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

The overall goal of this procedure is to describe a postauricular surgical approach that allows the rapid and direct delivery of virus into the mouse Cochlear S scale timi through the round window membrane. This is accomplished by first exposing the tympanic bulla. In the second step, the tissue is perforated and the edia artery and round window membrane are visualized.

The round window membrane is then punctured and the virus is injected into the spon. In the final step, the hole is sealed. Ultimately, the expression of the transgene protein and the recovery of the animal's hearing can be assessed by immunofluorescent microscopy and measurement of the auditory brainstem response respectively.

The main advantages of this technique over the existing methods like coch, ostomy, and ventral approach is that the post ular round window membrane injection is minimally invasive relatively fast, allow higher volume injections, and facilitate widespread transfection of variety of cells throughout the cochlear. Generally, individuals new to this technique will struggle because the procedure is heavily dependent on thorough understanding of the anatomy of the cochlear. After confirming anesthesia by toe, pinch shave the left postauricular region and disinfect the skin of a postnatal day, 10 to 12 mouse with 70%ethanol and povidone iodine.

Then cover the eyes with a protective ophthalmic ointment and position the animal with the neck extended on a heating pad. When the surgical preparation is complete, incise the subcutaneous tissue with small scissors to expose the postauricular muscle. After retracting the adipose tissue to the posterior side of the incision, separate the muscles to the right and left sides perpendicular to the incision.

To expose the temporal bone, use a 25 gauge needle to perforate the tympanic bulla, peeling the bone back with forceps as necessary to expand the hole and to allow access to the basal turn of the cochlea. Then widen the hole sufficiently to allow visualization of the staed artery and the round window membrane. Puncture the round window membrane gently in the center with a burro silicate, capillary pipette.

Dry the round window membrane with a sterile filter paper. Then wait five to 10 minutes for the fluid e flux to stabilize At this step. Take care when holding and advancing the pipette, so to make the round window membrane perforation as small as possible.

Depending on the microscope use, the pipette can be held by hand or by microm manipulator. As you can see here, we hold the pipette by hand as the microscope is too close to the mouse to allow for a micro manipulator. Now draw one to two microliters of the virus into a pulled glass pipette with a 15 micrometer diameter tip and micro inject the virus into the scale of timoni through the same hole previously made in the round window membrane.

Then pull out the pipette and quickly seal the round window niche with a small plug of muscle, securing it with a small drop of tissue adhesive. Cover the hole in the auditory bulla with adipose tissue and return the postauricular muscles and adipose tissue to their normal position. Next, using a 6.0 or smaller absorbable chromic suture, close the wound and disinfect the incision area with povidone iodine.

Then place the mouse in a warm cage, returning the pop to the mother after full recovery with daily monitoring. To assess cochlear function as early as four days after viral delivery, position a sedated transfected animal on a heating pad, and then place subdermal needle electrodes at the vertex below the pinna of the left ear and below the contralateral ear. Finally, start recording the auditory brainstem responses or a BR in a soundproof chamber as previously described, measuring wave one to analyze the activity of the cochlear nerve as seen in the cochlear hole mount.

This approach demonstrates successful transgene expression within inner hair cells, outer hair cells, and supporting cells without significant organ of COR T injury that different cell types expressing GFP can be observed in the cochlear hole. Mount demonstrates that the choice of adeno associated virus serotype must be compatible with the cell types to be transduced. The number of cells transfected varies depending on the concentration and the volume the virus injected.

In this case, the increase from 0.6 to one microliter resulted in an increase in the number of cells transfected to a level comparable to wild type V glute three transgene expression was restored in injected KO mice as seen in these images here, the effect of postauricular round window membrane delivery of V GL three virus into the inner ear of V gl three knockout mice on hearing rescue is demonstrated as seen in the graph. The A BR traces were restored in the VLU three rescued knockout mouse reaching thresholds comparable to those seen in wild type mice. While attempting this procedure, it is important to remember to take your time for each step to prevent damage of the inner and middle ear structures as well as the neck muscles.

After watching this video, you should have good understanding of how to perform a post surgical approach for the delivery of molecular pharmacologic viral experimental treatments into the MAs re skeleton pan through the round wind membrane.