Name: surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane
Uploaded: 2015-03-16T14:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

NOTE: All procedures and animal handling complied with NIH ethics guidelines and approved protocol requirements of the Institutional Animal Care and Use Committee of the University of California, San Francisco.
1. Preparing the Animal for Surgery
<ol>
	<li>Carry out surgical procedures in a clean, dedicated space. Autoclave all surgical instruments, sterilize with a glass-bead sterilizer&#160;prior to surgery. 
	NOTE: In this protocol, use postnatal day 10-12 (P10-12) FVB mice. Different ages and strains of mice can be used to meet the needs of a specific project. Mice older than P40 can be challenging because the bulla bone gets harder.</li>
	<li>Anesthetize the mice with intraperitoneal injection of a mixture of ketamine hydrochloride (100 mg/kg), xylazine hydrochloride (10 mg/kg), and acepromazine (2 mg/kg). Begin surgical preparation only after the animal no longer responds to painful stimuli, such as toe pinch. If necessary, administer a booster dose (one-fifth the original dose) of the anesthetic cocktail to restore the original anesthetic plane. 
	NOTE: Take care at this step because most of the mortality observed at this age is caused by anesthetic overdose.</li>
	<li>Cover the animal&#39;s eyes with a protective ophthalmic ointment to keep the eyes moist during anesthesia, suppressing the animal&#39;s blink reflex.</li>
	<li>Position the mouse with neck extended on a heating pad throughout the procedure until the mouse is totally awake, to prevent post-anesthesia hypothermia.</li>
	<li>Shave the left post-auricular region with a clipper and disinfect with 70% ethanol and povidone-iodine before surgical manipulation.</li>
</ol>
2. The Surgical Procedure and Vector Injection
<ol>
	<li>Use a post-auricular approach to expose the tympanic bulla. Incise the subcutaneous tissue with small scissors to expose the post-auricular muscle. After retracting the adipose tissue to the posterior side of the incision, separate the muscles to the right and left side perpendicular to the incision to expose the temporal bone. Ensure that this incision is long enough to work comfortably with adequate visualization.</li>
	<li>Perforate the tympanic bulla with a 25 G needle and expand the hole as necessary with forceps by peeling the bone back to allow access to the basal turn of the cochlea, and then widen sufficiently to visualize the stapedial artery and the round window membrane (RWM).</li>
	<li>Puncture the RWM gently in the center with a borosilicate capillary pipette. Observe fluid efflux through the RWM at this point; this is normal. Wait until the efflux has stabilized (the effluxed fluid from the cochlea is dried with a sterile filter paper). 
	NOTE: Take care when holding and advancing the glass needle so that the RWM perforation is as small as possible. Depending on the microscope used, hold the pipettes using a micromanipulator or by hand using a pipette holder.</li>
	<li>Prepare the pipettes for injection using a pipette puller the same way patch clamp pipettes are prepared. Ensure that the tip diameter is large (about 15 &#181;m in diameter) so that pipetting the virus in and out is done easily.</li>
	<li>Draw up 1 to 2 &#181;l of AAV1-VGLUT3, AAV1-GFP or AAV2-GFP into an injection pipette. After the efflux is stabilized (5 to 10 min), microinject this fixed volume into the scala tympani through the same hole previously made in RWM.</li>
	<li>Seal the RW niche quickly after pulling out the pipette with a small plug of muscle and secure it with a small drop of tissue adhesive placed on the muscle to avoid leakage from the round window. 
	NOTE : Seal this hole very well after the needle is removed with a small plug of muscle&#160;to prevent any perilymph leakage from the cochlea-this step is critical to preserve hearing. Failure to seal completely the RWM will result in hearing loss over time.</li>
