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Protective Efficacy of a Dietary Supplement Based on Forskolin, Homotaurine, Spearmint Extract, and Group B Vitamins in a Mouse Model of Optic Nerve Injury

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p < 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p < 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p < 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina.


Introduction
Glaucoma, an optic neuropathy that involves optic nerve (ON) head injury associated with visual field defects, is a leading cause of blindness worldwide [1]. The main risk of developing glaucoma is an increase in intraocular pressure (IOP), which results in a compression of the ON head at the level of the lamina cribrosa, triggering axon degeneration and further apoptotic death of RGC [2]. Therefore, pharmacological treatments focused on lowering IOP are currently used to counteract glaucoma progression [3]. However, these treatments are not completely effective in managing the disease and effective therapies without side effects have not been identified yet [4]. The typical feature of glaucoma is retinal ganglion cell (RGC) degeneration following RGC axon damage that is mainly induced by IOP elevation, although RGC death may also occur despite normal IOP [1]. In this respect, neuroprotective strategies independent from the use of IOP lowering drugs or as an adjuvant of these agents, remain a challenge.
Among rodent models of glaucoma, the optic nerve crush (ONC) model that mimics the IOP-induced compression at the ON head [5], is used to induce a rapid degeneration of RGC axons and an acute RGC injury, eventually leading to RGC death with relatively little inter-animal variability [6]. The mechanism of RGC death after ONC is not fully understood, although inflammatory events triggered by ON damage seem to play a major role [7]. In the inflammatory response, activated glial cells surrounding RGCs are characterized by the phosphorylation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), a critical regulator of inflammatory processes, which leads to increased expression of inflammatory cytokines as demonstrated in DBA/2J mice, a model of inherited glaucoma [8]. A major role of inflammation in RGC death is also supported by upregulated levels of inflammatory cytokines as found in the aqueous humor of glaucoma patients, suggesting that acute inflammatory processes are ongoing in eyes with increased IOP [9]. On its hand, inflammation acts as a switch activating apoptotic events ultimately leading to RGC death as demonstrated in both experimental models of glaucoma and glaucoma patients [10,11]. RGC loss results in altered RGC function that can be detected by pattern electroretinogram (PERG), which may be used to determine RGC dysfunction in concomitance with the progressive thinning of the RGC layer and the damage of both the inner nuclear layer (INL) and the optic nerve [12]. There are also several indications that the amplitude of the photopic negative response (PhNR) to light flashes is well correlated with the integrity of the RGC layer, thus providing an objective assessment of RGC functionality [13]. PhNR is a negative-going wave following the b-wave of the cone response that originates in the inner retinal layer and correlates well with the INL integrity and function [14]. In addition, PhNR can be easily recorded using conventional ERG thus allowing to determine the aand b-wave amplitudes that reflect the function of the retinal layers above RGCs that, instead, do not participate in ERG generation [15]. In the mouse model of ONC, reduced amplitude of PhNR is likely to be associated with damaged RGC function due to RGC death [16].
Although it is less clear why IOP elevation selectively compromises RGC survival, a major priority is to protect retinal neurons from death, which necessitates the elucidation of molecular/cellular mechanisms underlying RGC loss. In the ONC model, for instance, blocking the signaling pathway of the cytokine tumor necrosis factor α or activating autophagic processes by selective drugs have been demonstrated effective strategies in improving RGC survival [17,18]. In patients, glaucoma is treated by ocular hypotensive pharmacological approaches that reduce the production of eye fluid and/or improve how fluid drains from the eye [19].
In addition to pharmacological therapies, increasing evidence support the efficacy of nutritional supplements in the treatment of ocular pathologies associated with retinal cell degeneration. In glaucoma models, for instance, long-term dietary interventions with resveratrol or α lipoic acid have been demonstrated to be protective against RGC death [20,21]. In addition, there are several indications that dietary supplementation may play a role in the treatment or prevention of human glaucomatous related pathologies [22]. Particularly, some nutrients have proven capable of lowering IOP, increasing circulation to the optic nerve, modulating excitotoxicity, and promoting RGC survival [23]. The additional fact that dietary supplementation plays a main anti-inflammatory role [24] and that inflammatory processes are major triggers of retinal cell degeneration as in the case of glaucoma, allowed us to hypothesize that nutritional compounds may participate to protect retinal cells from degenerative processes. The present study was conducted to determine whether a diet supplementation with an association of forskolin, homotaurine, spearmint extract, and vitamins of the B group promotes the survival of RGCs in a mouse model of ONC injury and to investigate the neuroprotective mechanisms against RGC loss including an evaluation of the compound efficacy on ONC-associated inflammatory processes and apoptotic cascade.

