DR5 and DcR2 Expression in Human Lumbar Intervertebral Discs

IVD degeneration occurs frequently in adult humans and is associated with low back pain and disc herniation. Despite enormous clinical and research efforts, the mechanism of IVD degeneration is still not fully understood. Evidence has indicated that programmed cell death (PCD) plays an important role in some human degenerative diseases. Three members of the TNF-a ligand superfamily, Fas ligand, TNF-α and TRAIL (TNF-α-related apoptosis inducing ligand) have been identified to exhibit potent cytotoxic activity and induce apoptosis of susceptible cells. It is still unclear whether the TRAIL-R/TRAIL signal pathway contributes to the pathology of IVD degeneration. The goal of this study was to reveal the presence and distribution of DR5 and DcR2, and their relationship with apoptosis of lumbar IVD cells. Material and Methods Sixty (60) samples of herniated lumbar disc tissue were obtained from 60 patients undergoing surgical discectomy for persistent sciatic pain. Among them, there are 40 contained and 20 non-contained discs (2 from L3-L4, 34 from L4-L5, 24 from L5-S1). Patient ages ranged from 18 to 68 years (mean = 45 years) and the mean duration of sciatic pain ranged from 1 week to 18 years (mean = 16 weeks). Also, 22 samples of “normal” lumbar IVD tissue were obtained from 8 young victims of fatal accident within 12 hours of death. None of the victims had lumbar IVD herniations or family history of the disorder. All tissue was fixed in 10% neutral formalin and embedded in paraffin for immunohistochemical staining. Samples of surgical disc tissue were also extracted for molecular assessment. Immunohistochemistry Stains: Tissue sections (3μm thick) were prepared and mounted on polylysine-coated slides. Sections were dewaxed, hydrated in 85% ethanol and incubated in 3% hydrogen peroxide for 10 min, followed by four washes in phosphate buffered saline. The slides were incubated with rabbit polyclonal anti-DR5 (1:80 dilution) or rabbit polyclonal anti-DcR2 (1:200 dilution) for 70 min, and incubated for 20 min with a goat anti-rabbit secondary antibody. Slides were washed and stained with DAB, followed by a counterstain with haematoxylin. Samples of herniated lumbar disc tissue without primary antibody were used as negative controls, and lung carcinoma tissue was used as a positive control. The annulus fibrous (AF) cells and the nucleus pulposus (NP) cells were counted. The total number of disc cells and those positive for antibody staining were quantified and the percentage of positive disc cells to total disc cells was calculated. Statistic Analysis The percentage of HIC positively stained cells over the total cells in the region was calculated in the nucleus and annulus of each disc. Statistical comparison between the nucleus and annulus regions was performed by the Student T test using the SAS software package. A p value of less than 0.05 was considered as significant. Data were expressed as mean percentage ± standard deviation. In addition, IHC-positive percentage among normal, and herniated discs, and other variables such as age and duration of the disease were compared by oneway ANOVA with LSD post hoc multiple comparisons. Results Immunohistochemical localization: Significantly more DR5-positive stained cells were identified in herniated IVD tissues than in normal IVD tissues (Table I). As for the localization, higher percentages of DR5+ cells were present at NP region (Figure 1) than AF area (Table I).