Pathogenesis of Pregabalin-Induced Limb Defects in Mouse Embryos

Purpose. It is known that antiepileptic drugs might adversely affect neuronal function and thus influence brain development. However, we have reported that limb deformities are one of the most prominent disturbances caused by pregabalin (PGB) in the developing embryo. The aim of this work is to gain a better understanding of possible molecular mechanisms behind the musculoskeletal injuries and limb deformities associated with PGB. Methods: Pregnant mice divided into four groups. Each mouse received an intraperitoneal injection (IP) of 0, 20 (group I), 40 (group II) or 80 (group III) mg/kg/day of PGB during the organogenesis period. On gestational day 18, embryos were separated and their limbs were dissected. Levels of apoptotic proteins were analyzed by Western blotting. To establish whether apoptosis is present in the limbs, the specimens were examined by TUNEL. Pathological findings were also reported as a score ranging from 1 to 3 based on the level of differentiation. Results: Western blot analysis demonstrated that PGB in all PGB-treated groups significantly upregulated the levels of cleaved caspase-3, 8 and 9. Also, the results showed that PGB exposure increased the percentage of TUNEL positive cells in different limb tissues especially the mesenchymal tissue. The histopathological findings revealed that PGB administration to pregnant mice inhibited limb tissue differentiation, albeit to varying degrees. Conclusions: The result of our study revealed that apoptosis and inhibition of limb tissue differentiation play an important role in the pathogenesis of PGB-induced limb malformations. Both intrinsic and extrinsic caspase-dependent pathways of cell death are important in mediating the abnormal limb development triggered by insult with the PGB. Evaluating the effect of PGB on molecules involved in the cross-talk between intrinsic and extrinsic apoptotic pathways and cell adhesion, migration, proliferation, and differentiation during embryonic development can further help to identify and clarify the involved mechanisms.