Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

The stria vascularis functions to generate the endo cochlear potential and regulate eye and homeostasis in the cochlea. The auditory development and restoration program seeks to understand the underlying mechanisms connecting dysfunctional eye and homeostasis with hearing instability disorders, which are diseases where either fluctuation in or sudden hearing loss occurs. Work in my lab has established the transcriptional landscape of the stria vascularis and has contributed to our understanding of its importance in hearing.

We have used these data to draw parallels to poorly understood hearing instability disorders, including Meniere's disease. This protocol enables the dissection and isolation of whole mount stria vascularis for further molecular studies, including immunohistochemistry. Given that stria vascularis cell types are intricately intertwined with one another, the use of nuclear isolation and single nucleus RNA sequencing has facilitated our understanding of the distinct transcriptional machinery involved.

To begin, identify the inner ear within the temporal bone of a euthanized mouse. Scrape away the cranial nerves using number 55 forceps. Then use number 55 forceps to dissect out the bula and capsule, detaching them from surrounding bone.

Remove any remaining soft tissue from the inner ear surface. For SV dissection, use number 55 forceps to hold the specimen by the vestibular portion with the cochlear facing up. Pierce through the cochlear bone at the apex using the same forceps.

Scrape along the apical turn, applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall. Using number 55 forceps, remove cochlear bone pieces from the apical and middle turns.

Gently pushing down the lateral wall, pry and remove the bone wall pieces towards the middle turn to expose the lateral wall of the apical and middle turns. Push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of the apical and middle turns from the spiral ganglion along the outer hair cell layer.

Then remove pieces of spiral ganglion from inside the cochlear. Detach the cochlear from the vestibular portion of the temporal bone by inserting the forceps into the round and oval window and pushing down towards the vestibule to have better access to the lateral wall of the basal turn. After the cochlear is detached, remove the vestibular portion of the inner ear.

Prioritize preserving as much of the stria as possible. Continue removing til the bone of the middle turn is detached from the SV.Next, remove pieces of the remaining cochlear bone covering the basal turn. This can be accomplished by pushing the lateral wall layer under the bone to detach it.

Pry and gently pull the tissue to remove the farthest basal part of the lateral wall, being careful not to damage the soft tissue below. With the basal part of the lateral wall detached, separate the lateral wall from the remaining cochlear by tracing the forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall until it reaches the apex.

Then move the lateral wall to a fresh PBS. Lay the lateral wall flat revealing the SV as a darker layer of tissue on the internal side of the lateral wall. Gently pry the SV layer away from the lateral wall.

Push aside the detached SV, and advance the forceps along the lateral wall, attempting to detach the SV as one long ribbon, trying not to squeeze the SV.Use a tissue scoop to collect the fragile tissue. Cochlear were dissected from adult mouse aged greater than or equal to postnatal Day 30. Inner ear was extracted from the surrounding temporal bone.

SV was exposed after the removal of bone covering the middle turn. The SV was fully separated from the lateral wall after dissection. Homogenize the dissected stria vascularis tissue from mouse ear in a two milliliter Downs homogenizer with 10 to 20 strokes on ice.

Lice the tissue on ice for 25 minutes. Then filter the tissue through a 30 micron filter, and spin the filtrate at 500 G for five minutes at four degrees Celsius. Remove the supernatant and re-suspend the cell pallette in one milliliter of nuclei wash and resuspension buffer.

Next, filter the cells through a 10 micron filter and centrifuge at 500 G for five minutes at four degrees Celsius. Remove the supernatant and re-suspend in 50 microliters of nuclei wash and resuspension buffer. After trypan blue staining, dilute five microliters of the cell suspension into 50 microliters of 1x PBS and count the cells.

To prepare single nuclei captures, load the sample with the desired nuclear density onto the chip. Prepare for immuno staining by dissecting the stria vascularis from the mouse and add the tissue to a 24 well plate containing 200 microliters of 4%paraformaldehyde in 1x PBS. Incubate the tissue for 20 minutes at room temperature.

Perform two short washes of 1x PBS on an orbital shaker at low RPM. Remove the PBS. Then perform permeablization and blocking for a minimum of one hour at room temperature in 300 microliters of PBST solution.

Next, stain the tissue with the primary antibody overnight at four degrees Celsius. Then add a secondary antibody and incubate for two hours at room temperature on an orbital shaker at low RPM. Cover the plate to protect the fluorescently tagged secondary antibody from light.

Next, prepare a larger micro slide by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 by 18 millimeter glass cover slip. Place one drop of mounting reagent between the glue streaks. Using number 55 forceps, grab as little tissue as possible on one end of the SV and transfer it from PBS to the mounting reagent.

Place one end of the cover slip on the slide where one streak of glue was placed. Then gently release the cover slip to avoid creating air bubbles. Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount.

Label the specimen on the glass cover slip away from the visualization field. Visualize the SV tissue under a dissection microscope to ensure it is lying flat and not near any air bubbles. If the SV tissue is not oriented correctly, try to push out air bubbles or carefully remove the smaller glass cover slip and reposition the SV.A whole mount of SV from P 30 mouse was prepared and confocal imaging was performed.

ZS green expression was observed in the intermediate cells, GSIB-4 labeled endothelial cells, and DAPI labeled the nuclei.