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

We thank our co-authors from the original publications for their help and support. We thank Charlotte Emma Bamberger for helping to acquire the apotome images.

For the analysis of embryonic development and maturation, bovine discs were used. To evaluate degeneration of the IVD, human samples were analyzed.
Human IVD tissue was obtained from patients undergoing surgery for lumbar disc degeneration, disc prolapse, or spinal trauma in the Department of Orthopaedic Surgery, University Hospital of T&#252;bingen and the BG Trauma Centre T&#252;bingen. Full ethical committee approval was obtained before the commencement of the study (project number 244/2013...

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Using fluorescence microscopy augmented by mosaic imaging and optical sectioning, we evaluated the spatial arrangement of chondrocytes in the anulus of the lumbar IVD throughout development, maturation, and degeneration. While degenerative tissue could be harvested from patients receiving spine surgery for disc degeneration, analysis of the embryonic period and maturation phase required the use of a model organism (bovine). High cellular densities were noted in the anulus during early embryonic development. In the furthe...

The intervertebral disc (IVD) is a cartilage-based structure that biochemically and with respect to cellular architecture, at first sight, resembles in many ways the articular cartilage1. Indeed, both IVD degeneration and osteoarthritis (OA) of articular cartilage are characterized by joint space narrowing due to cartilage wear, subchondral cyst and osteophyte formation, and subchondral sclerosis2,3. Despite these seeming similarities architecture and functional role of both tissues differ. While the matrix of articular cartilage is mainly formed of an arcade-forming collagen type II network, the IVD consists of three different types of tissue: the collagen type II-rich nucleus pulposus in the center takes up axial loads and transmits them to an encompassing ring of densely packed circular collagen type I fibers which is called anulus fibrosus. Their function is to absorb the translated axial pressures received by the proteoglycan- and water-rich nucleus with their tensile longitudinal fiber strength. At the top and bottom of each nucleus and anulus a hyaline cartilaginous endplate forms the junction to the adjacent vertebrae4 (Figure 1).
In articular cartilage, four distinct spatial chondrocyte patterns can be found: pairs, strings, double strings, small respectively big clusters5,6,7 (Figure 2). Changes in this pattern are associated with OA onset and progression8,9. Spatial chondrocyte organization is also indicative for a direct functional property of cartilage, namely its stiffness, underlining the functional relevance of this image-based grading approach10,11. These patterns can additionally be identified with already existing clinically available technology12. Due to the similarities between the IVD and articular cartilage, it can be hypothesized that characteristic chondrocyte patterns are also present in the IVD. Cluster formation is a phenomenon also observed in the degenerated IVD13,14.
When trying to analyze spatial cellular organization in the IVD, it is necessary to overcome several technical difficulties that are not present when investigating articular cartilage:
First, processing of the tissue itself is much more challenging than with the homogeneous hyaline cartilage which is largely composed of collagen type II. The IVD&#39;s main fiber component is collagen type I, which makes it much more difficult to generate thin histologic sections. While in the hyaline articular cartilage even thick sections can easily be analyzed due to the &#34;glass-like&#34; nature of the tissue, the collagen type I network of the IVD is optically highly impenetrable. For this reason, a strong background noise is a common problem in the histology of the IVD. A fast and cheap way to penetrate this optically dense tissue is the use of an optical sectioning device e.g., by means of an Apotome. In such an Apotome, a grid is inserted in the illumination pathway of a conventional fluorescence microscope. In front of the grid a plane-parallel glass plate is placed. This tilts back and forth thus projecting the grid in the image in three different positions. For each z-position, three raw images with the projected grid are created and superimposed. By means of special software, the out of focus light can be calculated out. The underlying principle is that, if the grid is visible, that information is in focus, if not it is considered to be out of focus. With this technique, well focused and high-resolution images can be acquired in a reasonable amount of time.
Secondly, the tissue is hard to come by from human donors. When doing total knee replacement, the entire surface of the joint can be obtained for further analysis during surgery. Although osteoarthritis of a diarthrodial joint is also a disease of the whole joint, there are nevertheless strong focal differences in the quality of the cartilage with usually some areas of the joint still being intact, for example due to reduced loading in that area. This situation is different in the IVD, where surgery is usually only performed when the disc is globally destroyed. When obtaining tissue from human donors from the operation room, the tissue is also highly fragmented and it is necessary to correctly allocate the tissue to one of the three cartilage types of the IVD before doing further analyses. To allow more detailed analyses of also larger tissue sections and to look into the embryonic development of the IVD the choice of an animal model organism is, therefore, necessary.
When choosing such a model organism it is important to have a system which is comparable with the human disc with respect to its anatomy and dimensions, its mechanical loading, the present cell population as well as its tissue composition. For the purpose of the presented technique here we suggest the use of bovine lumbar disc tissue: A critical property of the human disc resulting in its low regenerative potential is the loss of notochordal cells during maturation in the nucleus. However, in numerous model organisms notochordal cells can be detected their entire life long. Most of the few animals which lose their notochordal cells such as sheep, goats or chondrodystrophig dogs have an IVD that is much smaller than human discs. Only lumbar bovine discs present with a comparable sagittal disc diameter to those of human IVDs15.
A key factor leading to early disc degeneration is excessive mechanical loading. The intradiscal pressures of a standing cow in the lumbar spine are around 0.8 MPa with the spine aligned horizontally. Surprisingly these pressures are comparable to the lumbar intradiscal pressures reported for the erect human spine (0.5 MPa)15,16. Also the amount of water and proteoglycans in bovine discs is comparable to that of the IVD from young humans17. Therefore, although the actual movement pattern of the motion segments might differ in quadrupedal animals from the bipedal human, with respect to total loading and disc characteristics, the cow is much closer to human biology than other established animal models for the IVD such as sheep and dogs.
In this protocol we present a technique how to analyze changes in the IVD from the point of view of spatial chondrocyte organization from early embryonic development to end stage degeneration.

