Interestingly, collagen type IX expression decreased in only the AF at 10 days. To investigate changes in expression of important proteoglycan matrix components of the tissues, we studied the expression pattern of aggrecan and fibromodulin and found a significant downregulation in both the NP and AF after 3 and 10 days in organ culture. selleck chemical Vorinostat To investigate whether a decrease in aggrecan expres sion is coupled with an increase in aggrecan break down, Western blot analysis of NP and AF tissue lysates was performed. Owing to the lower cellularity of the AF in comparison with the NP tissue, a smaller proportion of cellular protein was observed for AF. Figure 3C shows that, in treated samples, anti ARGxx antibody detects additional cleaved aggrecan core pro tein fragments in comparison with untreated controls.

Upregulation in catabolic gene expression We performed an analysis to investigate whether treat ment with cytokines and nutritional deficiency results in upregulation of genes encoding catabolic enzymes. Fig ures 4A and 4B show a significant induction in MMP 3 expression in NP and AF tissues at 3 and 10 days after treatment. Expression of MMP 3 after 3 days of treat Inhibitors,Modulators,Libraries ment was also analyzed by Western blot analysis. Interestingly, the increase in MMP 3 protein expression is more prominent Inhibitors,Modulators,Libraries in the AF tissue in com parison with the NP. Immuoflorescence analysis was used to confirm the expression and localization of MMP 3 in the treated discs. Figure 4D shows a positive Inhibitors,Modulators,Libraries staining of MMP 3 in NP and AF cells. Outer AF cells demonstrate enhanced expression of MMP 3 in compar ison with inner AF cells.

We then examined expression of MMP 9 and MMP 13 in treated discs. Similar to MMP 3, both MMP 9 and MMP 13 show increased mRNA expression in both of the tissues. Western blot analysis was also used to mea sure MMP 9 and MMP 13 expression in disc tissues. Figure 5C shows a significant induction in MMP 9 in AF cells of the treated Inhibitors,Modulators,Libraries disc and a smaller increase in MMP 9 expression in the NP tissue. In contrast to MMP 9 protein, MMP 13 protein shows a prominent increase in NP when compared with the AF tissue. Overall, these results demonstrated an enhanced expression of catabolic MMPs in the disc after the treatment. Changes in expression of genes that are associated with pain and inflammation Since the disc organ culture is an in vitro system, a direct assessment of pain and inflammation without the involvement of neuronal and immunological compo nents is not feasible.

However, to get an indirect assessment of whether Inhibitors,Modulators,Libraries the degenerative changes in the discal tissues elicit changes in the expression of genes that activate neuronal and immune components result ing in pain and inflammation changes in expression of marker genes such as nerve growth factor, TNF a, and IL SB203580 buy 1b were measured. Figures 6A and 6B show that 3 days of treatment results in increased NGF expression by the NP and the AF.