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.
