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

Summary

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Abstract

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level.

Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Introduction

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)¹. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)^1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea^{4,5,6,7,8,9,10,11}. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells¹² (Figure 1).

Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na⁺-K⁺-ATPase (NKA)^5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)^15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph¹². Pathology of the SV has been implicated in numerous otologic disorders^17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP^{19,20,21,22,23}. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells²², root cells^12,24, macrophages²⁵, pericytes²⁶, and endothelial cells²⁷, that have incompletely defined roles involving ionic homeostasis and the generation of EP²⁸.

In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells²⁹, and can be particularly useful when studying the heterogenous SV³⁰. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere's disease¹⁸. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.

In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

Protocol

All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All experimental protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All methods were carried out in accordance with relevant guidelines and regulations of the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.

1. Animal euthanasia

1. Use C57BL/6J or Kcnj10-ZsGreen transgenic mice, postnatal day 30+ (P30+), that weigh between 15-20 g.
2. Euthanize an adult mouse (age P30 or older) using an approved protocol (here CO₂ asphyxiation). Perform decapitation as a secondary step.
   NOTE: If immunostaining, some antibodies may have background signal due to nonspecific binding to blood in the capillaries of the SV. Cardiac perfusion with fixative can address this issue by removing blood from the capillaries of the SV. However, most antibodies can achieve good results without this method, and it will not be covered in this protocol.

2. Exposing bony labyrinth

1. Clean the mouse by spraying with 70% ethanol and wiping with a paper towel. Pay particular attention to the head region to reduce the risk of contamination. Remove the skin from the skull by pulling the skin toward the nose.
2. Using sharp scissors, split the skull along the midsagittal plane to separate the left and right portions.
3. Remove the brain from the skull to expose the bony labyrinth.

3. Inner ear extraction

1. Identify the inner ear within the temporal bone (Figure 2A) and scrape away the cranial nerves using #55 forceps.
   NOTE: The user should stabilize the specimen with their nondominant hand using a second set of #55 forceps. Different areas of the specimen may be grabbed with the stabilizing forceps throughout the dissection if critical areas of the specimen are avoided (e.g., do not crush the cochlea).
2. Using #55 forceps, dissect out the bulla and capsule, detaching them from surrounding bone. Remove any remaining soft tissue from the inner ear surface.
3. Transfer the specimen to a clear tissue culture dish containing 1x cold PBS (phosphate-buffered saline), pH 7.4, and place under a dissection scope (Figure 2B).

4. SV dissection

NOTE: With practice, it is possible to dissect the SV as one long piece resembling a ribbon. The SV is fragile, so if it breaks into pieces, these may be stored together. Alternatively, these can be stored in separately labeled wells according to their turn (e.g., basal, middle, apical).

1. If the dissection scope light sources can be moved, place one light above the dish and the second light source from the side, parallel to the dissecting surface. This allows for a better contrast of the tissue under the cochlear bone.
2. Using #55 forceps, hold the specimen by the vestibular portion with the cochlea facing up.
3. Using #55 forceps, pierce through the cochlear bone at the apex.
   NOTE: #5 forceps are thicker than #55 and may be used for breaking bone, but the increase in size/strength sacrifices the fineness of the forceps and the ability to manipulate small tissues. Consider using forceps made with a material such as inox or dumostar to avoid damage that may occur to more delicate forceps. Avoid pushing the forceps too deep under the bone to avoid damaging the SV.
4. Using #55 forceps, scrape along the apical turn (Figure 2C), applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall.
   NOTE: It may be helpful to think about this dissection as "removing everything away from the SV", rather than "dissecting the SV out of the cochlea".
5. Continue using #55 forceps to remove cochlear bone pieces from the apical and middle turns (Figure 2D,E). Gently pushing down the lateral wall, pry and remove bone wall pieces toward the middle turn to expose the lateral wall of the apical and middle turns.
6. Continuing using #55 forceps, gently push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of apical and middle turns from the spiral ganglion along the outer hair cell layer. Remove pieces of spiral ganglion from inside the cochlea.
   NOTE: #55 forceps are smaller and provide an advantage when manipulating small and delicate tissues. Extra care should be taken to avoid bending or damaging them.
7. To have better access to the lateral wall of the basal turn, detach the cochlea from the vestibular portion of the temporal bone using #55 forceps. Put the #55 forceps into the round and oval window and push down toward the vestibule. The cochlea will detach. Remove the vestibular portion of the inner ear.
8. Continuing using #55 forceps, now remove pieces of basal cochlear bone, starting from the middle turn area. Gently push the lateral wall layer under the bone to detach it.
9. Remove the farthest basal part of the lateral wall by prying and gently pulling the tissue. The bone in this region may be too thick to be removed without damaging the soft tissue below.
10. Now, with the basal part of the lateral wall detached, separate as much of the lateral wall from the cochlea as possible. This can be done by tracing the #55 forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall. This can be done all the way to the apex in one piece if the user is experienced. Move the lateral wall to fresh PBS.
    NOTE: Although larger pieces of SV may be easier to image, given the delicate nature of the tissues, smaller pieces can be taken (Figure 2F,G), and depending on the size, can also work for imaging.
11. Use #55 forceps to lay the lateral wall flat. The SV should be visible as a darker layer of tissue on the internal side of the lateral wall.
    NOTE: Users may find, particularly closer to the apical end, that some of the SV tissue incidentally detaches from the lateral wall throughout the dissection.
12. Gently pry the SV layer to detach it from the lateral wall at its basal end (Figure 2H). Gently push aside the detached SV and move the forceps further toward the apical end of the lateral wall, detaching the SV as one long ribbon if possible (Figure 2I). Do not squeeze the SV with forceps to pull it. If the tissue is too fragile to be grabbed, it can alternatively be collected using a tissue scoop, such as a chalazion curette.
    NOTE: Moving the SV should always be done under light microscopy to ensure it is not lost. Always be careful when grabbing using forceps due to the risk of damage. Users may find that using chalazion curette mitigates the risk of damage to SV tissue. For single-nucleus sequencing, proceed to section 5. For SV immunostaining, proceed to section 7.

5. SV single-nucleus suspension

NOTE: This protocol is adapted for SV tissue specifically from a published manufacturer's single-nucleus suspension protocol. Platforms from different manufacturers may be used³¹. Given platform-specific variation, it is recommended to review the manufacturer-specific protocol provided with the equipment. To achieve optimal results for sNuc-Seq and minimize RNA degradation, the faster the tissue dissection the better (recommended within 15-20 min from euthanasia). It may be helpful to euthanize one animal at a time and only when ready to dissect. Having multiple people simultaneously work on the dissections can also eliminate degradation time (e.g., one lab personnel working on the left ear while another works on right ear).

1. If the SV tissue will be used for sNuc-Seq, place the tissue in chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl², 0.005% nonidet P40 in nuclease free water).
   NOTE: If sequencing is to be done immediately following dissection, continue the protocol. If it is desired to pause the protocol, SV preservation is possible by placing the SV into RNAlater. Store overnight at 4 °C, then flash freeze in liquid nitrogen and store at -80 °C until ready to use. When ready to use, thaw and wash twice in PBS for 5 min each, then proceed with homogenization (step 5.2).
2. Homogenize the tissue in a 2 mL dounce homogenizer with 10-20 strokes on ice.
   NOTE: The glass cylinder will manually contact the bottom of the glass tube, fragmenting the SV. The SV is delicate, so 20 strokes usually achieves successful homogenization.
3. Lyse the tissue on ice for 25 min.
   NOTE: The lysis time varies according to the tissue type. A 25 min lysis in SV tissue may achieve a low cell viability (less than 4%), but the time may be adapted if the cell viability is too high (step 5.8).
4. Filter through a 30 µm filter and spin the filtrate at 500 x g for 5 min at 4 °C.
5. Remove the supernatant and resuspend the cell pellet in 1 mL of nuclei wash and resuspension buffer (1x PBS, 1% bovine serum albumin [BSA], 0.2U/µL RNase inhibitor).
6. Filter the cells through a 10 µm filter and centrifuge at 500 x g for 5 min at 4 °C.
7. Remove the supernatant and resuspend in 50 µL of nuclei wash and resuspension buffer.
8. Count the nuclei using a cell counter and trypan blue staining. Dilute 5 µL of the cell suspension into 50 µL of 1x PBS for cell counting; The viability should be minimal (3%-4%) at this point. If the viability is found to be higher, adapt the protocol for step 5.3 (lysis) and standardize the lysis time to ensure adequate lysis of the cells.
   NOTE: A high cell viability in a single nucleus preparation means there are more intact cells than desired, and that additional digestion may be required.
9. Load the sample with the desired nuclear density onto the manufacturer's apparatus/chip (see manufacturers guide). Single-nuclei captures are now ready.
   ​NOTE: From one adult mouse SV, 50 µL of the suspension at a concentration of 150,000 nuclei/mL is usually achieved.

