| Part one Effect of calcitonin on spontaneous intervertebral disc degeneration in DH guinea pig.Objective: To determine whether age related IVD degeneration would occur in DH guinea pig and to further investigate the effect and mechanism of calcitonin(CT) on intervertebral disc degeneration in DH guinea pig.Methods:1 Forty-two 3-month-old female guinea pigs were random Ly assigned into 2 groups as follows: Twenty-four were treated by normal saline as control group and sacrificed just before intervention(3-month-old), or continuing intervened for 3(6-month-old), 6(9-month-old), and 9 months(12-month-old), 6 animals at each time point. The other 18 were received CT(8μg/kg/day, Miacalcic, Novartis Ltd.) treatment at 3 months of age and sacrificed at the time of treatment for 3, 6 and 9 months respectively.2 The L4-6 segments of the lumbar spine(including the intervertebral disc) were fixed in neutral-buffered 10% formalin, decalcified and embedded in paraffin. Samples were then cut into 4-μm-thick sections and stained with van Gieson(VG) for light microscopic examination. Degenerative changes in the intervertebral discs were observed and the degrees of change in the stained sections were scored in blinded fashion. Disc height measurements were taken from endplate to endplate on histological samples from the L5-6 segments, for each image, the height was determined by an average of three measurements made in three areas of the disc space: on the left side, center and right side. The ratio of disc height to L6 vertebral body height was calculated. The porosity of bony endplate indicated the ratio of the volume of the pores in the bony endplate over the total volume of the bony endplate. The bone endplatearea and total endplate area were also measured and the ratio of bone endplate area to total endplate area was calculated.Results: 1 Body weight: the body weight of DH guinea pig increased with age from 3 to 12 months, with significant difference in comparison with the former time point, respectively(P < 0.05). 2 Normal histological appearance was observed in 6 months old or younger animals, the evidence of disc degeneration was present in 9 months old animals as the nucleus pulposus became narrowed, coupled with loss of notochordal cells, and these changes were aggravated as age increased to 12 months old. Loss of collagen, proliferation of fibrocartilaginous tissue and disruption of the nuclear-annular border were observed at the annulus fibrosus. In contrast to the control animals, CT treated animals showed wider disc and more notochordal cells in nucleus pulposus, coupled with more clear nuclear-annular border. 3 Accordingly, the histological score reflected pathological changes to the IVD. For the control groups, the scores increased with age, significant differences were observed between older(9 and 12 months) and younger(3 and 6 months) animals(P < 0.05). CT treatment markedly reduced the histological score at 12 months of age, in contrast to the control group(P < 0.05). 4 The disc height in guinea pig was increased from 3 months to 6 months of age, thereafter decreased with age, with significant difference between 12 and 9 months old animals(P < 0.05). At 12 months of age, CT treated animals showed significantly higher disc height when compared with control animals(P < 0.05). In addition to the changes observed by assessing the disc height, the ratio of disc height to vertebral body height showed more pronounced difference between 9 and 6 months old animals, which was not significant when assessed the disc height only. 5 The ratio of bony endplate area to total endplate area were much higher in older(9 and 12 months) animals than those of younger(3 and 6 months) animals(P < 0.05). CT treatment could increase this ratio in 12 months old animals(P < 0.05).Part two Effects of CT on the expression levels of Col-II, GAG and MMP-1 in IVD in DH guinea pig.Objective: 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:1 Forty-two 3-month-old female guinea pigs were random Ly assigned into 2 groups as follows: Twenty-four were treated by normal saline as control group and sacrificed just before intervention(3-month-old), or continuing intervened for 3(6-month-old), 6(9-month-old), and 9 months(12-month-old), 6 animals at each time point. The other 18 were received CT(8μg/kg/day, Miacalcic, Novartis Ltd.) treatment at 3 months of age and sacrificed at the time of treatment for 3, 6 and 9 months respectively.2 The L4-6 segments of the lumbar spine(including the intervertebral disc) were fixed in neutral-buffered 10% formalin, decalcified and embedded in paraffin. Samples were then cut into 4-μm-thick sections. Glycosaminoglycan, type II collagen and matrix metalloprotease(MMP)-1 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: 1 The protein levels of glycosaminoglycan, type II collagen and MMP-1 expression 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. Immunohistochemistry assay for type II collagen in the nucleus pulposus showed that the protein levels of type II collagen increased from 3 to 9 months of age and decreased at 12 months of age, CT treatment dramatically retarded the decrease(P < 0.05). 2 Immunohistochemistry assay for type II collagen in the annulus fibrosusshowed that the peak protein level of type II collagen at 6 months of age and thereafter decreased with age, CT treatment dramatically retarded the decrease(P < 0.05). 