In-vitro Evaluation of the Neuroprotective Effects of Forskolin on Retinal Ganglion Cells

Abstract

Purpose: The diterpene forskolin is the principal active molecule of the Coleus forskohlii plant and has been shown to process neuroprotective properties. The primary mechanism of action of forskolin is to directly increase the activity of the enzyme adenylyl cyclase and subsequently intracellular cyclic adenosine monophosphate (cAMP). The main aim of this study was to determine whether forskolin was neuroprotective to retinal ganglion cells (RGC) using three different apoptotic insults.

Methods: All experiments were performed in the RGC-5 cell line. Cell viability experiments (using the MTT assay) were conducted on RGC-5 using three different insults known to induce cell death through apoptosis: hydrogen peroxide, cobalt chloride and dimethyl sulfoxide. All data was analysed using unpaired two tailed T-test to assess statistical significance. Experiments using both Hoechst staining and propidium iodide staining were used to assess apoptosis pathways and necrosis in the presence of a caspase inhibitor (e.g. caspace-3 inhibitor).

Results: Forskolin (50uM) was associated with greater cell viability compared to the control in the presence of a hypoxia-inducing apoptotic agent (cobalt chloride 200, 250 and 300 μM) (n=3, p= 0.003, 0.013 and 0.02 respectively). Similar results were noted with forskolin 50 μM in the presence of dimethyl sulfoxide (DMSO) 4% and 6% concentrations (n=3, p= 0.008 and 0.0001 respectively). There was no significant difference in cell viability between forskolin and the control in the presence of hydrogen peroxide. It was also demonstrated that in the presence of hypoxia, forskolin appeared to exhibit a biphasic dose response with peak effect between 25-50 μM. Finally, cobalt chloride induced hypoxia was found to be caspase-3 independent.

Conclusions: Forskolin appears to have a neuroprotective role in RGC-5 particularly against hypoxia-induced apoptotic cell death. This may have implications in conditions where loss of retinal ganglion cells (RGC) and their axons is associated with ischaemia.

Keywords: 615 neuroprotection • 531 ganglion cells