Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc

Abstract

The fate of notochord cells during disc development and aging is still a subject of debate. Cells with the typical notochordal morphology disappear from the disc within the first decade of life. However, the pure morphologic differentiation of notochordal from non-notochordal disc cells can be difficult, prompting the use of cellular markers. Previous reports on these notochordal cell markers only explored the occurrence in young age groups without considering changes during disc degeneration. The aim of this study, therefore, was to investigate presence, localization, and abundance of cells expressing notochordal cell markers in human lumbar discs during disc development and degeneration. Based on pilot studies, cytokeratins CK-8, -18 and -19 as well as Galectin-3 were chosen from a broad panel of potential notochordal cell markers and used for immunohistochemical staining of 30 human lumbar autopsy samples (0-86 years) and 38 human surgical disc samples (26-69 years). In the autopsy group, 80% of fetal to adolescent discs (0-17 years) and 100% of young adult discs (18-30 years) contained many cells with positive labeling. These cells were strongly clustered and nearly exclusively located in areas with granular changes (or other matrix defects), showing predominantly a chondrocytic morphology as well as (in a much lesser extent) a fibrocytic phenotype. In mature discs (31-60 years) and elderly discs (≥ 60 years) only 25 and 22-33%, respectively, contained few stained nuclear cells, mostly associated with matrix defects. In the surgical group, only 16% of samples from young adults (≤ 47 years) exhibited positively labeled cells whereas mature to old surgical discs (>47 years) contained no labeled cells. This is the first study describing the presence and temporo-spatial localization of cells expressing notochordal cell markers in human lumbar intervertebral discs of all ages and variable degree of disc degeneration. Our findings indicate that cells with a (immunohistochemically) notochord-like phenotype are present in a considerable fraction of adult lumbar intervertebral discs. The presence of these cells is associated with distinct features of (early) age-related disc degeneration, particularly with granular matrix changes.

Fig. 1

Immunolocalization of pan CK AE1/AE3 (staining cytokeratin-8 and -19 among others) in disc tissue of various age groups (autopsy group). a Fetal nucleus pulposus and pre-anulus region (arrows), showing typical labeling of notochordal cells (28th week, disc level L3/4). b Young adult nucleus pulposus tissue (21 years, disc level L2/3), showing numerous positively stained cells that are exclusively labeled in granular matrix areas (arrows) and adjacent to matrix defects (×200)

Fig. 2

Immunolocalization of cytokeratin-18 (CK-18) in disc tissue of various age groups (autopsy group). a Fetal nucleus pulposus tissue (small arrows) and notochord sheath and pre-anulus region (big arrow), showing typical positive labeling of notochordal cells (28th week, disc level L3/4). b Young adult nucleus pulposus tissue (25 years, disc level L3/4), showing numerous positively labeled cells. c Old adult nucleus pulposus tissue (77 years, disc level L3/4), showing no positively stained disc cells (arrows) (×200)

Fig. 3

Immunolocalization of Galectin-3 in disc tissue of various age groups (autopsy group). a Fetal nucleus pulposus, showing typical positive labeling of notochordal cells (28th week, disc level L3/4) (×400). b Young adult nucleus pulposus tissue (25 years, disc level L3/4), showing numerous positive cells comparable to the cytokeratin pattern (×200)

Fig. 4

Immunohistochemical double labeling experiments. The simultaneous staining of CK-19 (red color) and Galectin-3 (brown color) shows exact cellular co-localization of both parameters (arrows) (×400)

Fig. 5

Morphometric evaluation of immunohistochemical reaction results for pan-CK, CK-18 and Galectin-3 in the autopsy series. The histogram shows the distribution of negative/positive cells and the extent of positive staining in the four age groups, juvenile/adolescent (0–17 years), young adult (18–30 years), mature adult (31–60 years) and elderly age group (≥60 years). Categorization: − no reaction, + few labeled cells, i.e. <10% of cells stained, ++ intermediate amount of stained cells with 10–50% of cells stained, +++ numerous labeled cells, i.e. >50% of cells stained

Fig. 6

Immunohistochemical labeling of amorphous granular matrix material. The immunostaining for pan-cytokeratin (AE1/AE3) (mature adult autopsy group, 31 years, disc level L4/5) reveals a specific positive labeling of part of the granular material (arrows) (×400)

Fig. 7

Immunolocalization of notochordal markers in the surgical material a Pan-cytokeratin (AE1/AE3) positive cells are present in a mature adult surgical disc sample (36 years, disc level L4/5) with clusters of positively labeled cells. b Galectin-3 positive cells are seen in a mature adult surgical disc sample (36 years, disc level L4/5) with clusters of positively labeled cells (×400)