preparation of stria vascularis single-nucleus suspension

用于单核测序或免疫染色的血管纹显微切割

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&#39;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.

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

解剖成年小鼠血管纹以进行单核测序或免疫染色

Watch this Scientific Journal Video about Preparation of Stria Vascularis Single-Nucleus Suspension at JoVE.com

Preparation of Stria Vascularis Single-Nucleus Suspension  

血管纹单核悬浮液的制备

在两毫升Dounce均质器中均质化小鼠耳朵解剖的血管纹组织，在冰上划10至20次。将组织在冰上裂解25分钟。然后，通过 30 微米过滤器过滤组织，并在 500G 下在 4 摄氏度下旋转滤液五分钟。除去上清液并将细胞沉淀重悬于一毫升细胞核洗涤液和重悬缓冲液中。接下来，通过10微米过滤器过滤细胞，并在4摄氏度下以500G离心五分钟。除去上清液并重悬于50微升细胞核洗涤和重悬缓冲液中。台盼蓝染色后，将5微升细胞悬液稀释到50微升1X PBS中并计数细胞。要制备单个细胞核捕获，请将具有所需核密度的样品加载到芯片上。

preparation-of-stria-vascularis-single-nucleus-suspension

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Biology

Preparation of Stria Vascularis Single-Nucleus Suspension

147 次观看. 在两毫升Dounce均质器中均质化小鼠耳朵解剖的血管纹组织，在冰上划10至20次。将组织在冰上裂解25分钟。然后，通过 30 微米过滤器过滤组织，并在 500G 下在 4 摄氏度下旋转滤液五分钟。除去上清液并将细胞沉淀重悬于一毫升细胞核洗涤液和重悬缓冲液中。接下来，通过10微米过滤器过滤细胞，并在4摄氏度下以500G离心五分钟。除去上清液并重悬于50微升细胞核洗涤和重悬...

视频: Preparation of Stria Vascularis Single-Nucleus Suspension  

Dissection of 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 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.