| Budget Amount *help |
¥3,920,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
The pathogenesis of intervertebral disc degeneration is poorly understood, but is known to be associated with a variety of cellular and biochemical changes. Vertebral endplate changes and bone marrow changes visible in magnetic resonance imaging (MRI), are closely associated with dipc degeneration. To investigate molecular mechanisms underlying the process of endplate degeneration, we tried to apply laser capture microdissetion combined with microarray analysis to vertebral endplate.
Although we have succeeded in extract total RNA from vertebral endplate, its quantity has not been enough for microarray analysis. Thus, we could not help abandon carry out the microarray analysis. However, we could acquire many skills for the analysis of hard tissues including bone and cartilage. Besides, we have developed novel disc degeneration model for rat tail.
We have already developed continuous weight bearing disc degeneration model for mouse tail. In this model, degradation of IVD was observed with
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in two weeks after operation. Using this model, we tested if weight loading, a most common cause of intervertebral disc (IVD) degeneration, induces Runx2 expression, which may cause chondrocyte hypertrophy. Indeed, weight loading of the IVD by compression for a day significantly increased Runx2 expression. Thus, we next examined the expression of Runx2 with other disc degradation markers in canine IVD, The expression of RUNX2, type-X collagen and MMP-13 mRNA in young intact and degenerated IVD were examined by semi-quantitative RT-PCR analysis. The localization of Runx2 and type-X collagen protein in control and degenerated IVD were examined by imunohistochemistly. The expression of Runx2 transcript and protein, in combination with type-X collagen and MMP-13, was enhanced in degenerated IVDs of the dog.
Next, we have investigated the relationship between expression patterns of Runx2 and the degree of intervertebral disc degeneration in canine NP cells through comparing the expression patterns to MMP-13 expression, a disc degeneration marker, and to MRI findings. Canine disc specimens were obtained from chondrodystrophoid dogs, including Dachshund and Beagle. RACE method indicated the canine Runx2 cDNA showed over 97% conservation with human RUNX2 and 95% with mouse. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that mRNA levels of Runx2 and MMP-13 were similarly increased in degenerated NP and herniated specimens. Immunohistochemical analysis indicated that T2/CSF ratio in MRI images was significantly correlated with Runx2 protein expression rate in various age beagle disc samples. Thus, our findings suggest Runx2 is a novel marker for disc degeneration.
As mentioned above, intervertebral disc degeneration induced by mechanical compression is an important issue in spinal disorder research. In this study, the biomechanical aspect of the rat tail model was also investigated. An external loading device equipped with super-elastic TiNi springs was developed to apply a precise load to the rat tail. By using this device, rat tail discs were subjected to compressive stress of 0.5 or 1.0 MPa for 2 weeks. Discs in the sham group received an attachment of the device but no loading. After the experimental period, first the intact tail with peripheral tissues (PT) such as tendon and skin and then the retrieved disc without PT were subjected to a uniaxial tension-compression test ; biomechanical characteristics such as range of motion (ROM), neutral zone (NZ), and hysteresis loss (HL) were evaluated. Furthermore, the loadbearing contribution of PT in the intact tail was estimated by comparing the load-displacement curves obtained by the mechanical tests performed with and without PT.The experimental findings revealed that the continuous compressive stress induced reduction in disc thickness. The intact tail demonstrated decreases in ROM and NZ as well as increases in HL.On the other hand, the retrieved disc demonstrated increases in ROM, NZ, and HL.Further, a significant increase in the load-bearing contribution of PT was indicated. These findings suggest that the load-bearing capacity of the disc was seriously deteriorated by the application of compressive stress of 0.5 or 1.0MPa for 2 weeks.
Lastly, we have developed novel LCM technique applied for hard tissues, such as articular cartilage. Using our techniques, we have been investigating the mechanisms of cartilage degeneration. Less
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