| BackgroundsDisc degeneration is a common disease, which the main cause of neck pain and back pain and the main cause of serious impact on the quality of life of people. Aging is an important factor in disc degeneration. According to the related research, the population who aged over 60 years have emergence of the radiographic osteophytes, disc space narrowing reach 60-80%. As a prerequisite and basis for a series of spine pathology degenerative diseases, disc is essential part of research the diseases of the spine. The mechanism caused disc degeneration is contained biomechanics, cells and extracellular matrix, and it is a vicious circle which interaction each other. The stability of the spine was closely related to abnormal mechanical load in the intervertebral disc, and maybe a contributing factor to disc degeneration. So, what are the structural of the spine stability? It is composition by passive and active stabilization system. Passive system of spinal stability was including vertebral facet joints, ligaments and the annulus, etc; while the surrounding muscles was the active system of stabilize the spine.Osteoporosis is an age-related disease, especially in postmenopausal women. It is a disease of microstructure of bone degeneration mainly caused loss of bone matrix and bone mineral. Estrogen deficiency may be an important factor in women after menopause caused by osteoporosis. Spine degeneration and osteoporosis both are age-related disorders, which will lead to reduced quality of life, increased disability and mortality, while the relationship between disc degeneration and osteoporosis is still unkown.In order to explore the relationship between the two diseases, our research team preliminary experiments have created an animal model of disc degeneration by excision neck and back muscles which may cause dynamic and static power imbalances force, and then observing typical disc degeneration. A composite model of disc degeneration and osteoporosis was also made, and observing osteoporosis may aggravate the degeneration of disc by the impact of the cartilage endplate. The model of disc degeneration observed typical phenomenon of degeneration of the spine through the removal of exogenous stabilizing muscle structure. However, static and dynamic force imbalance model will cause length segments degeneration of cervical, and it can not simulate common clinical segment degeneration.Since exogenous cut back muscles can cause neck cervical disc degeneration, then whether disrupt the endogenous structural stability of the spine can also cause degeneration or not? Therefore, this study removed cervical facet joint, destruction of cervical endogenous stable structure, to see whether it can cause disc degeneration or not, whether, in the condition of osteoporosis, is able to increase the disc degeneration, and further research in biochemical indicators. Finally, this study to verify whether aggravate disc degeneration by osteporosis is caused by a lack of estrogen.Objectives1. To establish an animal model of intervertebral disc degeneration by bilateral facetectomy, and evaluate the morphology of cartilage endplate lesion the microstructure of vertebral body, histological and biochemical changes of the disc.2. To evaluate whether osteoporosis could induce and accelerate the disc degeneration induced by facetectomy.Methods1. Twenty-eight Sprague-Dawley female rats at the age of 3 months were assigned randomly into the model and control groups (n=16 and 12). The C4/5 and C5/6 bilateral facets were resected in the model group. The C5/6 segments and C4/5 disc tissues were harvested at 12 and 24 weeks after surgery. The C5/6 segments were scanned using micro CT, stained with Safranine O/fast green, and the C4/5 disc tissues were measured with RT-PCR. Lesions of the cartilage endplate were identified on axial CT images, and ratio of the lesion area to the whole endplate area was calculated. Intervertebral disc height (IDH) was measured and, and disc degeneration (DD) was graded for each sample. The gene expression of Aggrecan、Col2 a 1 and MMP-3 were analyzed using a real-time PCR.2. Forty-four Sprague-Dawley female rats were elected, plus two groups in the above, and randomly divided into three groups as follows:the sham group (n=12), the OVX group (n= 16), the FR group (n=16), the FR-OVX group (n=16), the ERT group (n=16). Specimens of C4-6 segment were harvested at 12 and 24 weeks post-surgery. The OVX group was undertaking ovariectomy. The FR-OVX group was combination the FR and OVX. The ERT group was treated with estradiol in the FR-OVX group. Micro-CT analysis was performed to evaluate the cartilage endplates lesions (CEL), the intervertebral disc height (IDH) and the microstructural indices in the cervical spine. Histological analysis with Safranine O/fast green stain and histological score were performed to observe the characteristics of the degenerative discs. Immunohistochemistry and real-time PCR measurements for Aggrecan、Col2 a 1 and MMP-3 mRNA expressions on the disc were performed to assess the underlying molecular signaling changes in matrix metabolism during intervertebral disc degeneration.Results1. Evaluating the impact on bilateral facet resection of cervical disc degeneration in rats.The C5/6 disc height was remarkable decreased in the model group at the 12 weeks post-surgery, the disc height in model group is 0.51±0.02 mm, reducing 0.4 mm compared with sham group. And became more narrow with the time at 24 weeks. We can also observed obvious defects in the cartilage endplates in the model group at the 12 weeks post-operation, which was more severity at the 24 weeks.According to the results