Animals
The present work was performed in agreement with the guidelines of both the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This study also adheres to the European Communities Council Directive (2010/63/UE) and the Italian guidelines for animal care (DL 26/14). The Commission for Animal Wellbeing of the University of Pisa approved the experimental protocol (Permit Number: 0009069/2014). According to 3Rs principles for ethical use of animals in scientific research, all efforts were made to reduce both the number of mice and their suffering. Mice (C57BL/6J strain) were furnished by Charles River Laboratories Italy (Calco, Italy) and a breeding colony was established in the animal facility of the Department of Biology. Fifty-six mice either male or female (8 weeks old) were used.

Optic Nerve Crush (ONC)
ONC surgery was performed in agreement with Loskutova et al. [23]. After anesthesia with Avertin, the temporal conjunctiva of the mice was incised, and the lateral rectus muscle was dissected. Taking care to avoid damages to blood vessels or muscles, the optic nerve was exposed and clamped 1 mm posterior to the globe with Dumont no. 5 self-closing tweezers (Ted Pella Inc., Redding, CA, USA) for 5 seconds. The surgical site was then treated with an antibiotic ointment. Following surgery, mice did not exhibit any abnormal eating and drinking behavior. The optic nerves of both eyes were crushed in agreement with previous studies [25,26] and the effects of ONC in operated and in control mice were then compared. In this respect, there are some limitations to the use of the non-damaged eye as an internal control for a number of reasons. For instance, proliferating glial cells may migrate from the operated eye to the uninjured contralateral retina [27]. In addition, contralateral phagocytic microglial response has been observed after ON axotomy [28]. Moreover, contralateral response to ON injury seems to involve the expression of neurofilament markers that characterize damaged RGCs in the non-crushed eye, although RGC number did not significantly decrease [29].

Retinal Vasculature
As shown in previous studies, the crush of the ON performed distal to the globe seems to preserve retinal circulation [30]. To confirm that retinal blood flow was not altered by the crush, 3 crushed mice and 3 control mice underwent fluorangiography 1 hour after ONC. In particular, anesthetized mice were perfused with phosphate-buffered saline (50 mL) followed by 5 mg/mL fluorescein isothiocyanate dextran (20 mL). The eyes were then dissected, and after collection, retinas were flat mounted. Images were acquired as detailed below. Quantitative evaluation of vessel density was also performed as previously reported [31]. Vessel area was measured in 8 fields of 100 pixels x 100 pixels for each retina (4 in the central retina and 4 in the peripheral retina) using Adobe Photoshop CS3 (AdobeSystems, MountainView, CA, USA).

