RT-PCR Examination of Proinflammatory Cytokine Levels in NPY Knockout Mice

Objectives: Previous results from this group demonstrated that adjuvant-induced inflammation of the TMJ produced a significant increase in inflammatory mediators within the TMJ tissues. However, little is known about the involvement of the neurotransmitter neuropeptide y (NPY) in chronic pain and inflammation of the TMJ. Therefore, the goal of this study was to use RT-PCR to examine changes in the expression of the proinflammatory cytokines interleukin-1b (Il-1b) and tumor necrosis factor-a (TNF-a) during inflammation of the TMJ in NPY knockout mice. Methods: Twenty adult male NPY knockout mice were divided into two groups, of which one group of ten was injected with complete Freund's adjuvant (CFA, 30 mg in 6 ml of paraffin oil) into the TMJ to induce acute inflammation (KO+CFA) while the other group of ten mice did not receive CFA (KO only). In addition, twenty age matched wild type male mice were grouped similarly and received the same treatments (WT+CFA or WT only). All mice were sacrificed one week later and TMJ tissues (disc, retrodiscal, and capsule) were removed and homogenized. Subsequently, RNA was extracted and real time RT-PCR was run to examine changes in RNA expression for Il-1b and TNF-a. Results: RT-PCR results indicate that Il-1b and TNF-a mRNA was significantly increased in the inflamed TMJ tissues (p£0.01) relative to the uninjected controls for both KO+CFA versus KO only and WT+CFA versus WT only. However, mRNA levels for both Il-1b and TNF-a were significantly increased in the TMJ tissues of the KO+CFA mice in comparison to the WT+CFA mice (p£0.01). Conclusions: This study demonstrates that adjuvant-induced inflammation of the TMJ resulted in an up regulation of mRNA expression of the proinflammatory cytokines IL-1b and TNF-a. This increase was significantly exacerbated with the knockout of the NPY gene. Supported by the Baylor Oral Health Foundation.