	<li>After injection, cover the hole in the auditory bulla with adipose tissue, return the post-auricular muscles and adipose tissue to their normal position and suture the wound in layers with a 6-0 or smaller absorbable chromic suture.</li>
	<li>Disinfect the wound with povidone-iodine.</li>
</ol>
3. Postoperative Care
<ol>
	<li>Place mice in a warm cage and do not leave them unattended until they are fully recovered then&#160;move them back with the mother. It is recommended to remove the male from the cage before putting the pups back with the mother.</li>
	<li>Administer subcutaneous Carprofen (2 mg/kg) for analgesia postoperatively and every 24 hr thereafter for 3 days, to manage inflammation and pain. Monitor the animals daily for signs of distress, abnormal weight loss, pain, or infection. Generally by the 3rd day after the surgery all mice should be acting normally. If any signs of distress or disease appear in a mouse after the 3rd day, consider euthanizing the mouse as per institutional guidelines.</li>
</ol>
4. Assessment of Cochlear Function Following Viral Delivery Using Auditory Brainstem Response (ABR) Recordings
NOTE: The auditory brainstem response (ABR)&#160;is an&#160;auditory evoked potential&#160;extracted from ongoing electrical activity in the brain and recorded via electrodes placed under the scalp. The animal is stimulated with sound. The resulting recording consists of five waves that reflect the electrical activity of successive points in the auditory pathway in the first 10 msec after onset of an auditory stimulus.
<ol>
	<li>Anesthetize mice by intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine hydrochloride as described in section 1.2 of this protocol, except without acepromazine.</li>
	<li>Place the mouse on a heating pad throughout the hearing test until the mouse is totally awake, to prevent post-anesthesia hypothermia. 
	NOTE: Use the following sound stimuli in this experiment: click (5 msec duration; 31 Hz presentation rate).</li>
	<li>Place subdermal needle electrodes at the vertex, below the pinna of the left ear (reference), and below the contralateral ear (ground) and start recording ABRs as previously described in a sound-proof chamber27,28. Record ABR waveforms in 5 dB sound pressure level intervals down from maximum amplitude.</li>
	<li>Determine ABR thresholds postoperatively as early as 4 days after viral delivery 
	NOTE: Threshold is defined as the lowest stimulus level at which response peaks for waves I&#8211;V were clearly and repeatedly present on visual inspection.</li>
	<li>Measure Wave I to analyze the activity from the cochlear nerve. The lowest stimulus level that yields a detectable ABR waveform is defined as the threshold.</li>
</ol>
5. Cochlear Transgene Protein Expression Using Immunofluorescence
<ol>
	<li>Anesthetize mice by an overdose of intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine hydrochloride as described in section 1.2 of this protocol, except without acepromazine.</li>
	<li>Decapitate the head only after the animal no longer responds to painful stimuli, such as toe pinch.</li>
	<li>Dissect the cochleae out and process for whole-mount immunofluorescence as described26. A detailed protocol&#160;of cochlear whole-mount immunofluorescence using anti-VGLUT3 and anti-Myosin 7a antibodies is described in Akil et al., 201329.</li>
	<li>For GFP labeling, incubate the cochlear whole-mounts overnight at 4 &#176;C with a rabbit anti-GFP antibody at 1:250 dilution.</li>
	<li>Rinse the cochleae twice for 10 min with PBS and then incubate for 2 hours in goat anti-rabbit IgG conjugated to Cy2 diluted 1:4,000 in PBS.</li>
	<li>Rinse the cochleae with PBS twice for 10 minutes and incubate them with DAPI for 15 min.</li>
	<li>Mount the cochleae on glass slides and observe under a microscope with confocal immunofluorescence.</li>
</ol>

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M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <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+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

In this work, we describe in detail a technique that can be used for cochlear gene therapy, with the goal of restoring or rescuing normal auditory function that is compromised by a genetic defect. As it is typically atraumatic, this approach is safe for cochlear gene transfer or other potential molecular therapies30. Other approaches for cochlear therapy have been described, including a ventral approach24, cochleostomy31,32 and endolymphatic sac delivery33 in mouse and guinea p...

Gene therapy has long been suggested as a potential treatment for genetic hearing loss, but success in this area has remained elusive1. To date, virally mediated methodologies have predominated due the theoretic ability to target specific cell types within the relatively inaccessible cochlea. Both adenovirus (AV) and adeno-associated virus (AAV) &#160;have been used for cochlear gene delivery. AAVs are advantageous in the cochlea for a number of reasons. They are replication-deficient viruses and can efficiently transfer transgenic molecules to different cell types including neurons, an important target for a number of causes of hearing loss. AAV entry into the cell is mediated by specific receptors2; thus, the choice of a particular serotype must be compatible with the cell types to be transduced. AAVs can effectively transfect hair cells3 and incorporate into the host genome, resulting in stable, long-term expression of the transgenic protein and phenotypic change in the cell4. While not necessarily advantageous for short-term applications such as hair-cell regeneration, long-term expression is very important for stable rescue of genetic defects. Because AAVs are not associated with any human disease or infection and demonstrate no ototoxicity5,6,7, they are an ideal candidate for use in gene therapy for inherited forms of hearing loss8.
Transfer of exogenous genetic material into the mammalian inner ear using viral vectors has been studied over the last decade and is emerging as a promising technique for treating both genetic and acquired forms of hearing loss9. The cochlea is potentially an ideal target for gene therapy for several reasons: 1) its small volume necessitates&#160;a limited amount of the virus needed; 2) its relative isolation from other organ systems limits side effects; and 3) its fluid-filled chambers facilitate viral delivery throughout the labyrinth10,11,12,13,14,15.
Mouse models of congenital deafness allow for use of many methods of study to monitor development of the inner ear in a systematic, replicable way. While the small size of mouse cochleae does present some surgical difficulty, the mouse serves as an extremely important model in the study of genetic hearing loss, with several experimental advantages over other species16. Mouse models allow assessment of a range of characteristics through genetic linkage analysis, collection of detailed morphological observations, and simulating pathogenic scenarios; as such, they are good candidates for virally mediated gene therapy. Extensive genetic studies in mice combined with technological advances have made it possible to generate genetically modified mice in a reproducible way across laboratories17,18,19,20,21. Furthermore, there exist numerous models for both acquired and inherited hearing loss phenotypes in mice, allowing rigorous testing in this animal model22,23,24. Thus, correcting hearing using virally mediated gene therapy in a mouse model is an appropriate first step in the search for a cure for human disease.
We have previously shown that transgenic mice lacking vesicular glutamate transporter 3 (VGLUT3) are born deaf due to lack of glutamate release at the IHC ribbon synapse25. Because this mutation does not lead to a primary degeneration of the sensory hair cells, these mutant mice are &#160;potentially an excellent model in which to test cochlear gene therapy for congenital hearing loss.
To date, a number of viral delivery techniques for cochlear gene therapy have been described, including round window membrane diffusion, round window membrane injection, and delivery via a cochleostomy. There are potential advantages and disadvantages of each of these approaches9.
Here we report a surgical method for virally mediated gene delivery to the VGLUT3 KO mouse inner ear through the round window membrane (RWM). The post-auricular RWM injection method is minimally invasive with excellent hearing preservation, and is relatively fast. As we have previously published, in an effort to restore hearing in this mouse model, an AAV1 vector carrying the VGLUT3 gene (AAV1-VGLUT3) was introduced into the cochlea of these deaf mice at postnatal day 12(P!@), resulting in the restoration of hearing26. Hearing in the VGLUT3 KO mice was verified by auditory brainstem response (ABR), while transgene protein expression was verified using immunofluorescence (IF). This methodology thus demonstrates that virally-mediated gene therapy can correct a genetic defect that would otherwise results in deafness.

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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).

To verify the technical features and utility of the post-auricular approach for cochlear molecular therapy, AAV1-VGLUT3, AAV1-GFP and AAV2-GFP were delivered into P10-12 mice inner ear via the RWM. This approach demonstrates successful transgene expression within inner hair cells (IHC) (VGLUT3 Figure 1 and GFP Figure 2 and GFP Figure 3A), outer hair cells (OHC) (GFP Figure 2) and supporting cells (GFP Figure 2 and Figure 3A</stro...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

15.4K Views.  University of California, San Francisco. 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. 

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Developmental Biology

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.

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) ...

Genetics

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

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. ...

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

Summary

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Chapters in this video

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

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