Evidence of Nonnotochordal Origin in Chondrocyte-like Cells of the Nucleus Pulposus Appearing in Early Stage Disk Degeneration in the Mouse Model

Abstract

Introduction Although degeneration of the intervertebral disk (IVD) accounts for progression of various spinal diseases, not much has been clarified on the changes in cellular environment in the pathology of disk degeneration. Various animal models have been reported for use in different purposes; however, not much variation has been shown in mice. Development of a reproducible mouse model is important since recent molecular regulatory investigations are often performed using transgenic (Tg) mice. To define fate of cells in the IVD that originate from the notochord, we investigated the IVDs of double-transgenic (TG) mouse encoding Protein-0 (P0)-Cre/Floxed-EGFP. Cells originating from the neural crest or notochord express positivity for GFP in this TG mouse. We then developed a new disk degeneration model in this Tg mouse and investigated the changes in the cellular environment of the IVD. Materials and Methods A total of 40 P0-Cre/Floxed-EGFP mice (8 weeks old, female) were divided in four groups. A loop was created with wires in their tails which induced degeneration in the nucleus pulposus and annulus fibrosus (AF) by lateral compression in all mice except for the normal control group ( n = 10). Loop was loosened after 2 weeks and left to regenerate for 2 weeks in group R ( n = 10). Loop was kept tightened for four straight weeks in group D ( n = 10). Classical annulus puncture by a 27G needle was performed in group P( n = 10). All animals were sacrificed after 4 weeks. Histological evaluations were performed on HE and Safranin-O stained sections using disk degeneration grading system by Nishimura et al and Nomura et al. Immunohistochemistry was performed to detect GFP positive cells and CD24, a defined cell marker for nucleus pulposus cells in the IVD. Furthermore, immunohistochemistry was performed to detect brachyury, a specific marker for the notochord. Results When histological grades were evaluated as a sum score of AF and nucleus pulposus, Group D showed the most degenerative changes compared to Group R or P. The severity of disk degeneration showed a linear trend in the order of severity. Next, morphology of cells that compose the IVD was investigated, and found an appearance of chondrocyte-like cells in the very lateral portion of nucleus pulposus near inner AF. Unlike the AF cells, chondrocyte-like cells expressed CD24, however, did not express GFP and brachyury, suggesting a nonnotochordal origin of chondrocyte-like cells. Conclusion Transition of notochordal nucleus pulposus cells to chondrocyte-like cells have been documented by many researchers. Kim et al reported that chondrocyte-like NP cells in the nucleus pulposus originated and migrated from the cartilage endplate in vitro and in vivo. Choi and McCann suggest that in the newborn mouse, all cells in the NP region, including smaller chondrocyte-like NP cells, are of notochordal origin. However, combination of disk degeneration model and lineage tracking shows that some of the NP cells may not originate from the notochord in at least three mouse disk degeneration models. Further analysis of these models may prove to be important in understanding the pathology of disk degeneration and possibly regenerative techniques. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared