Trolox aids coenzyme Q₁₀ in neuroprotection against NMDA induced damage via upregulation of VEGF in rat model of glutamate excitotoxicity

Glutamate induced damage to retinal ganglion cells (RGCs) requires tight physiological regulation of the N-methyl-D-aspartate (NMDA) receptors. Previously, studies have demonstrated the neuroprotective abilities of antioxidants like coenzyme Q₁₀ (CoQ₁₀) and vitamin E analogs like α-tocopherol against neuropathies resulting from NMDA insult, but have failed to shed light on the effect of CoQ₁₀ and trolox, a hydrophilic analog of vitamin E, on glaucomatous neurodegeneration. In the current study, we wanted to investigate whether the combined effect of trolox with CoQ₁₀ could alleviate NMDA-induced death of retinal cells while also trying to elucidate the underlying mechanism in relation to the yet unexplained role of vascular endothelial growth factor (VEGF) in NMDA-mediated excitotoxicity. After successful NMDA-induced degeneration, we followed it up with the treatment of combination of Trolox and CoQ₁₀. The structural damage by NMDA was repaired significantly and retina retained structural integrity comparable to levels of control in the treatment group of Trolox and CoQ₁₀. Detection of ROS generation after NMDA insult showed that together, Trolox and CoQ₁₀ could significantly bring down the high levels of free radicals while also rescuing mitochondrial membrane potential (MMP). A significant increase in NMDA receptor Grin2A by CoQ₁₀ alone as well as by CoQ₁₀ and trolox was accompanied by a lowered Grin2B receptor expression, suggesting neuroprotective action of Trolox and CoQ₁₀. Subsequently, lowered VEGFR1 and VEGFR2 receptor expression by NMDA treatment also recovered when subjected to combined treatment of Trolox and CoQ₁₀. Western blot analyses also indicated the same whereby Trolox and CoQ₁₀ could increase the diminished levels of phosphorylated VEGFR2. Immunofluorescence studies also indicated a positive correlation between recovered VEGFR2 and NMDAR2A levels and diminished levels of NMDAR2D, confirming the results obtained by RT-PCR analysis. This is the first report in our knowledge that demonstrates the efficacy of trolox in combination with CoQ₁₀ highlighting the importance of maintaining VEGF levels that are lowered in ocular diseases due to NMDA-related toxicities.