Due to excess in glutamate levels, overstimulation of the glutamate receptors occurs and causes toxicity to retinal neurons resulting in a condition called excitotoxicity. The excitotoxic conditions are stimulated by a set of receptors known as N-methyl-D-aspartate receptors (NMDARs) (Ullian et al., 2004; Skerry and Genever, 2001) and the pathway for expression of glaucomatous neuropathy is mediated via NMDA-mediated excitotoxicity. Thus, in order to maintain normal retinal function, it is critical to remove glutamate from the extracellular space (Gillespie and Monje, 2020; Ishikawa, 2013a). Even though, current medical therapies are focused on lowering of intraocular pressure (IOP), RGC death in glaucoma is also a major risk factor for vision loss and occurs predominantly through apoptosis. Other contributing factors for RGC death are oxidative stress, calcium influx and glutamate release (Ishikawa, 2013b; Torriglia et al., 2016).

Since antioxidants have been widely used as remedial therapies for curing several diseases, studies on optic neuropathies have also employed the use of antioxidants both individually and in combination to combat the degenerative effects in such diseases (M, 2015) (Jung et al., 2020). One such antioxidant is coenzyme Q10 (CoQ10),an endogenous, ubiquitous, lipid-soluble molecule produced by mevalonate pathway (Martucci and Nucci, 2019a; Tsai et al., 2016). More often with increasing age it has been observed that levels of CoQ10 begin to decrease gradually which culminates in the beginning of several diseases causing physical dysfunctions (Huo et al., 2018). Various retinal diseases are caused by a deficiency of CoQ10 associated with glaucoma-related NMDAR degenerations (Adornetto et al., 2020; Fernández-Vega et al., 2020; Morrone et al., 2018; Zhang et al., 2017). It has been established that CoQ10 tends to be neuroprotective by inhibiting formation of the mitochondrial permeability transition pore and increasing survival ofRGCs (Nucci et al., 2007a). A number of experiments performed both in vivo and in vitro suggest the neuroprotective potential of CoQ10 against oxidative stress, elevated IOP, and excitotoxicity, and implicate CoQ10 uptake by the retina as the reason behind positive outcomes in a glaucomatous retina (Zhang et al., 2017; Martucci and Nucci, 2019b).

Another interesting antioxidant is Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an analogue of vitamin E, which acts actively by scavenging peroxyl and alkoxyl radicals. Trolox can slow down lipid peroxidation at lower doses much better than its counterparts (Rozanowska et al., 2019). In addition, trolox treatment successfully reduced apoptotic cell death, by reducing oxidative stress (Sharma and Sayyed, 2006) in a model of diabetes, a disease which also implicates glutamate excitotoxicity as one of the mechanisms for cell death (Ansari et al., 1998). Trolox at a concentration of 30 μM was found to be neuroprotective in one of the studies against rIL-1β-induced neuronal degeneration (Radesäter et al., 2003).

For some time now, studies stating the positive effects of CoQ10 when used alone or in combination with another antioxidant have been conducted and have shown brighter outcomes for ophthalmic neuropathies (Russo et al., 2008). CoQ10 when encapsulated into vitamin E micelles delivered micromolar concentrations of CoQ10 to the vitreous successfully and promoted cell survival (Ekicier Acar et al., 2019). Since, CoQ10 can stabilize mitochondrial membrane potential (MMP) by decreasing reactive oxygen species (ROS), it thereby induces ATP synthesis and leads to protection of neuronal cells (Doozandeh and Yazdani, 2016; Nakajima et al., 2008). Coqun eye drops, a combination of CoQ10 and vitamin E when administrated to glaucoma patients considerably improved retinal function in a clinical trial (Doozandeh and Yazdani, 2016). The resultant theory was that it appeared as if both the agents possess a synergistic antioxidant potential and the plausible cause could be the regenerative capacity of CoQ10 which augmented reduced α-tocopherol (LASS and SOHAL, 2000; Sohal, 2004) when given as a vitamin E/CoQ10 co-therapy (Constantinescu et al., 1994). Furthermore, minimized DNA fragmentation and retinal cell apoptosis were also reported when CoQ10 and vitamin E were topically administered in a rat model of highIOP (Davis et al., 2017; Nucci et al., 2007b) and to patients of open angle glaucoma (Parisi et al., 2014). In some reports, VEGF has been known to act as a trophic factor on non-vascular cells and contribute towards the maintenance and function of RGCs (Shen et al., 2017). It could do so by modulating the exchange between vascular and neuronal compartments, in turn promoting RGC survival in a report where VEGFD was supplemented in conditions of NMDA-induced degeneration (Schlüter et al., 2020). Another reported evidence of RGC survival by VEGFA showed that the growth factor could modulate VEGFR2 signalling via the PI3/Akt pathway and this finding was subsequently confirmed in animal models (Schlüter et al., 2020). Similarly, when RGCs were exposed to conditions of hypoxia, an increase in VEGF concentrations was recorded which promoted the survival of RGCs due to a probable protective role of VEGF (Choi et al., 2019) and suggested that RGCs were responsible for secreting VEGF (Froger et al., 2020). But on the other hand, a contrasting report suggested that VEGF results in neuronal death by Wnt pathway/COX-2 pathway in NMDA induced damage model of RGC loss (Ning et al., 2017). Hence, the underlying role of VEGF remains unclear in the pathogenesis of glaucoma as some reports claim it to possess a neuroprotective function while others suggest its apoptotic function. Due to this contradiction, it necessitates the determination of actual role played by VEGF in neurodegenerative diseases like glaucoma.

Even though elaborate, the above mentioned studies demonstrating the benefits of the combination of CoQ10 and vitamin E/Trolox, lack in their results since they have missed highlighting the underlying protective mechanisms responsible for the neuroprotective function of Trolox in combination with CoQ10 (Lee et al., 2014b). Hence, in the current study we aimed to diffuse the deleterious effects of NMDA and enhance RGC survival, without involving/aiming for IOP reduction, and also tried to elaborate on the role played by VEGF in NMDA-mediated excitotoxicity when using Trolox with CoQ10.