| Part I: rabbit lumbar intervertebral disc degeneration model buildingObjectives: To establish a rabbit model of intervertebral disc degeneration induced by puncturing the anulus fibrosus with needles of defined gaugesMethods: The L2/3 and L4/5 (L3 / 4 and L5 / 6 as self-control) intervetebral discs of New Zealand white rabbit in ordinary grade were stabbed by 16-gauge transfixion pin into a depth of 5 mm in the anterolateral annulus fibrosus magnetic resonance imaging scans of the stabbed discs and intact discs were performed preoperatively and at the2th,4th,8th.16th and 32th week af ter surgery. The histologic and immunohistochemical analyses were performed after the animals were killed and the materials were ext racted.Results: In the magnetic resonance imaging,the stabbed discs exhibited a progressive decrease of signal intensity in T2-weighted images which start at 2 week Statistically significant (P <0.05)after stabbing and last for 16 weeks.Immunohistochemical and histologic analyses revealed progressive decrease of chondrocyte-like cells and type II collagen (P<0.01).Conclusions: The stabbing approach results in a slowly progressive intervertebral disc degeneration in rabbit model.This model is available for studying the status intervertebral disc degeneration.Although the rabbits of the spine in bio-mechanics and anatomy of human existence with some differences, but the disc tissue structure similar to humans, it can be used as animal models of IDD research, and may for this kind of research a large sample of experimental data. Part II: different age groups of rabbit intervertebral disc cells in the distribution of types of researchObjectives: To evaluate notochordal cells and chondrocyte cell morphology and distribution with each stage of ages.Methods: The experimental group to take 10 New Zealand white rabbits, weighing 2±0.5kg, 2 years old, complete, remove the lumbar spine, rampant rabbit lumbar intervertebral disc annulus incision, complete remove the nucleus, into the petri dish, PPS lotion modulation PH7. 21% of low melting point agarose coverage, coverage, fixed tissue, and then implanted in 10% neutral formalin fixed, for the edge of nucleus pulposus and annulus fibrosus unclear degenerative disc three times, with 6mm diameter needle out disc central part of the agarose-embedded nucleus. Fluorescence quenching of anti-sealing tablet Fengpian laser confocal microscope observation, semi-quantitative.Results: Confocal microscopy can be in New Zealand white rabbit nucleus pulposus samples, we found two kinds of cells, notochord cells and chondrocytes in cell size between the two although there are obvious differences, but the 3-month-old when the intervertebral discs account for large-cell the main part, from the cell counts on the analysis of large cells accounted for all of the 80.92%, and the small cells have very different; in three years, when the sharp increase in the number of small cells, while the large number of cells is decreased, the proportion of 32.8% that large cell from 3 months to 3 years is to reduce the number, while a small number of cells increased faster. Vimentin immunofluorescence intensity of the average optical density of semi-quantitative analysis, and average optical density of CD44 immunofluorescence intensity of semi-quantitative analysis (P <0.01, P <0.05), there are statistically significant.Conclusions: Select notochord cells in this experiment the characteristic marker CD44 and Vimentin fluorescent mark was measured in notochord cells and cartilage-like nucleus pulposus cells, the average fluorescence intensity, results showed that: notochord cells expression intensity of these two proteins was significantly higher than medullary nucleated cells (P<0.01), and negatively correlated with age, younger, stronger expression. Tip cell maturation and aging as the notochord, and notochord cells, regulation and function-related protein expression gradually decreased. To play a role in its surrounding environment is also reduced gradually.Part III: notochord cells in the rabbit intervertebral disc degeneration in the process of the role and fateObjectives: To evaluate the roles of notochord cells in the process of disc degeneration.Methods: To take New Zealand white rabbits divided into 3-month-old and 3 years two groups of a total of 14, in March body weight 1.5±2.0kg, 3 year-old group of about 3.0-4.5kg body weight of 28. Each has two-line operation is not as normal controls. The remaining 12 rats in each group into the manufacture of disc degeneration model of operation. Lines at each time point imaging MRI confirmed disc degeneration, via ear vein injection of 10 mL of air experimental animals were sacrificed, access to surgery and the normal self-control segment of the intervertebral disc nucleus pulposus, surgical segmental degeneration of nucleus pulposus as a group, self - the absence of treatment gap for a control group. Remove the nucleus pulposus is divided into two parts, part of the line-free tissue sections stained with HE after aggrecan,Ⅱ-type collagen and PCNA staining, immunohistochemical staining. Another part of -70 degrees to save, RT-PCR detection.Results: Intervertebral disc degeneration models show that notochordal cells gradually disappear, being irregular cartilage-like matrix of nucleus pulposus cells and replaced in March of rabbit disc degeneration model, before 16 weeks of notochord cells showed PCNA expression, 16 weeks after the spinal cord cells were significantly reduced, did not express PCNA, group cartilage-like cells PCNA16 weeks after an increase in cartilage-like nucleus pulposus cells, showing positive staining; March group notochord cells PCNA-positive cells gradually increased the rate of eight weeks before 16 weeks, decreased gradually. Cartilage-like nucleus pulposus cells, PCNA-positive cells 4 weeks before the rate remained stable, increased gradually after 8 weeks. Nucleus pulposus cells, PCNA and aggrecan protein levels in March group was significantly higher than 3-year group, notochord cells in the first 2 weeks of basic stability, the latter dropped significantly. Cartilage-like nucleus pulposus cells in the first 8 weeks of basic stability, the latter slightly increased.Conclusions: The experimental two age groups in the event of proteoglycan and collagen typeⅡRT-PCR reduction in the level, MRI T2 signal and decreased significantly as the following time points after the same group in March compared with the histological observation of the 3-year group there are more notochord cells, Although the cellular protein level of PCNA and expression of aggrecan was no significant difference between the two groups, but the nucleus pulposus cells, PCNA and aggrecan protein levels in March group was significantly higher than 3-year group. Nucleus pulposus cells show a certain degree of proliferation and proteoglycan secretion capacity and the number of cells in the spinal cord. |
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