of micro-CT analysis, BV/TV and Tb.Sp have significantly difference between model group and sham group at the time of 12 weeks, while Tb.Th and Tb.N have no obviously change between these two groups. At the 24 weeks post-operation, all the indices of bone trabecular show no different. It is speculate that at the early time of bilateral facet resection the strength of the vertebral was increased.The histological results, at the time of 12 weeks, revealed that the C5/6 disc appeared moderate degeneration, which the nucleus pulposus was shrinking in size and the cells clustered, the annulus fibrosus was unorganized and have some cracks, the endplate was observed the defect in the marginal of endplate. While, the nucleus pulposus was more reduced in size and cells were become scarcer, the annulus fibrosus was showed more clefts, and the spindle shaped chondrocytes appeared earlier in the hyaline cartilage in 24 weeks post-operation. The histological scores are 7.1±1.3 and5.3±0.5 points in the sham and model groups, respectively at 12 weeks. There are significantly difference between two groups at 12 weeks, and which have a positive correlation with the time.The mRNA expression of Aggrecan and Col2 a 1 were decreased, which is just 0.45 and 0.44 times compared with sham group, and the expression of the MMP-3 mRNA was increased in model group at the time of 12 weeks which is 1.23 times to sham group. While, in the 24 weeks post-operation, the mRNA expression of above indices also decreased in Aggrecan and Col2 a 1, increased in MMP-3 in model group.2. To develop an innovative intervertebral disc degeneration model of rats, and investigate the relationship of osteoporosis and disc degeneration.At the time of 12 weeks, osteoporosis occurred after the OVX surgery and became severe afterwards. While, compared with OVX group, BV/TV, Tb.Th, Tb.N and Tb.Sp, treat with 17β-estradiol, showed significantly difference. But there is no difference compared with sham group.The C5/6 disc height was decreased in the FR group at the 12 weeks post-surgery, and became more narrow with the time at 24 weeks. Compared with the FR group, the IDH was significantly lesser in the FR-OVX group both in 12 and 24 weeks (P<0.05). In the ERT group, the IDH, at the time of 12 and 24 weeks, was similar to the FR group. These results were indicated that the OVX surgery would be accelerated decrease in the IDH.There were obvious defects in the cartilage endplates in the FR group. The CEL was appeared on the peripheral and central of endplates in C5-caudal, and on the ventral side of endplates in C6 cranial at 12 weeks after surgery. Moreover, these defects became expanded in size at the 24 weeks. While, combine with osteoporosis, the CEL on the both C5caudal and C6 cephalad endplates were obviously greater in the FR-OVX group than in FR group both in 12 and 24 weeks, indicating that the OVX surgery would resulted in larger endplates abration. These were significantly increased in the FR-OVX group compared to FR group at 12 and 24 weeks, but no obvious difference between FR group and ERT group.The histological results revealed that the C5/6 disc appeared, at the time of 12 weeks after surgery, moderate degeneration which the nucleus pulposus was shrinking in size and the cells clustered, the annulus fibrosus was unorganized and have some cracks, the endplate was observed the defect in the marginal of endplate in the FR group. Moreover, the disc degeneration was become deteriorated at 24 weeks. Meanwhile, the intervertebral disc in the FR-OVX group more severe compared with FR group at both 12 and 24 weeks. But there were no significantly difference between the FR group and ERT group both in 12 and 24 weeks.The histological scores of disc degeneration (DDS) were in accordance with the situation of the degenerate. Added the factor of OVX, the DDS was increased in 12 weeks, but show no different between the FR and FR-OVX groups. At the time of 24 weeks, the DDS was significantly higher in the FR-OVX group than FR group. With the estradiol therapy, the condition of DDS was consistent with the FR group. These results suggesting that OVX surgery could deteriorate the disc degeneration.At the time of 12 weeks, the density of Aggrecan and Col2 a 1 staining in the FR group markedly decreased compared with sham group. While, much stronger immunostaining was observed for Aggrecan and Col2 a 1 in the FR-OVX group. To contrary, the immunostaining of MMP-3 become gradually stronger in sham, FR and FR-OVX groups, respectively. And at the time of 24 weeks, these indices showed the same trend, but the difference was more obvious. There were no difference in all the indices between the FR group and ERT group both in 12 and 24 weeks.The mRNA expression of Aggrecan and type Ⅱ collagen were decreased, and the expression of the MMP-3 mRNA were increased in the FR and FRT-OVX group compared to sham group in 12 weeks, and becoming more obvious at the tine of 24 weeks. There were no significantly difference between the sham group and the OVX group in 12 weeks, but showed obviously difference at the time of 24 weeks. When it comes to the ERT group, the indices of mRNA expression were showed no different against to the FR group.Conclusions1. The present study suggested that bilateral facetectomy led to the disc degeneration in cervical spine, and guarantied a successful rat model for the disc degeneration.2. Combination of FR and OVX lead to more serious disc degeneration than any one thereof. We can obtained that osteoporosis was positively correlated with the degeneration of intervertebral discs in rats. |
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