用于三维电镜及X射线显微镜的完整小鼠耳蜗块染方法

En-bloc Staining of Intact Mouse Cochlea for Three-dimensional X-ray and Electron Microscopy

丁旭;王方方;王皓煜;梁雅坤;常海双;黄治物;华云峰;吴皓;

1:上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科

2:上海交通大学医学院耳科学研究所

3:上海市耳鼻疾病转化医学重点实验室

4:上海精准医学研究院

摘要
目的优化大尺寸脑组织样品的制备方法,将其用于小鼠耳蜗,获得适用于三维电镜和X射线显微镜成像的样品,用于耳蜗组织超微结构的病理及相关听觉疾病的研究。方法取3只野生型成年CBA/Ca小鼠耳蜗,经含0.08 M二甲胂酸盐缓冲液、2%多聚甲醛、2.5%戊二醛的4℃混合固定液灌注固定,添加了5%乙二胺四乙酸的混合液中低温短暂脱钙,采用还原性锇块染和铅染、脱水及树脂包埋。根据X射线显微镜和扫描电镜在不同尺度下的成像质量,对制备过程中步骤及时长进行了优化。结果 X射线显微镜下首次获得了具有毛细胞分辨率的完整小鼠耳蜗三维结构样品,其具有高染色对比度和均一性;扫描电镜及透射电镜下,样品中各结构,包括听神经髓鞘、传入传出突触、毛细胞静纤毛、线粒体结构形态均清晰可见。结论优化的完整小鼠耳蜗电镜块染样品制备方法使介观到微观的耳蜗三维超微结构的研究成为可能,为耳科疾病的病理结构研究提供了新方法。
关键词
耳蜗;超微结构;重金属块染;电镜;X射线显微镜
基金项目(Foundation):
国家自然科学基金(81800901)
作者
丁旭;王方方;王皓煜;梁雅坤;常海双;黄治物;华云峰;吴皓;
参考文献

1 Wan G,Corfas G.Transient auditory nerve demyelination as a new mechanism for hidden hearing loss[J].Nat Commun,2017,8:14487.

2 Wong AB,Rutherford MA,Gabrielaitis M,et al.Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis[J].EMBO J,2014,33:247.

3 Merchan-Perez A,Liberman MC.Ultrastructural differences among afferent synapses on cochlear hair cells:Correlations with spontaneous discharge rate[J].Journal of Comparative Neurology,1996,371:208.

4 Fuchs PA,Lehar M,Hiel H.Ultrastructure of cisternal synapses on outer hair cells of the mouse cochlea[J].J Comp Neurol,2014,522:717.

5 Shodo R,Hayatsu M,Koga D,et al.Three-dimensional reconstruction of root cells and interdental cells in the rat inner ear by serial section scanning electron microscopy[J].Biomed Res,2017,38:239.

6 Michanski S,Smaluch K,Steyer AM,et al.Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea[J].Proc Natl Acad Sci USA,2019,116:6415.

7 Bullen A,West T,Moores CA,et al.Association of intracellular and synaptic organization in cochlear inner hair cells revealed by 3D electron microscopy[J].Journal of Cell Science,2015,128:2529.

8 Anttonen T,Belevich I,Kirjavainen A,et al.How to bury the dead:elimination of apoptotic hair cells from the hearing organ of the mouse[J].J Assoc Res Otolaryngol,2014,15:975.

9 Mikula S,Denk W.High-resolution whole-brain staining for electron microscopic circuit reconstruction[J].Nat Methods,2015,12:541.

10 Kubota Y.Editorial:electron-microscopy-based tools for imaging cellular circuits and organisms[J].Front Neural Circuits,2019,13:64.

11 Hua Y,Laserstein P,Helmstaedter M.Large-volume en-bloc staining for electron microscopy-based connectomics[J].Nature Communications,2015,6:7923.

12 Briggman KL,Bock DD.Volume electron microscopy for neuronal circuit reconstruction[J].Curr Opin Neurobiol,2012,22:154.

13 El-Boustani S,Ip JPK,Breton-Provencher V,et al.Locally coordinated synaptic plasticity of visual cortex neurons in vivo[J].Science,2018,360:1349.

14 Hayworth KJ,Peale D,Januszewski M,et al.Gas cluster ion beam SEM for imaging of large tissue samples with 10 nm isotropic resolution[J].Nat Methods,2020,17:68.

15 Lin Y,Li LL,Nie W,et al.Brain activity regulates loose coupling between mitochondrial and cytosolic Ca2+ transients[J].Nat Commun,2019,10:5277.

16 Motta A,Berning M,Boergens KM,et al.Dense connectomic reconstruction in layer 4 of the somatosensory cortex[J].Science,2019,366:1.

17 Schmidt H,Gour A,Straehle J,et al.Axonal synapse sorting in medial entorhinal cortex[J].Nature,2017,549:469.

18 Denk W,Horstmann H.Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure[J].PLoS Biol,2004,2:e329.

19 Helmstaedter M.Cellular-resolution connectomics:challenges of dense neural circuit reconstruction[J].Nat Methods,2013,10:501.

20 Helmstaedter M,Briggman KL,Turaga SC,et al.Connectomic reconstruction of the inner plexiform layer in the mouse retina[J].Nature,2013,500:168.

21 Saliani A,Perraud B,Duval T,et al.Axon and myelin morphology in animal and human spinal cord[J].Front Neuroanat,2017,11:129.

22 Hequembourg S,Liberman MC.Spiral ligament pathology:a major aspect of age-related cochlear degeneration in C57BL/6 mice[J].Journal of the Association for Research in Otolaryngology,2001,2:118.

23 Francis HW,Rivas A,Lehar M,et al.Two types of afferent terminals innervate cochlear inner hair cells in C57BL/6J mice[J].Brain Res,2004,1016:182.

24 Stamataki S.Synaptic alterations at inner hair cells precede spiral ganglion cell loss in aging C57BL/6J mice[J].Hearing research,2006,221:104.

25 王根英,陆月良,颜永碧,等.耳蜗透射电镜标本制备技术[J].第二军医大学学报,1990(03):271.

26 张文丽,付兴华,李倩,等.耳蜗透射电镜超薄切片样品的制备技术改进[J].电子显微学报,2011,30:69.

27 Richter CP,Young H,Richter SV,et al.Fluvastatin protects cochleae from damage by high-level noise[J].Scientific Reports,2018,8:3033.

28 Topperwien M,Gradl R,Keppeler D,et al.Propagation-based phase-contrast x-ray tomography of cochlea using a compact synchrotron source[J].Sci Rep,2018,8:4922.

29 Buchanan J,Takeno M,Bodor A,et al.Using micro CT to evaluate stain penetration and establish fiducial marks for high throughput electron microscopy[J].Microscopy and Microanalysis,2019,25:1036.

30 Yi B,Hu S,Zuo C,et al.Effects of long-term salicylate administration on synaptic ultrastructure and metabolic activity in the rat CNS[J].Sci Rep,2016,6:24428.

31 Ye B,Wang Q,Hu H,et al.Restoring autophagic flux attenuates cochlear spiral ganglion neuron degeneration by promoting TFEB nuclear translocation via inhibiting MTOR[J].Autophagy,2019,15:998.