Abstract
Purpose :
There is increasing interest in creatine as a potential agent to provide neuroprotection. Our study aimed to investigate the effect of prophylactic creatine on cultured retinal cells subjected to mitochondrial electron transport chain inhibition as a model of metabolic and energetic dysfunction.
Methods :
Mixed rat retinal cultures comprising neurons and glia were established and treated at six days in vitro with a range of creatine concentrations (no creatine controls, 0.1mM, 0.5mM, 1.0mM, and 5.0mM) for 24 hours. Subsequent to this, half of each group was subjected to a 1-hour or a 24-hour incubation with sodium azide (10mM, N=12; and 1mM, N=4 respectively). Cells were then fixed and processed for immunocytochemistry. Antibodies labelling distinct neuronal populations (Calretinin and GABA) were used for quantification of viable cells in each group. Furthermore, potential mechanisms of protection were also investigated by assessing apoptosis (TUNEL assay) and levels of adenosine triphosphate (ATP). One-way ANOVA followed by Tukey multiple-comparison test was used for statistical analysis.
Results :
1-hour and 24-hour incubations with sodium azide resulted in losses of up to 85% and 98% respectively for both Calretinin-immunoreactive (IR) and GABA-IR neurons. In the 1-hour azide-treated group, prophylactic treatment of creatine at >0.5mM significantly prevented neuronal death by up to 55% for Calretinin-IR neurons (p=0.001) and 30% for GABA-IR neurons (p=0.002). Similarly, concurrent 24-hour incubations of creatine (>0.5mM) and sodium azide prevented neuronal death by up to 36% in the Calretinin-IR population (p=0.024). Application of creatine also led to a reduction in TUNEL-positive nuclei in cultures treated with sodium azide, but did not result in an increased cellular ATP.
Conclusions :
Prophylactic treatment of retinal cells with creatine provided a partial reduction in retinal neuronal loss resulting from metabolic compromise. As creatine was not shown to provide more ATP in this model, further investigations will be needed to elucidate this protection. Nevertheless, if confirmed by in vivo and clinical studies, creatine may be considered as a useful neuroprotective agent that may have a future role to play in the treatment of ocular diseases such as glaucoma or retinal vascular occlusions.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.