All trans retinoic acid modulates peripheral nerve fibroblasts viability and apoptosis

Following peripheral nerve injury, residing fibroblasts start to proliferate and accumulate at the injury site and may participate in neuroma tissue evolution. Retinoic acid has been shown to regulate many cellular processes and to display anti-proliferative and anti-fibrotic properties. The aim of this study was to investigate the impact of all trans retinoic acid (ATRA) on rat peripheral nerve fibroblasts.

Peripheral nerve fibroblasts and C166 cells were treated with increasing doses of ATRA (0.05 nM to 1 μM). The viability of cells was determined with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, the number of peripheral nerve fibroblasts was counted after two days of ATRA treatment and alternatively up to the end of next week. Acridine orange/ethidium bromide double staining was implemented to morphologically visualize the possible mechanism of cell death. For apoptosis, caspase 3/7 activity was measured using Caspase-Glo 3/7 assay kit.

MTT assay revealed that 0.05-1 nM of ATRA reduces fibroblasts viabilities. Then, almost a plateau state was observed from 1 nM to 1 μM of ATRA exposure. Additionally, a deceleration in peripheral nerve fibroblasts growth was confirmed via cell counting. Quantification of acridine orange/ethidium bromide staining displayed highly increased number of early apoptotic cells following ATRA administration. Amplified activation of caspase 3/7 was in favor of apoptosis in ATRA treated peripheral nerve fibroblasts.

The data from the present study demonstrate that ATRA could interfere in peripheral nerve fibroblasts viabilities and induce apoptosis. Although more investigations are needed to be implemented, our in vitro results indicate that retinoic acid can probably help the regeneration of injured axon via reducing of fibroblasts growth.