Applying Immunohistochemistry & Reverse Transcription Pcr to Intervertebral Disc Degeneration in an Animal Model

STUDY DESIGN: Devise a set of protocols for immunohistochemical (IHC) and gene expression analysis that will permit the measurement of changes that occur during degeneration in the lumbar intervertebral disc of a rat model. OBJECTIVES: Quantify how the composition of the disc changes with degeneration in the animal model and prove that these changes correspond to those within a degenerate human disc, supporting the rat as a valid model of spinal disc degeneration. In the future, use the model to determine mechanisms for the degenerative process. SUMMARY OF BACKGROUND DATA: Rodents have been widely used as models to study disc degeneration. The chemical changes within the human disc have also been extensively studied by various methods. However, the research to understand the same chemical changes in the rat has been limited. METHODS: IHC staining of the lumbar intervertebral discs of healthy adult rats (as a basis) began with fixing thin slices of paraffin-embedded tissue onto glass slides. The samples were then treated with an antibody specific to the chosen antigen that was then coupled to a biotinlabeled secondary antibody. Finally, the antigens were localized using colorimetric staining. Reverse transcription-polymerase chain reaction (RT-PCR) was also employed to understand gene expression changes within the disc. An RNA extraction was first performed, then RTPCR to create first-strand cDNA, and finally, standard PCR to amplify a desired gene. RESULTS: The nucleus pulposus on the slide experienced some degradation during the fixing and staining procedures. The collagen I staining proved the most problematic, with staining occurring within the inner annulus and nucleus, a result contradictory with