Dietary Supplementation
The diet supplement used in the present study is designated as Gangliomix®and is marketed in tablets by Sooft Italia SpA (Montegiorgio, Italy). The dose presently in use for humans is 10 mg/Kg taken once or twice per day. We have converted this dose, taking into account the different metabolism of the mouse [32]. Therefore, animals were treated with 2.5 mg/Kg twice per day, which corresponds to a dose in between the higher and lower amounts advised for humans. In particular, 125 mg of Gangliomix active components were suspended in 10 mL of vehicle (10% sucrose in water). This amount of Gangliomix corresponds to 86.7 mg of a dry extract of spearmint (containing 20.9 mg of total polyphenols and 12.6 mg of rosmarinic acid) designated as Neumentix, 19.3 mg of dry extract of Coleus forskohlii (titrated at 10% in forskolin), 14.5 mg of homotaurine, 2.7 mg of vitamin PP, 0.4 mg of vitamin B2, 0.4 mg of vitamin B6, 0.3 mg of vitamin B1, and 0.5 mg of vitamin B12. Neumentix, which is rich in polyphenols and rosmarinic acid, is believed to be most beneficial for cognitive support by providing neurons with trophic supply [33] while the additional components of Gangliomix are also known to play a neuroprotective role [34,35].
Gangliomix (200 µL of the suspension) was administered by oral gavage for 14 days before and 14 days after ONC. Although not allowing to discriminate between preventive and curative efficacy, this regimen is supported by previous findings in a mouse model of dry AMD, in which fatty acids supplementation after the model was established, was nearly inferior (however, not nihil) to the preand post-supplementation [36]. In this respect, food supplements cannot be intended as therapeutic agents to be taken to treat a certain pathology, but they are used to integrate the normal diet with elements that are not present in a sufficient amount. At most, given enough experimental evidence, they can be taken to participate in reacting against a pathological insult, cooperating with therapeutic drugs, or enhancing their efficacy.
Uncrushed and crushed mice, either unfed or fed with vehicle or diet supplements, were used. Evaluation of retinal function as detailed below did not differ between unfed controls and controls fed with Gangliomix. The parameters evaluated here did not significantly differ between unfed or vehicle-fed ONC mice. Twenty mice were used as controls (10 unfed mice and 5 mice in each of the feeding groups), while 24 mice were used in the ONC group (10 unfed mice and 7 mice in each of the feeding groups). Mice were further subdivided into smaller groups to be used in the experimental procedures detailed below. Except for electroretinography, in which the effects of Gangliomix on PhNR were tested in 7 animals for each group, the additional experiments were carried out on 3 or 4 mice for every experimental condition. In this respect, sampling/experimental design was carried out before conducting the study to optimize the sample size that would assure an adequate power to detect statistical significance as detailed below (paragraph 2.8).

Measurement of the Photopic Negative Response
Two weeks after ONC, RGC function was evaluated by measuring the PhNR as previously described [37]. Electroretinographic recordings were made using a Ganzfeld stimulator (Biomedica Mangoni, Pisa, Italy). Mice were dark adapted overnight and the electroretinographic responses were recorded using Ag/AgCl corneal electrodes. A reference electrode was placed on the forehead while a ground electrode was placed on the tail. ERG was recorded at the light intensity of 3 cd-s/m 2 in mice that were light adapted for 10 min on a background light intensity of 30 cd/m 2 . Ten waveforms were measured from each animal and results were averaged. The PhNR amplitude was recorded from the baseline to the trough following the b-wave. PhNR amplitude was compared among the experimental groups.

RGC Immunohistochemistry and Quantification
After anesthesia, 3 mice for each experimental condition were sacrificed and their eyes were isolated. Retinas were then explanted, fixed in 4% paraformaldehyde (dissolved in 0.1 M phosphate buffer -PB) for 90 min at 4 • C, and stored at 4 • C in 25% sucrose dissolved in 0.1 M PB. Retinas were then incubated in a primary antibody against Brn3 (1:100 dilution in PB containing 5% BSA and 2% TritonX-100; sc-6026; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h at 4 • C. After washing with PB, retinas were incubated overnight in a secondary antibody conjugated to AlexaFluor 488 (1:100 dilution; A-16001; Molecular Probes, Eugene, OR, USA) at 4 • C. After washing with PB, retinas were mounted on glass slides with the vitreous side facing up. RGCs labeled with Brn3 antibody were viewed with a fluorescence microscope (Ni-E; Nikon-Europe, Amsterdam, The Netherlands). Images were acquired with DS-Fi1c camera (Nikon-Europe) and Brn3-positive cells were counted using NIS-Elements software (Nikon-Europe).

