title: Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs: an ex vivo study.
abstract: METHODS
  Various amounts of static mechanical load were applied to mouse intervertebral discs in organ cultures. The apoptosis then was examined using nick end labeling. Two mitogen-activated protein kinase (MAPK) inhibitors were added to the medium.
  
  
  OBJECTIVE
  To establish an experimental model for detecting factors regulating chondrocyte apoptosis induced by mechanical stress, and to determine the role of MAPK and p38 in the stress-induced apoptotic pathway of endplate chondrocytes.
  
  
  BACKGROUND
  The cause of degenerative change in the cartilaginous endplate (CEP) remains unclear. The authors' previous findings using a mouse model suggested that apoptosis in the cartilaginous endplate may play a role in intervertebral disc degeneration, and that mechanical stress may induce apoptosis. If apoptosis of endplate chondrocytes is involved in the cascade of intervertebral disc degeneration, then how apoptosis is induced by mechanical stress should be important in preventing disc degeneration. However, the mechanism of apoptosis induced by mechanical stress remains unclear.
  
  
  METHODS
  Mouse coccygeal discs were harvested and organ cultured. Various static compression loads (0, 0.2, 0.4, 0.8, and 1.0 MPa) were applied on intervertebral discs placed in culture bottles for 24 hours. Paraffin-embedded sections of the harvested discs were stained using Safranin-O and the nick end labeling procedure. The apoptotic cells were counted in the cartilaginous endplate and junctional anulus fibrosus of each intervertebral disc. In addition, U0126 (MAPK inhibitor) and SB202190 (p38 inhibitor) were added to the culture medium to determine their regulatory roles in the apoptosis of endplate chondrocytes induced by mechanical load.
  
  
  RESULTS
  Histologically, loaded discs became bulged, and the disc space became narrow. Apoptosis was absent in discs without load, but was particularly noticeable in loaded discs (load weight, 1.0 MPa). The number of apoptotic cells increased depending on the weight of the load. The two MAPK inhibitors significantly increased the number of apoptotic cells.
  
  
  CONCLUSIONS
  Chondrocyte apoptosis was induced using a static mechanical load especially in the cartilaginous endplate in an organ culture. Apoptosis occurred similarly to previous findings using an in vivo model. This culture system thus reflected the apoptosis demonstrated in vivo. Because biologically active reagents such as MAPK inhibitors can be simply added to culture media, this system may be a useful method for detecting factors that influence apoptosis induced by mechanical stress. Both MAPK inhibitors increased the occurrence of apoptosis. This suggests that these two MAPKs can counteract the apoptotic pathway induced by mechanical stress.
authors.affiliation: Department of Orthopaedic Surgery, Kansai-Rosai Hospital, Hyogo, Japan. ariga-k@umin.ac.jp
authors.forename: Kenta
authors.initials: K
authors.lastname: Ariga
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yonenobu
authors.forename: Takanobu
authors.initials: T
authors.lastname: Nakase
authors.forename: Noboru
authors.initials: N
authors.lastname: Hosono
authors.forename: Shin'ya
authors.initials: S
authors.lastname: Okuda
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Meng
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Tamura
authors.forename: Hideki
authors.initials: H
authors.lastname: Yoshikawa
mesh_terms: D000818:Animals; D017209:Apoptosis; D002070:Butadienes / Q000494:pharmacology; D002452:Cell Count; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006132:Growth Plate / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D007093:Imidazoles / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007403:Intervertebral Disc / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D009924:Organ Culture Techniques; D011725:Pyridines / Q000494:pharmacology; D013314:Stress, Mechanical; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009570:Nitriles; D011725:Pyridines; C113580:U 0126; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole