Method Article

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

DOI:

10.3791/57351

⸱

May 25th, 2018

 ,  ,  ,  ,  ,  , 
1Department of Otolaryngology-Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, 2Department of Otolaryngology-Head and Neck Surgery, Shanghai First People's Hospital, Shanghai Jiao Tong University

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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.

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Protocol

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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

  1. To make the injection cannula (Figure 1A), connect polyimide tubing (inner diameter 114.3 µm, outer diameter 139.7 µm, length ~3 cm) to polyethylene tubing (inner diameter 280 µm, outer diameter 640 µm, 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.
  2. 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.
  3. Evacuate a 10 µL 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 µL/min and the volume to 1 µL for fast-green dye and AAV8-GFP, or 2 µL for streptomycin.
    NOTE: The recommended range of injection speed is 0.1−0.5 µL/min, and the recommended range of injection volume is 0.5−2 µL.
  4. Pull back the micro-syringe to 1 µL and extract the injection reagent. An air gap will form between the normal saline and the injected reagent (Figure 1C).
  5. 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.

2. Canalostomy in Adult Mice

  1. 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 5−10 min until the animal exhibits no response to painful stimuli (the toe-pinch reflex). Place the animal on a preheated electric pad after anesthesia. An analgesic, Meloxicam (1 mg/kg), was applied subcutaneously before surgery.
  2. Cover the animal'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.
  3. Place the animal on a preheated electric pad. Set the temperature of the electric pad to ~37°C. Place the animal in the right lateral position to facilitate surgery on the left ear.
  4. Make a 1-1.5-cm post-auricular incision ~3 mm from the left retroauricular groove (Figure 2A).
  5. 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'clock, the PSC and the LSC are typically located ~3 mm from the root of the pinna between 2 and 3 o'clock (Figure 2B).
  6. 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° 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).
  7. 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.
  8. Clean the effusion surrounding the hole of the PSC using a cotton pellet.
  9. Insert the tip of the polyimide tubing gently into the PSC toward the crus commune to a depth of 1−2 mm (Figure 2E). Start the injection by pressing the 'run' button on the pump.
  10. 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.
  11. 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.
  12. Position the animal on an electric pad preheated to ~37 °C. Place the animal in the right lateral position for recovery.
  13. Perform auditory brainstem response (ABR) measurements and swimming tests one week after the surgery18.
  14. If required, perform repeated injections in a different area of the PSC or LSC.

3. Canalostomy in Neonatal Mice

  1. 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. 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.
  2. The following steps differ in the neonate compared to the adult mice.
    1. Make a ~3-mm postauricular incision from ~2 mm posterior to the auricular crease (Figure 3A - B).
    2. 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).
    3. 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).
    4. Close the skin using a 6-0 suture.
    5. Perform ABR measurements and swimming tests at P3030.
  3. Parental cannibalism is a common problem after neonatal surgery. The following steps reduce the likelihood of parental cannibalism.
    1. Clean up the blood surrounding the incision with alcohol wipes after surgery.
    2. Ensure that the neonate can move freely before returning it to the dam.
    3. Smear neonates with soiled bedding from the mother's cage, and place the neonates back in the middle of the litter.
    4. Any that do not undergo surgery should receive a similar postauricular incision and suture.
    5. Separate the male from the breeding cage.

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Results

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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...

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Discussion

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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...

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Disclosures

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No conflicts of interest are declared.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Polymide TubingA-M Systems823400
Polyethylene TubingScientific Commodities Inc.BB31695-PE/1
10μl MicrosyringeHamilton Company80001
Xylazine HCLSigma-Aldrich Co. Llc.X-1251
Operating MiroscopeCarl Zeiss Optical LLC.Pico
Micro ForcepsDumont Dumostar10576
Fast-green DyeSigma-Aldrich Co. Llc.F7252
AAV8-GFPBioMiao Biological Technology Co. Ltd (Beijing, China)20161101Titer: 2×10e12 vg/mL
Streptomycin SulfateSigma-Aldrich Co. Llc.S9137
Microinjection PumpStoelting Co.789100S
Electric PadPet Fun11072931136
1 cc SyringeMishawa Medical Industries Ltd. (Shanghai, China)2011-3151258
Ketamine HCLGutian Pharmaceutical Co., Ltd. (Fujian, China)H35020148
Electric Animal ClipperCodos Electrical Appliances Co., Ltd. (Guangdong, China)CP-8000
Cotton PelletYatai Healthcare Ltd. (Henan, China)Yu-2008-1640081
SutureShanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China)Hu-2013-2650207
Eye OintmentBeijing Shuangji Pharmaceutical Ltd. (Beijng China)H11021270

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CanalostomyDrug DeliveryInner EarSemicircular CanalSurgical TechniqueAAV8 GFP InjectionNeonatal MiceAdult MiceConfocal ImagingHair Cell Transduction

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