscle piece for later use.
Locate the posterior semicircular canal or PSC, a fluid-filled duct within the inner ear.
Make a hole in the middle of the PSC and clean the area.
Take a cannula connected to a drug-filled micro-syringe, which is attached to a syringe pump.
Insert this cannula into the hole on the PSC and move it towards the crus commune, a region where the posterior canal merges with the anterior canal, to access the inner ear.
Inject the drug solution through the cannula into the inner ear, ensuring local drug delivery.
Post-delivery, remove the cannula and place the excised muscle piece into the hole to prevent leakage. Finally, close the incision with sutures.&#160; &#160;&#160;

canalostomy-for-local-drug-delivery-into-inner-ear-an-adult

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

114 Views. Fonte: Guo, J. et al., Canalostomia come approccio chirurgico alla somministrazione locale di farmaci nelle orecchie interne di topi adulti e neonatali.J. Vis. Exp. (2018) Questo video mostra la canalostomia, una procedura che consente la somministrazione locale di farmaci nell'orecchio interno di un topo attraverso il canale semicircolare con danni minimi all'udito e alla funzione vestibolare. Questo metodo consente l'inoculazione di vettori virali, prodotti farmaceutici e piccole molecole nel...

Video: Canalostomia per la somministrazione locale di farmaci nell'orecchio interno di un topo adulto - Concept

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.

Metodo chirurgico per Virally Mediated Gene consegna all&#39;orecchio mouse interno attraverso la finestra rotonda a membrana

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

Ricerca

JoVE Journal

Neuroscienze

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.

Approccio del canale semicircolare posteriore per consegna del Gene dell'orecchio interno in Mouse neonatale

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 ...

Medicina

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.

Uno studio comparato Drug metodi di consegna mirate per l&#39;orecchio interno mouse: Bullostomy<em&gt; Versus</em&gt; Iniezione transtimpanica

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. ...

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

Trascrizione

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