Ariga Kenta, Yonenobu Kazuo, Nakase Takanobu, Hosono Noboru, Okuda Shin'ya, Meng Wenxiang, Tamura Yuichi, Yoshikawa Hideki
Department of Orthopaedic Surgery, Kansai-Rosai Hospital, Hyogo, Japan.
Spine (Phila Pa 1976). 2003 Jul 15;28(14):1528-33.
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.
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.
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.
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.
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.
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.
在器官培养中，对小鼠椎间盘施加不同程度的静态机械负荷。然后使用缺口末端标记法检测细胞凋亡情况。向培养基中添加了两种丝裂原活化蛋白激酶（MAPK）抑制剂。
建立一个用于检测调节机械应力诱导软骨细胞凋亡因子的实验模型，并确定MAPK和p38在终板软骨细胞应激诱导凋亡途径中的作用。
软骨终板（CEP）退变改变的原因尚不清楚。作者先前使用小鼠模型的研究结果表明，软骨终板中的细胞凋亡可能在椎间盘退变中起作用，并且机械应力可能诱导细胞凋亡。如果终板软骨细胞的凋亡参与了椎间盘退变的级联反应，那么机械应力如何诱导凋亡对于预防椎间盘退变应该是很重要的。然而，机械应力诱导凋亡的机制仍不清楚。
采集小鼠尾椎间盘并进行器官培养。对置于培养瓶中的椎间盘施加不同的静态压缩负荷（0、0.2、0.4、0.8和1.0兆帕），持续24小时。对采集的椎间盘石蜡包埋切片进行番红O染色和缺口末端标记程序。对每个椎间盘的软骨终板和纤维环交界区的凋亡细胞进行计数。此外，将U0126（MAPK抑制剂）和SB202190（p38抑制剂）添加到培养基中，以确定它们在机械负荷诱导的终板软骨细胞凋亡中的调节作用。
组织学上，加载负荷的椎间盘膨出，椎间盘间隙变窄。未加载负荷的椎间盘未出现细胞凋亡，但在加载负荷的椎间盘（负荷重量为1.0兆帕）中细胞凋亡尤为明显。凋亡细胞数量随负荷重量增加而增加。两种MAPK抑制剂显著增加了凋亡细胞数量。
在器官培养中，特别是在软骨终板中，使用静态机械负荷诱导软骨细胞凋亡。细胞凋亡的发生与先前使用体内模型的研究结果相似。因此，这个培养系统反映了体内观察到的细胞凋亡情况。由于可以简单地向培养基中添加诸如MAPK抑制剂等生物活性试剂，该系统可能是检测影响机械应力诱导凋亡因子的一种有用方法。两种MAPK抑制剂均增加了细胞凋亡的发生率。这表明这两种MAPK可以对抗机械应力诱导的凋亡途径。
