轴向负载诱导小鼠椎间盘退变模型的建立

A mouse model of intervertebral disc degeneration induced by axial loads

目的:探讨建立轴向负载诱导小鼠椎间盘退变模型的有效方法。方法:将24只16月龄SPF级小鼠随机分为模型组和对照组,每组12只。每日10∶00、15∶00、19∶00将模型组小鼠放入自制热板鼠笼中,打开热板并设定温度为50℃,从小鼠开始跳跃至力竭约10min左右;对照组小鼠不做任何处理。分别于造模1个月和3个月后从模型组和对照组随机抽取6只小鼠,采用颈椎脱臼法处死,完整取下L3~4椎间盘和L3、L4椎体,制作连续冠状位切片后,分别行苏木素-伊红(hematoxylin-eosin,HE)染色和阿尔新蓝-苏木素(alcianblue-hematoxylin,ABH)染色,在光学显微镜下观察腰椎间盘组织形态学变化,通过免疫组织化学染色法在光学显微镜下观察腰椎间盘组织中Ⅱ型胶原蛋白的表达情况,并通过绘图软件测量椎间盘高度和软骨终板厚度。结果:①腰椎间盘组织形态学观察结果。HE染色显示造模1个月及3个月后,对照组腰椎间盘组织基本正常;模型组腰椎间盘组织可见退行性改变,其中造模3个月后较造模1个月后退变更严重,椎间盘纤维环出现裂隙,软骨终板分层结构紊乱、排列不规则,髓核破坏或皱缩,软骨终板内出现多个骨化中心。ABH染色结果显示造模1个月和3个月后,对照组软骨终板细胞排列整齐,软骨终板细胞数目较多,软骨基质染色较蓝,部分椎间盘软骨终板内存在较少呈红染的骨化中心,但骨化中心形态较小;模型组软骨终板细胞排列混乱,细胞数目明显减少,且软骨基质染色变浅,软骨终板出现多个骨化中心,且骨化中心形态较大。②腰椎间盘免疫组织化学观察结果。造模1个月和3个月后,对照组Ⅱ型胶原蛋白在髓核、纤维环内层和软骨终板均有阳性表达,染色较深;模型组Ⅱ型胶原蛋白的阳性表达在髓核中央及纤维环外层较对照组明显降低,且造模3个月后Ⅱ型胶原蛋白在髓核与纤维环外层的表达较造模1个月后降低更明显。③腰椎间盘形态学测量结果。造模1个月后,模型组椎间盘高度和软骨终板厚度均小于对照组[(0.211±0.063)mm,(0.289±0.050)mm,t=2.984,P=0.007;(0.074±0.011)mm,(0.097±0.015)mm,t=2.813,P=0.023];造模3个月后,模型组椎间盘高度与对照组比较,差异无统计学意义[(0.197±0.052)mm,(0.235±0.053)mm,t=1.478,P=0.163];模型组软骨终板厚度小于对照组[(0.060±0.012)mm,(0.093±0.018)mm,t=4.092,P=0.001]。结论:用热板诱导小鼠跳跃,可以有效建立轴向负载诱导小鼠椎间盘退变模型。

Objective:To explore the effective method for building mouse models of intervertebral disc degeneration induced by axial loads.Methods:Twenty-four 16-month-old SPF-grade mice were randomly divided into model group and control group,12 cases in each group.The mice in model group were put into a self-made heating plate cage at 10∶00,15∶00 and 19∶00 every day.The heating plate was turned on and its temperature was set to 50℃.Every time the mice kept jumping for about 10 minutes and were exhausted in the end.The mice in control group were not given any intervention.Six mice were randomly selected out from each group respectively and were executed by using cervical dislocation method after 1-and 3-month modeling respectively.Their L 3-4 intervertebral disc and bilateral adjacent vertebral bodies were fetched out completely and were sectioned continuously in coronal position for hematoxylin-eosin(HE)staining and alcian blue-hematoxylin(ABH)staining.The histological and morphological changes of lumbar intervertebral disc tissues were observed under the optical microscope,and the protein expression levels of typeⅡcollagen in lumbar intervertebral disc tissues were detected by using immunohistochemical staining,and the intervertebral disc height and cartilage endplate thickness were measured by using drawing software.Results:HE staining results demonstrated that lumbar intervertebral disc tissues of mice were basically normal in control group after 1-and 3-month modeling respectively,while degenerative changes were found in lumbar intervertebral disc tissues of mice in model group and degenerative changes were more visible after 3-month modeling.The degeneration presented with(1)cracks in intervertebral disc annulus fibrosus,(2)disordered and irregularly arranged layered structure of cartilage endplate,(3)damaged or wizened nucleus pulposus,and(4)multiple ossification centers in cartilage endplate.ABH staining results showed that after 1-and 3-month modeling,abundant and regularly arranged cartilage endplate cells were found in control group,and the cartilage matrix presented with blue staining,and a few littlish red stained ossification centers were found in cartilage endplate of some intervertebral discs;while cartilage endplate cells arranged disorderly and decreased significantly in model group,and cartilage matrix presented with light-coloured staining,and multiple largish ossification centers appeared in cartilage endplate.The immunohistochemical staining results of lumbar intervertebral disc tissues showed that positive expressions of typeⅡcollagen were found in nucleus pulposus,inner layer of fibrous rings and cartilage endplate in control group after 1-and 3-month modeling,and the cells were dark stained;while the positive expressions of typeⅡcollagen were obviously lower in center of nucleus pulposus and outer layer of fibrous rings in model group compared to control group,and more obvious decrease was found after 3-month modeling.The intervertebral disc height and cartilage endplate thickness were smaller in model group compared to control group after 1-month modeling(0.211+/-0.063 vs 0.289+/-0.050 mm,t=2.984,P=0.007;0.074+/-0.011 vs 0.097+/-0.015 mm,t=2.813,P=0.023).There was no statistical difference in intervertebral disc height between model group and control group after 3–month modeling(0.197+/-0.052 vs 0.235+/-0.053 mm,t=1.478,P=0.163).The cartilage endplate thickness was smaller in model group compared to control group(0.060+/-0.012 vs 0.093+/-0.018 mm,t=4.092,P=0.001).Conclusion:The mouse model of intervertebral disc degeneration induced by axial loads can be built effectively by using heating plate for forcing mouse to jump.