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.
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.
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 cochlea4,5,6,7,8,9,10,11. 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 disorders17,18. 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 EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, 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.
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
2. Exposing bony labyrinth
3. Inner ear extraction
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).
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).
6. SV single-nucleus sequencing
7. SV immunostaining and tissue mounting
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.
sNuc-Seq is useful in investigating the transcriptional profile of the various cells in the heterogenous SV. One way this may be visualized is by clustering on a 2D UMAP (Figure 3). The dataset generated from sNuc-Seq may be evaluated using different data analysis techniques, further outlined in the discussion.
SV whole mounting with GS-IB4 and DAPI immunolabeling, along with Kcnj10-ZsGreen fluorescence, are presented in Figure 4. Using florescence, the SV can be visualized after removing the surrounding tissues and mounting. In these mice, ZsGreen is expressed particularly in the SV intermediate cells. The vasculature of the SV can be visualized in red (endothelial cells, GS-IB4).
Figure 1: Schematic of the stria vascularis cellular heterogeneity and organization. Stria vascularis cellular heterogeneity and organization. Schematic of the stria vascularis (SV) and its relationship to structures in the cochlea. The SV is composed of three layers of cells and is responsible for generating EP and high potassium concentration in the endolymph-containing scala media. The relationship between the marginal, intermediate, and basal cells are demonstrated with the marginal extending basolateral projections to interdigitate with the intermediate cells, which have bidirectional cellular projections that interdigitate with both the marginal and basal cells. In addition to these cell types, other cell types, including spindle cells (yellow), endothelial cells, pericytes, and macrophages (not shown) are present in the SV. Used with permission from Korrapati et al.30. Please click here to view a larger version of this figure.
Figure 2: Dissection of an adult mouse cochlea at greater than or equal to postnatal day 30 (P30+). (A) Specimen during inner ear extraction from the surrounding temporal bone during step 3.2. The inner ear is outlined in purple. (B) Specimen during step 4.2 showing the surface of the cochlea and underlying pigmented SV. (C) Specimen during step 4.4 showing bone removed from the apical turn and the exposed SV (yellow arrow). (D) Specimen during step 4.5 showing the cochlea with more bone removed from the apical and middle turns. (E) Specimen during step 4.5 showing the removal of bone covering the middle turn, showing the underlying SV (yellow arrow). (F) Example of smaller pieces of SV (yellow arrow) and the lateral wall (blue arrow) during step 4.10. (G) Example of fluorescent ZsGreen SV while the remaining lateral wall remains dark. Kcnj10-ZsGreen is particularly expressed in the intermediate cells of the SV. (H) Example of larger pieces of SV (yellow arrow) and the lateral wall (blue) being separated during step 4.12. (I) The SV fully separated from the lateral wall during step 4.12. Please click here to view a larger version of this figure.
Figure 3: UMAP plot of sNuc-Seq datasets from an RNAlater-treated adult stria vascularis. Samples preserved with RNAlater treatment of adult SV tissue with subsequent nuclei isolation were clustered by a modularity-based clustering method with the Leiden optimization algorithm and visualized in a dimensionally reduced fashion by a 2D UMAP plot. Each dot represents a single cell, and cells with similar transcriptional profiles are clustered together. Cell types are colored based on their expression of known genes expressed by adult SV cell types. The samples were stored in RNAlater for no more than 6 months. The SV from about five CBA/J P30 mice were used to generate this dataset. Please click here to view a larger version of this figure.
Figure 4. Stria vascularis whole mounting from a P30 Kcnj10-ZsGreen mouse. Confocal image of SV whole mounting from a P30 mouse, demonstrating ZsGreen expression in the intermediate cells, GS-IB4 (red) labeling endothelial cells, and DAPI (white) labeling nuclei. Please click here to view a larger version of this figure.
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 transcriptional heterogeneity amongst SV cell types and can be used in the future to investigate the contribution of these cell types to SV function, including the generation and maintenance of EP. Datasets generated using sNuc-Seq can be displayed in a dimensionally reduced fashion to facilitate the understanding of overall transcriptional differences and compare different cell populations using a UMAP plot Figure 4. There are numerous ways to conduct bioinformatic analysis for the interpretation of sNuc-Seq datasets. These include but are not limited to: differential expression analysis, single-cell regulatory network inference and clustering (SCENIC), heatmap or grin violin plot construction, and regulon analysis18. Further computational analysis techniques for single-cell RNA sequencing are similar to sNuc-Seq, and are discussed in more detail in reviews by Hwang et al.32, Shafer33, and Chen et al.34.
The other major application of this dissection technique is immunostaining to better study selected structures within the SV. We can observe this in the setting of an SV whole mount that was imaged using confocal microscopy (Figure 3). The nuclei of all cells are visualized with DAPI labeling. The capillaries are seen via endothelial cell staining with GS-IB4 in red. Intermediate cells, in this case, have been genetically modified using an intermediate cell-specific promoter to express ZsGreen.
Limitations of sNuc-Seq include: (1) lack of spatial information, (2) biases against immune cell populations, and (3) exclusion of cytoplasmic RNA. Spatial transcriptomic techniques can provide more specific information about cellular subpopulations within tissue35. Given protocol differences between sNuc-Seq and single-cell RNA sequencing, such as tissue dissociation and storage, the sNuc-Seq-generated transcriptional profile may bias against immune cells while being more powerful at characterizing attached cell types36. Also, single-cell RNA sequencing includes cytoplasmic RNA such as mitochondrial RNA, while sNuc-Seq does not. Despite these instances of transcriptional differences between the two methods, it has been demonstrated that, by and large, sNuc-Seq has a similar transcriptional profile to whole single-cell RNA sequencing37, and in the SV in particular30.
For successful microdissection, there are several critical steps to be aware of. In the first few steps, one must properly extract the inner ear to avoid crushing the underlying cochlea. Observation of proper temporal bone extraction is shown in Figure 2A. Once the cochlea is properly exposed, care must be taken to gently scrape and break the cochlear bone to expose the underlying lateral wall and SV. Once the lateral wall and SV are extracted successfully, the user must carefully detach the SV from the lateral wall. Enough force in the correct plane should be applied to pry the SV from the lateral wall, while avoiding damage to the fragile SV. For SV microdissection, it may be helpful to think of “dissecting other structures away from the SV”, rather than “dissecting the SV out of the cochlea”.
This protocol can be modified for different ages of mice and for different mammalian species, such as rats. For younger mice, tissues will differ in texture and structures will be smaller in general. For different mammalian species, the cochlear anatomy will be relatively similar, with the size of the animal determining the majority of the difference.
In general, drawbacks of this protocol are similar to other cochlear microdissection techniques38. Isolating the delicate SV can be technically challenging and it can be broken into smaller pieces.
This technique is not optimal for saving the organ of Corti for whole mounting as it is often damaged with this protocol. There exists other dissection techniques specifically aimed at preserving the organ of Corti for whole mounting and immunostaining38, but these have their own drawbacks, considering sNuc-Seq must be done with fresh samples to avoid RNA degradation, not in the setting of tissue fixation and decalcification.
With time and repetition, users will become more comfortable with this SV microdissection protocol.
The authors have nothing to disclose.
This research was supported in part by the Intramural Research Program of the NIH, NIDCD to M.H. (DC000088)
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 |