Neuroprotective Effect of a Nutritional Supplement Containing Spearmint Extract, Forskolin, Homotaurine and Group B Vitamins in a Mouse Model of Transient Ocular Hypertension

**Background:** Glaucoma is a multifactorial neurodegenerative disease characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve damage. Elevated intraocular pressure (IOP) is the main risk factor, but IOP-lowering therapy alone often fails to halt disease progression, indicating a need for neuroprotective strategies. Nutritional supplements with antioxidant, anti-inflammatory, and neuroprotective properties have been proposed as adjuvants. The dietary supplement Gangliomix contains forskolin, homotaurine, spearmint extract (rich in polyphenols), and B vitamins (PP, B1, B2, B6, B12). This study evaluated its neuroprotective efficacy in a mouse model of acute glaucoma induced by transient ocular hypertension.

**Methods:** Male C57BL/6J mice were treated daily with Gangliomix (125 mg/kg) or vehicle via oral gavage for 10 days before and 7 days after ischemia. Transient ocular hypertension was induced by elevating IOP above 90 mmHg for 60 minutes via anterior chamber cannulation. RGC survival was assessed by retrograde FluoroGold labeling and Brn3a immunohistochemistry. Retinal structure was evaluated by H&E staining. Calpain activation was measured by immunoblotting for 150/145 kDa α-spectrin breakdown products (SBDPs) at 1 hour reperfusion. Apoptosis was assessed by caspase-3 activation and PARP cleavage at 24 hours. Astrogliosis was evaluated by GFAP expression at 7 days. IOP was monitored before, during, and after ischemia.

**Key Results:** Gangliomix significantly prevented RGC loss: FluoroGold-labeled RGC survival was 65 ± 6.3% in treated vs. 48 ± 4.2% in vehicle-treated ischemic retinas (p < 0.05). Brn3a-positive cell counts were also preserved. Retinal thickness and GCL cell density were partially maintained. Calpain activation (150/145 kDa SBDPs) was significantly reduced in Gangliomix-treated mice (p < 0.05). Caspase-3 activation and PARP cleavage were also significantly decreased (p < 0.05). GFAP upregulation was mitigated by Gangliomix. IOP values did not differ between groups, indicating an IOP-independent neuroprotective effect.

**Clinical Implications:** This study demonstrates that oral supplementation with Gangliomix provides neuroprotection in an acute glaucoma model by reducing excitotoxicity, apoptosis, and astrogliosis without affecting IOP. The multi-target approach—addressing oxidative stress, inflammation, mitochondrial dysfunction, and NAD+ metabolism—may offer synergistic benefits. These findings support the potential of Gangliomix as an adjuvant to IOP-lowering therapy in glaucoma patients. However, limitations include the acute nature of the model and the need for clinical trials to confirm efficacy and safety in humans.