PAX1 expression in canine intervertebral disc degeneration and nucleus pulposus cell cultivation

Abstract

Part 1 Background – Little is known about phenotype markers of the canine nucleus pulposus cell (NPC). Because of remarkable similarities regarding intervertebral disc (IVD) degeneration between dogs and humans, it is likely that the phenotype is alike as well. Paired box 1 (PAX1) is a marker for the healthy human NPC, whose expression decreases as the degree of degeneration increases. Nothing is yet known about the expression of PAX1 during canine IVD degeneration. Objective – To investigate PAX1 expression in different stages of IVD degeneration in dogs. Methods – Three decalcified canine IVD samples per Thompson grade were used, from both chondrodystrophic and non-chondrodystrophic dog breeds. Immunohistochemical staining was performed and PAX1 immunopositivity ratio was determined for every sample. Results – An increasing pattern in PAX1 immunopositivity from grade 1 towards grade 5 was visible. No significant differences in PAX1 expression were found between different Thompson grades. A significantly strong positive correlation between PAX1 immunopositivity ratio and IVD degeneration grade was found, indicating that the higher the Thompson score, the higher the PAX1 expression in canine NPCs. Conclusion – The correlation that was found in this study does not correspond to the human pattern. In fact, it is the opposite. This could either indicate that PAX1 is not a healthy canine NPC marker or that there is a reason for PAX1 expression to increase, such as a reparative response of the canine NPCs. Furthermore, low sample size could have affected the outcome of this study. Hence, more future research into the role of PAX1 and its expression in canine NPCs is required.

Part 2 Background – For canine nucleus pulposus cell (NPC) cultivation, expansion and redifferentiation media containing fetal bovine serum (FBS) are commonly used. However, due to several disadvantages of FBS, possibilities for reduction of FBS or alternatives are increasingly being sought. Fibroblast growth factor (FGF) might be such an alternative. Cultured cells should express healthy NPC markers. In human cells, Paired box 1 (PAX1) is such a healthy marker. Due to similarities between humans and dogs regarding intervertebral disc degeneration, PAX1 expression was evaluated in canine NPCs cultured in different conditions. Objective – To investigate which expansion medium and redifferentiation medium could be best combined in order to grow the healthiest canine NPCs, considering the amount of FBS during expansion and redifferentiation and the presence of FGF during expansion. Methods – Canine NPCs were collected from three Beagles and cultured in six different conditions; three different expansion media (10% FBS, 5% FBS + FGF and 5% FBS) and two different redifferentiation media (10% FBS and no FBS). Immunohistochemical staining was performed and PAX1 immunopositivity was evaluated for every condition. Results –PAX1 immunopositivity did not vary between different conditions in terms of expansion media. In terms of redifferentiation, cells cultured in media with FBS showed slightly more positive PAX1 staining. FGF did not have an effect on PAX1 immunopositivity or the amount of cells. Conclusion – Considering the aim of reducing the use of FBS, it would be best to use an expansion medium containing 5% FBS, since fewer FBS does not affect PAX1 expression. Regarding redifferentiation, a medium with 10% FBS could best be used for culturing canine NPCs. However, since it is uncertain whether PAX1 is a healthy canine NPC marker, other readouts should be considered as well.