Western Blot
Anesthetized mice (4 for each experimental condition) were sacrificed, their eyes were isolated, retinas were explanted, and stored at −80 • C. Each of the analyzed samples contained two retinas from two different mice. The RIPA buffer supplemented with phosphatase and proteinase inhibitor cocktails (Roche Applied Science, Indianapolis, IN) was used for retinal homogenization. The Micro BCA Protein Assay (Thermo Fisher Scientific, Whaltam, MA, USA) was used for protein quantification. Samples containing 30 µg proteins were electrophoresed under reducing conditions on 4%-20% SDS-PAGE gels. Proteins were then transferred on polyvinylidene difluoride membranes and blots were blocked with 5% skim milk (1 h at room temperature). Blots were then incubated overnight at 4 • C with the primary antibodies listed in Table S1. After washing, blots were then transferred to HRP-conjugated secondary antibodies (1:5000 dilution, 1 h at room temperature). Finally, blots were developed with the Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After images acquisition (ChemiDoc XRS + ; Bio-Rad Laboratories, Inc., Hercules, CA, USA), the optical density (OD) of the bands was evaluated (Image Lab 3.0 software; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Data were normalized to the corresponding OD of β-actin or NF-κB as appropriate. All experiments were performed in duplicate.

Statistical Analysis
Statistical significance was evaluated with Prism 5.03 (GraphPad Software, Inc., San Diego, CA, USA) using non-parametric tests. In particular, the Mann-Whitney test was used to evaluate differences in vessel density. In addition, the Kruskal-Wallis test followed by the Dunn's multiple comparison test was used to evaluate differences in PhNR amplitude, RGC numbers, and optical density ratio. Results were expressed as box plots. Differences with p < 0.05 were considered significant. According to the 3Rs principles for ethical use of animals in scientific research, an a priori power analysis was performed (G*Power 3.0.10, www.gpower.hhu.de). Sample size was calculated considering α = 0.05, a size effect of at least 1 (a size effect sufficiently high to evaluate relevant differences between groups), and a statistical power of at least 0.80. After data collection, a post hoc power analysis was performed to confirm the validity of our assumption. Power values are indicated in the figure legends.

The Effect of ONC on Retinal Vasculature
To confirm the successful establishment of the ONC model, crushed mice were perfused with fluorescein isothiocyanate-dextran 1 h after ONC and compared with controls to explore the possibility that intra-retinal vasculature would be impaired. As shown in Figure 1A,B, no area of capillary non-perfusion or vascular leakage as indicative of retinal ischemia could be observed after ONC. The quantitative evaluation in Figure 1C demonstrates that 1 h after ONC, no difference in vessel density could be observed, suggesting that no angiogenic processes were activated in the crushed retina at this time point.

Effects of Diet Supplementation with Gangliomix on PhNR Amplitude
To assess the efficacy of either Gangliomix on retinal function, we analyzed the amplitude of the PhNR. As shown in Figure 2, in mice with ONC supplemented with vehicle, the amplitude of the PhNR was lower than in control mice. Dietary supplementation with Gangliomix prevented the ONC-induced reduction of PhNR amplitude ( Figure 2A). As shown by the quantitative analysis in Figure 2B, the amplitude of the PhNR was significantly reduced in the ONC model by about 38% as compared to control mice (p < 0.001). In comparison to vehicle-fed mice, Gangliomix administration prevented the reduction in PhNR amplitude by almost 45% (p < 0.01).

Effects of Diet Supplementation with Gangliomix on PhNR Amplitude
To assess the efficacy of either Gangliomix on retinal function, we analyzed the amplitude of the PhNR. As shown in Figure 2, in mice with ONC supplemented with vehicle, the amplitude of the PhNR was lower than in control mice. Dietary supplementation with Gangliomix prevented the ONC-induced reduction of PhNR amplitude ( Figure 2A). As shown by the quantitative analysis in Figure 2B, the amplitude of the PhNR was significantly reduced in the ONC model by about 38% as compared to control mice (p < 0.001). In comparison to vehicle-fed mice, Gangliomix administration prevented the reduction in PhNR amplitude by almost 45% (p < 0.01).

