組織透明化在神經(jīng)科學(xué)領(lǐng)域中的應(yīng)用

Imaging large regions of the spinal cord of GFP-M mice

(a) Entire spinal cord of a GFP-M mouse cut into 3- to 4-mm-long segments, cleared and imaged with ultramicroscopy. (b) Drawing of the ultramicroscopy setup showing tissue positioning and the light path. (c) A spinal cord segment (length 4 mm, T12 to L2 spine level) of a GFP-M mouse scanned with ultramicroscopy shown in a horizontal view. (d) Cross-view projection (50-µm thickness) of the indicated region in c. White arrows in c,d mark individual axons in the white matter; red arrows mark cell bodies in the gray matter. (e) Traced white and gray matter boundaries and axon bundles (yellow arrows).

阿爾茲海默癥（AD）是一種起病隱匿的進(jìn)行性發(fā)展的神經(jīng)系統(tǒng)退行性疾病。臨床上以記憶障礙、失語、失用、失認(rèn)、視空間技能損壞、執(zhí)行功能障礙以及人格和行為改變等全面性癡呆表現(xiàn)為特征，細(xì)胞外β淀粉樣蛋白沉積和細(xì)胞內(nèi)tau蛋白磷酸化是AD的主要病理特征。Liebmann等人利用iDISCO技術(shù)對(duì)阿爾茲海默癥模型小鼠腦進(jìn)行透明化闡明了β淀粉樣蛋白沉積和小膠質(zhì)細(xì)胞及脈管系統(tǒng)之間的空間分布關(guān)系，這將對(duì)阿爾茲海默癥的科學(xué)研究和治療產(chǎn)生重大意義。

Double Labeling of β-Amyloid Plaques and Reactive Microgliosis, Axonal Dystrophy, or Vasculature in Cleared AD Brains with Volume Imaging
（A）confocal image of β-amyloid plaques and cell nuclei double staining in a 17-month-old2xTg AD cleared mouse brain. (B) Confocal optical slices of microglia stained with anti-Iba1 antibody. (C) 3D surface rendering of four isolated β-amyloid plaques and neighboring microglia cells from the AD brain described in (B). (D)Maximum intensity projection of β-amyloid plaques and neuro filament H staining in a10-month-old 2xTgAD cleared mouse cortex(500mmthick). (E)Magnified region from (D) showing labeled axons in absence of β-amyloid plaques. (F) Magnified region from (D) showing dystrophic axons surrounding labeled β-amyloid plaques. (G) Maximum intensity projection of optical section (1mm) from the cortex of an 11-month-old 2xTgAD mouse brain labeled for plaques (anti-b amyloid anti-bodies) and vasculature. (H)Maximum intensity projection of optical section (1mm) from the cortex and hippocampus of an 8-month-old 2xTgAD mouse brain labeled for plaques (Congored) and vasculature.

Chung等人通過CLARITY技術(shù)獲得透明而完整的小鼠大腦，利用Thy1–eYFP信號(hào)實(shí)現(xiàn)了對(duì)小鼠大腦神經(jīng)元進(jìn)行遠(yuǎn)距離投射、神經(jīng)回路、細(xì)胞關(guān)系、亞細(xì)胞結(jié)構(gòu)、蛋白質(zhì)復(fù)合物、 核酸和神經(jīng)遞質(zhì)的成像。展現(xiàn)了大腦中復(fù)雜的精細(xì)連接和分子結(jié)構(gòu)。

Intact adult mouse brain imaging

a, Cajal quote before CLARITY. b, Cajal quote after CLARITY: Thy1–eYFP line-H mouse brain after hydrogel–tissue hybridization, ETC and refractive-index matching. c, Fluorescence image of brain depicted in b. d, Dorsal aspect is imaged, then brain is inverted and ventral aspect imaged. e, Three-dimensional rendering of clarified brain imaged；f, Non sectioned mouse brain tissue showing cortex, hippocampus and thalamus；g–l, Optical sections from f showing negligible resolution；m, Cross-section of axons in clarified Thy1–channelrhodopsin2 (ChR2)–eYFP striatum；n, Dendrites and spines of neurons in clarified Thy1–eYFP line-H cortex.

MURAKAMI等人應(yīng)用CUBIC-X透明并膨大小鼠大腦，在亞細(xì)胞水平對(duì)整個(gè)小鼠大腦進(jìn)行成像，并繪制出了一張小鼠大腦圖譜。他們采用化學(xué)方法標(biāo)記了大腦中的每個(gè)細(xì)胞，然后在大腦透明化的同時(shí)將其尺寸擴(kuò)大了十倍，利用精密成像技術(shù)對(duì)神經(jīng)元進(jìn)行了三維重建，總計(jì)約7200萬個(gè)細(xì)胞。



Construction of a single-cell-resolution mouse brain atlas (CUBIC-Atlas)

a, Overview of construction of the CUBIC-Atlas. b, The CUBIC-Atlas. Horizontal, sagittal and coronal view of single-plane images (left) and volume-rendered images (right) of the CUBIC-Atlas. c–j, Major anatomical areas in the CUBIC-Atlas. k, Overview of whole-brain cell counting in C57BL/6N 8-week-old male mice. l, Cell numbers in each brain area.

總之，組織透明技術(shù)結(jié)合顯微成像技術(shù)，極大推動(dòng)了全身或全器官成像的進(jìn)程，有助于加強(qiáng)對(duì)完整生物系統(tǒng)的綜合理解，為神經(jīng)科學(xué)研究提供了更加細(xì)致的信息，是神經(jīng)科學(xué)研究領(lǐng)域強(qiáng)有利的工具。

目前組織透明化方法有油性，基于水凝膠和水性的組織透明化方法。其中油性組織透明化方法主要有BABB, 3DISCO, uDISCO，F(xiàn)DISCO和 PEGASOS等，基于水凝膠的透明化方法有CLARITY和SHIELD，水性組織透明化方法有CLearT/CLearT2,SeeDB, FRUIT, Scale和CUBIC等方法。而锘海研發(fā)的透明化試劑盒依托測樣服務(wù)中積累到的豐富而寶貴的經(jīng)驗(yàn)，對(duì)各種透明化方法進(jìn)行測試和比較后對(duì)試劑配方進(jìn)行了精心優(yōu)化，使其在組織熒光保護(hù)、樣本形態(tài)維持、降低自發(fā)熒光等方面表現(xiàn)出非常優(yōu)異的性能，是研究神經(jīng)科學(xué)的不二之選。


                                                    锘海組織透明化試劑盒  

參考文獻(xiàn)：

1. Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N, Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277.
2. Liebmann T, Renier N, Bettayeb K, Greengard P, Tessier-Lavigne M, Flajolet M. Three-Dimensional Study of Alzheimer's Disease Hallmarks Using the iDISCO Clearing Method. Cell Rep. 2016 Jul 26;16(4):1138-1152. doi: 10.1016/j.celrep.2016.06.060. Epub 2016 Jul 14. PMID: 27425620; PMCID: PMC5040352.
3. Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K. Structural and molecular interrogation of intact biological systems. Nature. 2013 May 16;497(7449):332-7. doi: 10.1038/nature12107. Epub 2013 Apr 10. PMID: 23575631; PMCID: PMC4092167.
4. Murakami TC, Mano T, Saikawa S, Horiguchi SA, Shigeta D, Baba K, Sekiya H, Shimizu Y, Tanaka KF, Kiyonari H, Iino M, Mochizuki H, Tainaka K, Ueda HR. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat Neurosci. 2018 Apr;21(4):625-637. doi: 10.1038/s41593-018-0109-1. Epub 2018 Mar 5. PMID: 29507408.
5. Tian T, Yang Z, Li X. Tissue clearing technique: Recent progress and biomedical applications. J Anat. 2021 Feb;238(2):489-507. doi: 10.1111/joa.13309. Epub 2020 Sep 16. PMID: 32939792; PMCID: PMC7812135.
 

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