6. SV single-nucleus sequencing

1. Send the samples to the core facility for sequencing.
   NOTE: This part of the protocol follows the general steps of (1) gel beads-in emulsion (GEM) generation and barcoding, (2) post-GEM-RT cleanup and cDNA amplification, and (3) 3' gene expression library construction. The remainder of the sNuc-Seq protocol is relatively lengthy, and it is recommended that readers use their manufacturer-specific protocol. The manufacturer guide will likely describe steps with specific details and accompanying images. There may also be 'how-to' videos on the manufacturer website. Datasets generated may be represented in a dimensional reduced fashion, such as a uniform manifold approximation and projection (UMAP; Figure 3). Many labs utilize a sequencing core that may perform this protocol for users.

7. SV immunostaining and tissue mounting

1. If the tissue will be used for immunostaining, transfer it to a 24-well plate, submerged in 200 µL of 4% paraformaldehyde (PFA) in 1x PBS, and incubate for 20 min at room temperature.
   NOTE: It is helpful to perform all liquid removal, washing, and immunostaining underneath a microscope. Visualization while pipetting the tissue will ensure it is not accidently lost during liquid removal. The volumes of the antibodies and washes may be adjusted as long as the volume is large enough to submerge the SV tissue within the solution.
2. After the fixation step, remove the PFA by performing two short washes in 1x PBS (approximately 500 µL) at 4 °C for 5 min each on an orbital shaker at low (30-50) RPM. If storing, the tissue can be stored in 1x PBS at 4 °C.
3. Remove the PBS and permeabilize and block for a minimum of 1 h at room temperature in approximately 300 µL of PBS-T solution (0.05 Tween20 in 1x PBS with 10% fetal bovine serum).
   NOTE: Primary and secondary antibodies should be diluted in this blocking solution.
4. Stain the tissue with primary antibody according to the specifications of the particular antibody used, usually overnight at 4 °C . Remove the remaining PBS and add the diluted primary antibody.
   NOTE: The dilution should be chosen based on the manufacturer suggestions, published data, previous experience, etc. Usually, the first concentration to test is a 1:100-200 dilution. For the example presented in this paper, the GS-IB4 and DAPI antibodies were each diluted at 1:200. If staining for more than one protein, multiple primary antibodies can be combined into the same solution, as long as each primary antibody has a different host to avoid cross reactivity of secondary antibodies.
5. Wash the primary antibody off the tissue using approximately 500 µL of 1x PBS twice for 10 min each on an orbital shaker at low RPM.
   NOTE: Continue to ensure that the SV tissue is submerged in the solution, and not stuck on the side of the well.
6. Stain with a secondary antibody according to the specifications of the particular antibody used, usually for 2 h at room temperature on an orbital shaker at low RPM. Cover the 24-well plate so the fluorescently tagged secondary antibody is protected from light.
   NOTE: If more than one secondary antibody is needed, combine the secondary antibodies into the same solution. Ensure to avoid cross labeling.
7. Wash the secondary antibody off the tissue using approximately 500 µL of 1x PBS four times for 5 min each on an orbital shaker at low RPM. Keep the 24-well plate covered to protect it from light.
8. Prepare the larger microslide (75 mm x 25 mm) by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 mm x 18 mm smaller glass coverslip. Place one drop of mounting reagent (approximately 50 µL) in between the glue streaks.
   NOTE: The glue creates a small amount of vertical space so that when the second coverslip is placed on top, the SV tissue sample can safely exist, but continue to remain in place. While the stria thickness is 30-40 microns, squeezing the tissue between the glass surfaces should be avoided to preserve morphology. Alternatively, clear tape may also be used.
9. Using #55 forceps, grab as little tissue as possible on one end of the SV and transfer from the PBS to the drop of mounting reagent on the larger microslide (75 mm x 25 mm). Place one end of a smaller glass coverslip (18 mm x 18 mm) on the slide where one streak of glue was placed and gently release to avoid creating air bubbles. The coverslip will fall to the other glue streak and will cover the SV tissue.
10. Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount. Label the specimen accordingly on the glass coverslip away from the visualization field. It is now ready for confocal microscopy (Figure 4).
11. Visualize the SV tissue using a dissection microscope to ensure it is laying flat and not near any air bubbles. If the SV tissue is not oriented correctly, the user can attempt to push out air bubbles or carefully remove the smaller glass coverslip and reposition the SV. This must be done gently to avoid breaking the glass coverslips.