Effects of Diet Supplementation with Gangliomix on RGC Survival
We next addressed the question of whether Gangliomix efficacy on retinal function was accompanied by a reduction of RGC loss that characterizes the ONC model [6]. To this purpose, the number of surviving RGCs two weeks after ONC was evaluated. This was done by immunostaining the RGCs with their marker Brn3. As shown in Figure 3, as compared to control mice ( Figure 3A), a significant RGC loss was observed in retinal whole mounts of mice with ONC fed with the vehicle ( Figure 3B). Gangliomix counteracted the ONC-induced RGC degeneration ( Figure 3C) as also confirmed by the high magnification images ( Figure 3D-F) of the boxed areas shown in Figure 3A-C. As shown in Figure 3G, RGC quantification demonstrates that in mice with ONC fed with the vehicle, the RGC number decreased by about 50% as compared to that measured in control mice (p < 0.001). Gangliomix supplementation significantly prevented the RGC loss (p < 0.01) with about 65% spared RGCs as compared to vehicle supplementation.

Effects of Diet Supplementation with Gangliomix on RGC Survival
We next addressed the question of whether Gangliomix efficacy on retinal function was accompanied by a reduction of RGC loss that characterizes the ONC model [6]. To this purpose, the number of surviving RGCs two weeks after ONC was evaluated. This was done by immunostaining the RGCs with their marker Brn3. As shown in Figure 3, as compared to control mice ( Figure 3A), a significant RGC loss was observed in retinal whole mounts of mice with ONC fed with the vehicle ( Figure 3B). Gangliomix counteracted the ONC-induced RGC degeneration ( Figure 3C) as also confirmed by the high magnification images ( Figure 3D-F) of the boxed areas shown in Figure 3A-C. As shown in Figure 3G, RGC quantification demonstrates that in mice with ONC fed with the vehicle, the RGC number decreased by about 50% as compared to that measured in control mice (p < 0.001). Gangliomix supplementation significantly prevented the RGC loss (p < 0.01) with about 65% spared RGCs as compared to vehicle supplementation.  Figure 4 shows the ONC-induced expression of inflammatory markers and the effects of Gangliomix supplementation on their levels. With respect to controls, ONC significantly increased the protein levels of GFAP, iNOS, and IL-6 as well as the phosphorylated form of the p65 subunit of NF-κB (p < 0.001). Gangliomix decreased the upregulated levels of all the above inflammatory markers by a value ranging from 44% for GFAP to 60% for iNOS (p < 0.001).  Figure 4 shows the ONC-induced expression of inflammatory markers and the effects of Gangliomix supplementation on their levels. With respect to controls, ONC significantly increased the protein levels of GFAP, iNOS, and IL-6 as well as the phosphorylated form of the p65 subunit of NF-κB (p < 0.001). Gangliomix decreased the upregulated levels of all the above inflammatory markers by a value ranging from 44% for GFAP to 60% for iNOS (p < 0.001).  Then, we evaluated whether apoptotic processes might be affected by Gangliomix. To this purpose, we measured the protein levels of the apoptotic markers Bax, Bcl-2, and caspase 3, as an increase in the Bax/Bcl-2 ratio is known to trigger an apoptotic cascade ultimately leading to the activation of those caspases responsible for the execution of cell death, including caspase 3. As shown by the representative blots in Figure 5A and the corresponding densitometric analysis ( Figure 5B,C), in ONC mice, the level of pro-apoptotic Bax was increased, while the level of anti-apoptotic Bcl-2 was decreased leading to an increased Bax/Bcl2 ratio. In addition, an increase in the protein levels of the active form of caspase 3 could be observed in ONC mice. As compared to ONC mice fed with the vehicle, Gangliomix was found to reduce the Bax/Bcl-2 ratio and active caspase 3 levels by about 68% and 50% (p < 0.001), respectively. Then, we evaluated whether apoptotic processes might be affected by Gangliomix. To this purpose, we measured the protein levels of the apoptotic markers Bax, Bcl-2, and caspase 3, as an increase in the Bax/Bcl-2 ratio is known to trigger an apoptotic cascade ultimately leading to the activation of those caspases responsible for the execution of cell death, including caspase 3. As shown by the representative blots in Figure 5A and the corresponding densitometric analysis ( Figure 5B,C), in ONC mice, the level of pro-apoptotic Bax was increased, while the level of anti-apoptotic Bcl-2 was decreased leading to an increased Bax/Bcl2 ratio. In addition, an increase in the protein levels of the active form of caspase 3 could be observed in ONC mice. As compared to ONC mice fed with the vehicle, Gangliomix was found to reduce the Bax/Bcl-2 ratio and active caspase 3 levels by about 68% and 50% (p < 0.001), respectively.

