解剖成年小鼠血管纹以进行单核测序或免疫染色
Summary
血管纹对于内耳蜗电位的产生至关重要。在这里，我们介绍了成年小鼠血管纹的解剖以进行单核测序或免疫染色。
Abstract
由血管纹产生的内耳蜗电位对于维持有利于适当的毛细胞机械转导和最终听力的环境至关重要。血管纹的病变可导致听力下降。解剖成体血管纹可以集中捕获单核，随后进行单核测序和免疫染色。这些技术用于在单细胞水平上研究血管纹的病理生理学。
单核测序可用于血管纹的转录分析。同时，免疫染色在识别特定细胞群方面仍然有用。这两种方法都需要适当的血管纹夹层作为先决条件，这可能被证明在技术上具有挑战性。
Introduction
耳蜗由三个充满液体的腔室组成，即前庭鳞片、中鳞片和鼓鳞片。前庭鳞片和鼓鳞片均含有外淋巴液，其具有高浓度的钠（138 mM）和低浓度的钾（6.8 mM）1。Scala介质含有内淋巴液，其具有高浓度的钾（154mM）和低浓度的钠（0.91mM）1,2,3。这种离子浓度的差异可以称为耳蜗内电位（EP），主要是由钾离子沿着耳蜗侧壁通过血管纹（SV）中的各种离子通道和间隙连接产生的4,5,6,7,8,9,10,11 .SV是一种异质的，高度血管化的组织，排列在耳蜗侧壁的内侧，包含三种主要细胞类型：边缘细胞，中间细胞和基底细胞12（图1）。
边缘细胞通过紧密连接连接，形成SV的最内侧表面。顶膜面向斯卡拉介质的内淋巴，并通过各种通道（包括KCNE1 / KCNQ1，SLC12A2和Na + – K + – ATP酶（NKA）5,10,13,14）促进钾离子转运到内淋巴中。中间细胞是位于边缘细胞和基底细胞之间的色素细胞，并使用KCNJ10（Kir 4.1）15,16促进钾通过SV运输。基底细胞靠近耳蜗侧壁，与螺旋韧带的纤维细胞密切相关，以促进外淋巴12的钾循环。SV的病理学与许多耳科疾病有关17,18。在主要SV细胞类型（如Kcnq1，Kcne1，Kcnj10和Cldn11）中表达的基因突变可导致耳聋和SV功能障碍，包括EP19,20,21,22,23的丢失。除了三种主要细胞类型外，SV中还有其他研究较少的细胞类型，例如梭形细胞22，根细胞12,24，巨噬细胞25，周细胞26和内皮细胞27，其作用不完全确定，涉及离子稳态和EP 28的产生。
与批量RNA测序相比，单核RNA测序（sNuc-Seq）提供有关细胞异质性的信息，而不是一组细胞29的mRNA平均值，并且在研究异质SV30时特别有用。例如，sNuc-Seq已经产生了转录分析，表明纺锤体和根细胞可能在EP生成，听力损失和梅尼埃病中发挥作用18。各种SV细胞类型的进一步转录表征可以为我们提供有关SV相关听力波动和听力损失的不同机制和亚型病理生理学的宝贵信息。这些精细的内耳结构的收获对于最佳组织分析至关重要。
在本研究中，描述了从成年小鼠耳蜗中获取和分离血管纹以进行sNuc-Seq或免疫染色的显微解剖方法。需要解剖成年小鼠SV以了解各种SV细胞类型并进一步表征它们在听力中的作用。
Protocol
所有动物实验和程序均按照美国国立神经系统疾病和中风研究所动物护理和使用委员会以及国立卫生研究院国家耳聋和其他沟通障碍研究所批准的协议进行。所有实验方案均由美国国立神经系统疾病和中风研究所动物护理和使用委员会以及美国国立卫生研究院国家耳聋和其他沟通障碍研究所批准。所有方法均按照美国国立神经系统疾病和中风研究所动物护理和使用委员会以及国立卫生研究院国家?…
Representative Results
我们提出了一种分离用于sNuc-Seq或免疫染色的SV的方法。耳蜗相对于SV的相关解剖结构（图1）可以帮助用户更好地了解SV的组织和解剖方案的步骤。 相关视频详细介绍了从P30鼠标显微解剖SV的每个步骤， 图2显示了这种解剖和分离SV的关键步骤的快照。 sNuc-Seq可用于研究异质SV中各种细胞的转录谱。一种可视化方?…
Discussion
在单细胞测序出现之前，许多研究人员使用批量组织分析，这只能分析跨细胞平均的转录组。特别是，单细胞和sNuc-Seq使得分离单细胞或单核的转录组成为可能，分别32。在这种情况下，可以鉴定边缘细胞、中间细胞和基底细胞以及梭形细胞的单核转录组30。这使得能够研究SV细胞类型之间的转录异质性，并可用于将来研究这些细胞类型对SV功能的贡献，包括EP的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国立卫生研究院校内研究计划，NIDCD到MH（DC000088）的部分支持
Materials
| 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 |
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Cite This Article
Strepay, D., Olszewski, R., Taukulis, I., Johns, J. D., Gu, S., Hoa, M. Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining. J. Vis. Exp. (194), e65254, doi:10.3791/65254 (2023).