| Part one The protential effects of salmon calcitonin on disc degeneration djacent to a lumbar fusion in ovariectomized ratsObjective:To establish a model of intervertebral disc degeneration adjacent to lumbar fusion in ovariectomized rats and to investigate the effects of salmon calcitonin on this model.Methods:50 female SD rats were include in this study. All rats received either Sham(n=20) surgery or bilateral ovariectomy(OVX)(n=30). Four weeks after surgery, all but ten 10 rats from each group underwent Posterolateral lumbar fusion(PLF) at L4–L5. OVX+PLF rats were treated by either(1) saline vehicle treatment(OVX+PLF+V, n=10); or(2) subcutaneous injection of salmon CT at a dose of 16 IU/kg per 2 days for 12 weeks(OVX+PLF+s CT, n=10). The remaining rats from each group received saline vehicle treatment and were divided into three groups of 10 each: Sham+V, OVX+V, and PLF+V. After 12 weeks of treatment, all animals were sacrificed. The L3~6 segment discs were removed, dissected of muscle, and assessed palpated manually. Then, separate the bilateral L4~5 intertransverse fusion site and L5~6 intervertebral disc, which decalcified, embedded, sectioned to dyeing in turn. Adjacent~segment intervertebral disc was stained with VG followed by the histological score. The height of the intervertebral disc endplate, endplate thickness and endplate calcification area ratio were measured based on the VG staining images.Results:1 Fusion evaluationResults of manual palpation: For manual palpation, the steel wire of all PLF~surgery rats remained intact and connected to the spinous process. There was no detectable movement at the levels of fusion on manual palpation in all PLF~surgery rats.Results of X~ray film: A lower radiographic density was observed in OVX+PLF+V rats compared with the PLF+V and OVX+PLF+s CT groups. The radiographic scores indicated a delayed fusion process in OVX+PLF+V rats, which was significantly lower than the PLF+V and OVX+PLF+s CT groups(P < 0.05).2 Histological observation and qutitative parameter analysis of intervertebral disc degenerationThe Sham+V group appeared to exhibit abundant notochordal cells and surrounding extracellular matrix in the nucleus pulposus, well~arranged outer annulus fibrosus, and the endplate was rich in hyaline cartilage. The OVX+V, PLF+V, and OVX+PLF+V groups experienced degeneration of the nucleus pulposus, annulus fibrosus, and endplate. Loss of cellularity of notochordal cells and their replacement by chondrocyte~like cells were found in the nucleus pulposus, and the matrix displayed mucoid degeneration. Loss of collagen, proliferation of fibrocartilaginous tissue, and disruption of the nuclear~annular border were observed in the annulus fibrosus. The endplate exhibited large areas of calcification to different degrees. These mineralized tissues became more obvious in the deep zone of the middle cartilage endplate. All degenerative changes were more obvious in the OVX+PLF+V group compared with the OVX+V or PLF+V groups.Accordingly, the histological score in the OVX+V, PLF+V, OVX+PLF+V, and OVX+PLF+s CT groups were markedly higher than the Sham+V group(P < 0.05); the highest value was found in the OVX+PLF+V group, which was significantly different compared with any other group(P < 0.05).The disc height in the OVX+V, PLF+V, and OVX+PLF+V groups were all decreased compared with the Sham+V group, and the opposite trends were found in endplate thickness and ratio of calcified area to total endplate area(P < 0.05). The OVX+PLF+V group showed the most serious degenerative changes, with the lowest disc height, highest endplate thickness, and largest ratio of calcified area to the total endplate area(P < 0.05). s CT treatment markedly prevented these degenerative changes, indicated by the significantly higher disc height, lower endplate thickness, and decreased ratio of calcified area to the total endplate area, compared with the OVX+PLF+V group(P < 0.05).Conclusion:Rats received either lumbar fusion or dual ovariectomy displayed intervertebral disc degeneration and