Discussion
Optic neuropathies are neurodegenerative diseases that affect the optic nerve characterized by the gradual damage of RGCs and their axons. Among optic nerve diseases, glaucoma, which represents the second most common cause of blindness in the world and is expected to affect about 80 million people worldwide by 2020, is more often associated with elevated IOP that leads to RGC degeneration with consequent alterations of the visual function [1]. Since RGCs play a pivotal role in the process of vision, their degeneration, which begins as an early focal injury to the axons and progresses to apoptotic death, results in a gradual loss of visual acuity that, if left untreated, leads to irreversible blindness [38].
Glaucoma is intensely investigated at the preclinical and clinical levels, but its pharmacological management is complex, and no targeted therapies are available to cure the disease. Presently, available first line medications used to prevent glaucoma progression only include IOP decreasing drugs such as beta blockers, carbonic anhydrase inhibitors, alpha-2 adrenergic agonists, and prostaglandin analogs [19].
The recent trend to use nutritional approaches in addition to drug therapy to possibly counteract eye diseases with a major inflammatory component is gaining more and more interest as an increasing amount of scientific data highlight the ability of diet supplements to cross the blood retinal barrier, and to modulate inflammatory pathways that account for neurodegenerative processes in the retina [24]. Most literature refers to the protective effects of omega-3 fatty acids supplementation on RGC death after optic nerve injury in mice [39]. Additionally, long-term dietary resveratrol treatment has been shown to protect the retina against RGC morphological changes after optic nerve injury [20].
Here, we demonstrate the efficacy of the association of forskolin, homotaurine, spearmint extract, and vitamins of the B group on ONC-induced retinal dysfunction and RGC survival. This is demonstrated on the one hand by the preserving effects of Gangliomix on the amplitude of the PhNR, a sensitive marker of functional RGC [15,[40][41][42][43][44]; and on the other hand, by the increased immunostaining with Brn3, a survival transcription factor of RGC [45] resulting in an improved ratio of Bax/Bcl2 and decreased expression of the apoptosis effector caspase-3. These findings suggest that Gangliomix prevents RGC death by blocking early events in the apoptotic cascade. This, in turn, may depend on the reduced levels of inflammatory markers after Gangliomix supplementation, which are likely to participate to RGC recovery. Among them, GFAP is a marker of glia activation, in particular of Müller cell gliosis [46]. Once activated after optic nerve damage, as in the ONC model or after IOP increase as in the inherited glaucoma mouse model, glial cells become hypertrophic and induce the

