Name: optical sectioning and visualization of the intervertebral disc from embryonic development to degeneration
Uploaded: 2021-07-08T15:00:00
Description: Watch this Scientific Journal Video about Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration at JoVE.com

我们感谢原始出版物的合著者的帮助和支持。我们感谢夏洛特·艾玛·班贝格（Charlotte Emma Bamberger）帮助获取了apotome图像。

为了分析胚胎发育和成熟，使用牛盘。为了评估IVD的变性，分析了人类样本。
从图宾根大学医院骨科和图宾根BG创伤中心接受腰椎间盘退行性变，椎间盘脱垂或脊柱创伤手术的患者那里获得了人IVD组织。在研究开始之前，已获得伦理委员会的全面批准（项目编号244/2013BO2）。在参与之前，所有患者都收到了书面知情同意书。这些方法是根据批准的指南进行的。
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使用荧光显微镜，加上镶嵌成像和光学切片，我们评估了软骨细胞在整个发育，成熟和变性过程中腰椎IVD肛门中的空间排列。虽然退行性组织可以从接受椎间盘变性手术的患者身上收获，但胚胎期和成熟期的分析需要使用模型生物（牛）。在早期胚胎发育期间，肛门中观察到高细胞密度。在进一步的发育过程中，可以观察到出生后生长和成熟细胞密度的显着降低。在具有晚期椎间盘变性的人体组?...

