Abstract
Introduction
Low back pain (LBP) affects 80% of the whole population around the world and causes great social-economic burden. Intervertebral disc (IVD) degenerates with aging and is a major contribution to LBP. Mature NP (nucleus pulposus, the center part of IVD) cells are similar to, but not the same as, chondrocytes. The search for NP markers has prompted the proposal of keratin-19 as one of the NP markers mainly based on its transcriptional level.1 In our previous study, among all the investigated. keratin-19 is the most downregulated gene in degenerated human NP. 2 In this study, we aim to track the changes in the protein expression of keratin-19 in disc degeneration and aging process with human clinical specimens and rodent models of induced degeneration and natural aging.
Material and Methods
The corresponding informed patient consent and institutional review board (IRB) approval and animal ethics approval was obtained from the relevant committee. Human IVD were collected from patients with disc degeneration (graded IV-V at the Schneiderman scale) undergoing discectomy or during corrective scoliosis surgery. Rat induced degeneration model was established by needle puncture as described previously.3 Mice ranged from date of birth to 2 years old were used to represent natural aging in rodents. The expression of keratin-19 was investigated by immunochemical staining. Keratin-19 positivity was determined as the number of keratin-19 positive cells divided by the total number of cells per whole section.
Results
Keratin-19 expression was detected in both NP and AF (annulus fibrosus) of degenerated and non-degenerated human IVD. The signal of Keratin-19 was exclusively intracellular. Cells within a cluster showed heterogeneous expression for Keratin -19. Degenerated human NP contains significantly less Keratin-19 positive cells when compared with non-degenerated human NP (D-NP versus ND-NP: 13.87 ± 1.27% versus 29.99 ± 4.68%, p = 0.016). In healthy rat, Keratin-19 was found to be strongly expressed in the NP but not in the AF. A minor portion of cells in the cartilaginous EP (endplate) was also Keratin-19 positive. Keratin-19 positivity remained at a similar level post injury in the NP, but were induced in the AF after puncture. In aging mice, at P0, Keratin-19 showed strong expression in the vacuolated notochordal NP cells and in the cartilage anlage of vertebrae bodies, with no expression in the AF. From P0 to 18 months of age, Keratin-19 remained expressed at a relatively constant level in the NP constituted by large vacuolated notochordal cells. By 24 months of age, Keratin-19 expression decreased in the NP.
Conclusion
We found that although Keratin-19 was significantly decreased in human degenerated NP, it only showed a mild decrease or otherwise unchanged expression in rodent models of progressive disc degeneration and aging. Since Keratin-19 is also a notochordal cell marker,4 it may reflect that notochordal cells are still present in the aged mouse NP and during the mild stage of degeneration in the rat model. To our knowledge, this is the first investigation into the expression and location of keratin-19 in different IVD aging/degeneration systems.
Acknowledgments
This work was supported by General Funding from National Science Foundation of China (NSFC, 81371993), Small Project Funding of The University of Hong Kong (201409176191), and General Research Fund of RGC in Hong Kong (17104815).
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