Discussion
Optic neuropathies are neurodegenerative diseases that affect the optic nerve characterized by the gradual damage of RGCs and their axons. Among optic nerve diseases, glaucoma, which represents the second most common cause of blindness in the world and is expected to affect about 80 million people worldwide by 2020, is more often associated with elevated IOP that leads to RGC degeneration with consequent alterations of the visual function [1]. Since RGCs play a pivotal role in the process of vision, their degeneration, which begins as an early focal injury to the axons and progresses to apoptotic death, results in a gradual loss of visual acuity that, if left untreated, leads to irreversible blindness [38].
Glaucoma is intensely investigated at the preclinical and clinical levels, but its pharmacological management is complex, and no targeted therapies are available to cure the disease. Presently, available first line medications used to prevent glaucoma progression only include IOP decreasing drugs such as beta blockers, carbonic anhydrase inhibitors, alpha-2 adrenergic agonists, and prostaglandin analogs [19].
The recent trend to use nutritional approaches in addition to drug therapy to possibly counteract eye diseases with a major inflammatory component is gaining more and more interest as an increasing amount of scientific data highlight the ability of diet supplements to cross the blood retinal barrier, and to modulate inflammatory pathways that account for neurodegenerative processes in the retina [24]. Most literature refers to the protective effects of omega-3 fatty acids supplementation on RGC death after optic nerve injury in mice [39]. Additionally, long-term dietary resveratrol treatment has been shown to protect the retina against RGC morphological changes after optic nerve injury [20].
Here, we demonstrate the efficacy of the association of forskolin, homotaurine, spearmint extract, and vitamins of the B group on ONC-induced retinal dysfunction and RGC survival. This is demonstrated on the one hand by the preserving effects of Gangliomix on the amplitude of the PhNR, a sensitive marker of functional RGC [15,[40][41][42][43][44]; and on the other hand, by the increased immunostaining with Brn3, a survival transcription factor of RGC [45] resulting in an improved ratio of Bax/Bcl2 and decreased expression of the apoptosis effector caspase-3. These findings suggest that Gangliomix prevents RGC death by blocking early events in the apoptotic cascade. This, in turn, may depend on the reduced levels of inflammatory markers after Gangliomix supplementation, which are likely to participate to RGC recovery. Among them, GFAP is a marker of glia activation, in particular of Müller cell gliosis [46]. Once activated after optic nerve damage, as in the ONC model or after IOP increase as in the inherited glaucoma mouse model, glial cells become hypertrophic and induce the activation of NF-κB, which promotes the early release of pro-inflammatory cytokines, including IL-6, and the accumulation of inflammatory mediators such as iNOS [8,47]. Interestingly, inflammatory cytokines have been also shown to promote RGC survival [48][49][50], in line with the notion that both reparative and pathogenic factors coexist in the inflammatory microenvironment [51]. In glaucoma, in particular, early stages of the disease are characterized by protective effects of the inflammatory response [52]. However, chronicization of the inflammatory process put RGC survival at high risk [53].
Among Gangliomix components, Neumentix is a spearmint extract rich in polyphenols and rosmarinic acid that has been demonstrated to improve working memory and attention by reducing oxidative stress thus possibly counteracting neuron degeneration [33]. No information is available on Neumentix efficacy in degenerative pathologies of the retina, although there are indications that polyphenols, acting as anti-inflammatory and antioxidant compounds, can aid anti-glaucoma therapy by providing metabolic support to the cells involved in glaucomatous injury [54]. Among polyphenols, flavonoids demonstrate protective effects on RGC death by reducing inflammation and oxidative stress in cell culture as well as in the ONC model [22]. A recent meta-analysis has shown no significant effect of flavonoids on lowering IOP in glaucoma patients [55], thus suggesting a pure neuroprotective effect. Among the additional components of Gangliomix, a major effect of forskolin has been demonstrated in glaucoma patients [56]. When provided in combination with beta blockers, prostaglandins or alpha-2 adrenergic agonists forskolin has the capacity to reduce IOP beyond the levels achieved with traditional therapy alone [56,57]. Forskolin not only exerts indirect beneficial neuroprotective effects on RGCs by reduction of the IOP, but also exerts direct neuroprotection through different mechanisms including the activation of trophic factor expression by astrocytes and vascular endothelial cells, the translocation of the neurotrophic factor receptor TrkB to the neuron cell membrane, and cAMP elevation that is known to reduce excitotoxic damage and to inhibit the resulting apoptotic cell death [58]. Additional components of Gangliomix may participate in its neuroprotective efficacy, for instance homotaurine and group B vitamins. Homotaurine is a natural aminosulfonate compound endowed with neuroprotective effects as demonstrated in a rat model of neurodegeneration and in primary retinal cell cultured in neurotoxic conditions [59,60]. In addition, vitamin supplementation may function as potential neuroprotective agents against oxidative stress in glaucoma patients, although the association between serum vitamin levels and glaucoma prevalence in humans remains controversial [22]. In particular, low intake of vitamin B1 is associated with an increased risk of developing glaucoma indicating that diet supplements containing B vitamins may be viewed as a protective strategy to prevent RGC death [61]. In this respect, a synthetic derivative of vitamin B1 displays neuroprotective effects on cultured retinal ganglion cells probably due to its anti-apoptotic properties [62]. The fact that multiple bioactive molecules found in natural compounds may synergistically interact to provide therapeutic efficacy that is higher than that of the individual molecules suggests the possibility that Gangliomix efficacy may depend on synergistic interaction among its different components. For instance, forskolin has been reported to act synergistically with homotaurine to protect RGC from death induced by increased IOP possibly by preventing calpain activation that is associated to neurodegenerative events [34]. In addition, in glaucoma patients with IOP compensated by topical drugs, food supplements containing forskolin, homotaurine, and group B vitamins have been found to increase both PERG amplitude and additional parameters related to RGC function [35]. Overall, literature data indicating the possible synergy between the different bioactive components of Gangliomix suggest that no single constituting element may have, per se, a neuroprotective activity comparable to that of the entire compound.