<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>
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			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich,US</td>
			<td>P5119</td>
			<td></td>
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			<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>
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optical sectioning and visualization of the intervertebral disc from embryonic development to degeneration

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 decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

Using mosaic images, the architecture of the IVD with its dense collagen fiber network in the anulus and the softer nucleus can clearly be recognized (Figure 4). A continuous decrease in cellular density can be observed during embryonic development (Figure 5). While in the early stages of IVD development a cell density of 11,435 cells/mm&#178; in the bovine anulus fibrosus and 17,426 cells/mm&#178; in the bovine nucleus pulposus can be found, these numbers decre...

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

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

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 ...

Intervertebral Disc degeneration leads to a high degree of impairment and pain. Our protocol allows us to investigate morphologically disc development and its changes in degeneration. The dense collagen type one network makes it usually difficult to analyze the Intervertebral Disc by microscopy.With optical sectioning, we can investigate the 3D arrangement of chondrocytes within the different tissues. Begin by placing intra-operatively obtained, Intervertebral Disc or IVD samples in DMEM supplemented with 2%volume by volume Penicillin-Streptomycin, and 1.2%volume by volume Amphotericin B, immediately post collection, store IVD samples at four degrees Celsius until further processing. If frozen thaw the human IVD samples at room temperature and identify the origin of the human IVD tissue based on microscopic properties such as;college density and orientation.To collect the bovine disc tissue, take the motion segment consisting of the IVD with its two adjacent vertebrae and using a surgical blade number 15, dissect the whole bovine disc from the subchondral bone, flip the disc to reach the centered areas. After identifying the different areas of the cartilage, use a surgical blade number 20 or 22 to cut out the area of interest from the whole disc. The IVDs from bovine embryos with a crown-rump length smaller than 20 centimeters should be processed without dissection.Performed decalcification of the tissue as described in the manuscript. Confirm successful decalcification, if a 20 gauge needle penetrates the disc tissue, or if applicable the vertebrae without notable resistance. Exact the tissue samples in the tenfold volume of 4%formaldehyde solution in PBS overnight at four degrees Celsius.On the next day, place the tissue in a water-soluble embedding medium onto the Cryotome knob, such that either an axial plane or a plane that perpendicular cuts the collagen type one normally is generated. Use the standard Cryotome to section the embedded tissue at a thickness of 70 micrometers in human samples and 40 micrometers in bovine samples. Collect the sections on a glass slide before rinsing the sections three times with PBS, add a suitable fluorescent dye followed by the mounting medium and cover the sections with a cover slip.Place a slide with a stain tissue section on the sample holder of the florescence microscope by starting the structure illumination device perform single field of view imaging with fluorescent filters and adequate illumination. Using an image optimization software compatible with the florescence microscope, process the pictures by optimizing the intensity and brightness. To visualize the section as a whole use the mosaic imaging technique by opening the 60 acquisition settings from the toolbar panel and adjusting the mosaic settings in mosaics register.To do so define the number of columns and rows of field of view images that shall later be merged into one overview image, press setup and adjust the focus correction of individual tiles. Post-process the pictures by optimizing the intensity and brightness. To analyze the spatial chondrocyte organization use the 3d function incorporated in the software by adjusting the Z-stack settings with the start/stop button, define the scanning parameters like the start and stop positions in the Z axis and the slice distance.Identify individual cellular patterns and use a cell count plugin for the quantitative analysis of the cellular patterns, calculate the cell density by dividing the counted cells by the size of the chosen region of interest. The architecture of the IVD with its dense collagen fiber network in the annulus and the softer nucleus can be recognized in the mosaic images taken in the axial and sagittal plane. In magnified areas from the mosaic images, different spatial organizational patterns of chondrocytes such as;single cells, pairs and strings were noticed.The study of different developmental and maturation stages of bovine annulus fibrosis displayed a continuous decrease in cellular density during embryonic disk development. On the other hand an increasing cellular density and cluster formation were observed in the adult human disc during degeneration. The cell density in the early stages of the IVD development in the bovine annulus fibrosis and the bovine nucleus pulposus decreased rapidly until birth.By channel imaging of the IVD with the Epitome allowed visualization of the 3D architecture of the spatial patterns such as cytoplasm and the nucleus, in the intact IVD single as well as pairs of chondrocytes were spotted. Whereas in the degenerated anulus cell clusters were found. The most challenging part is to allocate the tissue to its origin and to correct the embedded for sectioning.This is essential to obtain reliable results. Having correctly dissected and selected IVD tissue from different stages and origins allows us to deepen the analysis by further biochemical and biomechanical analysis, such as the ELISA or atomic force microscopy.

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