Results

We present a method to isolate the SV to be used for either sNuc-Seq or immunostaining. The relevant anatomy (Figure 1) of the cochlea relative to the SV can help users better understand the organization of the SV and steps of the dissection protocol.

Each step of this microdissection of SV from a P30 mouse is detailed in the associated video, and snapshots of the key steps of this dissection and isolation of SV are presented in Figure 2

Discussion

Prior to the advent of single-cell sequencing, many researchers used bulk tissue analysis, which only made it possible to analyze transcriptomes averaged across cells. In particular, single-cell and sNuc-Seq made it possible to isolate the transcriptome of a single cell or single nucleus, respectively³². In this instance, single-nucleus transcriptomes can be identified for marginal, intermediate, and basal cells, as well as spindle cells³⁰. This enables the investigation of...

Materials

Name	Company	Catalog Number	Comments
10-µm filter (Polyethylenterephthalat)	PluriSelect	#43-50010-01	Filter tissue during sNuc-Seq
18 x 18 mm cover glass	Fisher Scientific	12-541A	Cover slip to mount SV
30-µm filter (Polyethylenterephthalat)	PluriSelect	#43-50030-03	Filter tissue during sNuc-Seq
75 x 25 mm Superfrost Plus/Colorforst Plus Microslide	Daigger	EF15978Z	Microslide to mount SV on
C57BL/6J Mice	The Jackson Laboratory	RRID: IMSR_JAX:000664	General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.
Cell Counter	Logos Biosystems	L20001	Used for cell counting
Chalizon curette 5'', size 3 2.5 mm	Biomedical Research Instruments	15-1020	Used to transfer SV
Chromium Next GEM single Cell 3' GEM Kit v3.1	Chromium	PN-1000141	Generates single cell 3' gene expression libraries
Clear nail polish	Fisher Scientific	NC1849418	Used for sealing SV mount
Corning Falcon Standard Tissue Culture Dishes, 24 well	Corning	08-772B	Culture dish used to hold specimen during dissection
DAPI	Invitrogen	D1306, RRID: AB_2629482	Stain used for nucleus labeling
Dounce homogenizer	Sigma-Aldrich	D8938	Used to homogenize tissue for sNuc-seq
Dumont #5 Forceps	Fine Science Tools	11252-30	General forceps for dissection
Dumont #55 Forceps	Fine Science Tools	11255-20	Forceps with fine tip that makes SV manipulation easier
Fetal Bovine Serum	ThermoFisher	16000044	Used for steps of sNuc-Seq
Glue stick	Fisher Scientific	NC0691392	Used for mounting SV
GS-IB4 Antibody	Molecular Probes	I21411, RRID: AB-2314662	Antibody used for capillary labeling
KCNJ10-ZsGreen Mice	n/a	n/a	Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.
MgCl2	ThermoFisher	AM9530G	Used for steps of sNuc-Seq
Mounting reagent	ThermoFisher	#S36940	Mounting reagent for SV
Multiwell 24 well plate	Corning	#353047	Plate used for immunostaining
NaCl	ThermoFisher	AAJ216183	Used for steps of sNuc-Seq
Nonidet P40	Sigma-Aldrich	9-16-45-9	Used for steps of sNuc-Seq
Nuclease free water	ThermoFisher	4387936	Used for steps of sNuc-Seq
Orbital shaker	Silent Shake	SYC-2102A	Used for steps of immunostaining
PBS	ThermoFisher	J61196.AP	Used for steps of immunostaining and dissection
RNA Later	Invitrogen	AM7021	Used for preservation of SV for sNuc-Seq
Scizzors	Fine Science Tools	14058-09	Used for splitting mouse skull
Tris-HCl	Sigma-Aldrich	15506017	Used for steps of sNuc-Seq
Trypan blue stain	Gibco	15250061	Used for cell counting
Tween20	ThermoFisher	AAJ20605AP	Used for steps of sNuc-Seq
Zeiss STEMI SV 11 Apo stereomicroscope	Zeiss	n/a	Microscope used for dissections