aggravated when received both opertaion, which can be used as a model of intervertebral disc degeneration in post~menopause women with lumbar fusion operation. Calcitonin treatment can prevent intervertebral disc degeneration adjacent to lumbar fusion in ovariectomized rats. Part two Effects of calcitonin on the expression levels of Col~I, Col~II, ggrecan, MMP~13 and ADAMTS~4 in intervetebral disc djacent to a lumbar fusion in ovariectomized ratsObjective:To determine the age related changes and effect of Calcitonin on the extracelluar matrix metablism by assessing the expression levels of glycosaminoglycan, type II collagen and matrix metalloprotease(MMP)~1 in DH guinea pig by immunohistochestry analysis.Methods:50 female SD rats were include in this study. All rats received either Sham(n=20) surgery or bilateral ovariectomy(OVX)(n=30). Four weeks after surgery, all but ten 10 rats from each group underwent Posterolateral lumbar fusion(PLF) at L4–L5. OVX+PLF rats were treated by either(1) saline vehicle treatment(OVX+PLF+V, n=10); or(2) subcutaneous injection of salmon CT at a dose of 16 IU/kg per 2 days for 12 weeks(OVX+PLF+s CT, n=10). The remaining rats from each group received saline vehicle treatment and were divided into three groups of 10 each: Sham+V, OVX+V, and PLF+V. After 12 weeks of treatment, all animals were sacrificed. The L3~6 segment discs were removed, dissected of muscle, and assessed palpated manually. Tissue sections from L5 to L6 segments were deparaffinized in xylene and rehydrated in a reverse~graded series of ethanol, finally embeded in paraffin. Samples were then cut into 4~ m~thick sections. Col~I, Col~II, Aggrecan, MMP~13 and ADAMTS~4 expression levels in nucleus pulposus and annulus fibrosus were detected by Immunohistochemistry stanining. The sample appearing yellow or brownish yellow was considered as positive staining. All sections were semi~quantitatively analyzed by Image Pro Plus(IPP) version 6.0 software, and the integrated optical density(IOD) was measured by the staining in 6 fields in each section on the images at 400× magnification, the average IOD and used for statistical analysis.Results:The positive staining of Col~I, Col~II, Aggrecan, MMP~13 and ADAMTS~4 were both observed in the nucleus pulposus and annulus fibrosus were detected by immunohistochemical staining. Positive staining was confirmed as the intensity from faint yellow to brownish yellow.The difference of each between any two groups were as follows:Col~I: Immunohistochemistry assay and IOD for Col~I in the nucleus pulposus showed and annulus fibrosus showed similar trends that the protein levels of Col~I expressed lowest in Sham+V group, while highest expression level was observed in OVX+PLF+V group. The IOD of OVX+V group, PLF+V group and OVX+PLF+Vgroup were significantly higher than that of Sham+Vgroup, OVX+PLF+V group showed also markedly higher levels than those of OVX+V group, PLF+Vgroup and OVX+PLF+s CT group.Col~II: Immunohistochemistry assay and IOD for Col~II in the nucleus pulposus showed and annulus fibrosus showed similar trends that the protein levels of Col~II expressed highest in Sham+V group, while lowest expression level was observed in OVX+PLF+V group. The IOD of OVX+V group, PLF+V group and OVX+PLF+Vgroup were significantly lower than that of Sham+Vgroup, OVX+PLF+V group showed also markedly lower levels than those of OVX+V group, PLF+Vgroup and OVX+PLF+s CT group.Aggrecan: Immunohistochemistry assay and IOD for Aggrecan in the nucleus pulposus showed and annulus fibrosus showed similar trends that the protein levels of Aggrecan expressed highest in Sham+V group, while lowest expression level was observed in OVX+PLF+V group. The IOD of OVX+V group, PLF+V group and OVX+PLF+Vgroup were significantly lower than that of Sham+Vgroup, OVX+PLF+V group showed also markedly lower levels than those of OVX+V group, PLF+Vgroup and OVX+PLF+s CT group.MMP~13: Immunohistochemistry assay and IOD for MMP~13 in the nucleus pulposus showed and annulus fibrosus showed similar trends that the protein levels