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


METHODS
Twenty-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.


RESULTS
The 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.


CONCLUSIONS
This 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.
Takashi
T
Yurube
Hiroaki
H
Hirata
Kenichiro
K
Kakutani
Koichiro
K
Maeno
Toru
T
Takada
Zhongying
Z
Zhang
Koji
K
Takayama
Takehiko
T
Matsushita
Ryosuke
R
Kuroda
Masahiro
M
Kurosaka
Kotaro
K
Nishida
D000375:Aging / Q000473:pathology*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D055959:Intervertebral Disc Degeneration / Q000503:physiopathology*; D008297:Male; D009672:Notochord / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013314:Stress, Mechanical; D013623:Tail