椎间盘（IVD）是一种基于软骨的结构，乍一看，它在生物化学和细胞结构方面类似于关节软骨1。事实上，关节软骨的IVD变性和骨关节炎（OA）的特征都是由于软骨磨损，软骨下囊肿和骨赘形成以及软骨下硬化症2，3而导致的关节空间变窄。尽管有这些看似相似的东西，但两种组织的结构和功能作用还是不同的。虽然关节软骨的基质主要由拱廊形成的II型胶原蛋白网络组成，但IVD由三种不同类型的组织组成：中心富含II型胶原的髓核承担轴向负荷并将其传递到密集排列的圆形胶原I型纤维环中，称为肛门纤维。它们的功能是吸收富含蛋白聚糖和水的原子核所接收的平移轴向压力及其拉伸纵向纤维强度。在每个细胞核和肛门的顶部和底部，透明软骨终板形成与相邻椎骨4的连接（图1）。
在关节软骨中，可以找到四种不同的空间软骨细胞模式：成对，弦，双弦，小分别大簇5，6，7（图2）。这种模式的变化与OA的发病和进展有关8，9。空间软骨细胞组织也表明了软骨的直接功能特性，即其刚度，强调了这种基于图像的分级方法的功能相关性10，11。这些模式还可以用已经存在的临床可用技术12来识别。由于IVD和关节软骨之间的相似性，可以假设IVD中也存在特征性软骨细胞模式。簇的形成是一种现象，在退化的IVD13，14中也观察到。
当试图分析IVD中的空间细胞组织时，有必要克服在研究关节软骨时不存在的几个技术难题：
首先，组织本身的加工比主要由II型胶原蛋白组成的均质透明软骨更具挑战性。IVD的主要纤维成分是I型胶原蛋白，这使得产生薄的组织学切片变得更加困难。虽然在透明关节软骨中，由于组织的"玻璃状"性质，甚至厚厚的切片也很容易被分析，IVD的I型胶原蛋白网络在光学上是高度不可穿透的。因此，强烈的背景噪声是IVD组织学中的常见问题。穿透这种光学致密组织的一种快速而廉价的方法是使用光学切片装置，例如通过Apotome。在这样的Apotome中，在常规荧光显微镜的照明途径中插入网格。在网格的前面放置一个平面平行的玻璃板。这会来回倾斜，从而将图像中的网格投影到三个不同的位置。对于每个 z 位置，将创建并叠加三个具有投影网格的原始图像。通过特殊的软件，可以计算出失焦的光。基本原则是，如果网格可见，则该信息处于焦点中，如果不是，则被视为失焦。使用这种技术，可以在合理的时间内获得聚焦良好的高分辨率图像。
其次，组织很难从人类捐赠者那里获得。当进行全膝关节置换术时，可以获得关节的整个表面，以便在手术过程中进行进一步分析。虽然椎间关节的骨关节炎也是整个关节的疾病，但软骨的质量仍然存在强烈的局灶性差异，通常关节的某些区域仍然完好无损，例如由于该区域的负荷减少。这种情况在IVD中有所不同，其中手术通常仅在椎间盘被整体破坏时进行。当从手术室从人类供体获取组织时，组织也是高度碎片化的，在进行进一步分析之前，有必要将组织正确分配到IVD的三种软骨类型之一。因此，为了能够对更大的组织切片进行更详细的分析并研究IVD的胚胎发育，因此，选择动物模型生物是必要的。
在选择这样的模型生物体时，重要的是要有一个在解剖结构和尺寸，机械负荷，当前细胞群以及组织组成方面与人类椎间盘相媲美的系统。为了本文所提出的技术的目的，我们建议使用牛腰椎间盘组织：人椎间盘的一个关键特性导致其低再生潜力，即在细胞核成熟期间，赱骨细胞的损失。然而，在许多模型生物中，可以检测到其整个生命周期的赭骨细胞。大多数失去赱枝细胞的少数动物，如绵羊，山羊或嗜软骨病的狗，其IVD比人类椎间盘小得多。只有腰椎牛椎间盘的行散盘直径与人类IVDs15相当。
导致早期椎间盘退化的一个关键因素是机械负荷过大。站立的牛在腰椎中的椎间盘内压力约为0.8 MPa，脊柱水平对齐。令人惊讶的是，这些压力与直立人脊柱（0.5 MPa）报告的腰椎椎间盘内压力相当15，16。此外，牛盘中的水和蛋白聚糖的含量与年轻人类的IVD相当17。因此，尽管四足动物的运动片段的实际运动模式可能与两足动物不同，但在总负荷和椎间盘特征方面，牛比其他已建立的IVD动物模型（如绵羊和狗）更接近人类生物学。
在该协议中，我们提出了一种技术，如何从空间软骨细胞组织的角度分析IVD从早期胚胎发育到终末期变性的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Merck KGaA,&#160; Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Microscope Slides SuperFrost Plus</td>
			<td>R. Langenbrinck, Germany</td>
			<td>03-0060</td>
			<td></td>
		</tr>
		<tr>
			<td>ApoTome</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td>462000115</td>
			<td></td>
		</tr>
		<tr>
			<td>AxioVision Rel. 4.8 with Modul MosaiX</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Actin Tracking Stain</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>A57249</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems, US</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco, Life Technologies, Germany</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich, US</td>
			<td>60004</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Miscoscope - Axio Observer Z1 with Axio Cam MR3 and Colibri</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td>3834000604</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck KGaA,&#160; Germany</td>
			<td>104002</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J 1.53a, with Cell counter plugin</td>
			<td>National Insittute of Health (NIH), US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Alexa Fluor 568 Phalloidin</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic Cover Glasses</td>
			<td>R. Langenbrinck, Germany</td>
			<td>01-1818/1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen Liquid Blocker</td>
			<td>Science Sevices&#160; GmbH, Germany</td>
			<td>N71310</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich, US</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich,US</td>
			<td>P5119</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>pf medical AG, Germany</td>
			<td>2023-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T. Compound</td>
			<td>Sakura Finetek, Netherlands</td>
			<td>SA6255012</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical sectioning and visualization of the intervertebral disc from embryonic development to degeneration

椎间盘（IVD）变性是腰痛的主要原因，并且对受影响的个体造成高度损害。为了解码椎间盘变性并能够开发再生方法，对IVD的细胞生物学的透彻理解至关重要。这种生物学中仍未得到解答的一个方面是细胞如何在生理状态和变性过程中在空间上排列的问题。IVD的生物学特性及其可用性使这种组织难以分析。本研究调查了肛门纤维中从早期胚胎发育到终末期变性的空间软骨细胞组织。应用光学切片方法（Apotome）以牛胚胎组织作为动物模型和从接受脊柱手术的患者那里获得的人盘组织进行高分辨率染色分析。由于早期胚胎牛盘中的软骨细胞密度非常高，细胞数量在妊娠，生长和成熟期间减少。在人盘中，细胞密度的增加伴随着组织变性的进展。正如已经在关节软骨中证明的那样，集群形成代表了晚期椎间盘变性的特征。

使用马赛克图像，可以清楚地识别IVD的结构及其在肛门和较软的细胞核中的致密胶原纤维网络（图4）。在胚胎发育过程中可以观察到细胞密度的连续下降（图5）。虽然在IVD发展的早期阶段，可以发现牛肛裂纤维中的细胞密度为11，435个细胞/mm²，牛髓核中的细胞密度为17，426个细胞/mm²，但这些数字在出生前迅速下降到1，011个细胞/mm²（牛肛门纤维）和...

Watch this Scientific Journal Video about Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration at JoVE.com

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

我们提出了一种使用光学切片方法研究椎间盘肛门纤维化中空间软骨细胞组织的方法。

椎间盘从胚胎发育到退化的光学切片和可视化

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

椎间盘退行性变导致高度损伤和疼痛。我们的协议使我们能够研究形态学上的椎间盘发育及其变性的变化。致密的一型胶原蛋白网络通常难以通过显微镜分析椎间盘。通过光学切片，我们可以研究不同组织中软骨细胞的3D排列。首先将术中获得的椎间盘或IVD样品放入DMEM中，补充2%体积的青霉素 - 链霉素和1.2%体积的两性霉素B，收集后立即将IVD样品储存在4摄氏度，直到进一步处理。如果冷冻解冻，则在室温下解冻人类IVD样品，并根据微观特性（例如：大学密度和方向）确定人类IVD组织的起源。要收集牛盘组织，取由IVD及其两个相邻椎骨组成的运动段，并使用15号手术刀片，从软骨下骨上解剖整个牛盘，翻转椎间盘到达中心区域。在识别软骨的不同区域后，使用编号为20或22的手术刀片从整个椎间盘中切出感兴趣的区域。来自冠臀长度小于20厘米的牛胚胎的IVD应不进行解剖。如手稿中所述，对组织进行了脱钙。如果 20 号针穿透椎间盘组织，或者如果适用，椎骨没有明显的阻力，则确认脱钙成功。在PBS中将组织样品在PBS中十倍体积的4%甲醛溶液中精确四十倍，在4摄氏度下过夜。第二天，将组织置于水溶性包埋介质中，放在冷冻机旋钮上，这样就会正常产生垂直切割胶原的轴向平面或平面。使用标准冷冻切片机，在人类样品中以70微米的厚度和在牛样品中以40微米的厚度切割嵌入的组织。在用PBS冲洗切片三次之前，收集载玻片上的切片，添加合适的荧光染料，然后安装介质，并用盖玻片覆盖切片。通过启动结构照明装置，将带有染色组织切片的载玻片放在荧光显微镜的样品架上，使用荧光滤光片和足够的照明进行单视场成像。使用与荧光显微镜兼容的图像优化软件，通过优化强度和亮度来处理图像。要将部分作为一个整体可视化，请使用镶嵌成像技术，方法是从工具栏面板打开 60 个采集设置，然后调整镶嵌寄存器中的镶嵌设置。为此，请定义稍后应合并到一个概视图图像中的视野图像的列数和行数，请按设置并调整各个图块的焦点校正。通过优化强度和亮度对图片进行后期处理。要分析空间软骨细胞组织，请使用软件中包含的3d功能，通过使用开始/停止按钮调整Z-stack设置，定义扫描参数，如Z轴上的开始和停止位置以及切片距离。识别单个细胞模式并使用细胞计数插件对细胞模式进行定量分析，通过将计数的细胞除以所选感兴趣区域的大小来计算细胞密度。IVD的结构及其在环中致密的胶原纤维网络和较软的核可以在轴向和矢状平面拍摄的马赛克图像中识别。在马赛克图像的放大区域中，注意到软骨细胞的不同空间组织模式，例如单细胞，成对和弦。对牛环状纤维化不同发育和成熟阶段的研究显示，胚胎盘发育过程中细胞密度持续下降。另一方面，在变性期间在成人人盘中观察到细胞密度和簇形成的增加。IVD发育早期牛环纤维化和牛髓核细胞密度迅速下降，直至出生。通过对具有Epitome的IVD进行通道成像，可以可视化空间模式（例如细胞质和细胞核）的3D结构，在完整的IVD单个以及成对的软骨细胞中被发现。而在退化的肛门细胞簇中发现了。最具挑战性的部分是将组织分配到其原点并校正嵌入以进行切片。这对于获得可靠的结果至关重要。正确解剖和选择来自不同阶段和起源的IVD组织使我们能够通过进一步的生化和生物力学分析（例如ELISA或原子力显微镜）来深化分析。

Research

JoVE Journal

Biology

Proper Care and Cleaning of the Microscope

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Culture and Maintenance of Human Embryonic Stem Cells  

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Freezing and Thawing Human Embryonic Stem Cells

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...