3 Immunohistochemistry assay for GAG in the nucleus pulposus showed that the protein levels of GAG increased from 3 to 9 months of age and decreased at 12 months of age, CT treatment dramatically retarded the decrease(P < 0.05). 4 Immunohistochemistry assay for GAG in the annulus fibrosus: the peak protein level of type II collagen at 9 months of age and thereafter decreased with age, CT treatment dramatically increased GAG expression at 6 and 9 months of age(P < 0.05). 5 Immunohistochemistry assay for MMP-1 in the nucleus pulposus showed that the protein levels of MMP-1 increased from 3 to 9 months of age and decreased at 12 months of age, CT treatment dramatically decreased the MMP-1 expression at 9 months of age(P < 0.05). 6 Immunohistochemistry assay for MMP-1 in the annulus fibrosus showed that the protein levels of MMP-1 increased from 3 to 9 months of age and decreased at 12 months of age, CT treatment dramatically decreased the MMP-1 expressions(P < 0.05).Part three Effects of Calcitonin on bone mineral density, micro structure and biomechanical properties of the lumbar vertebrae in DH guinea pig.Objective: To determine the age related changes and effect of Calcitonin on bone mineral density, micro structure and biomechanical properties of the lumbar vertebrae.Methods:1 Forty-two 3-month-old female guinea pigs were random Ly assigned into 2 groups as follows: Twenty-four were treated by normal saline as control group and sacrificed just before intervention(3-month-old), or continuing intervened for 3(6-month-old), 6(9-month-old), and 9 months(12-month-old), 6 animals at each time point. The other 18 were received CT(8μg/kg/day, Miacalcic, Novartis Ltd.) treatment at 3 months of age and sacrificed at the time of treatment for 3, 6 and 9 months respectively. all lumbar vertebraes were harvested.2 Segments L3–L5 of each animal were removed, dissected free of muscles, and bone mineral density(BMD) measurements were performed on the anteroposterior plane by dual energy X-ray absorptiometry. 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(VOI), which was restricted to an inner cylinder with 1.5 mm diameter and the entire vertebra, excluding the region 1 mm from the proximal and distal growth plate. The following 3D morphometric parameters were calculated to describe the bone mass and microstructure: percent bone volume(BV/TV), trabecular number(Tb.N), trabecular thickness(Tb.Th), trabecular separation(Tb.Sp) and structural model index(SMI).3 The mechanical strength of the lumbar vertebra(L2) was measured by compression test. In this test, the planoparallel surfaces were obtained by removing the cranial and caudal ends of the vertebral specimen, thereby allowing for a uniform compression test to be performed on individual rodent vertebrae. From the vertebral body, a central cylinder with planoparallel ends and a height of approximately 5 mm was obtained. All compression tests were performed in the displacement-control mode at a crosshead speed of 0.5 mm/min to eliminate any strain rate effects. Maximum load, yield stress, maximum stress, and elastic modulus were obtained from compression tests of the vertebral bodies.Results: 1 The results of bone mineral density assessment: The bone mineral density of L3-5 were increased from 3 to 6 months of age, and maintain at stable levels until 12 months old, with significant difference in 6, 9 and 12 months old animals in comparison with that of 3 months old animals, CT treatment notably increased the bone mineral density in 12 months old animals(P < 0.05). 2 The results of micro-CT analysis: Trabecular bone loss was indicated in 12 months old animals as BV/TV was markedly lower than younger animals, while the Tb.Sp and SMI were markedly higher in 12 months old animals than those of younger animals. Compared with controlgroups, CT treatment notably increased the BV/TV and markedly suppressed the Tb.Sp in 12 months old animals(P < 0.05). 3 The results of biomechanical test: Maximum load, yield stress, maximum stress, and elastic modulus were all increased from 3 months to 6 months of age, thereafter maintained in stable levels untill 12 months old, of which 6, 9 and 12 months groups were significantly higher than that of 3 month group, while there is no significant difference between any two groups from 6, 9 and 12 months old control animals. CT treatment markedly increased maximum load, yield stress, maximum stress in all time points, but for elastic modulus, only 6 months old animals showed significant difference between CT treated and control animals(P < 0.05).Conclusions:1 Age-related spontaneous intervertebral disc degeneration occurred in Dunkin Hartley guinea pigs and aggravated with age. Dunkin Hartley guinea pig could be used as an animal model to study the etiological factor and mechanism of intervertebral disc degeneration as its spontaneous intervertebral disc degeneration shares similarities with human in some histological characteristics.2 In comparison with 9 months old animals, 12 months old DH guinea pigs showed lower levels of glycosaminoglycan and type II collagen, and higher levels of matrix metalloprotease(MMP)-1, Pretreatment with CT could inhibit these changes of extracellular matrix metabolism, which may contribute to the protective effect of CT on the IVD degeneration in this model.3 CT treatment could improve the microstructure and biomechanical properties of the vertebrae, thereafter be benefical for improving the biological environment, which may contribute to the protective effect of CT on the IVD degeneration in this model. |
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