Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model

Abstract

IntroductionThe intervertebral disc has a complex structure originating developmentally from both the mesenchyme and notochord. Notochordal cells disappear during adolescence, which is also when human discs begin to show degenerative signs. During degeneration later in life, disc cells decline because of apoptosis. Although many animal models have been developed to simulate human disc degeneration, few studies have explored the long-term changes in cell population and phenotype. Our objective was to elucidate the time-dependent notochordal cell disappearance and apoptotic cell death in a rat tail static compression-induced disc degeneration model.MethodsTwenty-four 12-week-old male Sprague–Dawley rat tails were instrumented with an Ilizarov-type device and loaded statically at 1.3 MPa for up to 56 days. Loaded and distal-unloaded discs were harvested. Changes in cell number and phenotype were assessed with histomorphology and immunofluorescence. Apoptosis involvement was determined with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunohistochemistry.ResultsThe number of disc nucleus pulposus and annulus fibrosus cells decreased with the loading period; particularly, the decrease was notable at day 7 in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, indicating notochordal origin. Subsequently, the proportion of cells positive for TUNEL and cleaved caspase-3, markers of apoptosis induction, increased from day 7 through day 56. Although the percentage of cells immunopositive for cleaved caspase-8, a marker of apoptosis initiation through the death-receptor pathway, increased only at day 7, the percentage of cells immunopositive for cleaved caspase-9 and p53-regulated apoptosis-inducing protein 1 (p53AIP1), markers of apoptosis initiation through the p53-mediated mitochondrial pathway, increased from day 7 through day 56. The percentage of cells immunopositive for B-cell lymphoma 2 (Bcl-2) and silent mating type information regulation 2 homolog 1 (SIRT1), antiapoptotic proteins, decreased consistently with compression.ConclusionsThis rat tail model mimics notochordal cell disappearance and apoptotic cell death in human disc aging and degeneration. Sustained static compression induces transient activation of apoptosis through the death-receptor pathway and persistent activation of apoptosis through the p53-mediated mitochondrial pathway in disc cells. The increased proapoptotic and decreased antiapoptotic proteins observed at all time points signify static compression-induced disc cell death and degeneration.

Autophagy and mTOR signaling during intervertebral disc aging and degeneration

“…8 Furthermore, a notably high incidence of apoptosis has been observed in human aged and degenerated discs 30 and rodent degenerative discs by static compression. 23,31 The incidence of irreversible cell growth arrest due to aging, or senescence, 32 also increases during human disc degeneration. 33,34 Understanding of this unique, harsh, low-glucose, low-oxygen, low-pH and high-osmolality environment and the load fluctuations that disc cells are exposed to is essential for designing new biological therapies to prevent disc aging and degeneration.…”

Large Cytoplasmic Vacuoles within Notochordal Nucleus Pulposus Cells: A Possible Regulator of Intracellular Pressure That Shapes the Cytoskeleton and Controls Proliferation

“…Although our hypothesis helps to elucidate the histocytological changes that occur at the very beginning of disc aging and degeneration, the equation P e = P m + P v takes no account of the nutrient deficiency [Guehring et al, 2009] or mechanical injuries [Yurube et al, 2014] that might reduce the viability of NNPC. Given that NNPC are more metabolically active and sensitive to nutrient disturbances [Guehring et al, 2009;Park et al, 2015], the possibility exists that the notochordal cell resources are exhausted by excessive apoptosis or senescence while the cells attracted from the CEP mediate the fibrocartilaginous changes within the NP [Kim et al, 2003[Kim et al, , 2009].…”

LRP5-deficiency in OsxCreERT2 mice models intervertebral disc degeneration by aging and compression

“…Immunohistochemical staining for osterix in the NP of compressed IVD noted a cell shift by the intensity of the staining and morphology of the cell. Tail compression is known to induce loss of notochordal cells in the NP prior to cell death (Yurube et al, 2014) and elevate chondrocyte-like cells. Currently, the role of osterix in the IVD is unclear but, in bone, osterix is a critical transcription factor that drives osteoblastic cell differentiation (RUNX2 before osterix and β-Catenin after osterix).…”

“…Chondrocyte-like cells of the IVD resemble articular chondrocytes in their appearance and transcriptional expression and cohabitate with notochordal cells in the nucleus pulposus (NP) (Clouet et al, 2009; Minogue et al, 2010), but are smaller and phenotypically distinct from notochordal cells (Chen et al, 2006; Minogue et al, 2010). Aging and IVD degeneration induce the disappearance of notochordal cells (Richardson et al, 2017), which are replaced by chondrocyte-like cells (Boos et al, 2002; Yurube et al, 2014), ostensibly via differentiation of prehypertrophic chondrocyte-like cells (Rutges et al, 2010). RUNX2 (Cbfa1), Sp7 (Osterix) and Ctnnb1 (β-Catenin) progressively drive skeletal progenitors to become osteoblasts and later osteocytes, but loss of osterix or WNT signaling diverts cell fate towards chondrogenesis (Long, 2011).…”