The Study Of FasL Maintain Immunological Privilege Of Intervertebral Disc And Induce Apoptosis Of Nucleus Pulposus Cells

Part 1. A Rabbit Model of Disc Degeneration by an Anulus Needle PunctureObjective : To establish a slowly progressive, reproducible rabbit model of intervertebral disc degeneration by puncturing the anulus with needles of defined gauges. Method: The L3/4, L4/5 and L5/6 lumbar intervertebral discs of 24 New Zealand White rabbits were stabbed by 18-gauge hypodermic needle to a depth of 5mm in the left anterolateral anulus fibrosus. Magnetic resonance imaging scans, Immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 control discs were performed preoperation or at 3, 6, 10weeks post surgery. Result: Form the magnetic resonance imaging,we detected the stabbed discs exhibited a progressive decrease in signal intensity of T2-weighted images starting at 3 weeks post stab and continuing through 10 weeks. Immunohistochemical and histologic analyses revealed progressive loss of chondrocyte-like cells and type II collagen(p<0.01).Conclusion: The Stabbing approach, resulted in a slowly progressive intervertebral disc degeneration of rabbit. This model would appear suitable for studying and testing safety and efficacy of novel treatments of intervertebral disc degeneration. Part 2. The expression of Fas ligand on normal and stabbed disc cells of rabbit IDD modelObjective To clarify the relation between physiologic barrier and the role of FasL,and investigate a possible pathogenesis of intervertebral disc degeneration (IDD).Methods The anular puncture model of rabbit were established using defined needle gauges and depths. The normal and the stabbed discs were harvested at each time period of 3,6,and 10 weeks after surgery. Immunohistochemical staining of these discs for Fas and FasL was performed by standard procedures. The mean apoptosis index of disc cells was analyzed by flow cytometry.Results The normal discs cells exhibited relatively weak immunopositivity, and the stabbed disc cells exhibited intense immunopositivity. A significant difference in the percentage of FasL-positive disc cells between the normal discs and the stabbed discs (P<0.001). The mean apoptosis indexes at 3 weeks, 6 weeks, 10 weeks post stab were significantly higher than normal disc (P<0.001,0.002 and 0.006, respectively). There was a significant correlation between the degree of FasL-positive expression and the degree of Fas-positive expression of the disc cells post stab (r = 0.571, P = 0.0036). Conclusions This observation indicates that the nucleus pulposus is a 'immune-privileged' sites. The immune privilege is maintained by FasL and the physiologic barrier together. When the physiologic barrier was damaged, the role of FasL changed, coexpressed with Fas induce apoptosis of disc cells. These results indicate that an autoimmune reaction may be a possible pathogenesis of IDD. Part 3. The expression of Fas ligand mRNA on human intervertebral disc.Objective. To clarify the existence of Fas ligand on intact intervertebral disc cells and herniated lumbar disc tissues.Methods. Sixteen herniated lumbar disc tissues (contained disc, n=8; noncontained disc, n=8)and four normal human disc tissues were examined to investigate the mRNA expression of FasL,Fas,TNF and IL by reverse transcription polymerase chain reaction method. The degeree of mRNA expression were compared.Results. The result of RT-PCR confirmed the existence of Fas ligand in normal and herniated disc tissues, but Fas,TNF and IL mRNA were expressed only in herniated disc tissues. A higher degree of FasL expression in herniated disc cells was found in noncontained discs than in contained discs ( P<0.05). Conclusion. We demonstrated the existence of Fas ligand on normal disc cells, which should play a key role in the potential molecular mechanism to maintain immune privilege of the disc. But after herniation, undergo inflammatory reaction and apoptotic cell death via autocrine or paracrine FasL mechanisms by the disc cells themselves.