of MMP~13 expressed lowest in Sham+V group, while highest expression level was observed in OVX+PLF+V group. The IOD of OVX+V group, PLF+V group and OVX+PLF+Vgroup were significantly higher than that of Sham+Vgroup, OVX+PLF+V group showed also markedly higher levels than those of OVX+V group, PLF+Vgroup and OVX+PLF+s CT group.ADAMTS~4: Immunohistochemistry assay and IOD for ADAMTS~4 in the nucleus pulposus showed and annulus fibrosus showed similar trends that the protein levels of ADAMTS~4 expressed lowest in Sham+V group, while highest expression level was observed in OVX+PLF+V group. The IOD of OVX+V group, PLF+V group and OVX+PLF+Vgroup were significantly higher than that of Sham+Vgroup, OVX+PLF+V group showed also markedly higher levels than those of OVX+V group, PLF+Vgroup and OVX+PLF+s CT group.Conclusion: Decreased expression levels of Col~II and aggrecan, along with increased levels of Col~I, MMP~13, and ADAMTS~4, were observed in adjacent level intervertebral disc in ovariectomized rats with lumbar fusion. In contrast, CT treatment thoroughly reversed these changes in expression levels, indicating that the protective effects of CT on adjacent~segment disc degeneration in this model were, at least in part, due to modulation of the synthesis and degradation of extracellular matrix. Part three Effects of calcitonin on the bone mass and ro~structure of umbar vertebrae adjacent to fusion site in ovariectomized ats.Objective:To investigate the effects of calcitonin on the bone mass and micro~structure of lumbar vertebrae adjacent to fusion site in ovariectomized rats.Methods:50 female SD rats were include in this study. All rats received either Sham(n=20) surgery or bilateral ovariectomy(OVX)(n=30). Four weeks after surgery, all but ten 10 rats from each group underwent Posterolateral lumbar fusion(PLF) at L4–L5. OVX+PLF rats were treated by either(1) saline vehicle treatment(OVX+PLF+V, n=10); or(2) subcutaneous injection of salmon CT at a dose of 16 IU/kg per 2 days for 12 weeks(OVX+PLF+s CT, n=10). The remaining rats from each group received saline vehicle treatment and were divided into three groups of 10 each: Sham+V, OVX+V, and PLF+V. After 12 weeks of treatment, all animals were sacrificed. The L3~6 segment were harvested, the bone mineral density(BMD) of L3 to L6 were analyzed by dual energy X~ray absorptiometry using a densitometer. Three~dimensional(3D) analysis was performed on the trabeculae of the cancellous tissue of the L6 vertebra, On the original 3D images, morphometric indices were directly determined from the volume of interest, which was defined as being restricted to an inner cylinder with a 1.5~mm diameter and a 3~mm length(1–4 mm proximal to the growth plate), excluding the cortex,. The following 3D morphometric parameters were calculated to describe the bone mass and microstructure: BMD, percent bone volume(BV/TV), trabecular number(Tb.N), trabecular thickness(Tb.Th), trabecular separation(Tb.Sp) and structural model index(SMI).Results:1 BMD of L3–L6 vertebrae:The same trends in the differences in BMD of the L3–L6 vertebrae were seen between any two of all five groups. Rats in the OVX+V and OVX+PLF+V groups exhibited significantly lower BMD compared with the Sham+V and PLF+V groups(P < 0.05); while OVX+PLF+s CT rats showed significantly higher BMD compared with OVX+PLF+V rats(P < 0.05).2 Microstructural parameters of the L6 vertebrae: Micro~computed tomography showed that BV/TV, Tb.Th, and Tb.N were significantly lower, and Tb.Sp and SMI were significantly higher in the OVX+V and OVX+PLF+V groups compared with the Sham+V and PLF+V groups(P < 0.05). CT~treated rats had markedly higher BV/TV, TB.Th, and Tb.N and lower Tb.Sp and SMI compared with the OVX+PLF+V group(P < 0.05).Conclusion: CT treatment could partially prevent the bone loss and deterioration of cancellous bone in adjacent vertebrae in ovariectomized rat with lumbar fusion, which may contribute to, at least in part, the protective effect of CT on the intervertebral disc degeneration in this model. |
PDF Full Text Request