Conclusions
Taken together, the present evidence that Gangliomix supplementation successfully ameliorates RGC dysfunction by acting as a neuroprotective compound can now offer new perspectives not only for improving our knowledge on the effectiveness of diet supplement in counteracting RGC loss in glaucoma, but also for eventually expanding a complementary nutritional intervention in ocular pathologies associated with RGC degeneration. As shown in the schematic representation of Figure 6, the present data demonstrate an ability of Gangliomix to significantly preserve visual dysfunction by substantially preventing RGC loss through a major anti-inflammatory and anti-apoptotic action.
pathologies associated with RGC degeneration. As shown in the schematic representation of Figure  6, the present data demonstrate an ability of Gangliomix to significantly preserve visual dysfunction by substantially preventing RGC loss through a major anti-inflammatory and anti-apoptotic action.
Extrapolating from animal data the therapeutic efficacy in humans must be approached with caution because animal studies are often poor predictors of human reactions. However, the fact that Gangliomix was indeed effective in the mouse model at a regimen around that presently in use in humans, suggests that Gangliomix may have some potential to show therapeutic efficacy in glaucoma patients or, at least, multi-treatment using Gangliomix in combination with current therapies may show marked improvement in RGC protection. Figure 6. Schematic diagram showing a possible mechanism through which Gangliomix may protect RGCs from loss and ameliorate visual dysfunction. ONC activates inflammatory processes leading to gliosis that, together with inflammation, triggers cytokine secretion ultimately leading to RGC death and visual dysfunction. Gangliomix reduces the levels of inflammatory and apoptotic mediators thus acting as a neuroprotectant, partially preventing RGC degeneration and counteracting visual dysfunction.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Table S1: Primary antibodies used in the Western blot analysis.  RGCs from loss and ameliorate visual dysfunction. ONC activates inflammatory processes leading to gliosis that, together with inflammation, triggers cytokine secretion ultimately leading to RGC death and visual dysfunction. Gangliomix reduces the levels of inflammatory and apoptotic mediators thus acting as a neuroprotectant, partially preventing RGC degeneration and counteracting visual dysfunction.
Extrapolating from animal data the therapeutic efficacy in humans must be approached with caution because animal studies are often poor predictors of human reactions. However, the fact that Gangliomix was indeed effective in the mouse model at a regimen around that presently in use in humans, suggests that Gangliomix may have some potential to show therapeutic efficacy in glaucoma patients or, at least, multi-treatment using Gangliomix in combination with current therapies may show marked improvement in RGC protection.